The Hypertrophic Response in C2C12 Myoblasts Recruits the G1 Cell Cycle Machinery*

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 cyclin-dependent 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 describe 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 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 describe 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.
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 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 content were performed on 100,000 collected events using CellQuest software (Becton Dickinson).
Cyclin-dependent Kinase Assays-Cdk2 and Cdk4 activities were measured by kinase assay using histone H1 (Roche) or GST-Rb (amino acids 769 -921; Santa Cruz Biotechnology) as substrates using procedures modified from standard methods (11). Cdk2 or Cdk4 was immunoprecipitated as described above. The immune complexes were washed three times with lysis buffer and three times with kinase buffer (25 mM HEPES, pH 7.4, 10 mM MgCl 2 , 1 mM dithiothreitol). Cdk2 activity was assayed by resuspending immune complexes in 40 l of kinase buffer containing 30 M ATP, 5 Ci of [␥-32 P]ATP, 5 g of histone H1 (Roche) and incubating for 30 min at 30°C. The reactions were stopped with sample buffer and heating at 85°C for 5 min. Cdk4 activity was assayed using the same procedure with 1 g of GST-Rb as substrate. Phosphorylated histone H1 or Rb was resolved by gradient SDS-PAGE and analyzed by autoradiography. Cdk4 activity was quantitated by liquid scintillography of [␥-32 P]Rb excised from the gel.
Transcriptional Reporter Assay-E2F4B-Luc (12) containing four consensus E2F-binding sites and E1B TATA upstream of Photinus pyralis luciferase was provided by F. Dick and N. Dyson (Massachusetts General Hospital Cancer Center). E2F1-Luc (13) containing the human E2F-1 promoter fused to P. pyralis luciferase was provided by W. Kaelin (Dana-Farber Cancer Institute). C2C12 cells were transiently transfected using LipofectAMINE PLUS (Invitrogen) according to the manufacturer's instructions, and luciferase activity was assayed in whole cell lysates using the dual luciferase reporter assay system (Promega) as described previously (14). After recovering from transfection, the cells were quiesced by serum withdrawal and then stimulated with 100 nM AngII or 10% fetal bovine serum. pRL-TK (Promega), encoding Renilla reniformis luciferase downstream of a herpes thymidine kinase promoter, was included in each transfection for normalization of transfection efficiency.

RESULTS AND DISCUSSION
AngII Stimulates Hypertrophy and G 1 Arrest in C2C12 Myoblasts-AngII has been shown to induce hypertrophy in cardiac myocytes and vascular smooth muscle cells (1, 2); however, its effect on skeletal muscle growth has not been demonstrated. To determine whether AngII stimulates hypertrophy in C2C12 myoblasts, we treated quiescent cells with AngII (100 nM) in serum-free medium. Protein synthesis, as measured by [ 3 H]leucine incorporation, increased 2.6-fold with AngII stimulation compared with quiescent cells, and 1.4-fold compared with proliferating cells (Fig. 1A). AngII had no significant effect, however, on [ 3 H]thymidine incorporation, an indicator of DNA synthesis (Fig. 1A). As a result, the [ 3 H]leucine/[ 3 H]thymidine ratio, an indicator of cellular hypertrophy, increased from 1.6 in serum-induced proliferating cells or 2.2 in quiescent myocytes to 5.0 in AngII-stimulated cells undergoing hypertrophy.
We also measured relative cell size by flow cytometry using forward angle light scatter. This analysis demonstrated a 12% increase in cell size in AngII-stimulated cells compared with quiescent cells (Fig. 1B), similar to the increase seen in AngIIstimulated vascular smooth muscle cells (15). Flow cytometry of Hoechst-stained myocytes also showed that AngII-stimu-lated cells were arrested in the G 0 /G 1 phase of the cell cycle (Fig. 1C). This was similar to results observed for both quiescent myoblasts and differentiating myocytes. Taken together, these findings suggest that AngII stimulates hypertrophy by increasing protein synthesis in the absence of cell division.
