Multiple Controls for the Synthesis of Muscle-specific Proteins in BC3 H1 Cells

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1 Published Online: 1 Februry, 1982 Supp Info: Downloded from jcb.rupress.org on July 1, 218 Multiple Controls for the Synthesis of Muscle-specific Proteins in BC3 H1 Cells ROBRT MUNSON, Jr., KNDRA L. CALDWLL, nd LUIS GLASR Deprtment of Biologicl Chemistry, Division of Biology nd Biomedicl Sciences, Wshington University School of Medicine, St. Louis, Missouri 6311 ABSTRACT The regultion of the synthesis of muscle-specific proteins hs been exmined in BCH1 cells, smooth muscle-like cell line isolted by Schubert et l. (l. Cell Biol., 1974, 61 : ). The synthesis of both cretine kinse nd the cetylcholine receptor pper to be under dul control, positive control due to cell-cell contct which increses the rte of synthesis of this protein, nd negtive signl, elicited by serum components, tht decreses the rte of synthesis of these proteins. Induction of muscle-specific proteins in BCH1 cells is reversible process nd cn be rrested fter prtil induction hs tken plce by the ddition of serum or high-moleculr-weight protein frction from serum to these cells. The highmoleculr-weight protein frction from serum is not by itself mitogenic for BCH1 cells nd cnnot be replced by vriety of known hormones (mitogenic fctors). The control of the synthesis of specific proteins during myogenesis hs been the subject of intensive investigtion in vrious tissue culture systems using myoblsts or estblished cell lines such s L-6 (1, 17, 2). Most of these systems use fusing cells, where the events relted to cell fusion cnnot lwys be redily seprted from the cell recognition events necessry for the induction of muscle-specific proteins (such s cretine kinse nd the cetylcholine receptor) (2, 7, 19, 21). Less informtion is vilble regrding the induction of muscle-specific proteins in smooth muscle cells (5). BC3H1 is smooth-muscle-like cell line isolted severl yers go by Schubert et l. (18). These cells do not fuse but induction of the synthesis of the muscle isoenyme of cretine kinse nd the cetylcholine receptor cn be demonstrted to tke plce under vriety of conditions (13, 18). In this communiction we present evidence to show tht in these cells the synthesis of muscle-specific proteins is regulted by the blnce between two ntgonistic signls: cell contct brings bout the induction of the synthesis of these proteins wheres serum fctors suppress the induction ofthese proteins. Induction of these muscle-specific proteins is reversible phenomenon. While induction of these muscle-specific proteins only occurs in quiescent cells, repression (inhibition of induction) is not obligtorily coupled to the resumption of cell growth. MATRIALS AND MTHODS BC,H1 cells, nonfusing muscle cell line, ws obtined from Dr. D. Schubert t the Slk Institute. The cells were grown t 37 C in 1% C2-enriched 35 tmosphere in Dulbecco's modified gle's medium (DMM) supplemented, where indicted, with 15% nutrient medium F12 nd the specified concentrtion of dilyed fetl clf serum or dilyed horse serum nd L-glutmine (.1 mg/ ml), penicillin ( U/ml), nd streptomycin (1 /+g/ml).cells were trnsferred from log phse cultures by trypsinition with crystlline trypsin, nd replted. Fresh cultures were strted from froen stock t 4- to 6-wk intervls. Medi were obtined from Gibco Lbortories (Grnd Islnd Biologicl Co., Grnd Islnd, NY) or the Wshington University Bsic Cncer Center. Ser were obtined from Gibco Lbortories nd K. C. Biologicls. In some experiments cells were grown on collgen-coted dishes. Dishes were coted by Nlls vpor precipittion of cid-soluble clf skin collgen (Sigm Chemicl Co., St. Louis, MO) (14). The dishes were stored dry fter sterilition with UV light. Before use, dishes were wshed twice with Hepes-buffered C''- nd Mg"-free Hnks' solution nd once with complete medium. Cretine kinse ws determined by the following modifiction of published procedures (11, 2). Cultures in 35-mm dishes were wshed once with 2 ml of phosphte-buffered sline (PBS) nd the cell lyer ws suspended in.5 ml of 1% nonidet P-4 (NP-4) in 5 glycyl-glycine, ph The cells were homogenied (1 to 15 strokes) in 1-ml Dounce homogenier. For ssy, the extrct ws ppropritely diluted in the sme buffer contining 1 mg/ml bovine