Myostatin Induces Cyclin D1 Degradation to Cause Cell Cycle Arrest through a Phosphatidylinositol 3-Kinase/AKT/GSK-3β Pathway and Is Antagonized by Insulin-like Growth Factor 1
2006; Elsevier BV; Volume: 282; Issue: 6 Linguagem: Inglês
10.1074/jbc.m610185200
ISSN1083-351X
AutoresWei Yang, Yong Zhang, Yanfeng Li, Zhenguo Wu, Dahai Zhu,
Tópico(s)Neurogenetic and Muscular Disorders Research
ResumoMyostatin is a transforming growth factor β superfamily member and is known as an inhibitor of skeletal muscle cell proliferation and differentiation. Exposure to myostatin induces G1 phase cell cycle arrest. In this study, we demonstrated that myostatin down-regulates Cdk4 activity via promotion of cyclin D1 degradation. Overexpression of cyclin D1 significantly blocked myostatin-induced proliferation inhibition. We further showed that phosphorylation at threonine 286 by GSK-3β was required for myostatin-stimulated cyclin D1 nuclear export and degradation. This process is dependent upon the activin receptor IIB and the phosphatidylinositol 3-kinase/Akt pathway but not Smad3. Insulin-like growth factor 1 (IGF-1) treatment or Akt activation attenuated the myostatin-stimulated cyclin D1 degradation as well as the associated cell proliferation repression. In contrast, attenuation of IGF-1 signaling caused C2C12 cells to undergo apoptosis in response to myostatin treatment. The observation that IGF-1 treatment increases myostatin expression through a phosphatidylinositol 3-kinase pathway suggests a possible feedback regulation between IGF-1 and myostatin. These findings uncover a novel role for myostatin in the regulation of cell growth and cell death in concert with IGF-1. Myostatin is a transforming growth factor β superfamily member and is known as an inhibitor of skeletal muscle cell proliferation and differentiation. Exposure to myostatin induces G1 phase cell cycle arrest. In this study, we demonstrated that myostatin down-regulates Cdk4 activity via promotion of cyclin D1 degradation. Overexpression of cyclin D1 significantly blocked myostatin-induced proliferation inhibition. We further showed that phosphorylation at threonine 286 by GSK-3β was required for myostatin-stimulated cyclin D1 nuclear export and degradation. This process is dependent upon the activin receptor IIB and the phosphatidylinositol 3-kinase/Akt pathway but not Smad3. Insulin-like growth factor 1 (IGF-1) treatment or Akt activation attenuated the myostatin-stimulated cyclin D1 degradation as well as the associated cell proliferation repression. In contrast, attenuation of IGF-1 signaling caused C2C12 cells to undergo apoptosis in response to myostatin treatment. The observation that IGF-1 treatment increases myostatin expression through a phosphatidylinositol 3-kinase pathway suggests a possible feedback regulation between IGF-1 and myostatin. These findings uncover a novel role for myostatin in the regulation of cell growth and cell death in concert with IGF-1. Myostatin, also known as growth differentiation factor 8, or GDF-8, is a transforming growth factor-β (TGF-β) 2The abbreviations used are: TGF, transforming growth factor; Rb, retinoblastoma; PI3K, phosphatidylinositol 3-kinase; IGF, insulin-like growth factor 1; PBS, phosphate-buffered saline; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRITC, tetramethylrhodamine isothiocyanate; GST, glutathione S-transferase. 2The abbreviations used are: TGF, transforming growth factor; Rb, retinoblastoma; PI3K, phosphatidylinositol 3-kinase; IGF, insulin-like growth factor 1; PBS, phosphate-buffered saline; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRITC, tetramethylrhodamine isothiocyanate; GST, glutathione S-transferase. superfamily member and is known to be involved in regulation of skeletal muscle mass (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3107) Google Scholar). Unlike most TGF-β/GDF family members, which are expressed nonspecifically in many tissue types, myostatin is highly expressed in developing and adult skeletal muscle cells, where it plays an essential role in limiting skeletal muscle growth (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3107) Google Scholar, 2Gonzalez-Cadavid N.F. Taylor W.E. Yarasheski