Cdk4, but Not Cdk2, Is Active in C2C12 Myoblasts Undergoing Hypertrophy-Because cell cycle re-entry and G 1 /S transit in proliferative cells commences with assembly and activation of Cdk4/6-cyclin D and subsequent activation of Cdk2cyclin E, we studied the effect of AngII stimulation on G 1 cyclin expression and Cdk activity in C2C12 cells. AngII had an up-regulatory effect on cyclin D1, whereas cyclin D3 was downregulated in AngII-stimulated cells, compared with quiescent cells (Fig. 2A). In differentiating C2C12 cells, cyclin D1 expression was repressed, whereas cyclin D3 levels were elevated, compared with proliferating cells (Fig. 2A). These findings are consistent with previous observations that cyclin D1 is a mitogen sensor and the limiting factor in the assembly of active Cdk4 complexes necessary for G 1 /S transit (16), whereas cyclin D3 contributes to the irreversible withdrawal of postmitotic cells from the cell cycle and is critical in the maintenance of a differentiated phenotype (17).
Although D-type cyclins control Cdk4/6 activity in early G 1 , cyclin E regulates Cdk2 activity near the G 1 /S transition (18). Cyclin E was repressed in differentiating cells compared with proliferating cells; however, we observed up-regulation of cyclin E expression in AngII-stimulated cells ( Fig. 2A). Because Cdk activities are controlled by association with regulatory cyclins and not at the level of expression, we were not surprised to find equivalent levels of Cdk2 or Cdk4 protein in AngIIstimulated cells compared with quiescent, proliferating, and differentiating cells (Fig. 2A). G 1 cyclin expression was induced in AngII-stimulated cells, suggesting cell cycle re-entry. Because these cells arrested in G 1 , however, we hypothesized that only Cdk4-cyclin D1 would be active during the hypertrophic response, because Cdk2 activity is associated with the G 1 /S transition. To test this, we measured Cdk activities in C2C12 cells following AngII stimulation. Cdk4 and Cdk2 were immunoprecipitated from cell lysate using anti-Cdk4 or anti-CDk2 antibody, and kinase activity was determined using GST-Rb and histone H1 as substrate, respectively. Cdk4 and Cdk2 activities were increased in proliferating myocytes and not in AngII-stimulated cells after 24 h (Fig. 2B). To determine whether kinase activities might be transient, we assayed Cdk4 and Cdk2 activities at 8, 12, 16, 20, and 24 h after stimulation with AngII. Although Cdk2 activity was not detected at any of these earlier time points (data not shown), a burst of Cdk4 activity was observed at 12 h (Fig. 2C). These results demonstrate an early increase in Cdk4 activity following AngII stimulation. The lack of Cdk2 activity in AngII-treated cells is consistent with their failure to progress through the G 1 -S transition.
Levels of Cdk4-bound Cdk Inhibitors, p21 Waf1/Cip1 and p27 Kip1 , Are Unchanged in Hypertrophic C2C12 Myoblasts-G 1 progression is controlled through Cdk activity, and this in turn is regulated in part by CKIs such as p21 Waf1/Cip1 and p27 Kip1 (19). To further examine the mechanism by which C2C12 cells recruit the cell cycle machinery but remain arrested in G 1 during the hypertrophic response, we determined the levels of p21 Waf1/Cip1 and p27 Kip1 expression following AngII treatment (Fig. 3A). As expected, levels of both proteins were down-regulated in proliferating myoblasts and up-regulated in differentiating myotubes, compared with quiescent cells. Expression of both proteins was elevated in AngII-treated myoblasts compared with quiescent cells, at levels similar to those observed in differentiating myotubes. Other investigators have observed elevated p27 Kip1 levels in AngII-stimulated, vascular smooth muscle cells, resulting in diminished Cdk2 activity and hypertrophy (15). In addition, up-regulation of p21 Waf1/Cip1 and p27 Kip1 has been observed in the mesangial cell hypertrophic response to high glucose (20,21). Our results are consistent with those reported for other cell types and extend a general role for these G 1 -active CKIs in the response to hypertrophic stimuli.
Although increased expression of CKIs may suggest a role for these proteins in AngII-stimulated hypertrophy in C2C12 cells, their effects as cell cycle regulators are delivered through interaction with Cdk complexes. To correlate the increased p21 Waf1/Cip1 and p27 Kip1 levels that we had observed in response to AngII with a downstream effect on Cdk4 activity and G 1 progression, we next measured their association with the Cdk4-cyclin D complex. We observed co-immunoprecipitation of p21 Waf1/Cip1 with Cdk4 complexes in differentiating cells, and this association appeared to be decreased in proliferating cells, as expected. Surprisingly, the p21 Waf1/Cip1 -Cdk4 interaction was not significantly disrupted in AngII-stimulated cells, compared with proliferating cells (Fig. 3B).
