Expression of Laminin Chains during Myogenic Differentiation*

THEJOURNAL OF B I O L ~ ~ ICHEMISTRY CAL 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 12, Issue of March 25, pp. 9270-9277, 1994 Printed in U.S.A.

Expression of Laminin Chains during Myogenic Differentiation* (Received for publication, September 27, 1993, and in revised form, December 10, 1993)

Todd G. KrollS, Barry P. Peters$, Carolyn Marziasz Hustadn, Peter A. Jonesn, Paul D. Killen[[**, and Raymond W.RuddonS $$@ From the program in Cellular and Molecular Biology and the lllepartment of Pathology, University ofMichigan Medical School, Ann Arbor, Michigan 48109, the $$?3ppleyInstitute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, the §Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606, and the Wenneth Norris Jr Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90033

Expression of two Ae-related chains of the extracellular matrix glycoprotein laminin was induced as multipotent C3HlOT1/2 mouse embryo fibroblasts differentiated into myoblasts and myofibers. C3HlOT1/2 fibroblasts expressed the Ble (M,= 215,000) and B2e(M, = 205,000) lamininchains based on metabolic radiolabeling, immunoprecipitation,peptide mapping, andmRNA analysis. In contrast, myoblasts derived from C3HlOT1/2 fibroblasts treated with DNA demethylating agents or transfected with the cDNA encoding MyoD expressed the Ae (M,= 400,000) and a novel Ae-related laminin chain (designatedAc3h, M, = 350,000) in addition to the Ble andB2e chains. Expression of the Ae and Ac3h chains paralleled the capacity for myofiber formation in six additional C3HlOT1/2 myoblast clones with varied potentials forterminal differentiation and coincided with a switch in laminin isoforms from those of M, = 850,000 synthesized by C3HlOT1/2 fibroblasts to those of M, = 900,000-950,000 synthesized byC3HlOT1/2myoblasts and myofibers. Culturesof mouse C2C12, mouse BC3H1, rat L6, and primary mouse myoblastsalso synthesized the Ae, AcSh, Ble, and B2e lamininchains. The results demonstrate that expression of the Ae and Ac3h laminin chains is associated with expression of MyoD and the mammalian myogenicdifferentiation program.

Glycoproteins of the extracellular matrix and their cell surface receptors are important regulatorsof morphogenesis and growth in normal tissues (1, 2). Laminins are glycoproteins deposited into specialized extracellular matrices, basement membranes, which are synthesized by epithelial, endothelial, nerve, and muscle cells. Knowledge of the structure and function of laminins is based primarily on studies of one laminin isoform identified in and isolated from the mouse EngelbrethHolm-Swarm (EHS)l tumor (3). EHS laminin is composed of three protein chains, Ae2 ( M , = 400,000), B l e ( M , = 215,000),

* This work was supported in part by National Institutes of Health Grants CA-36727 (to R. W. R.), CA-41359 (to B. P. P.), and CA-49758(to P. A. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisernent” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. ** Supported by Grants HL31963, DK39255, and DK44848. $9 To whom correspondence and reprintrequests should be addressed. Tel.: 402-559-4238;Fax: 402-559-4651. The abbreviations used are: EHS, Engelbreth-Holm-Swarm; 5-azaC, 5-aza-cytidine; MCA, 3-methylcholanthrene; DMEM,DulbecCO’S modified Eagle’smedium;5-azaCdR,5-aza-2‘-deoxycytidine; bp, base pairb); kb, kilobaseb). * We have used the nomenclature for laminin chains adopted recently by Engel and co-workers(81).Laminin derived fromthe EHS tumor is designated “Ae-Ble-BZe,” where “e” reflects the origin of the chains

and B2e ( M , = 205,000), which are assembled into a cruciform structure (Ae-Ble-B2e, M, = 950,000) stabilized by disulfide bonds (4, 5). Two additional laminin chains,one related to B l e (Bls, 6) andone related to Ae (Am, 7, 81, have been identified and studied, and other laminin-related proteins have been reported (7, 9-13). Although Ae-Ble-B2e laminin purified from the EHS tumor matrix is known to promote the adhesion, growth, and differentiation of multiple cell types (4, 14),little is known about the function of other laminin isoforms. It is difficult to study laminins synthesized in normal tissues because they are often expressed at low levels. For example, expression ofAe chainmRNAis not usually detectable (15-19) unless poly(A)-enriched RNA fractions are examined (20, 21). In addition, only Ae-Ble-B2e and Am-Ble-B2e laminin isoforms have been isolated from normal tissuesources (7, 22). As a result, tumors and tumor cell lines have been used often as model systems to study the expression and function of laminins (3, 15,23-26). One disadvantage of the tumorcell lines studied so far is that they tend to express laminin chains in a constitutive and/or coordinate fashion, whereas normal basement membranes contain varied levels of the Ae, Am, Ble, Bls, and B2e laminin chains (27) that are expressed in tissue- and development-specific patterns (18, 21, 28-31). The identification of cell culture systems that recapitulate these developmental patterns would add significantly t o the study of laminins and basement membrane extracellular matrices. C3HlOT1/2 mouse embryo fibroblasts treatedwith DNA demethylating agents suchas 5-aza-cytidine (5-azaC)differentiate intomyoblasts and myofibers (32). MyoD, a transcription factor that was isolated based on its elevated expression in 5-azaC-derived myoblasts compared to untreated C3HlOT112 fibroblasts (33), is involved in these differentiation events and regulates myogenesis in most, if not all, multicellular animals (34, 35). When overexpressed by transfection, MyoD converts C3HlOT1/2 fibroblasts into myoblasts that can fuse and synthesize muscle gene products (36). Here, theexpression of laminins wasexamined in untreated C3HlOT1/2 fibroblasts, clonal myoblasts derived from C3HlOT1/2 fibroblasts, established rodent myoblast cell lines, and primary mouse muscle cells. An induction of the Ae and Ac3h laminin chains was shown t o be associated with expression of MyoD and the mammalian muscle differentiation program. Expression of the Ae chain during C3HlOT1/2 myogenesis recapitulates cell in culture a pattern of laminin expression observed previously in differentiatingembryonic epithelial tisfrom the EHS tumor matrix (3). “Am”designatesthe Ae-related laminin chain named merosin or mouse heart laminin (7, 8). “Bls” designates the Ble-related chain localized t o synaptic and other basement membranes (6). “Ac3h designates the Ae-related laminin chain identified and characterized in this manuscript.

