©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Positive and Negative Regulation of D-type Cyclin Expression in Skeletal Myoblasts by Basic Fibroblast Growth Factor and Transforming Growth Factor
A ROLE FOR CYCLIN D1 IN CONTROL OF MYOBLAST DIFFERENTIATION (*)

(Received for publication, August 5, 1994; and in revised form, November 18, 1994)

Sunkara S. Rao D. Stave Kohtz

From the Department of Pathology(1194), Mount Sinai School of Medicine, New York, New York 10029

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Differentiation of skeletal myoblasts in culture is negatively regulated by certain growth factors, including basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGFbeta). We investigated the effects of bFGF and TGFbeta on D-type cyclin expression in skeletal myoblasts. When myoblasts were induced to differentiate in low mitogen medium, expression of cyclin D1 rapidly fell below detectable levels. In contrast, expression of cyclin D3 increased to levels exceeding those present in myoblasts. Expression of cyclin D1 was induced in myoblasts by bFGF and TGFbeta (albeit with different kinetics for each factor), while induction of cyclin D3 expression was inhibited by these growth factors. Although these results are consistent with other reports showing induction of cyclin D1 by growth factors, induction of cyclin D3 expression during terminal differentiation of myoblasts and inhibition of this induction by growth factors is surprising. These results suggest that cyclin D3, previously thought to be only a positive regulator of cell cycle progression, may also function in the cellular context of terminal differentiated muscle. Stable expression of cyclin D1 from an ectopic viral promoter inhibits C2C12 myoblast differentiation, but only in those clones where the level of cyclin D1 expression does not significantly exceed that present in control myoblasts stimulated by bFGF. Together, these result suggest that cyclin D1 expression functions in the inhibition of myoblast differentiation by certain growth factors.


INTRODUCTION

Growth and differentiation of skeletal myoblasts in culture is regulated by polypeptide growth factors. The responses of skeletal myoblasts to two of these factors, basic fibroblast growth factor (bFGF) (^1)(Lathrop et al., 1985; Spizz et al., 1986; Clegg et al., 1987) and transforming growth factor beta (TGFbeta) (Olson et al., 1986, Massague et al., 1986) have been well characterized. Both bFGF and TGFbeta inhibit myoblast differentiation, and some studies have suggested that this activity is not directly linked to their actions as mitogens (Spizz et al., 1986; Clegg et al., 1987). A central issue in understanding the mechanism by which specific growth factors inhibit myoblast differentiation involves their specific effects on the activity of the myogenic basic helix-loop-helix (bHLH) regulators (reviewed by Weintraub et al.(1991) and Olson (1992)). The myogenic (bHLH) regulators, also referred to as the myoD family, are muscle-specific transcriptional activators that were originally identified by their ability, when expressed ectopically, to transdetermine fibroblasts to the myogenic phenotype (Davis et al., 1987). Four myoD family members have been identified (myoD, myogenin, myf-5, and MRF4/myf-6/herculin; Davis et al. (1987), Wright et al.(1989), Braun et al. (1989), Rhodes and Konieczny(1989), Braun et al.(1990), and Miner and Wold(1990)), and these share a common structural motif, the bHLH, that mediates dimer assembly and site-specific binding to DNA. Post-translation mechanisms that control myogenic bHLH regulator activity operate either by abolishing the DNA binding activity of these regulators or by impeding their ability to activate transcription of muscle genes at stages downstream from DNA binding. The former mechanism functions in myoblasts responding to bFGF, which abrogates of the DNA binding activity of myogenic bHLH regulators by inducing phosphorylation of the basic region by protein kinase C (Li et al., 1992). In contrast, myogenic bHLH regulators in myoblasts blocked from differentiation by TGFbeta appear to retain DNA binding activity, but do not activate transcription of muscle genes (Brennan et al., 1991). The mechanisms that control myogenic bHLH regulator activity without affecting their ability to bind DNA, however, are not well understood.

