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MUSCLE CELL BIOLOGY AND CELL MOTILITY
Center for Exercise Science, Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida
Submitted 12 December 2007 ; accepted in final form 7 February 2008
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ABSTRACT |
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nitric oxide synthase; nuclear factor of activated t-cells; glycogen synthase kinase-3β; soluble guanylate cyclase; myosin heavy chain I/β
Nitric oxide (NO) is a ubiquitous signaling molecule produced enzymatically by NO synthases (NOS). Recently, it has been reported that NO is required for NFATc3 nuclear accumulation in mouse cerebral arteries in response to increased intravascular pressure and that this effect was dependent on inhibition of NFAT nuclear export (8). Since both constitutive isoforms of NOS (i.e., endothelial and neuronal isoforms, eNOS and nNOS, respectively) are expressed by skeletal muscle cells (25) and are regulated by calcium-calmodulin binding (25), we postulated that a similar mechanism could be important for NFAT-dependent transcriptional adaptations in this tissue.
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 (23). Since GSK-3β has been identified as an important regulator of NFATc1 nuclear export (24) and type I slow MHC expression (9) in muscle, we postulated that NO supports NFAT-dependent transcription, translocation, and nuclear accumulation in muscle cells by inhibiting GSK-3β activity. Therefore, we tested the following hypotheses: 1) endogenous NOS activity is necessary for calcium ionophore-induced slow MHC mRNA expression, 2) endogenous NOS activity is necessary for calcium ionophore-induced nuclear accumulation of NFAT and NFAT-dependent transcriptional activity, 3) NOS activity is necessary for calcium-ionophore-induced inhibitory phosphorylation of GSK-3β, 4) an NO donor is sufficient to affect GSK-3β phosphorylation, and 5) NO-dependent GSK-3β phosphorylation and calcium-ionophore-induced NFAT-dependent transcriptional activity are mediated by soluble guanylate cyclase (sGC) activity.
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METHODS |
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Cell Culture Mouse C2C12 myoblasts were obtained from American Type Culture Collection (Manassas, VA) and cultured at 37°C in 5% CO2-95% atmospheric air.
Myoblasts 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 and 1% penicillin-streptomycin. For the 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) experiment, C2C12 cells were plated on 24-well plates, grown to 70–80% confluency, and differentiation induced by switching to medium containing 2% horse serum for 5 days before treatment. For all luciferase experiments at 70–80% confluency, the growth medium was removed, and cultures were washed with serum-free media and transiently transfected with plasmid vectors as described below. For all Western blots and RNA isolation, C2C12 myotubes were plated on six-well plates, grown to 70–80% confluency, and differentiation induced by switching to medium containing 2% horse serum for 7 days. Myotubes were treated with one or more of the following chemicals in media containing 2% serum: A23187 [GenBank] , L-NAME, PAPA-NO, ODQ, YC-1, LiCl. Whenever treatments were used in combination, inhibitors of NOS (L-NAME) and guanylate cyclase (ODQ) were added 30 min before other treatments. Control groups were exposed to treatment vehicles in concentrations equal to experimental groups. When total protein extracts were harvested, cells were washed twice in ice-cold PBS and harvested in nondenaturing lysis buffer (NDL) containing 1% vol/vol Triton X-100, 0.3 M NaCl, 0.05 M Tris-base, 5 mM EDTA, 3.1 µM NaN3, 95 mM NaF, 22 µM Na3VO4. For isolation of nuclear proteins, cells were harvested in ice-cold PBS containing 1 µM Na3VO4 and 0.05% vol/vol protease inhibitors (catalog no. p-8340) and 0.5% vol/vol phosphatase inhibitors (catalog no. p-5726) from Sigma and centrifuged, and the resulting pellets were treated with NE-PER nuclear and cytosolic extraction reagents according to the manufacturer's procedures (Pierce Biotechnology, Rockford, IL). Both NDL and NE-PER buffers contained 0.1% vol/vol protease inhibitors and 1% vol/vol phosphatase inhibitors from Sigma. For RNA, cells were harvested in TRIzol reagent (Life Tech, Carlsbad, CA) according to manufacturer's instructions.
