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Originally published In Press as doi:10.1074/jbc.M211357200 on March 11, 2003

J. Biol. Chem., Vol. 278, Issue 23, 21221-21231, June 6, 2003
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The Mitogen-activated Protein Kinase Cascade Promotes Myoblast Cell Survival by Stabilizing the Cyclin-dependent Kinase Inhibitor, p21WAF1 Protein*

Olga Ostrovsky and Eyal Bengal {ddagger}

From the Department of Biochemistry, Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, P. O. Box 9649, Haifa 31096, Israel

Received for publication, November 7, 2002 , and in revised form, February 6, 2003.
   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
During myogenesis, proliferating myoblasts withdraw from the cell cycle and are either eliminated by programmed cell death or differentiate into mature myotubes. Previous studies indicate that mitogen-activated protein kinase (MAPK) activity is significantly induced with the onset of terminal differentiation of C2 myoblasts. We have investigated the part played by the MAPK pathway in the differentiation of C2 myoblasts. Specific activation of MAPK by expression of an active Raf1-estrogen receptor chimera protein reduced significantly the number of myoblasts undergoing programmed cell death in the differentiation medium. Activation of Raf1 prevented the proteolytic activation of the proapoptotic caspase 9-protein during differentiation. The antiapoptotic function of Raf1 correlated with accumulation of the p21WAF1 protein resulting from its increased stability. Antisense expression of p21 was used to determine whether the p21WAF1 protein mediated the antiapoptotic activity of Raf1. Reduction of p21WAF1 protein in muscle cells abolished the antiapoptotic activity of the MAPK pathway. We conclude that MAPK contributes to muscle differentiation by preventing apoptotic cell death of differentiating myoblasts and that this activity is mediated by stabilization of the p21WAF1 protein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
During myogenesis, proliferating myoblasts withdraw permanently from the cell cycle, express muscle-specific genes, and fuse into multinucleated myotubes. The induction of the cyclin-dependent kinase (cdk)1 inhibitor p21WAF1 followed by hypophosphorylation of the retinoblastoma (pRb) protein are key events in the establishment of the postmitotic state that leads to the subsequent differentiation (1). During the process of in vitro differentiation of myogenic cell lines a large fraction of myoblasts is lost through apoptotic cell death, but terminally differentiated myotubes are more resistant. It is generally accepted that myoblasts are exposed to apoptotic cell death during the gross changes occurring in the differentiation process. Those myoblasts that cannot complete the process because of incomplete withdrawal from the cell cycle are subjected to apoptotic cell death (1). The resistance of myoblasts to apoptosis was found to be correlated with the induction of p21WAF1 cdk inhibitor and hypophosphorylation of the retinoblastoma protein, molecules that participate in the withdrawal from the cell cycle (2). In addition, forced expression of p21WAF1 blocked apoptosis during the differentiation of C2 cells, whereas inhibition of p21WAF1 by antisense oligonucleotides induced frequent apoptosis (2). Also, mice deficient in both p21WAF1 and p57 cdk inhibitors have defective muscle formation and exhibit increased rates of myoblast apoptosis (3). The effect of p21WAF1 on myoblast survival is likely to be determined by its capacity to induce the activity of pRb. Consistent with this idea are results showing that pRb-deficient (Rb-/-) myoblast cells undergo higher rates of apoptosis during differentiation than their wild type counterparts (4). Moreover, transgenic mice expressing low levels of pRb display substantial cell death of muscle tissue prior to the onset of terminal differentiation (5). Taken together these studies suggest that defects in those proteins that induce permanent withdrawal of myoblasts from the cell cycle may trigger apoptotic cell death.

Although the function of insulin-like growth factors (IGFs) as inducers of muscle survival has been known for a long time, the intracellular signaling pathways have only recently begun to emerge (6). Two classes of intracellular pathways, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinases (MAPKs) are involved in different aspects of IGF-facilitated muscle differentiation (7, 8, 9, 10). Recent studies have focused mostly on the function of the PI3K pathway in the survival of differentiating myoblasts. By manipulating different kinases and using inhibitors of this pathway, it was demonstrated that it played a major role in protecting differentiating myoblasts from undergoing cell death (11, 12, 13).

A second signaling pathway induced by IGF-MAPK might also protect muscle cells from apoptotic cell death (10). It was recently shown that transient transfection of constitutively active Mek1, a specific activator of extracellular regulated kinases (ERKs), maintained myoblast viability in the absence of growth factors (14).

Several authors (15, 16) reported that the activity of ERK was significantly induced with the onset of myoblast terminal differentiation. We suggested that this activation is an intrinsic property of muscle cells. It is now well established that the MAPK pathway that was commonly regarded as mitogenic, can also induce withdrawal from the cell cycle and survival of cells depending on the magnitude and length of the signal and the specific cell type (17, 18, 19). We decided to investigate the role played by the MAPK pathway in the commitment and differentiation of myoblasts. Our results show that the activity of the MAPK pathway reduces the number of differentiating myoblasts undergoing apoptotic cell death. The MAPK pathway also induces the accumulation of the p21WAF1 protein by prolonging its half-life in differentiating cells. Reduction of p21WAF1 protein by antisense expression interferes with the antiapoptotic function of the MAPK pathway. We conclude that the MAPK pathway regulates the survival of differentiating myoblasts and that this activity is mediated by stabilization of the p21WAF1 protein.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
U0126 was supplied by Calbiochem. It was dissolved in Me2SO to a concentration of 10 mM and was added directly to the differentiation medium to a final concentration of 10 mM. {beta}-Estradiol was purchased from Sigma. Polyclonal antibodies to ERK1,2 and phospho-specific ERK1,2 were purchased from Cell Signaling Technology. A phospho-specific ERK1,2 monoclonal antibody used in immunostaining of cells was purchased from Sigma. A monoclonal antibody to human pRb was purchased from Pharmingen. A monoclonal antibody to myosin heavy chain (MF-20) was a gift from Dr. S. Tapscott. A monoclonal antibody to bromodeoxyuridine (BMC9318) was purchased from Roche Applied Science. Anti-p21WAF1 antibody was from Transduction Laboratories. An antibody to the estrogen receptor was from Santa Cruz Biotechnology. A polyclonal antibody to cleaved caspase 3 (#9661) was from Cell Signaling Technology. A monoclonal antibody to caspase 9 was a gift from Dr. T. Kleinberger.

Plasmids
p21-Luc was described before (20). Retroviral vectors pBP3{Delta}Raf1DD:ER and pBP3{Delta}Raf301:ER were a generous gift from Dr. M. McMahon (18, 21). The retroviral vector encoding for MD:ER was a generous gift from Dr. S. Tapscott (22). The pBABE-GFP retroviral vector was constructed by replacing the puromycin coding sequence with the EGFP coding sequence, which was PCR-amplified and cloned into the ClaI-HindIII sites of pBABE-puro. The mouse p21 cDNA fragment was cloned in the EcoRI site of pBABE-GFP, and a clone that contained the antisense orientation of p21 relative to the promoter was used for further studies.

Generation of Stable C2 Clones
C2 cells were a gift from Dr. D. Yaffe (23). Infection of C2 myoblasts with replication-defective retroviruses was used to generate C2 cell lines expressing the different chimera proteins. Retroviruses expressing the different proteins were generated by transfection of retroviral vectors and an expression vector of vesicular stomatitis virus, the glycoprotein, into viral packaging cells, 293gp, expressing the gag and pol genes (24). The medium of transfected 293gp cells containing retroviruses was used to infect C2 cells. Forty-eight hours later, cells were selected with puromycin (3 µg/ml). Resistant clones were pooled together a week later. The expression of the chimera proteins was determined in Western blots with an antibody to estrogen receptor.

Cell Culture
Cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 15% calf serum (HyClone), penicillin, and streptomycin (growth medium, GM). To induce differentiation, we used Dulbecco's modified Eagle's medium supplemented with 10 mg of insulin per ml and 10 mg of transferrin per ml (differentiation medium, DM). Differentiation of C2 cell lines expressing the fusion ER proteins was induced by the addition of DM. {beta}-Estradiol (10-6 M) was added to DM at different time periods as indicated, and U0126 (10 µM) was added to DM after 24 h.