K. Sinha-Hikim I. Ma K. Ezzat S. Shen R. Lalani R. Asa S. Mamita M. Nair G. Arver S. Bhasin S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14938-14943Crossref PubMed Scopus (486) Google Scholar). In addition, as low levels of myostatin expression have been observed in cardiac muscle, mammary gland, adipose, and preadipose tissues, it may also serve a broader biological function (3Morissette M.R. Cook S.A. Foo S. McKoy G. Ashida N. Novikov M. Scherrer-Crosbie M. Li L. Matsui T. Brooks G. Rosenzweig A. Circ. Res. 2006; 99: 15-24Crossref PubMed Scopus (130) Google Scholar, 4Sharma M. Kambadur R. Matthews K.G. Somers W.G. Devlin G.P. Conaglen J.V. Fowke P.J. Bass J.J. J. Cell Physiol. 1999; 180: 1-9Crossref PubMed Scopus (348) Google Scholar, 5Ji S. Losinski R.L. Cornelius S.G. Frank G.R. Willis G.M. Gerrard D.E. Depreux F.F. Spurlock M.E. Am. J. Physiol. 1998; 275: R1265-R1273Crossref PubMed Google Scholar). Indeed, myostatin null mice exhibited a phenotype that was characterized by a marked hypertrophy and hyperplasia of skeletal muscle and loss of fat mass (1McPherron A.C. Lawler A.M. Lee S.J. Nature. 1997; 387: 83-90Crossref PubMed Scopus (3107) Google Scholar). Similarly, enlarged skeletal muscle mass is evident in naturally occurring mutations of myostatin in humans and cattle (6Schuelke M. Wagner K.R. Stolz L.E. Hubner C. Riebel T. Komen W. Braun T. Tobin J.F. Lee S.J. N. Engl. J. Med. 2004; 350: 2682-2688Crossref PubMed Scopus (1074) Google Scholar, 7Grobet L. Martin L.J. Poncelet D. Pirottin D. Brouwers B. Riquet J. Schoeberlein A. Dunner S. Menissier F. Massabanda J. Fries R. Hanset R. Georges M. Nat. Genet. 1997; 17: 71-74Crossref PubMed Scopus (1183) Google Scholar). Recent evidence suggests that myostatin expression and activity may be involved in the development of metabolic disorders, such as muscular dystrophy, obesity, and type II diabetes mellitus (8Gonzalez-Cadavid N.F. Bhasin S. Curr. Opin. Clin. Nutr. Metab. Care. 2004; 7: 451-457Crossref PubMed Scopus (67) Google Scholar). With the aim of utilizing the inhibitory effects of myostatin upon multiple physiological processes and ultimately achieving an improved quality of life for obese and diabetic patients, various strategies for manipulating the biological activities of myostatin are under development (9Lee S.J. Annu. Rev. Cell Dev. Biol. 2004; 20: 61-86Crossref PubMed Scopus (624) Google Scholar).Cell fate determination and proliferation of myogenic cells are critical processes in skeletal muscle formation during early embryo development. Studies have demonstrated that myostatin can inhibit the proliferation of myoblasts, satellite cells, and rhabdomyosarcoma cells. Myostatin treatment prevented myoblast progression from the G1 phase to the S phase of the cell cycle by up-regulating expression of the Cdk (cyclin-dependent kinase) inhibitor p21Waf1, Cip1 and decreasing the levels and activity of Cdk2. Also, the retinoblastoma (Rb) protein was found to be present predominantly in the hypophosphorylated form in myostatin-stimulated myoblasts (10Thomas M. Langley B. Berry C. Sharma M. Kirk S. Bass J. Kambadur R. J. Biol. Chem. 2000; 275: 40235-40243Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar). Myostatin has also been implicated in the regulation of satellite cell activation, proliferation, and self-renewal that accompanies the up-regulation of p21Waf1, Cip1 (11McCroskery S. Thomas M. Maxwell L. Sharma M. Kambadur R. J. Cell Biol. 2003; 162: 1135-1147Crossref PubMed Scopus (570) Google Scholar). However, work by Langley et al. (12Langley B. Thomas M. McFarlane C. Gilmour S. Sharma M. Kambadur R. Oncogene. 2004; 23: 524-534Crossref PubMed Scopus (47) Google Scholar) showed that myostatin treatment inhibited rhabdomyosarcoma cell proliferation through an Rb-independent mechanism. Therefore, the antiproliferative effects of myostatin could be achieved by utilization of multisignal pathways.Experiments in vitro and in vivo have implicated activin receptor IIB, TGF-β receptor I (ALK5), and activin receptor IB (ALK4) as receptors in the mediation of myostatin signals (13Lee S.J. McPherron A.