Myoblast differentiation is restricted to a specific window in the G(1) phase of the cell cycle; after mitogen withdrawal, myoblasts outside of this window will either not differentiate, or continue to cycle until reaching this window, and then differentiate (reviewed in Olson(1992)). Specific parameters of cell cycle progression are apparently linked to the regulation of myogenic differentiation. The retinoblastoma protein (pRb; reviewed by Weinberg (1991), Marshall(1991), and Goodrich et al.(1991)) physically associates with the HLH subdomain of myoD family members, and the significance of this interaction is 2-fold. 1) Expression of myogenic bHLH regulators in some cells results in G(1) arrest, which may be mediated by binding of these regulators to pRb (Crescenzi et al., 1990; Sorrentino et al., 1990); and 2) myogenic bHLH regulators do not activate muscle gene transcription in SAOS-2 cells, which express only defective pRb (Gu et al., 1993), suggesting that association with pRb may be important to the function of myoD family members as transcriptional activators. Activation of cyclin-dependent kinases (cdk) has also been associated with repression of muscle gene transcription. C2C12 myoblasts expressing a temperature-sensitive mutant of SV40 T-antigen differentiate at the non-permissive temperature (T-antigen is inactive), but differentiated myotubes lose the muscle phenotype and re-enter the cell cycle when cultured at the permissive temperature. One of the initial events associated with loss of the myogenic phenotype in these cells is activation of cdk activity (Gu et al., 1993). Phosphorylation of histone H1 on at least one cdk site is abolished in differentiated myotubes (Cole et al., 1993). (^2)This was demonstrated in situ using a monoclonal antibody that reacts with an epitope on histone H1 only when it is phosphorylated by a cdk. Loss of histone H1 phosphorylation at this epitope was shown to be directly associated with the differentiation of myocytes rather than the acquisition of a quiescent state by undifferentiated mononuclear cells (Cole et al., 1993).

Cyclins are the regulatory subunits of the cdks, and their expression is modulated according to type during cell cycle progression. The activities of various cyclin-cdk complexes regulate progress through and transitions between phases of the cell cycle. Cyclins A and B are expressed at peak levels during S/G(2) and G(2)/M, respectively, and control the progress of events during S, G(2), and M phases of the cell cycle (Minshull et al., 1990; Pines and Hunter, 1990; Norbury and Nurse, 1992). The G(1) cyclins (cyclins C, E, and D-type cyclins) are expressed during the G(1) phase of the cell cycle, and control progress through that phase and the transition to S phase (Baldin et al., 1993; reviewed by Sherr(1993)). D-type cyclins are unique among reported cyclins in that their expression in some cell types is modulated by growth factor signals (Xiong et al., 1991; Matsushime et al., 1991a, 1991b; Inaba et al., 1992). Expression of D-type cyclins is thought to translate mitogenic signals transduced from growth factors into the language of regulators that control cell cycle progression. The presence of certain D-type cyclins may promote cell cycle progression in specific cell types, and their absence may signal an exit from the cell cycle (Cocks et al., 1992). In some cell types, such as myoblasts and myeloid cells, inducing differentiation and exiting the cell cycle are closely coupled. Indeed, ectopic expression of cyclin D2 and D3 inhibits differentiation of 32D myeloid cells into granulocytes (Kato and Sherr, 1993), while ectopic expression of cyclin D1 inhibits activation of muscle-specific gene transcription by myogenic basic helix-loop-helix regulators (Rao et al., 1994). In order to better understand the role of D-type cyclin expression in the regulation of differentiation by growth factors, we have studied the expression of cyclins D1 and D3 in differentiating myoblasts and in myoblasts blocked from differentiation by bFGF and TGFbeta. In addition, we show that ectopic expression of cyclin D1 at levels equivalent to those expressed in cells stimulated by bFGF inhibits differentiation of C2C12 myoblasts.


MATERIALS AND METHODS

Growth of Cultures

C2C12 myoblasts (Blau et al., 1983) were routinely passaged in high mitogen medium (Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum (HyClone), 0.5% chick embryo extract (Life Technologies, Inc.), minimal essential medium nonessential amino acids (Life Technologies, Inc.), minimal essential medium vitamins (Life Technologies, Inc.), 1 mM sodium pyruvate, and 100 µg/ml gentamicin). To induce differentiation, myoblasts were cultured in low mitogen medium (Dulbecco's modified Eagle's medium supplemented with 2-4% horse serum, minimal essential medium nonessential amino acids (Life Technologies, Inc.), minimal essential medium vitamins (Life Technologies, Inc.), 1 mM sodium pyruvate, and 100 µg/ml gentamicin). Subclone 10 of C2C12 cells (C2C12-10) was generated by plating C2C12 cells (acquired from ATCC) at clonal density for 14 days, removing colonies with cloning cylinders, and testing each clone for inhibition of differentiation by bFGF or TGFbeta. TGFbeta (R& Systems) was used at a concentration of 5 ng/ml. bFGF (Collaborative Biomedical Research) was used at a concentration of 20 ng/ml.

