Nitric Oxide Facilitates Calcium-Induced NFAT-Dependent Transcription in Myotubes

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Nitric Oxide Facilitates Calcium-Induced NFAT-Dependent Transcription in Myotubes
Simmons, Catherine
Criswell, David ( Mentor )
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Gainesville, Fla.
University of Florida
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Nitric Oxide Facilitates Calcium-Induced NFAT-Dependent Transcription
in Myotubes

Catherine G. Simmons


Chronic activation of skeletal muscle induces expression of slow-twitch genes via activation of the transcription

factor, nuclear factor of activated T-cells (NFAT). Nitric oxide synthase (NOS) activity is involved in exercise-

related induction of slow-twitch genes, in vivo. Therefore, we tested the hypothesis that nitric oxide synthase

activity is necessary for calcium-induced NFAT transcriptional activity in C2C12 myotubes. METHODS: Myoblasts

were transiently transfected with a reporter plasmid containing the firefly luciferase gene driven by an

NFAT-dependent promoter (pNFAT-luc, Stratagene). Differentiated myotubes were exposed to various

treatments and harvested for reporter gene assay. RESULTS: The calcium ionophore, A23187 (1pM for 9h),

or thapsigargin (2pM for 4h) increased NFAT transcriptional activity by 16 or 4-fold, respectively, while co-

treatment of myotubes with the NOS-inhibitor, N(G)-L-nitro-arginine methyl ester (L-NAME; 5mM), prevented

the effects of A23187 or thapsigargin on NFAT activity. Treatment of myotubes with various doses of the nitric

oxide donor, DETA-NO (1-50 pM), had no effect on NFAT activity alone. However, co-treatment with DETA-NO

and A23187 caused a dose-dependent synergistic effect such that NFAT activation in cells treated with 10 pM

DETA-NO + 0.4 pM A23187 was twice that in cells treated with A23187 (0.4 pM) alone. CONCLUSIONS: NOS

activity is necessary for calcium-induced NFAT-dependent transcription. Furthermore, although nitric oxide may

not be sufficient to induce NFAT-dependent transcription, it facilitates calcium-induced NFAT activity.


Adult vertebrate skeletal muscle consists of different fiber types, one slow (type I/3) and three fast (IIa, IIx,

and IIb), which differ in their contraction speed, strength, fatigability, and insulin sensitivityi. Skeletal

muscle exhibits a high degree of plasticity with transformations in fiber type occurring in response to

altered physiological demand and contractile load.

Tonic, low-frequency neural activity or electrical stimulation causes a shift from fast, glycolytic fibers to the

slow, oxidative phenotype2. The pathway by which low frequency muscle activation induces transcription of

slow-twitch genes involves sustained calcium [Ca2+] levels sufficient to stimulate calcineurin phosphatase

activity. Dephosphorylation of nuclear factor of activated t cells (NFAT) transcription factors by calcineurin

promotes its translocation from the cytoplasm to the nucleus, where it will bind to a nucleotide recognition

sequence and stimulate the transcription of target, slow-twitch genes2.

Although this pathway explains activity-induced activation of NFAT, overall transcriptional activity, and therefore

fiber type change is determined by the balance between activation and deactivation of this transcription

factor. Recent studies suggest that glycogen synthase kinase 3P (GSK3P) and casein kinase 1 or 2

(CK1/2) synergistically regulate nuclear export of NFAT in skeletal muscle fibers by phosphorylation of its

serine residues3.

Nitric oxide is a ubiquitous signaling molecule produced enzymatically from nitric oxide synthases (NOS).

Recently, Gonzalez-Bosc et al. (2004) reported that nitric oxide is required for NFATc3 nuclear accumulation in

mouse cerebral arteries in response to increased intravascular pressure, and that this effect was dependent

upon inhibition of NFAT nuclear export4.

Our lab has recently reported that inhibition of NOS prevents induction of slow myosin heavy chain (MHC)

gene expression during functional overload of the rat plantaris muscle. Since GSK-3B has been identified as

an important regulator of NFATcl nuclear export3 and type I slow MHC expression5 in muscle, we postulated

that nitric oxide supports NFAT-dependent transcription in muscle cells by inhibiting GSK-3B activity and

subsequent nuclear export of NFAT. Therefore, we tested the hypotheses that 1) endogenous NOS activity

is necessary for calcium ionophore-induced NFAT-dependent transcriptional activity, and 2) that a nitric oxide

donor will increase calcium ionophore-induced NFAT-dependent transcriptional activity.