Measurement of NO Production Intracellular NO was monitored with DAF-FM (Invitrogen, Carlsbad, CA), a pH-insensitive fluorescent dye that emits increased fluorescence after reaction with an active intermediate of NO formed during the spontaneous oxidation of NO to NO2–. C2C12 myotubes were incubated at 37°C for 30 min in phenol red-free, serum-free DMEM containing 10 µM of DAF-FM diacetate. After loading was completed, cells were rinsed three times with phenol red-free, serum-free DMEM and then placed in a SpectraMax M5 multi-detection reader (Molecular Devices, Sunnyvale, CA) for fluorometric analysis of live cells. NO fluorescence was measured using excitation and emission wavelengths of 488 and 520 nm, respectively. After measurement, cells were harvested for protein analysis and fluorometric intensity normalized to protein content for each well. Treatment groups before fluorometric analysis are described below in experimental treatments section.
Transient Transfections Myoblasts were transfected with either a reporter plasmid containing the firefly luciferase gene driven by a promoter sequence containing four repeats of a consensus NFAT binding site or a negative control plasmid (pNFAT-luc or pCIS-CK, Stratagene; 0.4 µg/well). Cells were cotransfected with a second plasmid (pRL-CMV, 0.02 µg/well; Promega, Madison, WI) to control for transfection efficiency. Plasmids were complexed with Lipofectin reagent (Invitrogen) and exposed to myoblasts in serum-free DMEM for 5 h. Parallel myotube cultures were transfected using identical procedures with a plasmid expressing green fluorescent protein (pEGFP-C1, BD Biosciences) to quantify transfection efficiency, which was consistently >60%. After transfection, cells were again placed in 10% FBS media for 16 h before being switched to differentiation media (DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin). Differentiation medium was refreshed every 48 h until confluent myotubes were formed (4–7 days).
Dual Luciferase Assay Immediately after treatment, myotube cultures were washed with ice-cold PBS and lysed by addition of 120 µl passive lysis buffer (Stratagene). Plates were rocked at room temperature for 15 min. The lysate was then transferred to microcentrifuge tubes and centrifuged for 5 min (4°C, 300 g) to sediment cellular debris. Supernatant 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 10-µl volume of cell lysate using the dual luciferase assay kit (Promega) according to the manufacturer's instructions and a luminometer (model FB12, Berthold) set to measure average light intensity in relative light units (RLU) over a 10-s 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.
RNA Expression by RT-PCR Concentration and purity of the extracted RNA were measured spectrophotometrically at A260 and A280 in 1x Tris-EDTA (TE) buffer (Promega). Reverse transcription (RT) was performed using the SuperScript III First-Strand Synthesis System for RT-PCR according to the manufacturer's instructions (Life Technologies). Reactions were carried out using 5 µg of total RNA and 2.5 µM oligo(dT)20 primers. First-strand cDNA was treated with two units of RNase H and stored at –80°C. Primers and probes for slow MHC (GenBank NM_012751, 1m Assay no. Rn00562597_m1) were obtained from the ABI Assays-on-Demand service and consisted of Taqman 5' labeled FAM reporters and 3' nonfluorescent quenchers. Primer and probe sequences from this service are proprietary and, therefore, are not reported. Primer and probe sequences also consisting of Taqman 5'-labeled FAM reporters and 3' nonfluorescent quenchers for hypoxanthine guanine phosphoribosyl transferase (HPRT) obtained from Applied Biosystems (Assays-by-Design) are the following: forward, 5'-GTTGGATACAGGCCAGACTTTGT-3'; reverse, 5'-AGTCAAGGGCATATCCAACAACAA -3'; Probe, 5'-ACTTGTCTGGAATTTCA-3'.
Quantitative real-time PCR was performed using the ABI Prism 7700 Sequence Detection System (ABI, Foster City, CA). Each 25-µl PCR reaction contained 1 µl of cDNA reaction mixture. In this technique, amplification of the fluorescently labeled probe sequence located between the PCR primers was monitored in real time during the PCR program. The number of PCR cycles required to reach a predetermined threshold of fluorescence (CT) was determined for each sample. Samples were quantified relative to the CT (using the 2–
CT method, where CT is threshold cycle) (20) for a normalizing gene (HPRT) determined separately in the same sample.