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Induction of Ae-related Laminin Chains sues, suggesting a role for the Ae chain in themorphogenesis of both epithelia and muscle. The results establish the C3HlOT1/2 system as a useful model with which to investigate the regulation and function of laminins at the molecular level. EXPERIMENTALPROCEDURES Cell Lines-The C3HlOTV2, clone 8, mouse embryo fibroblast cell line (37) was obtained from ATCC and Dr. Andrew Feinberg and Dr. Shirley Ranier (Dept. of Human Genetics, University of Michigan Medical School, Ann Arbor, MI).The ha-ClB myoblast cell line (clone B) was isolated previously from C3H10TV2 fibroblasts treated with 5-azaC (38). The CM1 myoblast and VECl fibroblast cell lines were isolated previously from C3H10T1/2 fibroblasts transfected with a vector expressing or not expressing MyoD, respectively (39). The transformed cell line MCA C1 15C1 was isolated previously from C3HIOT1/2 fibroblasts treated with 3-methylcholanthrene (MCA, Ref. 39). The 15-1A1, 15-5B6, 10-1A2, 10-1D4, and 10 VEC2 cell lines, which vary in their potential for terminal myogenic differentiation (39), were derived from MCAC1 15C1 cells either treated with 5-azaC (15-1A1 and 15-5B6), transfected with a vector expressing MyoD (10-1A2 and 10-1D4). or transfected with the vector not expressing MyoD (10 VEC2). The cell lines weregrown in Eagle's basal medium with Earle's salts, 1000 mgfliter glucose, 10-20% heat-inactivated fetal calf serum, 50 pg/ml streptomycin sulfate, 50 unitdml penicillin G sodium, and 500 mgfliter glutamine (all from Life Technologies, Inc.) in a humidified air atmosphere containing 5% CO, a t 37 "C. The rat L6 (40), mouse C2C12 (41), and BC3H1 (42) myoblast cell lines were obtained from ATCC and grown as above in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal calf serum. Myoblasts were induced to terminally differentiate into myofibers by changing the medium to DMEM containing 1% fetal calf serum. F9 mouse teratocarcinoma cells (43) were obtained from ATCC, grown as above in DMEM containing 10% fetal calf serum, and induced to differentiate into parietal endoderm-like cells by the addition of 1.0 trans-retinoic acid (Sigma) and 1.0 mM dibutyryl cyclic AMP (Sigma) to the culture medium (43). 5-Aza-2'-deozycytidine (5-azaCdR) lFeatment of C3HlOTl/2 Fibroblasts-C3HlOTl/2 fibroblasts were plated into 100-mm tissue culture dishes and exposed to medium containing 1.0 p~ 5-azaCdR (Sigma) 1 day later. After 24 h, the 5-azaCdR-containingmedium was removed, and the cells were maintained and subpassaged in medium without 5-azaCdR thereafter. Myofibers, adipocytes, and chondrocytes were present in the cultures 3 weeks after 5-azaCdR treatment as reported previously (32). Isolation of Primary Cultures of Neonate and Adult Mouse Muscle Cells-Muscle cells were isolated from the hind limbs of adult and neonate mice in a procedure similar to that described by Konigsberg and co-workers (44). Briefly, the mice were killed, and hind limb muscles were removed, dissected free of connective tissue, and minced into fine pieceswith a surgical scissors in ice-cold Dulbecco'sphosphatebuffered saline containing 100 pg/mlstreptomycin sulfate, 100 unitdml penicillin G sodium, and 2.5 pg/ml amphotericin B (Life Technologies, Inc.). The mince from adult muscles received 2.0% collagenase type I1 (Worthington),whereas the mince fromneonate muscles received 0.1% collagenase type I (Worthington).Mince-enzyme suspensions were incubated a t 37 "C for 20 min, and enzymes were inhibited by addition of ice-cold complete growth medium consisting of DMEM containing 20% fetal calf serum, 3% chick embryo extract, 100 pg/ml streptomycin sulfate, 100 unitdml penicillin G sodium, 2.5 pg/ml amphotericin B, and 500 mgfliter glutamine. Suspensions were filtered through sterile 100150-pm screens and aliquoted into gelatin-coated tissue culture dishes containing complete growth medium. The next morning, unattached cells, enriched for myoblasts relative to fibroblasts, were replated into new gelatin-coated dishes. Cultures were sometimes further enriched for myoblasts by trypsin treatment as described (44). Metabolic Radiolabeling-Cultures were washed with DMEM lacking methionine and/or cysteine and were incubated in the same medium supplemented with 10-20% heat-inactivated fetal calf serum at 37 "C for 30min. Cells were radiolabeled by incubation in DMEM containing 10-20% heat-inactivated fetal calf serum and 100-200 pCi/ml [35S]methionine and/or [3sSlcysteine (each >I100 Ci/mmol; Amersham or DuPont NEN). The labeling medium was removed, and cell layers were washed and either lysed or incubated in nonradioactive DMEM containing 1 6 2 0 %fetal calf serum for an additional 4-h chase period and then lysed. Cells were lysed in Dulbecco's phosphate-bufferedsaline, pH 7.4, 1%Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS),20 m~ EDTA, 5 m~ N-ethylmaleimide,and 0.04% sodium azide. Protease inhibitors were included in the lysis buffer: 2 m~ phe-

