Transforming Growth Factor-@ Stimulates the Expression of Q2

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistty and Molecular Biology, Inc.

Vol. 265, No. 5, Issue of February 15, pp. 2681-2667,199O Printed in U.S. A.

Transforming Growth Factor-@ Stimulates the Expression of Q2-Macroglobulin by Cultured Bovine Adrenocortical Cells*

(Received for publication, June 30, 1989)

De Li Shi$#, Catherine SavonaS, Jean GagnonV, Claude CochetS, Edmond M. Chambazl, and Jean- Jacques FeigeS 11 From the SLaboratoire de Biochimie des Rkgulations Cellulaires Endocrines, Institut National de la Sant6 et de la Recherche Medicale and the TLaboratoire de Biologie Structurule, Dkpartement de Recherche Fondamentale, Fkdiration des Luboratoires de Biologie, Centre d’Etudes Nucleaires de Grenoble, 85X, Grenoble Cedex 38041, France

Adrenocortical cell major secreted protein was purified from the conditioned medium of primary cultures of bovine adrenocortical (BAC) cells. Immuno- chemical analysis and N-terminal sequencing of the purified protein identified it to cya-macroglobulin ((Ye- M). It appeared that 15 out of the 17 N-terminal amino acids were conserved between adrenocortical cell major secreted protein and human CQ-M. Study of az-M production by BAC cells revealed that its secretion was stimulated severalfold by transforming growth factor- ,& (TGF-fil). The stimulation occurred in a time-dependent (reaching a plateau at 24 h) and dose-dependent (EDEO = 0.1 rig/ml TGF-bl) manner. It was blocked when BAC cells were exposed to 5,6-dichlorobenzimidazole riboside, a potent inhibitor of RNA polymerase II, suggesting that TGF-j31 acts as an activator of as-M gene expression at the transcriptional level. Northern blot analysis confirmed that the (Y~-M mRNA level was increased (4-fold) in BAC cells following TGF-@I treatment. TGF-82, TGF-P1,2, basic fibroblast growth factor, and angiotensin II also appeared able to stimulate (Ye- M secretion in BAC cells, whereas adrenocorticotropin was strongly inhibitory. Given the previous reports that TGF-fil is a potent inhibitor of adrenocortical steroidogenesis (Feige J. J., Cachet, C., Rainey, W. E., Madani, C., and Chambaz, E. M. (1987) J. Biol. Chem. 262,13491-13495) and that az-M is a TGF-fil-binding protein, these observations suggest that (Y~-M may play an important role in conjunction with hormones and growth factors in the homeostatic regulation of adrenocortical functions.

cYz-Macroglobulin (a*-M)l is one of the major plasma pro-

* This work was supported by Institut National de la Santa! et de la Recherche M6dicale Grant U-244. the Comissariat i 1’Eneraie Atomique (DRF-G), the Fondation pour la Recherche MLdicale, aid the Association pour la Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page _ - charges. This article must therefore be hereby m&ed “aduertise- ment” in accordance with 18 U.S.C. Section 1734 solelv to indicate this fact.

§ Supported by a fellowship from the Institut National de la Santk et de la Recherche Medicale.

11 To whom correspondence should be addressed. ‘The abbreviations used are: an-M, az-macroglobulin; al-M, 01~-

macroglobulin; a1-13, al-inhibitor 3; TGF-P, transforming growth factor-a; FGF, fibroblast growth factor: ILl. interleukin 1: IL6. interleukin 6; ACTH, adrenocorticotropinf ACMSP, adrenodortical cell major secreted protein; PBS, phosphate-buffered saline; DRB, 5,6- dichlorobenzimidazole riboside; BAC cells, bovine adrenocortical cells; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; kb, kilobase.

teins; its most remarkable property is its ability to function as a broad specificity protease inhibitor (1). It is a high molecular weight glycoprotein made of four identical subunits of approximate M, 180,000. Although this circulating protein mainly originates from the parenchymal cells of the liver, its synthesis has been observed in a number of other cell types (2-4).

