Involvement of PKR in the Regulation of Myogenesis1

Yosefa Kronfeld-Kinar, Shlomit Vilchik,Tehila Hyman, Flavio Leibkowicz, andSamuel Salzberg2

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900[Y. K-K., S. V., T. H., S. S.], and Laboratory of Microbiology,Rambam Medical Center, Haifa [F. L.], Israel

AbstractThe involvement of the double-stranded RNA-activatedprotein kinase PKR in the regulation of the myogenicprocess was investigated. For this purpose, the murinemyogenic cell line C2C12 was used. The cells werefirst cultivated in either growth medium ordifferentiation medium (DM), and the activation of PKRduring differentiation was determined by monitoring itsenzymatic activity and by immunoblot analysis. Asignificant increase in both parameters was detectedalready at 24 h in DM, whereas in cells grown ingrowth medium, the increase was evident only after96 h, when spontaneous differentiation was observedin highly crowded cultures. Consequently, weestablished the direct effect of PKR activation on themyogenic process. C2C12 cells were transfected withan expression vector harboring a cDNA moleculeencoding human PKR fused to the induciblemetallothionein promoter. One of the clones (clone 8)expressing high levels of PKR was selected and furtheranalyzed. In the presence of ZnCl2, which activates thepromoter, the rate of cell growth of the transfectedcells was clearly reduced compared to that of wild-type C2C12 cells transfected with only the neomycin-resistant gene (C2-NEO). In addition, alteredmorphology with partial fusion was observed.Biochemically, an increase in creatine kinase activityaccompanied by an increased rate of expression of themyogenic protein troponin T and the myogenictranscription factors myoD and myogenin was detectedin clone 8 cells exposed to ZnCl2. Most importantly, aninduction in the level of cyclin-dependent kinaseinhibitor p21WAF1 and an increase in the level of theunderphosphorylated active form of the tumorsuppressor protein pRb concomitant with the down-regulation of cyclin D1 and c-myc were also evident inthe transfected clones. These changes were similar to

those observed in normal C2C12 cells cultivated in DM.We conclude that PKR is an important regulatoryprotein participating in the myogenic process.

IntroductionPKR, a double-stranded RNA-activated serine-threonineprotein kinase, has been originally described as an IFN-inducible enzyme implicated in antiviral activity (1, 2).Upon activation, the enzyme molecule is first autophos-phorylated at several sites, followed by the phosphoryla-tion of the target molecules (3). Several such targets havebeen reported. The best characterized of these is the a

subunit of the translation initiation factor eIF-2. The factoris phosphorylated on serine residue 51; consequently, theexchange of the eIF-2-bound GDP with GTP by exchangefactor eIF-2B is blocked (4), resulting in the inhibition ofprotein synthesis. In addition, the transcription factor in-hibitor IkB (5) and the HIV-specific Tar-binding protein tat(6) were also described as possible (although not neces-sarily direct) targets phosphorylated by PKR. The genesencoding PKR both from human (7) and mouse (8) originwere isolated and characterized, and the structural do-mains present in the protein molecule were elucidated.The NH2-terminal portion of the enzyme appears to con-tain the double-stranded RNA binding motif and the abilityto interact with other protein molecules, including itself(9 –11). However, it is still not clear whether dimerization isindeed required for the PKR-mediated biological effects(12). The catalytic domains of the enzyme, on the otherhand, are all located in the COOH-terminal region (13). Ascan be judged from its mode of action, PKR is not involvedonly in antiviral activity but has a much broader biologicalsignificance. It has been clearly demonstrated that ectopicexpression of negative dominant mutants of PKR in NIH/3T3 mouse fibroblasts resulted in their malignant transfor-mation (14 –16), indicating that the enzyme has a tumor-suppressive effect and is most likely associated with theregulation of cell growth. In addition, overexpression ofwild-type PKR was reported to induce apoptosis in sus-ceptible cells, whereas expression of bcl-2 or mutatedPKR was able to protect the cells from this event (17, 18).Interestingly, PKR can modulate the function of the signaltransducer and activator of transcription STAT1 by asso-ciation or dissociation between these two proteins (19).Finally, PKR was reported to down-regulate the expres-sion of c-myc in growth-retarded M1 cells transfected withwild-type PKR (20). Taken together, these results suggestthat the enzyme fulfills a pivotal regulatory role within thecell. However, it is still not clear whether it is involved indifferentiation processes. To address this question, westudied the effect of the ectopic expression of PKR onmyogenesis.

Skeletal muscle cell cultures are an excellent experimentaltool for the study of differentiation in vitro. Upon withdrawal

Received 10/7/98; revised 12/7/98; accepted 1/22/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 Supported by grants from the Paula Better Estate, the Harvest CancerFund, the Bar-Ilan University Research Authority, and the Brazilian Friendsof the Israel Cancer Association.2 To whom requests for reprints should be addressed, at Faculty of LifeSciences, Bar-Ilan University, Ramat-Gan 52900, Israel. Phone: 972-3-5318220; Fax: 972-3-6356041; E-mail: salzbs@mail.biu.ac.il.

