1 EXPLORING MOLECULAR MECHANISMS OF LIVER TO PANCREATIC BETA CELL REPROGRAMMING By WILLIAM LEPAGE DONELAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 William LePage Donelan
3 To my financial advisor s and best friend s, Grandma Jane and Grandpa Billy
4 ACKNOWLEDGMENTS This work was su pported in part by grants from T he National Institutes of Health, NIDDK DK071831 and DK64054 (LJ Yang ). I would also like to thank T he University The Pathology Department, The Molecular Cell Biology Concentration, my graduate research committee and T he Laboratory of Dr LiJun Yang (with special thanks to Dr. Shi Wu Li) for supporting my graduate education Most importantly, I thank my friends and family for their love and support.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 12 Type 1 Diabetes Introduction ................................ ................................ .................. 12 Tissue Pathology and Cellular Pathology ................................ ......................... 12 Molecular Pathology ................................ ................................ ......................... 13 Susceptibility: Genetic and Environmental ................................ ....................... 14 Therapy and Treatment ................................ ................................ .................... 15 Diabetic Complications ................................ ................................ ..................... 17 MODY ................................ ................................ ................................ ..................... 18 Transcription Factors ................................ ................................ .............................. 20 Pdx1 ................................ ................................ ................................ ................. 21 Ngn3 ................................ ................................ ................................ ................. 22 Nkx6.1 ................................ ................................ ................................ .............. 23 ................................ ................................ ................................ .............. 24 ................................ ................................ ................................ .............. 35 HNF Re gulatory Circuit ................................ ................................ ..................... 38 2 PDX1 MEDIATED SUPPRESSION OF HNF1 DURING REPROGRAMMING OF HUMAN HEPATIC CELLS TOWARDS PANCREATIC BETA CELLS .............. 40 Introduction ................................ ................................ ................................ ............. 40 Materials and Methods ................................ ................................ ............................ 42 Cell Lines and Cell Culture ................................ ................................ ............... 42 Construction of Plasmids ................................ ................................ .................. 43 Lent iviral Vector (LV) Preparation, Titration, and Transduction ........................ 43 RT PCR and Real Time RT PCR Analysis ................................ ....................... 44 Western Blotting and Immunocytochemistry (ICC) ................................ ........... 45 Transfections and Luciferase Assay ................................ ................................ 45 Statistical Analysis ................................ ................................ ............................ 46 Results ................................ ................................ ................................ .................... 46 Pdx1 VP16 (PV) and Ngn3 Toge ther Strongly Induced Insulin Promoter Activity ................................ ................................ ................................ ........... 46 Pdx1 and Ngn3 Induced Expression of Genes Related to Endocrine Pancreas ................................ ................................ ................................ ....... 47 Quantitative Analysis of PTF Promoter Activities ................................ ............. 48 ................................ ....................... 49
6 in Hepatic Cells ................................ .. 49 Pdx1 Competitive Inhibition ................................ ................................ .................... 50 Discussion ................................ ................................ ................................ .............. 51 3 DISTINCT REGULATION OF HNF1 BY Nkx6.1 IN PANCREATIC BETA CELLS ................................ ................................ ................................ .................... 76 Introduction ................................ ................................ ................................ ............. 76 Materials and Methods ................................ ................................ ............................ 78 Cell Culture ................................ ................................ ................................ ....... 78 Plasmid Construc tion ................................ ................................ ....................... 78 Transient Transfection and Luciferase Assays ................................ ................. 79 Site Directed Mutagenesis ................................ ................................ ................ 79 Electrophoretic Mobility Shift Assay (EMSA) ................................ .................... 80 Chromatin Immunoprecipitation (ChIP) Assay ................................ .................. 80 Gene Expression and Quantitative RT PCR ................................ .................... 81 Statistical Analysis ................................ ................................ ............................ 81 Results ................................ ................................ ................................ .................... 81 .......................... 81 Beta Cells ................................ ................................ ................................ ...... 82 ................................ ................................ 82 ................................ .......................... 83 Promoter in Beta Cells ................................ ................................ .................. 84 .............................. 84 Nkx6.1 O ............. 85 ......... 86 Discussion ................................ ................................ ................................ .............. 86 4 NOVEL DETECTION OF PANCREATIC AND DUODENAL HOMEOBOX 1 (Pdx1) AUTOANTIBODIES (PAA) IN HUMAN SERA USING LUCIFERASE IMMUNOPRECIPITATION SYSTEMS (LIPS) ASSAY ................................ ......... 109 Introduction ................................ ................................ ................................ ........... 109 Materials and Methods ................................ ................................ .......................... 109 Plasmid Construction ................................ ................................ ..................... 109 Fusion Protein Lysate ................................ ................................ ..................... 110 Sera ................................ ................................ ................................ ................ 110 LIPS Assay ................................ ................................ ................................ ..... 111 Results ................................ ................................ ................................ .................. 111 Discussion ................................ ................................ ................................ ............ 113 5 P ANCREATIC AND DUODENAL HOMEOBOX 1 (PDX1) AUTOANTIBODIES (PAA) FROM HUMAN SERA DETECTED IN AUTOIMMUNE DISEASES AND CANCER ................................ ................................ ................................ ............... 122
7 Introduction ................................ ................................ ................................ ........... 122 Materials and Methods ................................ ................................ .......................... 122 Results ................................ ................................ ................................ .................. 123 Discussion ................................ ................................ ................................ ............ 123 6 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 127 LIST OF REFERENCES ................................ ................................ ............................. 134 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 158
8 LIST OF FIGURES Figure page 2 1 Confirmation of LV PTF gene expression by RT PCR.. ................................ ..... 56 2 2 Confirmation of LV PTF gene expression by western blotting.. .......................... 57 2 3 Confirmation of LV PTF gene expression by immunocytochemistry .................. 58 2 4 Activation of rat insulin I gene in Huh7 cells with Pdx1, PV, Ngn3 and combinations ................................ ................................ ................................ ...... 59 2 5 Time course activation of rat insulin I gene in Huh7 cells with Pdx1/Ngn3 or PV/Ngn3 .. ................................ ................................ ................................ ........... 60 2 6 RT PCR analysis of pancreatic gene expression. ................................ .............. 61 2 7 RT PCR analysis of liver specific gene expression ................................ ............ 62 2 8 Pax4 luciferase reporter gene analysis during reprogramming .......................... 63 2 9 NeuroD luciferase reporter g ene analysis during reprogramming ...................... 64 2 10 INS1 luciferase reporter gene analysis during reprogramming ........................... 65 2 11 Ngn3 luciferase reporter gene analysis during reprogramming. ......................... 66 2 12 Nkx2.2 luciferase reporter gene analysis during reprogramming. ...................... 67 2 13 Down regulation of and ATT in Huh7 cells over expressing Pdx1 by western blotting ................................ ................................ .................... 68 2 14 Down regulation of luciferase reporter in Huh7 cells over expressing Pdx1. ................................ ................................ ................................ ................. 69 2 15 Down regulation of by full length Pdx1. ................................ .................. 70 2 16 Pdx1 increases P2 transcripts tha t compete with P1 transcripts. .......... 71 2 17 Cell luc. ........... 72 2 18 Down regulation of luciferase reporter by Pdx1 in 3T3 cells. ................ 73 2 19 Down regulation of luciferase reporter by in 3T3 cells. ............ 74 2 20 A proposed mechanism of Pdx1 medeated hepatocyte to ward IPC reprogramming ................................ ................................ ................................ 75
9 3 1 ....................... 92 3 2 Deletion analysis of the ................................ ..... 93 3 3 ................... 94 3 4 ................................ ................................ ................ 95 3 5 oncentration dependent manner .... 96 3 6 ................................ ................................ ................ 97 3 7 HNF ................................ ................................ 98 3 8 r constructs ................................ ............... 99 3 9 Luciferase activity of Nkx6.1 binding site mutant cells. ................................ ................................ ................................ ................. 100 3 10 r by EMSA with INS1 cell lysate ................. 101 3 11 Nkx6.1 ChIP assay. ................................ ................................ ......................... 102 3 12 ................................ ................................ ........................... 103 3 13 ................ 104 3 14 with NIT1 cell lysate ................. 105 3 15 RT PCR for de termination of gene expression. ................................ ................ 106 3 16 Effect of Nkx6.1 o ver expression on mRNA levels ................................ ........... 107 3 17 Effect of Nk x6.1 knockdown on mRNA levels ................................ .................. 108 4 1 Detection of GAD A in human sera by LIPS assay. ................................ .......... 116 4 2 Detection of IA 2 A in human sera by LIPS assay ................................ ............. 117 4 3 Sensitivity and specificity of LIPS assay vs. RIPA for GADA and IA 2A.. ......... 118 4 4 Detection of PAA in human sera by LI PS assay ................................ ............... 119 4 5 Antigenic specificity of PAA using luciferase only control. ............................... 120 4 6 Antigenic specificity of PAA by competit ion with purified rP dx1 protein ........... 121 5 1 PAA detec ted in human T1D patient sera ................................ ........................ 125 5 2 PAA detected in human autoimmune disease and cancer patient sera. .......... 126
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy E XPLORING MOLECULAR MECHANISMS OF LIVER TO PANCREATIC BETA CELL REPROGRAMMING By William LePage Donelan August 2012 Chair: LiJun Yang Major: Medical Sciences Molecular Cell Biology Ectopic expression of pancreatic transcription factors (PTFs) reprograms hepatic cells toward pancreatic beta cells. However, molecular mechanisms underlying this process have not been well studied, particularly regarding suppression of the hepat ic phenotype. To study the mechanisms involved in hepatic to pancreatic beta cell reprogramming, we generated lentiviral vectors expressing PTFs (Pdx1, Pdx1 VP16, and Ngn3), transduced hepatocellular carcinoma Huh7 cells, and examined the early effects on expression of key pancreatic and hepatic genes. We demonstrated that P dx1 not only activates beta cell specific genes but also suppresses the key hepatic transcription factor HNF1 alpha and hepatic functional gene ALB by RT PCR and western blotting. L uciferase reporter assays further confirmed Pdx1 mediated suppression of HN F1 alpha in hepatic cells HNF1 alpha suppression may be, in part, due to up regulation of HNF4 alpha P2 isoforms, which was also observed following ectopic Pdx1 expression because these isoforms are weaker than P1 driven isoforms and they may compete for b inding the HNF1 alpha proximal promoter. We also cloned a 3kb proximal HNF1 alpha promoter element into the pGL3 luciferase reporter to further study its regulation We found that NK6 homeodomain 1 (Nkx 6.1) binds to a c is
11 regulatory element in the HNF 1 alpha promoter and is a major regulator of this gene in b eta cells. We identified an Nkx 6.1 recognition sequence in the distal region of the HNF 1 alpha promoter and demo nstrated specific binding of Nkx 6.1 in beta cells by EMSAs and ChIP assays. Site direc ted mutagen esis of the Nkx 6.1 core binding sequence eliminates Nkx 6.1 mediated activation and substantial ly decreases activity of the HNF 1 alpha promoter in beta cells. Over expression or siRNA mediated knockdown of Nkx6.1 gene expression results in increa sed or diminish ed HNF 1 alpha gene expression, respectively, in b eta cells. We conclude that Nkx6.1 is a novel regulator of HNF 1 alpha in pancreatic beta cells. develope d a sensiti ve, specific, and non radioactive liquid phase luciferase immunopre cipitation systems (LIPS) assay for detection of PAA W e screened sera from patients with type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, and various forms of cancer ( including pancreatic cancer) and detected positive PAA sera from all groups.
