Enhanced Synthesis and Secretion of Type IV Collagen and Entactin ...

Vol. 263, No. 31, Issue of November 5 , pp. 16163-16169,1988 Printed in U.S. A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Enhanced Synthesis and Secretion of Type IV Collagen and Entactin during Adipose Conversion of 3T3-Ll Cells and Production of Unorthodox Laminin Complex* (Received for publication, April 1, 1988)

Yasuaki ArataniS andYasuo Kitagawas From the Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa, Nagoya 464-01, Japan

Synthesis and secretionof basement membrane pro- review, see Ref. 13), such as insulin (14-17) and adrenergic teins by 3T3-Ll adipocytes were studiedby metabolic agonists (18-20). However, this cell line has not been studied labeling of the cells with [3SS]methionine.Enhanced for the ability to secrete extracellular matrix proteins during synthesis and secretion of many polypeptides of high its adipose conversion. molecular weight were observed by stimulating the Electron microscopic observations of mature fat cells (21adipose conversion of 3T3-Ll fibroblasts with dexa- 31) have revealed that the plasma membrane is surrounded methasone, l-methyl-3-isobutylxanthine, and insulin. by a region which is similar to basement membranes of other Among these polypeptides, al(IV) and a2(IV) chains of tissues. A t the boundary of this region, a network of collagen collagen were identifiedbased on specific immunopre- fibers which appeared to be inserting into it was often obcipitationand digestion withbacterial collagenase. served. According to Napolitano (27), who followed the adiSynthesis and secretion of al(IV) and m(IV) chains pocyte differentiation from fibroblasts in developing fat tissue were negligible in the fibroblasts, but remarkably enhanced in adipocytes. Based on specific immunoprecip- of newborn rats, the formation of a basement membrane-like itation of a sulfated polypeptide of 150 kDa, enhanced structure around fibroblasts was one of the first ultrastruc(6-fold) synthesis and secretion of entactin were also tural changes seen. The interaction of these basement memdemonstrated. Immunoprecipitation with anti-laminin branes with fat cells seemed loose. They did not conform to antiserum showed synthesis of three polypeptides with the decrease of size of depleted-fat cells in starved animals sizes corresponding to B subunits but failed to demon- (23, 26,32, 33). Instead, they became invaginated, forming strate synthesis of the A subunit. Synthesis of these loops and folds when cell size decreased, and did not follow the contour of cell surface (34). It has been postulated that laminin-related polypeptides remained constant during the conversion. Nonreducing sodium dodecyl sul- the extracellular matrix organizes fat cells into small lobules, fate electrophoresis showed intracellular assembly of observed in uiuo, by forming a network of reticular fibers (35). three laminin-related polypeptides into binary and ter-For further understanding of the function of these basement nary complexes in a similar sequence of ABlB2 for- membranes, analysis of proteins secreted from fat precursor mation via BIBz in embryonal carcinoma F9 (Morita, cells is needed. Kimata et al. presented a biochemical descripA., Sugimoto, E., and Kitagawa, Y. (1985) Biochem. tion of these basement membranes, demonstrating the synJ. 229, 259-264). The ternary complex of laminin in thesis of laminin, proteoheparan sulfate (36), and type IV 3T3-Ll cells had a size significantlysmallerthan collagen (37) in fat precursor cells of mouse mammary gland. ABIBz complex in F9 cells. In this complex, a novel Kuri-Harcuch et al. (38) reported histochemical studies of the subunit appears to take the place of the A subunit. organization of the extracellular matrix during adipose conThus, a novel laminin complex is produced by 3T3-Ll version of 3T3-F442A cells. cells. In this paper, we studied the production of extracellular proteins by 3T3-Ll cells during their differentiation. Enhanced secretion of type IV collagen and entactin was obThe 3T3-Ll fibroblast converts to an adipocyte-like cell served, whereas laminin remained constant duringthe adipose under certain culture conditions (1-5) and has often been conversion. We also suggest that 3T3-Ll cells produce an used as amodel for differentiation by studying its morpholog- “unorthodox” laminin complex, in which a novel subunit is ical and biochemical properties during the conversion. Con- substituted for the A subunit. comitant with the accumulation of triglycerides as intracelEXPERIMENTAL PROCEDURES lular lipid droplets, decreased expression of cytoskeletal proteins such as actin and tubulin has been demonstrated (6). Materials-Rabbit antiserumagainstentactin, purifiedfroma The cells acquire many lipogenic (4, 7-11) and lipolytic (11, mouse endodermal cell line (M1536-B3), was a generous gift of Dr. 12) enzymes and become sensitive to many hormones (for a A. E. Chung(University of Pittsburgh,Pittsburgh,PA).Rabbit * This investigation was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked“aduertisement”inaccordancewith 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of a Japan Society for the Promotion of Science Fellowship for Japanese Junior Scientists. 5 To whom reprint requests and correspondence should be addressed.