Transient Transfection Assays
Transfections were performed by calcium phosphate precipitation as described (25) or using the TransFast reagent of Promega according to the recommended protocol. Myoblasts in 6-cm TC dishes (Corning) were transfected with a total amount of 10 mg (or 5 mg, using TransFast) of luciferase reporter plasmid DNA and a control reporter gene expressing Renilla under the constitutive cytomegalovirus promoter. Following transfection, the medium was replaced with DM for another 24–48 h. {beta}-Estradiol was added to the cells as indicated. Protein extracts were prepared and used to measure luciferase and Renilla activities using the Luciferase Assay system from Promega. Luciferase activity was divided by Renilla activity of each reaction to correct for the transfection efficiency.

Immunohistochemical Staining
Cells were fixed and immunostained as described previously (16). The primary antibodies used were anti-phospho-ERK (Sigma), anti-p21WAF1, anti-cleaved caspase 3 (Transduction Laboratories), and monoclonal anti-MHC (MF-20). The immunochemically stained cells were viewed at x200 magnification under a fluorescence microscope (Olympus, model BX50) and photographed with a digital camera.

RNA Analysis
RNA was extracted using TRITM reagent (MRC Inc.) and analyzed by Northern blotting as described previously (16). Blots were hybridized with probes for MLC2 (PVZLC2), p21WAF1 (pCDNA-Waf1), and glyceraldehyde-3-phosphate dehydrogenase (pMGAP).

Western Analysis
Cells were lysed, and whole cell extracts were collected as described (16). Equal amounts of extracted proteins (30–100 µg) were loaded and separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. For detecting the different forms of pRb, proteins were separated over 7.5% SDS-PAGE before being transferred to membranes. Membranes were incubated in blocking buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20, 2% w/v nonfat dry milk) and then with the same buffer with the first and secondary antibodies. Between the second and third incubations, membranes were washed three times in 0.1% Tween 20 and 1x TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl). Immunoblotting was conducted with the following antibodies: anti-ERK and anti-phospho-ERK (Cell Signaling), 1:1000; anti-pRb (Pharmingen), 1:1000; anti-p21WAF1, 1:1000; anti-MHC, 1:2.5; and anti-caspase 9, 1:1000. Proteins were visualized using the enhanced chemiluminescence kit by Pierce Inc.

Bromodeoxyuridine Staining
Bromodeoxyuridine (BrdUrd) was added to cell media at 10 µM. After 2–3 h the cells were washed with PBS, fixed with methanol (10 min), and permeabilized in 0.25% Triton X-100 (10 min). Following a PBS wash, the cells were incubated in 2 N HCl solution for 1 h. The solution was neutralized by washing the cells three times in 0.1 M borate buffer (pH 8.5). Subsequently, the cells were incubated with 6 mg/ml anti-BrdUrd antibody in PBS containing 0.1% bovine serum albumin for 1.5 h. The remainder of the procedure was identical to the immunohistochemical staining of cells described earlier (16).

Apoptotic Cell Death Assays
TUNEL Assay—The assay kit was purchased from Roche Applied Science. The assay was performed according to the manufacturer's instructions.

Hoechst Staining—After washing with PBS, the cells were incubated with the DNA dye bisbenzimidine (Hoechst 33258) (10 µg/ml) for 30 min. Nuclear morphology was observed at x200 magnification under an upright fluorescence microscope (Olympus, model BX50) and photographed with a digital camera. The percentage of cells with condensed DNA was calculated.

DNA Fragmentation Assay—After washing with PBS, cells were collected and then resuspended in extraction buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA, pH 8.0, 20 µg/ml RNase A, 0.5% SDS). Samples were incubated at 37 °C for 1 h. Proteinase K (100 mg/ml) was added, and incubation was continued at 50 °C for 3 more h. DNA was then extracted with phenol/chloroform and precipitated with ethanol. Following a 70% ethanol wash, genomic DNA was resuspended in TE (10 mM Tris, 1 mM EDTA, pH 8.0). An aliquot of 30 µg of DNA was analyzed by electrophoresis in 1.8% agarose gels containing ethidium bromide.

Antisense Expression
Replication-defective retrovirus expressing mouse p21 antisense and the green fluorescent protein (GFP) or control retroviruses expressing the GFP protein were used to infect C2 cells. One day following infection, the medium was replaced by differentiation medium. {beta}-Estradiol was added to medium 24 h later, and cells were stained with Hoechst after 48 h in DM. Cells were viewed for the Hoechst and green fluorescence staining under the above fluorescence microscope.

In Vitro Kinase Assay for ERK
The assay was performed as described in Gredinger et al. (16).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Regulated Activation of ERK MAPK Pathway in C2 Myoblasts—In a previous study we observed that ERK MAPK activity was significantly induced during in vitro muscle differentiation (16). In this study we have found that an increase in phosphorylated ERK was observed after 24 h of C2 myoblasts growth in differentiation medium (DM), and it gradually accumulated as cells differentiated into myotubes (Fig. 1A). To study the functional significance of MAPK pathway activation during myoblast differentiation, we generated inducible C2 cell lines that expressed a conditional Raf1 protein. C2 myoblasts were infected with retroviruses containing either a fusion gene of an activated Raf1 and the hormone-binding domain of estrogen receptor ({Delta}Raf1DD:ER) or an inactivated Raf1 and the hormone-binding domain of estrogen receptor ({Delta}Raf301:ER) (21). Clones harboring the retroviral vectors were selected and further analyzed. These cells constitutively expressed the fusion proteins (not shown). Addition of {beta}-estradiol to C2 cells expressing the active Raf1 protein ({Delta}Raf1DD:ER) induced the phosphorylation of ERK1 by 3–5 fold (Fig. 1B). It did not affect the phosphorylation state of the closely related MAPKs, p38 and JNK (data not shown). Addition of hormone to C2 cells expressing the inactive Raf1 protein ({Delta}Raf301:ER) had no effect on the phosphorylation of MAPK (Fig. 1B). The in vitro kinase activity of ERK protein that was immunoprecipitated from cell extracts was also analyzed (Fig. 1C). Addition of {beta}-estradiol to C2 cells expressing the active Raf1 protein ({Delta}Raf1DD:ER) induced ERK activity by 3- to 5-fold (lanes 1–4), whereas its addition to C2 cells expressing the inactive Raf1 protein ({Delta}Raf301:ER) had no effect on ERK activity (lanes 5–8). ERK activity that was induced by exogenously activated Raf1 ({Delta}Raf1DD:ER) was only 1.5- to 2-fold higher than endogenous ERK activity in cells growing in DM for 48 h (compare lanes 2 and 4 to lane 11). Thus, the system enables us to phosphorylate and activate ERK in an Raf1-dependent manner. Activation of Raf1 induces ERK activities that are in the range of the endogenous activity of ERK in differentiating cells.