Northern Blot Analyses

Total cellular RNA was isolated from cultured cells by guanidinium/isothiocyanate denaturation and cesium chloride density centrifugation as described previously (Chirgwin et al., 1979). Messenger RNA was enriched by oligo(dT)-cellulose affinity chromatography and resolved by formaldehyde gel electrophoresis as described (Sambrook et al., 1989). The mRNA was transferred and cross-linked to GeneScreen Plus (DuPont NEN) using the protocol provided by the manufacturer. Blots were baked under vacuum at 80 °C for 2 h to reverse the formaldehyde reaction. Prehybridization and hybridization mixes contained 2 times SSC (1 times SSC is 0.15 M NaCl, 15 mM trisodium citrate, pH 7.0), 2% sodium dodecyl sulfate, 10% dextran sulfate, 50% formamide, 1 mg/ml sonicated denatured salmon sperm DNA, 100 µg/ml poly(A), 100 µg/ml yeast transfer RNA). Prehybridization was performed for 6 h at 51 °C, followed by an 18-h hybridization at 51 °C with 10^6 dpm/ml probe. The blots were washed twice with 2 times SSC, 1% SDS (55 °C, 30 min), then once with 0.2 times SSC (50 °C, 30 min). Autoradiography was performed with Kodak X-AR5 film and DuPont intensifying screens. Bands were quantified with either a PhosphorImager (Molecular Dynamics) or Microdensitometry Scanner (LDK).

The cDNA clones for cyclin D1 (Motokura et al., 1991) and cyclin D3 (Lew et al., 1991; Hinds et al., 1992) were obtained in pRc-CMV (Invitrogen) from Dr. Phil Hinds (Whitehead Institute), Dr. Robert Weinberg (Whitehead Institute), and Dr. Andrew Arnold (Harvard Medical School). The cDNA fragments were excised by HindIII and XbaI digestions, and purified by agarose gel electrophoresis. The plasmid cM113aR containing the mouse fast troponin I gene (Koppe et al., 1989) was obtained from Dr. Steve Koznieczny (Purdue University). A plasmid containing the cDNA for rat glyceraldehyde-3-phosphate dehydrogenase (Piechaczyk et al., 1984) was obtained from Dr. Jim Bieker (Mount Sinai School of Medicine). Radiolabeled probes were generated by the random hexamer priming method using a kit (New England Biolabs).

Western Blot Analyses

Myoblast cultures grown on T25 flasks were washed three times with phosphate-buffered saline (PBS), then lysed directly on the plates with sample buffer (100 mM Tris, pH 6.5, 2% SDS, 5% beta-mercaptoethanol, 10% glycerol, 0.025% phenol red). Lysates were heated to 100 °C prior to gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed according to a standard protocol, and proteins were transferred electrophoretically to nitrocellulose and blotted as described in detail elsewhere (Cole et al., 1993). A rabbit polyclonal cyclin D1 antibody and mouse monoclonal cyclin D3 antibody were obtained from Pharmingen. Hybridoma cells secreting mouse monoclonal sMHC antibody MF-20 (Bader et al., 1982) were obtained from the Developmental Studies Hybridoma Bank. I-Coupled anti-rabbit and anti-mouse antibodies were obtained from DuPont NEN.

Immunofluorescence Microscopy

Myoblasts to be used for immunofluorescence analysis were grown on 30-mm culture plates. The cells were washed 3 times with PBS (ambient temperature), then fixed with 100% methanol (-20 °C, 10 min). The cells were rehydrated in PBS (ambient temperature), then incubated with PBS containing 5% horse serum for 30 min (37 °C). Primary antibody was used at a concentration of 1:30 in PBS containing 5% horse serum, and was incubated on the cells in a humidified chamber for 1 h (37 °C). After 3 washes with PBS, the cells were incubated with a fluorescein-conjugated anti-mouse IgG/IgM antibody (Boehringer/Mannheim) for 1 h (37 °C). The cells were washed 3 times with PBS, then and bound antibody was visualized with a Nikon Diaphot inverted phase fluorescence microscopy.