12 CHAPTER 1 LITERATURE REVIEW Type 1 Diabetes Introduction Autoimmune (type 1) diabetes is a disease in which the immune system targets and destroys the insulin producing beta cells within the pancreatic islets of Langerhans resulting in the inability to produce sufficient insulin in response to elevated glucose levels 1, 2 Insulin replacement is the most widely used therapy f or controlling blood glucose concentration to prevent acute disease. Although this therapy provides short term benefit to patients with type 1 diabetes, euglycemia is extremely difficult to maintain. The serious acute complications of hypoglycemia, such as diabetic coma, and the difficulty of maintaining euglycemia lead type 1 diabetic patients to frequently have blood glucose levels that are elevated from the physiological norm. Unfortunately, long term hyperglycemia can damage the integrity of the vasc ulature and is commonly associated with disease of the heart, kidneys, and retinas, and extremities. Tissue Pathology and Cellular Pathology Type 1 diabetes is an autoimmune disease characterized by the immunological destruction of beta cells within the pa ncreatic islets of Langerhans 1 Islets are clusters of endocrine cells consisting of insulin secreting beta cells, glucagon secreting alpha cells, somatostatin secreting delta cells, pancreatic peptide secreting PP cells, and ghrelin producing epsilon cells. Beta cells account for about 70% of cells within the islet and are responsible for synthesizing and releasing insulin in order to maintain physiological blood glucose concentration. Insulin release allows for the cellular uptake and utili zation of glucose for energy. Clinical diagnosis of type 1 diabetes usually occurs after 80% 90% of pancreatic beta cell mass has been destroyed 3 At this time,
13 remaining beta cells often have enlarged nuclei and degranulated cytoplasm and isle ts become abnormally small due to the destruction of the majority of their cells 1 The mechanism by which beta cell destruction occurs is through autoimmune attack 1 This autoimmunity is associated with a chronic inflammatory inf iltrate known as insulitis consisting primarily of CD8+ T lymphocytes, although CD4+ T lymphocytes, B lymphocytes, macrophages, and natural killer cells are also involved in the response. The T cell mediated destruction of beta cells is due to autoreactiv e T cells for specific beta cell proteins including insulin, glutamic acid decarboxylase (GAD), insulinoma 2 associated protein (IA 2), and zinc transporter 8 protein (ZnT8) 4 6 Autoantibodies can serve as an early indicator for increased susceptibility to type 1 diabetes because they are present in 70% 80% of newly diagnosed patients as opposed to their presence in only 0.5% of the general population 1 Molecular Pathology Genes provide both susceptibility and protection for type 1 diabetes 1 3, 5 The greatest contributing genetic component is the major histocompatibility complex (MHC), located on hu man chromosome 6. The region containing the highly polymorphic human leukocyte antigen (HLA) genes has been determined to have the greatest influence on disease susceptibility. There are three classes of HLA genes differing in their function. HLA class I molecules form dimeric proteins composed of alpha chains in association with 2 microglobulin. These molecules are present on the surface of all nucleated cells and platelets and their function is to present antigenic peptide fragments to T cell recepto rs on CD8+ T lymphocytes. The HLA class II molecules consist of three subclasses (HLA DR, HLA DQ, and HLA DP) and are also dimeric proteins composed of alpha and beta chains. They are constitutively expressed in antigen presenting
14 dendritic cells and B l ymphocytes while expression can be induced in macrophages and endothelial cells. These molecules contain a binding site for peptide fragments of antigens and present these antigenic peptides to T cell receptors on CD4+ T lymphocytes. The HLA class III m olecules form a variety of structures with many differing functions ; examples include complement components, tumor necrosis factor, and heat shock protein Hsp70 Although the functions of HLA genes are well characterized, their specific contribution to disease pathogenesis is poorly understood 3, 5 It is obvious that the structural differences between suscepti ve and protective HLA proteins will functionally affect their interaction with antigens and autoreactive T lymphocytes. Several hypot hesized mechanisms have been proposed. It is possible that defective HLA molecules bind to beta cell specific antigens in the periphery and induce an autoreactive T cell response. Another explanation is that unstable HLA/self antigen complexes in the thy mus fail to induce self tolerance and release auto reactive T cells to the periphery. Yet a third hypothesis is that defective HLA molecules alter T cell activity by their interactions with T cell receptors and modulate their phenotype from regulatory to inflammatory. Susceptibility: Genetic and Environmental It is clear that there are both environmental and genetic components related to type 1 diabetes susceptibility 3 This is evident from its familial occurrence ; however, it does not follow a M e ndelian pattern of inheritance, likely because it is a multifactorial disease. In the United States there is increased lifetime susceptibility for first degree relatives of diabetics when compared to members of the general population (5% vs. 0.3%). Twin studies also demonstrate a higher concordance rate for monozygotic twins
15 when compared to dizygotic twins (30 50% vs. 6 10%) 3 Although there is a clear genetic relationship to type 1 diabetes susceptibility, the concordance rate for identical twi ns is very low relative to other genetic diseases Nutrition, viral infections, exposure to toxins, vaccinations, climate, and stress are all commonly implicated in the manifestation of the disease ; however, extensive research has been unable to determin e that any environmental factor actually causes the disease 2, 3, 5 It is therefore likely that genetic susceptibility for type 1 diabetes is inherited and that environmental factors influence or modify its development. Therapy and Treatment Currently, insulin replacement therapy is required to maintain euglycemia and to prevent serious acute disease. Unfortunately, this therapy is incapable of maintaining the equivalent stringent control of blood glucose levels provided by healthy beta cells. Without proper blood glucose regulation, several complications due to vascu lature degradation can occur. New therapies for the treatment or cure of type 1 diabetes must focus on two elements: beta cell replacement and recurrent autoimmunity 2, 7 Transplantation therapies are an option, but donor organs are difficult to acquire and require the use of long term immunosuppression in order to prevent allograft rejection and recurrent autoimmune destruction 2 In addition, average transplantation fails after five years. Islet transplantation is perhaps a safer treatment option, but requires several donor pancreata for each patient 8 10 In vitro differentiation of beta cells, however, could theoretically supply an indefinite source of islet tissue 8 Gene therapy for the generation of beta cells ( or insulin producing beta like cells ) may provide a source of cells for transplantation or a method of in vivo beta cell reprogramming 11 Many studies 12 28 have demonstrated that beta like cell differentiation
16 is inducible by modulating external conditions or delivering pancreatic transcriptional factors. Exposing beta cell precursors to a high glucose environment alone has the ability to shift the cells towards a beta cell gene expression profile 22, 25 It is also clear, from reprogramming studies, that various transcriptional factors exhibit a primary role in the induction of beta cell like differentiation and insulin gene regulation, m ost importantly the pancreatic and duodenal homeobox gene 1 (Pdx1). Depending on the conditions and transcriptional factors used, beta cell precursors can differentiate into glucose responsive insulin producing cells that are morphologically indistinguish able from true beta cells. Several sources for cells have been successfully utilized including hepatic cells, 12 21, 23 28 bone marrow cells, 22 and pancreatic exocrine cells 29 Transp lantation of beta like cells into chemically induced diabetic mice has been shown to restore euglycemia 12 14, 17, 19, 20 22 25, 27, 28 Several methods have been employed in successful gene therapy including direct protein delivery, lentiviral mediated delivery, and protein transduction technology. Protein transduction technology is of specific interest due to its rela tive safety and efficacy of transduction 30 36 Some of the transcriptional factors involved in beta cell like differentiation (Pdx1 31, 35, 36 and NeuroD 3 0, 32 ) contain protein transduction domains (PTDs) which are specific amino acid sequences, enriched with positively charged arginine and lysine residues, allowing for their transduction across cellular membranes by lipid raft mediated macropinocytosis. Molecules can also be engineered to utilize the same mechanism by addition of a PTD, as has been done for TAT mediated Ngn3 37 Following beta cell transplantation or regeneration requires either long term immunosuppression or an immune system modulation to elimi nate the possibility of
17 recurrent autoimmunity. Studies have been focusing on modulation of the T cell repertoire toward a regulatory status with the goal of establishing a T cell profile that consists of a majority of regulatory cells in order to establ ish long term tolerance 7, 38 Diabetic Complications The majority of the morbidity and mortality associated with type 1 diabetes is due to complications arising from poor glycemic control 39 In theory, mai ntaining euglycemia would effectively eliminate the complications related to diabetes, but this is nearly impossible to achieve. Hypoglycemia results in serious acute disease that can quickly lead to diabetic coma and possible death. Recurrent or long la sting episodes of hypoglycemia can lead to cognitive impairment and predisposes individuals to future hypoglycemic occurrences. The fear of hypoglycemia often leads patients to maintain hyperglycemia because the short term problems are much less severe. However, long term hyperglycemia leads to several complications as the result of microvascular and macrovascular disorders including nephropathy, retinopathy, neuropathy, and cardiovascular disease 39 Vascular dysfunction plays a major role in the aforementioned complications related to long term hyperglycemia. The damage to endothelial tissue results in organ and tissue failure. Hyperglycemia leads to the accumulation of reactive oxygen species such as superoxide, hydrogen peroxide, and hypochlorous acid and also reactive nitrogen species such as nitric oxide 40 The reactive oxygen species initiate lipid pe roxidation leading to inflammation. The increase in oxidative stress is due to advanced glycation end product accumulation which activates vascular NADPH oxidase. Mitochondrial uncoupling and activation of poly ADP ribose polymerase also plays a role res ulting in endothelial damage.
18 Endothelial progenitor cells play a major role in protecting the integrity of the vasculature system as well as promoting angiogenesis 41 They are derived from the bone marrow and can be mobilized through release of growth factors and cytokines to sites of ischemia and endothelial injury. At the site of injury endothelial progenitor cells adhere to the vessel walls where they begin to migrate through the extracellular matrix. Their expansion leads to the formation of new vasculatu re structures. Most type 1 diabetic patients have decreased levels of circulating endothelial progenitor cells and increased dysfunction of these cells. The accumulation of vascular ischemic sites prevents adequate blood supply to several tissues and can lead to organ failures and critical limb ischemia requiring amputation. MODY Monogenetic diabetes accounts for 1 2% of all diabetes cases and because it is caused by mutation in a single gene, it can be diagnosed by genetic testing 42 45 Maturity onset diabetes of the young (MODY) is caused by autosomal dominant mutation in one of at least six beta cell specific genes incl uding the enzyme glucokinase NeuroD1 that result in diabetes are respectively termed MODY1 6 46 49 MODY2 is characterized by hyperglycemia onset at birth that does not progress with age and is most often controlled by diet. MODY2 patients rarely develop complications due t o hyperglycemia. MODY3 is the most prominent form of MODY and accounts for about 70% of the disease. MODY1 is less common, accounting for about 5% of MODY but has similar disease progression as MODY3. These forms are characterized by adolescent onset of hyperglycemia that progressively worsens with age and most often
19 requires pharmacological treatment. Patients with MODY1 or MODY3 often suffer from is an important develo pmental transcription factor, MODY4 is characterized by a wide range of developmental pathologies that affect several systems and accounts for about 2% of MODY. The other transcription factor forms of MODY have only been found in a few families and accoun t for less than 2% of MODY. In general, they progress similarly to MODY1 and MODY3 but they are too rare to be well characterized. In more than 10% of patients who are considered to have MODY, the cause is unknown and suggests that other genes or unknown regulatory elements for the known MODY genes are likely to contribute to disease. The group comprising unknown MODY may also be higher than estimated due to misdiagnosis of type 1 or type 2 diabetes in these patients 50 52 Defining the etiology of MODY is essential for proper pharmacogenetic treatment of the disease 42 45 MO DY1 and MODY3 genes regulate the expression of a plethora of genes involved in glucose stimulated insulin secretion 48 mainly result in defective glucose metabolism and insulin secretion in the beta cell 48 For this reason, it is not surprising that MODY1 and MODY3 patients respond well to treatment with oral sulfonylureas given that these drugs bind the K ATP channel which is do wnstream of glucose metabolism in the insulin secretion pathway 44 Sulfonylurea therapy can cl ose the K ATP channel which increases intracellular Ca 2+ and stimulates insulin secretion by an ATP independent pathway. MODY1 and MODY 3 patients, who are initially misdiagnosed with type 1 or type 2 diabetes, have successfully transferred to sulfonylurea treatment without deterioration in glycemic control 50, 53, 54 Sulfonylurea
20 treatment often leads to better glycemic control than insulin especially for children and adolescents. Sulfonylurea therapy has been shown to be ineffective in treating other forms of MODY 42 45 Sulfonylurea treatment reduces the serious risk of hypoglycemia compared to insulin t reatment and patients who have switched from insulin to sulfonylurea report a great increase in their quality of life 44 However, current treatments for MODY3 are not sufficient because patients with MODY3 have hyperglycemia that often worsens with time and up to 40% require the use of insulin 46 Long term hyperglycemia in this disease can lead to the full scale of diabetic complications especially neuropathy and retinopathy 46 Transcription Factors DNA binding proteins have long been studied for their important roles in a variety of cellular processes including transcription 55 (For Review s regarding transcription see Refs 56 59 ). Trans cription factors play a crucial role during development and tissue differentiation 21, 60 63 and their role in the reprogramming of mature adult tissues into alternative tissue lineages has become an important topic of study in the field of regenerative medicine 64 66 Several transcription factors have been discovered that act as master switch genes and regulate a cascade of downstream gene expression leading to cellular differentiation, s uch as MyoD 67 for muscle cell differentiation and Pdx1 21, 60, 61 for pancreatic beta cells. Induced expression of MyoD alone in many differentiated cell types including primary fibroblasts, melanoma, ad ipocytes, and hepatocytes induces expression of muscle cell markers and adoption of the muscle cell phenotype 67 More recently, it has been found that transcription factors can lead to the reprogramming of both mouse 68 70 and human 71 73 fibroblasts into induced pluri potent cells that closely resemble embryonic stem cells with the capacity to differentiate into all
21 three germ layers. Transdifferentiation of cells occurs by shifting their gene expression and phenotype from one differentiated cell type to another 64 66 (Note: Originally, transdifferentiation was defined as direct conversion of o ne cell type to another without acquiring an intermediate state, whereas reprogramming required cellular dedifferentiation before converting to another cell type. H owever, current literature often uses these terms synonymously.) Transcription factors maintain cellular phenotype by working with epigenetic modulators 74 77 and by regulating cell specific gene expression 21 and are involved in many diseases including MODY 42 45 Pdx1 Pancreatic and duodenal homeobox 1 (Pdx1) also known as STF1, IDX1, IUF1, and IPF1, is a transcription factor necessary for pancreatic development and beta cell maturation 78 The Pdx1 gene 79 has been cloned independently by several labs 80 82 and in humans, encodes a 283 amino acid protein that is part of the ParaHox gene cluster 83 Pdx1 expression is first detected in the primitive foregut endoderm as the ventral and dorsal pancreatic buds develop 84, 85 which later merge to form the pancreas. Pdx1 expression later shifts to the endocrine compartment as islets form and the exocrine cells begin to appe ar in the pancreas. Later in development, Pdx1 becomes mostly restricted to the mature beta cells, with limited expression in other endocrine cells 80 82, 84 Homozygous mutations to the Pdx1 gene in mice lead to pancreatic agenesis 85, 86 (while all other internal organs appear normal including the gastrointestinal tract) leading to death shortly after birth 86 No pancreatic tissue or insulin gene expression was detected in these neonates. It has also been reported that a human with a homozygous inactivating mutation to the Pdx1 gene was born without a pancreas 87
22 Regulation of the Pdx1 gene has been well characterized by several reports ; 88 99 89 91, 94, 99 90 MafA 98 Pax6 94 NeuroD 95 Nkx2.2 97 USF 96 and Pdx1 90 itself. Pdx1 is also positively regulated by nutritional and ho rmonal factors such as glucose, GLP 1, insulin, T 3 HB EGF, and TNF 88 Pdx1 is involved in the expression of many genes (including insulin 82 somatostatin 80 Glut2 100 islet amyloid peptide 101 and glucokinase 102 ) and is considered the master transcriptional regulator of pancreatic beta cells 103 Pdx1 also inhibits expression of glucagon 104 Ectopic expression of Pdx1 has been shown to induce hepatic dedifferentiation by suppressing the expression of CCAAT/enhancer binding protein beta (CEBP ) 105 Pdx1 contain s an intrinsic protein transduction domain (PTD ) which is a specific amino acid sequence enriched with positively charged arginine and lysine residues, allowing for transduction across cellular membranes by lipid raft mediated macropinocytosis 31, 35, 36 It is also interesting to note that Pdx1 has been previously implicated in immune modulation by preventing development of hyperglyc emia in non obese diabetic (NOD) mice following intraperitoneal injection of Pdx1 protein 17 Ngn3 Neurogenin 3 (Ngn3) also known as Atoh5, bHLHa7, and Math4B, is expressed by all pancreatic endocrine cell precursors and regulates islet formation 106 It peaks in expression during endocrine cell development and is only detectable at low levels in adult pancreas. Ngn3 has bee n cloned and encodes a 214 amino protein that is a member of a family of basic helix loop helix transcription factors 107 Mice with homozygous mutations to the Ngn3 gene fail to develop endocrine cells or precursors and die from diabetes shortly after birth 106 Over expression of Ngn3 also leads to a
23 reduced endocrine mass 108 demonstrating the importance of strin gent regulation of this gene during development Ngn3 is positively regulated by HNF 1 109 HNF 3 109 and HNF 6 110 while Hes 1 represses Ngn3 expression through the Notch signaling pathway by binding to silencing elements in its proximal pro moter 109 Ngn3 is involved in the expression of many endocrine genes 111, 112 and is a direct regulator of NeuroD 113 and Pax4 114 Nkx6.1 NK6 homeobox 1 ( Nkx6.1 ), also known as NKX6A, is a homeodomain transcription factor involved in pancreatic differentiation and beta cell homeostasis 115 It has been cloned and enco des a 367 amino acid protein 116 In the mature human islet, it is exclusively expressed in the beta cell 117 and is required for normal glucose stimulated insulin secretion 118 Embryonic expression of Nkx6.1 is dependent on Nkx2.2 119, 120 and in the mature beta cell it is regulated by Pdx1 120 Nkx6.1 maintains beta cell phenotype in part by direct interaction with the glucagon promoter suppressing its activity 118, 121 Nkx6.1 inhibits glucagon expression by competing with Pax6 (glucagon activator) for occupancy of the G1 element on the glucagon promoter. Nk x6.1 has also been linked to beta cell proliferation by up regulating cyclins A, B, and E as well as many regulatory kinases 122 Over expression of Nk x6.1 has been shown to increase glucose stimulated insulin secretion in rat islets while in human islets it caused beta cell replication and maintained normal glucose stimulated insulin secretion 122 Nkx6.1 homeodomain constructs have been shown to bind sequences containing the core homeodomain binding site (5' TAAT 3' or 5' ATTA 3') and direct both gene repression and gene activation 123, 124 In its own promoter, it has been shown to bind a similar sequence (5' ATTT 3') to positively regulate its own expression 125 Nkx6.1 has the ability to function
24 as both a transcriptional activator and repressor which may be sequence dependent 125 The transcriptional repression domain has been isolated to the N terminus 124 while the tr anscriptional activation domain has been shown to be dependent on the C terminus 125 The C terminus has also been observed to interfere with DNA binding but greatly enhance specificity for homeodomain core containing sequences 126 It is also of interest that type 2 diabetic islets have been shown to have altered Nkx6.1 expression 127 The specific function of Nkx6.1 in glucose stimulated insulin secretion of the mature beta cell remains elusive. B1, TCF1, APF, and HP1) is an important gene involved in the pancreat ic beta cell transcriptional regulatory network 128 It is also involved in the regulation of important beta cell specific genes such as Pdx1, 90 IGF 1, 129 and insulin 130 Loss of function mutations 131, 132 in of monogenic diabetes in humans 46, 47, 49, 133, 134 MODY3 is characterized by impaired beta cell function caused by defective insulin secretion in response to glucose stimulation. While clinical presentation of the disease is often mild, hyperglycemia worsens with time and up to 40% of patients require treatment with insulin. Long term hyperglycemia can lead to the full range of diabetic complications, especially an incomplete dominant manner, disease occurs with the loss of function of a single allele. The haploinsufficiency of this gene demonstrates the critical importance of gene dosage to maintain normal beta cell function. Abnormal regulation of gene transcription to a defective insulin secretory pathway, loss of beta cell mass, or both.