9271

nylmethylsulfonyl fluoride, 1 pg/ml pepstatin A, 2 pg/ml antipain dihydrochloride, 1 pg/ml leupeptin, 1pg/ml aprotinin, and 10 pg/ml benzamidine hydrochloride. Cell lysates were collected with a syringe fitted with a 21-gauge needle and were frozen at -70 "C. Medium samples were collected from cultures prior to cell lysis and brought to the same concentration of lysis buffer as the cell lysates by addition of one-fourth volume of 5-fold concentrated lysis buffer stock solution. ImmunoprecipitationandSDS-Polyacrylamide Gel Electrophoresis-Frozen cell lysate or medium samples were thawed and spun at 100,000x g for 1h at 4 "C. Aliquots of the clarified supernatants were immunoprecipitated by addition of a specific antiserum or antibody. These included: 1)a rabbit antiserum raised against mouse EHS (AeBle-Bae) laminin or the corresponding rabbit preimmune serum (supplied by Dr.Randall Knibbs and Dr. Irwin Goldstein,University ofMichigan Medical School), 2) an affinity-purified rabbit antiserum raised against mouse EHS laminin (Sigma),3) rat a monoclonal antibody (AL-4) specific to the carboxyl-terminal globule of the Ae laminin chain (45, supplied by Dr. Amy Skubitz, University of Minnesota Medical School, Minneapolis, MN), or 4) a monoclonal antibody specific for skeletal muscle (fast)myosin heavychain (Sigma).Samples were incubated overnight at 4 "C, and resulting immune complexes were isolated by incubation with 200 pl of 10% Protein A-Sepharose (Sigma) per milliliter sample for 2 4 h at 4 "C. Protein A-Sepharose,which had been incubated with rabbit anti-rat IgG or rabbit anti-mouse IgG and then washed three times with lysis buffer, was usedto isolate immune complexes of the rat monoclonal and mouse monoclonal antibodies, respectively. Protein A-Sepharose pellets were collected and washed three times in 10 ml of cell lysis buffer containing 20 m~ EDTA. Conditions required for precipitation of 280% of the laminin forms with the polyclonal antiserum were determined in preliminary experiments and used in subsequent studies. Immunoprecipitated proteins were fractionated by electrophoresis on 3-7.5% or 3-10% gradient SDS-polyacrylamide gelsin the discontinuous buffer system (46). In absence of reducing agent in the sample buffer, the interchain disulfide bonds are intact, and isoforms composed of the laminin chains are resolved. In the presence of 2.5% 2-mercaptoethanol in the sample buffer, the interchain disulfide bonds are dissociated, and individual laminin chains are observed. Radiolabeled proteins were visualized by fluorography using ENHANCE (DuPont NEN), and synthesis of the laminin chains was quantitated by densitometry measurement of their levels on x-ray films using the BioImage 110s image analysis system equipped with a CCD camera and Sun-Sparc station 1+with BioImage wholeband softwareversion X (BioImage, Ann Arbor, MI). Peptide Mapping-Peptide maps were generated from the laminin chains in a protocol similar to that of Cleveland and co-workers (47). Immunoprecipitates containing the laminin chains were separated on 3-7.5% SDS-polyacrylamidegels under reducing conditions. Some immunoprecipitates were first treatedwith N-glycopeptidaseF (see below) to remove their asparagine-linked oligosaccharides. Gel slicescontaining the laminin chains were incubated in 0.125 M Tris-HC1, 0.1-0.5% SDS, pH = 6.8, containing 1.0-1.5 p g / d endoproteinase Lys-C (Boehringer Mannheim) for 20-30 min at 37 "C. The gel slices wererinsed in the same buffer containing 2% SDSand lacking the protease, incubated for 5-10 min at 23 "C in the same buffer containing 2.5% 2-mercaptoethanol, and applied to seconddimension 3-7.5% or 3-10% SDS gels to generate peptide maps. lFeatment oflmmunoprecipitates with N-Glycopeptidase F-Laminin immunoprecipitates were washed and equilibrated in reaction buffer consisting of 100 m~ NaH2P0,, 25 m~ EDTA, pH = 7.2, and thenboiled for 5 min in 50 pl of reaction buffer containing 0.2%SDS and 1% 2-mercaptoethanol. 5% Nonidet P-40 and 1.0 unit N-glycopeptidase F (Boehringer Mannheim) were added to the mixture, and the reaction was incubated a t 25 "C overnight. Isolation of RNA and Northern Blots-Total cellular RNA was collected by centrifugation of guanidine isothiocyanate cell lysates through a 5.7 M cesium chloride cushionat 174,000x g for 18-22 h at 20 "C (48). Poly(A)-enriched RNA fractions were isolated from total RNA on columns of oligo(dT)-cellulose(Pharmacia LKB Biotechnology Inc.; 49). 2.0-5 pg of the poly(A)-enriched RNA was fractionated on 0.7-1.0% agarose gels, transferred by capillary blotting to Zeta-Probe nylon membranes (Bio-Rad), and hybridized with radiolabeled cDNA probes as described by Church and Gilbert (50). cDNAprobes forthe Bleand B2e laminin chains and ornithine aminotransferase (51) were labeled to a specific activity of approximately log dpm/pg using [32PldCTP(DuPont NEN) and random primers (52). The cDNA probe for the Ae laminin chain was labeled using [32PldCTPin the polymerase chain reaction (53). The cDNA templates used in theradiolabeling reactions were: 1)a 1413-bp EcoRIISacIfragment encoding domain I and a portion of do-

