(Received for publication, August 5, 1994; and in revised form, November 18, 1994)
From the
Differentiation of skeletal myoblasts in culture is negatively
regulated by certain growth factors, including basic fibroblast growth
factor (bFGF) and transforming growth factor (TGF
). We
investigated the effects of bFGF and TGF
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 TGF
(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.
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) ()(Lathrop et al., 1985; Spizz et
al., 1986; Clegg et al., 1987) and transforming growth
factor
(TGF
) (Olson et al., 1986, Massague et
al., 1986) have been well characterized. Both bFGF and TGF
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 TGF
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 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
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). (
)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 and
G
/M, respectively, and control the progress of events
during S, G
, and M phases of the cell cycle (Minshull et al., 1990; Pines and Hunter, 1990; Norbury and Nurse,
1992). The G
cyclins (cyclins C, E, and D-type cyclins) are
expressed during the G
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 TGF
. 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.
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).
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 TGF (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 TGF
( 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 TGF (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 (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 TGF. 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 TGF
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 TGF
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 TGF. 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 TGF
(T lanes). Whole
cell lysates were then analyzed by Western blot for expression of sMHC,
cyclin D1, or cyclin D3. Protein from 2
10
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.
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
TGF ( 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 TGF
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.
Figure 6:
Inhibition of C2C12 myoblast
differentiation by ectopic expression of cyclin D1; I, colony assay.
C2C12 myoblasts were co-transfected with either pEMSVscribe2 (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 TGF 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 TGF (T), or low mitogen medium supplemented with bFGF (F)
at high density for 48 h.
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 TGF 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 TGF
was significantly delayed.
Initial accumulation of cyclin D1 mRNA was observed after 16 h in low
mitogen medium supplemented with TGF
, increasing to higher levels
at 32 and 64 h. The difference between the pattern of cyclin D1
expression induced by bFGF and TGF
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 TGF
is that the effect is actually mediated
by a second factor secreted in response to TGF
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 TGF) 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 TGF
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.