Our observation that Cdk4 activity is low or absent in differentiating cells and elevated in proliferating cells and at 12 h following AngII stimulation suggests that p21 Waf1/Cip1 may function through several mechanisms in the same cell line. Although p21 Waf1/Cip1 appears to associate with inactive Cdk4 complexes in differentiating and quiescent C2C12 cells, it also interacts with active Cdk4 in C2C12 cells responding to hypertrophic stimuli. It has been shown in other systems that despite its initial description as a Cdk inhibitor, p21 Waf1/Cip1 may be found in active Cdk complexes and may facilitate the assembly of active Cdk4-cyclin D1 complexes in particular (22). Our data suggest the possibility that both activities of p21 Waf1/Cip1 are present in skeletal muscle myoblasts responding to different stimuli. Although the interaction between p21 Waf1/Cip1 and Cdk4 in differentiating cells may be inhibitory, p21 Waf1/Cip1 also may stabilize Cdk4-cyclin D1 activity in cells undergoing hypertrophy, allowing for the brief but nonsustained Cdk4 activity observed.
In contrast, p27 Kip1 co-immunoprecipitated with inactive Cdk2 in differentiating and AngII-treated cells compared with proliferating C2C12 myoblasts (data not shown). This suggests that p27 Kip1 functions primarily as a Cdk2 inhibitor at the G 1 /S transition in hypertrophic C2C12 myocytes.
Rb Is Hyperphosphorylated in Hypertrophic C2C12 Myoblasts-Rb is an important in vivo substrate of Cdk4-cyclin D1. As a critical regulator of the G 1 /S transition of the cell cycle, Rb can exist in hypophosphorylated and hyperphosphorylated forms (23). Although the active, hypophosphorylated form binds to and inhibits E2F transcription factors required for expression of genes involved in DNA synthesis, the inactive, hyperphosphorylated form releases E2F, allowing for its activation (24). To determine whether the transient Cdk4 activity observed in cells undergoing hypertrophy inactivates Rb, we examined Rb phosphorylation in AngII-stimulated cells. Whereas hypophosphorylated (active) Rb persisted in quiescent myoblasts and differentiated myotubes, Rb phosphorylated on serine 780, which has been shown to be preferentially phosphorylated by Cdk4-cyclin D1 (25), was detected in both proliferating cells and in hypertrophic cells stimulated with AngII (Fig. 4A). Hyperphosphorylated Rb similarly was de-tected in C2C12 myoblasts treated with other known hypertrophic stimuli, including prostaglandin F 2␣ and 1,25-dihydroxyvitamin D3 (data not shown). These results suggest that Rb is inactivated by Cdk4 phosphorylation during the hypertrophic response in C2C12 cells and that this occurs regardless of the initial hypertrophic stimulus.
Rb Hyperphosphorylation by Cdk4-Cyclin D Is Associated with the Release of HDAC1-Rb plays a fundamental role in cell cycle progression through its association with the E2F FIG. 2. A transient increase in Cdk4 activity accompanies the hypertrophic response in AngII-stimulated C2C12 cells. The cells were treated as described in the legend to Fig. 1. A, cyclin and Cdk expression. The cell lysates were separated by SDS-PAGE and analyzed with indicated antibodies by immunoblot. A representative experiment is shown (n ϭ 3). Cyclins D1 and E were up-regulated, cyclin D3 was down-regulated, and G 1 Cdk levels were unchanged in AngII-stimulated cells, compared with differentiating or quiescent cells. B, Cdk activities with AngII stimulation. Cdk2 and Cdk4 were immunoprecipitated from cell lysates, and kinase activities were measured using histone H1 and GST-Rb as Cdk2 and Cdk4 substrates, respectively. 32 P-Labeled substrates were separated by SDS-PAGE and analyzed by autoradiography. A representative experiment is shown (n ϭ 3). Cdk2 and Cdk4 are active only in proliferating cells at this time point. C, Cdk4 activity over 24 h of AngII stimulation. Cdk4 activity was assayed at indicated time points (top panel). Kinase activity was quantitated as picomoles of 32 P incorporated/min/mg substrate (bottom panel). The data shown are the means Ϯ S.E. (n ϭ 3). A transient, ϳ3-fold increase in Cdk4 activity was measured following AngII stimulation for 12 h.