Stable Transfections

C2C12 myoblasts were transfected by the calcium phosphate precipitation method. A 50% confluent T75 flask of C2C12 myoblasts was co-transfected with 3 µg of pSV2Neo (Southern and Berg, 1982) and 10 µg of pEMSV-cyclin D1 (or pEMSVscribealpha2, the vector control). The cells were cultured with the precipitates for 18 h, then passed to two 15-cm culture dishes (per T75 flask). The cells were cultured in high mitogen medium in the presence of 500 µg/ml (effective concentration) G418 for 2-3 weeks, with the media being changed every 3 days. When the colonies reached macroscopic size, they were cultured for 48-72 h in low mitogen medium. The plates were then washed three times with PBS (ambient temperature), and fixed for 10 min in 70% ethanol, 10% formaldehyde, 5% acetic acid (-20 °C). The colonies were rehydrated and washed several times with PBS, then incubated for 2 h with 5 µg/ml monoclonal antibody MF-20 (Bader et al., 1982) in PBS supplemented with 5% horse serum (37 °C). The plates were then washed twice with PBS, and incubated 1 h (37 °C) with peroxidase-coupled anti-mouse IgG (Boehringer/Mannheim; 1:500). After three washes with PBS, the reactions were developed with 1 mg/ml diaminobenzidine and 0.15% H(2)O(2) in PBS. After being scored and photographed, the plates were stained with 0.25% Crystal Violet to score total G418-resistant colonies.


RESULTS

Induction of Cyclin D1 Expression in Skeletal Myoblasts by bFGF or TGFbeta Occurs with Different Kinetics

A subclone of C2C12 myoblasts (Blau et al., 1983) was selected that was inhibited from differentiation by bFGF (20 ng/ml) or TGFbeta (5 ng/ml) with high sensitivity (C2C12-10; referred to within as C2C12). These cells were plated at 20-25% confluence in high mitogen medium (Dulbecco's modified Eagle's medium, 15% fetal bovine serum, 0.5% chick embryo extract), and allowed to grow for 48 h to 80-100% confluence. The RNA extracted from myoblasts at this stage of their growth cycle was used as a 0 point. In the course of several experiments, variations in expression of cyclins at the 0-16-h points were observed that were associated with differences in the starting conditions; for example, subconfluent myoblasts or myoblasts cultured in high mitogen medium for less than 48 h displayed relatively extended expression of cyclin D1, continuing at high levels 4-8 h after switching the cells to low mitogen medium (data not shown). The selected starting conditions represent myoblasts arriving at confluence in a medium that they have partly depleted of mitogens, a state similar to early growth arrest in fibroblasts. These cultures were switched to either low mitogen medium (Dulbecco's modified Eagle's medium, 4% horse serum), low mitogen medium supplemented with 20 ng/ml bFGF, or low mitogen medium supplemented with 5 ng/ml TGFbeta. At specific times post-induction, mRNA was extracted from the cultures and analyzed by Northern blot.

Parallel expression of two cyclin D1 mRNA species (4.5 and 3.8 kilobase pairs) was observed in skeletal myoblasts. Expression of cyclin D1 dropped off from 0 point levels after switching the cultures to low mitogen medium ( Fig. 1and Fig. 2). Supplementing the low mitogen medium with bFGF resulted in induction of cyclin D1 expression after 4 h, with high level expression desisting 32 h post-induction ( Fig. 1and Fig. 2). In the experiment shown (Fig. 1), expression of cyclin D1 mRNA returned after 64 h; however, in other experiments, cyclin D1 expression was not observed at times longer than 32 h in low mitogen medium supplemented with bFGF. Supplementing low mitogen medium with TGFbeta (5 ng/ml) resulted in a relatively delayed induction of cyclin D1 expression, with high level expression beginning 16 h post-induction. Cyclin D1 expression persisted for 32 and 64 h in myoblasts cultured in low mitogen medium supplemented with TGFbeta ( Fig. 1and Fig. 2).


Figure 1: Northern blot analysis of cyclin D1 and D3 expression in differentiating myoblasts. Myoblasts were subcultured from a single parental pool as described in the text (0 lane), then switched in parallel to either low mitogen medium (C lanes), low mitogen medium supplemented with bFGF (F lanes), or low mitogen medium supplemented with TGFbeta (T lanes). After the indicated time (hours), mRNA was isolated and analyzed by Northern blot with probes for cyclin D1 (Motokura et al., 1991) and D3 (Lew et al., 1991), and subsequently by probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (as a loading control). 5 µg of poly(A) RNA was loaded per lane.




Figure 2: Quantitative summary of cyclin, cdk, and troponin I gene expression in myogenic cultures. The results shown in Fig. 1were quantified by PhosphorImager analysis, and cyclin D1 and D3 mRNA levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase signals for their lanes. Northern blot analyses were also performed in other similar experiments for cyclin-dependent kinase 4 (cdk 4; Matsushime et al., 1992) and troponin I(f) (Koppe et al., 1989), then quantified by microdensitometry scan and presented as values standardized to glyceraldehyde-3-phosphate dehydrogenase signals.