25 Therefore, precise regulation of this gene is necessary to prevent a prominent form of diabetes. exocr ine and endocrine cells and all islet cells of the murine pancreas 135, 136 (For bri ef 13 7, 138 ). The rat species 139 141 12q24.3 and 5F respectively 139, 141 Human and r at nucleotide sequences show an overall 94% homology in coding regions and overall amino acid homology of 95% 139, 140 Rat and mouse amino acid sequence homology was found to be 99% 141 There is even considerable homology within the intronic gene sequences, especially in the flanking regions of exons, betw een species 139 141 This demonstrates high conservation of g enomic structure and suggests that this protein acts in a very specific manner and contains critical structures required for natural function. The DNA sequence consists of a long open reading frame of 1884 bp and the sequence surrounding the first ATG cod GGAGCC ATG G consensus sequence 142 A / G CC ATG 143 There is also a TATA like box ( GATAAATA 144 with very close homology to the TATA box co nsensus sequence 145 TATA A / T AA A / G 144, 146 148 and this complex has been crystallized 149 One study 150 using a 497 bp proximal that this reporter was activate in hepatocytes, but not in rat insulinoma (INS1) cells.
26 The rat and mouse gene both contain 9 exons and 8 introns and the rat full length coding region is less than 40kb 140 The full length cDNA encodes a 628 amino acid 88kd dimeric protein and was first cloned from rat liver 142 dim ers in the presence or absence of DNA 151, 152 The N terminus contains a DNA binding site that is structurally, though distantly related to the homeobox domain 142, 151 (See Refs 153 155 for comprehensive reviews regarding the homeodomain and Ref 153 an additional 21 amino acid sequence loop connecting helix 2 and helix 3 with 100% homology between mouse and rat 141 The protein also shares some similarity to the POU family of transcriptional activators 156, 157 GTTAATNATTAAC 137 on 158 Three separat e isoforms have been identified ( ) by real time PCR and no evidence for additional transcripts was found 159 The different isoforms are generated through differential selection of polyadenylation and alternative splicing 159 The fact that distributed differently between tissues and during different time points in development suggests that they may control gene expression in a temporal and tissue specific manner 159 The alternate isoform ratios may also contribut e to differential gene regulation between cell types. Alternative splicing of transcripts increases versatility of function and several documented proteins act as activators and repressors from the same gene 160
27 Electron mobility shift assays (EMSA) demonstrate that the dimerize and bind DNA with similar affinity 159 Transient CAT transfection assays in n have a five fold 159 It is noteworthy that did not suggesting that this coactivator was not a limiting step in this experimental procedure. A few studies demonstrate the biochemical characterization of the full length 152, 161 The DNA binding domain is a tripartite homeodomain composed of 81 amino acids and contains an N terminal helix similar to part of m y osin 152 The N terminal 31 amino acids mediate dimerization with these helical regions essential for DNA binding 152 The A domain (amino acids 1 33) is responsible for DNA binding which can be abolished by subs titutions 152 The B domain (amino acids 100 184) shows weak similarity to the subregion of A of th e POU domain 157 and is important for efficient DNA binding. (See Refs 157, 162, 163 for brief reviews or Ref 164 for a comprehensive review of the POU homeodomain). Mutations in the POU binding 152 The highly divergent homeodomain is located in domain C (amino acids 198 281), and substitutions here can lead to loss of binding activity, ev en when using conserved substitutions 152 The c terminal 150 amino acids are rich in serine and th reonine 159 and deletion of the c terminus porti on DNA binding 142, 152 networks 128 (See Ref 58 for review of tissue specific gene regulation). It is involved in
28 the development of the pancreas and critical for insulin and glucose regulation in the adult 46, 49, 165 There are several excellent reviews and studies that document these transcriptional regulatory networks in liver 128, 163, 166 and pancreas 60, 128, 167, 168 tissue distribution is predominantly endodermal 141, 156 In the mouse 141 transcripts have been found in the liver, kidney, stomach, and intestine, but not in the ovary, brain, heart, or lung. In rats 156 transcripts have been found in t he liver 142 kidney, and at low level in the intestine, spleen and thymus, but not in the skin, lung, hea rt, or 159 liver 159, 169 intestine 159 thymus 159 adult and fetal pancreas 169 and isolated islets 169 but at almost null levels in skeletal muscle 169 and visce ral adipose 169 examined mRNAs in certain fetal and adult human tissue cell lines by a semi quantitative RT PCR method did not attempt to quantify absolute amounts of each mRNA but rather compared the ratios between each isoform at the RNA level 159 tios in a variety of cell lines. Pancreas was not examined in this study. The RNAse protection method was not sensitive PCR another g roup 169 the kidney and liver and all tissues examined (fetal liver was not examined). A
29 determined in skeletal muscle or visceral adipose as expected. It is interesting to note 60, 61 The role of miRNA in post translational gene regulation has been shown to be highly important in the regulation of gene expression. Traditionally, miRNA has been shown to be a translational repressor but in some cases it can function to increase gene expression 170 (See Ref 171 for a review of miRNA function). During intestinal epithelial differentiation in Caco miR 194 2 172, 173 of miRNAs, and provide greater u nderstanding of gene regulation in beta cells. null pups display no obvious phenotypic abnormalities required for embryonic development 174 in dedifferentiated hepatoma cell lines and hepatic extinguishing somatic hybrids 156, 175 178 developing murine pancreas 135 its transcriptional activation becomes dependent only in terminally differentiated cells 167 This may be due to the reciprocal expression of a that share dimerization domains and homeodomains, but not activation domains 179, 180 prominent in differentiated cells 156, 175 178, 180 182
30 heterodimers in vitro 18 0, 183 obvious abnormalities during embryonic development but following birth they fail to thrive and suffer from a progressive wasting syndrome 174 These mutant mice suffer from hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome 174 expression is important for beta cell function and maintaining glucose homeo stasis. glucose tolerance without loss of beta cell mass 184 Genes involved in beta ce ll 185 Decreased steady state mRNA levels were observed for genes encoding glucose transporter 2 (Glut2), neutral and basic amino acid transporter, liver pyruvate kinase (L Pk), and insulin NeuroD 185 loxP recombination system develop Laron dwarfism and non insulin dependent diabetes 186 ous and fasting hyperglycemia leading to the development of diabetes 174, 184 186 dependent operon speci fically in beta cells demonstrates that over expression also leads to a diabetic phenotype 187 ression compromises islet morphology and reduces beta cell mass. These mice show a threefold reduction in beta cells and many appear scattered near ductal structures. Over expression also
31 suppresses beta cell cycle activity and induces cell death. Ki67 staining shows that expression of cell cycle inhibitor p27. Apoptosis is induced in a significant density of r expression causes hyperglycemia at birth that progressively worsens with age. Other studies in mice show MODY3 like phenotype 188, 189 collectrin which facilitates S NARE complex formation and controls insulin exocytosis 190, 191 Studies in INS1 insulinoma cells over expressing a dominant negative mutant under control of a reverse tetracycline dependent transactivator ( or ) ll homeostasis 192 negative over expressing cells activated caspase 3 leading to apoptosi s. Apoptosis was accompanied by mitochondrial hyperpolarization, decreased expression of anti apoptotic Bcl xL, and mitochondrial release of cytochrome c and caspase 9. Apoptosis could be rescued with transient over expression of Bcl xL. Another study u sing the same dominant negative conditions showed pronounced decrease in insulin expression and other genes involved in glucose stimulated insulin secretion 193 Other interes ting studies in INS 1 insulinoma P291fsinC, with the most common MODY3 mutation, under control of the reverse tetracycline dependent transactivator 129, 194 This forces the
32 e xpression of several genes important for beta cell function. Insulin secretory responses to glucose were also impaired in these cells. chromatin structure and alters gene expression 75 In the liver, the phenylalanine hydroxylase (PAH) gene is activated prenatal and is regulated by several transcription livers 75, 174 Hepatic PAH expression could not be induce d by traditional activation enhancing hormones in these mice. Liver specific DNase I hypersensitivity sites of the was also associated with chromatin structure and gene expression of alpha 1 antitrypsin (AAT) and corticosteroid binding globulin (CBG) in hepatocytes 76 nucleosomal hyperacetylation in a tissue specific manner 74 and L type pyruvate kinase (pklr) in both pancreatic islets and liver cells of norm al mice. expression is maintained in liver cells. These expression patterns correlate directly with hyperacetylation of GLUT2 and pklr promoter nucleosomes in pancrea tic insulin producing cells, but not in liver cells. Studies in dedifferentiated M29 cells show that 195 These studies
33 epigenetic modification and that its cellular concentration may determine chromatin architecture. a t ranscriptional network that regulates cell type specific global gene expression and maintains normal cellular phenotype and function. Liver and pancreas share common embryonic lineage 196 and both are derived from the same endoderm tissue mass 61 63 They also share expression of several HNF transcription factors 60, 128, 163, 166 168, 197 G / A GTTA A / C TNNT C / T NNC A / C 177 One st target genes by computer assisted analysis and subsequent binding assays 198 In a genome scale promoter analysis in human liver and pancreatic islets 128 found to occupy the p romoters of at least 222 genes in liver and 106 in pancreas analyzed on a micro array of 13,000 genes (Hu13K array). The targeted promoter regions span 700 to +200 from the transcription start site, where most identified transcription factor binding site occur. Analysis of distal promoters would likely reveal individual ChIP experiments and stringent threshold criteria to have a high accuracy. es encode important biochemical proteins related to normal cellular function as well as important transcriptional regulators of gene expression. Transient transfection assays in HepG2 cells have demonstrated that an indirect negative autoregulatory feedba regulation 146
34 dependent genes 146 by a direct interaction with the AF 199, 200 also known as PHS or PCD is an 11kDa homotetrameric protein (45kDA) that selectively stabilizes mers and enhances transcriptional activity 201 204 The cDNAs from human, mouse, and rat encode a 104 amino acid protein 204 but transcriptional modification has been demonstrated in the liver of humans and rats 205 DCoH binds as a dimmer to the helical dimerization domain of di 206 It has been purified from human and displays identical protein primary structures to rat 205 Its crystal structure has been determined alone 202, 206 206, 20 7 leading to the biochemical characterization of some MODY3 mutations 207 Interestingly, mice that are DCoH null display mild glu cose intolerance, but do not develop diabetes 208 were reduced. It was also ob served that low levels of a second DCoH related gene (DCoH2) may have had a complementing activity. The crystal structure of DCoH2 has been determined and biochemical studies have found it to be partially redundant in enzymatic and transcriptional function s 209 GLUT2 promoter in HIT T15 cells 210 Coimmunoprecipitation assays demonstrate that histone acet yltransferases (HATs), CREB binding protein (CPB), p300/CPB associated factor (P/CAF0 ) Src 77 CBP was shown to
35 interact with the N terminal region and P/CAF, Src 1, and RAC3 interacted with the C terminal activation domain. The diverse functions of these coactivators suggest that ptional machinery recruitment. All of the aforementioned coactivators have been shown to acetylate surrounding nucleosomes allowing greater access for other transcription factors to their promoters. (For excellent reviews regarding transcriptional regul ation by nucleosomal histone acetylation see Refs 211, 212 ). Increased transcriptional activat ion fold above basal levels and co transfection with CBP, P/CAF, Src 1, and RAC3 expression vec tors increased activation by 20 fold, 44 fold, 26 fold, and 19 fold respectively. 1, and RAC3 increased activation respectively by 135 fold, 121 fold, and 89 Src 1 a nd RAC3 increased activation 132 fold and 152 fold respectively. The dramatic increase in activation after coactivator recruitment is clearly a synergistic reaction. The a coactivator for transcription 199 for cell specific regulation of gene expression and that these factors can positively regul 128, 134, 167, 168, 213, 214 the pancreas in mice 136 and in murine beta cells it is essential for the regulation of gene expression and for glucose metabolism 215 218 liver nuclear extra cts 219 and has been well characterized structurally and functionally by
36 several reports 149, 167, 1 99, 219 237 The A/B region contains an AF 1 strong terminal transactivation domain. The DNA binding region is in the C domain and the E region contains a ligand binding domain, dimerization domain, and AF 2 transactivation domain. The F region is va riable and some isoforms contain a coactivator recruiting 219 and binds DNA as a homodiomer 220 238 while th e mouse gene contains only 10 exons 235 The 1365 base pair open reading frame of huma different structural regions 219 Almost half of protein coding genes function by use of alternative promoters and the functional consequences of alternat ive promoter use in mammalian genomes have been reviewed 239 promoter usage and alternative transcript splicing 238 and they c an be organized into two 3) contain exon 1A with the strong AF 1 activation domain whereas isoforms generated from the P2 9) contain exon 1D, or exon 1D and 1E, and lack t his activation 6 223 isoforms may be unreliable and these isoforms are unlikely to exist as suggested by more recent reports 150, 224, 240 The two promoters are separated by approximately 46 kilobases 236 Both the P1 and 167, 221, 236 and the P2 promoter also contains Pdx1 236 and HNF6 221, 236 (also known as OC 2) binding sites. Similar to mice 167, 222 229, 236, 240, 241 is driven predominantly from the P2 promot er producing 9 isoforms (and potentially
37 12 238 ) in similar concentrations as demonstrated by RT PCR analysis of transcripts. Liver expression is predominantly driven by the P1 promoter 229, 236, 240, 241 In concordance wit h these findings, murine nucleosomal histones of the P2 region are hyperacetylated in pancreatic islets; P1 regions are hyperacetylated in liver 167 Another predominant transcripts in human beta cells 2 24 but also found very low levels of 6. It was previously shown 224, 229 232 To generalize the expression o in hepatocytes is most prominently expressed through the P1 promoter while in beta cell s most reports pts cannot be detected 238 Primarily P2 isoforms have been detected in adult beta cell s, and they have been associated with reduced transcriptiona l activation potential when compared to P1 isoforms 224, 229, 232 10 amino acid sequence in the F domain. Studies show deletion of the F domain 229, 242 and the 10 amino acid insert can help to abrogate this inhibitory effect 233, 234 The inhibitory effect caused by the F domain has been localized to a 14 amino acid residue (428 441) that is sufficient to repress activity of the AF 2 activ ation domain 230 however, 10 fold decreases were found in the pancreas 167 167 establishing a different