201Vol. 10, 201–212, March 1999 Cell Growth & Differentiation

from the cell cycle, myogenic committed cells known asmyoblasts fuse to form myotubes. This process is well con-trolled by a series of myogenic specific transcription factors,the myoD family. The family consists of myoD, myogenin,Myf-5, and MRF4, all of which contain a DNA-binding basicregion and a helix-loop-helix motif responsible for interactionwith other proteins (21), mostly with products of the E2Agene, such as E12 or E47 (22). This complex binds to anelement on the DNA termed the E box with the consensussequence CANNTG. Although myoD homodimers are as sta-ble as the myoD-E12 heterodimers, only the latter bind to theE box (23). It should be noted, however, that additionalproteins are involved in modulating the activity of the myoDfamily (24).

IFN3 has been previously shown to induce morpholog-ical and biochemical changes in several cell systems,including skeletal myogenic cell cultures (25–27). In addi-tion, the expression of both PKR and 2-5A synthetase,another IFN-induced protein, was reported to increaseduring myogenic differentiation in vitro (28). Furthermore,various agents that inhibit myogenesis were also effectivein interfering with the expression of these proteins (29, 30).Most recently, it has been shown that an additional IFN-inducible protein, p202, increased during skeletal muscle

3 The abbreviations used are: IFN, interferon; GM, growth medium; DM,differentiation medium; CDK, cyclin-dependent kinase.

Fig. 1. Activation of PKR dur-ing myogenic differentiation ofC2C12 cells. The cells wereseeded in GM, and the mediumwas replaced with either GM(DIVISION) or DM (DIFFEREN-TIATION) 24 h later. At the indi-cated times thereafter, cell ex-tracts were prepared, and PKRwas identified either by immu-noblot analysis (A) or by its en-zymatic activity (B). Densitom-etry of A and B is presented in Cand D, respectively.

202 Involvement of PKR in Myogenesis

differentiation (31). However, these elevated levels may befortuitous, and more direct evidence is needed to clarifywhether PKR plays a role in the myogenic process. In thisreport, we show that ectopic expression of PKR in myo-genic cells results in morphological, molecular, and bio-chemical alterations associated with skeletal muscle dif-ferentiation.

ResultsActivation of PKR during Myogenic Differentiation ofC2C12 Cells. We had to establish first whether PKR is spe-cifically activated during induction of differentiation of themyogenic cell line C2C12. The cells were cultivated in eitherGM or DM. Cell extracts were prepared at different times,and the presence of PKR was identified by immunoblot anal-ysis using polyclonal antibodies directed against humanPKR. The PKR protein appeared as a broad band of 66–68kDa (Fig. 1A). In parallel, the level of the biologically activeprotein was determined by monitoring its enzymatic activityunder the same conditions. The 32P-labeled autophospho-rylated form of PKR, which was 67 kDa in size, was anindication of enzymatic activity (Fig. 1B). The basal level ofthe PKR protein at the initiation of the experiment (zero time)was low (data not shown) and did not change within the first24 h in GM (Fig. 1A, DIVISION, lane 1; Fig. 1C). However, agradual increase was observed with time in culture up to144 h (Fig. 1 A, C, DIVISION). It should be noted that a fewmyotubes were always visible in crowded C2C12 cells evenwhen cultivated in GM, an indication of spontaneous differ-entiation. The kinetics of PKR enzymatic activity obtainedwith C2C12 dividing cells (Figs. 1, B and D, DIVISION) wassimilar to that observed with the level of the PKR protein,demonstrating that the gradual increase in this activity re-flects an increase in the total amount of PKR. In contrast, inthe case of C2C12 cells grown in DM, a significant increasein both the level of PKR and its enzymatic activity was

already evident at 24 h after the medium change (Fig. 1, Aand B, DIFFERENTIATION). The level of both parametersremained constant up to 96 h and was followed by a de-crease. At this time, the cultures were fully differentiated.

Transfection of PKR into C2C12 Cells. The fact thatPKR is induced during differentiation of C2C12 cells does notnecessarily imply that the enzyme plays a role in myogenesis.To show its direct involvement in the process, we con-structed plasmid pMPKR, which harbors a cDNA encodinghuman PKR fused to the metallothionion promoter (see “Ma-terials and Methods”). This plasmid was cotransfected intoC2C12 cells with pSVneo, and 25 neomycin-resistant cloneswere isolated and expanded. Eight clones expressed highlevels of PKR in response to the presence of ZnCl2 (whichactivates the promoter). The results obtained with a repre-sentative clone, clone 8, are presented in Fig. 2; however twoadditional clones that were analyzed similarly yielded com-parable results that were not included for the sake of sim-plicity. For control cells, we used a clone transfected withpSVneo only. This clone was designated C2-NEO. Immuno-blot analysis as well as a determination of PKR enzymaticactivity indicated that the basal level of PKR in either C2-NEOor clone 8 cells was rather low; however, only clone 8 cellsexposed to ZnCl2 for 24 h responded with an elevated levelof PKR protein (Fig. 2A, lane 6) and an increase in its enzy-matic activity (Fig. 2B, lane 6). In contrast, both C2-NEO andclone 8 cells responded equally well to a 24-h treatment withIFN (Fig. 2, A and B, lanes 2 and 5), an indication that theendogenous PKR-encoding gene is functional in these typesof cells.