(Y~-M is constitutively expressed at high levels in humans. In rats, its blood concentration is dramatically increased during acute and chronic inflammation occurring in response to tissue damage or infections. In this species, it appears to be the major acute-phase protein (5, 6). Besides its well- known protease inhibitor activity, CQ-M also acts as a plasma carrier protein for metal ions, small basic polypeptides, and several growth factors including platelet-derived growth factor (7), nerve growth factor (8), interleukin 1 (ILl) (9), fibroblast growth factor (FGF) (lo), and transforming growth factor-p (TGF-P) (11).

The TGF-P family of growth and differentiation regulatory molecules comprises several factors found in insects and vertebrates (reviewed in Refs. 12 and 13). Within this family, the TGF-@s represent a group of highly homologous (70-85%) disulfide-linked dimers of 25 kDa. Up to now, five homodi- merit forms of TGF-P, named TGF-& to TGF-&,, have been structurally characterized. In addition, the heterodimer com- posed of one p1 chain and one p2 chain has been found in porcine platelets (14). These various homologous forms have been shown to act as growth stimulators, growth inhibitors, or differentiation modulators on numerous cell types derived from a variety of origins (12, 13, 15).

As an example, we have previously shown that TGF-P1 is a very potent inhibitor of basal as well as hormone-stimulated adrenocortical cell steroidogenesis (16, 17). The factor has been shown to act at several levels in the biosynthetic pathway of corticosteroids, the main effect being the transcriptional regulation of the 17a-hydroxylase (cytochrome P-45017,) (16, 17).* Moreover, bovine adrenocortical (BAC) cells possess TGF-P receptors, whose expression is regulated by the trophic hormone ACTH (19). These cell secrete TGF-pl as a latent inactive form, the molecular structure of which is under current study. TGF-PI thus appears as a potential regulator of adrenocortical differentiated functions, possibly acting through an autocrine loop.

In several cell types, TGF-P, has been reported to decrease the expression of several proteases including transin/stromelysin and collagenase (20, 21) and to stimulate the expression of protease inhibitors such as plasminogen activator inhibitor-l and tissue metalloproteinase inhibitor (21-23).

* 0. Pascal, G. Defaye, A. Perrin, J. J. Feige, and E. M. Chambaz, J. Biol. Chem., submitted for publication.

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Stimulation of Adrenocortical az.M Secretion by TGF-PI

These effects have been proposed to participate in the coor- dinated stimulation of extracellular matrix formation induced by TGF-PI (15). Indeed, TGF-PI has been shown to increase expression of various types of collagen (24-27), as well as that of fibronectin (24,25,27,28), tenascin (29), fibronectin receptor (28,30,31), vitronectin receptor (32), and glycosaminogly- cans (33).

In the present study, we report the characterization of adrenocortical cell major secreted protein (ACMSP) and its identification to cuz-M following purification and N-terminal protein sequencing. Study of ACMSP production by BAC cells revealed that its secretion was stimulated severalfold by TGF-/3i. The stimulation occurred in a time-dependent (reaching a plateau at 24 h) and dose-dependent (EDso = 0.1 rig/ml TGF-@J manner. TGF-&-stimulated (Y~-M secretion was blocked when BAC cells were exposed to 5,6dichloro- benzimidazole riboside (DRB), a potent inhibitor of RNA polymerase II. This observation strongly suggested that TGF- pi is acting as an activator of (Y~-M gene expression at the transcriptional level. Actually, Northern blot analysis confirmed that the (Y*-M level was increased 4-fold in BAC cells following TGF-P1 treatment. The effects of other adrenocortical effecters were also investigated.