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  		FIG. 1.
Regulated activation of the MAPK pathway in muscle cells by a chimera Raf1-estrogen receptor protein affects the structure of myotubes. A, C2 cells were grown in GM and then in DM for the indicated time periods, and proteins were extracted and separated over 10% SDS-polyacrylamide gels. Total and phosphorylated forms of ERK were detected by Western analysis. B, two myoblast cell lines expressing either {Delta}Raf1DD:ER or {Delta}Raf301:ER proteins were generated as described under "Experimental Procedures." Cells were grown in GM, and then the medium was replaced with DM with or without {beta}-estradiol (10-6 M) or Mek inhibitor, U0126 (10 µM). Cells were grown for 24 h in DM before proteins were extracted and separated over SDS-PAGE. Total ERK proteins and their phosphorylated forms were analyzed by Western blotting. C, in vitro kinase assay. The two cell lines described in B were grown in DM with or without {beta}-estradiol for the indicated time periods. Cells were harvested, and fix amounts of extracts were used to determine the in vitro ERK activity. Briefly, ERK proteins were immunoprecipitated and were used to phosphorylate myelin basic protein in vitro as described under "Experimental Procedures." The -fold induction calculated for each {beta}-estradiol treatment was relative to cells grown in the exact same conditions but in the absence of the hormone. D, {beta}-estradiol was added to C2 myoblasts expressing the {Delta}Raf1DD:ER protein together with DM or 36 h following the addition of DM and cells were fixed and immunostained with an antibody to MHC after a total growth period of 72 h in DM. The nuclei were stained with DAPI. The average number of nuclei per cell was calculated by analyzing at least 40 myotubes (positively stained MHC cells) for each treatment. Values presented in the histogram are means from four independent experiments. Error bars represent standard errors. E, {beta}-estradiol was added to C2 myoblasts expressing the {Delta}Raf1DD:ER protein together with DM or 36 h following the addition of DM, and cells were grown for a total time in DM as indicated. Cell extracts were prepared and used in a Western analysis to identify the protein levels of myosin heavy chain (MHC) and phosphorylated MAPK (P-MAPK). {alpha}-Tubulin was used as a control of protein loading in each lane.

 

Several studies suggest that the activation of MAPK inhibits muscle differentiation (26, 27, 28). To study whether MAPK affected muscle differentiation, {beta}-estradiol was added to cells together with the differentiation medium (0 h), during myoblast proliferation, or 36 h following the addition of differentiation medium (36 h). ERK was phosphorylated in each case of Raf1 activation (not shown). First, we investigated the structure of myotubes after growing them for 72 h in DM by immunostaining with an antibody to myosin heavy chain and found that ERK activation affected the size of myotubes (Fig. 1D). Early activation of ERK (0 h) usually reduced the size of myotubes relative to control cells (Fig. 1D, middle panel). Late activation of ERK (36 h) was followed by the appearance of larger myotubes with more nuclei per myotube relative to control cells (Fig. 1D, right panel). The number of nuclei per myotube was calculated (Fig. 1D, histogram). Early activation of Raf1 slightly reduced the average number of nuclei per myotube, whereas late activation increased this number by 2-fold relative to control cells. To find out whether these differences in myotube structure were also reflected in the expression of the structural protein MHC, the levels of MHC were analyzed at several time points following early or late activation of Raf1 (Fig. 1E). Early activation of Raf1 (0 h) mildly reduced (<2-fold) MHC expression after 8, 24, and 48 h of growth in DM relative to untreated cells (lanes 2–7). However, after 72 h of growth in DM, MHC levels were similar between treated and untreated cells (lanes 9 and 10). Late activation (36 h) of Raf1 did not affect the expression levels of MHC at 48 and 72 h of growth in DM relative to untreated cells (compare lanes 6–8 and 9–11). Thus, differences in the structure of myotubes, especially those resulted from late Raf1 activation are probably not due to any significant changes in muscle-specific expression.

MAPK Does Not Induce Proliferation of Differentiating MyoBlasts—One obvious consequence of Raf1 activation was the higher density of nuclei in {beta}-estradiol-treated cultures relative to untreated cultures (Fig. 1D, compare the left panel to the middle and right panels). This difference raised the possibility that MAPK could promote myoblast proliferation during these stages. The percentage of cells in S phase was analyzed by the bromodeoxyuridine labeling assay (Fig. 2A). Activation of Raf1, at different times after myoblasts were induced to differentiate in DM, did not induce any significant proliferation beyond the levels observed in control cells grown in DM for the same period of time (Fig. 2A). The phosphorylation state of the retinoblastoma protein can serve as an indicator for the proliferation state of muscle cells. Two major phosphorylated forms of pRb exist in replicating myoblasts, whereas only one underphosphorylated form is found in postmitotic cells. The phosphorylation of pRb is expected if resting muscle cells are induced to re-enter the cell cycle. We analyzed how the activation of MAPK affected the phosphorylation status of pRb (Fig. 2B). The two forms of pRb were present in proliferating myoblasts, whereas only the underphosphorylated form was found in myoblasts growing in DM (lanes 1 and 2). Activation of MAPK in myoblasts growing in DM for short (24 h) or long (72 h) periods did not change the phosphorylation pattern of pRb; namely, the protein remained in its underphosphorylated form, indicating that resting myoblasts did not re-enter the cell cycle (lanes 3 and 5). On the whole, the results presented in Fig. 2 suggest that activation of the MAPK pathway does not reverse the withdrawal of myoblasts from the cell cycle.



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  		FIG. 2.
Activation of {Delta}Raf1DD:ER protein does not induce proliferation in differentiating myoblasts. A, C2 {Delta}Raf1DD:ER cells were grown for several time periods in DM as indicated, in the absence or presence of {beta}-estradiol that was added 24 h before the cells were analyzed. To identify the nuclei involved in DNA synthesis, bromodeoxyuridine (BrdU) was added to DM 2 h before the cells were fixed and immunostained, as described under "Experimental Procedures." The total number of nuclei was identified by DAPI staining. Representative microscopic fields were photographed. Each field was photographed twice, for BrdUrd and for DAPI staining. The histogram on the right side of the picture represents the average of five different fields that were counted to calculate the percentage of cells in the S phase. The percentage of cells in the S phase was determined by dividing the number of BrdUrd-stained nuclei by the number of DAPI-stained nuclei in each microscopic field. B, C2 {Delta}Raf1:ER cells were grown in GM (0 h in DM) and then in DM for the indicated time periods. {beta}-Estradiol was added to cells 8 h before proteins were extracted. Proteins were separated over SDS-PAGE. Western blotting identified the different forms of the pRb protein and the total and phosphorylated forms of MAPK as described under "Experimental Procedures." Abbreviations: pRb, underphosphorylated form of retinoblastoma; ppRb, hyperphosphorylated form of retinoblastoma; MAPK, total ERK proteins; P-MAPK, phosphorylated ERK proteins.

 

Activation of MAPK Prevents Apoptotic Cell Death of Differentiating Myoblasts—A high proportion of myoblasts undergoes programmed cell death (PCD) during in vitro differentiation (1). First, we wanted to validate that, in the C2 cell line, myoblasts were undergoing PCD at these stages. For that purpose, we immunostained cells grown in DM for 48 h with an antibody to cleaved caspase 3 (active form) to detect ongoing apoptosis and with an antibody to MHC to detect myotubes (Fig. 3A). Most (above 95%) of the cells that were stained for the expression of cleaved caspase 3 did not stain for myosin heavy chain (Fig. 3A, see "Merge"). A higher magnification of a portion of the microscopic field presented in Fig. 3A shows that staining of caspase 3 was in most cases cytoplasmic (Fig. 3B). Occasionally, staining of cells appeared nuclear, although it could reflect false identification of cells found in advance stages of apoptosis with their cytoplasm collapsed and structure deformed. We can conclude that at these stages the majority of cells undergoing PCD are myoblasts and not myotubes. We calculated the number of cells positively stained for cleaved caspase 3 relative to the total number of myoblast nuclei and found that 29% of the cells were undergoing PCD.



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  		FIG. 3.
Myoblasts but not myotubes express the active form of caspase 3. A, C2 cells were grown for 48 h in DM. Cells were then fixed and immunostained with antibodies to cleaved caspase 3 (green staining) and to myosin heavy chain (red staining). Cell nuclei were stained with DAPI. B, a higher magnification of a section of the microscopic field presented in panel A. Arrows point toward cells that are stained positively to cleaved caspase 3, and their staining is cytoplasmic.