FIG. 3. The p21 Waf1/Cip1 -Cdk4 association is maintained in AngII-stimulated C2C12 myoblasts. The cells were treated as described in the legend to Fig. 1. A, CKI expression with AngII stimulation. Cell lysates were separated by SDS-PAGE and analyzed with the indicated antibodies by immunoblot. A representative experiment is shown (n ϭ 3). The levels of p21 Waf1/Cip1 and p27 Kip1 proteins were elevated in AngII-treated cells. B, CKI-Cdk4 association with AngII stimulation. Cdk4 was immunoprecipitated (IP) from cell lysates. The complexes were separated by SDS-PAGE and analyzed with antibodies against p27 Kip1 or p21 Waf1/Cip1 by immunoblot. Although the association between p21 Waf1/Cip1 and Cdk4 was increased in differentiating and quiescent (0h) cells, compared with proliferating cells, there was no significant change in the p21 Waf1/Cip1 -Cdk4 association from 0 h (inactive Cdk4) to 12 h (active Cdk4) of AngII treatment.

FIG. 4. Rb is phosphorylated and HDAC1 is released from the
Rb complex in AngII-treated C2C12 myoblasts. The cells were treated as described in the legend to Fig. 1. A, Rb is phosphorylated at S780. Rb was immunoprecipitated (IP) from cell lysates, separated by SDS-PAGE, and detected by immunoblot using an anti-Rb antibody that recognizes both hypophosphorylated and hyperphosphorylated Rb (top panel), or a monoclonal antibody directed against Rb phosphorylated at Ser 780 (bottom panel). A representative experiment is shown (n ϭ 3). Hyperphosphorylated Rb (ppRb) was readily detected in proliferating cells, as well as in cells treated with AngII by 12 h, compared with quiescent (0h) and differentiated cells, in which mostly hypophosphorylated Rb (pRb) was observed (top panel). This pattern of phosphorylation specifically was detected at Ser 780 , a preferred site for Cdk4cyclin D1 (bottom panel). B, HDAC1 is released from the Rb complex. HDAC1 was immunoprecipitated from cell lysates after 12 h. The complexes were separated by SDS-PAGE and analyzed by immunoblot with anti-Rb antibody. The HDAC1-Rb interaction was markedly reduced in AngII-treated cells.
family of transcription factors (5). These in turn regulate expression of genes important for G 1 /S transition and DNA synthesis (26). Rb represses transcription from promoters containing E2F binding sites by complexing with HDAC enzymes (27)(28)(29) and SWI/SNF nucleosome remodeling factors (30). The first molecular event for cell cycle re-entry caused by mitogenic stimuli in nonmyogenic, proliferating cells is activation of Cdk4/6-cyclin D1 followed by initial phosphorylation of Rb by activated Cdk complexes. Phosphorylation of Rb displaces HDAC1 from the inhibitory HDAC1-Rb-SWI/SNF complex (31). This partial disruption of the Rb inhibitory complex relieves repression of the cyclin E gene (32). Cyclin E expression and assembly with Cdk2 lead to formation of active Cdk2-cyclin E complexes, which hyperphosphorylate Rb, further disrupting the inhibitory complex (24). This leads to the complete release of E2F and transcriptional activation of a wide variety of E2F target genes necessary for DNA replication and mitosis (8). The association between Rb and HDAC1 recently has been shown to coincide with myogenic differentiation and myocyte cell cycle withdrawal (33). Because of this and our observation of Cdk4 activity (Fig. 2C) without subsequent Cdk2 activity and Rb hyperphosphorylation (Fig. 4A) in C2C12 cells undergoing hypertrophy, we wanted to determine the downstream effect of this transient Cdk4 activity on HDAC1 association with the Rb inhibitory complex. To accomplish this, we performed co-immunoprecipitation experiments of Rb with HDAC1 in AngIItreated C2C12 cells (Fig. 4B). Although Rb co-immunoprecipitated with HDAC1 in quiescent and differentiating C2C12 cells, indicating active repression by the Rb inhibitory complex, the amount of HDAC1 associated with the Rb complex in AngII-treated cells was markedly reduced. These data show for the first time that HDAC1 is released upon phosphorylation of Rb by Cdk4-cyclin D complexes in myogenic cells during the hypertrophic response.
