Access the full text.
Sign up today, get DeepDyve free for 14 days.
Loading next page...
/lp/unpaywall/the-mitogen-activated-protein-kinase-cascade-promotes-myoblast-cell-xpAbUQaCpF
References
References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.
- Publisher
- Unpaywall
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.m211357200
- Publisher site
- See Article on Publisher Site
Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 23, Issue of June 6, pp. 21221–21231, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Mitogen-activated Protein Kinase Cascade Promotes Myoblast Cell Survival by Stabilizing the Cyclin-dependent Kinase Inhibitor, WAF1 p21 Protein* Received for publication, November 7, 2002, and in revised form, February 6, 2003 Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M211357200 Olga Ostrovsky and Eyal Bengal‡ 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 of in vitro differentiation of myogenic cell lines a large fraction During myogenesis, proliferating myoblasts withdraw from the cell cycle and are either eliminated by pro- of myoblasts is lost through apoptotic cell death, but terminally grammed cell death or differentiate into mature myo- differentiated myotubes are more resistant. It is generally ac- tubes. Previous studies indicate that mitogen-activated cepted that myoblasts are exposed to apoptotic cell death dur- protein kinase (MAPK) activity is significantly induced ing the gross changes occurring in the differentiation process. with the onset of terminal differentiation of C2 myo- Those myoblasts that cannot complete the process because of blasts. We have investigated the part played by the incomplete withdrawal from the cell cycle are subjected to MAPK pathway in the differentiation of C2 myoblasts. apoptotic cell death (1). The resistance of myoblasts to apo- Specific activation of MAPK by expression of an active WAF1 ptosis was found to be correlated with the induction of p21 Raf1-estrogen receptor chimera protein reduced signif- cdk inhibitor and hypophosphorylation of the retinoblastoma icantly the number of myoblasts undergoing pro- protein, molecules that participate in the withdrawal from the grammed cell death in the differentiation medium. Ac- WAF1 cell cycle (2). In addition, forced expression of p21 blocked tivation of Raf1 prevented the proteolytic activation of apoptosis during the differentiation of C2 cells, whereas inhi- the proapoptotic caspase 9-protein during differentia- WAF1 bition of p21 by antisense oligonucleotides induced fre- tion. The antiapoptotic function of Raf1 correlated with WAF1 WAF1 quent apoptosis (2). Also, mice deficient in both p21 and accumulation of the p21 protein resulting from its p57 cdk inhibitors have defective muscle formation and exhibit increased stability. Antisense expression of p21 was WAF1 WAF1 increased rates of myoblast apoptosis (3). The effect of p21 used to determine whether the p21 protein medi- on myoblast survival is likely to be determined by its capacity ated the antiapoptotic activity of Raf1. Reduction of WAF1 p21 protein in muscle cells abolished the antiapo- to induce the activity of pRb. Consistent with this idea are ptotic activity of the MAPK pathway. We conclude that results showing that pRb-deficient (Rb ) myoblast cells un- MAPK contributes to muscle differentiation by prevent- dergo higher rates of apoptosis during differentiation than ing apoptotic cell death of differentiating myoblasts and their wild type counterparts (4). Moreover, transgenic mice that this activity is mediated by stabilization of the expressing low levels of pRb display substantial cell death of WAF1 p21 protein. 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 During myogenesis, proliferating myoblasts withdraw per- the cell cycle may trigger apoptotic cell death. manently from the cell cycle, express muscle-specific genes, Although the function of insulin-like growth factors (IGFs) as and fuse into multinucleated myotubes. The induction of the inducers of muscle survival has been known for a long time, the 1 WAF1 cyclin-dependent kinase (cdk) inhibitor p21 followed by intracellular signaling pathways have only recently begun to hypophosphorylation of the retinoblastoma (pRb) protein are emerge (6). Two classes of intracellular pathways, phospho- key events in the establishment of the postmitotic state that inositide 3-kinase (PI3K) and mitogen-activated protein ki- leads to the subsequent differentiation (1). During the process nases (MAPKs) are involved in different aspects of IGF-facili- tated muscle differentiation (7–10). Recent studies have * This work was supported by a grant from the Israel Science Foun- focused mostly on the function of the PI3K pathway in the dation (to E. B.), by a grant from United States-Israel Binational Sci- survival of differentiating myoblasts. By manipulating differ- ence Foundation (to E. B.), by funds from the Rappaport Foundation for ent kinases and using inhibitors of this pathway, it was dem- Medical Research, and by funds from the Foundation for the Promotion of Research in the Technion, Israel Institute of Technology. The costs of onstrated that it played a major role in protecting differentiat- publication of this article were defrayed in part by the payment of page ing myoblasts from undergoing cell death (11–13). charges. This article must therefore be hereby marked “advertisement” A second signaling pathway induced by IGF-MAPK might in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. also protect muscle cells from apoptotic cell death (10). It was ‡ To whom correspondence should be addressed. Tel.: 972-4-8295- 287; Fax: 972-4-8553-299; E-mail: [email protected]. recently shown that transient transfection of constitutively The abbreviations used are: cdk, cyclin-dependent kinase; PI3K, active Mek1, a specific activator of extracellular regulated ki- phosphoinositide 3-kinase; MAPK, mitogen-activated protein kinase; nases (ERKs), maintained myoblast viability in the absence of pRb, retinoblastoma; IGF, insulin-like growth factor; ERK, extracellu- growth factors (14). lar signal-regulated kinase; GFP, green fluorescent protein; EGFP, enhanced GFP; GM, growth medium; DM, differentiation medium; ER, Several authors (15, 16) reported that the activity of ERK estrogen receptor; MHC, myosin heavy chain; BrdUrd, bromodeoxyuri- was significantly induced with the onset of myoblast terminal dine; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleoti- differentiation. We suggested that this activation is an intrin- dyl transferase-mediated dUTP nick end labeling; PCD, programmed sic property of muscle cells. It is now well established that the cell death; IAP, inhibitors of apoptosis; JNK1, c-Jun NH -terminal kinase 1; DAPI, 4,6-diamidino-2-phenylindole. MAPK pathway that was commonly regarded as mitogenic, can This paper is available on line at http://www.jbc.org 21221 This is an Open Access article under the CC BY license. 