Western Blotting Protein concentrations were measured using the DC Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA). Aliquots of cell lysates (8–15 µg) were run in 4–20% and 12% SDS-PAGE gels for phospho-GSK-3β, total GSK, NFATc1, β-actin, and histone. Nuclear extracts (11 µg) were run in 12% SDS-PAGE gels for NFAT blots. Protein was transferred to nitrocellulose membrane and blocked with Odyssey blocking buffer for 1 h. The primary antibodies used were the following: phospho-GSK-3β (Ser-9): sc-11757 (goat), 1:1,000 dilution, GSK-3β (H-76): sc-9166 (rabbit), 1:1,000 dilution, NFAT c1 (F-1) sc-8405 (mouse), 1:500 dilution, (Santa Cruz Biotechnology, Santa Cruz, CA), β-actin (mouse), 1:4,000 dilution, (Abcam, Cambridge, MA) and anti-histone H2B, (rabbit), 1:5,000 dilution (Upstate, Lake Placid, NY). The membranes were incubated at 4°C overnight in primary antibody diluted with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE), TBS, and 0.01% Tween-20, and then washed with TBS-T four times and incubated for 35 min in secondary antibody, Odyssey blocking buffer, and TBS-T. Secondary antibodies used were the following: IRDye 800CW rabbit anti-goat (LI-COR, 1:5,000), IRDye 680 mouse anti-rabbit (LI-COR, 1:2,500), and IRDye 680 rabbit anti-mouse (LI-COR, 1:5,000). Membranes were washed four times with TBS-T and once with TBS before being scanned and detected using the Odyssey infrared imaging system (LI-COR).
Experimental Treatments NO production. EXPERIMENT 1. One hour after DAF-FM loading, myotube cultures were treated with one of the following nine treatments: 1) DMSO vehicle control, 2) 1 µM MAHMA-NO, 3) 0.1 µM A23187 [GenBank] , 4) 0.4 µM A23187 [GenBank] , 5) 1 µM A23187 [GenBank] , 6) 5 mM L-NAME, 7) 0.1 µM A23187 [GenBank] + L-NAME, 8) 0.4 µM A23187 [GenBank] + L-NAME, or 9) 1 µM A23187 [GenBank] + L-NAME. Groups 6–9 were pretreated with L-NAME for 30 min before addition of other compounds. Fluorometric analysis was then performed as described under "Measurement of NO Production".
MHC-I/β expression. EXPERIMENT 2. Myotubes were treated for 24 h with one of the following four chemicals: 1) DMSO vehicle control, 2) 0.4 µM A23187 [GenBank] , 3) A23187 [GenBank] + L-NAME, and 4) 5 mM L-NAME. RT-PCR was then performed as described in detail above.
NFAT transactivating potential. Dose and time course experiments were conducted to obtain the optimal calcium ionophore treatment to evoke NFAT transcriptional activity.
EXPERIMENT 3. Transfected myotube cultures were exposed to one of the following eight treatments for 9 h: 1) DMSO vehicle control, 2) 5 mM L-NAME, 3) 1 µM CsA to inhibit calcineurin activity, 4) 1 µM A23187 [GenBank] , 5) L-NAME + A23187 [GenBank] , 6) CsA + A23187 [GenBank] , 7) 5 mM of GSK-3β inhibitor LiCl, or 8) LiCl + A23187 [GenBank] + L-NAME. The optimal dose of A23187 [GenBank] was used in this experiment (1 µM). However, all subsequent A23187 [GenBank] NFAT transactivating experiments were conducted at an effective but submaximal dose of 0.4 µM to increase sensitivity of the signaling pathway to potential modifiers.
EXPERIMENT 4. Transfected myotube cultures were exposed to one of the following four treatments for 9 h: 1) DMSO vehicle control, 2) 5 mM L-NAME, 3) 2 µM thapsigargin, or 4) L-NAME + thapsigargin.
EXPERIMENT 5. Transfected myotube cultures were exposed to a range of concentrations of the NO donor DETA-NO (0, 1, 10 and 50 µM) for 9 h, with or without the calcium ionophore A23187 [GenBank] (0.4 µM).