Induction of Ae-related Laminin Chains

9272 A.

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FIG.2. Induction of the Ae and Ac3h laminin chains in clonal myoblasts derived from C3H10T1/2 fibroblasts. Laminin chains " were immunoprecipitated from radiolabeled lysates of C3H10T1/2 fi~ 4 0 0 broblasts, clone B myoblasts, which were derived from C3HlOT1/2 fibroblasts treated with 5-azaC, clone CM1 myoblasts, which were de" " 6200 rived from C3H10TV2 fibroblasts transfected with a vector expressing MyoD, and clone VEC1 fibroblasts, which were derived from 5-azaCdR:' (-) ' (+) ' C3H10T1/2 fibroblaststransfectedwiththe vector not expressing Flc:. 1. Induction of laminins in C3HlOT1/2 fibroblast cultures MyoD, andwereseparated on SDS gels under reducing conditions. treated with 5-azaCdR. Cultures of C3H10T1/2 fibroblastswere Cultures of C3H10T1/2 (lane 2 ) and VECl (lane 3 ) fibroblasts synthetreated with the DNA dcmethylating agent 5-azaCdRfor 24 h, allowed sized the Rle ( M , = 215,000) and R2e ( M , = 205.000). hut little Ae ( M , to differentiate intomyofiber, adipocyte, andchondrocyte cells (32). and = 400.000t orAc3h ( M , = 350,000) laminin chains.In contrast, cultures radiolabeledwith I:'"Slmethionine ina 1-h pulse/4-h chase protocol. of clone R (lane Z ) and CMl (lane 4 ) myoblasts synthesized theAe and Laminins were immunoprecipitatedfrom the 1-h pulse cell lysate (lanes Ac3h in addition to the Ble and B2e laminin chains. I and 4 ), 4-h chase cell lysate lianas 2 and 5 ), and 4-h chase medium tlanes 3 and 6 ) samples and were fractionated on SDS gels under either reducing ( A ) or nonreducing ( B )conditions. A , cultures of untreated JAR choriocarcinoma (24) cells (data CBH10T1/2 fibroblasts synthesized(lanes Z and 2 ) and secreted (lane 3 ) teratocarcinoma (43), and R l c ( M , = 215.000t and R2e ( M , = 205,000). hut littleAe ( M , = 400,000) not shown). The identityof the four laminin chains was deteror Ac3h ( M , = 350,000) laminin chains. In contrast, cultures of treated mined in experiments shown below. C3H10T1/2 fibroblasts synthesized(lanes 4 and 5 t and secreted(lane 6 ) The Ble, B2e, and Ac3h laminin chains derived from cell Ac and Ac3h in addition toRle and B2e chains. B , cultures ofuntreated C3H10T1/2 fibroblasts synthesized(Ianes I and 2 ) and secreted (lane 3 ) lysates (Fig.lA, lane 4 ) migrated more quicklyon gels than the laminin isoforms of M , = 850,000.whereascultures of treated B le , B2e, and Ac3h chains, respectively, derived from culture C3H10T1/2 fibroblasts synthesized(lanes 4 and .5 1 and secreted (lane 6 ) medium (Fig. l A , lane 6 ). This is likely the result of processing laminin isoforms of M , = 900,000-950,000 in addition to those of M , = on the 850.000. Fihronectin proteinst Fn t were also precipitatedby the Protein of the high mannose asparagine-linked oligosaccharides intracellular laminin chains to complex sialic acid-containing A-Sepharose immunoadsorbent ( A and B , lanes 14, see Ref. 9). I -a

I

I

main G of the Ae laminin chain (bases 5233-6645 in Ref. 54t, 2) a 1718-bp EcoRICYhaI fragment encoding a portion of domain I and the 3'-untranslated region of the R2e laminin chain (bases 4408-6125 in Ref. 55). 3)a 4566-bp GcoRIIHindIII fragment encodingthe Rle laminin chain (bases 565-5130 in Ref. 56). and 4 ) a 1938-bp EcoRIIEcoRI fragmentencodingornithineaminotransferase(51). Radiolabeled cDNA fragments were separated from unincorporated [:'2PldCTP using QuickSpin G-50 Sephadex columns (Roehringer Mannheim). Expression of laminin chain mRNA was quantitated by densitometry using the RioImage 110s image analysis system described above. RESULTS