serum lbumin (BSA). A two-stge ssy for cretine kinse is used. In the first stge, the rection mixture contins l mm ADP, 1 mm MgC1%.2% BSA,.1 mm P',P didenosine, 5' pentphosphte (Boehringer Mnnheim Biochemicls, Indinpolis, IN), 2.5 mm dithiothreitol, 25 mm phosphocretine, 3 MM D-glucose nd 2 U/ ml hexokinse, nd extrct in totl volume of 4 Il. Controls lck phosphocretine. The rection is initited by the ddition of extrct. After incubtion t 37 C for 3 min, the rection is stopped by the ddition of 1 Wl of 1 N NOH. After incubtion t 25 C for 1 min, the rection mixture is diluted to.8 ml with 5 mm imidole buffer, ph 7., followed by the ddition of NADP nd glucose 6-P-dehydrogense to finl concentrtion of.82 mm nd 1.7 U/ml, respectively, in totl volume of 1 ml. After incubtion t 25 for 1 min, the fluorescence due to NADPH is determined in Frrnd Fluorimeter (11), nd vlues obtined in the bsence ofphosphocretine were subtrcted. The ssy is liner with time, nd up to 1 nmol of NADPH formed during the ssy period. TH JOURNAL OF CLL BIOLOGY " VOLUM 9L rbruary The Rockefeller University Press /82/2/35/7 $1.

2 At lest two concentrtions of ech extrct were tested to insure linerity, nd ll results re the verge ofduplicte dishes. Cretine kinse ws normlied to cell numbers determined in duplicte dishes with Coulter counter or to cell protein obtined by growing cells for minimum of two genertions in medium supplemented with rdioctive mino cid usully with l ILCi/ml of [3 H]-leucine, nd mesuring the rdioctivity in protein by precipittion with trichlorocetic cid in the presence of deoxycholte (15) in the sme extrcts s were used for the ssy of cretine kinse. Although both protein nd cell number were determined in ech experiment, the results were lwys identicl; therefore, in most experiments we present only the dt bsed on cell numbers. All regents, unless otherwise indicted, were obtined from Sigm Chemicl Co. It is importnt tht the ADP used be s free ofatp s possible. The cetylcholine receptor ws determined by mesuring the binding of [ iss I)-bungrotoxin (13). -Bungrotoxin (Sigm Chemicl Co.) ws iodinted using immobilied lctoperoxidse (Bio-Rd nymobeds ; Bio-Rd Lbortories, Richmond, CA); the specific ctivity ofthe lbeled toxin preprtions used verged 25 ACi/Pg. Nonspecific binding ws determined in the presence of 1 crbmylcholine..1 lg of ["Il-bungrotoxin ws dded directly to 35-mm culture dishes, which were then incubted for 5 min t 37 C. Control dishes received crbmylcholine (I mm finl concentrtion) 1 min before the ddition of -bungrotoxin.at the end of the incubtion, the dishes were subjected to six rpid wshes with 2 ml of DM contining 2% horse serum. The cell lyer ws solubilied in 2 ml of 1% SDS (in PBS) nd counted in Beckmn gmm counter (Beckmn Instruments, Inc., Fullerton, CA). Fetl clf serum (Gibco Lbortories, Lot C68829) ws frctionted t 4 C s follows.the serum ws dilyed overnight ginst 1 volof.l MTris-Cl, ph ml of dilyed serum ws pplied to 2.4 x 2-cm column of CM- Sephdex C-5 equilibrted with the sme buffer. Serum proteins were eluted from the column with the sme buffer until the OD.m ws below.6 (16). The elute ws concentrted to the originl volume, using Pellicon PTGC 1, moleculr weight exclusion filter (Millipore Corp., Bedford, MA) in n Amicon concentrtor (Amicon Corp., Lexington, MA), nd dilyed ginst PBS. 2-ml liquots were pplied to 2.5 x 137-cm Sephcryl S-2 column equilibrted with PBS. The smple ws eluted t flow rte of.53 ml/min. 5.3-ml frctions were collected nd pooled nd concentrted s indicted in Fig. 9 (see below). Frctions were dilyed ginst DMM nd filter-sterilied before use. All experiments described below hve been done t lest three times with results similr or identicl to those illustrted. RSULTS Cretine kinse cn be induced in BC3H1 cells under severl conditions. As shown in Fig. 1, if the cells re llowed to grow in medium contining 2% serum without medium chnges (13), they cese to grow t x 15 cells/cm'. After the cesstion of growth, the level of cretine kinse in these cells rises. If the medium is chnged dily (n experiment which cn only be crried out on collgen-coted dishes to which confluent cells dhere firmly), then the cells grow to slightly higher density but the rte of induction of cretine kinse fter cesstion of growth is lmost unffected. Compre, in