The Hypertrophic Response in C2C12 Myoblasts Recruits the G1Cell Cycle Machinery

Myint Hlaing, Xun Shen, Paul Dazin, Harold S. Bernstein
2002 Journal of Biological Chemistry  
Hypertrophy occurs in postmitotic muscle as an adaptive response to various physiological and pathological stresses. Studies in vascular smooth muscle cells and primary cardiomyocytes suggest that angiotensin II-mediated hypertrophy activates signaling pathways associated with cell proliferation. Regulation of cyclindependent kinase (Cdk)-cyclin activities is essential to cell size control in lower eukaryotes, yet their role in the hypertrophic response in muscle is incompletely understood. We
more » ... escribe an in vitro model of hypertrophy in C2C12 skeletal myoblasts and demonstrate that induction of hypertrophy involves transient activation of Cdk4, subsequent phosphorylation of Rb, and release of HDAC1 from the Rb inhibitory complex. We also demonstrate that E2F-1 becomes transcriptionally active yet remains associated with Rb. We propose a model whereby partial inactivation of the Rb complex leads to derepression of a subset of E2F-1 targets necessary for cell growth without division during hypertrophy. Hypertrophy occurs in postmitotic cardiac and skeletal muscle as a fundamental adaptive process in response to various stresses in both physiological and pathological situations. A number of studies indicate that AngII 1 acts as a hypertrophic stimulus in vascular smooth muscle cells (1) and in primary cultures of cardiomyocytes (2). Recently, AngII has been shown to be required for optimal overload-induced skeletal muscle hypertrophy (3). These observations suggest that AngII can act as a hypertrophic stimulus both in vitro and in vivo across myogenic cell types. The signaling events responsible for AngII-mediated hypertrophy have been extensively studied. AngII has been shown to induce several immediate-early genes, such as c-fos, c-jun, egr-1, and c-myc, primarily through the G protein-coupled angiotensin receptor subtype 1 in both myogenic and nonmyogenic cells (2, 4), indicating that mitogenic and hypertrophic stimuli appear to share certain intracellular responses. Cell cycle entry and G 1 progression are controlled primarily by Cdk-cyclin complexes through their actions on the E2F-1-Rb complex (5). Cdk4 and Cdk6, assembled with their regulatory subunits, the D-type cyclins, are activated in response to mitogenic stimuli, heralding cell cycle entry and G 1 progression (6). Active Cdk4/6-cyclin D1 phosphorylates Rb during early G 1 ; this leads to the up-regulation of cyclin E, its assembly with Cdk2, and activation of the Cdk2-cyclin E complex, which in turn hyperphosphorylates Rb (7) . Hyperphosphorylated Rb releases and thereby activates the transcription factor E2F-1, allowing the expression of genes necessary for DNA replication and mitosis (8) . Although regulation of cyclin-Cdk activities is essential to cell size control in lower eukaryotes (6), their role in the hypertrophic response in skeletal muscle is incompletely understood. In this study, we describe an in vitro model of muscle cell hypertrophy using C2C12 cells. We demonstrate for the first time that the hypertrophic response in these cells involves the transient activation of Cdk4, but not Cdk2, with subsequent phosphorylation of Rb, release of HDAC1 from the Rb inhibitory complex, and activation of the transcription factor, E2F-1. We propose a model by which partial inactivation of the Rb complex leads to the derepression of a subset of E2F targets necessary for cell growth during hypertrophy. EXPERIMENTAL PROCEDURES Antibodies-Monoclonal antibodies to cyclin D1, cyclin D3, p21 Waf1/Cip1 , p27 Kip1 , and E2F-1, and polyclonal antibodies against cyclin E Cdk2 , Cdk4, and HDAC1 were obtained from Santa Cruz Biotechnology. Monoclonal antibody against Rb, which recognizes hypophosphorylated and hyperphosphorylated Rb, was obtained from Pharmingen. Polyclonal phospho-Rb (S780) antibody was obtained from Cell Signaling Technology. Cell Culture-Actively growing C2C12 skeletal myoblasts (ATCC) were maintained in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum (Invitrogen). The cells were made quiescent by serum withdrawal for 48 h and stimulated to proliferate by adding 20% fetal bovine serum. Myogenic differentiation was induced in subconfluent cultures by the addition of 2% horse serum. Cellular hypertrophy was stimulated with AngII (100 nM; Sigma). DNA and Protein Synthesis Assays-Quiescent cells were stimulated with AngII or 20% dialyzed fetal bovine serum for 24 h. The cells were pulse-labeled for 2 h with 2 Ci/ml [ 3 H]thymidine to measure DNA synthesis or 1 Ci/ml [ 3 H]leucine (PerkinElmer Life Sciences) to measure protein synthesis and then suspended in cold 10% trichloroacetic acid for 30 min at 4°C. The trichloroacetic acidprecipitable material was washed with cold 5% trichloroacetic acid, cold 70% ethanol, and deionized water and then solubilized in 0.1 M NaOH. [ 3 H]Thymidine and [ 3 H]leucine incorporation was measured by liquid scintillography. Flow Cytometry-All of the cultures were analyzed after 24 h of treatment, as described previously (9). Briefly, 10 6 cells were stained with propidium iodide to exclude dead cells from evaluation and Hoechst 33342 (Molecular Probes) to measure DNA content in living cells. Flow cytometry was performed using a Becton Dickinson VANTAGE SE with dual argon ion lasers at 488-and 363-nm light output. Propidium iodide and Hoechst signals were acquired using 630/22-and 457/10-mm band pass filters, respectively. All of the analyses of DNA
doi:10.1074/jbc.m201980200 pmid:11967266 fatcat:w4amnfuwhfauzma6drocaq225e