Cell Culture

C2C12 myoblasts (ATCC) cells were plated on 24-well collagen-coated plates and proliferated in Dulbecco's

Modified Eagle's Medium (DMEM) growth media containing 10% Fetal Bovine Serum (FBS) and 1%

penicillin/streptomycin. At 70-80% confluency, the GM was removed and cultures were washed with serum

free media and transiently transfected with plasmid vectors as described below.

Transient Transfections

Myoblasts were transfected with either a reporter plasmid containing the firefly luciferase gene driven by a

promoter sequence containing 4 repeats of a consensus NFAT binding site, or a negative control plasmid (pNFAT-

luc or pCIS-CK, Stratagene; 0.4 pg per well). Cells were co-transfected with a second plasmid (pRL-CMV,

Promega; 0.02 pg per well) to control for transfection efficiency. Plasmids were completed with Lipofectin

reagent (Invitrogen) and exposed to myoblasts in serum-free DMEM for five hours. After transfection, cells

were again placed in 10% FBS media for 16h before switching to differentiation media (DM) (DMEM

supplemented with 10% horse serum and 1% penicillin/streptomycin). DM was refreshed every 48 hours

until confluent myotubes were formed (4 days).

Experimental Treatments

Myotubes were exposed to various treatments in DM and harvested for assay of firefly and renilla luciferase

activities using the dual luciferase assay (Promega), according to the manufacturer's instructions. Dose and

time course experiments were conducted to obtain the optimal calcium ionophore treatment to evoke

NFAT transcriptional activity. Experiment 1. Transfected myotube cultures were exposed to one of the following

6 treatments for 9h: 1) No supplement control, 2) 5 mM N-nitro-L-arginine methyl ester (L-NAME) to inhibit

NOS activity, 3) 1 pM Cyclosporin A (CsA) to inhibit calcineurin activity, 4) 1 pM of calcium ionophore, A23187, 5)

L-NAME + A23187, or 6) CsA + A23187. Experiment 2. Transfected myotube cultures were exposed to one of

the following 4 treatments for 4h: 1) No supplement control, 2) 5mM L-NAME, 3) 2pM Thapsigargin, or 4) L-NAME

+ Thapsigargin. Experiment 3. Transfected myotube cultures were exposed to a range of concentrations of the

NO donor, DETA-NO (0, 1, 10, and 50pM; Cayman Chemical) for 9h, with or without the calcium ionophore,

A23187 (0.4pM).

Dual Luciferase Assay

Immediately after treatment, myotube cultures were washed with ice-cold PBS and lysed by addition of 120

pl passive lysis buffer. Plates were rocked at room temperature for 15 min. The lysate was then transferred

to micorcentrifuge tubes and centrifuged for 5min (40C, 300g) to sediment cellular debris. Supernatent

was transferred to new tubes and kept on ice during the assay.

Firefly luciferase (originating from transcriptional activity of the pNFAT-luc or pCIS-CK vectors) and renilla

luciferase activities (originating from the constitutively active uptake-control plasmid; pRL-CMV) were

measured sequentially in the same 10pl volume of cell lysate using the dual luciferase assay kit (Promega)

according to the manufacturer's instructions and a luminometer (Berthold, Model FB12) set to measure average

light intensity in relative light units (RLU) over a 10s measurement period. NFAT-dependent transcriptional

activity for each sample was taken as the raw firefly luciferase activity (RLU) divided by the renilla luciferase

activity (RLU). For each experiment, all values were expressed relative to the average of the control group.

Statistical Analyses

Normalized values were analyzed for each experiment using a 2-way ANOVA (calcium ionophore x L-

NAME, thapsigargin x L-NAME, or calcium ionophore x DETA-NO concentration; SPSS v. 12.0.1). Tukey's test

was applied post-hoc to determine individual group differences where main effects were found. Significance

was established at P<0.05.


L-NAME inhibits NFAT-dependent transcriptional activity induced by intracellular calcium

Treatment with the calcium ionophore, A23187, caused a 16-fold increase in NFAT transcriptional activity.
Cyclosporin A (CsA), a calcineurin inhibitor, blocked the effect of the calcium ionophore. The non-isoform-
specific inhibitor of NOS, L-NAME, also blocked the effect of the calcium ionophore on NFAT transcriptional
activity (Figure 1).

15 *

I Control
I 10- A23187 (1pA


Control L-NAME CsA
(5mM) (lpM)
Figure 1. NFAT dependent transcriptional activity relative to uptake control in C2C12 myotubes
harvested immediately after a 9 hour treatment with either 5 mM N-nitro-L-arginine methyl ester
(L-NAME), 1 pM Cyclosporin A (CsA), or a no supplement control; with or without co-treatment with
the calcium ionophore; A23187. Asterisk denotes significant difference from control (P<0.05).