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9306-9311Crossref PubMed Scopus (1251) Google Scholar). The binding of myostatin to heteromeric receptor complexes typically induces phosphorylation and activation of the associated intracellular signal transducers (Smad2/3), which then translocate into the nucleus and together with co-Smad (Smad4) regulate the expression of target genes (14Rebbapragada A. Benchabane H. Wrana J.L. Celeste A.J. Attisano L. Mol. Cell Biol. 2003; 23: 7230-7242Crossref PubMed Scopus (453) Google Scholar). In addition, it has been suggested that p38 and Akt pathways involved in myostatin signal transduction (3Morissette M.R. Cook S.A. Foo S. McKoy G. Ashida N. Novikov M. Scherrer-Crosbie M. Li L. Matsui T. Brooks G. Rosenzweig A. Circ. Res. 2006; 99: 15-24Crossref PubMed Scopus (130) Google Scholar, 15Philip B. Lu Z. Gao Y. Cell. Signal. 2005; 17: 365-375Crossref PubMed Scopus (109) Google Scholar). Recently, we have also found that the Erk1/2 mitogen-activated protein kinase pathway participated in the regulation of muscle cell growth and differentiation by myostatin (16Yang W. Chen Y. Zhang Y. Wang X. Yang N. Zhu D. Cancer Res. 2006; 66: 1320-1326Crossref PubMed Google Scholar).Cyclins are essential components of the cell cycle machinery; each binds and activates specific Cdk partner proteins. Normal progression through the G1 phase of the cell cycle requires both D- and E-type cyclins. The cyclin-Cdk complexes formed during G1 phase initiate phosphorylation of the Rb family of tumor suppressor proteins (pRb, p107, and p130) and thereby abrogate their inhibitory activity (17Pei X.H. Xiong Y. Oncogene. 2005; 24: 2787-2795Crossref PubMed Scopus (137) Google Scholar). The D-type cyclins primarily activate Cdk4 and -6, whereas cyclin E activates Cdk2. Given the crucial role that D-type cyclins play in the progression through G1 of the cell cycle, it is not surprising that their expression is frequently down-regulated in cells stimulated with anti-proliferative cytokines, such as TGF-β family members. This down-regulation generally manifests both at the level of mRNA transcription and protein stabilization. Diehl et al. (18Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1850) Google Scholar) showed that the cyclin D1 proteolysis was accelerated via the phosphatidylinositol 3-kinase (PI3K)/Akt/GSK-3β pathway. GSK-3β phosphorylates cyclin D1 at threonine 286, which triggers its nuclear export, ubiquitination, and subsequent degradation. In contrast, mitogens, such as insulin-like growth factor 1 (IGF-1), inhibit GSK-3β kinase activity and stabilize the cyclin D1 protein by activating the PI3K/Akt pathway (19von Wichert G. Haeussler U. Greten F.R. Kliche S. Dralle H. Bohm B.O. Adler G. Seufferlein T. Oncogene. 2005; 24: 1284-1289Crossref PubMed Scopus (37) Google Scholar).Although different models have been proposed to explain how myostatin causes cell cycle arrest, most studies are based on correlative results or overexpression. The mechanism underlying the function of myostatin remains unclear. In this study, we have demonstrated that myostatin augments cyclin D1 protein degradation. The PI3K/Akt/GSK-3β signaling cascade participates in myostatin-regulated cyclin D1 degradation and cell proliferation inhibition through a proteasome-dependent pathway. We report biochemical and cellular evidence indicating that these effects of myostatin on cyclin D1 expression and cell proliferation can be blocked by treatment with IGF-1, a survival and proliferation promotion factor. Most significantly, we found that blocking the IGF-1/PI3K/Akt survival pathway caused C2C12 cells to undergo apoptosis in response to myostatin. Finally, our data indicate that myostatin expression in C2C12 cells is induced by IGF-1 treatment through the PI3K/Akt pathway.EXPERIMENTAL PROCEDURESMaterials—The following antibodies and reagents were used in this study: anti-cyclin D1, anti-cyclin E, anti-Cdk2, anti-p27, anti-p21, anti-Cdk4, anti-β-actin, anti-tubulin, anti-c-Myc, and anti-hemagglutinin antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-Smad3, anti-Akt, anti-phospho-Akt (Ser473), anti-GSK-3β, and anti-phospho-GSK-3β (Ser9) antibodies and wortmannin from Cell Signaling (Beverly, MA); LiCl and MG132 from Sigma; recombinant IGF-1 and antibody against IGF-1 receptor from R&D (Minneapolis, MN). Recombinant myostatin and monoclonal antibody against myostatin were prepared as previously reported (20Yang W. Wang K. Chen Y. Zhang Y. Huang B. Zhu D.H. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao. 2003; 35: 1016-1022PubMed Google Scholar, 21Yang W. Zhang Y. Ma G. Zhao X. Chen Y. Zhu D. Biochem. Biophys. Res. Commun. 2005; 326: 660-666Crossref PubMed Scopus (32) Google Scholar).DNA Constructs—The expression vector for GST-Rb-(773–928) was generously provided by Dr. Fang Liu (State University of New Jersey). The wild type and T286A mutant cyclin D1 were constructed by using pHIT-Myc3 retroviral vector with the following primers (5′-CGG AAT TCA TGG AAC ACC AGC TCC TGT G-3′ (upper) and 5′-CCC AAG CTT TCA GAT GTC CAC GTC CCG CAC GTC GGT GGG TGT GCA AGC-3′ (lower) for the wild type; 5′-CGG AAT TCA TGG AAC ACC AGC TCC TGT G-3′ (upper) and 5′-CCC AAG CTT TCA GAT GTC CAC GTC CCG CAC GTC GGT GGG TGC GCA AGC-3′ (lower) for the T286A mutant).Cell Culture and Transfection—Mouse C2C12 cells were from ATCC and maintained in Dulbecco's modified Eagle's medium supplemented with 4.5 g/liter glucose, 4 mml-glutamine, 10% fetal bovine serum (Hyclone, Logan, UT), and penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere. Primary fibroblasts isolated from Smad3 knock-out or wild type mice were provided by Dr. Xiao Yang. The inducible Akt overexpression C2C12 cell line was generously provided by Dr. Zhenguo Wu. During exponential growth, cells were treated with myostatin at a final concentration of 500 ng/ml for the indicated time, and cells used as control were treated with the same amount of phosphate-buffered saline (PBS). For electroporation transfection, cells were rinsed with PBS and trypsinized with 1 ml of trypsin/EDTA buffer for 1–2 min at 37 °C. After centrifugation, the cells were resuspended in a 600-μl culture medium (Dulbecco's modified Eagle's medium) and incubated with plasmid DNA for 10 min at 4 °C. The cell suspension was then transferred into a 4-mm gap electroporation cuvette and electroporated at 350 V, 16 ms, 1 pulse using a Bio-Rad electroporator.Retrovirus Production and Infection—The retrovirus was produced by transient transfection of retroviral constructs into the Phoenix helper-free retrovirus producer cell line (a gift from Dr. Gary Nolan, Stanford University) using the calcium phosphate method according to the standard protocol. For the infection of C2C12 myoblasts, the retroviral supernatant (48 h after transfection) was filtered and added into each C2C12 plate, with the addition of 3 μg/ml polybrene, and the cells were incubated overnight at 37 °C for infection.Immunoblotting—For cell lysate preparation, monolayer cells on 100-mm plates were lysed with 1.2 ml of lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, 50 mm NaF, 1 mm Na3VO4, 5 mm β-glycerophosphate, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride). The lysate was clarified by centrifuging at 14,000 × g for 20 min. Boiled samples with 2× SDS loading buffer were loaded onto a 10–12% polyacrylamide gel, and, after electrophoresis, the proteins were transferred onto a polyvinylidene difluoride membrane (PALL, East Hills, NY). The resulting blots were blocked with 1% bovine serum albumin for phosphoprotein antibodies and 10% milk for nonphosphoprotein antibodies for 1 h and then incubated with the primary antibody overnight at 4 °C. The secondary antibody used in the immunoblot was a 1:3000 dilution of horseradish peroxidase-linked anti-IgG. The ECL reagent (Amersham Biosciences) was used as the substrate for detection, and the membrane was exposed to an x-ray film for visualization.Immunoprecipitation-coupled Western Blot—C2C12 cells with various treatments were lysed in a buffer containing 50 mm Tris, pH 7.5, 100 mm NaCl, 2 mm EDTA, 1% Nonidet P-40, 10 mm iodoacetamide, and protease inhibitors. This was followed by centrifugation at 14,000 × g for 10 min. The clarified lysates were immunoprecipitated with either the anti-Cdk4 antibody or the anti-β-actin antibody as negative control. Protein A/G plus agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added and washed with the lysis buffer. Samples