 

To investigate the role of MAPK in preventing apoptotic cell death of myoblasts, we asked how activation or repression of the pathway affected cell viability by Hoechst and by TUNEL staining of nuclei (Fig. 4A). A significant percentage of myoblasts growing in DM for 48 h undergo apoptosis as can be seen by chromatin condensation observed by the dense staining of DNA (Fig. 4A). Addition of {beta}-estradiol to C2-{Delta}Raf1DD:ER cells after 24 h of myoblasts growth in DM for an additional period of 24 h largely prevented cell death. Conversely, treatment of cells with Mek inhibitor, U0126 increased dramatically cell death (Fig. 4A). The addition of {beta}-estradiol to the control C2-{Delta}Raf301:ER cells did not change the number of cells undergoing PCD, suggesting that the antiapoptotic effect was specific to Raf activity (Fig. 4A). Apoptotic cell death was also analyzed by TUNEL staining of fragmented DNA (Fig. 4B). Activation of the {Delta}Raf1DD:ER protein reduced while the addition of U0126 increased PCD to similar values observed in the Hoechst staining.



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  		FIG. 4.
Induction of Raf1 activity protects myoblasts from apoptotic cell death by preventing the activation of caspase 9. A, C2 {Delta}Raf1DD:ER or C2{Delta}Raf301:ER myoblast cells were grown for 48 h in DM in the absence or presence of {beta}-estradiol or U0126 that were added during the last 24 h of growth. Nuclei of living cells were stained with Hoechst dye for 30 min before cells were photographed under a fluorescence microscope. Dense staining of chromatin identified apoptotic cells. The graph summarizes the counts of five different fields. The experiment was repeated five times with similar results. B, C2 {Delta}Raf1DD:ER myoblasts were grown as described in A. Cells were fixed, and TUNEL staining of fragmented DNA was performed as described under "Experimental Procedures." The graph summarizes counting of 200 cells in three independent experiments. C, DNA fragmentation assay: C2 {Delta}Raf1DD:ER myoblasts were grown in DM for 72 h in the absence or presence of U0126 or {beta}-estradiol that were added after 24 h in DM. Fragmentation of genomic DNA was analyzed as a marker of apoptotic cell death. D, C2 {Delta}Raf1DD:ER myoblasts were grown as indicated in A. Proteins were extracted and separated over gels, and caspase 9 protein was analyzed by Western blotting. Two forms of caspase 9 were identified; an unprocessed inactive form and a processed active form. The blot was reacted also with antibodies to total MAPK and its phosphorylated forms.

 

Fragmentation of genomic DNA that appears as a typical ladder in gel electrophoresis can serve as a hallmark of PCD. Identical amounts of genomic DNA were separated over an agarose gel. No ladder was observed in DNA from proliferating myoblasts, however, a ladder was noticeable after growth of myoblasts in DM for 72 h (Fig. 4C, compare lanes 1 and 2). If the same cells were treated with U0126 for 48 h, after 24 h of myoblasts growth in DM the staining intensity of the ladder was increased significantly (Fig. 4C, lane 3). Treatment with {beta}-estradiol to increase MAPK activity during the same period of time decreased the intensity of the DNA ladder relative to untreated cells (compare lanes 2 and 4).

During the process of apoptotic cell death certain caspases are being activated by proteolytic cleavage (29, 30). We analyzed the possible involvement of caspase 9 in PCD of myoblasts and searched whether the MAPK pathway affected its activation. In proliferating myoblasts the inactive pro-caspase was the only noticeable form (Fig. 4D, lane 1). During differentiation of myoblasts, in addition to the inactive form of caspase 9, the active shorter form was observed after 24 h in DM, and its amount was increased after 48 h (Fig. 4D, lanes 2 and 3). Myoblasts growing for 48 h in DM and treated with U0126 expressed more of the active form than control cells not treated with the inhibitor (compare lanes 3 and 4). On the other hand, induction of Raf1 activity reduced the levels of active caspase 9 (compare lanes 3 and 5). These results suggest that the MAPK pathway regulates the activation of caspase 9 in the apoptotic pathway of myoblasts.

Activation of Raf1 in Muscle Cells Induces the Expression of p21WAF1—It was shown before that the expression of cyclin-dependent kinase inhibitor, p21WAF1, was necessary to prevent PCD of myoblasts (2). Next we tested whether MAPK was involved in the expression of p21WAF1 in muscle cells. Addition of {beta}-estradiol to C2-{Delta}Raf1DD:ER cells growing in GM induced protein levels of p21WAF1, whereas treatment of the same cells with Mek inhibitor, U0126, reduced its levels as observed by Western blotting (Fig. 5A). By immunostaining of cells treated with {beta}-estradiol, we could observe that myoblasts expressing the phosphorylated form of MAPK, also expressed p21WAF1 in their nuclei (Fig. 5B). Hence, MAPK is involved in the expression of p21WAF1 in proliferating myoblasts. The cdk inhibitor p21WAF1 is normally induced during early stages of muscle differentiation as myoblasts exit the cell cycle (20, 31). We followed the expression profile of p21WAF1 protein during the differentiation of C2-{Delta}Raf1DD:ER cells (Fig. 5C). Levels of p21WAF1 protein were low in proliferating myoblasts, induced after 24 h in DM, and gradually declined during their further growth in DM (Fig. 5C, lanes 1–5). Addition of {beta}-estradiol after growth of 36 h in DM induced the levels of p21WAF1 protein observed at later periods of cell growth (compare lanes 4 and 5 to lanes 6 and 7), whereas U0126 added to cells after growth of 36 h in DM reduced p21WAF1 protein to almost undetectable levels after further growth of cells (compare lanes 4 and 5 to lanes 8 and 9). These results indicate that MAPK could be involved in the expression of p21WAF1 in differentiating myoblasts in addition to other factors.



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  		FIG. 5.
The MAPK pathway induces the expression of p21WAF1 protein in C2 muscle cells. A, C2 {Delta}Raf1DD:ER myoblasts were grown in GM and {beta}-estradiol or U0126 were added for 24 h before proteins were extracted, separated over SDS-PAGE, and analyzed by Western blotting. The blot was reacted repeatedly with antibodies to p21WAF1, phosphorylated MAPK, and total MAPK. B, C2 {Delta}Raf1DD:ER myoblasts were grown as described in A. Cells were then fixed and immunostained with antibodies to p21WAF1 and phosphorylated MAPK. Cell nuclei were stained with DAPI. C, C2 {Delta}Raf1DD:ER myoblasts were grown in DM for the indicated time periods. {beta}-Estradiol or U0126 were added as indicated after 36 h of cell growth in DM. Proteins were extracted, separated over SDS-PAGE, and analyzed by Western blotting. The blot was reacted repeatedly with antibodies to p21WAF1, phosphorylated MAPK, and total MAPK.

 

Activation of Raf1 Extends the Half-life of the p21WAF1 Protein—In muscle cells, the MyoD protein functions to induce the transcription of p21WAF1 during differentiation (20). To find out how MAPK affected the expression of p21WAF1, we analyzed the transcript levels of p21WAF1 following activation or repression of MAPK (Fig. 6A). The levels of p21WAF1 were affected neither by the activation of MAPK caused by adding {beta}-estradiol to myoblasts growing for 24 h in DM nor by its inhibition following treatment with U0126, suggesting that MAPK did not affect transcription of p21WAF1 in muscle cells (Fig. 6A, lanes 2 and 3, Northern blot). However, in the same experiments, the levels of the p21WAF1 protein were changed by the MAPK pathway (Fig. 6A, Western blot). To further test whether MAPK could regulate the transcription of the p21 gene, a reporter gene containing the promoter sequences of p21 was transfected into C2 cells expressing {Delta}Raf1DD:ER (Fig. 6A, graph). {beta}-Estradiol was added following 24 h of growth in DM to induce and U0126 to repress MAPK activity, and luciferase activity was analyzed after growth of 72 h in DM. The activity of MAPK did not change in any significant way the expression levels of the p21 promoter-reporter gene (Fig. 6A, graph). These results suggest that MAPK functions post-transcriptionally to induce the levels of the p21WAF1 protein. The changes in the levels of the p21WAF1 protein could reflect alterations in protein stability. The half-life of the p21WAF1 protein was analyzed in a pulse-chase labeling experiment that was performed in {Delta}Raf1DD:ER-expressing cells grown for 48 h in DM. The half-life of p21WAF1 was about 30 min in cells grown in the absence of {beta}-estradiol (Fig. 6B). However, the level of p21WAF1 did not change when cells were grown in the presence of {beta}-estradiol even after 120 min of chase (Fig. 6B). In contrast, incubating the cells with U0126 reduced p21WAF1 half-life to less than 10 min (not shown). We conclude that the MAPK pathway contributes to the stability of the p21WAF1 protein in muscle cells. Next we wanted to find out whether activation of {Delta}RafDD:ER had a general effect on protein stability or that it stabilized p21WAF1 in a specific manner. For that purpose we analyzed the protein levels of another cdk inhibitor family member, p27KIP1, following the induction or inhibition of MAPK phosphorylation with {beta}-estradiol or U0126, respectively (Fig. 6C, lower panel). The steady-state levels of p27KIP1 were not affected, whereas those of p21WAF1 were changed as expected. We conclude that the MAPK pathway affects the protein levels of p21WAF1 but not of another family member, p27KIP1.