Treatment of myotubes with thapsigargin (2pM) for 4h to induce release of calcium from the sarcoplasmic
reticulum caused a 4-fold increase in NFAT transcriptional activity. Similar to the calcium ionophore experiment,
co-treatment with L-NAME inhibited this effect (Figure 2). Myotubes transfected with the negative control
vector, pCIS-CK, which contains the luciferase gene but lacks the NFAT-responsive promoter sequence, did
not respond to thapsigargin or L-NAME treatments (Figure 2).

f I, IControl
S| Thapsigargin (2pM)
S4- EZL-NANE (5rrn
- M3 Thapsig.+L-NAME


(negative control vector)

Figure 2. NFAT dependent transcriptional activity relative to uptake control in C2C12 myotubes

harvested immediately following a 4 hour treatment with either 5 mM L-NAME, 2 pM Thapsigargin,

L-NAME + Thapsigargin, or a no supplement control. Results compared to transfection with a

negative control vector. Asterisk denotes significant difference from control conditions (P<0.05).

The NO-donor, DETA-NO, augments A23187-induced NFAT-dependent transcriptional activity

Treatment of myotubes with DETA-NO (1-50pM) did not affect NFAT transcriptional activity alone. However,

co-treatment with A23187 produced a synergistic effect (Figure 3).

12- **

10. =Control
8 1 A23187 (0.4 M)


0 1 10 50
DETA-NO Concentration ( pIM

Figure 3. NFAT-dependent transcriptional activity relative to uptake control in C2C12 myotubes

harvested immediately after a 9 hour treatment of varying concentrations of DETA-NO (0, 1, 10, and

50 pM) with an without the treatment of a calcium ionophore. Asterisk denotes significant

difference from control (P<0.05). Double asterisk denotes significant difference from treatment

with calcium ionophore with no DETA-NO (P<0.05).


Nitric oxide has been found through multiple studies to be an important signaling molecule in muscle. It is

produced enzymatically from nitric oxide synthase (NOS) and increased during muscle contraction6 to

support multiple acute and chronic adaptive responses, such as glucose transport and mitochondrial biogenesis6.

A recent study reported that nitric oxide is required for NFATc3 nuclear accumulation in vascular tissue4.

However, our study is the first to report a relationship between nitric oxide and NFAT-dependent

transcriptional activity in skeletal muscle cells. We have found that nitric oxide is necessary for calcium-induced

NFAT dependent transcriptional activity, and NOS inhibition blunted these actions. Additionally, a nitric oxide

donor amplifies the effect of a calcium ionophore to enhance NFAT-dependent transcriptional activity in

cultured myotubes.

Our lab has previously shown that nitric oxide synthase is necessary for overload-induction of mRNA for the

slow isoform of myosin heavy chain (MHC I/p) in the rat plantaris muscle7. The current data extends that

observation to a controlled cell-culture environment and identifies NFAT-dependent transcription as a mechanism

of this effect. Groundbreaking research from the 1990s defined the pathway by which activation-induced

calcium signaling will activate calcineurin to dephosphorylate NFAT and instigate its nuclear import and

subsequent promoter activity of slow-specific genes2. Therefore, nitric oxide could influence MHC I/p expression

via involvement in one or more of the following steps: 1) dephosphorylation of NFAT and nuclear translocation,

2) DNA binding and promoter activation, 3) re-phosphorylation and nuclear export of NFAT, and/or 4) regulation

of MHC I/p mRNA stability or translational efficiency. Given our current data using an NFAT-dependent reporter

gene, we conclude that nitric oxide's involvement in MHC I/p gene regulation occurs at the transcriptional level

via NFAT regulation.

Meissner et al. (2006) has recently described the assembly of a transcriptional complex including NFATcl,

MyoD, MEF2D, and p300, on the MHC I/p promoter in response to calcium ionophore treatment1. Although

nitric oxide could affect assembly of this transcriptional complex, the current experiments show that nitric

oxide affects activity of an engineered promoter driven only by 4 repeats of a consensus NFAT-binding

element. Therefore, it is unlikely that our results, and the previously reported effects of nitric oxide on MHC I/

p mRNA, are due to nitric oxide effect on transcriptional complex formation. More likely, nitric oxide is involved

in nuclear import or export of NFAT.