38 regulatory network than in hepatocyt es 128, 166, 213 repress P2 activity in transfection a ssays in rodent liver 221 only knock in mice were generated to examine the in vivo role of the AF 1 activation domain 222 Both mouse types show no obvious phenotypic abnormalities although HN total knockouts which are embryonic lethal 243, 244 and also demonstrates the embryonic functional redundancy of the two isoforms. own transcription and that it can also repress the 146, 200 200 thereby functioning as both an activator and a repressor to regulate gene expression. HNF Regulatory Circuit HNF transcription factors are part of a complex regulatory circuit that is known to maintain hepatocyte phenotype 163 and maintain insulin secretion in response to glucose stimulation in the beta cell 134, 167, 168 In 245, 246 regulate each other as a feedback loop 166 A genome scale promoter analysis 128 in human liver and pancreatic islets using a micro array of 13,000 genes (Hu13K array) ccupied the promoters of at least 1575 genes in hepatocyte s and 1428 in pancreatic islet s, least 222 genes in hepatocyte s and 106 in the pancreatic islet s Promoter occupancy of ase II for 80% of hepatic genes and 73% of islet genes
39 2% of islet genes, suggests that a large number of genes are actively transcribed by
40 CHAPTER 2 PDX1 MEDIATED SUPPRESSION OF HNF1 DURING REPROGRAMMING OF HUMAN HEPATIC CELLS TOWARDS PANCREATIC BETA CELLS Introduction Reprogramming of hepatocytes into pancreatic beta cell like insulin producing cells (IPCs) by ectopic expression of pancreatic transcripti on factors (PTFs) has been well established through detailed in vitro characterization of pancreatic beta cell gene expression 12, 15, 16, 18, 19, 23 25, 27, 28 and in vivo restoration of normoglycemia in chemically induced diabetic mice 12, 13, 17, 19, 20, 23 25, 27, 28 This has significant clinical implications due to its therapeutic implication for type 1 diabetes (T1D). Transdifferentiation studies aimed at the treatment of T1D have mainly focused on using the liver 12, 13, 15 20, 23 25, 27, 28 as a tissue source due to its tissue abundance and high regenerative capacity 247 common developmental kinship with the pancreas 196 and the glucose sensing system shared by hepatocytes and pancreatic beta cells 248, 249 Since the liver and pancreas share a remarkably similar gene expression profile, including the expression of many specific tran scription factors 128, 245 and both tissues are responsive to glucose 248, 249 hepatocytes are an excellent target for reprogramming into pancreatic beta like IPCs. Although much attention has been given to understanding how to activate genes related to pancreatic beta cell development, understanding of the molecular mechanisms involved in shutting down the hepatocyte phenotype during PTF mediated hepatocyte to beta like IPC conversion remains quite limited 64 66 Hepatocytes make up 70 80% of the mass of the liver and perform an astonishing number of metabolic, endocrine, and secretory functions 63, 250 The liver plays a major role in metabolism, glycogen storage, plasma protein synthesis, hormone production, and detoxification. Ectopic expression of Pdx1 triggers hepatocyte
41 dedifferentiation by down regulating several liver specific functional genes including albumin (ALB), alcohol dehydrogenase phosphatase (G6PC), 1 antitrypsin (AAT), and hexokinase 2 (Hxk 2) 105, 251 However, the molecular links between Pdx1 over expression and down regulation of th ese hepatic genes are not entirely understood. Several transcription factors are necessary for the differentiation and maintenance of hepatocyte phenotype 128, 163, 166, 252, 253 In particular, h epatocyte nuclear factor 1 alpha (HNF1 ) and 4 alpha (HNF4 ) are important liver enriched transcription factors that play an important role in establishment and maintenance of the hepatocyte phenotype 128, 185, 245 downstream target genes in both hepatocytes and beta cells 128 and the regulatio n of specific isoforms and levels of expression by alternative splicing differ significantly between hepatocytes and beta cells 128, 240, 245 A developmental switch in the relative een the fetal and adult pancreas 169, 240 ontains two promoters (P1 and P2) that drive the expression of P1 3) or P2 derived 9) by alternative splicing and alternate usage of the promoters 236 The different promoters are used in different tissues and at different times during isoforms are exclusively detected in adult pancreatic islets 236, 240, 241 isoforms are present in developing human fetal pancreas 240 In contrast, P1 derived isoforms are most abundant in adult hepatic tissues with relatively low levels of P2 isoforms 222, 240, 241 Due to the tissue
42 transactivation capacities than P1 isoforms 224, 229, 232 Pdx1, which is not normally to the P2 promoter 236 i soforms following ectopic expression of Pdx1 may be responsible for hepatic cell dedifferentiation and transdifferentiation toward pancreatic beta cells. Here, we tested mediated hepatic dedifferentiation. In this study, we constructed lentiviral vectors to deliver PTFs into human hepatocellular carcinoma cells (Huh7 cell line), examined early molecular events related to the activation of pancreatic endocrine genes and suppression of hepatic genes, and ve inhibition. This work provides some insights into the mechanism of reprogramming from hepatocytes into pancreatic beta like IPCs. Understanding the molecular events during cell type conversion may help to elucidate the mechanisms underlying tissue reg eneration and plasticity. Materials and Methods Cell Lines and Cell Culture H uman hepatocellular carcinoma cell line Huh7, 3T3 mouse fibroblast cells, and 293 human embryonic kidney cells were purchased from ATCC and were cultured in DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin in a 37C incubator
43 with 100% humidity and 5% CO 2 Rat insulinoma (INS 1) cells were cultured in RMPI 1640 supplemented with 200mM L glutamine, 100mM sodium pyruvate, 2 mercaptoethanol, and 1% Penicillin/Streptomyci n in the same incubator. Construction of Plasmids The human Pdx1 expression plasmid (pCMV6 XL5) was purchased from Origene. The mouse Pdx1 expression plasmid was constructed by insertion of mouse Pdx1 cDNA into the BamHI/XbaI sites of the pCDNA3 vector (I nvitrogen). The truncated mouse Pdx1 expression vectors were constructed as previously described 254 The Sport6) was purchased from Open Biosystems. expression plasmids code for proteins lacking the 41 amino acids from the extended exon 8). The pRL TK expression vector was purchased from Promega. The mouse 255 The rat insulin I promoter (RIP) luciferase reporter was constructed by removing the TK promoter from the pRL TK plasmid and cloning the RIP promoter into this site using the BglII/HindIII restriction sites. The Pax4, NeuroD, Ngn3, and Nkx2.2 luciferase reporters (pFOXluc) were generous gifts from Michael German, the University of California, San Francisco, CA Lentiviral Vector (LV) Preparation, Titration, and Transduction The RIP driving the green f luorescence protein ( RIP GFP ) reporter was constructed as previously described 12 The LV containing the mouse Pdx1 VP16 (PV)
44 fusion gene was constructed as previously published 12, 22 The LV containing rhGFP and mouse Pdx1 were constructed as previously described 24 The LV containing mouse Ngn3 was constructed by inserting the cDNA of mouse Ngn3 into the pTYF vector cassette under the control of elongation factor 1 alpha (EF were generated and titrated as previously described 256 258 For lentiviral transduction, Huh7 cells were transduced with different LVs as indicated at a multiplicity of infection (MOI) of 10 in th efficiency was monitored by transducing Huh7 cells with LV encoding rhGFP. The insulin 1 promoter activities in Huh7 cells were detected by observing RIP GFP expression under a fluorescence m icroscope. RT PCR and Real Time RT PCR Analysis Total RNA was extracted from Huh7 cells transduced with different combinations of transcription factors or from human islets (generous gift from Dr. Xiaoping Deng at The University of Pennsylvania) using Triz Gene expression was detected by RT PCR. The forward and reverse PCR primers were designed (IDT Technologies) to be located in different exons. Amplification was performed for 35 cycles at 94C for 30s, 56C f or 30s, and 72C for 30s, and followed by 72C for 7 min. The PCR products were separated in 2% agarose gels by electrophoresis in TAE buffer. Digital images were captured and analyzed with a Qua n tity O ne Imager (BioRad). All of the PCR products were co nfirmed by Big Dye DNA sequence analysis in an ABI 377 sequencer (Global Medical Instrumentation, Inc.) PCR was performed on selected samples, collected as described above, using SYBR Green PCR Master Mi x (Applied
45 terminal modifications by detecting exon 9, exon9+, or exon 8+. Primer sequences are available upon request. Western Blotting and Immunocytochemistry (ICC) Huh7 cell lysates were harvested at day 4 post LV Pdx1, LV PV, LV Ngn3, or LV GFP treatment. The cellular proteins were quantified and standardized. Pdx1, PV, alpha 1 anti trypsin to our previously published methods 12, 24 In brief, cellular proteins were separated by SDS PAGE using 12% Tris HCl gels ( Bio Rad) and transferred to the filter membrane. Proteins were blotted with rabbit anti anti VP16 (1:200, BD Pharmagen), anti ALB (1:200, Santa Cruz), and anti AAT (1:200, Sant a Cruz) followed by HRP conjugated secondary antibody (1:20 000). All proteins were visualized by enhanced chemiluminescence (ECL) using a western blotting detection kit (Amersham Bioscience). For ICC, cytospin slides were prepared from Huh7 cells and Ng n3 protein was detected by rabbit anti serum against mouse Ngn3 (a generous gift from Michael S. German, University of California, San Francisco) at a dilution of 1:3000 according to our previously published methods 12, 24 Transfections and Luciferase Assay Cells were plated in 12 well plates and transfected with 0.1 DNA (as indicated) using Lipofectamine 2000 Reagent (Invitrogen) according to Luc plasmid was used as a transfection efficiency control in all experiments. Cell lysates were harvested and measured 24 hours post transfection using the Dual Luciferase Reporter Kit (Promega) according to
46 only 50l of each substrate reagent was used (as optimized by our lab) to read samples using a Lumat LB 9507 Luminometer (Berthold Technologies). All luciferase assays were done in duplicate or triplicate as indicated. All results are expressed as fold/c ontrol using pcDNA3 null expression vector following standardization by TK Luc for transfection efficiency. Statistical Analysis The statistical significance of our experimental findings was analyzed by using test (2 tailed, assuming equal vari ance). P values represent comparison to controls unless otherwise indicated. = p<0.05 and ** = p<0.001 Results Pdx1 VP16 (PV) and Ngn3 Together Strongly I n duced Insulin P romoter A ctivity We first assessed if our LVs designed to express PTFs were functional. We confirmed the transgene expressions of Pdx1, PV, and Ngn3 at the mRNA level by RT PCR (Fig. 2 1 ) and protein levels by western blotting for Pdx1 and PV (Fig. 2 2 ) and ICC for Ngn3 (Fig. 2 3 ). In order to monitor the activation of the insulin gene during the reprogramming process from hepatic cells toward pancreatic beta like IPCs, we constructed and produced LV RIP GFP as a reporter for monitoring RIP activity. The transduction efficiency of Huh7 cells was determined by LV GFP and more than 99% of transduced Huh7 cell expressing GFP was observed at day 2 (data not shown). RIP GFP was co transduced in to Huh7 cells with Pdx1, PV, Ngn3, or with combinations of Pdx1 /Ngn3 or PV/Ngn3 as indicated. At 96 hours of transduction, the expression of GFP was examin ed ( Fig. 2 4 ). Green fluorescence was observ ed in rare Huh7 cells transduced with single gene Pdx1, PV, or Ngn3, therefore Pdx1, PV, or Ngn3 alone cannot effectiv ely activate the insulin promoter RIP GFP at day 4 post transduction.
47 Green fluorescence was observed in nearly 50% of Huh7 cells transduced with Pdx1/Ngn3 and in more than 90% of Huh7 cells transduced with PV/Ngn3, indicating a synergistic effect of Ngn3 and Pdx1 in activating RIP GFP. This result is consistent with previous studies demonstrating that the insulin promoter can be more effectively activated by PV, a super active form of Pdx1 12, 16 To further compare the effect of Pdx1 and PV, we consecutively observed GFP expression at 48, 72, a nd 96 hours after Huh7 cells were transduced with Pdx1/Ngn3 or PV/Ngn3 (Fig. 2 5 ) The results show that PV is much more effective than Pdx1 to activate RIP GFP. Green fluorescence was observed in 50% of Huh7 cells transduced with PV/Ngn3 at day 2 compar ed with only 2% of cells treated with Pdx1/Ngn3. Pdx1 and Ngn3 Induced Expression of Genes Related to Endocrine P ancreas Given the limitations imposed by the chromatin structure on endogenous genes, we next asked whether the activation of the RIP GFP repor ter might be applicable to the endogenous human insulin gene. As shown in Fig. 2 5 although transduction of PV/Ngn3 led to activation of the ectopic promoter RIP GFP reporter as early as 24 hours, the activation of the endogenous human insulin gene could only be detected at 96 hours by RT PCR (Fig. 2 6 ). Notably, the absolute level of activation of the human insulin gene in the cells still remains low, implying that limitations may be due to the inaccessible chromatin structure of the endogenous gene. To investigate the gene expression profile of Huh7 cells with different treatment after 96 hours, cellular RNA samples were collected and RT PCR analysis was performed (Fig. 2 6 and 2 7 ). We screened several endocrine pancreas functional genes and found t hat endogenous expression of Pdx1 and Nkx6.1 could be activated by all treatments. Cells treated with Ngn3, Pdx1/Ngn3, and PV/Ngn3 activated NeuroD,
48 Pax4, and Isl 1 genes which are involved in pancreatic endocrine and beta cell development, and this is co nsistent with previous findings that NeuroD 113 and Pax4 114 are direct targets of Ngn3 112 All treatments using Ngn3 activated insulin gene expression. PV, not Pdx1, alone or combined with Ngn3 induced expression of somatostat in (SS) Pancreatic type glucokinase (P GK) was only expressed in cells transduced with Pdx1/Ngn3 or PV/Ngn3. No glucagon (GLUC) was detected in any sample s. Huh7 cells did have basal expression of Pax6, GLUT M). Huh7 cells regulated observed in cells with over expressi on of Pdx1 or PV. Quantitative Analysis of PTF Promoter A ctivities Using the dual luciferase assay we quantitatively analyzed the promoter activity of PTFs in the Huh7 cells undergoing reprogramming. Huh7 cells were treated with LVs carrying Pdx1, PV, Ng n3, Pdx1/Ngn3 or PV/Ngn3 for 72 hours and then transfected separately with PTF luciferase reporter plasmids (Pax4, NeuroD, INS1, Ngn3, or Nkx2.2). The relative luciferase activities are shown in Fig. 2 (8 12) Consistent with published data and our own g ene expression results, Ngn3 alone can directly activate Pax4, NeuroD, INS1, and Nkx2.2 genes, and suppress its own activation. Ngn3 combined with Pdx1 or PV did not show a synergistic effect on the Ngn3 mediated activation of Pax4, NeuroD, and Nkx2.2 gen es, but showed a significantly synergistic activation of INS1 (6 fold in Pdx1/Ngn3, and 16 fold in PV/Ngn3). In addition, Pdx1, but not PV, could effectively activate Pax4, Nkx2.2, and Ngn3 promoter reporters, indicating
49 that PV, the super active form of Pdx1, has no activation effect in this experimental setting. Pdx1 S in Hepatic C ells Previous studies have shown that several hepatic genes are down regulated following over expression of Pdx1 in hepatic cells 105, 251 In our current study, we have shown a down in Huh7 cells following treatment with Pdx1 or PV by RT PCR (Fig. 2 7 ). To confirm this finding, proteins of cell lysates harvested from day 4 LV Pdx1 or LV GFP treated Huh7 cells were separated by SDS/PAGE and probed by western blotting using antibodi es against down treated Huh7 cells (Fig. 2 13 ). This prompted us to evaluate the effect of Pdx1 on the expression of luciferase reporter and various concentrations of human Pdx1 or mouse Pdx1 expression plasmid as promoter activity in a concentration dependent manner (Fig. 2 14 ). With 0.8ug Pdx1 fact that C terminal truncated Pdx1 constructs did not show any inhibitory activity (Fig. 2 15 ). Pdx1 I Transcripts in Hepatic C ells 147 complex gene regulated by two distinct promoters (P1 and P2) and alternative
50 splicing 232, 236 238 that shows tissue specific expression 222, 240, 241 In the adult human adult human pancreas and islets, P2 driven isoforms are predominant 222, 240, 241 I n binding site in the P2 proximal promoter 236 To test our hypothesis, we performed real time RT PCR in Huh7 cells following treatment with LV PV to examine the effects on 9) isoform expression. As expected, we observed a pronounced i 2 16 ). 2 16 ). Pdx1 soforms S Via a Competitive I nhibition transactivators of their target genes 224, 229, 232 We therefore investigated the 1 and P2 promoters in Huh7, INS luciferase reporter (Fig. 2 17 ). In all of their corresponding (same c terminal modification) P2 drive n isoforms. However, 1 cells. In 1 cells, the strongest activators are the isoforms containing the trun cated C terminal end To further investigate the mechanism by which Pdx1 influences down regulation of