were separated by 12% SDS-PAGE and immunoblotted with anti-cyclin D1, anti-p27, and anti-Cdk4 antibodies.Small Interfering RNA (siRNA)—The target sequences of double-stranded nucleotides used for siRNA knockdown are GGC TCA GCT CAT GAA CGA C for ActRIIb (Ambion, Austin, TX), GAA CCG AGA GCT CCA GAT C for GSK-3β (number 1) (RIBOBIO, Guangzhou, Guangdong, China), GTA ACC CCC CTC TGG CCA C for GSK-3β (number 2) (RIBOBIO), GGA CAT TGG AGG AGA AGC C for IGF-1 receptor (number 1) (RIBOBIO), TCT CAT CTT AGG AGA GGA G for IGF-1 receptor (number 2) (RIBOBIO), and ATCCAATGGCACCGTCAAG for GAPDH (RIBOBIO). Cells cultured in a 6-well plate (2 × 105 cells/well) were transfected with 70 μm siRNA with Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were treated with myostatin or PBS for 2 h. The total protein extracts from the cells were used for Western blot analysis.Proliferation Assay—About 2 × 105 cells were seeded in 6-well plates and maintained in a Dulbecco's modified Eagle's medium culture medium for retrovirus infection and myostatin stimulation. During the last 12 h of each treatment, 5 μCi of [3H]thymidine was added to the culture medium. The cells were washed three times with PBS and trypsinized for radioactivity measurement in scintillation vials. The assay was performed in three replicates and repeated three times for statistic analysis.In Vitro Kinase Assay—C2C12 cells were lysed in a buffer containing 50 mm Tris, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm dithiothreitol and protease and phosphatase inhibitors. Antibodies against Cdk4 and β-actin (negative control) were used for immunoprecipitations. The kinase assay was carried out for 1 h in a 30-μl reaction containing 50 mm HEPES, pH 7.4, 15 mm MgCl2, 1 mm EGTA, 0.1% Tween 20, 1 mm dithiothreitol, 50 mm ATP, 5 μCi of [γ-32P]ATP (3000 Ci mmol), and affinity-purified GST-Rb-(773–928) as substrates at 30 °C. The reaction mixtures were then separated in a 12% polyacrylamide gel, and proteins phosphorylated by the immunoprecipitant were visualized by autoradiography.Reverse Transcription-PCR Analysis—Total RNA was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Semiquantitative reverse transcription-PCR was performed according to standard protocol to measure the expression of p110 with the primers p110-U (5′-CCC CCA CGA ATC CTA GTG GAA TGT TTA C-3′) and p110-L (5′-TGA AAA AGC CGA AGG TCA CAA AGT CGT C-3′).Immunofluorescence—The cells were fixed with 4% (w/v) paraformaldehyde in PBS, washed three times with a permeabilization buffer (0.3% Triton X-100 in PBS) for 10 min each time, and then blocked with 3% (w/v) bovine serum albumin (Calbiochem) in PBS. The samples were incubated for 2 h at room temperature with either the anti-GSK-3β (1:100) antibody or the anti-cyclin D1 antibody and for 1 h with fluorescein isothiocyanate- or TRITC-conjugated secondary antibodies against mouse IgG (Santa Cruz Biotechnology). Finally, the cells were washed with PBS and mounted onto microscope glass slides. 4′,6-Diamidino-2-phenylindole staining was performed simultaneously to show the position of the nuclei.RESULTSMyostatin Promotes Proteasome-dependent Cyclin D1 Degradation—Myostatin has been known as an inhibitor of skeletal muscle cell proliferation. To examine the possible mechanism of myostatin-induced cell cycle arrest during the G1 phase, the level of G1 regulators was assessed in C2C12 myoblasts exposed to myostatin (Fig. 1a). The results revealed that myostatin stimulation significantly reduced the steady level of cyclin D1 protein in a time-dependent manner. Meanwhile, the level of cyclin E was slightly down-regulated by myostatin. Consistent with previous reports, myostatin also increased the level of p21 in this study. However, the expression patterns of Cdk2 and Cdk4 were not significantly altered by myostatin treatment. The rapid decrease of cyclin D1 protein suggests that the protein stability may be regulated by myostatin. Blockade of the protein degradation pathway with the proteasome inhibitor MG132 almost completely inhibited the cyclin D1 protein degradation induced by myostatin (Fig. 1b). These results suggest that the proteasome-dependent pathway mediates myostatin-stimulated cyclin D1 