antiserum against laminin, purified from EHS sarcoma,l was kindly providedbyDr. K.Kimata(NagoyaUniversity, Nagoya, Japan). Anti-type IV collagen was raisedin the rabbit against type IV collagen (EHS sarcoma) from Bethesda Research Laboratories. DMEM and MEM, from Nissui Pharmaceutical Co. (Tokyo, Japan), FCS from The abbreviations usedare: EHS sarcoma,Engelbreth-HolmSwarm mouse sarcoma; DMEM,Dulbecco’s modified Eagle’s medium; MEM, Eagle’s minimal essentialmedium; FCS, fetalcalf serum; MIX, 1-methyl-3-isobutylxanthine; SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; RER, rough endoplasmic reticulum.

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M. A. Bioproducts (Walkersville, MD), and culture plates from Nunc (Roskilde, Denmark) were used for cell culture. Insulin and dexamethasone from Sigma, MIX from Aldrich, monensin from Behring Diagnostics, Protein A-Sepharose CL-4B from Pharmacia LKB Biotechnology Inc, tunicamycin from Wako Chemical Co. (Osaka, Japan), bacterial collagenase from Advanced Biofactures Co. (Form 111; Lynbrook, NY), ~-[~‘S]methionine (1200 Ci/mmol) from Amersham Corp., [35S]H~S0, (carrier free)and EN3HANCE from Du Pont-New England Nuclear, D-[2-3H]mannose (30 Ci/mmol) from ICN Radiochemicals (Irvine, CA), and XAR-5 x-ray films from Kodak were used. All other reagents wereof the highest quality commercially available. Cell Cultures-3T3-Ll cells provided by Dr. H. Green (Harvard Medical School, Boston, MA) were cultured essentially as described (20, 39). The cells were inoculated into 24-well plates and grown in DMEM containing 10% FCS, 50 units/ml penicillin, and 50 pg/ml streptomycin (DMEM-FCS). Cultureswere refed everyother day and maintained at 37 “C under humidified 5% CO, and 95% air until they became confluent (Day 0). Adipose conversion was stimulated by feeding the cells on DMEM-FCS containing 0.5 mM MIX, 0.25 p~ dexamethasone, and 10 pg/ml insulin. Two days later, the medium was changed to DMEM-FCS and the cells were allowed to differentiate. Parietal endoderm-like F9 cells were prepared as described (4043). Labeling Cells withRadioactive Precursors-Cultures in 24-well plates at theindicated stage of differentiation were washed once with methionine-free MEM and incubated in 200plof methionine-free MEM containing 0.5 mCi/ml [35S]methionineat 37 “C for 4 h under humidified 5% COZ and 95% air. The labeling of cultures with [3’S] H,S04 (1.6 mCi/ml) or with ~-[2-~H]mannose mCi/ml) (5 was done as above except that DMEM was used instead of methionine-free MEM. To study the effect of monensin (10 p ~ or) tunicamycin (10 pg/ml), the cultures were preincubated for 120 min in DMEM-FCS containing the drug and then labeled as above. Trichloroacetic acidinsoluble radioactivity incorporated into total cellular protein was measured by a filter-disc method (44). Incorporation of [35S]methionine into cellular protein was found to be enhanced during the course of adipose conversion of 3T3-Ll (see “Results”). Monensin and tunicamycin reduced the incorporation to 95 and 70% of that in their absence, respectively. To normalize these variations of incorporated radioactivity, aliquots of labeled medium or cell lysate corresponding to thesame amount of [3’S]methionineincorporated into total cellular protein were used for immunoprecipitation. For pulse-chase experiments with [35S]methionine,the cultures were preincubated with methionine-free MEM for 30 min and then pulse-labeled for 15 min with the same medium containing 1.66 mCi/ml [35S]methionineas above. The radiolabeled cells were “chased” by replacing the medium with DMEM and incubating for the indicated period. Labeled medium was collected and centrifuged before analysis, and thecells were lysed with 200 p1 of an “immunoprecipitation buffer” containing 10 mM Tris-HC1 (pH 8.0), 2 mM EDTA, 1%(v/v) Triton X-100, 0.4% (w/v) SDS, 1 mM PMSF, and 0.4 M NaCl. Collagenase Treatment-Aliquots (60 pl) of labeled medium from 3T3-Ll cells at Day 4 of the adipose conversion were adjusted to 2.5 mM N-ethylmaleimide and 3 mM PMSF, and digested with 0, 1.4, or 5.6 units of bacterial collagenase for 4 h at 37 “C. The digestion was terminated by adding an equal volume of 2 X concentrated immunoprecipitation buffer. Immunoprecipitation-Probably due to the affinity of basement membrane components to each other, immunoprecipitation of a component tended to precipitate other components. In order to reduce this coprecipitation, we used relatively high concentration of SDS and NaCl in the immunoprecipitation buffer (see above). Aliquots of labeled medium or cell lysate diluted to 500 pl with the immunoprecipitation buffer were preliminarily incubated with 30 pl of a 10% (w/v) suspension of Protein A-Sepharose for 30 min with shaking, and then the Sepharose beads were spun out. This procedure was effective in removing many of the polypeptides which had affinity only for the Protein A-Sepharose beads. Thesupernatants were incubated with 1 p1 of antiserum for 30 min, followedby further incubation with 30 p1 of Protein A-Sepharose for 30 min for shaking. All incubations were carried out at room temperature. Packed Sepharose beads were washed three times with the immunoprecipitation buffer and extracted by boiling in a buffer containing 6% (w/v) SDS, 134 mM Tris-HC1 (pH 6.8), 10% (v/v) glycerol, 4% (v/v) 2-mercaptoethanol, and 0.006% (w/v) bromphenol blue (2 X concentrated SDS sample buffer). Samples for two-dimensional electrophoresis of lam-