EXPERIMENTAL PROCEDURES

Materials-TGF-/3,, TGF-/3,, and TGF-& purified from human or porcine platelets were purchased from R & D Systems (Minneapolis, MN). Human recombinant basic FGF was a generous gift from Dr. L. Cousens (Chiron Co., Emeryville, CA). Synthetic /3.zI ACTH and angiotensin II were provided by Ciba-Geigy (Basel, Switzerland). Bovine plasma and sera used for cell culture were from Boehringer, Mannheim. Polyclonal antibodies to human ol,-M were purchased from Sigma as were DRB, insulin, and all other chemicals (highest purity grade available). The 3.5-kb rat (Y~-M cDNA probe (pRLAQM/ 295) (34) was kindly provided to us by Dr. G. H. Fey (Scripps Clinic, La Jolla, CA). The 1.3-kb rat glyceraldehyde-3-phosphate dehydrogenase cDNA probe (pRGAPD-13) (35) was the generous gift of Dr. J.-M. Blanchard (Centre Paul Lamarque, Montpellier, France). They were amplified and 32P-labeled by random primer extension following standard protocols (36). Protein A-Sepharose CL-4B was obtained from Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden). [35S] Methionine (>37 TBq/mmol), Nai*sI, and [a-32P]dCTP (222 TBq/ mmol) were from Amersham Corp. Protein A (Pharmacia) was 1251- labeled using the chloramine-T method.

Cell Culture-Bovine adrenocortical fasciculata cell suspensions were prepared from freshly collected adrenal glands and grown in Ham’s F-12 medium (Gibco, Cergy-Pontoise, France) supplemented with 12.5% horse serum and 2.5% fetal calf serum as previously described (37). Cells were seeded either in 24-well Falcon culture plates (8 x lo* cells/well) or lo-cm culture dishes (8 X lo5 cells/dish) under an air/Cop (95/5) atmosphere. They were cultured for 5 days until confluency. At the onset of each experiment, the cells were incubated for 2 h in serum-free medium and the media were subsequently replaced with fresh serum-free medium containing the indicated concentrations of growth factors or hormones. After indicated periods of time, the media were collected and centrifuged at 5000 X g for 5 min to remove cell debris.

Purification and N-terminal Sequencing of ACMSP-Conditioned medium corresponding to a 24-h incubation with 40 X lo6 adrenocortical cells was concentrated 80 times by ultrafiltration through a PM- 10 Amicon membrane. Concentrated medium was then applied to a Suuerose 12 column (Pharmacia) equilibrated in PBS. The column was eluted in the same buffer and -0.5-ml fractions were collected. The proteins present in these fractions were analyzed by SDS-PAGE. Fractions containing ACMSP were pooled, dialyzed against water, and lyophilized. An aliquot (approximately 400 pmol) of the protein was submitted to N-terminal sequencing on a gas-phase sequenator (Applied Biosystems, model 477A) equipped with an on-line phenyl- thiohydantoin analyzer (Applied Biosystems, model 12OA).

~5S/Methionine Labeling-Cells grown in 24-well plates were labeled with 50 &i/ml of [35S]methionine during the last 3 h in serum- free medium. At the end of the labeling period, the medium was collected and centrifuged at 5000 x g for 5 min to remove cell debris.

For gel electrophoresis, the medium was concentrated using a Cen- tricon 10 microconcentrator (Amicon). Alternatively, proteins present in the culture medium were precipitated by addition of 100% trichloroacetic acid to a final concentration of 10%. Samples were dissolved in Laemmli’s sample buffer (38) and analyzed on a 7.5% SDS-PAGE, Molecular weight markers (Sigma) were myosin (Mr 205,000), fl-galactosidase (M, 116,000), phosphorylase 6 (M, 97,000). bovine serum albumin (M, SS,OOO), and ovalbumin (A4, 45,000). Fractionated proteins were either stained with Coomassie Blue followed by autoradiography or transferred to nitrocellulose sheets for Western blotting.