degradation and that the down-regulation of cyclin D1 may play a very important role during myostatin-induced proliferation suppression.Myostatin Inhibits Cdk4 Activity and Rb Phosphorylation through Down-regulation of Cyclin D1—To test whether myostatin-induced cyclin D1 protein degradation alters Cdk4 function, we assayed Cdk4 kinase activity in immunoprecipitates from C2C12 cells in the presence or absence of myostatin. Specifically, phosphorylation of exogenous GST-Rb-(773–928) was measured, and the immunoprecipitate from a nonrelated IgG was used as a negative control. Cdk4 kinase activity was readily detected in PBS-treated cells, whereas only negligible Cdk4 kinase activity was detected in myostatin-treated cells (Fig. 1c). The myostatin-induced loss of Cdk4 kinase activity due to the accelerated cyclin D1 protein degradation was further confirmed by immunoprecipitation-coupled Western blot analysis. As shown in Fig. 1d, cyclin D1 was co-immunoprecipitated with Cdk4 from the extracts of PBS-treated cells. However, in myostatin-stimulated C2C12 cells, no association of Cdk4/cyclin D1 could be detected. To verify the expression of these CDK4 and cyclin D1 proteins, input control of total cell lysate was blotted with antibodies against CDK4 and cyclin D1, respectively (Fig. 1d). These findings provide experimental evidence indicating that myostatin inhibits myoblast proliferation by inhibiting Cdk4 kinase activity via promotion of proteasomedependent cyclin D1 degradation.Attenuation of Myostatin-induced Growth Inhibition by Overexpression of Cyclin D1—To directly test whether cyclin D1 down-regulation is a determining factor in myostatin-induced cell proliferation inhibition, cyclin D1 was overexpressed in proliferating C2C12 cells by retrovirus-mediated gene transfer. A virus carrying an empty vector was used as a control. Results in Fig. 2a indicated that the levels of cyclin D1 were much higher in C2C12 cells overexpressing this gene than in the cells carrying the vector control, although myostatin still had effects on the down-regulation of overexpressed cyclin D1. The virus-transduced cells were also used in a proliferation assay employing the [3H]thymidine incorporation method. Consistent with the immunoblotting data, the cells overexpressing cyclin D1 not only grew faster than control cells transfected with the vector alone but also showed more resistance to the anti-proliferative effects of myostatin (Fig. 2b). These data provide direct evidence indicating that cyclin D1 is a crucial target for the growth-inhibitory effects of myostatin in myoblasts.FIGURE 2Attenuation of myostatin-induced growth inhibition by overexpression of cyclin D1. Immunoblots (a) and [3H]thymidine incorporation assay (b) were carried out in C2C12 cells infected with retroviruses carrying a mock vector or cyclin D1 gene to measure the effects of myostatin on cyclin D1 expression and cell proliferation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)GSK-3β Is Required for Myostatin-induced Cyclin D1 Degradation—To examine the possibility that GSK-3β may be involved in myostatin-mediated inhibition of cell proliferation, GSK-3β activity in C2C12 cells was inhibited with the chemical inhibitor lithium chloride (LiCl) in the presence or absence of myostatin. Inhibition of GSK-3β kinase activity by LiCl attenuated myostatin-induced cell proliferation suppression (Fig. 3, a and b). We then tested whether myostatin-mediated cyclin D1 degradation is also regulated by GSK-3β with the use of LiCl and synthetic siRNAs. Pretreatment of C2C12 cells with LiCl suppressed the myostatin-induced cyclin D1 degradation (Fig. 3c). A similar effect was achieved when cells were transfected with synthetic siRNAs before myostatin treatment (Fig. 3d). Compared with the GAPDH siRNA control, myostatin failed to promote cyclin D1 protein degradation in the absence of endogenous GSK-3β (Fig. 3d).FIGURE 3GSK-3β kinase activation is required for myostatin-induced cyclin D1 degradation and proliferation suppression. a, proliferating C2C12 cells were treated with PBS or 500 ng/ml myostatin for 24 h in the presence or absence of 5 mm LiCl. b, cell proliferation in various treatment conditions described