inin subunits were extraced by the same buffer without 2-mercaptoethanol. SDS-Polyacrylamide Gel Electrophoresis-The discontinuous buffer system of Laemmli (45) was used with 3% (w/v) acrylamide for the stacking gel and 4% (w/v) acrylamide for the separation gel. Two-dimensional electrophoresis of laminin subunitswas carried out by the method of Dulis et al. (46) as modified (41), except that the incubation in SDS sample buffer before the second dimension was for 3 h. Addition of 50 mM iodoacetic acid to theimmunoprecipitation buffer did not change the patternof two-dimensional electrophoresis. This implies that any artificial disulfide bond was not formed during nonreducing SDS electrophoresis of the first dimension. After electrophoresis, the gels were impregnated with EN3HANCE, dried, and exposed to x-ray film at -80 “C. For molecular weight standards, we used [35S]methionine-labeledmedium (without immunoprecipitation) of parietal endoderm-like F9 cells (40-42). This medium contains laminin subunit A (450 kDa), B, (240 kDa), B, (230 kDa), and entactin (150 kDa) as major components (47,48).Migration positions of these standards are indicated at the margin of each fluorogram. For semiquantitative estimation of the radioactivity incorporated into basement membrane proteins, densitometry of the fluorogram was performed with a Shimazu dual-wavelength scanner, and relative densities of peptide bands were calculated.