Morphological Alterations and Growth Characteristicsof PKR-expressing C2C12 Cells. To establish whetherPKR is indeed involved in the initiation of the myogenicprocess, it was important to study the effect of its ectopicexpression on a variety of biological parameters. First, C2-NEO and clone 8 cells were grown in GM in the presence or

Fig. 2. Expression of PKR intransfected clone 8 cells. C2-NEO and clone 8 cells weretreated with either IFN or ZnCl2(Zn21) for 24 h. One group ofcultures remained untreated (C).Cell extracts were prepared, andPKR was identified either by im-munoblot analysis (A) or by en-zymatic activity (B). Densitome-try of A and B is presented in Cand D, respectively.

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absence of ZnCl2 for 96 h, and changes in cell morphologywere examined microscopically. Exposure of control cells toZnCl2 did not result in any alterations in the morphology ordensity of treated cells compared to those of untreated cells(compare Fig. 3, A and C). On the other hand, a strikingdifference was observed when clone 8 cells were similarlyanalyzed. Cell density was greatly reduced in ZnCl2-treatedcultures, most of the cells developed extended processes,and about 30% formed short myotubes comprised of two tothree cells (compare Fig. 3, B and D). The retarded growthrate of clone 8 cells exposed to ZnCl2 was confirmed in theexperiments described in Fig. 5. The growth rates of normalC2C12 cells grown in GM or DM were compared. Vital cellcounts were performed daily after the medium change. Cellswere labeled in parallel for 1.5 h with [3H]thymidine, and therate of DNA synthesis was determined. As expected, bothparameters were significantly reduced in differentiating ver-sus dividing C2C12 cells (Fig. 4, A and B). We then comparedthe rates of growth and DNA synthesis of C2-NEO and clone8 cells cultivated in GM in the presence or absence of ZnCl2.It is clearly shown that whereas C2-NEO cells were hardly

affected by exposure to ZnCl2, the growth rate (Fig. 4C) andkinetics of DNA synthesis (Fig. 4D) were decreased in clone8 cells treated with ZnCl2. We conclude that cultivation in DMand ectopic expression of PKR induce similar effects on thegrowth characteristics of C2C12 cells.

Appearance of Muscle-specific Proteins and Tran-scription Factors in C2C12 Transfected Cells. The re-duced rate of proliferation of muscle cultures is accompaniedby the expression of muscle-specific proteins. This was con-firmed in our study by the following observation. The levels ofcreatine kinase activity and troponin T gradually increasedwith time up to 120 h when C2C12 cells were grown in DM.In contrast, when cells were cultivated in GM, the level ofthese muscle-specific proteins remained low, and an in-crease became evident only after 96–144 h (Fig. 5, A, I andB, I). At this late time, spontaneous differentiation was ob-served in highly crowded cultures. We next determinedwhether the growth inhibition detected in clone 8 cells ex-pressing PKR is also accompanied by an elevated level ofmuscle-specific proteins. C2-NEO and clone 8 cells weregrown in the presence and absence of ZnCl2, and the levels

Fig. 3. Effect of ZnCl2 on the morphology of C2C12 cell variants. C2-NEO (A and C) and clone 8 (B and D) cells were seeded in GM (2Zn21) (A and B).Some cultures were exposed to ZnCl2 3 h later (1Zn21) (C and D). After an additional 96 h, the cell morphology was examined with a phase-contrastmicroscope (3125).

204 Involvement of PKR in Myogenesis

of both creatine kinase activity and troponin T were deter-mined at daily intervals. The results demonstrate that thepresence of ZnCl2 did not affect the level of either protein inC2-NEO cells (Fig. 5, A, II and B, II). However, the appear-ance of both proteins was significantly accelerated in ZnCl2-treated versus untreated clone 8 cells (Fig. 5, A, II and B, III),indicating that the ectopic expression of PKR induces thesynthesis of muscle-specific proteins.

