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Regulation of the Levels of Small Heat-Shock Proteins during Differentiation of C2C12 Cells
Experimental Cell Research 266, 213–221 (2001) doi:10.1006/excr.2001.5220, available online at http://www.idealibrary.com on
Regulation of the Levels of Small Heat-Shock Proteins during Differentiation of C2C12 Cells Hidenori Ito, 1 Keiko Kamei, Ikuko Iwamoto, Yutaka Inaguma, and Kanefusa Kato Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya, Kasugai, Aichi 480-0392, Japan
Levels of the small heat-shock proteins (sHSPs) HSP27 and ␣B-crystallin during differentiation of mouse C2C12 cells were determined using specific immunoassays. Increases of these proteins were about 3-fold and 10-fold, respectively. Under the same conditions, however, the level of HSP70 in C2C12 cells barely increased, indicating selective accumulation of HSP27 and ␣B-crystallin with differentiation. While expression of mRNA for ␣B-crystallin was also markedly increased and that for HSP27 was but to a lesser extent, mRNA for HSP70 could barely be detected during differentiation. Activation of the heat-shock factor was not observed, in contrast to the case with heatstressed undifferentiated cells. Various inhibitors of protein kinases affected the differentiation and the associated increase of sHSPs. Rapamycin, an inhibitor of p70 S6 kinase, completely inhibited the differentiation and suppressed the accumulation of HSP27 and ␣B-crystallin. SB203580, an inhibitor of p38 MAP kinase, also inhibited differentiation, but the accumulation of ␣B-crystallin was rather enhanced. PD98059, an inhibitor of MAP kinase kinase, significantly increased expression of a differentiation marker for muscle cells, creatine kinase M isozyme, as well as accumulation of ␣B-crystallin. These results suggest that accumulation of sHSPs during differentiation of C2C12 cells is regulated in a complex manner. © 2001 Academic Press
Key Words: crystallin; differentiation; HSP27; muscle; stress response.
INTRODUCTION
Small heat-shock or stress proteins (sHSPs), 2 which have a molecular mass of about 15–30 kDa, share 1 To whom correspondence and reprint requests should be addressed. Fax: ⫹81-568-88-0829. E-mail: itohide@inst-hsc. pref.aichi.jp. 2 Abbreviations used: CKM, creatine kinase M isozyme; HSPs, heat-shock or stress proteins; sHSPs, small HSPs; HSF, heat-shock factor; HSE, heat-shock element; PBS, phosphate-buffered saline; MEK, MAP kinase kinase.
several features: a homologous carboxyl-terminal amino acid sequence called the “␣-crystallin domain” [1]; formation of large oligomers that have an apparent molecular mass of ⬎500 kDa [2, 3]; induction by various stresses, which include heat stress, oxidative stress, and chemical stress [4, 5]; and phosphorylation in response to various stresses or growth factors [6 – 8]. It is also reported that the expression and the phosphorylation pattern of sHSPs change during differentiation of some types of mammalian cells, including HL-60 cells [9], rat olfactory neurons [10], and embryonic stem cells [11]. HSP27 and ␣B-crystallin, representative sHSPs, are present not only in stressed cells or tissues but also sometimes under nonstressed conditions, especially in the skeletal muscle [12, 13]. It has been reported that HSP27 is an actin-binding protein that modifies actin polymerization [14, 15] and that ␣B-crystallin interacts with actin and desmin directly and is located in the central regions of the I bands (Z-lines) in cardiomyocytes [16]. We previously reported that levels of HSP27 and ␣B-crystallin in rat hindlimb muscles change developmentally and under pathological conditions [17]. Neufer and Benjamin described expression of HSP27 and ␣B-crystallin in skeletal muscle as changing during continuous contractile activity [18]. Recently, a single mutation in ␣B-crystallin, R120G, was found in a French family suffering from desminrelated myopathy, which has an autosomal dominant myopathy that results in weakness of the proximal and distal limb muscles [19]. These findings imply that sHSPs play important roles in maintenance of the function of muscles, although their exact physiological functions remain unclear. C2C12 mouse myoblast cells are well known for their ability to differentiate into multinucleated myotubes and are often used as a model for myogenesis [20]. In this study, we determined changes in expression of sHSPs in C2C12 cells during differentiation and attempted to clarify protein kinase cascades that play regulatory roles.
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0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Cell cultures and treatments. Mouse C2C12 cells (obtained from the American Type Culture Collection) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 20% fetal calf serum (EquitechBio, Inc., Ingram, TX) at 37°C in a humidified atmosphere of 95% air and 5% CO 2. For differentiation, they were grown in dishes to confluence and then the culture medium was replaced with DMEM containing 5% heat-inactivated horse serum (GIBCO, Grand Island, NY). In some experiments, protein kinase inhibitors such as 10 M SB203580, 10 M PD169316, 0.1 M rapamycin, 50 M PD98059 (all of which were purchased from Calbiochem, La Jolla, CA), or 10 M Uo 126 (Promega, Madison, WI) were added to the culture medium. Cells in each dish were rinsed with phosphate-buffered saline (PBS, containing 8 g of NaCl, 0.2 g of KCl, 1.15 g of Na 2HPO 4䡠12H 20, and 0.2 g of KH 2PO 4 in 1000 ml of H 2O) at various time periods, frozen at 80°C for a few days, collected, suspended in 0.3 ml of PBS, sonicated, and centrifuged at 125,000g for 20 min at 4°C. Supernatants were used for quantitation of HSP27, ␣B-crystallin, HSP70, and creatine kinase M isozyme (CKM). Quantitation of HSP27, ␣B-crystallin, and CKM. Concentrations of HSP27 [17], ␣B-crystallin [12], and CKM [21] in extracts of cells were estimated by specific immunoassays as described previously. Electrophoresis and Western blot analysis. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed by the method of Laemmli [22] in a 10 or 12.5% polyacrylamide slab gel. For Western blot analysis, proteins in polyacrylamide gels were transferred electrophoretically to nitrocellulose membranes and immunostained with affinity-purified antibodies against rat HSP27 or against the carboxyl-terminal decapeptide of ␣B-crystallin and peroxidase-labeled goat antibodies against rabbit IgG, as described previously [23]. For detection of HSP70, a mouse monoclonal antibody specific for the inducible form of HSP70 (C92F3A-5; StressGen Biotech., Victoria, BC, Canada) and peroxidase-labeled antibodies raised in a goat against mouse IgG (Medical and Biological Labs. Co., Nagoya, Japan) were used. Peroxidase activities on the nitrocellulose membranes were visualized on X-ray film using a Western blot chemiluminescence reagent (Renaissance; Dupont NEN, Boston, MA). Isolation of RNA and Northern blot analysis. Total RNA was isolated from cells with a QuickPrep total RNA Extraction kit (Amersham Pharmacia Biotech, Tokyo, Japan). Twenty micrograms of total RNA was subjected to electrophoresis on a 0.9% agarose–2.2 M formaldehyde gel and blotted onto nitrocellulose membranes. For Northern blot analysis, membranes were allowed to hybridize with cDNA probes that had been labeled with 32P using a Multiprime DNA labeling system (Amersham Pharmacia Biotech), as described previously [23]. A BamHI-HindIII fragment of cDNA for mouse HSP27 [24] was kindly provided by Dr. L. F. Cooper, University of North Carolina, and a PstI fragment of cDNA for bovine ␣B-crystallin [25] by Dr. H. Bloemendal, University of Nijmegen, The Netherlands. An EcoRI fragment of cDNA for HSP70 was obtained from StressGen Biotech. For determination of the stability of mRNA for ␣B-crystallin during differentiation, 40 g (for control cells) or 20 g (for differentiated cells) of total RNA was electrophoresed, hybridized, and autoradiographed. Relative densities of bands were quantified using an NIH image program (National Institutes of Health, Bethesda, MD). Gel shift assays of the heat-shock transcription factor. Cells were harvested at 0°C in 20 mM Hepes–KOH buffer, pH 7.9, that contained 25% (v/v) glycerol, 0.5 M NaCl, 1.5 mM MgCl 2, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol, 0.5 g/ml pepstatin A, and 0.3 mg/ml Pefablock SC (Boehringer Mannheim, Mannheim, Germany). The suspension was sonicated and centrifuged at 4°C at 125,000g for 20 min, and supernatants that contained 15 g protein were incubated at 0°C for 20 min with
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P-labeled oligonucleotide (heat-shock element of Drosophila HSP70; 5⬘-GCCTCGAATGTTCGCGAAGTTTCG-3⬘) [26] in 10 mM Tris–HCl buffer, pH 8.0, containing 0.5 g poly(dI– dC) (Amersham Pharmacia Biotech), 50 mM NaCl, 1 mM EDTA, 5% glycerol, and 0.5 mM dithiothreitol. Reaction mixtures were then subjected to electrophoresis in 4% polyacrylamide gels and autoradiographed with X-ray film. Other methods. Concentrations of protein in soluble extracts were estimated with a protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. Rat HSP27 [17], ␣B-crystallin [12], and CKM [21], used as standards for immunoassays, were purified from skeletal muscle.
RESULTS
Changes in Levels of HSP27, ␣B-Crystallin, and CKM during Differentiation of C2C12 Cells The C2C12 cells began to fuse at 2–3 days after the medium was changed to induce differentiation, and the surface of the culture dish became covered with multinucleated myotubes after 5– 6 days (Fig. 1A). Although CKM was undetectable in undifferentiated cells (⬍1 ng/mg protein), its concentration markedly increased after 3 to 6 days of differentiation (Fig. 1B), as reported previously [27]. The level of ␣B-crystallin in undifferentiated cells was also low (about 50 –100 ng/mg protein) but increased after 3 days of differentiation and this continued to day 6 (Fig. 1C). The magnitude varied between experiments but the level of ␣B-crystallin reached about 5- to 10-fold of that of control myoblasts in 6-day-differentiated cells (Fig. 1C). The level of HSP27 in control myoblasts was relatively high (about 1000 ng/mg protein) and increased during differentiation (Fig. 1D). The magnitude again varied with the experiment, with the level of HSP27 reaching about 1.5- to 4-fold of that in the control cells after 6 days (Fig. 1D). Western Blot Analyses of HSP27, ␣B-Crystallin, and HSP70 To determine whether another representative stress protein, HSP70, was also increased during differentiation of C2C12 cells, we performed Western blot analyses of cell extracts. This confirmed that the level of ␣B-crystallin markedly increased during differentiation (Fig. 2). The amount of HSP27 also tended to increase, although not to the same degree as that of ␣B-crystallin (Fig. 2). The level of HSP70 in control myoblasts was very low and increased only slight during differentiation, in contrast to its marked expression with heat (Fig. 2). These results indicate the differentiation-dependent accumulation to be preferential for sHSPs, especially ␣B-crystallin, in C2C12 cells. Northern Blot Analyses of HSP27, ␣B-Crystallin, and HSP70 We estimated changes in the levels of mRNAs for HSP27, ␣B-crystallin, and HSP70 during differentia-
DIFFERENTIATION OF MUSCLE CELLS AND sHSPs
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FIG. 1. Differentiation of C2C12 cells (A) and associated changes in the intracellular levels of CKM (B), ␣B-crystallin (C), and HSP27 (D). When cells reached confluence, the medium was changed to induce differentiation as described under Materials and Methods. The cells were harvested at the indicated times and the levels of three proteins estimated using specific immunoassays. Each point shows the mean ⫾ SD of results for four dishes.
tion of C2C12 cells using Northern blot analyses. Consistent with the results for protein levels, the mRNA for ␣B-crystallin in differentiated cells was clearly increased (Fig. 3A). This was also the case for HSP27, to a lesser extent (Fig. 3A). The mRNA for the inducible form of HSP70 (top band), which was induced by heat treatment, was not induced after differentiation, although the mRNA for a constitutive form of HSP70 (bottom band) was detected in control myoblasts as described previously [28] (Fig. 3A). To clarify the mechanism of the differentiation-dependent increase of ␣Bcrystallin, we estimated the stability of mRNA for ␣Bcrystallin using an inhibitor of transcription, actinomycin D. As shown in Figs. 3B and 3C, levels of mRNA for ␣B-crystallin decreased after actinomycin D treatment both in control and in differentiated cells. The decrease of mRNA in differentiated cells was slightly slower than that in control cells, although the
FIG. 2. Results of Western blot analyses of HSP70, HSP27, and ␣B-crystallin. When cells reached confluence, they were cultured with differentiation medium for the indicated periods. Aliquots of cell extract containing 20 g of protein for detection of HSP70 or ␣B-crystallin or 10 g of protein for detection of HSP27 were subjected to SDS–PAGE with subsequent Western blot analysis with antibodies against HSP70, HSP27, and ␣B-crystallin. Hs, undifferentiated cells heated at 42°C for 30 min and then cultured at 37°C for 8 h.