RESULTS

Enhanced Synthesis and Secretion of Type IV Collagen, Entactin, and Laminin by 3T3-Ll Adipocytes-Spontaneous conversion of 3T3-Ll cells into adipocytes occurs about 1 month after cultures reach confluence (2). To accelerate this conversion, we stimulated confluent cultures with a combination of dexamethasone, MIX, and insulin during the first2 days (Day 1 and 2) and then maintained cells in the usual culture medium (DMEM-FCS) for an additional 4 days. By thistreatment, most adipocyte phenotypic characteristics were expressed at maximum levels within 6 days after initiating the stimulation (20, 39). When cultures at different stages of the conversion were labeled with [35S]methionine, linear incorporation of radioactivity was observed for 2 h and declined thereafter. However, 70% incorporation of the extrapolated value of the initial linear part was still observed after 4 h of labeling. Secretion of radioactive proteins intothe medium was observed after a 1-h lag period and increased linearly thereafter for 2 h. Based on these observations, an estimation of synthesis and secretion of proteins was made on samples labeled for 4 h. [35S]Methionineincorporation/ well increased immediately after stimulating the conversion, reached a maximum on Day 3 or 4, and declined thereafter. Incorporation into secreted proteins/well changed roughly in parallel. The cell number increased %fold during thisprocess (results not shown). For immunoprecipitation, we took aliquots of labeled media or cell lysates corresponding to equal radioactivity incorporated into cellular protein, in order to normalize the variation during the conversion. This normalization gives the ratio of [35S]methionineincorporated into individual proteins to that of total proteins, and results become independent of the change in cell number, protein synthesizing activity/cell, or intracellular specific activity of [35S]methionine. Depending on adipose conversion, enhanced secretion of many polypeptides of molecular mass larger than 130 kDa was observed by SDS electrophoresis of labeled medium before immunoprecipitation. Polypeptides immunoprecipitated with anti-type IV collagen and anti-entactin antisera were included among major proteins secreted from 3T3-Ll adipocytes. These bands gave almost the same densities on the fluorogram of labeled media before and after immunoprecipitation (results not shown). This indicated that the immunoprecipitation method used wasquantitative. Because polypeptides precipitated with anti-laminin antiserum were minor components, we could not estimate the recovery. However,

of 3T3-Ll Adipocytes

Basement Membrane Proteins

16165

quantitative precipitationwith the same antiserum of laminin Immunoprecipitates with anti-laminin antiserum from mesubunits from labeled medium of embryonal carcinoma F9 dium of Day 1 (Fig. IC, lane under Medium, I ) showed three cells (40-43) suggested that the methodused was quantitative laminin-related polypeptides (indicated by solid triangles) also on the 3T3-Llsystem. with similar migration to laminin B subunits of F9 cells. Of Immunoprecipitation with anti-type IV collagen antiserum the additionalpolypeptides (indicated by open triangles), the showed two polypeptides with estimatedsizes of 185-200 kDa polypeptide with greater migration may correspond to entacin labeled medium from 3T3-Ll on Days 3-6 of the conversion tin, which coprecipitated with the laminin-related polypep(Fig. L4, lanes under Medium). These polypeptides had mi- tides due to potential affinity (48). Concerning another polygration distancessimilar to (uI(IV) and (u,(IV) collagen of peptide with lesser migration, denser band was observed beembryonalcarcinomaF9, and were sensitive to bacterial fore we improved the immunoprecipitationmethod by adding collagenase (results not shown). The immunoprecipitation the stepof preincubation with Protein A-Sepharose (see "Exfrom cell lysates showed two intracellular labeled polypeptides perimental Procedures"). This suggested that precipitationof with faster migration (Fig. L4, lanes under Cell). These may these bands was due to adsorption to Protein A-Sepharose. correspond to the precursors before getting terminalglycosyl- Secretion of the laminin-related polypeptides was enhanced ation of oligosaccharide side chains. Synthesis of these poly- on Day 1and 3. Immunoprecipitates from labeled cell lysates peptides was negligible in preadipocytes (Day 0) and markedly (Fig. IC, lanes under Medium) gave three bands with faster enhanced by adipose conversion, reachingthe maximum level mobility than those secreted into medium. The densities of on Day 3 (Fig. lA).Densitometric analysisof these bands of these intracellular bands remained virtually constant Day 0 and Day 3 suggested approximately 50-fold stimulation throughout the conversion. Another important observation of synthesis. concerning laminin of 3T3-Ll cells is that no labeled polyImmunoprecipitationwithanti-entactinantiserum from peptides corresponding to the A subunit (see the position of [35S]methionine-labeled medium showed a band of 150 kDa F9A at right of Fig. IC)was detected either within or secreted (Fig. lB, lanes under Medium). This band was labeled with from 3T3-Ll cells. ["S]H2S04 as well (not shown). The density of the band in Unorthodox Laminin Complex Production by 3T3-Llsamples from the medium increased 6-fold from Day 0 to Day Laminin was originally described as being composed of two 1. When the increase of total [35S]methionine incorporation polypeptides GP-1 and GP-2, containing 10-15% carbohyduring adipose conversion was taken into thecalculation, the drate and rich in half-cystine, which were found in basement enhancement calculated was 30-fold on Day 3. Secretion of entactin decreased during the later stage of adipose conver- membrane of an endodermal cell line (M1536-B3) (49, 50). The same proteins were found in Reichert's membrane (51) sion. However, the level on Day 6,whenmostadipocyte phenotypic characteristics had been expressed (20, 39), was and purified from EHS mouse sarcoma (52). Laminin was much higherthan that on Day 0. This indicates that enhancedlater found to consist of three polypeptides: subunit A (450 secretion of entactin is a characteristic of adipocytes. Immu- kDa), B1 (240 kDa), andBa (230 kDa) (47,53),assembled into noprecipitate from cell lysates showed the same band (Fig. a cross-shaped structure of 950 kDa stabilized by disulfide lB, lanes under Cell),but the density of the bandwas low and bonds (54). We have not detected any polypeptide correspondremainedvirtually constant throughout the course of the ing to lamininA in immunoprecipitatesfrom 3T3-Ll. Instead, three polypeptides with estimated molecular masses of 200, conversion. This suggests that entactin issecretedrapidly after the synthesis. The bands observed at the range corre- 210, and 230 kDa were observed in the medium, and their sponding to laminin B subunits may be due to affinity of putative precursors with estimated masses of 180, 190, and laminin to entactin(48). 210 kDa were detected in cell lysates (Fig. 1). Intracellular