Next, we wanted to provide evidence that the myogenictranscription factor myoD (32) is also expressed in clone 8cells expressing PKR. Total RNA was therefore extractedfrom ZnCl2-treated and untreated clone 8 cultures, andNorthern blot analysis was performed using a myoD-specificprobe. For comparison, a similar analysis was performed ondividing and differentiating C2C12 cells. The results demon-strate that whereas a significant increase in the level ofmyoD-specific RNA transcripts was observed in C2C12 cellsgrown in GM only after 120 h (Fig. 6, A, I), a major increasewas detected in differentiating cells cultivated for 24 h in DM,followed by a decrease only at 144 h (Fig. 6, A, II). Thus, asexpected, differentiation of C2C12 cells is accompanied by

an increased expression of the myoD-encoding gene. Inparallel, it is clearly shown that in ZnCl2-treated clone 8 cells(ectopically expressing PKR), an elevated expression ofmyoD was observed at 72–120 h (Fig. 6, B, II), whereas incontrol (untreated) cells, as in the case of dividing C2C12cells, an increased expression of myoD was detected only at120 h (Fig. 6, B, I). These results were confirmed by immu-noblot analysis in which the level of the myoD protein as wellas that of myogenin, a second myogenic transcription factor,was established. The results indicate that the amount of bothproteins increased in differentiating C2C12 cells, with a peakobserved at 48 h in DM, followed by a decrease thereafter. Individing cells, on the other hand, the amount remained low,and an increase was observed only at 120–144 h (Fig. 7, A,I and B, I). We then performed a similar analysis on clone 8cells grown in GM in the presence or absence of ZnCl2, usingC2-NEO cells grown under similar conditions as an addi-tional control. As expected, no effect of ZnCl2 was observedon the level of myogenin or myoD in C2-NEO cells (Fig. 7, A,II and B, II). However, in the case of clone 8 cells, the level ofboth proteins increased at least 24 h earlier in ZnCl2-treated

Fig. 4. Kinetics of cell growth and DNA synthesis. C2C12 cells were cultured in GM, and after 24 h, the medium was replaced with either GM (DIVISION)or DM (DIFFERENTIATION). At the indicated times thereafter, viable cell counts were performed (A). The cultures were labeled in parallel with [3H]-thymidine,and acid-insoluble radioactivity was determined (B). Similarly, C2-NEO and clone 8 cells were seeded in GM (Ct). Some of the cultures were treated withZnCl2 3 h later (Zn). The rates of cell growth (C) and DNA synthesis (D) were determined as described above.

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cells compared to untreated cells (Fig. 7, A, III and B, III). Weconclude that activation of PKR in transfected cells en-hances the synthesis of myogenic transcription factors.

Effect of Ectopic Expression of PKR on Cell Cycle-associated Proteins. Myogenic cells withdraw from the cellcycle during terminal differentiation. This process is associ-ated with changes in the expression of cell cycle-regulatedproteins (for a review, see Walsh and Perlman, Ref. 33). Toexplore whether ectopic expression of PKR is accompaniedby some of these changes, we studied the kinetics of c-myc,cyclin D1, the CDK inhibitor p21WAF1, and pRb synthesis,also known to be affected by IFN (34, 35). In agreement withearlier reports (33), the induction of p21WAF1 synthesis, thedown-regulation of cyclin D1, and the accumulation of theunderphosphorylated form of pRb were observed in C2C12differentiating cells after 24 h in DM. In dividing cells culti-

vated in GM, these changes occurred only at 120 h (Fig. 8,A–C, I). In a similar analysis performed on C2-NEO cellsgrown in the presence or absence of ZnCl2, a comparablepattern of the expression of p21WAF1, cyclin D1, or pRb wasevident in these two cell populations (Fig. 8, A–C, II). How-ever, in ZnCl2-treated clone 8 cells, we observed an in-creased synthesis of p21WAF1, as indicated by a 3.5- and5.3-fold increase over the background level at 72 and 96 hafter exposure to ZnCl2, respectively (Fig. 8, A, III). This is incontrast to a 1.3- and 1.2-fold increase in the level ofp21WAF1 detected at 72 and 96 h, respectively, in ZnCl2-treated C2-NEO cells (Fig. 8, A, II). In addition, the down-regulation of cyclin D1 and the accumulation of pRb (under-phosphorylated) were accelerated and occurred 24–48 hearlier in ZnCl2-treated versus untreated (control) C2-NEOcells (Fig. 8, B and C, III). Similarly, the reduction in c-mycexpression characteristic for C2C12 differentiating cells (Fig.9I) was detected in ZnCl2-treated clone 8 cells at least 24 hearlier than in control cells (Fig. 9III). It is thus concluded thatPKR is involved in the regulation of cell cycle-associatedproteins.

DiscussionThe IFN system as a whole has been previously shown toexhibit antiproliferation properties against a variety of celltypes (34). Furthermore, it seems that molecular events as-sociated with the cell cycle are some of the major targetsaffected by IFN. Thus, the reduction in c-myc expression, theaccumulation of the underphosphorylated form of pRb (36–39) with the concomitant down-regulation of cyclins andCDKs (40–42), the reduced levels of the active E2F family oftranscription factors (38, 40, 43), and the induction of CDK-inhibitory proteins (38, 41, 42) were reported to be the resultof IFN activity. However, most of the IFN-induced biologicalactivities are mediated by a variety of proteins activated byIFN via a unique signal transduction pathway (44). Accord-ingly, PKR has been shown to mediate IFN-induced c-mycsuppression in M1 myeloid leukemia cells (20). In addition,the ratio of the transcriptional activator of IFN-induced genes(IRF-1) to a suppressor of these genes (IRF-2) is high ingrowth-arrested cells and low in proliferating cells. Further-more, deletion of the IRF-1-encoding gene or overexpressionof IRF-2 may result in the development of tumors, includingthose of human origin (45). Finally, it has been demonstratedrecently that ectopic expression of 2-5A synthetase in mye-loid cells induces cell growth arrest, a reduction in c-mycexpression, an accumulation of the underphosphorylatedform of pRb, and the appearance of a myeloid differentiationmarker (46).