Fig. 1, the open squres (nonfed culture) with the closed squres (culture fed dily). If lte in the logrithmic phse of growth the medium is chnged to.5% serum (dy 3 in the experiment in Fig. 1), cell growth ceses within 24 h, nd within 48 h, one observes rpid increse in the level of cretine kinse (closed circles in Fig. 1). Note the reproducibility ofthese observtions in the experiments in which we compre cells on collgentreted dishes nd cells on plstic (open squres nd open circles in Fig. 1). These experiments rise the question whether the induction of cretine kinse t high cell density is due to the depletion by the cells of medium components required for growth, or is due to n event relted to cell contct. If depletion of the medium is the cuse of the induction of cretine kinse, it must be extremely rpid, becuse dily chnges of medium dely but do not bolish the induction of cretine kinse. To nswer this question, we hve used medium obtined from confluent cells ctively synthesiing cretine kinse (such s those indicted by filled squres in Fig. 1) nd dded this medium to sprse growing cells, to scertin whether these sprse cells would continue to grow in this medium or whether they would cese to grow nd rpidly initite the synthesis of cretine kinse. If the former occurred, then depletion of the medium components would be considered s likely cuse of the induction of cretine kinse t high cell density, but if sprse cells continued to grow in medium conditioned by cells t high density, then depletion of medium components cnnot by itself mount for the induction of cretine kinse t high density. The dt in Fig. 2 show tht cells trnsferred to conditioned medium continue to grow for severl dys nd do not immeditely initite the synthesis of cretine kinse ; thus, medium depletion cnnot ccount for the induction of cretine kinse t high cell density. In contrst, sprse cells t the sme density, trnsferred to medium contining 1% serum, rpidly induce cretine kinse. The induction of cretine kinse in sprse cells, fter chnge to medium contining 1% serum, is lso dependent on cell density. As shown in Fig. 3, no induction is observed t very low cell density, where 1% serum will support cell growth nd will notled to the induction of cretine kinse. In contrst, dily chnges of medium contining 1% serum t higher cell densities (tringles in Fig. 3) do not prevent the induction of cretine kinse. Similr observtions hve been mde with cells plted t 2 x 14 cells/35-mm dish, corresponding to the open squres in Fig. 3, where dily feeding of the cultures does not lter the kinetics of induction of cretine kinse (see Fig. 5 below). The dt suggest tht cell contct is required for the cesstion of growth nd the induction of cretine kinse. Fig. 4 illustrtes typicl res in cultures t densities in which cretine kinse cn (pnels d-j) nd cnnot (pnels -c) be induced by trnsfer of the culture to medium contining 1% serum. BC3H1 cells tend to ggregte in ptches, nd even t modest cell densities the cells show extensive cell-to-cell contct nd spindly morphology under conditions where cretine kinse is induced. Pnels, b, d, nd e represent cultures t the time they were trnsferred to 1% serum (dy 2). Note tht the cell densities re such tht cell-cell contct is very limited in R 3 v w 2 Y 2 1 U TIM (Dys) FIGUR 1 Induction of cretine kinse in BC3H1 cells. Cells were plted in 6-mm Flcon dishes t density of 7 x 14 cells/dish in DM - 2% fetl clf serum -.1 mg/ml glutmine, 1 U/ml penicillin nd 11g/ml Streptomycin. Cells were plted on collgen-coted dishes (I "), or directly on plstic (O "). In the series designted the medium ws chnged dily ; in ll other cultures the medium ws not chnged. On dy 3 (designted by the rrow), one set of cultures ws trnsferred to medium contining.5% fetl clf serum ("). Cell number nd cretine kinse concentrtion were determined s described in Mterils nd Methods. Protein ws mesured colorimetriclly (6). All determintions re the verge of duplicte determintions. B MUNSON, CALDWLL, AND GLASR Control of Muscle-specific Proteins 351