Western blot analyses for cyclin D1 protein were performed on whole cell lysates from myoblasts cultured in low mitogen medium, low mitogen medium supplemented with bFGF, and low mitogen medium supplemented with TGFbeta. Expression of sarcomeric myosin heavy chain (sMHC) is used as a marker of differentiation, and in its absence cells cultured in low mitogen medium supplemented with either bFGF or TGFbeta indicated that the myoblasts were blocked from differentiation. Changes in cyclin D1 protein levels directly reflected the observed changes in mRNA levels ( Fig. 3and Fig. 4). Accumulation of cyclin D1 protein peaked 4-8 h after the cells were introduced to low mitogen medium supplemented with bFGF, whereas accumulation of cyclin D1 protein in response to TGFbeta was observed after a 16-32-h delay.


Figure 3: Western blot analysis of cyclin D1 and D3 expression in differentiated myocytes and myoblasts blocked from differentiation by bFGF and TGFbeta. Myoblasts were cultured for 2, 4, 8, 16, 32, and 64 h in low mitogen medium (D lanes), low mitogen medium supplemented with bFGF (F lanes), or low mitogen medium supplemented with TGFbeta (T lanes). Whole cell lysates were then analyzed by Western blot for expression of sMHC, cyclin D1, or cyclin D3. Protein from 2 times 10^5 cells (nuclear count) was loaded per lane.




Figure 4: Quantitative summary of cyclin D1, cyclin D3, and sMHC protein expression in myogenic cultures. The results shown in Fig. 3were quantified by microdensitometry scan.



Enhanced Expression of Cyclin D3 Is Associated with Differentiation of Skeletal Myoblasts

Expression of the cyclin D3 gene was detected by Northern blot of mRNA isolated from skeletal myoblasts cultured in high mitogen medium. When myoblasts were cultured in low mitogen medium or low mitogen medium supplemented with TGFbeta for 2-4 h, expression of the cyclin D3 gene was reduced significantly, but after 8 h returned to levels equivalent to those observed in the 0-point myoblasts ( Fig. 1and Fig. 2). After 32 h in low mitogen medium, expression of the cyclin D3 gene increased significantly, persisting at a level greater than that of the 0 point through 64 h post-induction. In contrast, after 64 h in low mitogen supplemented with TGFbeta, accumulation of cyclin D3 mRNA had increased minimally in comparison to the level observed in 0 point myoblasts (Fig. 2). Myoblasts cultured in low mitogen medium supplemented with bFGF did not display a reduction in cyclin D3 mRNA levels between 2 and 8 h post-induction. By 64 h post-induction, the level of cyclin D3 mRNA was lower than that observed in myoblasts cultured in low mitogen medium alone, but greater than that in myoblasts cultured in low mitogen medium supplemented with TGFbeta. Although bFGF was less effective in preventing accumulation of cyclin D3 mRNA than TGFbeta, both factors prevented accumulation of sarcomeric transcripts (such as troponin I; Fig. 2) equivalently. To summarize, the cyclin D3 gene in myoblasts responded in a biphasic manner to mitogen withdrawal and induction of differentiation: 2-4 h post-induction, cyclin D3 mRNA quantitatively decreased from 0 point levels; 32-64 h post-induction, cyclin D3 mRNA accumulated to levels greater than those observed in 0 point myoblasts. Supplementing the low mitogen medium with bFGF inhibited the reduction at 2 and 4 h post-induction, and partially inhibited the later increase in cyclin D3 mRNA levels; supplementing the low mitogen with TGFbeta inhibited only the increase in cyclin D3 mRNA at 32 and 64 h post-induction.

Western blot analyses for cyclin D3 protein accumulation were performed on whole cell lysates from myoblasts cultured in low mitogen medium, low mitogen medium supplemented with bFGF, and low mitogen medium supplemented with TGFbeta ( Fig. 3and Fig. 4). Low levels of cyclin D3 protein were detected at most time points. A significant increase in cyclin D3 protein accumulation was observed in myoblasts induced to differentiate in low mitogen medium for 64 h. Inhibiting myoblast differentiation by supplementing the low mitogen medium with either bFGF or TGFbeta prevented this increase in cyclin D3 protein accumulation. Cyclin D3 protein levels changed more significantly during myoblast differentiation than did mRNA levels, suggesting that translational or post-translational regulatory mechanisms are important in controlling cyclin D3 expression in muscle cells.

Differentiated C2C12 myoblast cultures consist of multinucleated syncytia that express sarcomeric proteins and quiescent mononuclear cells that do not display a muscle phenotype. Immunofluorescence microscopy with a cyclin D3 antibody was used to determine which cell population expresses cyclin D3. A punctate nuclear distribution of cyclin D3 was observed in terminally differentiated myotubes (Fig. 5). Individual nuclei within each myotube would often display different intensities of cyclin D3 immunofluorescence, and cyclin D3 was also detected in some quiescent mononuclear cells (albeit at lesser intensity).