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FIG. 3. Northern blot analyses of HSP70, HSP27, and ␣B-crystallin (A) and estimation of the stability of mRNA for ␣B-crystallin (B and C). (A) Cells at different differentiation time points were harvested for isolation of total RNA. Twenty-microgram aliquots of RNA from each sample were subjected to electrophoresis with subsequent Northern blot analysis with cDNA probes for HSP70, HSP27, and ␣B-crystallin. Lanes 1 and 2, control cells; lanes 3 and 4, 3-day-differentiated cells; lanes 5 and 6, undifferentiated cells heated at 42°C for 30 min and then cultured at 37°C for 4 h. Bands for 28S rRNA are shown for reference. (B) Control cells and 5-days-differentiated cells were treated with 1 g/ml actinomycin D and were harvested at indicated times. Forty- (for control cells) or twenty-microgram (for differentiated cells) aliquots of RNA from each sample were subjected to Northern blot analysis using a cDNA probe for ␣B-crystallin. Bands for 28S rRNA are shown for reference. (C) The densities of bands shown in (B) were quantified and graphed. The data were shown as relative density to the cells at time 0.
extent of the difference between them was not much. This result suggests that the accumulation of ␣B-crystallin during differentiation may be mainly due to the transcriptional activation.
kinase cascade and a rapamycin-sensitive pathway [29]. We also examined the effects of protein kinase inhibitors on their differentiation-dependent accumulation of sHSPs. Consistent with the previous report
Heat-Shock Transcription Factor Is Not Activated during Differentiation of C2C12 Cells It is well known that activation of heat-shock gene transcription is mediated by the heat-shock transcription factor (HSF). Its involvement in the differentiation-dependent increase of sHSPs was therefore examined. As shown in Fig. 4, activation of HSF was not observed at any stage of differentiation of C2C12 cells, although it was clear when undifferentiated cells were subjected to heat stress. These results indicate that the differentiation-dependent increase of sHSPs in C2C12 cells is not due to activation of HSF and that another mechanism must be involved. Effects of Protein Kinase Inhibitors on the Formation of Myotubes and the Differentiation-Dependent Accumulation of sHSPs Recently, Cuenda and Cohen reported differentiation of C2C12 cells to be regulated by the p38 MAP
FIG. 4. HSE-binding activity in extracts of differentiating cells and heat-stressed undifferentiated cells. Cell extracts were reacted with 32P-labeled oligonucleotide, which contained the HSE sequence, at 0°C for 20 min, and the reaction mixtures were subjected to acrylamide gel electrophoresis. The gel was dried and autoradiographed with X-ray film. Hs, undifferentiated cells treated at 43°C for 30 min.
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FIG. 5. Effects of protein kinase inhibitors on the formation of myotubes. C2C12 cells were grown in medium that contained 20% fetal calf serum (FCS) until reaching confluence and then differentiated with medium contained 5% horse serum in the presence or absence of 10 M SB203580, 0.1 M rapamycin, or 50 M PD98059 for 3 or 6 days.
[29], addition to the differentiation medium of 10 M SB203580, an inhibitor of p38 MAP kinase, or more particularly, 0.1 M rapamycin, an inhibitor of p70 S6 kinase, caused suppression of the formation of myotubes (Fig. 5). In contrast, addition of 50 M PD98059, an inhibitor of MAP kinase kinase (MEK) caused formation of myotubes the shapes of which were different from those of control myotubes (Fig. 5). The differentiation-dependent increase in the level of CKM, a differentiation marker of muscle cells, was also suppressed by the addition of SB203580 or rapamycin to the medium (Fig. 6A), consistent with the degree of formation of myotubes. Addition of PD98059
did not suppress but rather increased the level of CKM (Fig. 6A). Addition of Rapamycin to the medium also caused the suppression of a differentiation-dependent increase of HSP27 and ␣B-crystallin in C2C12 cells (Figs. 6B and 6C). Surprisingly, SB203580 and PD98059 both markedly increased the accumulation of ␣B-crystallin and slightly increased that of HSP27 at early stages of differentiation (Figs. 6B and 6C). We also determined the effect of PD169316, another inhibitor of p38 MAP kinase, and Uo 126, a second inhibitor of MEK, on the accumulations of CKM and ␣B-crystallin, the results being similar to those with SB203580 and PD98059 (Figs. 6A and 6B), respectively (Table 1).
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FIG. 6. Effects of protein kinase inhibitors on the accumulation of CKM (A), ␣B-crystallin (B), and HSP27 (C). Cells were grown in medium that contained 20% FCS until reaching confluence and then cultured in medium that contained 5% heat-inactivated horse serum in the presence (closed symbols) or absence (open circles) of 10 M SB203580 (closed circles), 0.1 M rapamycin (closed squares), or 50 M PD98059 (closed triangles) for the indicated periods. Cells were harvested and estimated for levels of three proteins using specific immunoassays. Each point shows the mean ⫾ SD of results for 4 –5 dishes.