21222 MAPK Prevents Myoblast Apoptosis luciferase reporter plasmid DNA and a control reporter gene expressing also induce withdrawal from the cell cycle and survival of cells Renilla under the constitutive cytomegalovirus promoter. Following depending on the magnitude and length of the signal and the transfection, the medium was replaced with DM for another 24 – 48 h. specific cell type (17–19). We decided to investigate the role -Estradiol was added to the cells as indicated. Protein extracts were played by the MAPK pathway in the commitment and differ- prepared and used to measure luciferase and Renilla activities using entiation of myoblasts. Our results show that the activity of the the Luciferase Assay system from Promega. Luciferase activity was MAPK pathway reduces the number of differentiating myo- divided by Renilla activity of each reaction to correct for the transfec- tion efficiency. blasts undergoing apoptotic cell death. The MAPK pathway WAF1 also induces the accumulation of the p21 protein by pro- Immunohistochemical Staining longing its half-life in differentiating cells. Reduction of Cells were fixed and immunostained as described previously (16). WAF1 p21 protein by antisense expression interferes with the The primary antibodies used were anti-phospho-ERK (Sigma), anti- antiapoptotic function of the MAPK pathway. We conclude that WAF1 p21 , anti-cleaved caspase 3 (Transduction Laboratories), and the MAPK pathway regulates the survival of differentiating monoclonal anti-MHC (MF-20). The immunochemically stained cells myoblasts and that this activity is mediated by stabilization of were viewed at 200 magnification under a fluorescence microscope WAF1 (Olympus, model BX50) and photographed with a digital camera. the p21 protein. RNA Analysis EXPERIMENTAL PROCEDURES TM Materials RNA was extracted using TRI reagent (MRC Inc.) and analyzed by Northern blotting as described previously (16). Blots were hybridized U0126 was supplied by Calbiochem. It was dissolved in Me SO to a WAF1 with probes for MLC2 (PVZLC2), p21 (pCDNA-Waf1), and glycer- concentration of 10 mM and was added directly to the differentiation aldehyde-3-phosphate dehydrogenase (pMGAP). medium to a final concentration of 10 mM. -Estradiol was purchased from Sigma. Polyclonal antibodies to ERK1,2 and phospho-specific Western Analysis ERK1,2 were purchased from Cell Signaling Technology. A phospho- Cells were lysed, and whole cell extracts were collected as described specific ERK1,2 monoclonal antibody used in immunostaining of cells (16). Equal amounts of extracted proteins (30 –100 g) were loaded and was purchased from Sigma. A monoclonal antibody to human pRb was separated by 10% SDS-PAGE and transferred to nitrocellulose mem- purchased from Pharmingen. A monoclonal antibody to myosin heavy branes. For detecting the different forms of pRb, proteins were sepa- chain (MF-20) was a gift from Dr. S. Tapscott. A monoclonal antibody to rated over 7.5% SDS-PAGE before being transferred to membranes. bromodeoxyuridine (BMC9318) was purchased from Roche Applied Sci- WAF1 Membranes were incubated in blocking buffer (20 mM Tris-HCl, pH 7.4, ence. Anti-p21 antibody was from Transduction Laboratories. An 150 mM NaCl, 0.1% Tween 20, 2% w/v nonfat dry milk) and then with antibody to the estrogen receptor was from Santa Cruz Biotechnology. the same buffer with the first and secondary antibodies. Between the A polyclonal antibody to cleaved caspase 3 (#9661) was from Cell Sig- second and third incubations, membranes were washed three times in naling Technology. A monoclonal antibody to caspase 9 was a gift from 0.1% Tween 20 and 1 TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl). Dr. T. Kleinberger. Immunoblotting was conducted with the following antibodies: anti-ERK and anti-phospho-ERK (Cell Signaling), 1:1000; anti-pRb (Pharmin- Plasmids WAF1 gen), 1:1000; anti-p21 , 1:1000; anti-MHC, 1:2.5; and anti-caspase p21-Luc was described before (20). Retroviral vectors pBP3Raf- 9, 1:1000. Proteins were visualized using the enhanced chemilumines- 1 :ER and pBP3Raf301:ER were a generous gift from Dr. M. McMahon DD cence kit by Pierce Inc. (18, 21). The retroviral vector encoding for MD:ER was a generous gift from Dr. S. Tapscott (22). The pBABE-GFP retroviral vector was con- Bromodeoxyuridine Staining structed by replacing the puromycin coding sequence with the EGFP Bromodeoxyuridine (BrdUrd) was added to cell media at 10 M. After coding sequence, which was PCR-amplified and cloned into the ClaI- 2–3 h the cells were washed with PBS, fixed with methanol (10 min), HindIII sites of pBABE-puro. The mouse p21 cDNA fragment was and permeabilized in 0.25% Triton X-100 (10 min). Following a PBS cloned in the EcoRI site of pBABE-GFP, and a clone that contained the wash, the cells were incubated in 2 N HCl solution for 1 h. The solution antisense orientation of p21 relative to the promoter was used for was neutralized by washing the cells three times in 0.1 M borate buffer further studies. (pH 8.5). Subsequently, the cells were incubated with 6 mg/ml anti- BrdUrd antibody in PBS containing 0.1% bovine serum albumin for Generation of Stable C2 Clones 1.5 h. The remainder of the procedure was identical to the immunohis- C2 cells were a gift from Dr. D. Yaffe (23). Infection of C2 myoblasts tochemical staining of cells described earlier (16). with replication-defective retroviruses was used to generate C2 cell lines expressing the different chimera proteins. Retroviruses express- Apoptotic Cell Death Assays ing the different proteins were generated by transfection of retroviral TUNEL Assay—The assay kit was purchased from Roche Applied vectors and an expression vector of vesicular stomatitis virus, the Science. The assay was performed according to the manufacturer’s glycoprotein, into viral packaging cells, 293gp, expressing the gag and instructions. pol genes (24). The medium of transfected 293gp cells containing ret- Hoechst Staining—After washing with PBS, the cells were incubated roviruses was used to infect C2 cells. Forty-eight hours later, cells were with the DNA dye bisbenzimidine (Hoechst 33258) (10 g/ml) for 30 selected with puromycin (3 g/ml). Resistant clones were pooled to- min. Nuclear morphology was observed at 200 magnification under an gether a week later. The expression of the chimera proteins was deter- upright fluorescence microscope (Olympus, model BX50) and photo- mined in Western blots with an antibody to estrogen receptor. graphed with a digital camera. The percentage of cells with condensed DNA was calculated. Cell Culture DNA Fragmentation Assay—After washing with PBS, cells were Cell lines were maintained in Dulbecco’s modified Eagle’s medium collected and then resuspended in extraction buffer (10 mM Tris, pH 8.0, supplemented with 15% calf serum (HyClone), penicillin, and strepto- 0.1 mM EDTA, pH 8.0, 20 g/ml RNase A, 0.5% SDS). Samples were mycin (growth medium, GM). To induce differentiation, we used Dul- incubated at 37 °C for 1 h. Proteinase K (100 mg/ml) was added, and becco’s modified Eagle’s medium supplemented with 10 mg of insulin incubation was continued at 50 °C for 3 more h. DNA was then ex- per ml and 10 mg of transferrin per ml (differentiation medium, DM). tracted with phenol/chloroform and precipitated with ethanol. Follow- Differentiation of C2 cell lines expressing the fusion ER proteins was ing a 70% ethanol wash, genomic DNA was resuspended in TE (10 mM induced by the addition of DM. -Estradiol (10 M) was added to DM at Tris, 1 mM EDTA, pH 