Figure 5: Immunofluorescence microscopy of cyclin D3 in differentiated myocytes. Myoblasts were cultured on plastic dishes and induced to differentiate in low mitogen medium for 48 h. The cells were processed for immunofluorescence microscopy using a monoclonal antibody to cyclin D3 (a) or an unrelated mouse monoclonal antibody (c). a and c show immunofluorescence; b and d show phase microscopy of the same field. Bar indicates 10 µm.



Dose-specific Inhibition of C2C12 Myoblast Differentiation by Ectopic Expression of Cyclin D1

C2C12 myoblasts were co-transfected with pEMSV-cyclin D1 (or pEMSVscribealpha2 vector control) and pSV2Neo. G418-resistant colonies were selected, cultured in low mitogen medium, and differentiated colonies were scored after immunostaining for sMHC with monoclonal antibody MF-20 (Bader et al., 1982). Subsequently, G418-resist-ant colonies were scored by Crystal Violet stain. The results of three independent experiments are compiled in Table 1. In experiments 1 and 2, only colonies larger than 3 mm in diameter were scored. In these experiments, transfection of pEMSV-cyclin D1 increased the percentage of differentiation-defective clones 3.2- and 3.5-fold, respectively, over transfection of the expression vector (pEMSVscribealpha2). In experiment 3, all colonies were scored, and since the smaller colonies differentiate poorly, a greater frequency of spontaneous differentiation-defective colonies was scored. In experiment 3, transfection of pEMSV-cyclin D1 increased the percentage of differentiation-defective colonies 2.1-fold. The results of experiment 3 are also shown in Fig. 6. In this experiment, the differentiation-defective clones in the pEMSVscribealpha2 transfection are mostly <3 mm in diameter, and similar colonies comprise about half of the differentiation-defective colonies in the pEMSV-cyclin D1 transfection. The frequency of large (>3 mm diameter) differentiation-defective colonies was significantly greater in pEMSV-cyclin D1 than in pEMSVscribealpha2 transfected cells (Fig. 6).




Figure 6: Inhibition of C2C12 myoblast differentiation by ectopic expression of cyclin D1; I, colony assay. C2C12 myoblasts were co-transfected with either pEMSVscribealpha2 (Vector control) and pSV2Neo or pEMSV-cyclin D1 and pSV2Neo (Cyclin D1) as described under ``Materials and Methods.'' The selected colonies were induced to differentiate in low mitogen medium, then stained with a monoclonal antibody (MF-20; Bader et al., 1982) to sMHC. After being photographed, the colonies were stained with Crystal Violet. Inhibition of differentiation is scored when a colony of significant size appears in the Crystal Violet stain but is not apparent in the sMHC stain. A quantitative analyses of this experiment is given in Table 1.



In separate experiments, Northern blot analysis of recovered colonies indicated that >90% of the G418-resistant colonies expressed the integrated pEMSV-cyclin D1 construct(s). The level of ectopic cyclin D1 gene expression varied among the clones. A series of clones expressing differing levels of cyclin D1 transcripts was selected for further analysis (Fig. 7). Western blot analysis indicated that differences in cyclin D1 mRNA levels manifested as differences in levels of cyclin D1 protein. Western blot analysis of sMHC protein levels in myoblasts cultured in low mitogen medium for 48 h indicated that ectopic expression of cyclin D1 affected differentiation in a biphasic manner: at expression levels equivalent to or below those observed in myoblasts stimulated by bFGF, ectopic cyclin D1 inhibited differentiation; at expression levels profoundly exceeding those in myoblasts stimulated by bFGF, cyclin D1 had no effect on myoblast differentiation (as determined by sMHC expression; Fig. 7). Visual inspection of the clones indicated that inhibition of myoblast fusion and myotube assembly paralleled changes in sMHC expression (not shown). These results indicate that appropriate physiological levels of cyclin D1, such as those produced after stimulation by bFGF (or even lower levels), inhibit myoblast differentiation. Substantially higher levels do not inhibit differentiation, possibly because the proper assembly of a trimolecular complex consisting of cyclin D1 and two associated molecules (e.g. cdk 4 and a directed substrate) is unfavorable.