EXPERIMENT 6. Transfected myotube cultures were exposed to one of the following four treatments for 9 h: 1) DMSO vehicle control, 2) 0.4 µM A23187 [GenBank] , 3) 10 µM of sGC inhibitor ODQ, or 4) A23187 [GenBank] + ODQ.
NFAT translocation. EXPERIMENT 7. Myotubes were treated with one of the following six treatments for 4 h: 1) DMSO vehicle control, 2) 0.4 µM A23187 [GenBank] , 3) A23187 [GenBank] + L-NAME + LiCl, 4) A23187 [GenBank] + L-NAME, 5) 5 mM L-NAME, or 6) 5 mM LiCl. As described above, nuclear and cytosolic proteins were then separated for Western blot analysis (details above).
GSK-3β phosphorylation. EXPERIMENT 8. To determine the role of NOS in affecting GSK-3β phosphorylation, myotubes were treated for 4 h as follows: 1) DMSO vehicle control, 2) 0.4 µM A23187 [GenBank] , 3) A23187 [GenBank] + L-NAME, or 4) 5 mM L-NAME. Total protein extracts were then measured by Western Blot analysis.
EXPERIMENT 9.
The role cGMP plays downstream of NO in affecting GSK-3β was investigated by treating myotubes for 1 h with the following: 1) DMSO vehicle control, 2) 1 µM PAPA-NO, 3) PAPA-NO + ODQ , 4) PAPA-NO + YC-1, 5) 10 µM ODQ, or 6) 200 µM of sGC enhancer YC-1. PAPA-NO was used in this 1-h experiment due to its faster half-life for NO release (
15 min at 37°C, Cayman Chemical).
Statistical Analyses Normalized values were analyzed for each experiment using either one-way or two-way ANOVAs. Tukey's test was applied post hoc to determine individual group differences where main effects were found. Significance was established at P < 0.05.
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RESULTS |
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NFAT Nuclear Translocation is NOS Dependent Western blot analysis showed an increase in NFAT nuclear accumulation in A23187 [GenBank] -treated cells (4 h) compared with control. However, L-NAME blocked the calcium ionophore effect. Consistent with our data from the pNFAT transfection experiments, treatment with A23187 [GenBank] , L-NAME, and LiCl resulted in significantly higher NFAT nuclear accumulation than A23187 [GenBank] + L-NAME. Separation of nuclear and cytosolic protein fractions was confirmed by immunoblotting for histone and β-actin (Fig. 7).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Previous studies have described a calcium-calcineurin-NFAT-dependent pathway regulating fiber type-specific gene expression (11, 17). However, our study reports the novel finding that MHC-I/β expression is not only calcium dependent, but NOS dependent as well. In fact, our results show that the calcium ionophore A23187 [GenBank] requires NOS activity to induce MHC-I/β mRNA (Fig. 2). This is also the first evidence of a relationship between NO and NFAT-dependent transcriptional activity in skeletal muscle cells. We report that NOS activity is necessary for calcium ionophore-induced nuclear accumulation of NFATc1 (Fig. 7) and induction of NFAT-dependent transcriptional activity (Fig. 3). Also, we found that the NOS inhibitor L-NAME showed a similar attenuating affect on NFAT transcriptional activity in myotubes treated with the calcium-releasing chemical thapsigargin (Fig. 4). Additionally, an NO donor amplified the effect of a calcium ionophore to enhance NFAT-dependent transcriptional activity in cultured myotubes (Fig. 5). The current data extends our previous in vivo data (23) to a controlled cell culture environment and identifies regulation of NFAT-dependent transcription as a mechanism of the effect of NO on MHC-I/β expression.
Chin et al. (3) defined the pathway by which calcium signaling in muscle fibers will activate calcineurin to dephosphorylate NFAT and instigate its nuclear import and subsequent promoter activity of slow-specific genes. NO could influence MHC-I/β expression via involvement in one or more of the following steps: 1) dephosphorylation of NFAT and nuclear translocation/accumulation, 2) DNA binding and promoter activation, 3) rephosphorylation and nuclear export of NFAT, and/or 4) regulation of MHC-I/β mRNA stability or translational efficiency. Our data suggest that the involvement of NO in MHC-I/β gene regulation occurs at the transcriptional level via NFAT accumulation in the nucleus.