8.0). An aliquot of 30 g of DNA was analyzed by different time periods as indicated, and U0126 (10 M) was added to DM electrophoresis in 1.8% agarose gels containing ethidium bromide. after 24 h. Antisense Expression Transient Transfection Assays Replication-defective retrovirus expressing mouse p21 antisense and Transfections were performed by calcium phosphate precipitation as the green fluorescent protein (GFP) or control retroviruses expressing described (25) or using the TransFast reagent of Promega according to the GFP protein were used to infect C2 cells. One day following infec- the recommended protocol. Myoblasts in 6-cm TC dishes (Corning) were tion, the medium was replaced by differentiation medium. -Estradiol transfected with a total amount of 10 mg (or 5 mg, using TransFast) of was added to medium 24 h later, and cells were stained with Hoechst MAPK Prevents Myoblast Apoptosis 21223 after 48 h in DM. Cells were viewed for the Hoechst and green fluores- 48 h of growth in DM relative to untreated cells (lanes 2–7). cence staining under the above fluorescence microscope. However, after 72 h of growth in DM, MHC levels were similar between treated and untreated cells (lanes 9 and 10). In Vitro Kinase Assay for ERK Late activation (36 h) of Raf1 did not affect the expression The assay was performed as described in Gredinger et al. (16). levels of MHC at 48 and 72 h of growth in DM relative to untreated cells (compare lanes 6 – 8 and 9 –11). Thus, differ- RESULTS ences in the structure of myotubes, especially those resulted Regulated Activation of ERK MAPK Pathway in C2 Myo- from late Raf1 activation are probably not due to any signif- blasts—In a previous study we observed that ERK MAPK icant changes in muscle-specific expression. activity was significantly induced during in vitro muscle differ- MAPK Does Not Induce Proliferation of Differentiating Myo- entiation (16). In this study we have found that an increase in Blasts—One obvious consequence of Raf1 activation was the phosphorylated ERK was observed after 24 h of C2 myoblasts higher density of nuclei in -estradiol-treated cultures relative growth in differentiation medium (DM), and it gradually accu- to untreated cultures (Fig. 1D, compare the left panel to the mulated as cells differentiated into myotubes (Fig. 1A). To middle and right panels). This difference raised the possibility study the functional significance of MAPK pathway activation that MAPK could promote myoblast proliferation during these during myoblast differentiation, we generated inducible C2 cell stages. The percentage of cells in S phase was analyzed by the lines that expressed a conditional Raf1 protein. C2 myoblasts bromodeoxyuridine labeling assay (Fig. 2A). Activation of Raf1, were infected with retroviruses containing either a fusion gene at different times after myoblasts were induced to differentiate of an activated Raf1 and the hormone-binding domain of estro- in DM, did not induce any significant proliferation beyond the gen receptor (Raf1 :ER) or an inactivated Raf1 and the DD levels observed in control cells grown in DM for the same period hormone-binding domain of estrogen receptor (Raf301:ER) of time (Fig. 2A). The phosphorylation state of the retinoblas- (21). Clones harboring the retroviral vectors were selected and toma protein can serve as an indicator for the proliferation further analyzed. These cells constitutively expressed the fu- state of muscle cells. Two major phosphorylated forms of pRb sion proteins (not shown). Addition of -estradiol to C2 cells exist in replicating myoblasts, whereas only one underphospho- expressing the active Raf1 protein (Raf1 :ER) induced the DD rylated form is found in postmitotic cells. The phosphorylation phosphorylation of ERK1 by 3–5 fold (Fig. 1B). It did not affect of pRb is expected if resting muscle cells are induced to re-enter the phosphorylation state of the closely related MAPKs, p38 the cell cycle. We analyzed how the activation of MAPK af- and JNK (data not shown). Addition of hormone to C2 cells fected the phosphorylation status of pRb (Fig. 2B). The two expressing the inactive Raf1 protein (Raf301:ER) had no ef- forms of pRb were present in proliferating myoblasts, whereas fect on the phosphorylation of MAPK (Fig. 1B). The in vitro only the underphosphorylated form was found in myoblasts kinase activity of ERK protein that was immunoprecipitated growing in DM (lanes 1 and 2). Activation of MAPK in myo- from cell extracts was also analyzed (Fig. 1C). Addition of blasts growing in DM for short (24 h) or long (72 h) periods did -estradiol to C2 cells expressing the active Raf1 protein not change the phosphorylation pattern of pRb; namely, the (Raf1 :ER) induced ERK activity by 3- to 5-fold (lanes 1– 4), DD protein remained in its underphosphorylated form, indicating whereas its addition to C2 cells expressing the inactive Raf1 that resting myoblasts did not re-enter the cell cycle (lanes 3 protein (Raf301:ER) had no effect on ERK activity (lanes 5– 8). and 5). On the whole, the results presented in Fig. 2 suggest ERK activity that was induced by exogenously activated Raf1 that activation of the MAPK pathway does not reverse the (Raf1 :ER) was only 1.5- to 2-fold higher than endogenous DD withdrawal of myoblasts from the cell cycle. ERK activity in cells growing in DM for 48 h (compare lanes 2 Activation of MAPK Prevents Apoptotic Cell Death of Differ- and 4 to lane 11). Thus, the system enables us to phosphorylate entiating Myoblasts—A high proportion of myoblasts under- and activate ERK in an Raf1-dependent manner. Activation of goes programmed cell death (PCD) during in vitro differentia- Raf1 induces ERK activities that are in the range of the endog- tion (1). First, we wanted to validate that, in the C2 cell line, enous activity of ERK in differentiating cells. myoblasts were undergoing PCD at these stages. For that Several studies suggest that the activation of MAPK inhib- purpose, we immunostained cells grown in DM for 48 h with an its muscle differentiation (26 –28). To study whether MAPK antibody to cleaved caspase 3 (active form) to detect ongoing affected muscle differentiation, -estradiol was added to cells apoptosis and with an antibody to MHC to detect myotubes together with the differentiation medium (0 h), during myo- (Fig. 3A). Most (above 95%) of the cells that were stained for the blast proliferation, or 36 h following the addition of differen- expression of cleaved caspase 3 did not stain for myosin heavy tiation medium (36 h). ERK was phosphorylated in each case chain (Fig. 3A, see “Merge”). A higher magnification of a portion of Raf1 activation (not shown). First, we investigated the of the microscopic field presented in Fig. 3A shows that stain- structure of myotubes after growing them for 72 h in DM by ing of caspase 3 was in most cases cytoplasmic (Fig. 3B). Oc- immunostaining with an antibody to myosin heavy chain and casionally, staining of cells appeared nuclear, although it could found that ERK activation affected the size of myotubes (Fig. reflect false identification of cells found in advance stages of 1D). Early activation of ERK (0 h) usually reduced the size of apoptosis with their cytoplasm collapsed and structure de- myotubes relative