51 transcription factors related to our system (data n ot shown). We first demonstrated that luciferase reporter (Fig. 2 18 ). We then demonstrate that Pdx1 can then suppress this activation i n a concentration dependent manner (Fig. 2 18 promoter, we hypothesized that it may function indirectly by altering the regulation of its isoforms, we set up a competition assay in 3T3 cells to examine the effect of expressing 2 19 luciferase nd then examined the effect of the same C terminal modification). As expected and similar to the effect of Pdx1, luciferase r eporter in a concentration expressed Pdx1 in hepatic cells enhances the expression of H that are able to compete with P1 driven isoforms for the same binding site on the contributes to the early events of dedifferentiation of hepatic phenoty pe during liver to beta cell reprogramming. Discussion In the present study, we have established an effective model by LV expression of Pdx1, PV, Ngn3, and combinations of Pdx1/Ngn3 or PV/Ngn3 in Huh7 cells in which to study the early events of hepatic gen e down regulation and pancreatic gene up
52 regulation during the process of Pdx1 mediated hepatic reprogramming into beta like IPCs. We have demonstrated that coexpression of Ngn3 with Pdx1 is important for the activation of several endocrine pancreas genes such as Pax4, NeuroD, Isl 1, and Nkx2.2. We also examined the role of a Pdx1 fusion protein (PV) that is known to strongly activate insulin gene expression and increase the efficiency of hepatic to pancreatic reprogramming 12, 16, 23 Previous studies have shown that this modified form of Pdx1 carrying the VP16 transcriptional activation domain from the herpes simplex virus more efficiently induces insulin gene expression in the human HepG2 cell line and the rat WB cell line 12, 16 but whether this attribute was a general characteristic of other PTF genes or limited only to insulin gene was not explored. Our results show that insulin promoter a ctivity was about 3 fold higher in cells treated with PV/Ngn3 than in those treated with Pdx1/Ngn3. However, the Pdx1 modified with VP16 showed lower activation on Pax4, Ngn3, and Nkx2.2 promoters when compared to Pdx1 alone. Therefore, the addition of V P16 restricts Pdx1 transactivation in some contexts, suggesting the mechanism of activation of insulin by Pdx1 may be different from activation of the Pax4, Ngn3, and Nkx2.2 genes. The liver is largely composed of hepatocytes, which occupy 70 80% of parenc hymal liver volume in the rat 63, 250 Hepatocytes carry out the primary functions of the liver such as m etabolism, detoxification, and protein synthesis of several essential compounds including serum ALB, fibrinogen, and transferrin. It has been suggested that the dominant mechanism for controlling the expression of hepatocyte specific genes is at the trans criptional level 259 however, the molecular mechanism by which Pdx1 regulates the expression of these hepatic genes is not well established. Using our
53 reprogramming model, we have demonstrated that over expression of Pdx1 can down t genes (ALB and AAT) in hepatic cells by RT wide array of hepatic genes 128, 252 it may play a fundamental role in the process of dedifferentiation of hepatocyte phenotype during the reprogramming toward IPCs. Our findings are consistent w ith a previous study 105 where adenovirus mediated expression of Pdx1 led to down regulation of several mature hepatocyte specific genes including ALB ADH1B, G6PC, GLUL, and AAT. Several key hepatic genes are direct fibrinogen, transthyretin, and pyruvate kinase 260 266 Therefore, Pdx1 induced down ssion can affect these downstream target genes and may be important in the process of dedifferentiation of hepatic cells by down regulating the expression of an array of genes that determine hepatocyte phenotype. Our results also suggest that the mechanis m for down http://www.sladeklab.ucr.edu/HNF4.shtml). We demonstrated that spe isoforms function differently in hepatocytes and in beta cells (Fig. 2 17 ). In hepatic terminal modification that results from using different promoters (P1 vs. P2). HNF4 that are expressed using the P1 promoter contain exon 1A and are the strongest activators in hepatic cells. The C terminal modifications that result from alternative ur
54 experimental setting. In pancreatic beta cells the C terminal modifications appear to 2 17 ). P1 driven isoforms are still stronger activators in beta cells when compared to their corresponding P2 driven isoforms (with similar C terminal ends) but having the truncated C terminal end that stops in exon 8 increases the activity independent of the first exon. The isoform driven than specific function. According to our real time RT PCR data (Fig. 2 16 that are stronger transcriptional activators containing exon 1A (AF 1 activation domain) or n exon regulated. Exons 9 and 10 comprise the inhibitory F domain which inhibits transactivation potential by blocking coactivator binding 234 requires stronger activators (P1 driven) than in beta cells which predominantly rely on P2 driven isoforms and this may be due to cell specific mechanisms for regulating this gene. For example, the PTF Nkx6.1, which is expressed in beta cells but not in hepatocytes 14 inding to its distal promoter 255 We also show evidence (Fig. 2 19 ) that promoter specific isoforms can compete with each other for activation of their target genes. It is possible that induced expression of
55 process of hepatocyte toward IPCs, as the opposite has been shown during rodent liver development 221 These findings are similar t 246 Taken together, we propose the following molecular mechanism that may be in part responsible for Pdx1 me diated dedifferentiation of hepatocyte phenotype in transition toward pancreatic beta like IPCs (Fig. 2 20 P1 its downstream target genes and maintains hepatic phenotype. Following ectopic regulation of isoforms for the same DNA binding site P2 isoforms, may be a driving force in the dedifferentiation of the hepatic phenotype by suppressing downstream target gene s.
56 Figure 2 1. Confirmation of LV PTF gene expression by RT PCR. Total RNA was collected from Huh7 cells following transduction with expression vectors for Pdx1, PV Ngn3, or combinations as indicated. Indicated gene expression was measured by RT PCR.
57 Figure 2 2. Confirmation of LV PTF gene expression by western blotting. Huh7 cells were transduced with expression vectors for Pdx1, PV Ngn3, or combinations as indicated. Following transduction, Huh7 cells were scraped off and lysed in RIPA buffer. Equal amount s of cell lysates were separated on 12% polyacrylamide gels by SDS PAGE and immunoblotted with polyclonal antibody against Pdx1 or PV as indicated. INS 1 cell lysates were used as a positive control for Pdx1
58 Figure 2 3. Confirmation of LV PTF gene expression by immunocytochemistry. Huh7 cells were transduced with expression vectors for Pdx1, PV Ngn3, or combinations as indicated. Following transduction, cytospin slides made from Huh7 cells were air dried, fixed an d stained with Anti Ngn3 antibod ies using ICC
59 Figure 2 4. A ctivation of rat insulin I gene in Huh7 cells with Pdx1, PV, Ngn3 and combinations. Huh7 cells were transduced with RIP GFP reporter and Pdx1, PV Ngn3, or combinations as indicated for 96 h. The expression of GFP was observed under a fluorescence microscop e
60 Figure 2 5. Time course a ctivation of rat insulin I gene in Huh7 cells with Pdx1/Ngn3 or PV/Ngn3 Huh7 cells were transduced with LV Pdx1/Ngn3 or LV PV/Ngn3 in LVs in the presence of LV RIP GFP reporter The expression of GFP at 48h, 72h, and 96h was recorded by fluorescence microscopy.
61 Figure 2 6. RT PCR analysis of pancreatic gene expression. Huh7 cells were treated with LV transgenes ( Pdx1, PV Ngn3, or combinations as indicated ) for 96h. Equal amounts of total RNA/ cDNA sample were used for RT PCR analysis with gene specific primers.
62 Figure 2 7. RT PCR analysis of liver specific gene expression. Huh7 cells were treated with LV transgenes ( Pdx1, PV Ngn3, or combinations as indicated ) for 96h. Equal amounts of total RNA/ cDNA sample were used for RT PCR analysis with gene specific primers.
63 Figure 2 8. Pax4 l uciferase reporter gene analysis during reprogramming. Relative activity was measured for the Pax4 luciferase reporter following Huh7 cell transduction with LVs for Pdx1 PV Ngn3, or combinations as indicated for 72h. Huh7 cells transduced with LV GFP were used as a control. All experiments were done in triplicate and repeated independently at least three times. All p values are compared to non treated controls. ** = p<0.001
64 Figure 2 9. NeuroD l uciferase reporter gene analysis during reprogramming. Relative activit y was measured for the NeuroD luciferase reporter following Huh7 cell transduction with LVs for Pdx1 PV Ngn3, or combinations as indicated for 72h. Huh7 cells transduced with LV GFP were used as a control. All experiments were done in triplicate and re peated independently at least three times. All p values are compared to non treated controls. ** = p<0.001
65 Figure 2 10. INS1 l uciferase reporter gene analysis during reprogramming. Relative activity was measured for the INS1 luciferase reporter following Huh7 cell transduction with LVs for Pdx1 PV Ngn3, or combinations as indicated for 72h. Huh7 cells transduced with LV GFP were used as a control. All experiments were done in triplicate and repeated independently at least three times. All p values are compared to non treated controls. ** = p<0.001
66 Figure 2 11. Ngn3 l uciferase reporter gene analysis during reprogramming. Relative activit y was measured for the Ngn3 luciferase reporter following Huh7 cell transduction with LVs for Pdx1 PV Ngn3, or combinations as indicated for 72h. Huh7 cells transduced with LV GFP were used as a control. All experiments were done in triplicate and repe ated independently at least three times. All p values are compared to non treated controls. ** = p<0.001
67 Figure 2 12. Nkx2.2 l uciferase reporter gene analysis during reprogramming. Relative activity was measured for the Nkx2.2 luciferase reporte r following Huh7 cell transduction with LVs for Pdx1 PV Ngn3, or combinations as indicated for 72h. Huh7 cells transduced with LV GFP were used as a control. All experiments were done in triplicate and repeated independently at least three times. All p values are compared to non treated controls. ** = p<0.001
68 Figure 2 13. Down regulation of and ATT in Huh7 cells over expressing Pdx1 by western blotting Huh7 cells were transduced with or without LV Pdx1 or LV rhGFP for 96h. Following transduction, Huh7 cells were lysed in RIPA buffer. Equal amount of cell lysate s were separated on 12% SDS polyacrylamide gels and immunoblotted with antibod ies against Pdx1, ALB ATT, or a ctin.
69 Figure 2 14. Down regulation of luciferase reporter in Huh7 cells over expressing Pdx1. Huh7 cells were transfected with 1.0g of luciferase reporter plasmid and 0.2 0.8g Pdx1 expression plasmid as indicated pcDNA3 was used as a DNA quantity control. All values are signi ficant (p<0.05) compared to mock transfection controls.
70 Figure 2 15. Down regulation of by full length Pdx1. Huh7 cells were transfected with 1.0g of luciferase reporter plasmid and 0.8g of each Pdx1 truncated (120, 160, or 200 ami no acids) or full length (283 amino acids) plasmid with pcDNA3 as a DNA quantity control. ** = p<0.001
71 Figure 2 16. Pdx1 increases P2 transcripts that compete with P1 transcripts. Total RNA was collected from Huh7 cells following transduction with LV PV or control ( LV GFP ). Indicated gene expression was measured by real time RT PCR. Primers were designed to detect expression from the P1 promoter (Exon 1A) or the P2 promoter (Exon 1D) as well as the three C terminal modifications. This experiment was done in triplicate and repeated independently at least 3 times.
72 Figure 2 17. Cell luc. Relative act ivity was measured for the mouse luciferase reporter (1g /well) following transduction of various isoforms (1g /well) in Huh7, INS 1, and 293 cells. Activity was normalized in each cell type to the pcDNA3 empty vector control. This experiment was done in triplicate and repeated independently at least 3 times. All values are significant (p<0.05) compared to mock transfection controls.
73 Figure 2 18. Down regulation of luc iferase reporter by Pdx1 in 3T3 cells. Relative activity w as measured for the mouse luc iferase reporter (1g /well) following transduction of and Pdx1 alone, and in combination. + indicates 1 g/well and the concentration gradient shows three concentrations (0.1, 0.5 and 1.0 g) per well. All activation and suppression values are significant (p<0.05) except for activation by Pdx1 alone. Experiments were done in triplicate and repeated independently at least three times.
74 Figure 2 19. Down regulation of luc iferase reporter by in 3T3 cells. Relative activity was measured for the mouse luciferase reporter (1 g/well) following transduction of and alone, and in combination. + indicates 1 g/well and the concentration gradient shows three concentrations ( 0.1, 0.5, and 1.0 g)/well. All activation and suppression Experiments were done in triplicate and repeated independently at least three times.
75 Figure 2 20. A proposed mechanism of Pdx1 medi ated hepatocyte toward IPC reprogramming This cartoon depicts our proposed molecular mechanism for Pdx1 mediated down regulation of during hepatocyte to IPC reprogramming. The left side shows the molecular events involved in normal hepatocytes and the right side shows how these molecular events may be altered following ectopic expression of Pdx1 (based on our cumulative data). The HN and genes and promoters are shown in order to understand how their relationship and downstream gene targeting is affected by the expression of Pdx1 during reprogramming from hepatocytes toward IPCs. We propose that the competitive inhibition of HNF4a P1 isoforms, by P2 isoforms, suppresses and its downstream target gene expression and promotes hepatocyte dedifferentiation.
76 CHAPTER 3 DISTINCT REGULATION OF HNF1 BY Nkx 6.1 IN PANCREATIC BETA CELLS Introduction Hepatic nuclear factor glucose stimulated insulin secretion (GSIS) and is the major factor involved in most cases of maturity onset diabetes of the young (MODY) which accounts for about 1% of worldwide diabetes cases 46, 49 to maintain normal beta cell function and both under expression and over expression have been shown to lead to diabetes 75, 174, 184 187, 192 of the liver, pancreas, kidneys, stomach, and intestines 138, 141, 156, 1 69, 267 This transcription factor is essential for control of mature cellular phenotype in these tissues. In pancreatic beta involved in glycolysis and glucose stimula ted insulin secretion such as insulin 130 Glut2 glucose transporter 210 pyruvate kinase 268 aldolase B 269 167 167 regulat ion in beta cells has not been studied in detail, and this gene is assumed to be regulated based on mechanistic studies from hepatocytes 245 be sufficient for its activation in hepatocytes 144, 147 but evidence exists to suggest that expressed as three isoforms (A, B, and C) that have tissue specific distribution ratios 169 This research was originally published in The Journal of Biological Chemistry. William Donelan, Vijay Koya, Shi Wu Li, and Li Jun Yang. Distinct Regulation of Hepatic Nuclear Factor 1 by NKX6.1 in Pancreatic Be ta Cells. The Journal of Biological Chemistry. 2010; Vol 285:12181 12189. the American Society for Biochemistry and Molecular Biology.
77 pote 159 Second, the major regula predominantly expressed from the P1 promoter in hepatocytes, whereas in the pancreas only transcripts from the P2 promoter can be detected according to most reports 238, 240, 241 have a truncated N terminal region containing the transactivation domain and these isoforms have bee n shown to have lower transactivation potential when compared to isoforms expressed from the P1 promoter 224, 229, 232 the pancreas. Third, Huang et al 150 show that while a 497 bp pr luciferase reporter was shown to be activated in hepatocytes, it failed to be activated in site used for activation in hepatocytes. The aforementioned data may utilize a different mechanism for gene transcription in beta cells than previously identified for hepatocytes. NK6 homeodomain 1 (Nkx6.1) is a homeodomain transcription factor involved in pancreatic differentiation and beta cell ho meostasis 115 In mature human islets, it is exclusively expressed in beta cells 117 and is required for normal GSIS 118 Over expression of Nkx6.1 has been shown to increase GSIS in rat islets 122 It is also of interest that islets isolated from type 2 diabetic patients have altered Nkx6.1 expression 127 However, the specific function of Nk x6.1 in GSIS of mature beta cells remains elusive.