Immunoprecipitation-Cells were labeled with [?S]methionine as described above. Equal aliquots (1 ml) of radiolabeled culture medium were collected and-centrifuged to remove cell debris. 200 ~1 of 6% Triton X-100 in PBS was then added to the final concentration of 1%. The cell layers were rinsed with cold PBS for 5 min. Extraction of cell layers was done on ice under agitation using 1 ml of Triton X- 100 (1% in PBS). Prior to immunoprecipitation, unsolubilized cell debris was removed by centrifugation at 10,000 X g for 5 min. Protein A-Sepharose CL-4B beads (Pharmacia) were incubated with rabbit anti-a*-M IgG at a ratio of 5 mg of beads for 200 Pg of IgG in immunoprecipitation buffer (0.5 i NaCl, 60 mM Tris-HCl, 2 mM EDTA. 1 mM nhenvlmethvlsulfonvl fluoride. nH 7.6). After 1 h of incubation, the beads were sedimented by centrifugation and rinsed four times in immunoprecipitation buffer. They were then incubated with the radiolabeled materials overnight at 4 “C. At the end of this incubation the beads were again washed four times in immunoprecipitation buffer and the specific immunoprecipitates obtained were sedimented by centrifugation. The radiolabeled proteins in the final pellet were released by addition of Laemmli’s sample buffer and heated to 100 “C for 3 min. The supernatants were cleared by centrifugation at 10,000 X g for 5 min. They were then analyzed by 7.5% SDS-PAGE. Gels were processed for fluorography using Amplify (Amersham) and dried for autoradiography using Kodak X-AR5 films and enhancing screens.

Western Blotting-Culture medium was concentrated as described above. Cell layers were solubilized by adding directly 100 ~1 of Laemmli’s sample buffer to the culture. Following electrophoresis, proteins were electrophoretically transferred to nitrocellulose sheets. After transfer, the nitrocellulose was reversely stained with 2% Pon- ceau S in 0.2% trichloroacetic acid to control the transfer recovery. The nitrocellulose sheets were then incubated for 1 h with 3% bovine serum albumin in PBS to block nonspecific binding sites. They were rinsed in PBS containing 0.2% Tween 80 before incubation with the rabbit antibodies to a,-M (diluted l/25 in PBS) for 1 h at room temperature. The nitrocellulose sheets were washed overnight at 4 “C with the same buffer and the bound antibodies were visualized by ‘Y-protein A.

Inhibition of mRNA Synthesis-Cells grown in 24-well plates were incubated with 50 pg/ml DRB for 10 h in the presence or absence of TGF-0. [?S]Methionine (50 @Z/ml) was added to the culture during the last 3 h of incubation. The medium was collected and cell layers were solubilized with 1% Triton X-100 in PBS. All samples were analyzed by immunoprecipitation.

Estimation of c+M mRNA (Northern Blot Ana[ys$-BAC cells I -

were grown in 75-cm* plates and switched to the defined conditions for the indicated amount of time. Total cellular RNA was isolated according to Chomczymski and Sacchi (39). After extraction with phenol and chloroform, the RNA was precipitated, dissolved in water, and stored frozen. Quantitation and purity were assessed by meas- urement of absorbance of 260, 280, and 310 nm. For Northern blot analysis, 20 pg of RNA was fractionated on 1% agarose, 1.0 M formaldehvde gels, and transferred by blotting onto a nylon membrane according to the manufacturer’s recommendations (Gene- Screen Plus, Du Pont-New England Nuclear). Equal amounts of RNA were loaded on the basis of absorbance at 260 nm and equiva- lency of samples was verified by the intensity of staining of the 28 S and 18 S rRNA bands. Efficiency of transfer was judged by viewing the nylon membrane under UV light. The cDNA probes were radiolabeled with [o13”P]dCTP using random primers and Klenow frag- ments as described by Feinberg and Vogelstein (40) to a specific activity of 1.8 x lo9 cpm/rg. The blots were prehybridized for 4 h at 50 “C in a solution of 5 x SSC (1 x SSC = 0.15 M sodium chloride, 0.15 M sodium citrate), 5 x Denhardt’s (1 X Denhardt’s = 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.5% sodium dodecvl sulfate, 10% dextran sulfate, and 0.1 mg/ml denatured salmon sperm DNA. Hybridization was carried out in the same solution plus 2 x lo6 cpm of [32P]cDNA for 15 h at 50 “C. Filters


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Stimulation of Adrenocortical crz.M Secretion by TGF-fil 2883

were washed at 50 “C successively in 4 x SSC, 0.5% SDS; 3 x SSC, 0.5% SDS; 2 x SSC, 0.5% SDS, and 1 x SSC, 0.5% SDS. They were then exposed to x-ray films at -80 “C using intensifying screens.