Western Blot and Northern Blot Analyses of Cells That Had Been Treated with Protein Kinase Inhibitors To confirm the effects of protein kinase inhibitors on the accumulation of ␣B-crystallin during differentiation of C2C12 cells, we performed Western blot analyses of cell extracts. Consistent with immunoassay results, SB203580 and PD98059 enhanced, while rapamycin suppressed, the accumulation (Fig. 7A). Northern blot analyses revealed similar effects on
TABLE 1 Effects of PD169316 and Uo 126 on the DifferentiationDependent Accumulation of CKM and ␣B-Crystallin in C2C12 cells Concentration a (ng/mg protein) Treatment Day Day Day Day
0 6 6 ⫹ PD169316 6 ⫹ Uo 126
CKM
␣B-crystallin
0.861 ⫾ 0.113 1460 ⫾ 115 547 ⫾ 74.8 2320 ⫾ 256
19.8 ⫾ 6.99 591 ⫾ 53.7 937 ⫾ 114 3640 ⫾ 316
a Concentrations of CKM and ␣B-crystallin in extracts of cells were determined by specific immunoassays. Each value is the mean ⫾ SD of the results from three to five dishes.
FIG. 7. Western blot analysis (A) and Northern blot analysis (B) of C2C12 cells treated with various protein kinase inhibitors. (A) Cells were grown in medium with 20% FCS until reaching confluence and then cultured in medium with 5% heat-inactivated horse serum in the presence (lanes 5–10) or absence (lanes 3 and 4) of 10 M SB203580 (lanes 5 and 6), 50 M PD98059 (lanes 7 and 8), or 0.1 M rapamycin (lanes 9 and 10) for 6 days. Aliquots of cell extracts containing 10 g of protein were subjected to SDS–PAGE with subsequent Western blot analysis with antibodies against ␣B-crystallin. Lanes 1 and 2, control undifferentiated myoblasts. (B) Cells at 4 days after differentiation in the presence (lanes 3 to 5) or absence (lane 2) of 10 M SB203580 (lane 3), 50 M PD98059 (lane 4), and 0.1 M rapamycin (lane 5) were harvested for isolation of total RNA. Twenty micrograms of RNA from each sample was subjected to electrophoresis with subsequent hybridization with a cDNA probe for ␣B-crystallin (top panel). Bands for 28S rRNA are shown for reference. Lane 1, control undifferentiated myoblasts.
DIFFERENTIATION OF MUSCLE CELLS AND sHSPs
mRNA levels (Fig. 7B), suggesting that protein kinase inhibitors regulate the expression of ␣B-crystallin during differentiation of C2C12 cells at the transcriptional level. DISCUSSION
In the present study, concentrations of HSP27 and especially ␣B-crystallin were found to increase during differentiation of C2C12 cells into multinucleated myotubes. Recently, Sugiyama et al. reported similar results, describing increases of sHSPs such as MKBP/HSPB2, HSPB3, and ␣B-crystallin during differentiation of C2C12 cells, although the levels of HSP27 remained unchanged [30]. Under our experimental conditions, undifferentiated C2C12 cells already expressed HSP27 to a relatively high level and an increase during differentiation was only clearly detectable with immunoassay and not Western blot analysis. The regulation of sHSP induction is not fully understood, but it is believed that activation by heat-shock factor is important because heat-shock elements (HSEs) are present in the promoter regions of the HSP27 and ␣B-crystallin genes [31, 32]. Activation of transcription is induced after binding of the trimeric HSF to HSEs [33]. However, other mechanisms may be involved [34, 35]. During differentiation of C2C12 cells in the present experiments, the levels of sHSPs and their mRNAs, especially with ␣B-crystallin, increased without a significant increase of HSP70 (Figs. 2 and 3A). In addition, we could not observe any activation of HSF, in clear contrast to the heat-treatment case (Fig. 4). These results indicate that the mechanism(s) underlying the differentiation-dependent increase of sHSPs is quite different from that with stress induction. Addition of actinomycin D, an inhibitor of transcription, caused the decrease of mRNA for ␣B-crystallin both in control cells and in differentiated cells. The decrease in control cells was slightly faster than that in differentiated cells but the degree of the difference between them was not large (Figs. 3B and 3C). This result suggests that the increase of mRNA for ␣Bcrystallin may mainly be due to the transcriptional activation. It has been reported that a consensus muscle regulatory factor-binding site called MRF is present in the ␣B-crystallin gene [36]. Investigation of whether it might be involved in the differentiation-dependent increase of ␣B-crystallin under our experimental conditions is clearly warranted. There are several reports of a relationship between the differentiation of muscle cells and protein kinase action. Coolican et al. demonstrated that IGF-stimulated differentiation of L6A1 myoblasts was accelerated by PD98059 and inhibited by rapamycin [37]. Gredinger et al., on the other hand, showed that
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PD98059 inhibited the differentiation of C2C12 cells [38]. Zetser et al. reported inhibition of the differentiation of L8 cells by SB203580 [39]. Cuenda and Cohen found the same compounds SB203580 and rapamycin to inhibit the differentiation of C2C12 cells, although PD 98059 had no effect [29]. In this study, we demonstrated that rapamycin and SB203580 inhibited or delayed the differentiation of C2C12 cells. However, PD98059 tended to increase the level of CKM, a differentiation marker for muscle cells (Fig. 6A), and also alter the shape of myotubes formed (Fig. 5). In terms of the levels of sHSPs, the effects of SB203580 and rapamycin were quite different (Figs. 6B and 6C). Rapamycin was the more powerful inhibitor of myotube formation under our experimental conditions. The mechanism of the effect of SB203580 on the differentiation-dependent increase of ␣B-crystallin (Fig. 6B) is not clear. Since the level of HSP27 during differentiation was barely affected (Fig. 6C), addition of SB 203580 may not be by itself a stress and there may exist intrinsic transcriptional regulation for ␣B-crystallin. As described above, addition of protein kinase inhibitors during differentiation of C2C12 cells affected myotube formation and accumulation of sHSPs in a complex manner. These results indicate that the p38 MAP kinase pathway, the p70S6 kinase pathway, and the p44/42 MAP kinase pathway may play different roles in differentiation of muscle cells and accumulation of sHSPs during differentiation of C2C12 cells. The functions of sHSPs in muscle cells are obscure, although there are several reports of relationships with pathology or physiology. The precise role of sHSPs in differentiation of C2C12 cells also remains to be clarified. It has been reported that resistance to apoptosis is conferred during differentiation of C2C12 cells [40] and that sHSPs exert an anti-apoptotic effect in mammalian cells [41]. Recently, Davidson and Morange reported HSP27 involvement in differentiation of cardiomyocytes [42]. They showed an increase of the level of HSP27 during myocardial differentiation of P19 cells and suppression with an antisense approach blocked differentiation. Thus, sHSPs may play important roles in the maintenance of muscle cell functions. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan.