E2F-1 Remains Associated with Rb but Is Transcriptionally Active in Hypertrophic C2C12 Cells-Although AngII-stimulated cells arrested in G 1 (Fig. 1C) and did not demonstrate Cdk2 activity, Rb phosphorylation coincident with Cdk4 activity was observed (Figs. 4A and 2C). In addition, HDAC1 was partially released from the Rb inhibitory complex (Fig. 4B), and cyclin E expression was induced ( Fig. 2A). This suggested that E2F-1, which regulates cyclin E expression (32) and is regulated by the Rb complex, may be active at a subset of target promoters during the hypertrophic response. To test this, we first examined whether E2F-1 was released from Rb upon AngII stimulation. Surprisingly, co-immunoprecipitation of E2F-1 with Rb in proliferating and AngII-stimulated cells demonstrated that E2F-1 remained associated with Rb after AngII stimulation, although perhaps to a lesser extent than in quiescent, unstimulated cells (Fig. 5A).
To determine whether E2F-1 in complex with Rb but after HDAC1 release was transcriptionally active, we performed reporter assays in AngII-stimulated C2C12 cells. We used one reporter containing four tandem repeats of the E2F-1 binding site (E2F4B-luc) to examine E2F-1 DNA binding (12) and another containing the entire E2F-1 promoter (E2F1-luc) to evaluate transcriptional activation (13), as E2F-1 positively regulates its own expression. With both reporters, we observed a modest but significant increase (ϳ3-fold) in activity in C2C12 cells stimulated with AngII for 12 h (Fig. 5B), compared with unstimulated, quiescent cells. Although this represents only one-third of the E2F-1 activity seen in proliferating cells, it coincided with the increase in cyclin E expression (Fig. 5B), as well as the peak of Cdk4 activity (Fig. 2C), and Rb phosphorylation (Fig. 4A) also observed. These data demonstrate for the first time that during the hypertrophic response, E2F-1 is FIG. 5. E2F-1 becomes transcriptionally active in AngII-stimulated C2C12 cells. The cells were treated as described in the legend to Fig. 1. A, E2F-1 remains associated with Rb. Rb was immunoprecipitated (IP) from cell lysates. The complexes were separated by SDS-PAGE and analyzed by immunoblot with anti-E2F-1 or anti-Rb antibodies. There was a modest decrease in Rb-associated E2F-1 after 12 h of AngII stimulation, although the association persisted at levels higher than in proliferating cells. B, E2F-1 becomes transcriptionally active. The cells were transfected with luciferase reporter plasmids containing four tandem repeats of the E2F-1 binding site (E2F4B-luc) or the E2F-1 promoter (E2F1-luc). Cyclin E expression was measured as described in the legend to Fig. 2. A ϳ3-fold increase in E2F-1 reporter activity was observed in AngII-stimulated cells at 12 h (top panel), coincident with an increase in cyclin E expression (bottom panels). transcriptionally active without Rb hyperphosphorylation by Cdk2 or complete release from the Rb inhibitory complex.
The G 1 Cell Cycle Machinery Plays an Important Role in Skeletal Muscle Cell Hypertrophy-We have shown that AngII stimulation of C2C12 cells simulates the hypertrophic cell growth and G 1 arrest observed in primary cardiomyocytes and vascular smooth muscle cells. We have used this model system to elucidate the mechanisms by which the G 1 cell cycle regulatory apparatus controls the hypertrophic response in myoblasts. The early G 1 Cdk4-cyclin D1 complex is transiently activated in response to AngII in C2C12 cells, whereas the later G 1 Cdk2-cyclin E complex is not. Although p27 Kip1 is associated with inactive Cdk2 during this process, p21 Waf1/Cip1 may have two functions in these cells: inhibiting Cdk4 activity in differentiating cells and permitting a transient burst of Cdk4 activity during hypertrophy, perhaps through stability effects. Coincident with peak Cdk4 activity, Rb is hyperphosphorylated, resulting in the release of HDAC1 from the Rb inhibitory complex. Although E2F-1 remains associated with Rb, it becomes transcriptionally active, inducing the expression of at least one target gene, cyclin E.
Based on these observations, we propose a model in which hypertrophy results from the derepression of a subset of E2F-1 targets necessary for cell growth during the hypertrophic response (Fig. 6). The identification of this subset of E2F-1regulated genes and other components of the Rb complex that also may mediate partial E2F-1 activity will further validate such a model and provide potential targets for manipulating the hypertrophy response in muscle tissues.