Induction of Laminins in C3HlOTI 12 Fibrohlasts Dented with 5-AzaCdR-Three weeksafter a 24-h treatment of C3HlOT1/2 mouse embryo fibroblasts with 1PM 5-azaCdR, the culturescontainedmanymultinucleatedskeletalmyofibers, few lipid-filled adipocytes, and few polygonal chondrocytes in addition to the undifferentiated C3HlOT112 fibroblasts (data not shown, Ref. 32). Both treated and untreated C3HlOT112 cultures were radiolabeled with [""Slmethionine a in1-h pulse/ 4-hchase protocol, andlamininswereimmunoprecipitated a polyclonal from the cell lysate and medium samples with antiserum raised against Ae-Ble-B2e laminin ( M , = 950,000) derived from the mouse EHS tumor. The laminins were fractionated on SDS gels in the presence (Fig. lA)or absence (Fig. 1B) of reducing agent. The untreated C3HlOT1/2 fibroblast cultures synthesized B l e ( M , = 215,000) and B2e ( M , = 205,000), but little Ae-like ( M , = 350,000-400,000) chains (Fig. lA, lanes 1 3 ) and assembled M , = 850,000 isoforms of laminin (Fig. lB,lanes 1 3 ). In contrast, the treatedC3HlOT1/2 fibroblast cultures synthesized two Ae-like laminin chains, designatedAe ( M , = 400,000) and Ac3h ( M , = 350,000), in addition to the B l e a n dB2e chains (Fig. LA, lanes 4 4 ) and assembled M , = 900,000-950,000 in addition to M , = 850,000 isoforms of laminin (Fig. 1B, lanes 44).The M , = 900,000-950,000 isoforms co-migrated on gels with Ae-Ble-B2e laminin synthesized by EHS tumor (3), F9

oligosaccharides on thematuresecretedglycoproteins,as shown previously for laminin synthesized by JAR cells (24). In contrast to the Ac3h chain, the Ae chain derived from the cell lysates (Fig. lA, lane 4 ) varied little in migration from the Ae chain derived from the culture medium (Fig.l A , lane 6).Thus, the Ae a nd Ac3h laminin chains appeared todiffer in the number and/or degree of processing of their asparagine-linked oligosaccharides. Synthesis of fibronectin proteins was also apparent in this experiment (Fig. 1,A and B, F n ) because the fibronectins bind non-immunospecifically to the Protein A-Sepharose immunoadsorbent (9). Removal of thefibronectins by treatment of or by immunoprecipitation sampleswithgelatin-Sepharose with a fibronectin antiserumdid not affect subsequent laminin immunoprecipitations (data not shown). Induction of Laminin Chains in Clonal Myoblasts Derived from C3HIOTI 12 Fibroblasts-Myoblasts and myofibers were thepredominantdifferentiated cells presentincultures of C3HlOT1/2 fibroblasts treated with 5-azaCdR (data not shown, Ref. 32), suggesting that synthesis of the Ae and Ac3h laminin chains was associated with activation of the myogenic differentiation pathway in this system.To test this possibility, synthesis of laminin chains was examined inclonal myoblast cell lines derived from C3HlOT112 fibroblasts treated with 5-azaC (clone B myoblasts, Ref. 38) or transfected with a vector expressing MyoD (CM1 myoblasts, Ref. 39). Whereas control cultures of C3HlOT1/2 fibroblasts synthesized only B le a nd B2e laminin chains during a 1-h radiolabeling with [%]methionine (Fig. 2,lane 2 ), cultures of clone B myoblasts synthesized the Ae and Ac3h laminin chains in addition to the Ble and B2e chains under identical cultureconditions (Fig. 2,lane I ). Cultures of CM1 myoblasts synthesized the Ae, Ac3h, B le , a ndB2e laminin chains (Fig.2, lane 4 ) in a manner similar to clone B myoblasts (Fig. 2, lane I ), whereas cultures of control VECl fibroblasts,which were derived from C3HlOT1/2 fibroblasts transfected with the expression vector alone, synthesizedonly the Ble and B2e laminin chains (Fig. 2,