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Regulation of the Levels of Small Heat-Shock Proteins during Differentiation of C2C12 Cells Hidenori Ito, 1 Keiko Kamei, Ikuko Iwamoto, Yutaka Inaguma, and Kanefusa Kato Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, 713-8 Kamiya, Kasugai, Aichi 480-0392, Japan
Levels of the small heat-shock proteins (sHSPs) HSP27 and ␣B-crystallin during differentiation of mouse C2C12 cells were determined using specific immunoassays. Increases of these proteins were about 3-fold and 10-fold, respectively. Under the same conditions, however, the level of HSP70 in C2C12 cells barely increased, indicating selective accumulation of HSP27 and ␣B-crystallin with differentiation. While expression of mRNA for ␣B-crystallin was also markedly increased and that for HSP27 was but to a lesser extent, mRNA for HSP70 could barely be detected during differentiation. Activation of the heat-shock factor was not observed, in contrast to the case with heatstressed undifferentiated cells. Various inhibitors of protein kinases affected the differentiation and the associated increase of sHSPs. Rapamycin, an inhibitor of p70 S6 kinase, completely inhibited the differentiation and suppressed the accumulation of HSP27 and ␣B-crystallin. SB203580, an inhibitor of p38 MAP kinase, also inhibited differentiation, but the accumulation of ␣B-crystallin was rather enhanced. PD98059, an inhibitor of MAP kinase kinase, significantly increased expression of a differentiation marker for muscle cells, creatine kinase M isozyme, as well as accumulation of ␣B-crystallin. These results suggest that accumulation of sHSPs during differentiation of C2C12 cells is regulated in a complex manner. © 2001 Academic Press
Key Words: crystallin; differentiation; HSP27; muscle; stress response.
INTRODUCTION
Small heat-shock or stress proteins (sHSPs), 2 which have a molecular mass of about 15–30 kDa, share 1 To whom correspondence and reprint requests should be addressed. Fax: ⫹81-568-88-0829. E-mail: itohide@inst-hsc. pref.aichi.jp. 2 Abbreviations used: CKM, creatine kinase M isozyme; HSPs, heat-shock or stress proteins; sHSPs, small HSPs; HSF, heat-shock factor; HSE, heat-shock element; PBS, phosphate-buffered saline; MEK, MAP kinase kinase.
several features: a homologous carboxyl-terminal amino acid sequence called the “␣-crystallin domain” [1]; formation of large oligomers that have an apparent molecular mass of ⬎500 kDa [2, 3]; induction by various stresses, which include heat stress, oxidative stress, and chemical stress [4, 5]; and phosphorylation in response to various stresses or growth factors [6 – 8]. It is also reported that the expression and the phosphorylation pattern of sHSPs change during differentiation of some types of mammalian cells, including HL-60 cells [9], rat olfactory neurons [10], and embryonic stem cells [11]. HSP27 and ␣B-crystallin, representative sHSPs, are present not only in stressed cells or tissues but also sometimes under nonstressed conditions, especially in the skeletal muscle [12, 13]. It has been reported that HSP27 is an actin-binding protein that modifies actin polymerization [14, 15] and that ␣B-crystallin interacts with actin and desmin directly and is located in the central regions of the I bands (Z-lines) in cardiomyocytes [16]. We previously reported that levels of HSP27 and ␣B-crystallin in rat hindlimb muscles change developmentally and under pathological conditions [17]. Neufer and Benjamin described expression of HSP27 and ␣B-crystallin in skeletal muscle as changing during continuous contractile activity [18]. Recently, a single mutation in ␣B-crystallin, R120G, was found in a French family suffering from desminrelated myopathy, which has an autosomal dominant myopathy that results in weakness of the proximal and distal limb muscles [19]. These findings imply that sHSPs play important roles in maintenance of the function of muscles, although their exact physiological functions remain unclear. C2C12 mouse myoblast cells are well known for their ability to differentiate into multinucleated myotubes and are often used as a model for myogenesis [20]. In this study, we determined changes in expression of sHSPs in C2C12 cells during differentiation and attempted to clarify protein kinase cascades that play regulatory roles.
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0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Cell cultures and treatments. Mouse C2C12 cells (obtained from the American Type Culture Collection) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical Co., Tokyo, Japan) supplemented with 20% fetal calf serum (EquitechBio, Inc., Ingram, TX) at 37°C in a humidified atmosphere of 95% air and 5% CO 2. For differentiation, they were grown in dishes to confluence and then the culture medium was replaced with DMEM containing 5% heat-inactivated horse serum (GIBCO, Grand Island, NY). In some experiments, protein kinase inhibitors such as 10 M SB203580, 10 M PD169316, 0.1 M rapamycin, 50 M PD98059 (all of which were purchased from Calbiochem, La Jolla, CA), or 10 M Uo 126 (Promega, Madison, WI) were added to the culture medium. Cells in each dish were rinsed with phosphate-buffered saline (PBS, containing 8 g of NaCl, 0.2 g of KCl, 1.15 g of Na 2HPO 4䡠12H 20, and 0.2 g of KH 2PO 4 in 1000 ml of H 2O) at various time periods, frozen at 80°C for a few days, collected, suspended in 0.3 ml of PBS, sonicated, and centrifuged at 125,000g for 20 min at 4°C. Supernatants were used for quantitation of HSP27, ␣B-crystallin, HSP70, and creatine kinase M isozyme (CKM). Quantitation of HSP27, ␣B-crystallin, and CKM. Concentrations of HSP27 [17], ␣B-crystallin [12], and CKM [21] in extracts of cells were estimated by specific immunoassays as described previously. Electrophoresis and Western blot analysis. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was performed by the method of Laemmli [22] in a 10 or 12.5% polyacrylamide slab gel. For Western blot analysis, proteins in polyacrylamide gels were transferred electrophoretically to nitrocellulose membranes and immunostained with affinity-purified antibodies against rat HSP27 or against the carboxyl-terminal decapeptide of ␣B-crystallin and peroxidase-labeled goat antibodies against rabbit IgG, as described previously [23]. For detection of HSP70, a mouse monoclonal antibody specific for the inducible form of HSP70 (C92F3A-5; StressGen Biotech., Victoria, BC, Canada) and peroxidase-labeled antibodies raised in a goat against mouse IgG (Medical and Biological Labs. Co., Nagoya, Japan) were used. Peroxidase activities on the nitrocellulose membranes were visualized on X-ray film using a Western blot chemiluminescence reagent (Renaissance; Dupont NEN, Boston, MA). Isolation of RNA and Northern blot analysis. Total RNA was isolated from cells with a QuickPrep total RNA Extraction kit (Amersham Pharmacia Biotech, Tokyo, Japan). Twenty micrograms of total RNA was subjected to electrophoresis on a 0.9% agarose–2.2 M formaldehyde gel and blotted onto nitrocellulose membranes. For Northern blot analysis, membranes were allowed to hybridize with cDNA probes that had been labeled with 32P using a Multiprime DNA labeling system (Amersham Pharmacia Biotech), as described previously [23]. A BamHI-HindIII fragment of cDNA for mouse HSP27 [24] was kindly provided by Dr. L. F. Cooper, University of North Carolina, and a PstI fragment of cDNA for bovine ␣B-crystallin [25] by Dr. H. Bloemendal, University of Nijmegen, The Netherlands. An EcoRI fragment of cDNA for HSP70 was obtained from StressGen Biotech. For determination of the stability of mRNA for ␣B-crystallin during differentiation, 40 g (for control cells) or 20 g (for differentiated cells) of total RNA was electrophoresed, hybridized, and autoradiographed. Relative densities of bands were quantified using an NIH image program (National Institutes of Health, Bethesda, MD). Gel shift assays of the heat-shock transcription factor. Cells were harvested at 0°C in 20 mM Hepes–KOH buffer, pH 7.9, that contained 25% (v/v) glycerol, 0.5 M NaCl, 1.5 mM MgCl 2, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol, 0.5 g/ml pepstatin A, and 0.3 mg/ml Pefablock SC (Boehringer Mannheim, Mannheim, Germany). The suspension was sonicated and centrifuged at 4°C at 125,000g for 20 min, and supernatants that contained 15 g protein were incubated at 0°C for 20 min with
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P-labeled oligonucleotide (heat-shock element of Drosophila HSP70; 5⬘-GCCTCGAATGTTCGCGAAGTTTCG-3⬘) [26] in 10 mM Tris–HCl buffer, pH 8.0, containing 0.5 g poly(dI– dC) (Amersham Pharmacia Biotech), 50 mM NaCl, 1 mM EDTA, 5% glycerol, and 0.5 mM dithiothreitol. Reaction mixtures were then subjected to electrophoresis in 4% polyacrylamide gels and autoradiographed with X-ray film. Other methods. Concentrations of protein in soluble extracts were estimated with a protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. Rat HSP27 [17], ␣B-crystallin [12], and CKM [21], used as standards for immunoassays, were purified from skeletal muscle.
RESULTS
Changes in Levels of HSP27, ␣B-Crystallin, and CKM during Differentiation of C2C12 Cells The C2C12 cells began to fuse at 2–3 days after the medium was changed to induce differentiation, and the surface of the culture dish became covered with multinucleated myotubes after 5– 6 days (Fig. 1A). Although CKM was undetectable in undifferentiated cells (⬍1 ng/mg protein), its concentration markedly increased after 3 to 6 days of differentiation (Fig. 1B), as reported previously [27]. The level of ␣B-crystallin in undifferentiated cells was also low (about 50 –100 ng/mg protein) but increased after 3 days of differentiation and this continued to day 6 (Fig. 1C). The magnitude varied between experiments but the level of ␣B-crystallin reached about 5- to 10-fold of that of control myoblasts in 6-day-differentiated cells (Fig. 1C). The level of HSP27 in control myoblasts was relatively high (about 1000 ng/mg protein) and increased during differentiation (Fig. 1D). The magnitude again varied with the experiment, with the level of HSP27 reaching about 1.5- to 4-fold of that in the control cells after 6 days (Fig. 1D). Western Blot Analyses of HSP27, ␣B-Crystallin, and HSP70 To determine whether another representative stress protein, HSP70, was also increased during differentiation of C2C12 cells, we performed Western blot analyses of cell extracts. This confirmed that the level of ␣B-crystallin markedly increased during differentiation (Fig. 2). The amount of HSP27 also tended to increase, although not to the same degree as that of ␣B-crystallin (Fig. 2). The level of HSP70 in control myoblasts was very low and increased only slight during differentiation, in contrast to its marked expression with heat (Fig. 2). These results indicate the differentiation-dependent accumulation to be preferential for sHSPs, especially ␣B-crystallin, in C2C12 cells. Northern Blot Analyses of HSP27, ␣B-Crystallin, and HSP70 We estimated changes in the levels of mRNAs for HSP27, ␣B-crystallin, and HSP70 during differentia-
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FIG. 1. Differentiation of C2C12 cells (A) and associated changes in the intracellular levels of CKM (B), ␣B-crystallin (C), and HSP27 (D). When cells reached confluence, the medium was changed to induce differentiation as described under Materials and Methods. The cells were harvested at the indicated times and the levels of three proteins estimated using specific immunoassays. Each point shows the mean ⫾ SD of results for four dishes.
tion of C2C12 cells using Northern blot analyses. Consistent with the results for protein levels, the mRNA for ␣B-crystallin in differentiated cells was clearly increased (Fig. 3A). This was also the case for HSP27, to a lesser extent (Fig. 3A). The mRNA for the inducible form of HSP70 (top band), which was induced by heat treatment, was not induced after differentiation, although the mRNA for a constitutive form of HSP70 (bottom band) was detected in control myoblasts as described previously [28] (Fig. 3A). To clarify the mechanism of the differentiation-dependent increase of ␣Bcrystallin, we estimated the stability of mRNA for ␣Bcrystallin using an inhibitor of transcription, actinomycin D. As shown in Figs. 3B and 3C, levels of mRNA for ␣B-crystallin decreased after actinomycin D treatment both in control and in differentiated cells. The decrease of mRNA in differentiated cells was slightly slower than that in control cells, although the
FIG. 2. Results of Western blot analyses of HSP70, HSP27, and ␣B-crystallin. When cells reached confluence, they were cultured with differentiation medium for the indicated periods. Aliquots of cell extract containing 20 g of protein for detection of HSP70 or ␣B-crystallin or 10 g of protein for detection of HSP27 were subjected to SDS–PAGE with subsequent Western blot analysis with antibodies against HSP70, HSP27, and ␣B-crystallin. Hs, undifferentiated cells heated at 42°C for 30 min and then cultured at 37°C for 8 h.