Figure 7: Inhibition of C2C12 myoblast differentiation by ectopic expression of cyclin D1; II, analysis of individual clones. C2C12 myoblasts were stably transfected with pEMSV-cyclin D1 and pSV2Neo. G418-resistant colonies were expanded and analyzed by Northern blot. Nine clones expressing different levels of cyclin D1 mRNA from pEMSV-cyclin D1 were selected for further study (arbitrary clone numbers are indicated). These clones were grown to confluence, then cultured in low mitogen medium for 48 h. Cell lysates were then analyzed for cyclin D1 and sMHC expression by Western blot. Shown are the results of microdensitometry scans of Northern and Western blots analyses of cyclin D1 expression (upper panel) and Western blot analyses of sMHC expression (lower panel). Control C2C12 myoblast cultures were also cultured in low mitogen medium (Dif.) or low mitogen medium supplemented with bFGF (+FGF), and analyzed by Western blot for cyclin D1 or sMHC chain expression.



The appearance of increased quantities of multinucleated syncytia in high mitogen medium cultures suggested that myoblast clones expressing the highest level of ectopic cyclin D1 also had an enhanced frequency of spontaneous differentiation. Consequently, the responses of two of these clones (clones 9 and 11; Fig. 7) to bFGF and TGFbeta were tested. As determined from the expression of sMHC, clone 11 (and to a lesser extent clone 9) partially differentiated in the presence of bFGF (Fig. 8), suggesting that ectopic expression of excess cyclin D1 interfered with inhibition of myoblast differentiation by bFGF. This observation is consistent with the notion suggested above that expression of excess cyclin D1 interferes with its assembly into a multimeric complex that functions in inhibition of myoblast differentiation. As a result, the effects of endogenous cyclin D1 expression induced by bFGF are abrogated in the cellular context of a myoblast expressing excess ectopic cyclin D1. The ability of myoblast clones expressing excess cyclin D1 to partially differentiate in the presence of bFGF suggests that the induction of cyclin D1 expression by bFGF is a significant functional parameter in the inhibition of myoblast differentiation.


Figure 8: Myoblast clones expressing excess cyclin D1 partially differentiate in the presence of bFGF. C2C12 myoblasts and stably transfected clones 9 and 11 (described in Fig. 7) were analyzed by Western blot for sMHC chain expression after being cultured in high mitogen medium at low density (P), or in low mitogen medium (D), low mitogen supplemented with TGFbeta (T), or low mitogen medium supplemented with bFGF (F) at high density for 48 h.




DISCUSSION

Expression of the cyclin D1 gene in myoblasts is abolished when the cells are induced to differentiate in low mitogen medium. Supplementing low mitogen medium with either bFGF or TGFbeta inhibits differentiation of C2C12 myoblasts, and induced expression pattern of the cyclin D1 gene. Confluent myoblasts cultured in low mitogen medium supplemented with bFGF displayed a significant increase in cyclin D1 gene expression 4-8 h post-induction. At 16 h, cyclin D1 mRNA levels in these cultures fell significantly, and by 32 h they returned to initial levels. The pattern of induction of the cyclin D1 gene in myoblasts by bFGF is characteristic of the induction of early response genes, such as c-myc (Greenberg and Ziff, 1984), to certain growth factors. In comparison to induction by bFGF, induction of the cyclin D1 gene in myoblasts by TGFbeta was significantly delayed. Initial accumulation of cyclin D1 mRNA was observed after 16 h in low mitogen medium supplemented with TGFbeta, increasing to higher levels at 32 and 64 h. The difference between the pattern of cyclin D1 expression induced by bFGF and TGFbeta strongly suggests that the signaling pathways activated by these two factors in myoblasts diverge significantly. Another possible explanation for the delayed response of the cyclin D1 gene to TGFbeta is that the effect is actually mediated by a second factor secreted in response to TGFbeta by the myoblasts. Further experiments are warranted to explore this hypothesis.

Previously, we observed that ectopic expression of cyclin D1 prevents activation of muscle gene transcription by myogenic basic helix-loop-helix regulators in transient expression assays (Rao et al., 1994). As shown in this report, however, only a fraction of myoblast clones stably transfected with pEMSV-cyclin D1 are inhibited from differentiation. Northern and Western blot analyses showed that expression of ectopic cyclin D1 was observed in most clones. Individual clones expressed different amounts of cyclin D1 mRNA from the transfected construct, and in general cyclin D1 protein accumulated to quantities corresponding to the relative level of mRNA. Presumably, variations in cyclin D1 expression among individual clones are directly associated with differences in the copy number of transfected expression constructs. Myoblast differentiation, as determined morphologically by the assembly of myotubes and biochemically by expression of sMHC, was inhibited in clones expressing cyclin D1 at levels similar to those observed in cultures stimulated by bFGF. Clones expressing cyclin D1 at significantly higher levels were not inhibited by differentiation. This observation is consistent with a mechanism of inhibition requiring the assembly of a trimolecular complex consisting of cyclin D1 bound to two other components, such as cdk4 and a directed substrate. Inhibition of myoblast differentiation only at appropriate levels of expression (and not higher levels) has important implications for the interpretation of other experiments involving ectopic expression of cyclin D1. In particular, it is possible that certain functions of cyclin D1 may be masked by overexpression of the ectopic expression construct.