to control cells (Fig. 1D, middle panel). formed. We can conclude that at these stages the majority of Late activation of ERK (36 h) was followed by the appearance cells undergoing PCD are myoblasts and not myotubes. We of larger myotubes with more nuclei per myotube relative to calculated the number of cells positively stained for cleaved control cells (Fig. 1D, right panel). The number of nuclei per caspase 3 relative to the total number of myoblast nuclei and myotube was calculated (Fig. 1D, histogram). Early activa- found that 29% of the cells were undergoing PCD. tion of Raf1 slightly reduced the average number of nuclei per To investigate the role of MAPK in preventing apoptotic cell myotube, whereas late activation increased this number by death of myoblasts, we asked how activation or repression of the 2-fold relative to control cells. To find out whether these differences in myotube structure were also reflected in the pathway affected cell viability by Hoechst and by TUNEL stain- ing of nuclei (Fig. 4A). A significant percentage of myoblasts expression of the structural protein MHC, the levels of MHC were analyzed at several time points following early or late growing in DM for 48 h undergo apoptosis as can be seen by activation of Raf1 (Fig. 1E). Early activation of Raf1 (0 h) chromatin condensation observed by the dense staining of DNA mildly reduced (2-fold) MHC expression after 8, 24, and (Fig. 4A). Addition of -estradiol to C2-Raf1 :ER cells after DD 21224 MAPK Prevents Myoblast Apoptosis 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 Raf1 :ER or Raf301:ER proteins were generated as described under “Experimental Procedures.” Cells were grown in GM, DD and then the medium was replaced with DM with or without -estradiol (10 M) or Mek inhibitor, U0126 (10 M). 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 -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 -estradiol treatment was relative to cells grown in the exact same conditions but in the absence of the hormone. D, -estradiol was added to C2 myoblasts expressing the Raf1 :ER protein together with DM or 36 h following the addition of DM and cells were fixed and immunostained with DD 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, -estradiol was added to C2 myoblasts expressing the Raf1 :ER protein DD 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). -Tubulin was used as a control of protein loading in each lane. 24 h of myoblasts growth in DM for an additional period of 24 h the antiapoptotic effect was specific to Raf activity (Fig. 4A). largely prevented cell death. Conversely, treatment of cells with Apoptotic cell death was also analyzed by TUNEL staining of Mek inhibitor, U0126 increased dramatically cell death (Fig. 4A). fragmented DNA (Fig. 4B). Activation of the Raf1 :ER protein DD The addition of -estradiol to the control C2-Raf301:ER cells did reduced while the addition of U0126 increased PCD to similar not change the number of cells undergoing PCD, suggesting that values observed in the Hoechst staining. MAPK Prevents Myoblast Apoptosis 21225 FIG.2. Activation of Raf1 :ER protein does not induce proliferation in differentiating myoblasts. A,C2 Raf1 :ER cells were DD DD grown for several time periods in DM as indicated, in the absence or presence of -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 Raf1:ER cells were grown in GM (0 h in DM) and then in DM for the indicated time periods. -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, hyperphospho- rylated form of retinoblastoma; MAPK, total ERK proteins; P-MAPK, phosphorylated ERK proteins. 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 magnifi- cation of a section of the microscopic field presented in panel A. Arrows point to- ward cells that are stained positively to cleaved caspase 3, and their staining is cytoplasmic. Fragmentation of genomic DNA that appears as a typical untreated cells (compare lanes 2 and 4). ladder in gel electrophoresis can serve as a hallmark of PCD. During the process of apoptotic cell death certain caspases Identical amounts of genomic DNA were separated over an are being activated by proteolytic cleavage (29, 30). We ana- agarose gel. No ladder was observed in DNA from proliferating lyzed the possible involvement of caspase 9 in PCD of myo- myoblasts, however, a ladder was noticeable after growth of blasts and searched whether the MAPK pathway affected its myoblasts in DM for 72 h (Fig. 4C, compare lanes 1 and 2). If activation. In proliferating myoblasts the inactive pro-caspase the same cells were treated with U0126 for 48 h, after 24 h of was the only noticeable form (Fig. 4D, lane 1). During differ- myoblasts growth in DM the staining intensity of the ladder entiation of myoblasts, in addition to the inactive form of was increased significantly (Fig. 4C, lane 3). Treatment with caspase 9, the active shorter form was observed after 24 h in -estradiol to increase MAPK activity during the same period DM, and its amount was increased after 48 h (Fig. 4D, lanes 2 of time decreased the intensity of the DNA ladder relative to and 3). Myoblasts growing for 48 h in DM and treated with 21226 MAPK Prevents Myoblast Apoptosis FIG.4. Induction of Raf1 activity protects myoblasts from apoptotic cell death by preventing the activation of caspase 9. A,C2 Raf1 :ER or C2Raf301:ER myoblast cells were grown for 48 h in DM in the absence or presence of -estradiol or U0126 that were added during DD 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 Raf1 :ER myoblasts were grown as described in A. Cells were fixed, and TUNEL staining of DD 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 Raf1 :ER myoblasts were grown in DM for 72 h in the absence or presence of U0126 or -estradiol DD that were added after 24 h in DM. Fragmentation of genomic DNA was analyzed as a marker of apoptotic cell death. D,C2 Raf1 :ER myoblasts DD 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. WAF1 U0126 expressed more of the active form than control cells not p21 —It was shown before that the expression of cyclin-de- WAF1 treated with the inhibitor (compare lanes 3 and 4). On the other pendent kinase inhibitor, p21 , was necessary to prevent hand, induction of Raf1 activity reduced the levels of active PCD of myoblasts (2). Next we tested whether MAPK was WAF1 caspase 9 (compare lanes 3 and 5). These results suggest that involved in the expression of p21 in muscle cells. Addition the MAPK pathway regulates the activation of caspase 9 in the of -estradiol to C2-Raf1 :ER cells growing in GM induced DD WAF1 apoptotic pathway of myoblasts. protein levels of p21 , whereas treatment of the same cells Activation of Raf1 in Muscle Cells Induces the Expression of with Mek inhibitor, U0126, reduced its levels as observed by MAPK Prevents Myoblast Apoptosis 21227 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 Raf1 :ER (Fig. 6A, DD graph). -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 WAF1 induce the levels of the p21 protein. The changes in the WAF1 levels of the p21 protein could reflect alterations in protein WAF1 stability. The half-life of the p21 protein was analyzed in a pulse-chase labeling experiment that was performed in Raf1 :ER-expressing cells grown for 48 h in