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FIG. 3. Northern blot analyses of HSP70, HSP27, and ␣B-crystallin (A) and estimation of the stability of mRNA for ␣B-crystallin (B and C). (A) Cells at different differentiation time points were harvested for isolation of total RNA. Twenty-microgram aliquots of RNA from each sample were subjected to electrophoresis with subsequent Northern blot analysis with cDNA probes for HSP70, HSP27, and ␣B-crystallin. Lanes 1 and 2, control cells; lanes 3 and 4, 3-day-differentiated cells; lanes 5 and 6, undifferentiated cells heated at 42°C for 30 min and then cultured at 37°C for 4 h. Bands for 28S rRNA are shown for reference. (B) Control cells and 5-days-differentiated cells were treated with 1 g/ml actinomycin D and were harvested at indicated times. Forty- (for control cells) or twenty-microgram (for differentiated cells) aliquots of RNA from each sample were subjected to Northern blot analysis using a cDNA probe for ␣B-crystallin. Bands for 28S rRNA are shown for reference. (C) The densities of bands shown in (B) were quantified and graphed. The data were shown as relative density to the cells at time 0.
extent of the difference between them was not much. This result suggests that the accumulation of ␣B-crystallin during differentiation may be mainly due to the transcriptional activation.
kinase cascade and a rapamycin-sensitive pathway [29]. We also examined the effects of protein kinase inhibitors on their differentiation-dependent accumulation of sHSPs. Consistent with the previous report
Heat-Shock Transcription Factor Is Not Activated during Differentiation of C2C12 Cells It is well known that activation of heat-shock gene transcription is mediated by the heat-shock transcription factor (HSF). Its involvement in the differentiation-dependent increase of sHSPs was therefore examined. As shown in Fig. 4, activation of HSF was not observed at any stage of differentiation of C2C12 cells, although it was clear when undifferentiated cells were subjected to heat stress. These results indicate that the differentiation-dependent increase of sHSPs in C2C12 cells is not due to activation of HSF and that another mechanism must be involved. Effects of Protein Kinase Inhibitors on the Formation of Myotubes and the Differentiation-Dependent Accumulation of sHSPs Recently, Cuenda and Cohen reported differentiation of C2C12 cells to be regulated by the p38 MAP
FIG. 4. HSE-binding activity in extracts of differentiating cells and heat-stressed undifferentiated cells. Cell extracts were reacted with 32P-labeled oligonucleotide, which contained the HSE sequence, at 0°C for 20 min, and the reaction mixtures were subjected to acrylamide gel electrophoresis. The gel was dried and autoradiographed with X-ray film. Hs, undifferentiated cells treated at 43°C for 30 min.
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FIG. 5. Effects of protein kinase inhibitors on the formation of myotubes. C2C12 cells were grown in medium that contained 20% fetal calf serum (FCS) until reaching confluence and then differentiated with medium contained 5% horse serum in the presence or absence of 10 M SB203580, 0.1 M rapamycin, or 50 M PD98059 for 3 or 6 days.
[29], addition to the differentiation medium of 10 M SB203580, an inhibitor of p38 MAP kinase, or more particularly, 0.1 M rapamycin, an inhibitor of p70 S6 kinase, caused suppression of the formation of myotubes (Fig. 5). In contrast, addition of 50 M PD98059, an inhibitor of MAP kinase kinase (MEK) caused formation of myotubes the shapes of which were different from those of control myotubes (Fig. 5). The differentiation-dependent increase in the level of CKM, a differentiation marker of muscle cells, was also suppressed by the addition of SB203580 or rapamycin to the medium (Fig. 6A), consistent with the degree of formation of myotubes. Addition of PD98059
did not suppress but rather increased the level of CKM (Fig. 6A). Addition of Rapamycin to the medium also caused the suppression of a differentiation-dependent increase of HSP27 and ␣B-crystallin in C2C12 cells (Figs. 6B and 6C). Surprisingly, SB203580 and PD98059 both markedly increased the accumulation of ␣B-crystallin and slightly increased that of HSP27 at early stages of differentiation (Figs. 6B and 6C). We also determined the effect of PD169316, another inhibitor of p38 MAP kinase, and Uo 126, a second inhibitor of MEK, on the accumulations of CKM and ␣B-crystallin, the results being similar to those with SB203580 and PD98059 (Figs. 6A and 6B), respectively (Table 1).
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FIG. 6. Effects of protein kinase inhibitors on the accumulation of CKM (A), ␣B-crystallin (B), and HSP27 (C). Cells were grown in medium that contained 20% FCS until reaching confluence and then cultured in medium that contained 5% heat-inactivated horse serum in the presence (closed symbols) or absence (open circles) of 10 M SB203580 (closed circles), 0.1 M rapamycin (closed squares), or 50 M PD98059 (closed triangles) for the indicated periods. Cells were harvested and estimated for levels of three proteins using specific immunoassays. Each point shows the mean ⫾ SD of results for 4 –5 dishes.
Western Blot and Northern Blot Analyses of Cells That Had Been Treated with Protein Kinase Inhibitors To confirm the effects of protein kinase inhibitors on the accumulation of ␣B-crystallin during differentiation of C2C12 cells, we performed Western blot analyses of cell extracts. Consistent with immunoassay results, SB203580 and PD98059 enhanced, while rapamycin suppressed, the accumulation (Fig. 7A). Northern blot analyses revealed similar effects on
TABLE 1 Effects of PD169316 and Uo 126 on the DifferentiationDependent Accumulation of CKM and ␣B-Crystallin in C2C12 cells Concentration a (ng/mg protein) Treatment Day Day Day Day
0 6 6 ⫹ PD169316 6 ⫹ Uo 126
CKM
␣B-crystallin
0.861 ⫾ 0.113 1460 ⫾ 115 547 ⫾ 74.8 2320 ⫾ 256
19.8 ⫾ 6.99 591 ⫾ 53.7 937 ⫾ 114 3640 ⫾ 316
a Concentrations of CKM and ␣B-crystallin in extracts of cells were determined by specific immunoassays. Each value is the mean ⫾ SD of the results from three to five dishes.