78 gene expression exists in pancreatic beta cells. Here we report a novel finding that ssion in beta cells, which may provide insight into the understanding of the regulation of GSIS in beta cells. Materials and Methods Cell Culture and Huh7 human hepatocarcinoma cells were cultured in DMEM supplemented with 10% FBS, 1% Penicillin/Streptomycin, and 0.1% Kanamycin in a 37 incubator with 100% humidity and 5% CO 2 Plasmid Construction into the pGL3 vector (Promega) using the EcoRI and BglII restriction sites. pGL TK: The pGL TK expression vector was purchased from Promega. pcDNA3: The pcDNA3 vector was purchased from Invitrogen. CMV Pdx1: The human Pdx1 expression plasmid ( pCMV6 XL5 ) was purchased from Origene. Nkx6.1: The human Nkx6.1 expression plasmid (pBAT12) was a generous gift from Dr. Michael German from the University of California, San Francisco, CA. Ngn3: The mouse Ngn3 expression plasmid was a generous gift from Dr. Mark o Horb at the Institute de Recherches Cliniques de Montral, Montral, QC Canada. Mouse Ngn3 cDNA was cloned into the pcDNA3 vector using the BamHI restriction site. MafA: Human MafA cDNA was cloned into the pTYF lentiviral vector cassette under control of the elongation factor 1 alpha promoter using the BamHI and SpeI restriction sites. NeuroD1: Mouse NeuroD1 cDNA was cloned into the pcDNA3.1 CT GFP TOPO vector. Pax6: The xenopus Pax6 expression plasmid (a
79 generous gift from Dr. Marko Horb) was derived by inserting Pax6 cDNA into the cDNA was cloned into the pCMV plasmid (pCMV Sport6) was purchased from Open Biosystems. Pbx1: Mouse Pbx1 was cloned into the pcDNA3 vector using the BamHI and XbaI restriction sites. Transient Transfection and Luciferase Assays Cells were cultured as previously indicated and transfected with 0.1 (as indicated) using Lipofectamine 2000 Reagent (Invitrogen) according to Luc plasmid wa s used as a transfection control in all experiments. Cell lysates were harvested and measured 24 hours post transfection using the Dual Luciferase Reporter Kit (Promega) according to protocol was used (as optimized by our lab) to read samples using a Lumat LB 9507 Luminometer (Berthold Technologies). All luciferase assays were done in triplicate. Site Directed Mutagenesis Primers were designed (IDT Technologies) to induce a block mutation of 4 6 base pairs as indicated at the core sequence inducing a restriction enzyme site for easy confirmation of mutation. Primer sequences are as follows: Nkx6.1, (F:5' GGACCTGTT CCTCGAGGAAATGTGACACTTTAC 3') and (R:5' GTAAAGTGTCACATTTCCTCGAG GAACAGGTCC 3') HN CTTGCA AGGCTGAAGTCCGGCCGTCAGTCCCTTCCT AAGCGCAC 3') and (F:5'
80 GCTTAGGAAG GGACTGACGGCCGGACTTCAGCCTTGCAAGTGCAG 3'). Mutations were induced using PCR on relevant plasmid with Pfu polymerase enzyme. PCR products were incubated for 1 hour with DpnI (New England Biolabs) methylation sensitive restriction enzyme to remove template plasmid and transformed into competent E. coli. Positive colonies were inoculated in LB medium (MP Biomedicals) and plasmid was purified using a Qiagen plasmid purification maxi kit. Electrophoretic Mobility Shift Assay (EMSA ) Biotin labeled probes were designed (IDT Technologies) spanning the cis regulatory element identified. Sequences are as follows: (F:5' GAAGGATGGACCTGTTCCTAATGG AAATGTGACACTTTA 3') and (R:5' TAAA GTGTC ACATTTCCATTAGGAACAGGTCCATCCTTC 3'). Nuclear lysate from beta cell lines PER nuclear generous gift from Dr. Christopher Ne wgard at the Duke University Medical Center, Durham, NC. Anti Nkx6.1 polyclonal antibodies (Santa Cruz) were used. Binding reactions were performed using the LightShift Chemiluminescent EMSA Kit (Promega) according to manufacturer's protocol. Binding re actions were resolved by polyacrylamide gel electrophoresis using a 7.5% Tris HCl polyacrylamide gel (Biorad). Complexes were detected with the Chemiluminescent Nucleic Acid Detection Module (Pierce) according to manufacturer's protocol. Chromatin Immunop recipitation (ChIP) Assay ChIP assay was performed using the chromatin immunoprecipitation assay kit (Millipore) according to manufacturer's protocol. Beta cells cultured in 10cm culture dishes were used both with and without indicated transfection. Spec ific polyclonal
81 Following DNA isolation, sequences were evaluated by PCR using primers (IDT Technologies) flanking respective cis regulatory elements as indicated. Amplificatio n primer sequences are as follows: Nkx6.1, (F:5' CCCATCCAGGATGAAGTGAG 3') and (R:5' GACAAGGAGTTCTGGGCTAG TCACTCCCAATTGCAAGCCATG 3') and (F:5' TGCTGCTCTGTTTACATTGG 3'). Gene Expression and Quantitative RT PCR Cells were cultured as previous ly indicated and transfected with DNA or siRNA (as indicated) using Lipofectamine 2000 Reagent (Invitrogen) according to protocol. Nkx6.1 specific siRNA and control siRNA was purchased from Santa Cruz. Total RNA was extracted from cells us ing Trizol Reagent (Invitrogen) and RT PCR and real time RT PCR were performed as previously described(38). All sequences of primer pairs are available upon request. Statistical Analysis Statistical analysis was carried out using the two sample Student's t test. A P value < 0.01 was considered significant. All indicated significant values are in comparison to controls. Results ells ocytes. Transcription is regulated through a TATA like box ( 21 to binding site ( 47) 144, 147, 148 binding site) is fully active in hepatocytes but not in rat insulinoma INS1 cells 150 providing evidence that its regulation may be controlled by alternative elements in other
82 tissue types. To test this hypothesis, we have cloned a 2772 base pair (bp) region of am of the firefly luciferase gene in a pGL3 has full activity in both beta cells (Fig. 3 1) and hepatic cells (Fig. 3 2 ). We created tructs by 5' deletion analysis (Fig. 3 1 ) using cells is conferred through a more distal regulatory element ( 2772/ 1820). Loss of this regulatory region significantly dimi cells (Fig. 3 1 ) but not in hepatic cells (Fig. 3 2 ). NIH 3T3 cells serve as a control in regulatory region emplo C ells binding site (Fig. 3 6 ). As previously reported 144, 147, 148 transactivation in hepatic cells (Fig. 3 3 3 3 ). This demonstrates that beta cells are reg ulated by a separate response element that is different from hepatic cells. ctivated by Nkx6.1 2772/ 1820) that is beta cell specific, we were interested in discovering which factors regula te transcription at this site. Several beta cell specific transcription factors were screened (Pdx1, Ngn3, MafA,
83 rase reporter (Fig. 3 4 144, 147, 148 and we have found that Nkx6.1 is also a strong activator. Nkx6.1 can ac 3 5 ) and is likely to be the regulator of the cis regulatory element that is unique to beta cells. Mutational A nalysis of HNF romoter The literature pertaining to Nkx6.1 binding suggests that the most common core DNA sequence utilized by Nkx6.1 for binding is: (5' TAAT 3')(49 54) or its compliment (5' ATTA 3') and it has also been shown to bind a similar sequence (5' ATTT 3')(55). transactivation in beta cells ( 2772/ 1820) and found there is a single (5' TAAT 3') DNA sequence in this entire region (Fig. 3 6 ). An alignment of this promoter region between mouse and rat shows a very high level of homology similar to alignment of the promoter region near 7 ). This prompted us to use site directed mutagenesis to induce a mutation (Fig. 3 6 ) at this potential Nkx6.1 binding site in order to examine the effect on transactivation by Nkx6.1. We also gene rated a double mutant Nkx6.1 binding site mutation cells (Fig. 3 8 ). Results were as still be activated by Nkx6.1. Similarly, the Nkx6.1 binding site mutant failed to respond
84 Nkx6.1 binding site that is used for ini Beta C ells Since the Nkx6.1 binding site mutation was sufficient to prevent Nkx6.1 mediated beta cells. Transfection of the Nkx6.1 binding site mutant construct shows approxim 3 9 ). Four other random (5' TAAT 3') mutant constructs were generated and had no change in activity when compared to the normal promoter construct in similar assays (data not shown). Activity was not completely diminished in the Nkx6.1 binding site mutant construct suggesting that other beta cell specific This r cells. lement To further evaluate DNA promoter element ( 2772/ 1820) we designed biotin labeled oligonucleotide probes spanning the potential Nkx6.1 binding site (5' TAAT 3'). Using EMSA, we have demonstrated that Nkx6.1 protein from beta cell lysate can bind to our probe (Fig. 3 10 Lane 2). Addition of Nkx6.1 polyclona l antibody caused a disruption and supershift of the DNA protein band (Lane 3) confirming the protein is indeed Nkx6.1. Unlabeled cold probe is able to compete for binding Nkx6.1 (Lanes 4 & 5) while nonspecific probe is
85 unable to compete (Lane 6). This r esult confirms that Nkx6.1 protein is capable of ells were grown normally ( ) or transfected with the Nkx6.1 expression plasmid (+). Following chromatin immunoprecipitation with Nkx6.1 polyclonal antibody, 3 11 ). The ted with the Nkx6.1 expression plasmid show an increase in the intensity of the band confirming Nkx6.1 binding at this site. Our cumulative data able to positively regulate its expression. assay (Fig. 3 12 n normally ( ) or transfected with the specific isoform) shows a weak amplification band indicating that the binding site may still have function to allow its detection. It may also b
86 n Pancreatic Beta C ells mouse beta cell using (Fig. 3 13) and NIT (Fig. 3 14) cell lysate. Next, we confirmed that both beta cell lines expressed key important for maintaining beta cell function (Fig. 3 15 ). T o determine whether Nkx6.1 O ver expression of Nkx6.1 in n 3 fold over control ( Fig. 3 16 ), while Nkx6.1 specific siRNA knocked down endogenous expression of Fig. 3 17 ), providing further support that Nkx6.1 is a ntrol because it has been previously shown to be regulated by Nkx6.1 through distal promoter binding 122 ta cells. Discussion cells. Different from hepatocytes, we demonstrate that Nkx6.1 is a key regulator for cells. We by EMSA and ChIP assays. Furthermore, we also demonstrated that the endogenous best of pancreatic beta cells.
87 Nkx6.1 is known to be involved in pancreatic differentiation and beta cell function 115, 117, 118 Embryonic expression of Nkx6.1 is dependent on Nkx2.2 119, 120 and in mature beta cells it is regulated by Pdx1 120 Nkx6.1 maintains beta cell phenotype in part by direct interaction with the glucagon promoter, suppressing its activity 118, 121 Nkx6.1 inhibits glucago n expression by competing with Pax6 (glucagon activator) for occupancy of the G1 element on the glucagon promoter. Nkx6.1 has also been linked to beta cell proliferation by up regulating cyclins A, B, and E as well as many regulatory kinases 122 Studies in knockout mice reveal that Nkx6.1 gene is required for beta cell development, terminal differentiation, and biological function 119 Over expression of Nkx6.1 has been shown to increase GSIS in rat islets 122 To address what happens to gene expression in beta cells when Nkx6.1 expression is altered, we performed functional studies in beta cell lines to show the effects of Nkx6.1 over expression (Fig. 3 16) and knockdown (F ig. 3 17 ). As expected, Nkx6.1 over expression l ed to an time PCR while Nkx6.1 knockdown by siRNA led to decreased expression. In addition, Uchizono et al. 2009 270 shows that mice leading to significant changes in blood glucose levels. The same paper, by exploring homozygous deficient mice, reveals decreased Nkx6.1 gene expression. It also shows chang es in expression of genes involved in beta cell growth and proliferation, providing cell functional studies using islets, Newgard's group has published a study 122 showing
88 that over expression of Nkx6.1 increases GSIS in rat and human islets by inducing beta cell replication. In contrast, knockdown of Nkx6.1 in human islets leads to impaired GSIS 122 A recent report indicates that mutations affecting the expression of B lymphocyte kinase (BLK) gene was found to be responsible for some patients with MODY s ymptoms but without mutations in known MODY genes 271 BLK is a previously unidentified modulator of insulin synthesis and GSIS in beta cells by the mechanism of enhancing the expression of Nkx6.1, providing further evidence that Nkx6.1 may be a new candidate gene involved in MODY. Nkx6.1 homeodomain constructs have been shown to bind sequences containing the core homeodomain bindin g site (5' TAAT 3' or 5' ATTA 3') and direct both gene repression and gene activation 123, 124 In its own promoter, it has been shown to bind a similar sequence (5' ATTT 3') to positively regulate its own expression 125 Nkx6.1 h as the ability to function as both a transcriptional activator and repressor which may be sequence dependent 125 The transcriptional repression domain has been isolated to the N terminus 124 while the transcriptional activation domain has been shown to be dependent on the C terminus 125 The C terminus has also been observed to interfere with DNA binding but greatly enhance specificity for homeodomain core containing sequences 126 t alternative isoforms of 159, 169 Three separate isoforms confirmed by real time PCR and no evidence for additional forms was found 159 The
89 different isoforms are generated through differential selection of polyadenylation and alternative splicing generat 159 nscription does not use an alternative promoter as demonstrated by 5' RACE analysis 159 The time points in development suggesting that they may control gene expression in a temporal and tissue specific manner. The alternate isoform ratios may also contribute to differential gene regulation between cell types. Alternative splicing of transcripts increases versatility of function of products and several documented proteins act as activators and repressors from the s ame gene 160 mice develop many symptoms of diabetes 186 knockout mice marked by hyperglycemia and impaired glucose tolerance without loss of beta cell mass 184 Genes involved in beta cell regulation and metabolism are expressed the islet enriched transcription facto 185 abnormal beta cell gene expression, impaired glucose tolerance, and fasting hyperglycemia leading to the development of diabetes 174, 184 186 Mice eng ineered to 187 marked by compromised islet morphology and expression 75
90 MODY is a form of monogenetic diabetes caused by an autosomal dominant mutation in one of several genes. The most prominent form of this disease (>70%) is 42, 48, 50, 272 MODY3 is characterized by adolescent onset of hypergl ycemia that progressively worsens with a ge and most often requires pharmacological treatment. Patients with MODY3 often suffer from diabetic complications. Defining the etiology of MODY is essential for proper pharmacogenetic treatment of the disease 42 45 GSIS 48 and mutations mainly result in defective glucose metabolism and insulin secretion in beta cells 48 For this reason, it is not surprising that MODY3 patients respond well to treatment with oral sulfon ylureas given that these drugs bind the K ATP channel which is downstream of glucose metabolism in the insulin secretion pathway 44 Sulfonylurea therapy can close the K ATP channel which increases intracellular Ca 2+ and stimulates insulin secretion by an ATP independent pathway. Patients who are misdiagnosed with type 1 or type 2 diabetes have succes sfully transferred to sulfonylurea treatment without deterioration in glycemic control 50, 53, 54 Following etiologic identification of MODY3, patients have been transferred from insulin to oral sulfonylurea treatment and show improved glycemic control, reduced risks of hypoglycemia, and delay or prevention of diabetic complications 50, 53, 54 However, etiologic identification is essential to alter treatment in MODY patients because sulfonylureas are not effective f or all other forms of this disease 42 45 Current screening ximal promoter 43, 272
91 In up to 20% of MODY patients, the etiology of the disease is unknown, making it very difficult to predict the clinical cour se of the disease or provide the most effective this regulatory system is unique to beta cells. Our work provides potential novel regulatory elements that should be include d when screening for mutations that effect pancreas 117 but not expressed in the liver 14 This novel regul atory network may provide potential new targets for diagnosis and MODY and possibly implicate a new gene (Nkx6.1) involved in this disease. Identification of novel molecular targets that cause MODY has the potential to greatly improve treatment and the qu ality of life for a great deal of diabetic patients.