In agreement with the concept that IFN-induced proteinsplay a role in the regulation of cell growth and differentiation,we show in the present report that PKR is activated duringC2C12 myogenic cell differentiation. These results supportearlier findings on the induction of PKR activity in rat primaryskeletal muscle cultures (28) or in differentiating rat L8 myo-genic cells (30). It is not surprising to see that, even individing C2C12 cells, an elevated level of PKR was observedat 96 h after the initiation of the experiment (Fig. 1) becausespontaneous differentiation is common in crowded cultures.

Fig. 5. Determination of muscle-specific proteins. C2C12 cells weregrown in GM (DIVISION) or DM (DIFFERENTIATION). Creatine kinaseactivity was determined at the indicated times by an enzymatic assay (A,I), and troponin T was identified by immunoblot analysis (B, I). In parallel,C2-NEO (A, II and B, II) and clone 8 cells (A, II; B, III) cultivated in GM inthe absence (control, C) or presence of ZnCl2 were similarly analyzed.

206 Involvement of PKR in Myogenesis

This was accompanied by cell growth arrest (Fig. 4, A and B)and by elevated levels of muscle-specific proteins (Fig. 5, A,I and B, I); Fig. 7) detected at late times in C2C12 cellscultivated in GM. However, the most striking phenomenondemonstrated in our study was the fact that ectopic expres-sion of PKR in myogenic cells exposed to ZnCl2 induced avariety of morphological, biochemical, and molecularchanges characteristic of myogenic differentiation. Thus, al-though complete elongated myotubes were not detected inthese cultures, a change in cell morphology and the forma-tion of short myotubes consisting of three cells were com-mon (Fig. 3D). In addition, a retardation of cell growth (Fig. 4,C and D) coupled with the accelerated appearance of mus-cle-specific proteins creatine kinase and troponin T (Fig. 5, A,II and B, II) and myogenic transcription factors myoD andmyogenin (Fig. 6B, II; Fig. 7, A, III and B, III) was evident intransfected cells expressing PKR. Finally, an induction of theexpression of p21WAF1, accompanied by a reduction in thelevels of cyclin D1 and c-myc as well as an accumulation ofthe underphosphorylated form of pRb, was also observed in

these cells (Fig. 8, A–C, III; Fig. 9, III). According to our view,PKR is most likely involved in the down-regulation of geneexpression, possibly by the specific inhibition of the transla-tion of certain mRNA molecules. The induction of gene ex-pression in myogenesis, on the other hand, may then followor be the result of an independent signal transduction path-way and therefore is not directly related to PKR activity.

Recently, Datta et al. (31) reported an additional IFN-in-duced protein, p202, whose level is increased during thedifferentiation of C2C12 cells. However, in contrast to theresults obtained in our report with PKR, ectopic expressionof the gene encoding p202 in C2C12 cells inhibited ratherthan enhanced muscle differentiation. Thus, overexpressionof p202 reduced the level of myoD and inhibited the tran-scriptional activation of both myoD and myogenin. The dis-crepancy between the observed elevated level of p202 dur-ing differentiation and the inhibition of myoD and myogeninactivation by ectopic expression of p202 is explained byDatta et al. (31) to be the result of early expression in thetransfected cells. PKR, on the other hand, seems to be

Fig. 6. Detection of myoD-spe-cific RNA transcripts. Total RNAwas extracted from C2C12 cellsgrown in GM (A, I) or DM (A, II) andfrom untreated (B, I) or ZnCl2-treated (B, II) clone 8 cells. Theamount of RNA in individual sam-ples is shown in the ethidium bro-mide-stained gels presented ineach section. Northern blot analy-sis was performed using a myoD-specific probe.

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sufficient to induce an increase in muscle-specific proteins.Therefore, it must be concluded that p202 and PKR operateon two different levels, although both are activated duringmuscle differentiation.