9273

Induction of Ae-related Laminin Chains 1

2 3 4 5

6

b

7

FIG.3. The Ae and Ac3h chains are immunologically related but distinct laminin proteins. Laminin chains were immunoprecipitated from radiolabeled lysates of C3H10T1/2 fibroblast and clone B myoblast cultures and separated on SDS gels under reducing conditions.Arat monoclonal antibody (AL-4,Ref. 45) specific for the carboxylterminal globule of the Ae laminin chain immunoprecipitated both the Ae ( M , = 400,000) andAc3h ( M . = 350,000) laminin chainsfrom clone B myoblast culture lysates (/ane 1 ). Ble ( M , = 215,000) and B2e ( M , = 205,000) chains were precipitated by virtue of their association with the Ae and Ac3h chains (lane I ). Control rat I g G precipitated only fihronectins proteins under identical conditions (datanot shown). Laminin immunoprecipitates obtained using polyclonal laminin antiserum were treated with N-glycopeptidaseF to remove their asparagine-linkedoligosaccharides. TheAe and Ac3h laminin chains synthesizedby cultures of clone B myoblasts migrated separatelyfrom one anotherbefore (lane 2 ) and after (Lane 3)their treatment with N-glycopeptidase F. In contrast, the Ble andB2e laminin chains synthesized by cultures of clone B myoblasts (lanes 2 and 3 ) co-migrated with those synthesized by - * *. cultures of C3H10T1/2 fibroblasts (lanes4 and Fi ), before (lanes 2 and 4 ) F9 MYOC3H and after(lanes 3 and 5 )their treatmentwith N-glycopeptidaseF. The FIG.4. The Ae and AcSh laminin chains generate distinct pepdeglycoslyated Ae, Ble,and B2e lamininchainssynthesized by were C3HlOT1/2 myoblast cultures (lane 6 )co-migrated with the deglycosy- tide maps. Gel slices containing the radiolabeled laminin chains lated Ae, Ble, and R2e laminin chains, respectively, synthesized by F9 treated with endoproteinase Lys-C, and maps were generated on SDS gels from the resulting peptides (see “Experimental Procedures”). A, cells (lane 7). peptide maps generated by the Ae ( M , = 400,000) and Ac3h (M,= 350,000) chains synthesized by cultures of clone B myoblasts (MY01 lane 3 ) in a manner similar to C3HlOT1/2 fibroblasts (Fig. 2, were distinct from onc another. B,peptide maps generated by the Ble ( M , = 215,000) and B2e ( M , = 205,000) laminin chains synthesized by lane 2 ). Characterization of the Laminin Chains-The Ae, Ac3h,Ble, clone B myoblasts (MY01were indistinguishablefrom those generated by theBleand B2e lamininchains, respectively, synthesized by and B2e laminin chains were further characterized. The R l e C3H10T1/2 fibroblast (C31-I)and F9 cells ( F ~ I .

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and B2e chains synthesized by C3HlOT1/2 fibroblast cultures and the Ble,B2e, Ae, andAc3h chains synthesized by clone B myoblast cultures all reacted on immunoblots with polyclonal lane 6 ) co-migrated with the deglycosylated Ae, Ble, and B2e chains, respectively, synthesized by F9 cells (Fig. 3, lane7 ) . antisera raised against Ae-Ble-B2e laminin derived from the EHS tumor (data not shown). In addition, the Blc, B2e,Aeand Peptide maps of the Ae, Ac3h, Blc, and B2e laminin chains chains synthesizedby C3HlOT1/2 fibroblasts and clone B myo- were generated by endoproteinase Lys-C treatment of SDS gel blasts co-migrated on gels with the Ble, B2e, and Ae chains, slices containing these chains (see “Experimental Procedures”). respectively,synthesized by EHS, F9, and JAR tumor cells Maps generatedby the Ae andAc3h laminin chains synthesized by clone B myoblast cultures ( M Y O ) were distinct from one (data not shown). To investigate the relationship between the Ae and Ac3h another (Fig.4A ), whereas maps generatedby t h e B l e a nB2e d laminin chains, cultures of clone B myoblasts were radiolabeled chains synthesized by clone B myoblast cultures were indistinfor 10 h with [“Slmethionine and [‘“SSJcysteine,and the radio- guishable from those generated by the Ble and B2e chains, labeled cell lysateswereimmunoprecipitatedwith a mono- respectively, synthesized by C3H10T1/2 fibroblast (C3H) and clonal rat antibody (AL-4, Ref. 45) specific for the carboxyl- F9 (F9) cells (Fig.4B ). Peptide maps generatedby thc Ac lamiterminal globule of the Ae chain ofAe-Ble-B2e (EHS) laminin. nin chain synthesized by clone B myoblast cultures were also Themonoclonalantibodyimmunoprecipitatedboth Ae and similar to those generated by the Ae chain synthesized by F9 Ac3h laminin chains from clone B myoblast lysates (Fig. lane 3, cells (data not shown). The degree of glycosylation of the lamiI ) in a manner similar to the polyclonal antisera (Fig. 2, lane nin chains had a small effect on the proteolytic cleavage patI ). Ble and B2e laminin chains were also precipitated by virtue ternsobserved,andsimilarpeptidemapswereobtained I), whether or not the laminin chains were first treated with Nof their association with the Ae and Ac3h chains (Fig. 3, lane and mature forms of the Ac3h, Ble, and B2e chains were also glycopeptidase F (data not shown). apparent (Fig. 3, lane I ). Fibronectin proteins (Fn) were preExpression of Laminin Chain mRNA during C3HlOTl I2 cipitated using control rat IgG in place of the monoclonal an- Myogenesis-Steady state levels of mRNA encoding the Ae, tibody under the same conditions (data not shown). Ble, and B2e laminin chains were measured. Poly(A)-enriched The Ae and Ac3h laminin chains were next compared after RNA fractions were isolated from cultures of C3H10T1/2 fibroF (see “Experimen- blast, clone B myoblast, CM1 myoblast, and VECl fibroblast their deglycosylation with N-glycopeptidase tal Procedures”). TheAe and Ac3h chains synthesized by cul- cells, and blots containing the RNA were hybridized withDNA tures of clone B myoblasts migrated distinctly from one another fragments complimentary to the mouse Ae, Ble, andB2e tran3 ) removal of their scripts. Cultures of VECl fibroblasts (Fig. 5A, lanes I and 5 ) before (Fig. 3,lane 2 ) and after (Fig. 3, lane and C3H10T1/2 fibroblasts (Fig.5A, lanes 3 and 7 ) synthesized asparagine-linked carbohydrate chains, suggesting that they differed in their protein not oligosaccharide structures. On the little of a 9.5-kb transcript that hybridized to the Ae laminin probe. On the other hand, cultures of CM1 myoblasts (Fig.5A, other hand, the deglycosylated Ble and B2e laminin chains synthesized by cultures of clone B myoblasts (Fig. 3, lane 3 ) lanes 2 and 6) and clone B myoblasts (Fig. 5A, lanes 4 and 8 ) synthesized much higher levels of the 9.5-kb transcript. The co-migrated with the deglycosylated B l e a n d B2e chains, respectively, synthesized by C3H10T1/2 fibroblasts (Fig. 3, lane 9.5-kb transcript synthesized by the myoblasts (Fig. 50, lane 2 ) co-migrated on blots with the9.5-kb Ae transcript synthesized 5).In addition, the deglycosylated Ae, Ble, and B2e laminin chains synthesized by C3H10T1/2 myoblast cultures (Fig. 3, I ). C3H10T1/2 myoblast cultures also by F9 cells (Fig. 50, lane