92 Figure 3 1. in 3T3 and (3T3 and were transfected full length constructs that were arbitrarily set to 1.0. For transactivation was set equal. Bracket indicates beta cell specific cis response element. S amples were measured 24 hours following transfection and experiments were repeated independently at least three times. (*** p<0.001 )
93 Figure 3 2. Deletion analysis of in Huh7 cells. Huh7 c ells were as indicated. Luciferase values are relative to the full length constructs that were arbitrarily set to 1.0. S amples were meas ured 24 hours following transfection and experiments were repeated independently at least three times.
94 Figure 3 3. and Huh7 (open box) and 1.0. S amples were measured 24 hours following transfection and experiments were repeated independe ntly at least three times.
95 Figure 3 4. Full length ( cotransfected with various beta cell specific transcription factor expression S amples were measured 24 h ours following transfection and experiments were repeated inde pendently at least three times.
96 Figure 3 5. Full length ( in 3T3 cells with increasing amounts of Nkx6.1 expression plasmid as indicated. pcDNA3 was used as a control to equalize DNA quantity used for each transfection. S amples were measured 24 hours following transfection and experiments were repeated indepen dently at least three times.
97 Figure 3 6. sites are indicated in boxes on the promoter. Sequences below the shaded boxes show the induced mutations.
98 Figure 3 7. omoter species alignment. Mouse and rat (GenBank accession #: upper alignment lower alignment ) binding sites are shown. Vertical lines indicate the close homology between species. The shaded boxes indicate core biding sequences.
99 Figure 3 8. Normal ( top left panel ), top right panel ), mutant Nkx6.1 binding site ( bottom left panel ), or double mutant ( bot tom right panel mutation and open boxes indicate normal sequence. pcDNA3 was used as a control and the relative value obtained was arbitrarily set to 1.0. S amples were measured 24 hours following transfection and experiments were repeated independently at least three times.
100 Figure 3 9. promoter in beta cells. 1.0. S amples we re measured 24 hours following transfection and experiments were repeated independently at least three times. (*** p<0.001 )
101 Figure 3 10. INS1 cell lysate EMSAs were conducted with INS1 cell lysate an oligonucleotide (probe) as indicated. Arrows indicate Nkx6.1 binding and supershift. In competition assays, DNA binding reactions were preincubated with 10 fold (10x) or 100 on specific (NS) oligonucleotide as indicated.
102 Figure 3 11. Nkx6.1 ChIP assay. Nkx6.1 binding experiments are shown with (+) or without ( ) Nkx6.1 over expression in cells Representative images are shown following agarose gel electrophoresis of PCR products. Quantification of bands was done with Microsoft Photoshop quantification tool.
103 Figure 3 12. ChIP assay. without ( in cells Representative images are shown following agarose gel electrophoresis of PCR products. Quantification of bands was done with Microsoft Photoshop quantification tool.
104 Figure 3 13. lysate. promoter oligonucleotide (probe) as indicated. Arrows indicate Nkx6.1 binding and supershift. In competition assays, DNA binding reactions were preincubated with 10 fold (10x) or 100 f oligonucleotide or non specific (NS) oligonucleotide as indicated.
105 Figure 3 14. with NIT1 cell lysate EMSAs were conducted with NIT1 oligonucleotide (probe) as indicated. Arrows indicate Nkx6.1 binding and supershift. In competition assays, DNA binding reactions were preincubated with 10 fold (10x) or 100 specific (NS) oligonucleotide as indicated.
106 Figure 3 15. RT PCR for determination of gene expression. Total RNA was isolated from indicated cell lines and expression of pancreatic genes was measured by RT PCR.
107 Figure 3 16. Effect of Nkx6.1 over expression on mRNA levels. NIT1 mouse beta cells were transfected with CMV Nkx6.1 expression plasmid for 48 hours. Total RNA was isolated and subjected to reverse transcription. Target gene expression was quantified by real time PCR and expressed as fold over control as in dicated. Empty vector plasmid (pcDNA3) transfection was used for a negative control for all tested genes and arbitrarily set to 1.0.
108 Figure 3 17. Effect of Nkx6.1 knockdown on mRNA levels. NIT1 mouse beta cells were transfected with Nkx6.1 siRN A for 48 hours. Total RNA was isolated and subjected to reverse transcription. Target gene expression was quantified by real time PCR and expressed as fold over control as indicated. Empty vector plasmid (pcDNA3) transfection was used for a negative contr ol for all tested genes and arbitrarily set to 1.0.
109 CHAPTER 4 NOVEL DETECTION OF PANCREATIC AND DUODENAL HOMEOBOX 1 (Pdx1) AUTOANTIBODIES (PAA) IN HUMAN SERA USING LUCIFERASE IMMUNOPRECIPITATION SYSTEMS (LIPS) ASSAY Introduction Pancreatic and duodenal homeobox 1 (Pdx1) is a key transcription factor for pancreatic development and beta cell maturation and function, and it also plays important roles pancreatic beta cell survival and regeneration 273 Pdx1 autoantibodies (PAA) have recently been identified in serum from both non obese diabetic (NOD) mice and human type 1 diabetes (T1D) patients 254 Other autoantibodies in T1D such as insulin autoantibodies (I AA) 274 islet cell autoantibodies (ICA) 275 glutamic acid decarboxylase (GAD) autoantibodies (GADA) 276 and insulinoma 2 (IA 2) associated autoantibodies (IA 2A) 277 are useful markers for diagnosis as well as for predicting disease onset and may have a role for the timing of interventions 6 Clinical liquid phase radioimmunoprecipitation assays (RIPA) for these autoantibodies are available However, a non radioactive alternative assay has been developed for detection of GADA 278, 279 and IA 2A 279, 280 with similar sensitivity and specificity know as the luminescence immunoprecipitation system (LIPS) assay 281, 282 Here, we report a LIPS assay for detecting PAA in human sera using a Pdx1 luciferase fusion protein produced in mammalian cells. This new liquid phase assay provides a non radioactive means of detecting PAA in human sera. Materials and Methods Plasmi d C onstruction The human Pdx1 renilla luciferase fusion plasmid was constructed by cloning the renilla luciferase gene (from the pRen2 plasmid) upstream of the Pdx1 gene in pCMV
110 XL5 (Open Biosystems) using HindIII/BamHI restriction sites for expression in site directed mutagenesis to construct the luciferase only expression control. Renilla luciferase GAD65 278 and renilla luciferase IA2 280 fusion plasmids were generous gifts from Dr. Peter Burbelo (NIH, Bethesda, MD). All plasmids were purified using the Plasmid Maxi Kit (Qiagen ). Fusion Protein L ysate Mammalian fusion protein lysates were prepared by transfecting human embryonic kidney (293) cells with each plasmid using Lipofectamine 2000 Reagent (Invitrogen) 2 culture dishes. Fort y eight hours following transfection, lysates were harvested using 1ml Passive Lysis Buffer (Promega) per dish and supernatant (lysate) was collected following centrifugation. 293 cells were cultured at 37C in DMEM containing 10% fetal bovine serum and 1 % Penicillin/Streptomycin. Sera Healthy human donor sera (n = 10) with no known history of autoimmune diseases were used to establish the cut off between positive and negative. Fifty four serum samples from the University of Florida Pathology Laboratori es, Endocrine Autoantibody Laboratory were used to validate the LIPS assay, 29 of which are clinically tested triple positive for ICA (by indirect immunofluorescence), GADA, and IA 2A (by RIPA). The ICA assay has been validated previously and was the basis for the Diabetes Prevent ion Trial 1 study 283 and is used in the current T1D TrialNet studies 284 The GADA and IA 2A assays were manufactured by Kronus, Inc. (Star, Indiana) and validated previously. In addition, four sera from T1D patients awaiting renal transplants (that were previously
111 identified as PAA positive) were used for determination of PAA antigenic specificity. All sera were measured blindly LIPS A ssay LIPS assays were performed similarly to the previously published protocols 281, 282 wit h minor modifications. Pdx1 7 RLUs in Berthold Lumat LB9507) was incubated with 10 l human serum for PAA (or 1 l serum for GADA and IA 2A) in 96 well round bottom plates at a total volume of 100 l in PBS overnight with agitation. Healthy normal donor control sera were used to establish a normal range. Samples were then transferred to 96 well filter plates containing 10 l Immobilized Protein A/G plus Beads (Pierce) of 50% concentration (beads/volume) and incubated at 4C for 2hrs with agitation. All samples were washed 8 times with Buffer A as previously described 281, 282 20ul PBS was added to each well before reading in a LUMIstar Omega plate reader (BMG Labtech). Competition assays used purified recombinant Pdx1 protein (rPdx1) 285 or BSA at indicated concentrations and were incubated with Pdx1 luciferase fusion cell lysate overnight. Due to limited sera volumes, all assays were performed in singlet. We obtained extra sera for negative controls and some strong PAA positives that were used for competition assays to determine antigenic specificity. Results Because PAA are a recent discovery, there is no standard assay for detecting these antibodies in the clinical setting. To validate the LIPS assay, we first compared our detection of GADA (Fig. 4 1 ) and IA 2A (Fig. 4 2 ) by LIPS assay to clinical data previ ously determined by RIPA. Sera from patients (n = 54) and control subjects (n = 10) were screened for GADA or IA 2A by LIPS assay and a positive cutoff was set at 3
112 standard deviations (SD) above the mean of control subjects. For GADA, LIPS identified 28 out of 29 (97%) RIPA positive sera and 6 additional LIPS positive sera that were RIPA negative. For IA 2A, LIPS identified all 29 (100%) RIPA positive sera and one additional LIPS positive serum that was RIPA negative However, the degree of positivity for individual samples varied greatly between RIPA and LIPS assay without a linear correlation. For example, a particular sample may yield a high positive value by RIPA but a low to moderate value by LIPS assay, or vice versa. Compared to clinical RIPA, LIPS assay shows 97% sensitivity and 76 % specificity for GADA detection and 100% sensitivity and 96% specificity for IA 2A detection (Fig. 4 3 ) However, since the accuracy of the reference assay (RIPA) is unknown, the low specificity could alternatively indicate that the LIPS assay is actually more sensitive than RIPA. Taken together, these results suggest that the LIPS assay is comparable to the clinical RIPA for GADA and IA 2A in our hands. Next, we tested the above serum samples (54 patients and 10 he althy control s) for PAA by LIPS assay (Fig. 4 4 ). We arbitrarily set the positive cutoff as the mean +3 SD of 10 healthy control sera and defined any sample above this cutoff as positive for PAA. Using this cutoff, 7 out of 54 sera were positive for PAA (13%). Among the PAA positive samples, 6 out of 29 triple positive serum samples were PAA positive (21%) and 1 out of 25 triple negative serum samples was PAA positive (4%). Despite the abundance, PAA did not reach statistical significance in association with triple positive T1D To validate the antigenic specificity of the LIPS assay for PAA, we selected serum samples from T1D patients awaiting renal transplants that previously tested neg ative,
113 medium, and high positive for PAA by LIPS assay To exclude the possibility of antibody binding to luciferase, immuno reactivity with both Pdx1 luciferase antigen and with luciferase antigen alone was tested, demonstrating that the PAA positive sera bound only to Pdx1 luciferase (Fig. 4 5 ). We next confirmed Pdx1 antigenic specificity using a competition assa y with purified rPdx1 (Fig. 4 6 ). We selected our high est PAA positive sample for LI PS assay to detect PAA by incubating serum with various concentrations of rPdx1 or bovine serum albumin (BSA) as control. The PAA signal was reduced with increasing concentrations of rPdx1 but not with BSA, confirming that the PAA signal is specific for P dx1 protein. BSA non specifically reduced the signal but was unable to block detection of PAA even at high concentrations (0.125mg/ml). Discussion We have developed a liquid phase, non radioactive assay to detect PAA in human sera and confirmed t he prese nce of PAA in 7 out of 54 samples While developing our assay, we first performed LIPS assays to detect GADA (Fig. 4 1) and IA 2A (Fig. 4 2) from samples that had been previously evaluated clinically by RIPA. If RIPA is held as the reference standard, on e would conclude that our LIPS assay has nearly identical sensitivity but a reduced specificity for detection of these autoantibodies. This would suggest that we have identified several false positive sera. However, another interpretation of the data is that our assay is more sensitive than RIPA and that these RIPA. We conclude that the LIPS assay has similar sensitivity to RIPA and because it does not involve the use o f radioisotopes, it is safer and more convenient. Thus, the LIPS assay may be valuable in the clinical setting for detecting autoantibodies
114 Insulin is considered to be an early autoantigen in T1D and it has been proposed to be the primary antigen relat ed to T1D 286 288 This is ba sed on the early detection of IAA in people later developing T1D and by the fact that insulin is uniquely expressed and secreted by the beta cell whereas other autoantigens are not unique to the beta cell (e.g., GAD or IA 2A). Pdx1 could also be an early autoantigen with respect to T1D development and s ome evidence suggest s that Pdx1 may be a primary autoantigen related to T1D. In our previous report 254 we detected PAA in NOD mice by ELISA, western b lotting, and RIPA We also followed the PAA titer of several female NOD mice during the development of diabetes and found that PAA titers tended to peak prior to the onset of hyperglycemia and then dropped to undetectable levels after several weeks. This may be due to a lack of Pdx1 antigen stimulation following destruction of beta cells. To determine whether this is the case for humans will require evaluation of PAA in human T1D patients throughout disease development. Serum from individual patients sh ould be measured for PAA at various time points during disease development, from pre diabetic to clinical manifestation of diabetes. were not significantly associated with the presence of triple positive (ICA, GADA, and IA 2A) T1D related autoantibodies ( p = 0.10) Since our study only examined sera that was triple positive (or negative) for other T1D related autoantibodies and since the p value was relatively low, PAA association with other T1D related autoantibodi es merits further investigation. Future studies should examine whether PAA are significantly associated with sera from patients with only one or two T1D related autoantibodies and in patients with other T1D related autoantibodies such as zinc transporter 8 protein (ZnT8) islet autoantibodies
115 (ZnT8A) 289 or IAA 274 In addition, T1D patients that are negative for all othe r known T1D related autoantibodies could be examined. Due to our limited resources, several questions remain unanswered regarding PAA. Large scale systematic, longitudinal studies will be required to determine the prevalence of PAA in the normal populatio n, T1D patients, high risk or prediabetic populations, and in other diseases as well as to identify if there exists any clinical value of PAA for prediction, diagnosis, or monitoring of T1D patients.
116 Figure 4 1. Detection of GADA in human sera by LIPS assay. Sera (n = 54) that were tested for ICA, GADA and IA 2A clinically by RIPA or healthy normal donor control sera (n = 10) were used and the transverse line is 3SD above control mean. RLU (relative light units) are expressed as fold increase over cont rol mean (left y axis). Standard LIPS assay as performed with renilla luciferase GAD65 fusion protein lysate and compared to c linical RIPA data (right y axis).
117 Figure 4 2. Detection of IA 2A in human sera by LIPS assay. Sera (n = 54) that were tested for ICA, GADA and IA 2A clinically by RIPA or healthy normal donor control sera (n = 10) were used and the transverse line is 3SD above control mean. RLU (relative light units) are expressed as fold increase ove r control mean (left y axis). Standard LIPS assay as performed with renilla luciferase IA 2 fusion protein lysate and compar ed to clinical RIPA data (right y axis).