in a was quantified by cell number counts. c, C2C12 cells were pretreated with 20 mm LiCl for 30 min and then treated with 500 ng/ml myostatin for 3 h. Cyclin D1 levels were then measured by Western blots. d, five siRNA oligonucleotides for GSK-3β knock-down were designed, and the most efficient two siRNAs were chosen for the experiments. The siRNA-targeting GAPDH housekeeping gene was used as the control. Forty-eight hours after transfection, the C2C12 cells were treated with myostatin or the same volume of vehicle solution (PBS) for 4 h. Protein levels of cyclin D1 and GSK-3β were assessed by Western blots. Tubulin protein served as the sample loading control. e, c-Myc-tagged wild type or T286A mutant cyclin D1 was expressed in proliferating C2C12 cells via a retroviral delivery system. The cells were then treated with 500 ng/ml myostatin or the same volume of vehicle solution (PBS) for another 3 h. The stability of wild type and T286A mutant cyclin D1 was measured by Western blots probed with anti-c-Myc antibody. f and g, C2C12 cells were plated on coverslips and incubated with myostatin (500 ng/ml) or PBS for 6 h. Immunofluorescent labeling was performed with anti-GSK-3β and anti-cyclin D1 antibodies, linked to fluorescein isothiocyanate and TRITC, respectively. Parallel experiments lacking the primary antibodies were used as negative controls. Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. The results are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The phosphorylation of cyclin D1 at threonine 286 by GSK-3β is required for its degradation (18Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1850) Google Scholar, 20Yang W. Wang K. Chen Y. Zhang Y. Huang B. Zhu D.H. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao. 2003; 35: 1016-1022PubMed Google Scholar). To further confirm the requirement of GSK-3β in cyclin D1 degradation induced by myostatin, either the Myc-tagged wild type or T286A mutant cyclin D1 was introduced into C2C12 cells using retrovirus-mediated gene transfer. We found that down-regulation of cyclin D1 induced by myostatin was threonine 286-dependent (Fig. 3e); these data provide direct biochemical evidence that GSK-3β plays a functional role in myostatin-mediated cyclin D1 protein degradation.GSK-3β has been shown to remain in the cytoplasm in an inactivated form. However, once activated, it is translocated into the nucleus to phosphorylate cyclin D1 at threonine 286, and then the phosphorylated cyclin D1 is in turn translocated from the nucleus to the cytoplasm, where the phosphorylated cyclin D1 is degraded through a proteasome-mediated pathway (18Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1850) Google Scholar). We assayed the intracellular translocation of GSK-3β and cyclin D1 proteins with immunofluorescent labeling of C2C12 cells treated with myostatin. As shown in Fig. 3f, GSK-3β was most abundant in the cytoplasm of PBS-treated control C2C12 cells but was then translocated into the nucleus after myostatin treatment. In contrast, cyclin D1 could hardly be detected in myostatin-treated C2C12 cells, presumably due to its reduced stability. Blockade of the proteasome-mediated degradation pathway with MG132 revealed that myostatin induced cyclin D1 translocation from the nuclei to the cytoplasm in C2C12 cells (Fig. 3g). By using fluorescence intensity quantification software, we assessed that over 90% of cyclin D1 exists in the nucleus in PBS treatment control. However, almost 95% of cyclin D1 was exported from nucleus to cytosol in the presence of myostatin plus MG132. Altogether, these findings provide biochemical and cellular evidence showing that GSK-3β is required for myostatin-induced cyclin D1 degradation.Myostatin Activates GSK-3β by Inhibiting the PI3K/Akt Pathway—Ser9 of GSK-3β can be phosphorylated by multiple kinases, principally from the PI3K/Akt pathway (22Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4324) Google Scholar). Therefore, to elucidate the involvement of GSK-3β upstream molecules in myostatin-regulated cyclin D1 degradation, we assessed the phosphorylation status of Akt and GSK-3β following myostatin treatment and
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