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  		FIG. 6.
The MAPK pathway stabilizes the p21WAF1 protein. A, C2 {Delta}Raf1DD:ER myoblasts were grown in DM for 48 h, and {beta}-estradiol or U0126 were added for 24 h before RNA and proteins were extracted. Upper panel, a Northern blot that was sequentially hybridized with probes to p21 and to glyceraldehyde-3-phosphate dehydrogenase. Middle panel, a Western blot that was reacted repeatedly with antibodies to p21WAF1, phosphorylated MAPK, and total MAPK. Lower panel, luciferase assay: a reporter gene containing the transcription regulatory sequences of the p21 gene was used to transfect the C2 {Delta}Raf1DD:ER myoblasts, which were then were grown in DM for 48 h, and {beta}-estradiol or U0126 were added for 24 h before proteins were extracted and luciferase activity measured. Luciferase activity was adjusted to 1 unit in control cells growing in the absence of {beta}-estradiol or U0126. Values are means from three independent experiments. Error bars represent standard errors. In B: upper panel, C2 {Delta}Raf1DD:ER myoblasts were grown in DM for 48 h in the absence or presence of {beta}-estradiol. Cells were metabolically labeled with [35S]methionine for 1 h. Labeling was chased by replacing the medium with unlabeled medium for different periods of time, as indicated, before cells were lysed and proteins were extracted. The p21WAF1 protein was immunoprecipitated and resolved by SDS-PAGE. Lower panel, the bands were quantified, and relative values are presented in the graph. C,C2 {Delta}Raf1DD:ER myoblasts were grown in DM for 48 h, and {beta}-estradiol or U0126 were added for 24 h before proteins were extracted, separated over SDS-PAGE, and analyzed by Western blotting. The blot was reacted repeatedly with antibodies to p27 (Kip1), p21WAF1, phosphorylated MAPK, and tubulin. In D: upper panel, C2 MD:ER myoblasts were grown in DM for 48 h in the absence or presence of {beta}-estradiol or U0126 as indicated, and proteins were extracted, separated over SDS-PAGE, and analyzed by Western blotting. The blot was reacted repeatedly with antibodies to p21WAF1, phosphorylated MAPK, and total MAPK. Lower panel, C2 MD:ER cells were grown as described above, and staining of the nuclei of living cells with Hoechst dye for 30 min identified apoptotic cells. Apoptotic cells were identified under a fluorescence microscope, and the histogram summarizes the counts of five different fields. The experiment was repeated twice with similar results.

 

To investigate the possible interplay between transcriptional regulation by MyoD and post-transcriptional regulation by MAPK in determining the levels of p21WAF1 protein, we generated C2 myoblast cells that in addition to the endogenous MyoD, expressed an inducible form of the MyoD protein (C2-MyoD:ER). Addition of {beta}-estradiol to these cells induced higher transcript levels of p21WAF1, suggesting that the MyoD:ER protein induced the transcription of the p21 gene (data not shown). Induction of the exogenous MyoD:ER protein also increased the amount of p21WAF1 protein above its level in control cells (Fig. 6D, compare lanes 1 and 3). In contrast, treatment of cells with U0126, significantly reduced the level of the p21WAF1 protein (Fig. 6D, lane 2). However, inhibition of MAPK with U0126 did not reduce p21WAF1 protein if during that time, {beta}-estradiol was added to induce the activity of the MyoD:ER protein (Fig. 6D, lane 4). The amount of cells undergoing apoptotic cell death was inversely correlated with the expression levels of p21WAF1 (Fig. 6D, graph). Myoblasts expressing high levels of p21WAF1 resulting from the activation of MyoD:ER showed reduced PCD (lane 3), whereas those treated with Mek inhibitor expressing very low levels of p21WAF1 showed enhanced PCD (lane 2). Myoblasts expressing intermediate levels of p21WAF1 protein, obtained from the simultaneous activation of MyoD:ER and suppression of Mek, exhibited intermediate levels of PCD (lane 4). Taken together, these results suggest a possible interplay, between the transcriptional and post-transcriptional activities of MyoD and MAPK, respectively, in establishing the protein levels of p21WAF1 during muscle differentiation, may exist and that p21WAF1 levels correlate directly with the survival of differentiating myoblasts.

The Antiapoptotic Function of the MAPK Pathway in Muscle Cells Is Mediated by p21WAF1—To determine whether p21WAF1 was required for MAPK-mediated cell survival, we infected the C2-{Delta}Raf1DD:ER cells with a retrovirus encoding for an antisense p21 mRNA (p21WAF1AS) and the marker protein EGFP or with a control retrovirus encoding for EGFP only. Infected cells were grown in differentiation medium, and Raf1 was induced 12 h later. After 36 h in DM, the percentage of apoptotic cells that were positive for EGFP expression was analyzed by Hoechst staining (Fig. 7A). Some myoblasts infected with the control virus underwent apoptotic cell death, however, the majority of cells survived. In contrast, the majority of myoblasts, infected with the virus expressing antisense p21, underwent apoptotic cell death regardless of whether {beta}-estradiol was added to induce the {Delta}Raf1DD:ER protein or not (Fig. 7A). In addition to the condensed chromatin, these cells lost their normal elongated structure and adopted a small spherical structure. Immunostaining of differentiating myoblasts indicated that the expression of the antisense p21-encoding virus prevented the expression of endogenous p21WAF1 in most of the infected cells (Fig. 7B). These results indicate that MAPK promotes muscle cell survival by inducing the protein levels of p21WAF1.



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  		FIG. 7.
Antisense p21 expression prevents the MAPK-mediated antiapoptotic activity in differentiating cells. A, C2 {Delta}Raf1DD:ER myoblasts were infected with a replication-defective retroviral vector expressing AS-p21 and GFP (AS-p21-GFP) or with a retrovirus expressing only GFP (GFP-Control). One day following infection, the cell medium was replaced with DM, and {beta}-estradiol was added to some of the plates 12 h later. Cells grew in DM for 48 h before their nuclei were stained with Hoechst dye. The green-stained cells are those that were infected by the retroviruses. Dense chromatin staining identified apoptotic cells. The percentage of apoptotic cells was calculated by counting the number of apoptotic cells (Hoechst) out of about 200 infected cells (GFP-stained). B, C2 {Delta}Raf1DD:ER myoblasts were infected as described in A. Cells were grown in DM for 48 h. Cells were then fixed and immunostained using an antibody to the p21WAF1 protein. Cells that were EGFP-stained (green) were analyzed for p21WAF1 staining (red). The graph summarizes the counts of stained cells. The percentage of cells expressing p21WAF1 was calculated by dividing the cells stained green and red by the total number of green cells.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Late Activation of MAPK Affects Postmitotic Growth of Muscle Cells—The ERK MAPK pathway has been implicated in the control of myogenesis. Several studies have proposed that the pathway functioned by inducing proliferation and, therefore, inhibited muscle differentiation (8, 26, 27, 32, 33, 34, 35). Our previous work and that of others showed that ERK phosphorylation and activity were significantly induced during the terminal differentiation of myoblasts (15, 16). We suggested that the pathway was intrinsic to muscle cells and could stimulate the differentiation process. In the present work we investigated the mode of ERK involvement during the differentiation process. Our results indicate that the activation of ERK plays a crucial role in the survival of differentiating myoblasts.