3 N w V FIGUR 2 ffect of conditioned medium on growth of BC3H1 cells. Cells were plted in 35-mm collgen-coted dishes in DM contining 2% fetl clf serum, glutmine, nd ntibiotics. On dy, the cells were fed with fresh medium concontining 2% fetl clf serum (/), fresh medium contining 1% fetl clf serum ("), or medium tht hd been incubted for 24 h either with confluent BC3H1 cells (--~-) or with collgen-coted dishes lcking cells (----). Assys for cell number nd cretine kinse were crried out s described in Mterils nd Methods. Conditioned medium ws obtined from three consecutive dys, corresponding to dys 5, 6, nd 7 of Fig. 1 (filled squres), nd ws froen t the time of collection, then pooled nd filtered before use. The control medium from empty dishes ws prepred t the sme time nd hnded in the sme mnner. B 4 m, 2. Y 1, V FIGUR 3 Density dependence of the induction of cretine kinse in BC3H1 cells. BC3H1 cells were plted in 35-mm dishes t the densities indicted in DM with ntibiotics, glutmine, nd 2% horse serum. At the rrow (dy 2), the cells were wshed nd the medium ws chnged to medium contining 1% serum. Pnel A documents the rte of cell growth in these cultures. Pnels Bnd C illustrte the rte of induction of cretine kinse, normlied to cell nuer (pnel 8) nd to protein (pnel C). As described in Mterils nd Methods, the cultures were grown in the presence of [ 3H]- leucine (1 gci/ml), nd the reltive protein content ws determined from the mount of trichlorocetic cid-precipitble [ 3 H]leucinelbeled protein. Medium ws chnged every second dy in ll cultures, except in culture indicted by () where the medium ws chnged dily. the low-density cultures. Pnels c nd f represent the ppernce of these cultures t the end of the experiment (dy 6). The high-density cells show the morphology chrcteristic of such induced cultures, consistent with the biochemicl dt (open C tringles, Fig. 3). The low-density cultures remin fibroblstic in ppernce. Moreover, they hve continued to proliferte during the 4-d period without ny significnt increse in cretine kinse ctivity or [ 125 I]-bungrotoxin binding. By dy 6, they hve reched density where cell islnds hve formed, nd they re beginning to show the biochemicl differentition (open circles, Fig. 3). If BC3H1 cells re plted t densities of -- 2 x 14 cells/dish nd if fter 2 d the medium is chnged to 1% serum, induction of cretine kinse cn be detected by dy 4; the kinetics of induction re the sme whether medium is chnged dily or every second dy (Fig. 5). If the medium recovered from these cultures is dded to cells growing in 1% serum t lower cell density, the kinetics of growth or induction of cretine kinse re the sme in conditioned medium s in fresh medium contining 1% serum. Thus, induction of cretine kinse in these cells cnnot be due to depletion of medium component, or secretion by the cells of "inducing component" which is stble in the growth medium. Cell-to-cell contct ppers to be the most likely positive ffect or for the induction of cretine kinse in these cultures.' Arrest of cell growth is not by itself sufficient to induce cretine kinse. Cells rrested with hydroxyure do not show ny induction ofcretine kinse nor do they show the morphologicl chnges ssocited with differentition (dt not shown). Arrest with hydroxyure is less thn perfect control for rrest of cell growth by low serum nd contct since the former occurs t the Gi-S interphse in the cell cycle wheres the ltter re likely to ffect cell growth erly in Gl phse of the cell cycle. The experiments presented cn be rtionlied by the ssumption tht the induction of cretine kinse in BC 3H1 is determined by blnce between induction signls derived from cell-to-cell contct nd repression due to one or more components present in serum. To further test this model, we hve sked whether the induction of cretine kinse once initited cn be rrested by the ddition of serum. The observtions in Fig. 6 show tht ddition of serum2 (or serum frctions, see below) to BC 3H1 cells tht hve initited the induction of cretine kinse will prevent further synthesis of this enyme; compre, in Fig. 6, closed circles nd dshed line (inducing conditions) with open circles nd solid line (repressing conditions). The induction process thus ppers to be reversible. The reversibility of the induction of protein chrcteristic of differentited cells is unique phenomenon, nd it prompted us to exmine whether the synthesis of other muscle-specific proteins by BC 3H 1 cells ws subject to similr control mechnism. Fig. 7 illustrtes the induction of the cetylcholine receptor (defined by its bility to bind "51_-bungrotoxin) in BC3H1 cells under two sets of conditions, high cell density in 2% serum nd low cell density in 1% serum. For comprison, the induction of cretine kinse hs lso been mesured in prllel cultures. Under both conditions, synthesis of the cetylcholine receptor is induced, nd in ech cse its ppernce precedes ' Although we prefer to interpret the density effects on enyme induction to reflect cell-to-cell contct, we cnnot rigorously exclude tht high cell density results in loclied chnge in the cell environment which ffects enyme induction. s The induction of cretine kinse cn be rrested by the ddition of either fetl clf serum or horse serum. All the initil experiments were crried out with fetl clf serum, but lter experiments used horse serum with identicl results. Both of these ser support ctive growth of BC3H 1 cells. 352 TH JOURNAL OF CLL BIOLOGY " VOLUM 92, 1982

4 FIGUR 4 Morphology of BC 3 H1 t different densities. The cells shown in this figure re from prllel experiment to tht shown in Fig. 3. Cells shown in pnels -c re plted t 3 x 13 cells/35-mm dish, corresponding to open circles in Fig. 3. Pnels nd b show, t two different mgnifictions, cells on dy 2, the dy on which the medium is chnged to 1% serum. Pnel c shows the culture t the higher mgnifiction on dy 6. In Pnels d-f the cells were plted t 5 x 1 cells/35-mm dish corresponding to the open tringles in Fig. 3, pnels d nd e re on dy 2 nd pnel fon dy 6. Note, in pnel f, the spindly morphology chrcteristic of the cells tht re differentiting nd the fct tht, even on dy 2 (pnel e), cells plted t this density re primrily present s cell clusters rther thn isolted cells. In contrst, the cells shown in pnels b nd c remin flt, re well seprted, nd remin undifferentited lmost until the end of the culture period. Cultures were photogrphed with n inverted Nikon phse microscope. Br, 1 gm. by t lest one dy the ppernce of high levels of cretine kinse ctivity in the sme cultures. Thus, for exmple, when culture tht hs been mintined in 2% serum for 2 d nd hs reched 15 cells/dish in 2% serum is trnsferred to 1% serum, n immedite induction of the synthesis of the cetylcholine receptor is observed (open tringles in Fig. 7) where the rise in the ctivity of cretine kinse does not occur until dy 4. Thus, if trnsfer to 1% serum initites developmentl progrm in these cells, then the progrm is one in which induction of the cetylcholine receptor occurs significntly erlier thn the induction of cretine kinse ctivity. Similr temporl differences in the induction of these two musclespecific proteins hve been observed with myoblsts (see, for exmple, 18). If BC 3H1 cells in which prtil induction of the cetylcholine receptor hs tken plce in 1% serum re trnsferred to 2% serum, then we observe decrese in the concentrtion of the cetylcholine receptor present on the surfce of these cells with hlf-life of- 4 h (Fig. 8). In control experiments using cycloheximide to inhibit protein synthesis, we observe hlflife for the cetylcholine receptor of 32 h3 (dt not shown). The decrese in the level of the cetylcholine receptor in these cells fter ddition of serum strongly suggests tht serum prevents further synthesis (or surfce expression) of this differentited protein in prtilly induced cells nd does not simply prevent the recruitment of new cells into the differentition progrm, becuse if the ltter were the cse, the level of the surfce cetylcholine receptor in the culture would either increse slowly or remin constnt. The ddition of 2% serum to prtilly induced cells results in slow reinitition of growth (Fig. 6); it becomes importnt to consider whether the reinitition of cell growth is required to repress the synthesis of muscle-specific proteins. We hve in 'Previous mesurement of the turnover rte of the cetylcholine receptor in BC 3H 1 cells hve been mde by ddition of cycloheximide to confluent cells plted in 2 lo serum nd mintined without medium chnges. Under these conditions the hlf-life of the cetylcholine receptor ws -- 1 h (13). It seems likely tht the different culture conditions ccount for this difference in hlf-life. MUNSON, CALDWLL, AND GLASR Control of Muscle-specific Proteins 353