DM. The half- DD WAF1 life of p21 was about 30 min in cells grown in the absence WAF1 of -estradiol (Fig. 6B). However, the level of p21 did not change when cells were grown in the presence of -estradiol even after 120 min of chase (Fig. 6B). In contrast, incubating WAF1 the cells with U0126 reduced p21 half-life to less than 10 min (not shown). We conclude that the MAPK pathway con- WAF1 tributes to the stability of the p21 protein in muscle cells. WAF1 FIG.5. The MAPK pathway induces the expression of p21 Next we wanted to find out whether activation of Raf :ER DD protein in C2 muscle cells. A,C2 Raf1 :ER myoblasts were grown DD had a general effect on protein stability or that it stabilized in GM and -estradiol or U0126 were added for 24 h before proteins WAF1 p21 in a specific manner. For that purpose we analyzed were extracted, separated over SDS-PAGE, and analyzed by Western WAF1 the protein levels of another cdk inhibitor family member, blotting. The blot was reacted repeatedly with antibodies to p21 , KIP1 phosphorylated MAPK, and total MAPK. B,C2 Raf1 :ER myoblasts DD p27 , following the induction or inhibition of MAPK phos- were grown as described in A. Cells were then fixed and immunostained phorylation with -estradiol or U0126, respectively (Fig. 6C, WAF1 with antibodies to p21 and phosphorylated MAPK. Cell nuclei were KIP1 lower panel). The steady-state levels of p27 were not af- stained with DAPI. C,C2 Raf1 :ER myoblasts were grown in DM for DD WAF1 fected, whereas those of p21 were changed as expected. We the indicated time periods. -Estradiol or U0126 were added as indi- cated after 36 h of cell growth in DM. Proteins were extracted, sepa- conclude that the MAPK pathway affects the protein levels of rated over SDS-PAGE, and analyzed by Western blotting. The blot was WAF1 KIP1 p21 but not of another family member, p27 . WAF1 reacted repeatedly with antibodies to p21 , phosphorylated MAPK, To investigate the possible interplay between transcriptional and total MAPK. regulation by MyoD and post-transcriptional regulation by WAF1 MAPK in determining the levels of p21 protein, we gen- Western blotting (Fig. 5A). By immunostaining of cells treated erated C2 myoblast cells that in addition to the endogenous with -estradiol, we could observe that myoblasts expressing MyoD, expressed an inducible form of the MyoD protein (C2- WAF1 the phosphorylated form of MAPK, also expressed p21 in MyoD:ER). Addition of -estradiol to these cells induced higher their nuclei (Fig. 5B). Hence, MAPK is involved in the expres- WAF1 WAF1 transcript levels of p21 , suggesting that the MyoD:ER sion of p21 in proliferating myoblasts. The cdk inhibitor protein induced the transcription of the p21 gene (data not WAF1 p21 is normally induced during early stages of muscle shown). Induction of the exogenous MyoD:ER protein also in- differentiation as myoblasts exit the cell cycle (20, 31). We WAF1 WAF1 creased the amount of p21 protein above its level in control followed the expression profile of p21 protein during the cells (Fig. 6D, compare lanes 1 and 3). In contrast, treatment of differentiation of C2-Raf1 :ER cells (Fig. 5C). Levels of DD WAF1 WAF1 cells with U0126, significantly reduced the level of the p21 p21 protein were low in proliferating myoblasts, induced protein (Fig. 6D, lane 2). However, inhibition of MAPK with after 24 h in DM, and gradually declined during their further WAF1 U0126 did not reduce p21 protein if during that time, growth in DM (Fig. 5C, lanes 1–5). Addition of -estradiol after WAF1 -estradiol was added to induce the activity of the MyoD:ER growth of 36 h in DM induced the levels of p21 protein protein (Fig. 6D, lane 4). The amount of cells undergoing ap- observed at later periods of cell growth (compare lanes 4 and 5 optotic cell death was inversely correlated with the expression to lanes 6 and 7), whereas U0126 added to cells after growth of WAF1 WAF1 levels of p21 (Fig. 6D, graph). Myoblasts expressing high 36 h in DM reduced p21 protein to almost undetectable WAF1 levels of p21 resulting from the activation of MyoD:ER levels after further growth of cells (compare lanes 4 and 5 to showed reduced PCD (lane 3), whereas those treated with Mek lanes 8 and 9). These results indicate that MAPK could be WAF1 WAF1 inhibitor expressing very low levels of p21 showed en- involved in the expression of p21 in differentiating myo- hanced PCD (lane 2). Myoblasts expressing intermediate levels blasts in addition to other factors. WAF1 WAF1 of p21 protein, obtained from the simultaneous activation Activation of Raf1 Extends the Half-life of the p21 Pro- of MyoD:ER and suppression of Mek, exhibited intermediate tein—In muscle cells, the MyoD protein functions to induce the WAF1 levels of PCD (lane 4). Taken together, these results suggest a transcription of p21 during differentiation (20). To find out WAF1 possible interplay, between the transcriptional and post-tran- how MAPK affected the expression of p21 , we analyzed WAF1 scriptional activities of MyoD and MAPK, respectively, in es- the transcript levels of p21 following activation or repres- WAF1 WAF1 tablishing the protein levels of p21 during muscle differ- sion of MAPK (Fig. 6A). The levels of p21 were affected WAF1 entiation, may exist and that p21 levels correlate directly neither by the activation of MAPK caused by adding -estradiol to myoblasts growing for 24 h in DM nor by its inhibition with the survival of differentiating myoblasts. The Antiapoptotic Function of the MAPK Pathway in Muscle following treatment with U0126, suggesting that MAPK did WAF1 WAF1 WAF1 not affect transcription of p21 in muscle cells (Fig. 6A, Cells Is Mediated by p21 —To determine whether p21 was required for MAPK-mediated cell survival, we infected the lanes 2 and 3, Northern blot). However, in the same experi- WAF1 ments, the levels of the p21 protein were changed by the C2-Raf1 :ER cells with a retrovirus encoding for an anti- DD 21228 MAPK Prevents Myoblast Apoptosis WAF1 FIG.6. The MAPK pathway stabilizes the p21 protein. A,C2 Raf1 :ER myoblasts were grown in DM for 48 h, and -estradiol or DD U0126 were added for 24 h before RNA and proteins were extracted. Upper panel, a Northern blot that was sequentially hybridized with probes WAF1 to p21 and to glyceraldehyde-3-phosphate dehydrogenase. Middle panel, a Western blot that was reacted repeatedly with antibodies to p21 , 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 Raf1 :ER myoblasts, which were then were grown in DM for 48 h, and -estradiol or U0126 were added DD 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 -estradiol or U0126. Values are means from three independent experiments. Error bars represent standard errors. In B: upper panel, C2 Raf1 :ER myoblasts were grown in DM for 48 h in the absence or presence of -estradiol. Cells were metabolically labeled with DD [ S]methionine for 1 h. Labeling was chased by replacing the medium with unlabeled medium for different periods of time, as indicated, before WAF1 cells were lysed and proteins were extracted. The p21 protein was immunoprecipitated and resolved by SDS-PAGE. Lower panel, the bands were quantified, and relative values are presented in the graph. C,C2 Raf1 :ER myoblasts were grown in DM for 48 