FIG. 7. Western blot analysis (A) and Northern blot analysis (B) of C2C12 cells treated with various protein kinase inhibitors. (A) Cells were grown in medium with 20% FCS until reaching confluence and then cultured in medium with 5% heat-inactivated horse serum in the presence (lanes 5–10) or absence (lanes 3 and 4) of 10 M SB203580 (lanes 5 and 6), 50 M PD98059 (lanes 7 and 8), or 0.1 M rapamycin (lanes 9 and 10) for 6 days. Aliquots of cell extracts containing 10 g of protein were subjected to SDS–PAGE with subsequent Western blot analysis with antibodies against ␣B-crystallin. Lanes 1 and 2, control undifferentiated myoblasts. (B) Cells at 4 days after differentiation in the presence (lanes 3 to 5) or absence (lane 2) of 10 M SB203580 (lane 3), 50 M PD98059 (lane 4), and 0.1 M rapamycin (lane 5) were harvested for isolation of total RNA. Twenty micrograms of RNA from each sample was subjected to electrophoresis with subsequent hybridization with a cDNA probe for ␣B-crystallin (top panel). Bands for 28S rRNA are shown for reference. Lane 1, control undifferentiated myoblasts.
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mRNA levels (Fig. 7B), suggesting that protein kinase inhibitors regulate the expression of ␣B-crystallin during differentiation of C2C12 cells at the transcriptional level. DISCUSSION
In the present study, concentrations of HSP27 and especially ␣B-crystallin were found to increase during differentiation of C2C12 cells into multinucleated myotubes. Recently, Sugiyama et al. reported similar results, describing increases of sHSPs such as MKBP/HSPB2, HSPB3, and ␣B-crystallin during differentiation of C2C12 cells, although the levels of HSP27 remained unchanged [30]. Under our experimental conditions, undifferentiated C2C12 cells already expressed HSP27 to a relatively high level and an increase during differentiation was only clearly detectable with immunoassay and not Western blot analysis. The regulation of sHSP induction is not fully understood, but it is believed that activation by heat-shock factor is important because heat-shock elements (HSEs) are present in the promoter regions of the HSP27 and ␣B-crystallin genes [31, 32]. Activation of transcription is induced after binding of the trimeric HSF to HSEs [33]. However, other mechanisms may be involved [34, 35]. During differentiation of C2C12 cells in the present experiments, the levels of sHSPs and their mRNAs, especially with ␣B-crystallin, increased without a significant increase of HSP70 (Figs. 2 and 3A). In addition, we could not observe any activation of HSF, in clear contrast to the heat-treatment case (Fig. 4). These results indicate that the mechanism(s) underlying the differentiation-dependent increase of sHSPs is quite different from that with stress induction. Addition of actinomycin D, an inhibitor of transcription, caused the decrease of mRNA for ␣B-crystallin both in control cells and in differentiated cells. The decrease in control cells was slightly faster than that in differentiated cells but the degree of the difference between them was not large (Figs. 3B and 3C). This result suggests that the increase of mRNA for ␣Bcrystallin may mainly be due to the transcriptional activation. It has been reported that a consensus muscle regulatory factor-binding site called MRF is present in the ␣B-crystallin gene [36]. Investigation of whether it might be involved in the differentiation-dependent increase of ␣B-crystallin under our experimental conditions is clearly warranted. There are several reports of a relationship between the differentiation of muscle cells and protein kinase action. Coolican et al. demonstrated that IGF-stimulated differentiation of L6A1 myoblasts was accelerated by PD98059 and inhibited by rapamycin [37]. Gredinger et al., on the other hand, showed that
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PD98059 inhibited the differentiation of C2C12 cells [38]. Zetser et al. reported inhibition of the differentiation of L8 cells by SB203580 [39]. Cuenda and Cohen found the same compounds SB203580 and rapamycin to inhibit the differentiation of C2C12 cells, although PD 98059 had no effect [29]. In this study, we demonstrated that rapamycin and SB203580 inhibited or delayed the differentiation of C2C12 cells. However, PD98059 tended to increase the level of CKM, a differentiation marker for muscle cells (Fig. 6A), and also alter the shape of myotubes formed (Fig. 5). In terms of the levels of sHSPs, the effects of SB203580 and rapamycin were quite different (Figs. 6B and 6C). Rapamycin was the more powerful inhibitor of myotube formation under our experimental conditions. The mechanism of the effect of SB203580 on the differentiation-dependent increase of ␣B-crystallin (Fig. 6B) is not clear. Since the level of HSP27 during differentiation was barely affected (Fig. 6C), addition of SB 203580 may not be by itself a stress and there may exist intrinsic transcriptional regulation for ␣B-crystallin. As described above, addition of protein kinase inhibitors during differentiation of C2C12 cells affected myotube formation and accumulation of sHSPs in a complex manner. These results indicate that the p38 MAP kinase pathway, the p70S6 kinase pathway, and the p44/42 MAP kinase pathway may play different roles in differentiation of muscle cells and accumulation of sHSPs during differentiation of C2C12 cells. The functions of sHSPs in muscle cells are obscure, although there are several reports of relationships with pathology or physiology. The precise role of sHSPs in differentiation of C2C12 cells also remains to be clarified. It has been reported that resistance to apoptosis is conferred during differentiation of C2C12 cells [40] and that sHSPs exert an anti-apoptotic effect in mammalian cells [41]. Recently, Davidson and Morange reported HSP27 involvement in differentiation of cardiomyocytes [42]. They showed an increase of the level of HSP27 during myocardial differentiation of P19 cells and suppression with an antisense approach blocked differentiation. Thus, sHSPs may play important roles in the maintenance of muscle cell functions. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan.
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