RESULTS

Q-M Is the Major Protein Secreted by Bovine Adrenocortical Cells in Primary Culture-The proteins secreted by primary cultures of bovine adrenocortical cells were analyzed by SDS- PAGE of serum-free conditioned medium under reducing conditions. Coomassie Blue staining revealed the presence of a major 66-kDa protein and an additional band at 180 kDa (Fig. 1, lane 1). In order to discriminate between proteins synthesized by the cells and proteins derived from the serum- rich medium that might have been adsorbed to the cells before deprivation, we labeled cells with [35S]methionine and analyzed 35S-labeled proteins released into the medium (Fig. 1, lane 2). It appeared that the 180-kDa protein previously detected by Coomassie Blue staining was the major radiolabeled band. No radioactivity was detected at the level of the 66-kDa protein stained by Coomassie Blue. This probably represents serum albumin from the serum-supplemented medium but which remained stuck to the cells despite their preincubation (2 h at 37 “C) in serum-free medium before conditioning. Under nonreducing conditions, the 180-kDa ACMSP behaved as a very large molecular weight complex which did not enter the SDS-polyacrylamide gel (data not

FIG. 1. The major protein secreted in the conditioned medium of BAC cells is immunologically related to az-M. BAC cells were cultured in 24-well plates for 5 days under standard conditions and subsequently incubated for 24 h in serum-free medium. To analyze synthesized proteins present in the medium, cells were labeled for 3 h with 50 &X/ml of [35S]methionine (lane 2). At the end of the incubation, media were concentrated as described under “Ex- perimental Procedures” and analyzed by 7.5% SDS-PAGE under reducing conditions. Proteins were visualized by Coomassie Blue staining (lane I) or fluorography (lane 2). (r2-M immunoreactive proteins were detected by Western blotting using rabbit polyclonal antibodies to human LY~-M (lane 3). The quantity of secreted proteins analyzed on lanes 1 and 3 originated from 3 x lo6 cells.

89 101112 151821

20X0-

FIG. 2. Purification of ACMSP from BAC cell-conditioned medium. 40 ml of BAC cell-conditioned medium (obtained from a 24-h incubation on 40 x lo6 cells as described under “Experimental Procedures”) were concentrated to 0.5 ml by ultrafiltration through a PM-10 membrane. This concentrate was then loaded onto a Super- ose 12 column equilibrated in PBS and eluted with the same buffer at a flow rate of 0.5 ml/min. After collection of 8 fractions of 1 ml, the size of the following fractions was reduced to 0.5 ml. A280 moni- tored during the chromatography is represented. 5 ~1 aliquots of indicated fractions were analyzed by 7.5% SDS-PAGE under reducing conditions and proteins were stained by Coomassie Blue (insert). Fractions comprised between the arrows were pooled for N-terminal sequencing.

LNGNS KlYMV LV PSIQILIY

FIG. 3. Comparative N-terminal sequences of ACMSP and proteins structurally related to at-M. After chromatographic purification (Fig. 2), fractions containing ACMSP were pooled and treated as described under “Experimental Procedures” for N-terminal sequencing. The sequence of the 17 N-terminal amino acids of ACMSP was aligned with those of human LY~-M (38, 39), rat (YZ-M (37), rat al-M (36), and rat (~~-13 (40). Regions of total homology are boxed.

