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Nitric Oxide Facilitates Calcium-Induced NFAT-Dependent Transcription
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
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.
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.
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).
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,
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.
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).
I 10- A23187 (1pA
Control L-NAME CsA
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).
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
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
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).
Call P_ lw
NO sGC - cGMP
Figure 4. Proposed mechanism of NFAT activation and deactivation and proposed action of nitric
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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