h, and -estradiol or U0126 DD were added for 24 h before proteins were extracted, separated over SDS-PAGE, and analyzed by Western blotting. The blot was reacted repeatedly WAF1 with antibodies to p27 (Kip1), p21 , 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 -estradiol or U0126 as indicated, and proteins were extracted, separated over SDS-PAGE, and analyzed by Western WAF1 blotting. The blot was reacted repeatedly with antibodies to p21 , 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. sense p21 mRNA (p21WAF1 ) and the marker protein EGFP induced 12 h later. After 36 h in DM, the percentage of apo- AS or with a control retrovirus encoding for EGFP only. Infected ptotic cells that were positive for EGFP expression was ana- cells were grown in differentiation medium, and Raf1 was lyzed by Hoechst staining (Fig. 7A). Some myoblasts infected MAPK Prevents Myoblast Apoptosis 21229 FIG.7. Antisense p21 expression prevents the MAPK-mediated antiapoptotic activity in differentiating cells. A,C2 Raf1 :ER DD 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 -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 Raf1 :ER myoblasts were infected as described in A. Cells were DD WAF1 grown in DM for 48 h. Cells were then fixed and immunostained using an antibody to the p21 protein. Cells that were EGFP-stained (green) WAF1 WAF1 were analyzed for p21 staining (red). The graph summarizes the counts of stained cells. The percentage of cells expressing p21 was calculated by dividing the cells stained green and red by the total number of green cells. with the control virus underwent apoptotic cell death, however, example, transformation of fibroblasts depends on the activi- the majority of cells survived. In contrast, the majority of ties of several pathways, including MAPK and phosphoinositol myoblasts, infected with the virus expressing antisense p21, 3-kinase (PI3K), whereas cell cycle arrest is induced by the underwent apoptotic cell death regardless of whether -estra- activation of the MAPK pathway only (37); 3) quantitatively diol was added to induce the Raf1 :ER protein or not (Fig. different levels of MAPK activity elicit different responses. For DD 7A). In addition to the condensed chromatin, these cells lost example, transient activation of MAPK in PC12 cells induces their normal elongated structure and adopted a small spherical proliferation, whereas prolonged activation of MAPK triggers structure. Immunostaining of differentiating myoblasts indi- neurite outgrowth (38). These models, in total and individually, cated that the expression of the antisense p21-encoding virus can explain the conflicting roles of MAPK in myogenesis. The WAF1 prevented the expression of endogenous p21 in most of the basal phosphorylation and activity of MAPK is low in prolifer- infected cells (Fig. 7B). These results indicate that MAPK pro- ating C2 myoblasts relative to its sustained and significant motes muscle cell survival by inducing the protein levels of induction in myoblasts and myotubes growing in differentia- WAF1 p21 . tion medium. Therefore, levels of MAPK induction as well as the cell context and additional signaling pathways may explain DISCUSSION the differences in the way that the same signaling pathway Late Activation of MAPK Affects Postmitotic Growth of Mus- affects cell growth at different stages of differentiation. Inter- cle Cells—The ERK MAPK pathway has been implicated in the estingly, significant activation of MAPK both during early and control of myogenesis. Several studies have proposed that the late phases of differentiation did not induce any proliferation of pathway functioned by inducing proliferation and, therefore, myoblasts (Fig. 2). This result is in conflict with other studies inhibited muscle differentiation (8, 26, 27, 32–35). Our previ- (27, 28) and may be explained in the following two ways: (a)We ous work and that of others showed that ERK phosphorylation induced MAPK under serum starvation conditions that pro- and activity were significantly induced during the terminal moted withdrawal from the cell cycle. Under these conditions, differentiation of myoblasts (15, 16). We suggested that the cell context and signaling pathways may cooperate with MAPK pathway was intrinsic to muscle cells and could stimulate the in arresting cell cycle. In other studies, the MAPK pathway was differentiation process. In the present work we investigated inhibited in myoblasts growing in high serum. Under these the mode of ERK involvement during the differentiation conditions the pathway was involved in cell proliferation. (b)In process. Our results indicate that the activation of ERK plays a muscle cells, high levels of MAPK activity induce withdrawal, crucial role in the survival of differentiating myoblasts. whereas lower levels induce cell cycle progression. These pos- How can one explain the different effects of the MAPK path- sibilities deserve further investigation. way during myogenesis? Several models can be suggested to Antiapoptotic Activity of MAPK during Myoblast Differenti- explain the multiple responses mediated by the MAPK path- ation—In cell cultures many myoblasts undergo PCD under way: 1) the cellular response is dictated by the cellular context. conditions that promote differentiation (1). Based on several This model suggests that one pathway may affect many pro- different approaches we find that the MAPK pathway is in- cesses by regulating different sets of transcription factors that are available in different tissues or at a given time point in a volved in protecting myoblasts from undergoing PCD (Fig. 4). Signals that induce apoptosis culminate in the activation of certain tissue (36); 2) the combined activities of different signal transduction pathways determine the biological response. For caspases, which are the ultimate effectors of PCD. To find out 21230 MAPK Prevents Myoblast Apoptosis 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 in- creased 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 tran- scription factor CREB (41). The role of MAPK in cell survival FIG.8. A model for the involvement of MAPK in muscle cell survival. was also explored in Drosophila, known to express a group of proteins, REAPER, HID, and GRIM, that activate caspase view of its role as a myoblast survival factor (2). In proliferating processing and other proteins known as inhibitors of apoptosis myoblasts where the levels of phosphorylated MAPK and (IAPs) that inhibit caspase processing. The latter group can WAF1 p21 are low, activation of Raf1 induced the expression of directly bind to activated caspases and block the proteolytic WAF1 the p21 protein (Fig. 5, A and B). After 24 h of growth of C2 chain reaction. The first group of proteins binds directly to IAP WAF1 cells in DM the level of p21 protein increased but gradu- and antagonizes its activity, thereby allowing the proteolytic ally declined during further growth (Fig. 5C). This happens activation of caspases and apoptosis to proceed. In Drosophila, despite the normal induction of MAPK occurring during differ- MAPK phosphorylates the HID protein and inhibits its inter- entiation. Further activation of MAPK via the exogenously action with IAP and consequently its proapoptotic activity (42, expressed Raf1 :ER protein during these stages induced 43). Although the functional mammalian homologue of Hid has DD WAF1 higher levels of p21 protein, whereas inhibition of Mek not yet been identified, its existence was suggested (44, 45). with U0126 reduced its levels. Therefore, MAPK plays a role in One might speculate also that in mammalian cells, including WAF1 maintaining the levels of p21 during late phases of differ- muscle cells, MAPK may directly affect an HID-like protein in entiation. Some studies indicate that other factors such as preventing the proteolytic activation of caspases. MyoD and the PI3K pathway also affect the expression of MAPK Functions Independently of the PI3K Pathway in Pro- WAF1 p21 (13, 20). Thus, the balance between different factors, tecting Myoblasts from PCD—Recently, several works demon- including the MAPK pathway, may determine the absolute strated the involvement of the phosphoinositol 3-kinase (PI3K) WAF1 levels of p21 in differentiating myoblasts. As the activities pathway in the survival of differentiating myoblasts (11, 13). It of MyoD and PI3K are reduced during later stages of differen- was suggested that Akt, a kinase in the pathway, phosphoryl- tiation, enhanced activity of MAPK may substitute for these ated the mitochondrial BAD protein known to protect cells from WAF proteins in maintaining the expression of p21 . The vital undergoing PCD (46, 47). In light of our results it is of interest WAF1 role of p21 in cell cycle withdrawal and myoblast survival to know whether the PI3K and the MAPK pathways exhibit may explain the multiple pathways and factors involved in its cross-talk in their antiapoptotic functions. We found that mod- expression. ulation of MAPK activity did not affect the phosphorylation WAF1 MAPK Stabilizes the p21 Protein—In some cellular sys- state of Akt during muscle differentiation. Therefore, it is tems MAPK regulates p21 transcriptionally (50 –52), whereas likely that the antiapoptotic function of MAPK is not mediated in others it also affects the post-transcriptional processes (53). by Akt. Our conclusion is further supported by a recent study We studied the regulation of p21 by MAPK in muscle cells and that suggested IGF-I- and platelet-derived growth factor-in- found that the activation or repression of the pathway did not duced myoblast survival via two independent signaling path- affect the transcripts levels or the promoter activity of p21. ways (14). According to this work, IGF-I induced the PI3K Nevertheless, MAPK significantly affected the protein levels of pathway, whereas platelet-derived growth factor induced the WAF1 p21 , suggesting changes in protein synthesis or break- MAPK pathway, and each of the pathways was sufficient to down. The finding, that activation of Raf1 dramatically ex- promote muscle cell survival. tended while inhibition of Mek with U0126 significantly re- The activities of PI3K and MAPK do not overlap in the WAF1 duced the half-life of the p21 protein, indicates that the differentiation process. Moreover, Akt was shown to phospho- WAF1 MAPK pathway regulates the stability of the p21 protein. rylate and inhibit Raf1 in muscle cells (48). Whereas PI3K and WAF1 The p21 protein is degraded by the proteasome in a proc- Akt are induced at early stages of differentiation, MAPK acti- WAF1 ess that does not involve ubiquitination of p21 (54). Recent vation occurs at later stages. Therefore, it is possible that each WAF1 studies have emphasized that the turnover of p21 protein pathway functions independently to protect myoblasts from is regulated by several signaling pathways affecting cell PCD at different stages of the differentiation process. growth. Rac1/CDC42 activates the degradation of p21 (55), Several Factors, Including the MAPK Pathway, Maintain the WAF1 whereas p38 MAPK, JNK1 (56), and protein kinase B/Akt (57) Expression of p21 in Differentiating Myoblasts—MAPK WAF1 stabilize the protein by phosphorylating several of its residues. induces the expression of p21 in many cellular systems WAF1 One study suggested that the ERK MAPK pathway is required to and causes cell cycle arrest (49). The induction of p21 WAF stabilize p21 mRNA and p21 protein during the withdrawal expression during muscle differentiation plays at least two of primary hepatocytes from the cell cycle (53). All in all, these fundamental roles in the withdrawal of myoblasts from the cell WAF1 studies suggest that the stability of the p21 protein is af- cycle and in their survival (1). MyoD is involved in the tran- fected by multiple signaling events, implicating it as a major scriptional induction of p21 (20). In the present work we asked WAF1 WAF1 mechanism regulating the levels of p21 protein in cells. whether MAPK contributed to the expression of p21 ,in MAPK Induces Hypertrophic Growth of Myotubes—In the present study we observed that MAPK was also involved in the O. Ostrovsky and E. Bengal, unpublished results. determination of myotube size and the number of nuclei per MAPK Prevents Myoblast Apoptosis 21231 10. Sarbassov, D. D., and Peterson, C. A. (1998) Mol. Endocrinol. 12, 1870 –1878 myotube (Fig. 1). Similar observations were reported by others 11. Fujio, Y., Guo, K., Mano, T., Mitsuuchi, Y., Testa, J. R., and Walsh, K. (1999) (10, 58). In those studies, as well as in ours, MAPK activity Mol. Cell. Biol. 19, 5073–5082 12. Lawlor, M. A., and Rotwein, P. (2000) J. Cell Biol. 151, 1131–1140 increased the size of myotubes and prevented the collapse that 2 13. Lawlor, M. A., and Rotwein, P. (2000) Mol. Cell. Biol. 20, 8983– 8995 usually occurs after several days of growth in DM. Presently 14. Lawlor, M. A., Feng, X., Everding, D. R., Sieger, K., Stewart, C. E., and we don’t know if this is a direct or indirect effect of the MAPK Rotwein, P. (2000) Mol. Cell. Biol. 20, 3256 –3265 15. Sarbassov, D. D., Jones, L. G., and Peterson, C. A. (1997) Mol. Endocrinol. 11, pathway. A direct activity of MAPK could target the transla- 2038 –2047 tional apparatus and induce hypertrophic growth (59) or, alter- 16. Gredinger, E., Gerber, A. N., Tamir, Y., Tapscott, S. J., and Bengal, E. (1998) natively, induce fusion of myoblast cells to multinucleated myo- J. Biol. Chem. 273, 10436 –10444 17. Ravi, R. K., Weber, E., McMahon, M., Williams, J. R., Baylin, S., Mal, A., tubes. An indirect effect could for example be a result of the Harter, M. L., Dillehay, L. E., Claudio, P. P., Giordano, A., Nelkin, B. D., better survival of myoblasts induced by MAPK that allows the and Mabry, M. (1998) J. Clin. Invest. 101, 153–159 18. Woods, D., Parry, D., Cherwinski, H., Bosch, E., Lees, E., and McMahon, M. recruitment of more competent myoblasts to fuse and form dif- (1997) Mol. Cell. Biol. 17, 5598 –5611 ferentiated myotubes. These possibilities deserve further studies. 19. Ballif, B. A., and Blenis, J. (2001) Cell Growth & Differ. 12, 397– 408 Interestingly, from studies with Rb mice it is apparent that 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 pRb protein may affect muscle growth in a way similar to MAPK 21. McMahon, M. (2001) Methods Enzymol. 