An important finding in our study is the enhanced accu-mulation of the underphosphorylated form of pRb in PKR-expressing C2C12 cells (Fig. 8C, III). As mentioned above,this is accompanied by a reduction in the level of both cyclinD1 and CDK4 and an increase in the synthesis of p21WAF1.pRb seems to be an essential component in the terminaldifferentiation of C2C12 cells because expression of anti-sense Rb-1 RNA inhibits this process (47). In addition,p21WAF1 is markedly induced during muscle differentiation(33, 48), indicating that pRb must retain its active underphos-phorylated form during terminal differentiation. Based on the

data presented in our report, we conclude that in C2C12cells, PKR is sufficient to initiate a differentiation processcharacterized by cell growth arrest, the appearance of mus-cle-specific proteins, changes in the level of cell growth-associated factors, and the partial fusion of myoblasts. Al-though the development of the embryo appears to be normalin PKR knockout mice (49), it is likely that a PKR homologuethat has not yet been identified or other redundant proteinswith similar functions (2-5A synthetase, for example) areactivated in this case. This notion may not apply to commit-ted myogenic cells, such as C2C12 cells because transfec-tion of these cells with a dominant negative mutant of PKRleads to a significant inhibition of the myogenic process.5

Thus, taken together, our data suggest that PKR is an im-portant element in the regulation of myogenesis.

Materials and MethodsCell Cultures and Treatments

Murine myogenic C2C12 cells were maintained in DMEM supplementedwith 15% FCS (Biological Industries, Beth Haemek, Israel; GM) under10% CO2 at 37°C. The cultures were split every 3–4 days by removing thecells with a mixture of 0.25% trypsin and 0.05 M EDTA in PBS. Freshcultures were prepared from frozen cell pellets every 2 months. Unlessotherwise mentioned, the cells were seeded for experimental purposes at2 3 105 cells/10-cm tissue culture dish in GM. After 1 day, some of thecultures were shifted to DMEM supplemented with 10% horse serum and1 mg/ml insulin (DM).

Mouse a/b IFN (Access BioMedical, San Diego, CA; specific activity,9.8 3 106 IU/mg) was added to untreated cultures at a concentration of240 IU/ml.

A stock solution of 100 mM ZnCl2 (Sigma Chemical Co., St. Louis, MO)in 50 mM HEPES (pH 6.0) was prepared and kept at 220°C. Whereverapplicable, cells were seeded at 2 3 105 cells/10-cm dish in GM, andZnCl2 (final concentration, 100 mM) was added 3 h later.

Construction of Plasmids

The Bluescript KS plasmid harboring cDNA encoding human PKR (2.6 kb)was kindly supplied by B. R. G. Williams (Cleveland Clinic Foundation,Cleveland, OH).

The cDNA fragment was excised from the vector by HindIII. Thisfragment was then subcloned in the HindIII site of Bluescript SK, resultingin two possible orientations. To distinguish between the two constructs,several plasmid preparations were digested with SphI and XbaI. Ligationin the sense orientation was obtained when the resulting fragments were0.4- and 5-kb long. In the final stage, one of these plasmids (pBS-SK-PKR)was digested with SalI and XbaI, and the PKR-containing fragment wasligated into the polylinker SalI-XbaI site of plasmid pMSa (50). This plasmidcontains the metallothionein promoter. Before the final step, an extra SalIsite in pMSa was removed by SphI, followed by self-ligation. The finalconstruct, pMPKR, was used in this study.

Transfection

pMPKR was cotransfected with pSVneo (this plasmid contains the activeneomycin resistance gene fused to the early SV40 promoter; Ref. 51) intoC2C12 cells by electroporation. Approximately 2 3 107 cells/ml weresuspended in 250 ml of GM to which 200 ml of sucrose buffer [272 mM

sucrose and 7 mM Na3PO4 (pH 7.4)] and 50 ml of DNA containing 15 mg ofpMPKR and 1 mg of pSVneo were added. Electroporation was performedat 400 V and a capacitance of 500 mF using the Bio-Rad gene pulsar IIapparatus (Bio-Rad Laboratories, Hercules, CA). The cells were thentransferred into 10-cm dishes containing DMEM supplemented with 20%FCS. After 48 h of incubation in GM, the cultures were subdivided at a ratio

4 Unpublished results. 5 S. Salzberg et al., manuscript in preparation.

Fig. 7. Determination of myogenic transcription factors. C2C12 (I), C2-NEO (II), and clone 8 (III) cells were grown in the appropriate mediumindicated in the figure, as described in Fig. 5. The levels of myogenin (A)and myoD (B) were determined by immunoblot analysis.

208 Involvement of PKR in Myogenesis

of 1:10, and G418 (Calbiochem-Novabiochem Corp., La Jolla, CA) (800mg/ml) was added 24 h later. After an additional 14 days, most of the cellsdied, and single colonies were visible. About 30 clones were removed bytrypsin-EDTA solution, resuspended in GM with G418, and expanded. Fora negative control, C2C12 cells were transfected with 1 mg of pSVneoonly. Eight clones were similarly isolated. A representative clone, C2-NEO,was used throughout this study.