It is established that the protein phosphatase activity of calcineurin leads to dephosphorylation and

nuclear localization of NFAT proteins2. Therefore, any enhancing effect of nitric oxide on NFAT nuclear

accumulation would have to involve an interplay with calcineurin. Co-treatment of a nitric oxide donor with

calcium showed a dose-dependent effect of NFAT transcriptional activity. In the absence of the calcium

ionophore, the NO donor treatment did not alter or increase NFAT activity. This reveals that NO interplays and

works synergistically with calcium signaling, but does not directly turn on or augment the phosphatase activity

of calcineurin.

Nuclear NFAT concentrations are dependent on a balance between import and export (activation or

deactivation). Kinases such as c-Jun N-terminal protein kinase (JNK) and glycogen synthase kinase 3 (GSK3)

are known to phosphorylate NFAT3, cause its nuclear export3,4, inhibit DNA binding5, and blunt is

transactivating potential3. Nitric oxide was shown to inhibit JNK in an in vitro kinase assay8, however, GSK3 is

known to be a primary inhibitor of NFAT in skeletal muscle3,5. Overexpression of GSK3 promotes nuclear export

of NFAT in T cells, but when GSK3 is inhibited in culture, this export was reduced5. We are currently investigating

the role of nitric oxide in deactivation of GSK. Our current data is consistent with the hypothesis that

calcium/calcineurin-dependent NFAT activation is accompanied by nitric oxide production, which inhibits NFAT

nuclear export, perhaps via inhibition of GSK, to therefore enhance its transactivation potential (Figure 4).

Calcineurin '

Call P_ lw

\ -

Muscle Fiber

Figure 4. Proposed mechanism of NFAT activation and deactivation and proposed action of nitric
oxide synthase.


Skeletal muscle is extremely plastic and is known to adapt to physical load and stress. Slow and tonic
muscle contractions are known to activate the slow-twitch phenotype of skeletal muscle.This slow phenotype
exhibits greater endurance potential and insulin sensitivity corresponding to a greater potential for glucose
utilization. In our society today, insulin resistance syndrome has reached epidemic proportions, however,
every negative symptom and side effect of this disorder can be ameliorated by physical activity in human subjects.
In order to maximize the positive effects of exercise prescription, it is critical to understand the mechanism by
which muscle contractions result in muscle adaptation, particularly to the slow-twitch phenotype. Knowing the role
of nitric oxide in the regulation of muscle fiber type will enable us to maximize the potential of exercise
prescription and aid in the development of more effective treatments for metabolic disorders that plague so
many people today.


1. Meissner JD, Umeda PK, Chang KC, Gros G, Scheibe RJ. Activation of the 3 Myosin Heavy Chain Promoter by MEF-
2D, Myo D, p300, and the calcineurin/NFATcl pathway. J. Cell. Physiol. 211: 138-148, 2007.

2. Chin ER, Olson, EN, Richardson JA, Yang Q, Humphries C, Shelton JM, Wu H, Zhu W, Bassel-Duby R, Williams, RS.
A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type. Genes and Development.
12: 2499-2509, 1998.

3. Shen, T, Cseresnyes Z, Liu Y, Randall WR, Schneider MF. Regulation of the nuclear export of the transcription
factor NFATcl by protein kinases after slow fibre type electrical stimulation of adult mouse skeletal muscle fibers.
SPhysiol. 579.2: 535-551, 2007.

4. Gonzalez Bosc LV, Wilkerson MK, Bradley KN, Eckman DM, Hill-Eubanks DC, Nelson MT. Intraluminal pressure is
a stimulus for NFATc3 nuclear accumulation. J Biol Chem. 279(11): 10702-10709, 2004.

5. Jiang H, Li H, DiMario JX. Control of slow myosin heavy chain 2 gene expression by glycogen synthase kinase

activity in skeletal muscle fibers.Cell Tissue Res. 323: 489-494, 2006.

6. Stamler JS, Meissner G. Physiology of nitric oxide in skeletal muscle. Physiol Rev. 81(1): 209-237, 2001.

7. Sellman JE, DeRuisseau KC, Betters JL, Lira VA, Soltow QA, Selsby JT, Criswell DS. In vivo inhibition of nitric

oxide synthase impairs up regulation of contractile protein mRNA in overloaded plantaris muscle. JAppl Physiol.

100: 258-265, 2006.

8. Park HS, Huh SH, Kim MS, Kim DY, Gwag BJ, Cho SG, Choi EJ. Neuronal nitric oxide synthase (nNOS) modulates

the JNK1 activity through redox mechanism: A cGMP independent pathway. Biochem Biophys Res Commun. 346

(2): 408-414, 2006.


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