How can one explain the different effects of the MAPK pathway during myogenesis? Several models can be suggested to explain the multiple responses mediated by the MAPK pathway: 1) the cellular response is dictated by the cellular context. This model suggests that one pathway may affect many processes by regulating different sets of transcription factors that are available in different tissues or at a given time point in a certain tissue (36); 2) the combined activities of different signal transduction pathways determine the biological response. For example, transformation of fibroblasts depends on the activities of several pathways, including MAPK and phosphoinositol 3-kinase (PI3K), whereas cell cycle arrest is induced by the activation of the MAPK pathway only (37); 3) quantitatively different levels of MAPK activity elicit different responses. For example, transient activation of MAPK in PC12 cells induces proliferation, whereas prolonged activation of MAPK triggers neurite outgrowth (38). These models, in total and individually, can explain the conflicting roles of MAPK in myogenesis. The basal phosphorylation and activity of MAPK is low in proliferating C2 myoblasts relative to its sustained and significant induction in myoblasts and myotubes growing in differentiation medium. Therefore, levels of MAPK induction as well as the cell context and additional signaling pathways may explain the differences in the way that the same signaling pathway affects cell growth at different stages of differentiation. Interestingly, significant activation of MAPK both during early and late phases of differentiation did not induce any proliferation of myoblasts (Fig. 2). This result is in conflict with other studies (27, 28) and may be explained in the following two ways: (a)We induced MAPK under serum starvation conditions that promoted withdrawal from the cell cycle. Under these conditions, cell context and signaling pathways may cooperate with MAPK in arresting cell cycle. In other studies, the MAPK pathway was inhibited in myoblasts growing in high serum. Under these conditions the pathway was involved in cell proliferation. (b)In muscle cells, high levels of MAPK activity induce withdrawal, whereas lower levels induce cell cycle progression. These possibilities deserve further investigation.

Antiapoptotic Activity of MAPK during Myoblast Differentiation—In cell cultures many myoblasts undergo PCD under conditions that promote differentiation (1). Based on several different approaches we find that the MAPK pathway is involved in protecting myoblasts from undergoing PCD (Fig. 4).

Signals that induce apoptosis culminate in the activation of caspases, which are the ultimate effectors of PCD. To find out whether MAPK could affect the proteolytic activation of caspases, we followed the activation of caspase 9 (Fig. 4D). The processed form was detected during myoblast differentiation. Ectopic activation of Raf1 reduced the relative amount of the processed form, whereas inhibition of Mek with U0126 increased its relative levels. Therefore, MAPK affects the process at this stage or at stages that precede caspase activation. Recent studies suggested that in Rat1 fibroblast cells the MAPK pathway conferred protection against apoptosis at the level of cytosolic caspase activation and not in the earlier stage of cytochrome c release from the mitochondria (39, 40). Another study showed that MAPK promoted cell survival of neurons by phosphorylation of the proapoptotic protein BAD and the transcription factor CREB (41). The role of MAPK in cell survival was also explored in Drosophila, known to express a group of proteins, REAPER, HID, and GRIM, that activate caspase processing and other proteins known as inhibitors of apoptosis (IAPs) that inhibit caspase processing. The latter group can directly bind to activated caspases and block the proteolytic chain reaction. The first group of proteins binds directly to IAP and antagonizes its activity, thereby allowing the proteolytic activation of caspases and apoptosis to proceed. In Drosophila, MAPK phosphorylates the HID protein and inhibits its interaction with IAP and consequently its proapoptotic activity (42, 43). Although the functional mammalian homologue of Hid has not yet been identified, its existence was suggested (44, 45). One might speculate also that in mammalian cells, including muscle cells, MAPK may directly affect an HID-like protein in preventing the proteolytic activation of caspases.

MAPK Functions Independently of the PI3K Pathway in Protecting Myoblasts from PCD—Recently, several works demonstrated the involvement of the phosphoinositol 3-kinase (PI3K) pathway in the survival of differentiating myoblasts (11, 13). It was suggested that Akt, a kinase in the pathway, phosphorylated the mitochondrial BAD protein known to protect cells from undergoing PCD (46, 47). In light of our results it is of interest to know whether the PI3K and the MAPK pathways exhibit cross-talk in their antiapoptotic functions. We found that modulation of MAPK activity did not affect the phosphorylation state of Akt during muscle differentiation.2 Therefore, it is likely that the antiapoptotic function of MAPK is not mediated by Akt. Our conclusion is further supported by a recent study that suggested IGF-I- and platelet-derived growth factor-induced myoblast survival via two independent signaling pathways (14). According to this work, IGF-I induced the PI3K pathway, whereas platelet-derived growth factor induced the MAPK pathway, and each of the pathways was sufficient to promote muscle cell survival.

The activities of PI3K and MAPK do not overlap in the differentiation process. Moreover, Akt was shown to phosphorylate and inhibit Raf1 in muscle cells (48). Whereas PI3K and Akt are induced at early stages of differentiation, MAPK activation occurs at later stages.2 Therefore, it is possible that each pathway functions independently to protect myoblasts from PCD at different stages of the differentiation process.

Several Factors, Including the MAPK Pathway, Maintain the Expression of p21WAF1 in Differentiating Myoblasts—MAPK induces the expression of p21WAF1 in many cellular systems and causes cell cycle arrest (49). The induction of p21WAF1 expression during muscle differentiation plays at least two fundamental roles in the withdrawal of myoblasts from the cell cycle and in their survival (1). MyoD is involved in the transcriptional induction of p21 (20). In the present work we asked whether MAPK contributed to the expression of p21WAF1, in view of its role as a myoblast survival factor (2). In proliferating myoblasts where the levels of phosphorylated MAPK and p21WAF1 are low, activation of Raf1 induced the expression of the p21WAF1 protein (Fig. 5, A and B). After 24 h of growth of C2 cells in DM the level of p21WAF1 protein increased but gradually declined during further growth (Fig. 5C). This happens despite the normal induction of MAPK occurring during differentiation. Further activation of MAPK via the exogenously expressed {Delta}Raf1DD:ER protein during these stages induced higher levels of p21WAF1 protein, whereas inhibition of Mek with U0126 reduced its levels. Therefore, MAPK plays a role in maintaining the levels of p21WAF1 during late phases of differentiation. Some studies indicate that other factors such as MyoD and the PI3K pathway also affect the expression of p21WAF1 (13, 20). Thus, the balance between different factors, including the MAPK pathway, may determine the absolute levels of p21WAF1 in differentiating myoblasts. As the activities of MyoD and PI3K are reduced during later stages of differentiation, enhanced activity of MAPK may substitute for these proteins in maintaining the expression of p21WAF. The vital role of p21WAF1 in cell cycle withdrawal and myoblast survival may explain the multiple pathways and factors involved in its expression.

MAPK Stabilizes the p21WAF1 Protein—In some cellular systems MAPK regulates p21 transcriptionally (50, 51, 52), whereas in others it also affects the post-transcriptional processes (53). We studied the regulation of p21 by MAPK in muscle cells and found that the activation or repression of the pathway did not affect the transcripts levels or the promoter activity of p21. Nevertheless, MAPK significantly affected the protein levels of p21WAF1, suggesting changes in protein synthesis or breakdown. The finding, that activation of Raf1 dramatically extended while inhibition of Mek with U0126 significantly reduced the half-life of the p21WAF1 protein, indicates that the MAPK pathway regulates the stability of the p21WAF1 protein. The p21WAF1 protein is degraded by the proteasome in a process that does not involve ubiquitination of p21WAF1 (54). Recent studies have emphasized that the turnover of p21WAF1 protein is regulated by several signaling pathways affecting cell growth. Rac1/CDC42 activates the degradation of p21 (55), whereas p38 MAPK, JNK1 (56), and protein kinase B/Akt (57) stabilize the protein by phosphorylating several of its residues. One study suggested that the ERK MAPK pathway is required to stabilize p21 mRNA and p21WAF protein during the withdrawal of primary hepatocytes from the cell cycle (53). All in all, these studies suggest that the stability of the p21WAF1 protein is affected by multiple signaling events, implicating it as a major mechanism regulating the levels of p21WAF1 protein in cells.