shown). These properties being reminiscent of those of (Y*-M, we used polyclonal anti-human an-M antibodies to detect the presence of this protein by Western blotting of adrenocortical cell-conditioned medium. Fig. 1 (lane 3) shows that a 180- kDa protein with an electrophoretic mobility identical to that of ACMSP was detected by anti-cr*-M antibodies, suggesting that ACMSP was identical to cuz-M. Since the polyclonal anti-


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2884 Stimulation of Adrenocortical a1.M Secretion by TGF-/31

TGF-B @g/ml)

0 0,Ol 0,05 0,l 0,5 1 2 0 4

Time (hours)

8 10 26

FIG. 4. Effects of human TGF-8, on the secretion of metabolically labeled az-M. A, BAC cells (1.6 x loj cells/45 cm’) were cultured for 5 days in Ham’s F-12 medium followed by 24 h in serum-free medium containing the indicated concentrations of human TGF-PI. The cells were metabolically labeled with 50 &i/ml of [?S]methionine during the last 3 h of culture. The media were concentrated and analyzed directly by 7.5% SDS-PAGE under reducing conditions followed by fluorography. B, cells were incubated with 2 rig/ml human TGF-0, for the indicated periods of time; 50 &i/ ml of [35S]methionine was added during the last 3 h of culture. The media were analyzed as in A.

TGF-6 (nglml) 0 0.01 0,05 0,l 1

205 -

Medium

97

‘L--W

66

45 -

A Dose response Time course

0 0,Ol 0,05 0.1 1

Cell layers

FIG. 5. Effects of human TGF-8, on the accumulation of C-Q- M in medium and cell layers. BAC cells were cultured for 24 h in serum-free medium containing the indicated concentrations of human TGF-0,. Media were collected and concentrated; cell layers were solubilized directly in gel electrophoresis sample buffer. Proteins from both media and cell lavers were seoarated bv 7.5% SDS-PAGE under reducing conditions. Western blotting wai performed using rabbit antibodies to human (Y*-M (diluted l/25). The nitrocellulose sheets were probed with “‘I-protein A.

LY~-M antibodies could well cross-react with proteins structurally related to (Y~-M such as cul-macroglobulin (al-M), al- inhibitor 3 (al-Is), or pregnancy zone protein which all share several common epitopes with CQ-M (34, 41-45), we decided to purify ACMSP from adrenocortical cell-conditioned medium to characterize it further. Given the high molecular weight of ACMSP, it could easily be separated from serum albumin and other proteins present in the conditioned medium using gel filtration fast protein liquid chromatography on a Superose 12 column (Fig. 2). The first peak of protein running out of the column appeared to contain exclusively the 180-kDa ACMSP moiety (Fig. 2, insert). This purified material was subjected to N-terminal protein sequencing and the identification of the 17 N-terminal amino acids was obtained (Fig. 3). The sequence of bovine an-M is not available in literature except for its C terminus (46). So we compared the N-terminal sequence obtained for ACMSP with those of human a2-M (42, 43), rat an-M (34), rat cur-M (41), and rat

205 c

116

97

46 50 75 43 297 160 471 152 Medium Cell

FIG. 6. Effects of DRB on as-M synthesis and secretion. BAC cells were treated with DRB (50 rg/ml) for 10 h in the presence or absence of human TGF-0, (2 rig/ml). They were metabolically labeled with [Wlmethionine (50 rCi/ml) during the last 3 h of incubation. The media were collected and the cell layers were extracted with 1% Triton X-100 in PBS. Immunoprecipitations were performed using rabbit antibodies to human 02-M and protein A-Sepharose. The immunoprecipitates were analyzed by 7.5% SDS-PAGE and fluorography. Autoradiograms were densitometrically scanned (Shimadzu laser densitometer) and densitometric values obtained for each spot are indicated in arbitrary units at the bottom of each lane.

al-I3 (44) (Fig. 3). It appeared that 15 out of 17 amino acid residues were conserved between ACMSP and human az-M, and 12 out of 17 were conserved between ACMSP and rat CQ- M. Comparison with rat crl-M and rat al-Is showed a cluster of 7 consecutive residues conserved between ACMSP and these two proteins. However, the N terminus of ACMSP, which was similar to that of rat and human LY~-M differed to that of al-M and (~~-1~. Only 10 out of 15 residues of cul-M and 8 out of 16 residues of the al-Is N termini could be aligned with the N-terminal sequence of ACMSP.