5 C n n.6 Z X m 6 FIGUR 5 Induction of BC31-11 cells in medium conditioned by sprse cells. BC31-11 cells were plted in 35- dishes s described. On dy 2, the medium ws chnged to 1% serum (") nd chnged either dily (---) or every 2 d (-). The time scle for this culture is shown by the upper numbers on the bsciss. Medium ws recovered from the cultures fed dily nd used to grow second series of cultures shown by the open symbols. Three sets of cultures were prepred nd grown fter dy 2 (lower numbers on bsciss) either in 1% serum (O--~), or in conditioned medium s bove (O---O) or in control conditioned medium, which hd been exposed to plstic dishes with no cells for 24 h. All of these cultures show low induction of cretine kinse chrcteristic of these sprse cultures, but no difference ws detected between the three medi. FIGUR 7 Induction of cetylcholine receptor in BC cells. BC31-11 cells were plted in 35-mm collgen-coted dishes in 2% serum. On dy 2, some of the dishes were trnsferred to medium contining 1% serum. Pnel A, cell density : ("), 2% serum, (O), 1% serum. Pnel 8, cretine kinse (") nd specific -bungrotoxin binding () to cells in 1% serum. Pnel C, cretine kinse (/) nd specific -bungrotoxin binding (Q to cells in 2% serum. Nonspecific binding ws <.1 pg/12 cells. Note tht both in 1% serum (Pnel B) nd t high cell density (Pnel C) the induction of the cetylcholine receptor precedes the onset of cretine kinse induction by t lest 1 dy. Z Z m Z X H DAYS FIGUR 6 ffect of serum ddition on prtilly induced cells. BC31-11 cells were plted in 2% serum on dy 1. On dy 2, some dishes were trnsferred to medium contining 1% serum ("---) while others ( ) remined in 2% serum. On dy 4, some dishes in 1% serum were trnsferred bck to 2% serum (O-O). In this prticulr experiment, reinitition of cell growth in 2% serum ws extremely rpid. In cultures mintined longer in 1% serum, vrible lg period is observed before reinitition of growth (see, for exmple, Fig. 1). preliminry experiments frctionted serum s described in Mterils nd Methods by removl of proteins tht bind to CM-Sephdex followed by gel exclusion chromtogrphy (Fig. 9). The high moleculr weight frction from serum will repress the synthesis of cretine kinse, without reinititing cell growth, wheres the lower moleculr weight frctions hve no effect (Figs. 1 nd 11). Compre, in Fig. 11, pnel B, the high FIGUR 8 ffect of serum ddition on the level of the cetylcholine receptor. BC3H1 cells were plted in 2% horse serum ; fter 24 h the medium ws chnged to 1% serum. On dy 4, the medium ws chnged to 2% serum, nd t the times indicted the bility of the cells to bind [' 251]--bungrotoxin ws determined s described in Mterils nd Methods. The level of specific -bungrotoxin binding t the time of the chnge of the medium to 2% serum ws.2 pg/ 12 cells. 6 7 FRACTION NO. FIGUR 9 Frctiontion of fetl clf serum. Fetl clf serum ws frctionted s described under Mterils nd Methods, first by removl of proteins tht re retined on CM-Sephdex, nd next by gel exclusion chromtogrphy on Sephcryl S-2. Frctions were pooled s indicted in the figure nd ssyed for their bility to block the induction of cretine kinse when dded to cells in 1% serum t level equivlent to tht present in 2% serum. The results of these experiments re shown in Figs. 1 nd TH JOURNAL OF CLL BIOLOGY " VOLUM 92, 1982

6 v FIGUR 1 ffect of serum nd serum frctions on the induction of cretine kinse. BC 3 H1 cells were plted s described in Mterils nd Methods, using 1% dilyed fetl clf serum. After 2 d the dishes were wshed with DM nd incubted with DM contining 15% F12 nd 2% dilyed fetl clf serum (") or dilyed 1% fetl clf serum (O). On dy 4, cells were fed with the pproprite medium ; on dy 5, some of the dishes in 1% serum were trnsferred to medium contining 2% serum (A) or 1% serum plus Frction I (Fig. 9) t concentrtion equivlent to tht present in 2% serum (O). All cultures were mintined on 2-d feeding schedule, nd cretine kinse nd cell numbers were determined s described in Mterils nd Methods. All cultures lso contined 1 IiCi/ml of [35S] -methionine. The results in pnel B show cretine kinse normlied to cell numbers. Identicl results were obtined if the cretine kinse ws normlied to cell protein, mesured s trichlorocetic cid-insoluble [ 35 S] counts. moleculr weight frction, with pnel D, the low moleculr weight frction. The ddition of the high moleculr weight frction