MAPK Induces Hypertrophic Growth of Myotubes—In the present study we observed that MAPK was also involved in the determination of myotube size and the number of nuclei per myotube (Fig. 1). Similar observations were reported by others (10, 58). In those studies, as well as in ours, MAPK activity increased the size of myotubes and prevented the collapse that usually occurs after several days of growth in DM.2 Presently we don't know if this is a direct or indirect effect of the MAPK pathway. A direct activity of MAPK could target the translational apparatus and induce hypertrophic growth (59) or, alternatively, induce fusion of myoblast cells to multinucleated myotubes. An indirect effect could for example be a result of the better survival of myoblasts induced by MAPK that allows the recruitment of more competent myoblasts to fuse and form differentiated myotubes. These possibilities deserve further studies.

Interestingly, from studies with Rb-/- mice it is apparent that pRb protein may affect muscle growth in a way similar to MAPK (5). These mice die after birth with specific muscle defects, including increased myoblast PCD prior to myocyte fusion; the surviving myotubes are shorter, have less nuclei, and express reduced levels of late muscle-specific genes. The similarities between pRb and MAPK and the direct effect of MAPK on p21WAF1 protein suggest that these proteins may share the same pathway affecting muscle differentiation and survival.

Recently, we observed that the MAPK pathway was absolutely necessary for the differentiation of skeletal muscle during early development of Xenopus laevis (60). In this model system, MAPK affected the expression of late markers of differentiation. We could also demonstrate that activated Mek induced the levels of the MyoD protein in explants from injected embryos. Therefore, during early development of Xenopus, distinctly from the cell culture system used in the present study, MAPK directly affects myogenesis through the MyoD protein. However, it is possible that, like in the tissue culture model, MAPK functions to augment skeletal muscle differentiation by preventing myoblast cell death during Xenopus development.

A Model for the Antiapoptotic Activity of the MAPK Pathway in Muscle—As myoblasts undergo their terminal differentiation, many of the cells that cannot complete this process successfully are eliminated by PCD (Fig. 8). The cell cycle machinery and, specifically, the p21WAF1 cdk inhibitor regulate this process (61). p21WAF1 may serve as an indicator for the "decision" of myoblasts whether to proceed with the differentiation process or to undergo cell death. The protein levels of p21WAF1 are induced and maintained by several factors and signaling pathways during differentiation. MyoD, whose activity is the first to be induced during muscle differentiation, activates the transcription of the p21 gene. During these early stages, the PI3K-Akt pathway is also activated in a transient fashion to further induce the levels of p21WAF1. Later, as PI3K-Akt activity drops, the MAPK pathway is induced and functions to maintain the p21WAF1 protein during a later phase of differentiation.



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  		FIG. 8.
A model for the involvement of MAPK in muscle cell survival.

 


   FOOTNOTES
 
* This work was supported by a grant from the Israel Science Foundation (to E. B.), by a grant from United States-Israel Binational Science Foundation (to E. B.), by funds from the Rappaport Foundation for Medical Research, and by funds from the Foundation for the Promotion of Research in the Technion, Israel Institute of Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 972-4-8295-287; Fax: 972-4-8553-299; E-mail: bengal{at}tx.technion.ac.il.

1 The abbreviations used are: cdk, cyclin-dependent kinase; PI3K, phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; pRb, retinoblastoma; IGF, insulin-like growth factor; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; EGFP, enhanced GFP; GM, growth medium; DM, differentiation medium; ER, estrogen receptor; MHC, myosin heavy chain; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; PCD, programmed cell death; IAP, inhibitors of apoptosis; JNK1, c-Jun NH2-terminal kinase 1; DAPI, 4',6-diamidino-2-phenylindole. Back