A Medium

B Cell

Modlum

C Cell

Medium

( b Y ) 0 13 4 0 0 1 3 4 0( b Y ) O l3 4 6 0 1 3 4 6

*i

(bY)O

I

Cell

1 3 4 8 0 1 3 4 6

-F9 A

FIG. 1. Enhanced synthesis and secretion of basement membrane proteins during adipose conversion of 3T3-Ll cells. 3T3-Ll cells on 24-well plates were cultured in DMEM-FCS until they reached confluence (Day 0). The adipose conversion was stimulated by changing the medium to DMEM-FCS containing dexamethasone (0.25 PM), MIX (0.5 mM), and insulin (10 pglml). Two days later, the medium was changed to DMEM-FCS and cells were allowed to differentiate. On the day shown, cells were labeled with ["S]methionine (0.5 mCi/ml) for 4 h at 37 "C. Portions of radiolabeled medium corresponding to 3.0 X lo5 cpm of incorporated radioactivity, or cell lysate corresponding to 4.9 X lo6 cpm, were immunoprecipitated with anti-type IV ( A ) ,anti-entactin ( B ) ,or antilaminin (C) antiserum. Immunoprecipitates were applied to SDSelectrophoresis and a fluorogram of the dried gel was taken. Migration positions of laminin A subunit (FgA), B subunits (F9B),and entactin ( F 9 E ) in the medium of parietal endoderm-like F9 cells are indicated by arrowheads. Solid triangles at the track of Medium, 1 in C indicate laminin-related polypeptides, and open triangles indicate coprecipitated polypeptides described in the text.

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Basement Membrane Proteins of 3T3-Ll Adipocytes

processing, transport, and assembly of these laminin-related polypeptides were further analyzed in the following experiments. Fig. 2 shows the results of pulse labeling with [“‘Slmethionine for 15 min and chasing of 3T3-Ll cultures on Day1 of conversion. No radiolabeled band correspondingto laminin A was detected even after 15 minof pulse labeling (Fig. 2, lane under Cell 0). This excluded the possibility that laminin A was rapidly degraded during thelong period of labeling (4 h) in former experiments(Fig. 1).By chasing thelabeled cultures for 90 min or more, the intensitiesof three intracellular bands slowly decreased (Fig. 2, lanes under Cell), and they seemed secreted after processing into larger “mature” species (lanes under Medium). Compared with the rapid secretion of laminin as an ABIBP complex from parietal endoderm-like F9 cells (41,42), the results inFig. 2 show that secretion of lamininrelated polypeptides from 3T3-Ll is extremely slow. Although this experiment suggested a precursor-product relation between bands in the cell lysate and in themedium, it did not identify which cellularband is the precursor of which secreted band. Among three cellular bands, the 180-kDa band disappeared most rapidly from cell lysate. T o explore the oligosaccharide side chain processing and transport of these polypeptides, the effectsof monensin and tunicamycin were tested in the experiment presented inFig. 3. The mobilities of two intracellular bandswere not affected by monensin, but the mobility of the180-kDaband was increased (lane under Monensin, C ) . Monensin caused accumulation of all three labeled bands but the effect wasstrongest on the180-kDa band. These differences presumably reflect a difference between the 180-kDa band and the other two in its intracellular processing. Their response to monensin, together with their estimatedmolecular masses, suggest that the 190-

-F9 A

-F9B

-F9 E

FIG.3. Intracellular oligosaccharide side chain processing and transportof laminin-related polypeptidesin3T3-Ll cells. 3T3-Ll cells at Day 1 of adipose conversion were labeled with [““SI methionine in the absence or presence of tunicamycin (10 pg/ml) or . cell lysates ( C )correspondingto the monensin (10 p ~ )Radiolabeled same amount of incorporated radioactivity were immunoprecipitated. were immunopreCorresponding aliquots of radiolabeled media ( M ) cipitated. Other details are the same as in Figs. 1 and 2.