Cell Extracts

Cytoplasmatic (S10) Extracts. Cell extracts were prepared after theappropriate treatment by removing the cultured cells with a rubber po-liceman in PBS. The cells were then centrifuged at 800 3 g and resus-pended in an ice-cold lysis buffer containing 20 mM HEPES (pH 7.5), 5 mM

magnesium acetate, 2.5% NP40, and 1 mM DTT. The extracts werecentrifuged at 10,000 3 g for 10 min, and the soluble fractions (S10) werestored at 270°C until use. These extracts were used for determination ofPKR enzymatic activity.

Total Extracts. Cells were washed twice in cold PBS and centrifugedat 800 3 g for 10 min, and the pellets were thawed in 4 volumes of bufferW containing 10 mM HEPES (pH 7.9), 0.4 M NaCl, 0.1 mM EDTA, 1 mM DTT,5% (v/v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 0.1mM sodium vanadate, 10 mM sodium molybdate, 100 mg/ml leupeptin, 4mg/ml aprotinin, 2 mg/ml chymostatin, 1.5 mg/ml peptasin, and 2 mg/mlantipain. After repeated pipetting, the lyses were centrifuged at 10,000 3g for 20 min, and the supernatant was frozen in liquid N2. These extractswere used for the identification of the following proteins by immunoblotanalysis: PKR; troponin T; myogenin; myoD; c-myc; and pRb.

Nuclear Extract. Frozen pellets were prepared as described for totalextracts, thawed in buffer W with 10 mM NaCl only, and centrifuged at10,000 3 g for 20 min. The nuclear pellet was resuspended in originalbuffer W and centrifuged again. The supernatant was collected and keptin liquid nitrogen. These extracts were used for the determination of cyclinD1 by immunoblot analysis.

Determination of PKR Activity

Cell extracts (S10) were prepared as described above. Heparin (50–100units/ml) was added to samples containing 500 mg of protein each. Themixtures were incubated at 4°C for 10 min. An equal volume of poly(I):poly(C)-Sepharose beads was added at room temperature for 30 min, withoccasional gentle mixing. The beads were washed several times withbuffer B [50 mM KCl, 2 mM magnesium acetate, 7 mM 2-mercaptoethanol,

Fig. 8. Determination of cell cy-cle-associated proteins. The lev-els of p21WAF1 (A), cyclin D1 (B),and pRb (C) were determined byimmunoblot analysis of cell ex-tracts prepared from culturestreated as indicated in the figureand described in Fig. 5.

Fig. 9. Determination of c-myc synthesis. The rate of synthesis of c-mycwas determined by immunoblot analysis in extracts prepared from C2C12(I), C2-NEO (II), and clone 8 (III) cells treated as indicated in the figure.

209Cell Growth & Differentiation

20% glycerol, and 10 mM HEPES (pH 7.6)] and then once with buffer C(buffer B supplemented with 5 mM MnCl2). The final pellet was resus-pended in buffer C supplemented with 1 mCi of [g-32P]ATP (50–100Ci/mmol; Amersham Life Sciences, Ltd., Little Chalfont, United Kingdom)and incubated for 30 min at 30°C. After centrifugation, the pellet waswashed three times with buffer C and resuspended in 0.66 volume bufferC and 0.33 volume electrophoresis sample buffer containing 6% SDS(w/v), 30% glycerol (v/v), 0.02% bromphenol blue (w/v), 200 mM Tris-HCl(pH 6.8), and 250 mM 2-mercaptoethanol. The supernatants were col-lected and analyzed on 10% polyacrylamide slab gels containing SDS.The gel was dried, and the phosphorylated proteins were detected byautoradiography on Fuji RX film. Densitometry was determined by theScion-Image program.

Protein Analysis by Immunoblotting

Total or nuclear cell extracts were prepared as described above. Samples(20 mg) were loaded on polyacrylamide-SDS gel and analyzed by immu-noblotting. We used the rainbow-colored proteins as a molecular weightmarker (Amersham International).

Electrophoresis was carried out at 200 V for 1 h at 4°C. Transfer tonitrocellulose sheets was performed in the minitrans-blot cell (Bio-RadLaboratories) at 4°C in a buffer containing 25 mM Tris (pH 8.3), 192 mM

glycine, and 20% methanol for 1 h at 200 mA. The nitrocellulose sheet wasimmersed in blocking solution containing 10 mM Tris (pH 7.5), 100 mM

NaCl, 0.1% Tween 20, 5% FCS, and 3% nonfat milk in PBS for 1 h at roomtemperature. It was then transferred to a blocking solution supplementedwith the following preparation of antibodies: polyclonal antibodies di-rected against human PKR (dilution, 1:2000, supplied by Dr. A. Vojdani,Immunosciences Laboratory, Inc., Beverly Hills, CA); anti-c-myc mono-clonal antibodies (AB-3; dilution, 1:50, Calbiochem-Novabiochem Corp.);anti-Rb monoclonal antibodies (G3-245; dilution, 1:250; PharMingen, SanDiego, CA); anti-myoD (SC-760; dilution, 1:400; Santa Cruz Biotechnol-ogy, Santa Cruz, CA); anti-myogenin (SC-576; dilution, 1:400; Santa CruzBiotechnology); anti-troponin (T-6277; dilution, 1:2000; Sigma); anti-cyclinD1 (SC-6281; dilution, 1:400; Santa Cruz Biotechnology); and anti-p21WAF1 (SC-6246; dilution, 1:250; Santa Cruz Biotechnology). The mix-ture was incubated overnight at 4°C and then washed three times with asolution containing 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20in PBS. As a secondary detection antibody, we used peroxidase-labeledantimouse or antirabbit antibodies (Jackson ImmunoResearch; WestGrove, PA), and detection was performed by the enhanced chemilumi-nescence Western blotting procedure as described by the supplier (Am-ersham International). Light emission was detected by a 2-min exposureto Fuji RX film. Densitometry was determined by the Scion-Image pro-gram.