from serum (Frction 1, Fig. 9) does not result in reinitition of cell growth, not only s determined by cell number (Fig. 1), but lso s determined by the incorportion of [3H]-thymidine into DNA, much more sensitive mesurement of the movement of cells out of the Gl phse of the cell cycle nd into S phse (Tble I). We conclude from these preliminry experiments tht reinitition of cell growth is not necessry for the repression of differentition by serum components. By contrst, vriety of known mitogenic compounds including the pltelet-derived growth fctor, epiderml growth fctor, dexmethsone nd insulin do not repress differentition. Neither fibronectin nor cold-insoluble globulin cn replce the high moleculr weight frction from serum s inhibition of the induction of cretine kinse.' DISCUSSION The observtions presented re interpreted to indicte tht, under our culture conditions, the induction of the cretine kinse nd of the cetylcholine receptor in BC3H1 cells is reversible process. Induction of these two muscle-specific proteins is under dul control. Serum contins one or more ' The following compounds were tested t the concentrtions indicted. Insulin (1 Ag/ml), dexmethsone (1- ' M), trnsferrin (5 & 25 1Ag/ ml), fibroblst growth fctor (Collbortive Reserch, Inc.) (2ng/ml), humn lung fibronectin (4 fig/ml), dibutyryl camp (3 x 1-4 M), nd crude pltelet-derived growth fctor (t levels sturting for mutgenic response for 3T3 cells). These fctors were tested either lone or in vrious combintions ; none inhibited induction of muscle-specific proteins in BC 3 Hl cells in 1% serum. components which inhibit differentition wheres cell-to-cell contct' induces differentition. For the purpose of this discussion, differentition is defined s the synthesis of cretine kinse nd the cetylcholine receptor. In other systems, plsm membrnes dded to cells hve been successfully used to mimic cell contct phenomen (22); experiments re in progress to test whether membrnes, when dded to BC 3 H1 cells t low density, will induce the synthesis of muscle-specific proteins. Inhibition ofdifferentition cn tke plce in the bsence of cell growth (Fig. 1), nd the proteins present in the high moleculr weight frction of serum re not by themselves mitogenic for BC3 H1 cells. Whether the functionl molecules re truly high moleculr weight serum proteins or whether they re bound to proteins present in the high moleculr weight frction in serum remins to be determined. The inhibition by serum or serum frctions of the induction ofcretine kinse is prticulrly striking in light ofthe observtions in Fig. 7. These 8 v6 O 4 ~2 o N 9 8 Y V 4 FIGUR 11 ffect of serum frctions on the induction of cretine kinse. Culture conditions used were s in Fig. 1. The cells were grown for 2 d in 1% serum, then switched to 1% serum on dy 2. The cretine kinse of cultures in 1% serum is shown by (") in ech pnel. In pnel A, (O), re shown the results of dding, on dy 5, the proteins not retined on CM-Sephdex to cells. In pnel 8, (O), is shown the results of dding Frction I (Fig. 9) to the cells ; in pnel C, (O), Frction III, nd in pnel D, Frction IV. None of these serum frctions stimulted cell growth. Growth conditions xponentilly growing cells in 2% serum A c f t o TIM DAYS TABL I DNA Synthesis in BC3Hl Cells Rte of DNA synthesis Dy 5-6 Dy 6-7 Dy 7-8 A) 1% Serum B) Cells in A chnged to 2% serum on dy 5 C) Cells in A chnged to 1% serum + serum frction Iondy5 The experiment ws crried out in prllel to tht shown in Fig. 1. Approprite cultures were incubted for the 24-h periods indicted with 1 IACi/ml (.67 mm) [3 H]-thymidine to determine the rte of DNA synthesis. The results re expressed s rtio of [3H]/[ 35571, the ltter derived from [35 5]-methionine incorported into protein. Becuse the cells hd been chroniclly lbeled with [ 35 S]-methionine, the ltter is mesure of totl cell protein (22). B D t r 6 f3hl/l 35s1 2.7 r3hj/[355' - I3H I /f35s 1 - MUNSON, CALDWLL, AND GLASR Control of Muscle-specific Proteins 355