* F9A

.F

230kDa

19OkDa= 21 OkDa 180kt)a

F9B 2lOkDa 200 kDa

F9E

FIG.2. Pulse labeling and -chasing of laminin-related peptides in3T3-Ll cells. 3T3-Ll cells at Day 1 of adipose conversion were pulse-labeled for 15 min with [“S]methionine and chased for the indicated periods. Radiolabeled media or cell lysates corresponding to a half of culture in a well of a 24-well plate were immunoprecipitated by anti-laminin antiserum. The molecular masses of the polypeptides indicated were estimated based on reported values of laminin A subunit, €3 subunit, and entactin (47,48). Other details are the same as in Fig. 1.

and 210-kDa bands correspondto the precursors of B1and BP in F9 cells (termed B1. and Bzx in Refs. 41 and 42). In the presence of tunicamycin, a series of bands with faster mobility was observed (lane under Tunicamycin, C ) . These may correspond to unglycosylated precursors to which cotranslational transfer of N-linked high-mannose oligosaccharides did not occur ( 5 5 ) . The intensity of labeling of all three bands was weaker than for glycosylated precursors observed in the absence of tunicamycin (lanes under None, C and Monensin, C ) . This may be due to a protective function of high-mannose oligosaccharide side chains which allows the protein to resist proteolytic degradation (46, 56). These unglycosylated precursors did not seem to be secreted (lane under Tunicamycin, M). All three cellular laminin bands in 3T3-Ll cells became radiolabeled after incubating the culture with D-[2-3H]mannose for 4 h. When the ratio of incorporated ‘H to 35S (from [3sS]methionine) was compared among three bands, the 210two bands kDa bandgave a &fold higher ratio than the other intheabsence of monensin or tunicamycin(resultsnot shown). T o explore the assembly of laminin-related polypeptides in 3T3-Ll cells, immunoprecipitates from cell lysates (Fig. 4A) and medium (Fig. 4B) were analyzed by two-dimensional electrophoresis, in which nonreducing and reducing electro-

16167

Basement Membrane Proteinsof 3T3-Ll Adipocytes

A

. - .”.

B .

-F9A

-F9 B

*

Entwtin

/

“E Entactin/

FIG. 4. Nonreducing and reducing SDS electrophoresis of laminin-related polypeptides of 3T3-LI cells. Radiolabeled cell lysate ( A ) or medium ( B ) from 3T3-Ll at Day 1of adipose conversion was immunoprecipitated with anti-laminin antiserum. Precipitates were electrophoresed from left to right under nonreducing conditions and from top to bottom under reducing conditions as described in the text. The estimated molecular mass of laminin-related polypeptides is indicated. The spot corresponding to entactin, which may be coprecipitated due to an affinity to laminin (see text),is also indicated. Asterisk indicates possible component of the large laminin complex in 3T3-Ll cells. Other details are the same as in Figs. 1 and 2.