Znduction of Ae-relatedChains Laminin

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tated from lysatesof the radiolabeledcell clones and separated on SDS gels under reducing conditions. Synthesisof the laminin chains was calculated by densitometry measurement of B2e"8.0 1 -6.5 Bletheir levels on x-ray films using the BioImage analysis system (Table I). C. OAT!-2.2 Clone B myoblasts synthesized43-fold higher levelsof the Ae chain and 13-fold higher levels of the Ac3h chain than did D. 1 2 kb C3HlOT1/2 fibroblasts and scored 704, in an assay measuring 77 c9.5 the percentageof cell colonies containing multinucleatedmyo-7.5 fibers. On the other hand, C3H10T1/2 fibroblasts and clone MCA CI 15C1 fibroblasts, which were derived from C3H10T1/2 fibroblaststransformedwith MCA, synthesizedlittle Ae or FK. 5 . Steady state levelsof mRNA encoding theAe, Ble, and Ac3h laminin chains and did not form myofibers in the colony B2e laminin chains. Poly(A)-enriched RNA fractions were purified assay. Two clones were derived from MCA CI 15C1 cells transfrom total cellular RNA and 2.5 pg (lanes 1 4 ) or 5 pg (lanes 5-8) fected with a vector expressing MyoD. The first (10-1A2) synaliquots were separated on agarose gels, transferred to nylon membranes, and hybridized with radiolabeledcDNA probes complimentary thesized 26-fold higher levelsof the Ae chain and18-fold higher to the Ae, Ble, B2e, or ornithine aminotransferase (OAT)mRNA. A, levels of the Ac3h chain than didMCA C1 15C1 cells and scored cultures of VECl (lanes I and 5 ) and C3HlOT1/2 (lanes 3 and 7 ) fibro- 58% in the myofiber assay. The second (10-1D4) synthesized blasts expressed littleof a 9.5-kb Ae mRNA transcript, whereas cultures little Ae or Ac3h lamininchainsin a mannersimilarto of CM1 (lanes 2 and 6 )and clone B (lanes 4 and 8 )myoblasts expressed C3HlOT1/2, MCA CI 15C1, and 10 VEC2 fibroblasts, which higher levels of this 9.5-kb Ae transcript. B,cultures of VECl (fanesI werederivedfrom MCA C1 15C1cellstransfectedwiththe and 5 )and C3H10T1/2 ([ones3 and 7 ) fibroblasts and CM1 (fanes2 and 6 )and clone R (lanrs 4 and 8 )myoblasts expressed similar levelsof the vector not expressingMyoD. 10-1D4 and 10 VEX2 cells scored 6.5-kb Rle and 8.0-kb R2e mRNA transcripts. C , cultures of VECl ~1% and. 0, respectively, inthe myofiber colony assay. Two other (lanrs I and 5 ) and C3HIOT1/2 (lanes 3 and 7 ) fibroblasts and CM1 cloneswerederivedfrom MCA C1 15C1cellstreatedwith (lanes 2 and 6 ) and clone B (lanes 4 and 8 ) myoblastsexpresssed comparable levelsof a 2.2-kbmRNA transcript encoding ornithine ami- 5-azaC. The first (15-1A1) scored 20% in the myofiber colony notransferase, a mitochondrial enzyme ( 5 1 ) .U , the 9.5-kb Ae transcript assay but synthesized little Ae or Ac3h laminin chains. Howsynthesized by C3H10T1/2 myoblasts (lane.2)co-migrated on blots with ever, 15-1A1 cells did not terminally differentiate or synthesize the 9.5-kb Ae transcript synthesized by F9 cells (lanr I ) . A 7.5-kb or subsequent experiments (data muscle-specific myosin in this transcript synthesized by C3H10T1/2 myoblasts (lane 2 ) but not F9 not shown), suggesting i t had reverted to a phenotype resemcells (lane I ) also hybridized to the Ae cI)NA probe. bling C3H10T1/2 and MCA CI 15C1 fibroblast cells sometime synthesized a 7.5-kb transcript that hybridized to the Ae probe after the colony assay had been conducted. The second clone 5 0 , lane 2).The (15-5B6) scored 25% in the myofibercolony assay and syntheunder the same stringent conditions (Fig. 7.5-kb transcript was not apparent in F9 (Fig. 5 0 , lane 1 ) or sized 19-fold higher levels of the Ae chain and 11-fold higher C3H10T1/2 fibroblast cells (data not shown), suggesting i t en- levels of the Ac3h chain than didMCA CI l 5 C l cells. Synthesis of the Ble and B2e laminin chains varied less 2-fold than in all coded the Ac3h laminin chain. VECl fibroblasts (Fig. 5B, lanes 1 and .5),C3HlOT1/2 fibro- the myoblast and fibroblastcell clones. Synthesis of Laminin Chains by Established Myoblast Cell blasts (Fig. 5B, lanes 3 and 7). CM1 myoblasts (Fig. 5B, lanes 2 and 6), and clone B myoblasts (Fig. 5B, lanes 4 and 8 )all Lines and Primary Mouse Muscle Cells-Synthesis of laminin of established myoblastcell synthesized similar transcripts encoding the B l e (6.5 kb) and chains was also studied in cultures B2e (8.0 kb) laminin chains. The Ble and B2e transcripts co- lines and primary mouse muscle cells. Mouse C2C12, mouse migrated on blots with Ble and B2e transcripts, respectively, BC3H1, and rat L6 myoblasts were grown to 7 0 4 0 % conflusynthesized by F 9 cells (data not shown), and a control 2.2-kb ence in medium containing 20% fetal calf serum and were intranscript encoding the mitochondrial enzyme ornithine amin-duced to terminally differentiateby switching to medium confetal calf serum. Three days later, the cultures were otransferase varied little betweenthe fibroblast (Fig. 5C, lanes taining 1%. I , 3, 5, and 7 ) and myoblast (Fig. 5C, lanes 2, 4, 6, and 8 ) radiolabeled with I""S1methionine and [""Slcysteine for 1 h, and laminin chains and muscle-specific myosin heavy chain cultures. were immunoprecipitated fromcell lysates and fractionated on Expression of the laminin mRNA transcripts was quantiSDS gels under reducing conditions (Fig. 6, lanes 1 3 ). Primary tated by densitometry measurement of their levels on x-ray films using the BioImage 110s image analysis system (see "Ex- cultures of adult and neonate mouse myoblasts were grown untilmyoblastcoloniescontainedmultinucleatedmyofibers perimental Procedures"). Expression of Ae chain transcripts andwereradiolabeledfor 1 h with[:%Jmethionineand was determined to be 230-fold higher in C3HlOT1/2 myoblasts compared to C3HlOT1/2 fibroblasts after normalization to the [:%]cysteine. Laminin chains and myosin heavy chain were level of ornithineaminotransferaseexpressionineach cell then immunoprecipitated from the culture lysates and separated on SDS gels under reducing conditions (Fig. 6, lanes 4 type. In contrast, expression of the Ble and B2e chain tranand 5).The cultures of C2C12 (Fig. 6,lane 1 ), L6 (Fig. 6,lane scripts varied ~3-fold in the myoblast and fibroblast cells. lane 3 ), primary mouse adult (Fig. 6, lane 4 1, Synthesis of Laminin Chainsin Myoblast Clones with Varied 2 ), BC3H1 (Fig. 6, Potentials for Terminal DifferentiationSynthesis of laminin and primary mouse neonate (Fig. 6,lane 5 )myoblasts all synthesized theAe, Ac3h, Ble, andB2e laminin chains and myosin chains was measured in C3H10T1/2 cell clones with varied heavy chain (Fig. 6, lanes 1-5, M H ) . potentials for terminal myogenic differentiation. These clones werederivedpreviouslyfromC3H10T1/2fibroblaststransDISCUSSION or transformed withMCA and then either treated with 5-azaC In this manuscript, the expression and nature of four chains fected with the gene encoding MyoD to induce myogenicdifferentiation (39).The myogenic potential of each clone is thought of the extracellular matrix glycoprotein laminin were characto be determined by the interactions of cellular factors that terized as multipotent C3H10T1/2 mouse embryo fibroblasts control transformation and proliferation inducedby MCA with differentiated into myoblasts and myofibers. The Ae, Ble, and B2e laminin chains were identified based on: 1)their co-migracellular factors that control myogenic differentiation induced by 5-azaC and MyoD. Laminin chains were immunoprecipition on SDS gels and immunoblots with the Ae, Ble, and B2e 1 2 3 4 5 6 7 8

A.

Ae-

1

'

0

h

kb: c9.5

Induction of Chains Laminin Ae-related

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TABLEI Relative synthesis of the Ae, AcJh, Ble, a n d B2e laminin chains in C3HlOTll2 cell clones with varied potentials for terminal myogenic differentiation Cell line

C3H10Tl/2 Clone B MCA C1 15C1 lo-1A2 10-1D4 15-1A1 15-5B6 10 v E c 2

Derivation"

Mouse embryo C3H10T112 + 5-azaC C3HlOT1/2 + MCA MCA C115C1+ MYOD MCA C115C1+ MYOD MCA C1 15C1 + 5-azaC MCA C1 15C1 + 5-azaC MCA C1 15C1+ VEC

Percent colonies with myofibers"

0 70 0 58