Some myoblast clones expressing excess ectopic cyclin D1 acquired the ability to partly differentiate in the presence of bFGF. This observation suggests that cyclin D1 induced to physiological levels in normal myoblasts by bFGF may function in the inhibition of differentiation by this growth factor. The function of cyclin D1 in the inhibition of myoblast differentiation by bFGF would most likely lie downstream of the effects of this growth factor on protein kinase C phosphorylation of the myogenic bHLH proteins (Li et al., 1992). Induction of protein kinase C activity by bFGF and subsequent phosphorylation of myogenic bHLH regulators may occur rapidly and transiently. Inhibition of myoblast differentiation by bFGF at later time points could be associated with the induction of cyclin D1 expression. We have shown previously that the activity of a myogenin basic-region mutant lacking the protein kinase C site (Li et al., 1992) is inhibited by ectopic cyclin D1 even though it is refractory to inhibition by bFGF (Rao et al., 1994). This observation supports the notion that inhibition of myogenic bHLH regulator activity by cyclin D1 operates downstream from protein kinase C phosphorylation. Experiments in progress will determine whether myogenic bHLH proteins suppress cyclin D1 gene expression, and the possible role of protein kinase C phosphorylation may play to counter this suppression and mediate the induction of cyclin D expression in myoblasts cultured with bFGF. These observations would show that the activity of myogenin basic-region mutants in the presence of bFGF is associated with two events: lack of basic region phosphorylation by protein kinase C and, as a consequence, maintained suppression of cyclin D1 expression.

The cyclin D3 gene exhibited a biphasic pattern of expression in myoblasts induced to differentiate by low mitogen medium. Between 2 and 8 h post-induction, the levels of cyclin D3 mRNA was reduced in comparison to that in 0-point myoblast cultures. By 16 h post-induction, cyclin D3 mRNA levels had returned to those observed in 0 point cultures, and after 32 h (the onset of overt myogenic differentiation) were severalfold higher than starting point values. One interpretation of these observations is that the cyclin D3 gene is both induced by growth factors in myoblasts, and induced during differentiation of the myoblasts into myotubes. The initial drop in cyclin D3 levels observed in low mitogen medium (or in low mitogen supplemented with TGFbeta) could be associated with loss of a positive growth factor signal; the elimination of this drop from myoblast cultures in low mitogen medium supplemented with bFGF supports this interpretation. Differentiation of myoblasts after 32 h in low mitogen medium may be a prerequisite for later induction of the cyclin D3 gene; attenuation of this induction in myoblasts cultured in low mitogen medium supplemented with either TGFbeta or bFGF supports this interpretation. The fundamental point of this model is that positive regulation of the cyclin D3 in myoblasts and myotubes result from different molecular mechanisms rather than quantitative differences in the same mechanism. Analysis of the structure of the regulatory region of the cyclin D3 gene should yield further insights into the mechanism(s) controlling its expression in myogenic cells.

Finally, it is surprising that a cell cycle regulator (cyclin D3) is present in terminally differentiated myotubes, and that it is present at higher levels than in proliferating myoblasts. The suggested conclusion of this observation, that cell cycle regulators may have extended functions in some cells outside of cell cycle regulation, is intriguing. In future studies, it may be possible to demonstrate that some cyclins regulate changes in cell physiology that do not bear directly on proliferation, such as hypertrophy or cell death. It is also possible that the D-type cyclins are functionally antagonistic in some cell types, such that induction of cell cycle progression by one D-type cyclin may be competitively inhibited by expression by another D-type cyclin in the same cell. In this case, expression of cyclin D3 in terminal myocytes may have the unexpected function of preventing entry into the cell cycle rather than promoting it.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: bFGF, basic fibroblast growth factor; TGFbeta, transforming growth factor beta; bHLH, basic helix-loop-helix; cdk, cyclin-dependent kinase; PBS, phosphate-buffered saline; sMHC, sarcomeric myosin heavy chain.

(^2)
G. Shue and D. Kohtz, unpublished results.


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