phoresis are combined. In this technique, monomeric polypeptides lie on a diagonal, and polypeptides which participate in a disulfide-bonded complex give rise to spots ona vertical line. Analysis of cell lysates (Fig. 4A) showed monomer pools of the 210-, 190-, and 180-kDa polypeptides, although separation of 190- and 180-kDa spots was poor. This analysis also demonstrated existence of a dimer of 210- and 190-kDa polypeptides. This is comparable with the B1B2 binary complex formation in F9cells and suggests that the210- and 190-kDa polypeptides in 3T3-Ll cells are equivalent to laminin B1 and B2 subunits in F9 cells. Assembly of laminin-related polypeptides into a complex larger than the 210/190-kDa binary complex was also shown. This large complex contained the 210- and 190-kDa polypeptides. Although poor resolution made it difficult to detect additional components, reproducible results suggested that it contained a polypeptide larger than the 210-kDa (indicated by asterisk) and the180-kDa polypeptides (Fig. 4A). When the mobility of the large complex in 3T3-Ll was directly compared with that of intracellular precursor of ABlB2in F9cells by one-dimensional electrophoresis undernonreducingcondition (Fig. 5), it was significantly greater than that of the precursor ofABlB2. This made it difficult to consider the complex to be composed of four or more polypeptides with the sizes of 200 kDa. The composition of 190 kDa/(210 kDa)* or(190 kDa)2/210 kDa could approach the suggested mass of the large complex in 3T3-Ll cells, but the relative density of spots corresponding to 210- and 190kDa polypeptides (Fig. 4A) did not agree with either of these compositions. It therefore seemed that 180-kDa polypeptide itself or its processed product was in thecomplex. Considering rapid processing of the 180-kDa polypeptide in the pulsechase experiment (Fig. 2), the spot larger than 210 kDa (Fig. 4A, indicated by asterisk) may be a processed product of the 180-kDa polypeptide, which could be resolved partially only by two-dimensional electrophoresis. These indicate that the large complex in 3T3-Ll cells is the mixture of 180/190/210kDa and (180-kDa product)/190/210 kDa. Two-dimensional electrophoresis of radiolabeled medium showed that almost all labeled spots were in the large complex. Since secreted species of laminin-related polypeptides had the masses of 200-, 210-, and 230 kDa (Fig. 2), the putative three compo-

1.. ,.

Blx* &X-

@

-21OkDa 1 9 0kDa

@

-Entactin

FIG. 5. Mobility of the large laminin complex in 3T3-Ll cells compared to the laminin complex of F9 cells on SDS electrophoresis under nonreducing conditions. Radiolabeled cell lysates from 3T3-Ll cells on Day 1 of adipose conversion (3T3-Ll) or fromparietal endoderm-like F9 cells (F9) were immunoprecipitated with anti-laminin antiserum and separated on SDS electrophoresis under nonreducing conditions. A,, B,., and BZ.indicate precursors of laminin subunits observed in F9 cells (see Ref. 41). Notice that the largest complex in 3T3-Ll cellshas significantly greater mobility than that of the ArB~rB2. complex in F9 cells.

nents of the intracellular large complex should be further processed to larger mature species in this secreted complex. Only extremely small amounts of radioactivity were found at positions corresponding to monomers and dimer. This indicates that no more than a small fraction of laminin-related

16168

Basement Membrane Proteins of 3T3-Ll Adipocytes

polypeptides in 3T3-Ll can be secreted without completing the assembly into a ternary complex.