332, 401– 417 (5). These mice die after birth with specific muscle defects, in- 22. Hollenberg, S. M., Cheng, P. F., and Weintraub, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8028 – 8032 cluding increased myoblast PCD prior to myocyte fusion; the 23. Yaffe, D., and Saxel, O. (1977) Nature 270, 725–727 surviving myotubes are shorter, have less nuclei, and express 24. Naviaux, R. K., Costanzi, E., Haas, M., and Verma, I. M. (1996) J. Virol. 70, reduced levels of late muscle-specific genes. The similarities be- 5701–5705 WAF1 25. Davis, R. L., Cheng, P. F., Lassar, A. B., and Weintraub, H. (1990) Cell 60, tween pRb and MAPK and the direct effect of MAPK on p21 733–746 protein suggest that these proteins may share the same pathway 26. Coolican, S. A., Samuel, D. S., Ewton, D. Z., McWade, F. J., and Florini, J. R. affecting muscle differentiation and survival. (1997) J. Biol. Chem. 272, 6653– 6662 27. Jones, N. C., Fedorov, Y. V., Rosenthal, R. S., and Olwin, B. B. (2001) J. Cell. Recently, we observed that the MAPK pathway was abso- Physiol. 186, 104 –115 lutely necessary for the differentiation of skeletal muscle dur- 28. Tortorella, L. L., Milasincic, D. J., and Pilch, P. F. (2001) J. Biol. Chem. 276, 13709 –13717 ing early development of Xenopus laevis (60). In this model 29. Thornberry, N. A., and Lazebnik, Y. (1998) Science 281, 1312–1316 system, MAPK affected the expression of late markers of dif- 30. Nunez, G., Benedict, M. A., Hu, Y., and Inohara, N. (1998) Oncogene 17, ferentiation. We could also demonstrate that activated Mek in- 3237–3245 31. Skapek, S. X., Rhee, J., Spicer, D. B., and Lassar, A. B. (1995) Science 267, duced the levels of the MyoD protein in explants from injected 1022–1024 embryos. Therefore, during early development of Xenopus, dis- 32. Ramocki, M. B., Johnson, S. E., White, M. A., Ashendel, C. L., Konieczny, S. F., tinctly from the cell culture system used in the present study, and Taparowsky, E. J. (1997) Mol. Cell. Biol. 17, 3547–3555 33. Samuel, D. S., Ewton, D. Z., Coolican, S. A., Petley, T. D., McWade, F. J., and MAPK directly affects myogenesis through the MyoD protein. Florini, J. R. (1999) Horm. Metab. Res. 31, 55– 64 However, it is possible that, like in the tissue culture model, 34. Weyman, C. M., Ramocki, M. B., Taparowsky, E. J., and Wolfman, A. (1997) Oncogene 14, 697–704 MAPK functions to augment skeletal muscle differentiation by 35. Tombes, R. M., Auer, K. L., Mikkelsen, R., Valerie, K., Wymann, M. P., preventing myoblast cell death during Xenopus development. Marshall, C. J., McMahon, M., and Dent, P. (1998) Biochem. J. 330, A Model for the Antiapoptotic Activity of the MAPK Pathway 1451–1460 36. Gauld, S. B., Blair, D., Moss, C. A., Reid, S. D., and Harnett, M. M. (2002) in Muscle—As myoblasts undergo their terminal differentia- J. Immunol. 168, 3855–3864 tion, many of the cells that cannot complete this process suc- 37. Sheng, H., Shao, J., and DuBois, R. N. (2001) J. Biol. Chem. 276, 14498 –14504 cessfully are eliminated by PCD (Fig. 8). The cell cycle machin- 38. Marshall, C. J. (1995) Cell 80, 179 –185 WAF1 39. Erhardt, P., Schremser, E. J., and Cooper, G. M. (1999) Mol. Cell. Biol. 19, ery and, specifically, the p21 cdk inhibitor regulate this 5308 –5315 WAF1 process (61). p21 may serve as an indicator for the “deci- 40. Tashker, J. S., Olson, M., and Kornbluth, S. (2002) Mol. Biol. Cell 13, 393– 401 41. Bonni, A., Brunet, A., West, A. E., Datta, S. R., Takasu, M. A., and Greenberg, sion” of myoblasts whether to proceed with the differentiation M. E. (1999) Science 286, 1358 –1362 WAF1 process or to undergo cell death. The protein levels of p21 42. Kurada, P., and White, K. (1998) Cell 95, 319 –329 are induced and maintained by several factors and signaling 43. Bergmann, A., Agapite, J., McCall, K., and Steller, H. (1998) Cell 95, 331–341 44. Verhagen, A. M., and Vaux, D. L. (2002) Apoptosis 7, 163–166 pathways during differentiation. MyoD, whose activity is the 45. Haining, W. N., Carboy-Newcomb, C., Wei, C. L., and Steller, H. (1999) Proc. first to be induced during muscle differentiation, activates Natl. Acad. Sci. U. S. A. 96, 4936 – 4941 the transcription of the p21 gene. During these early stages, 46. del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R., and Nunez, G. (1997) Science 278, 687– 689 the PI3K-Akt pathway is also activated in a transient fashion 47. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, WAF1 to further induce the levels of p21 . Later, as PI3K-Akt M. E. (1997) Cell 91, 231–241 48. Rommel, C., Clarke, B. A., Zimmermann, S., Nunez, L., Rossman, R., Reid, K., activity drops, the MAPK pathway is induced and functions to Moelling, K., Yancopoulos, G. D., and Glass, D. J. (1999) Science 286, WAF1 maintain the p21 protein during a later phase of 1738 –1741 differentiation. 49. Crespo, P., and Leon, J. (2000) Cell. Mol. Life Sci. 57, 1613–1636 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 Acknowledgments—We thank Dr. M. McMahon for the Raf1 retrovi- 51. Ravi, R. K., McMahon, M., Yangang, Z., Williams, J. R., Dillehay, L. E., Nelkin, ral vectors, Dr. N. Somia and Dr. I. M. Verma for retroviral vectors and B. D., and Mabry, M. (1999) J. Cell. Biochem. 72, 458 – 469 a packaging cell line, Dr. S. J. Tapscott for the MyoD:ER vector and an 52. Liu, Y., Martindale, J. L., Gorospe, M., and Holbrook, N. J. (1996) Cancer Res. antibody to MHC, and Dr. T. Kleinberger for the antibody to caspase 9. 56, 31–35 We thank Bianca-Raikhlin-Eisenkraft for critical reading of the 53. Park, J. S., Qiao, L., Gilfor, D., Yang, M. Y., Hylemon, P. B., Benz, C., manuscript. Darlington, G., Firestone, G., Fisher, P. B., and Dent, P. (2000) Mol. Biol. Cell 11, 2915–2932 REFERENCES 54. Sheaff, R. J., Singer, J. D., Swanger, J., Smitherman, M., Roberts, J. M., and Clurman, B. E. (2000) Mol. Cell 5, 403– 410 1. Walsh, K. (1997) Prog. Cell Cycle Res. 3, 53–58 55. Bao, W., Thullberg, M., Zhang, H., Onischenko, A., and Stromblad, S. (2002) 2. Wang, J., and Walsh, K. (1996) Science 273, 359 –361 Mol. Cell. Biol. 22, 4587– 4597 3. Zhang, P., Wong, C., Liu, D., Finegold, M., Harper, J. W., and Elledge, S. J. 56. Kim, G. Y., Mercer, S. E., Ewton, D. Z., Yan, Z., Jin, K., and Friedman, E. (1999) Genes Dev. 13, 213–224 (2002) J. Biol. Chem. 277, 29792–29802 4. Wang, J., Guo, K., Wills, K. N., and Walsh, K. (1997) Cancer Res. 57, 351–354 5. Zacksenhaus, E., Jiang, Z., Chung, D., Marth, J. D., Phillips, R. A., and Gallie, 57. Li, Y., Dowbenko, D., and Lasky, L. A. (2002) J. Biol. Chem. 277, 11352–11361 58. Wu, Z., Woodring, P. J., Bhakta, K. S., Tamura, K., Wen, F., Feramisco, J. R., B. L. (1996) Genes Dev. 10, 3051–3064 6. Stewart, C. E., and Rotwein, P. (1996) J. Biol. Chem. 271, 11330 –11338 Karin, M., Wang, J. Y., and Puri, P. L. (2000) Mol. Cell. Biol. 20, 3951–3964 59. Stefanovsky, V. Y., Pelletier, G., Hannan, R., Gagnon-Kugler, T., Rothblum, 7. Xu, Q., and Wu, Z. (2000) J. Biol. Chem. 275, 36750 –36757 8. Weyman, C. M., and Wolfman, A. (1998) Endocrinology 139, 1794 –1800 L. I., and Moss, T. (2001) Mol. Cell 8, 1063–1073 9. Tureckova, J., Wilson, E. M., Cappalonga, J. L., and Rotwein, P. (2001) J. Biol. 60. Zetser, A., Frank, D., and Bengal, E. (2001) Dev. Biol. 240, 168 –181 Chem. 276, 39264 –39270 61. Walsh, K., and Perlman, H. (1997) Curr. Opin. Genet. Dev. 7, 597– 602
Journal
Journal of Biological Chemistry – Unpaywall
Published: May 30, 2003