Determination of Specific RNA Transcripts

For each treatment, three 10-cm tissue culture dishes were used. TotalRNA was extracted with Tri-reagent (Molecular Research Center, Inc.,Cincinnati, OH) according to the protocol supplied by the manufacturer.Samples containing 30 mg of RNA were analyzed on 1% agarose gels inrunning buffer containing formaldehyde, followed by blotting onto nitro-cellulose membrane filters (NitroPlus; MSI, Westboro, MA), as describedpreviously (52). The ethidium bromide-stained 18S and 28S bands ofrRNA in each lane were detected on both gels and filters by UV light. Nosignificant differences in intensity between the lanes were observed. Forhybridization, the nitrocellulose filters were first prehybridized at 42°C for2 h in prehybridization buffer as described by Sambrook et al. (52), withthe addition of 0.1 mg/ml single-stranded salmon sperm DNA. The labeledprobe was then added at 1–2 3 106 cpm/ml. Incubation was for 24 h at42°C, and then the filters were washed once with 1 3 saline-sodiumphosphate-EDTA (15 mM NaH2PO4, 150 mM NaCl, and 1 mM EDTA) and0.5% SDS for 30 min at room temperature and once with 0.1 3 saline-sodium phosphate-EDTA and 0.1% SDS for 30 min at 50°C. Filters weredried and exposed for autoradiography.

Probes

For the detection of myoD-specific transcripts, a 1.8-kb fragment excisedwith EcoRI from plasmid pEMC11S was used. This plasmid, generously

provided by J. Pierce (Laboratory of Cellular and Molecular Biology,National Cancer Institute, Bethesda, MD) harbors the murine myoD-en-coding sequences. The probes were labeled with [a-32P]CTP (specificactivity, 3000 Ci/mmol; Amersham) using the rapid multiprime DNA label-ing kit as recommended by the supplier (RAN. 1601, Amersham). Thespecific activity was 2–8 3 108 cpm/mg.

Determination of Growth Characterization

In the case of wild-type C2C12 cells, 1 3 105 cells/5-cm tissue cultureplate were seeded in GM. One day later, the medium was replaced witheither GM (for dividing cells) or DM (for differentiating cells). This wasconsidered zero time. With the transfected clones, the cells were seededat 1 3 105 cells/5-cm tissue culture plate in GM, and some of the cultureswere treated 3 h later with ZnCl2 (zero time). In all cases, the determinationof the growth rate or thymidine incorporation was performed as describedbelow.

At the appropriate times, groups of three plates/point were collected,the medium was removed, the plates were washed twice with PBS, andthe cells were collected with trypsin-EDTA, resuspended in PBS, centri-fuged, and resuspended again in 0.4% trypan blue in PBS (Sigma). Vitalcells were counted in a hemocytometer, using five different fields/count.The SD of all the counts per time (total, 15 counts) was determined.

Thymidine Incorporation. Cultures were prepared as describedabove. At the indicated times, the medium was removed from the plateand replaced with fresh medium containing 1 mCi/ml [3H]thymidine (Am-ersham International) for 1.5 h. The cultures were then washed three timeswith cold PBS. The cells were lysed with 1% SDS for 10 min at 37°C, andthe lysates were moved to test tubes. An equal volume of 20% trichloro-acetic acid was added, and the tubes were kept on ice for 20 min. Thesamples were then filtered through Whatman 25 mm GF/c filters (suppliedby Tamar, Ltd., Jerusalem, Israel). The filters were dried, placed in tolu-ene-based scintillation fluid, and counted in Packard 1600 TR liquidanalyzer. Each point represents the average of three different measure-ments.

Determination of Creatine Kinase Activity

Cultures were washed with Ca21- and Mg21-free PBS and homogenizedin 0.1 M sodium phosphate buffer (pH 7.0) supplemented with 0.1% TritonX-100. The enzymatic activity was determined as described by Shainberget al. (53). The ATP formed by the interaction of ADP with creatinephosphate phosphorylates glucose in the presence of hexokinase, yield-ing glucose-6 phosphate. The latter reduces NADP to NADPH, which isdetermined by recording the absorption at 340 nm.

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212 Involvement of PKR in Myogenesis