Meissner et al. (16) has recently described the assembly of a transcriptional complex, including NFATc1, MyoD, MEF2D, and p300, on the MHC-I/β promoter in response to calcium ionophore treatment. Although NO could affect assembly of this transcriptional complex, the current experiments show that NO affects activity of an engineered promoter driven only by four repeats of a consensus NFAT-binding element. Therefore, it is unlikely that our results, and the previously reported effects of NO on MHC I/β mRNA, are due to an NO effect on transcriptional complex formation. More likely, NO is involved in nuclear import or export of NFAT. Theoretically, NO could directly influence calcineurin phosphatase activity. An NO donor augmented the effect of a calcium ionophore on NFAT-dependent transcriptional activity (Fig. 5). However, in the absence of the calcium ionophore, NO donor treatment did not increase NFAT activity (Fig. 5). This suggests that NO (at the doses tested) works synergistically with calcium signaling but is not sufficient to independently augment the phosphatase activity of calcineurin.
NO and GSK-3β Nuclear NFAT concentrations are dependent on a balance between import and export (activation or deactivation). Kinases are known to phosphorylate NFAT (24), cause its nuclear export (8, 24), inhibit DNA binding (9), and blunt its transactivating potential (24). Protein kinase G and AMP-associated protein kinase (13) are activated by NO and inhibit NFAT signaling in cardiac muscle (7, 12). Nevertheless, our data indicate that NO augments NFAT-dependent transcription, suggesting that NO effects on NFAT activity in skeletal muscle are not mediated by these kinases. Recent reports indicate that GSK-3β is an important inhibitor of NFAT in skeletal muscle (9, 24). Overexpression of GSK-3β in avian skeletal muscle promotes nuclear export of NFAT, whereas inhibition of GSK-3β augments NFAT transactivating potential and enhances MHC-I/β expression (9). Therefore, we investigated the role of NO in deactivation of GSK-3β. We report that an NO donor induces inhibitory phosphorylation of GSK-3β. Furthermore, inhibition of GSK-3β by LiCl causes nuclear accumulation of NFATc1 and stimulates NFAT-dependent transcription. It is unclear why LiCl treatment alone was sufficient to induce NFAT-dependent transcription, whereas DETA-NO treatment only augmented the calcium ionophore effect. We postulate that either LiCl has effects beyond inhibition of GSK-3β that stimulate NFAT or DETA-NO at the doses tested inhibited GSK-3β to a lesser extent than LiCl. Nevertheless, the effects of LiCl on NFAT, unlike A23187 [GenBank] effects, are not inhibited by L-NAME, suggesting that GSK-3β inhibition occurs downstream of NOS activity. Our current data are consistent with the hypothesis that calcium/calcineurin-dependent NFAT activation is accompanied by NO production, which lessens NFAT nuclear export (Fig. 5) via inhibition of GSK-3β (Fig. 6), enhancing its transactivation potential (Fig. 8).
Influence of NO-cGMP Pathway Stimulation of sGC and the resultant accumulation of cGMP mediates many of the signaling functions of NO and regulates complex signaling cascades through immediate downstream effectors, including cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels (15). Guanylate cyclases and cGMP-mediated signaling cascades play a central role in the regulation of diverse physiological processes (10, 15). We show that the sGC inhibitor ODQ effectively blocks calcium-induced nuclear accumulation of NFATc1 and NFAT-dependent transcription. Furthermore, the NO-independent activator of sGC YC-1 induced phosphorylation of GSK-3β, and this effect was not augmented by concurrent treatment with an NO donor. This suggests that NO and sGC are part of the same pathway, rather than exerting separate effects on GSK-3β.
Interestingly, all pharmacological treatments used in this study support our working model (Fig. 10). Two calcium-modifying agents (A23187 [GenBank] and thapsigargin), one calcineurin inhibitor (CsA), one NOS inhibitor (L-NAME), three NO donors (DETA-NO, MAHMA-NO, and PAPA-NO), one sGC inhibitor (ODQ), one sGC enhancer (YC-1), and one GSK-3β inhibitor (LiCl) all provided complementary results, suggesting our findings are not due to nonspecific drug effects. Nevertheless, further studies employing genetic manipulations should confirm these proposed pathways.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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