2 O. Ostrovsky and E. Bengal, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. McMahon for the Raf1 retroviral vectors, Dr. N. Somia and Dr. I. M. Verma for retroviral vectors and a packaging cell line, Dr. S. J. Tapscott for the MyoD:ER vector and an antibody to MHC, and Dr. T. Kleinberger for the antibody to caspase 9. We thank Bianca-Raikhlin-Eisenkraft for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Walsh, K. (1997) Prog. Cell Cycle Res. 3, 53-58[Medline] [Order article via Infotrieve]
  2. Wang, J., and Walsh, K. (1996) Science 273, 359-361[Abstract]
  3. Zhang, P., Wong, C., Liu, D., Finegold, M., Harper, J. W., and Elledge, S. J. (1999) Genes Dev. 13, 213-224[Abstract/Free Full Text]
  4. Wang, J., Guo, K., Wills, K. N., and Walsh, K. (1997) Cancer Res. 57, 351-354[Abstract/Free Full Text]
  5. Zacksenhaus, E., Jiang, Z., Chung, D., Marth, J. D., Phillips, R. A., and Gallie, B. L. (1996) Genes Dev. 10, 3051-3064[Abstract/Free Full Text]
  6. Stewart, C. E., and Rotwein, P. (1996) J. Biol. Chem. 271, 11330-11338[Abstract/Free Full Text]
  7. Xu, Q., and Wu, Z. (2000) J. Biol. Chem. 275, 36750-36757[Abstract/Free Full Text]
  8. Weyman, C. M., and Wolfman, A. (1998) Endocrinology 139, 1794-1800[Abstract/Free Full Text]
  9. Tureckova, J., Wilson, E. M., Cappalonga, J. L., and Rotwein, P. (2001) J. Biol. Chem. 276, 39264-39270[Abstract/Free Full Text]
  10. Sarbassov, D. D., and Peterson, C. A. (1998) Mol. Endocrinol. 12, 1870-1878[Abstract/Free Full Text]
  11. Fujio, Y., Guo, K., Mano, T., Mitsuuchi, Y., Testa, J. R., and Walsh, K. (1999) Mol. Cell. Biol. 19, 5073-5082[Abstract/Free Full Text]
  12. Lawlor, M. A., and Rotwein, P. (2000) J. Cell Biol. 151, 1131-1140[Abstract/Free Full Text]
  13. Lawlor, M. A., and Rotwein, P. (2000) Mol. Cell. Biol. 20, 8983-8995[Abstract/Free Full Text]
  14. Lawlor, M. A., Feng, X., Everding, D. R., Sieger, K., Stewart, C. E., and Rotwein, P. (2000) Mol. Cell. Biol. 20, 3256-3265[Abstract/Free Full Text]
  15. Sarbassov, D. D., Jones, L. G., and Peterson, C. A. (1997) Mol. Endocrinol. 11, 2038-2047[Abstract/Free Full Text]
  16. Gredinger, E., Gerber, A. N., Tamir, Y., Tapscott, S. J., and Bengal, E. (1998) J. Biol. Chem. 273, 10436-10444[Abstract/Free Full Text]
  17. Ravi, R. K., Weber, E., McMahon, M., Williams, J. R., Baylin, S., Mal, A., Harter, M. L., Dillehay, L. E., Claudio, P. P., Giordano, A., Nelkin, B. D., and Mabry, M. (1998) J. Clin. Invest. 101, 153-159[Medline] [Order article via Infotrieve]
  18. Woods, D., Parry, D., Cherwinski, H., Bosch, E., Lees, E., and McMahon, M. (1997) Mol. Cell. Biol. 17, 5598-5611[Abstract]
  19. Ballif, B. A., and Blenis, J. (2001) Cell Growth & Differ. 12, 397-408[Free Full Text]
  20. Halevy, O., Novitch, B. G., Spicer, D. B., Skapek, S. X., Rhee, J., Hannon, G. J., Beach, D., and Lassar, A. B. (1995) Science 267, 1018-1021[Abstract/Free Full Text]
  21. McMahon, M. (2001) Methods Enzymol. 332, 401-417[Medline] [Order article via Infotrieve]
  22. Hollenberg, S. M., Cheng, P. F., and Weintraub, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8028-8032[Abstract/Free Full Text]
  23. Yaffe, D., and Saxel, O. (1977) Nature 270, 725-727[CrossRef][Medline] [Order article via Infotrieve]
  24. Naviaux, R. K., Costanzi, E., Haas, M., and Verma, I. M. (1996) J. Virol. 70, 5701-5705[Abstract/Free Full Text]
  25. Davis, R. L., Cheng, P. F., Lassar, A. B., and Weintraub, H. (1990) Cell 60, 733-746[CrossRef][Medline] [Order article via Infotrieve]
  26. Coolican, S. A., Samuel, D. S., Ewton, D. Z., McWade, F. J., and Florini, J. R. (1997) J. Biol. Chem. 272, 6653-6662[Abstract/Free Full Text]
  27. Jones, N. C., Fedorov, Y. V., Rosenthal, R. S., and Olwin, B. B. (2001) J. Cell. Physiol. 186, 104-115[CrossRef][Medline] [Order article via Infotrieve]
  28. Tortorella, L. L., Milasincic, D. J., and Pilch, P. F. (2001) J. Biol. Chem. 276, 13709-13717[Abstract/Free Full Text]
  29. Thornberry, N. A., and Lazebnik, Y. (1998) Science 281, 1312-1316[Abstract/Free Full Text]
  30. Nunez, G., Benedict, M. A., Hu, Y., and Inohara, N. (1998) Oncogene 17, 3237-3245[CrossRef][Medline] [Order article via Infotrieve]
  31. Skapek, S. X., Rhee, J., Spicer, D. B., and Lassar, A. B. (1995) Science 267, 1022-1024[Abstract/Free Full Text]
  32. Ramocki, M. B., Johnson, S. E., White, M. A., Ashendel, C. L., Konieczny, S. F., and Taparowsky, E. J. (1997) Mol. Cell. Biol. 17, 3547-3555[Abstract]
  33. Samuel, D. S., Ewton, D. Z., Coolican, S. A., Petley, T. D., McWade, F. J., and Florini, J. R. (1999) Horm. Metab. Res. 31, 55-64[Medline] [Order article via Infotrieve]
  34. Weyman, C. M., Ramocki, M. B., Taparowsky, E. J., and Wolfman, A. (1997) Oncogene 14, 697-704[CrossRef][Medline] [Order article via Infotrieve]
  35. Tombes, R. M., Auer, K. L., Mikkelsen, R., Valerie, K., Wymann, M. P., Marshall, C. J., McMahon, M., and Dent, P. (1998) Biochem. J. 330, 1451-1460
  36. Gauld, S. B., Blair, D., Moss, C. A., Reid, S. D., and Harnett, M. M. (2002) J. Immunol. 168, 3855-3864[Abstract/Free Full Text]
  37. Sheng, H., Shao, J., and DuBois, R. N. (2001) J. Biol. Chem. 276, 14498-14504[Abstract/Free Full Text]
  38. Marshall, C. J. (1995) Cell 80, 179-185[CrossRef][Medline] [Order article via Infotrieve]
  39. Erhardt, P., Schremser, E. J., and Cooper, G. M. (1999) Mol. Cell. Biol. 19, 5308-5315[Abstract/Free Full Text]
  40. Tashker, J. S., Olson, M., and Kornbluth, S. (2002) Mol. Biol. Cell 13, 393-401[Abstract/Free Full Text]
  41. Bonni, A., Brunet, A., West, A. E., Datta, S. R., Takasu, M. A., and Greenberg, M. E. (1999) Science 286, 1358-1362[Abstract/Free Full Text]
  42. Kurada, P., and White, K. (1998) Cell 95, 319-329[CrossRef][Medline] [Order article via Infotrieve]
  43. Bergmann, A., Agapite, J., McCall, K., and Steller, H. (1998) Cell 95, 331-341[CrossRef][Medline] [Order article via Infotrieve]
  44. Verhagen, A. M., and Vaux, D. L. (2002) Apoptosis 7, 163-166[CrossRef][Medline] [Order article via Infotrieve]
  45. Haining, W. N., Carboy-Newcomb, C., Wei, C. L., and Steller, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4936-4941[Abstract/Free Full Text]
  46. del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R., and Nunez, G. (1997) Science 278, 687-689[Abstract/Free Full Text]
  47. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241[CrossRef][Medline] [Order article via Infotrieve]
  48. Rommel, C., Clarke, B. A., Zimmermann, S., Nunez, L., Rossman, R., Reid, K., Moelling, K., Yancopoulos, G. D., and Glass, D. J. (1999) Science 286, 1738-1741[Abstract/Free Full Text]
  49. Crespo, P., and Leon, J. (2000) Cell. Mol. Life Sci. 57, 1613-1636[CrossRef][Medline] [Order article via Infotrieve]
  50. Wang, P. H., Schaaf, G. J., Chen, W. H., Feng, J., Prins, B. A., Levin, E. R., and Bahl, J. J. (1998) Biochem. Biophys. Res. Commun. 245, 912-917[CrossRef][Medline] [Order article via Infotrieve]
  51. Ravi, R. K., McMahon, M., Yangang, Z., Williams, J. R., Dillehay, L. E., Nelkin, B. D., and Mabry, M. (1999) J. Cell. Biochem. 72, 458-469[CrossRef][Medline] [Order article via Infotrieve]
  52. Liu, Y., Martindale, J. L., Gorospe, M., and Holbrook, N. J. (1996) Cancer Res. 56, 31-35[Abstract/Free Full Text]
  53. Park, J. S., Qiao, L., Gilfor, D., Yang, M. Y., Hylemon, P. B., Benz, C., Darlington, G., Firestone, G., Fisher, P. B., and Dent, P. (2000) Mol. Biol. Cell 11, 2915-2932[Abstract/Free Full Text]
  54. Sheaff, R. J., Singer, J. D., Swanger, J., Smitherman, M., Roberts, J. M., and Clurman, B. E. (2000) Mol. Cell 5, 403-410[CrossRef][Medline] [Order article via Infotrieve]
  55. Bao, W., Thullberg, M., Zhang, H., Onischenko, A., and Stromblad, S. (2002) Mol. Cell. Biol. 22, 4587-4597[Abstract/Free Full Text]
  56. Kim, G. Y., Mercer, S. E., Ewton, D. Z., Yan, Z., Jin, K., and Friedman, E. (2002) J. Biol. Chem. 277, 29792-29802[Abstract/Free Full Text]
  57. Li, Y., Dowbenko, D., and Lasky, L. A. (2002) J. Biol. Chem. 277, 11352-11361[Abstract/Free Full Text]
  58. Wu, Z., Woodring, P. J., Bhakta, K. S., Tamura, K., Wen, F., Feramisco, J. R., Karin, M., Wang, J. Y., and Puri, P. L. (2000) Mol. Cell. Biol. 20, 3951-3964[Abstract/Free Full Text]
  59. Stefanovsky, V. Y., Pelletier, G., Hannan, R., Gagnon-Kugler, T., Rothblum, L. I., and Moss, T. (2001) Mol. Cell 8, 1063-1073[CrossRef][Medline] [Order article via Infotrieve]
  60. Zetser, A., Frank, D., and Bengal, E. (2001) Dev. Biol. 240, 168-181[CrossRef][Medline] [Order article via Infotrieve]
  61. Walsh, K., and Perlman, H. (1997) Curr. Opin. Genet. Dev. 7, 597-602[CrossRef][Medline] [Order article via Infotrieve]

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