118 Sensitivity Specificity GADA 97% 76% IA 2A 100% 96% Figure 4 3. Sensitivity and specificity of LIPS assay vs. RIPA for GADA and IA 2A. Sera (n = 54) that were tested for ICA, GADA and IA 2A clinically by RIPA or healthy normal donor control sera (n = 10) were used. Standard LIPS assays GAD65 fusion protein lysate or renilla luciferase IA 2 fusion protein lysate Sensitivity and specificity were calculated using RIPA as a reference assay.
119 Figure 4 4. Detection of PAA in human sera by LIPS assay. Sera (n = 54) that were tested for ICA, GADA and IA 2A clinically by RIPA or healthy normal donor con trol sera (n = 10) were used to detect PAA using a standard LIPS assay with Pdx1 luciferase fusion protein lysate T he transverse line is 3SD above control mean. RLU (relative light units) are expressed as fold increase over control mean AA b = autoantibody.
120 Figure 4 5. Antigenic specificity of PAA using luciferase only control. were negative (Ctrl), medium (A & B), or high signal positive ( C ) for PAA (from T1D patients awaiting renal transplants) were used in a standard LIPS assay using either Pdx1 luciferase antigen or luciferase only antigen for detection
121 Figure 4 6. Antigenic specificity of PAA by competition with purified rPdx1 protein. A standard LIPS assay was performed to detect PAA from our high est signal positive serum incubated with indicated concentrations of rPdx1 or BSA.
122 CHAPTER 5 P ANCREATIC AND DUODENAL HOMEOBOX 1 (PDX1) AU TOANTIBODIES (PAA) FROM HUMAN SERA DETE CTED IN AUTOIMMUNE DISEASES AND CANCER Introduction In our previous report, we developed a liquid phase non radioactive luciferase immunoprecipitation systems (LIPS) assay 281, 290 for detection of pancreatic and duodenal homeobo x 1 (Pdx1) autoantibodies (PAA) 254 in human sera and we determined that PAA are not significantly associated with the presence of triple positive T1D autoantibodies (to ICA, GADA, and IA2A) In the present repo rt we have assessed the presence of PAA in patient sera with recent onset (RO) T1D and age matched non T1D control s and longstanding (LS) T1D in order to determine the relationship of PAA with T1D. We also assessed the presence of PAA in patient sera with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and various forms of cancer (including pancreatic cancer) in order to determine if PAA are unique to T1D. Materials and Methods The human Pdx1 renilla luciferase fusion plasmid was constr ucted and purified and m ammalian fusion protein lysates were prepared and used for LIPS assay as previously described. All sera were measured blindly and consisted of samples from patients with RO T1D (n = 100) and age matched non T1D control s (n = 100) LS T1D (N = 50), SLE ( n = 48), RA ( n = 30), or various forms of cancer (n = 70). Healthy human donor sera (n = 10) with no known history of autoimmune diseases were used as normal controls for setting up the cut off between positive and negative (3SD abov e control mean)
123 Results Using a cutoff of three standard deviations above the mean of norm human control sera, we detected positive PAA sera (Fig. 5 1 and 5 2 ) as follows: 7% from RO T1D patients (n = 100) and 13% from age matched non T1D control s (n = 100) 20% from LS T1D patients (N = 50), 48% from SLE patients (n=48), 3% from RA patients (n=30), and 16% from cancer patients (n=70). Sera that produced high positive signal for detection were assayed several times whenever possible (based on serum volu me) and consistent results were produced. It is interesting to note that a high PAA positive signal was detectable in a patient with pancreatic cancer (Fig. 5 2 cancer plot). Discussion Based on our data, the relationship of PAA to T1D is unclear. Altho ugh PAA were found in 7% of RO T1D patients, we surprisingly found PAA in 13% of age matched non T1D controls and also identified PAA in 20% of LS T1D patients. The high prevalence of PAA in the non T1D age matched controls is confounding. One possibilit y for this data is that the age matched control group may not actually be a normal population, and this is supported by the fact that our control group (used to establish PAA positive cutoff) was completely negative for PAA. Another possibility is that th is is an anomaly due to small sample size and larger scale studies may be required to identify the true prevalence of PAA in T1D vs normal patient sera. Finally, PAA could be the result of cross reactive autoantibodies to another antigen 291 This is the first report of PAA association with diseases other than T1D. We have observed a high prevalence (48%) of PAA in patients with SLE. Since double stranded DNA autoantibodies are common among SLE patients 292 and because Pdx1 is a DNA binding protein 293 we have assessed whether detection of our PAA positive signal from
124 SLE patient samples was actually due to double stranded DNA autoantibodies. A standard radioimmunoprecipitation assay was used to assess the presence of double stranded DNA autoantibodies in the SLE patient sera. Double stranded DNA autoantibodies were only detected in PAA negative sera (data not shown) confirming that PAA and double stranded DNA autoantibodies are distinct and have no association. One of the most intriguing pieces of data we found was that one of the highest signal s of detection for PAA was from a pancreatic cancer patient. We hypothesize that the resulting lesion from pancreatic cancer could cause leaking of Pdx1 protein and subsequent presentation to the immune system leading to production of PAA. If this is true, o ver production of Pdx1 from pancreatic cancer cells could lead to high titer PAA production by B cells. If PAA is directly associated with pancreatic cancer, detection of PAA could be useful for screening and diagnosing pancreatic cancer which remains one of the most elusive forms of cancer with a poor prognosis and 5 year survival rate less than 5% 294 PAA are detected in human sera from patients with autoimmune diseases or cancer by LIPS assay, but whether there is an association of PAA with each disease is unknown. Large scale sy stematic, and longitudinal studies are still required to determine the clinical value of PAA for prediction, diagnosis, or monitoring of T1D patients or patients with other diseases.
125 Figure 5 1. PAA detected in human T1D patient sera. Sera from recent onset (RO) T1D patients (n = 100) and non T1D age matched controls (n = 100) long standing (LS) T1D patient s (n = 50), or healthy normal donor controls (Ctrl, n = 10) were screened for PAA by stand ard LIPS assay using mammalian produced Pdx1 luciferase fusion protein lysate. The transverse line is 3SD above control mean. RLU (relative light units) are expressed as fold increase over control mean.
126 Figure 5 2. PAA detected in human autoimmune disease and cancer patient sera. Sera from healthy normal donor controls (Ctrl, n = 10) and patients with systemic lupus erythematosus (SLE, n = 48), rheumatoid arthritis (RA, n = 30), and various forms of cancer (Cancer, n = 70) were screened for PAA by standard LIPS assay using mammalian produced Pdx1 luciferase fusion protein lysate. The transverse line is 3SD above control mean. RLU = relative light units and are expressed as fold over control mean.
127 CHAPTER 6 C ONCLUSIONS AND FUTUR E DIRECTIONS Before the discovery of insulin by Banting and Best in 1921 295 T1D was a fatal disease. I nsulin replacement therapy is not a cure for T1D because t he difficulty of maintaining normal blood glucose concentration causes most T1D patients to have a hyperglycemic condition leading to vascular degradation and subsequent tissue damage and organ failure 1 However, in 2012, insulin replacement therapy continues to be the standard treatment for T1D. This provide s the rationale for the work in this dissertation with the ultimate goal of prov iding adequate control of blood glucose to T1D patients without any harmful side effects. Successful pancreas 296, 297 and islet transplantations 8, 298, 299 have o ccurred but are not suitable for the entire T1D population because they require use of immunosuppressants that cause harmful side effects and they are not permanent cures. For these reason s investigators have focus ed on alternative methods for generatin g beta cells Success in these attempts may still not be sufficient to provide a cure for T1D. As mentioned before, the pathogenesis of diabetes is two fold, and surrogate beta cells will face possible destruction from recurrent autoimmunity 297 Enhanced knowledge of t he mechanisms involved in T1D pathogenesis and beta cell differentiation will be important in developing a cure. Previous studies aimed at beta cell generation have used many different tissue sources and many different factors to investigate the reprogram ming process. We have focused our work on studying the mechanism s involved in reprogramming liver to pancreatic beta cells We chose this system based on previous reports that demonstrate the extraordinary ability of hepatic cells to transform into insul in producing beta cells. We investigated the reprogramming of liver into beta cells by ectopic
128 expression of pancreatic beta cell specific transcription factors Pdx1 and Ngn3 because they are fundamental factors that are shown to be essential for beta cel l development. Most of the other factors used in similar reprogramming studies are downstream targets of Pdx1 and Ngn3 or expressed during later stages of development 60, 293 Using these factors allowed us to study the fundamental early events involved in reprogramming liver to beta cells. Discovering suppression of HNF1 in Huh7 cells followi ng ectopic expression of Pdx1 was a key finding in our early studies and prompted us to further investigate the mechanism as to how this occurred. Although we cannot rule out the possibility that many mechanisms account for Pdx1 mediated HNF1 suppression we did discover major changes in the regulation and expression of its major activator HNF4 and beta cells used primarily P1 and P2 promoter respe 2 40 event in Pdx1 mediated liver to beta cell reprogramming. The fact that Pdx1 is a known regulator of the P2 promoter specifically in beta cells 236 further supports this notion. In order to study this process in greater detail, we cloned approxi mately 3kb of the reprogramming is a major focus of our lab, we have produced or acquired many transcription factor expression plasmids related to beta cells. Having these tools available pr ompted us to luciferase construct for potential transcriptional activators that are beta cell specific. Another driving factor in this exploration was the fact that, previously,
129 mal promoter) was found to be active in hepatic cells but completely inactive in rodent INS1 beta cells 150 and this beta cell specific transcription regulator s And i ndeed, Nkx6.1, which is not expressed in hepatic cells. Future studies should investigate the beta cell specific regulation of HNF1 greater detail. One of the main point s left unresolved from our work is the role of the line 150 cell line 255 However, much of our luciferase reporter data obtained from both liver specific isoforms generated from the P1 promoter and beta cell specific is o forms generated from the P2 promoter Although all the same DNA binding domain differences may allow for interactions with other molecu les that prevent them from functioning in a similar manner. The N exon 1D or exon 1D and exon 1E. These unrelated domains (1A vs 1D) have the potential to interact with other proteins in a cell type specific manner and form protein reporter was not active in the INS1 beta cell line, even considering that the cell line
130 expressed the major activator It is possible that exon 1D is not sufficient alone to recruit the necessary molecules for transcription, while exon 1A may be sufficient. It is possible that protein DNA complexes (normally prese nt in the endogenous context) are not available to interact with architecture of expressed in beta cells may not confer transactivation al one, but may require the participation from proteins or protein complexes that bind the distal regions of the DNA complexes may not be present or functional in hepatic cells but may be required for proper regulation in beta cells regulation of target genes The specific mechanisms that govern can lead to three potential C are believed to have the potential to acquire any of the three possible C terminal consequences for its regulation of target genes because it affects the interactions with other proteins and complexes. Since Pdx1 is a known r promoter in beta cells, it is possible that Pdx1 has influence on the specific mechanism s of alternative splicing and thus Pdx1 may play a role in the regulation of specific It is also possible that in general, the data from luciferase transfection assays is not representative of endogenous regulatory events since the gene architecture and
131 expression will make these s tudies difficult, but understanding cell specific mechanisms of regulation will certainly enhance our understanding of beta cell function and may provide additional markers useful for following liver to beta cell reprogramming. The discovery of PAA was ver y interesting within the context of my work, considering that Pd x1 is the primary transcription factor used during the reprogramming of hepatic cells into beta cells and because Pdx1 expression in hepatic cells leads to Our lab initially discovered PAA 254 by accident following our development of a system to produce rPdx1 in Pichia pastoris 285 Pdx1 is a transcription factor that contains a protein transduction domain (PTD ) which is a specifi c amino acid sequence enriched with positively charged arginine and lysine residues, allowing for its t ransduction across cellular membranes by lipid raft mediated macropinocytosis 31, 35, 36 For this reason, our lab injected NOD mice direct ly with Pdx1 protein in order to stimulate beta cell expansion and hepatic reprogramming into insulin producing cells to prevent the onset of diabetes 17 As a control, we generated a mutant non functional rPdx1 protein with the PTD (as well as DNA binding domain) removed. Although injecting NOD mice with mutant rPdx1 did not stimulate beta cell expansion or hepatic reprogramming, it prevented the onset of diabetes similarly to normal Pdx1 (unpublished data). We hypothesized that this protection was induced by antigen specific immunotherapy 300 following rPdx1 or mutant rPdx1 injection prompting us to examine the presence of Pdx1 antibodies in the serum of these mi ce. We also examined the serum of NOD mice for the presence of Pdx1 antibodies before treatment leading to the discovery of PAA in NOD mice (which spontaneously develop autoim mune diabetes) but not in other strains examined, incl udi ng NOD scid, C57/B6,
132 and BALB /c. It is interesting to note that, in general, the appearance of PAA in NOD mice peaked before the onset of hyperglycemia and then tapered off to very low or undetectable levels by ELISA. In this report 254 we also discovered the presence of PAA in human serum by western blotting, but were unable to detect human PAA from the same samples by ELISA which is possibly due to distortion of the PAA specific epitope. For this reason, I developed the liquid phase luciferase immunoprecipitation systems (LIPS) assay for detection of PAA in human sera. In regard to our work on PAA, a utoantibodies related to T1D are useful markers for diagnosis as well as for predicting disease onset 6 Although we have demonstrated that PAA are detected in human sera, it is unclear w hat their relationship is to T1D, if any. While a search of the scientific literature for autoantibodies in T1D patient sera will yield hundreds of results, few studies are able to demonstrate any association with disease and most fail to have any clinica l utility. While our follow up study demonstrated more PAA positive sera from non T1D patients compared to T1D patients our initial work did find 6 out of 29 triple positive serum (ICA, GADA, and IA 2A) samples were PAA positive (21%) and 1 out of 25 tri ple negative serum samples was PAA positive (4%) Although this finding is not statistically significant (p = 0.10) according to Fisher s exact test, the relatively low p value for a limited sample number merits further investigation of PAA in order to de termine their clini cal value for prediction, diagnosis, or monitoring of T1D patients or patients with other diseases. Perhaps the most interesting data from this study is the discovery of PAA from a patient with pancreatic cancer ( 5 year survival rate le ss than 5% 294 ). Pdx1 and pancreatic cancer are colocalized to the pancreas and it is reasonable to hypothesize that the molecular instability within the cancer tissue
133 could lead to presentation of Pdx1 to the immune system resu lting in PAA production by B cells. Due to the severity of pancreatic cancer, this single discovery merits further investigation to determine if PAA are a marker for pancreatic cancer as it could lead to earlier detection and improve prognosis for patien ts. In conclusion, this work sheds further insight into the molecular mechanisms involved in liver to pancreatic beta cell reprogramming and also presents a system that will be useful for determining the clinical utility of PAA. Future studies will be required to determine and employ other molecular mechanisms involved in the reprogramming process in order to successfully restore beta cell mass for the growing diabetic population.
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157 300. Shuhong Han, Wi lliam Donelan, Westley Reeves & Li Jun Yang (2011). Autoantigen Specific Immunotherapy, Type 1 Diabetes Pathogenesis, Genetics and Immunotherapy, David Wagner (Ed.), ISBN: 978 953 307 362 0, InTech, Available from: http://www.intechopen.com/books/type 1 diabetes pathogenesis genetics and immunotherapy/autoantigen specific immunotherapy
158 BIOGRAPHICAL SKETCH William LePage Donelan was born in Adams, M assachusetts in the United States of America on May 10, 1982. Early in life, h e moved to New York and gradua ted from Saratoga Springs High School in 2000. He attended the Crane School of Music and t he State University of New York (SUNY) at Potsdam unt il 2006 and received a B.A. in m usic and a B.S. i n b iology. Will then moved to Gainesville Florid a to attend gr aduate school at t he University of Florida College of Medicine. He was accepted to the Interdisciplinary Program in Biomedical Sciences and worked in the laboratory of Dr. Li Jun Yang until grad uating in 2012 with a Ph.D. in medical s cience s