subunit may be relatively ineffective in allowing the laminin complex t o leave RER for further processing within Golgi cisternae and forsecretion. DISCUSSION The absence of the A subunit may also affect the interaction between basementmembranesandfat cells. Because the Mouse fibroblastssuchas Swiss 3T3 (47, 52, 53)and BALB/3T3 (57) are known already to produce low levels of laminin complex is suggested to interact with cells at the of the A type IV collagen, entactin, and laminin in addition to fibro- junction of A, B1, and Bz subunits (70), substitution nectin, type I and I11 collagen. Synthesis of laminin, proteo- subunit by anothersubunit maydamage this“orthodox” heparan sulfate (36), and typeIV collagen (37) by immature interaction. This difference can be related to the electron adipocytes of the mouse mammary gland has been demon- microscopic observations on fat tissue, which indicate that cells is loose strated.Thepresentinvestigationon a culture model of interaction of basement membranes with fat adipocyte differentiation suggested that the ability of fibro- (34). A fundamental question worth addressing is whether 3T3blasts to secrete basement membrane proteins is enhanced during their differentiation into adipocytes. Our results give L1 cells have a chromosomal gene coding for the A subunit, a biochemicalbasis for previous histochemical observation on or the cells have another gene coding for the novel subunit. Since the novel subunit behaved like the A subunit during extracellular matrix organization during adipose conversion the assembly of the laminin complex, it is possible that the of a similar cellline,3T3-F442A (38). These observations version of the gene coding for theA subunit. altogether suggest that synthesisof basement membrane pro- gene is an altered Another possibility isthatthemRNA codingfor the A‘ teins is importantfor morphogenesis of adipose tissue. Many effects of hormones on the synthesis of basement subunit in 3T3-Ll cells is a truncated form of laminin A membrane proteins have been suggested. An increase in the mRNA, which may be produced by alternative splicing of the amount of total basement membrane collagen occurs in dia- primary transcript of the orthodox gene of the A subunit. Relevant to this is that the mRNAcoding for the A subunit betic tissues (58-60), and this is considered to underly the was hardlydetectableinmostadultmurinetissueswhen pathogenesis of diabetic microangiopathy, which is characterized by thickening of the basement membranes in renal glo- Kleinman et al. (71) tried Northern blot analysis using their meruli and capillaries. A suppressive effect of dexamethasone laminin A cDNA clone isolated from a cDNA library of the on type IV collagen gene expression (61) and its metabolism EHS sarcoma. Although they did notdescribe details of their (62) hasbeen proposed. In our investigation, 3T3-Ll cultures cDNA clone (71), it might encodeonly a partial sequence were treated during the first 2 days with a combination of (probably the 3‘ end) of laminin A mRNA, since cloning of dexamethasone, MIX, and insulin to accelerate the adipose such a long sequence (10 kilobases) is usually difficult. One possible explanation of their result is that the laminin A conversion. High levels of type IV collagen andentactin mRNA in many murine adult tissues are truncated and do synthesis continued even to Day 6 of the conversion, when the cultures had been maintained for 4 additional days in the not contain the sequences in their A subunit cDNA. This interpretation suggests that we must obtain clones encoding absence of hormones. The enhancement of type IV collagen the full-lengthsequences of A-related polypeptides, to explore synthesis was far greater than theeffects observed by insulin the possibility of a variety of laminin complex in various (63) andwas comparable to the expression of many adipocyte tissues. phenotypes such as lipogenic (4, 7-11) and lipolytic enzymes (11, 12; for a review, see Ref. 64) during adipose conversion Acknowledgments-We thank Dr. Alan M. Tartakoff (Case Westof 3T3-Ll and 3T3-F442A. ern Reserve University,Cleveland, OH) forhis comments on the The laminin complex produced by 3T3-Ll cells was found manuscript. Thanks are also due to Dr. HowardGreen (Harvard to be unorthodox in that it did not contain the A subunit. It Medical School, Boston, MA) for his gift of 3T3-Ll cells, Dr. Albert was assembled intracellularly intoa complex with a molecular E. Chung (University of Pittsburgh, Pittsburgh, PA) for his antimass significantly smaller than the ABIBz complex found in entactin antiserum, and Dr. Koji Kimata (Nagoya University, Nagoya) for his anti-laminin antiserum. F9 cells. The complex of 3T3-Ll cells appears to contain a novel subunit, similar toB1 and Bz subunits in itsmolecular REFERENCES mass but distinct from them in its intracellular processing 1. Todaro, G . J., and Green, H. (1963) J. Cell Biol. 17, 299-313 and assembly. This subunit did not seem to be laminin M 2. Green, H., and Kehinde, 0. (1974) Cell 1, 113-116 (65), because it migrated greater on SDS electrophoresis. The 3. Green, H., and Meuth, M. (1974) Cell 3, 127-133 absence of an A subunit in secreted laminins haspreviously 4. Green, H., and Kehinde, 0. (1975) Cell 5, 19-27 5. Green, H., and Kehinde, 0. (1976) Cell 7,105-113 been suggested in a variety of cultured cells (37, 66-69). 6. Spiegelman, B. M., and Farmer, S. R. (1982) Cell 29,53-60 However, no subunit which can substitute for the A subunit 7. Mackall, J. C., Student, A.K., Polakis, S. E., and Lane, M. D. during assembly of the laminin complex has previously been (1976) J . Biol. Chem. 251,6462-6464 described. In our studyof the posttranslationalassembly and 8. Kuri-Harcuch, W., and Green, H. (1977) J. Biol. Chem. 252, transport of laminin subunits in F9 cells (41), we demon2158-2160 strated thatB1 and Bz are first assembled into B1B2,and then 9. Pairaut, J., and Green, H. (1979) Proc. Nutl. Acud. Sci. U. S. A. 76,5138-5142 the A subunit subsequently joins them to make a n AB1B2.We also suggested that it is only after assembly of ABIBz that 10. Weiss, G. H., Rosen, 0. M., and Rubin, C. S. (1980) J. Biol. Chem. 255,4751-4757 laminin subunits canleave the RER. If a similar mechanism 11. Grimaldi, P., Negrel, R., and Ailhaud, G. (1978) Eur. J. 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Basement Membrane Proteinsof 3T3-Ll Adipocytes

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