7 dt show tht the cetylcholine receptor is induced in these cells well before significnt induction of cretive kinse cn be detected. Thus, induction of cretive kinse ctivity is reltively lte event in the differentition sequence of these cells nd yet it ppers to be totlly reversible. The fct tht the induction of the cetylcholine receptor precedes the induction of cretive kinse by one dy suggests tht cells tht re inititing the synthesis ofcretive kinse re lredy committed to the synthesis of t lest one set of muscle-specific proteins ; thus, cell prtilly induced for cretive kinse is lmost fully induced for the cetylcholine receptor. Similr differences in the time of ppernce of muscle-specific proteins hve been noted, for exmple, with chick myoblsts (19). There is similrity between these observtions nd previous reports on the induction of S-1 protein in C-6 cells. The induction of S-1 in these cells occurs t high cell density but is lso repressed by serum components tht re pprently derived from pltelets nd which re not mitogenic for C-6 cells (8). More recently, Orly et l. (12) hve demonstrted the repression by serum of differentited function of grnulos cells. It is not lwys cler to wht extent observtions mde with estblished cell lines cn be pplied to more "norml" primry cells in culture nd ultimtely to cells in the whole niml. Nevertheless the observtions with BC 3H1 cells represents n idel model to study the role of cell-cell interctions nd of serum components in controlling certin spects of cellulr differentition. BC3H1 cells re n estblished cell line with stble properties. Becuse the cells do not fuse, it hs been possible to show tht, t lest for those cells, differentition is not n irreversible phenomenon. Although prtilly differentited cells cn reinitite growth, repression of differentition cn tke plce without reinitition of cell growth. These observtions re in contrst to those mde by mny lbortories with myoblsts, where cesstion of growth is requirement for differentition, but where the process of differentition, once initited, is irreversible (see for exmple, reference 1). An extensive series of investigtions hve been crried out with primry cultures of smooth muscle cells (for review, see Chmley-Cmpbell et l., reference 5). The min observtion relevnt to this communiction is tht cell division cn occur t lest in some differentited smooth muscle cells, nd tht, in culture, growing cells will lose mny chrcteristics of smooth muscle cells but regin these t high cell density when growth ceses (3, 4). 5 These observtions re fully comptible with the observtions on BC 3H1 cells nd suggest tht BC 3H1 cells my be good model for the study of the synthesis of muscle-specific protein in smooth muscle cells. One would expect, from the observtions reported with BC 3H 1 cells, tht smooth muscle cells would not differentite t low density, nd would do so t s Prolonged culture of smooth muscle cells t low density results in irreversible differentition or modultion (Chmley-Cmpbell et l. (5]). This my reflect the selection by prolonged growth of cells which cn no longer differentite t high cell density. Tht the origin of these cells in smooth muscle rther thn fibroblsts cn be shown in the ntibodies specific for smooth muscle ctin, which indicte tht cells tht hve lost the bility to differentite t high cell density so s to show the morphologicl chrcteristics of smooth muscle cells, still synthesie smooth muscle ctin. confluence s result of cell-to-cell contct. Whether serum components influence differentition of primry smooth muscle cells is not known t the present time. BC3H1 cells differ from primry cultures of smooth muscle cells in their bility to mintin the cpcity to differentite fter repeted subculture nd in the fct tht the pltelet-derived growth fctor is not mitogen for these cells. A more extensive comprison of the differentition of BC3H1 cells nd smooth muscle cells in culture is clerly wrrnted, including the ssessment of the role of serum components in the differentition of both types of cells. Becuse serum lots my differ in the concentrtion of molecules tht inhibit cell differentition, side-by-side comprison of the effects of different serum lots on smooth muscle cells nd BC 3H1 cells will hve to be crried out. We re grteful to Mr. D. Broid of Sigm Chemicl Co. for the preprtion of specil lot of ADP free of ATP, Dr.M.A. Liebermn (Hrvrd University, Boston, MA) for gift of epiderml growth fctor, nd Dr. T. Deuel (Wshington University, Settle, WA) for smple of prtilly purified pltelet-derived growth fctor. 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