Taken together, these data strongly support the suggestion that ACMSP represents bovine a2-M since the N-terminal sequence obtained shows more homology to that of a2-M from other species. The amount of ACMSP/a2-M secreted in 24 h by BAC cells into their serum-free culture medium could be estimated to approximately 1 pg/lO’ cells.


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Stimulation of Adrenocortical a2.M Secretion by TGF-P1 2885

9-M

Timethours)

0 5 15 25

q

b

*“1+ 5.2 kb

GAPDH 1.3kb

FIG. 7. Effects of TGF-& on az-M mRNA levels. BAC cells (6 x lo6 cells/75 cm’) were cultured for 5 days in Ham’s F-12 medium until confluency. The cells were then incubated for 2 h in serum-free medium and the media were subsequently replaced with fresh serum- free medium and the media were subsequently replaced with fresh serum-free medium containing 2 rig/ml human TGF-P, for the indicated periods of time. Total cellular RNA was prepared as described under “Experimental Procedures.” 20 pg of RNA from each condition were subjected to electrophoresis in 1% agarose-formaldehyde gels followed bv Northern blot analvsis to estimate the 012-M mRNA levels. The same blot was also probed with a glyceraldehyde-3- phosphate dehydrogenase cDNA as a control housekeeping gene. The figure represents the autoradiograms of the blots hybridized with the 32P-labeled LY*-M and glyceraldehyde-3-phosphate dehydrogenase cDNA probes. The sizes of the transcripts were 5.2 and 1.3 kb for CQ- M and glyceraldehyde-3-phosphate dehydrogenase, respectively.

FIG. 8. Effects of growth factors and hormones on (YZ-M synthesis and secretion; comparison with human TGF-B1. Cells were incubated for 24 h in serum-free medium containing no additions (control), porcine TGF-Pi (2 rig/ml), porcine TGF-Pz (2 &ml), porcine TGF-Pi, (2 rig/ml), basicfibroblast growth factor (10 &ml), anaiotensin II (0.3 nn/mll. and ACTH (1 ne/mll. l?SlMethionine (50 &$ml) was added &ring the last 3 h of incubatibn. -The media were then collected and analyzed directly by 7.5% SDS-PAGE followed by fluorography.

Given the recent report that (Y~-M was able to bind TGF- fil, suggesting that the protein may play a role in the regulation of the latency of TGF-P1 in plasma (ll), we investigated the possibility that az-M synthesis could be conversely regulated by TGF-Pi.

aZ-M Synthesis Is Stimulated by TGF$, in Bovine Adre- nocortical Cells-Adrenocortical cells were treated with various concentrations of human TGF-PI for 15 h and labeled with [35S]methionine during the last 3 h of incubation. 35S- Labeled proteins present in the culture medium were subsequently analyzed by SDS-PAGE followed by autoradiography (Fig. 4A). It appeared that TGF-fi, was stimulating ACMSP/ CQ-M secretion up to &fold. The stimulation was detected for concentrations as low as 0.01 rig/ml (0.4 PM) and was maximal at 0.5 rig/ml (20 PM). In a time course study, we observed that the stimulation of CY~-M secretion required between 4 and 8 h of TGF+% treatment and increased up to 26 h of treatment. These kinetics suggest a transcriptional rather than a post- translational control of (Y~-M secretion. However, one could

ask the questions as to whether TGF-P, stimulated the secretion of CY~-M stored in BAC cells or its de nouo synthesis or acted on both processes. We addressed this question by esti- mating on Western blots the quantities of CQ-M present both in the cells and in the medium following TGF-P1 treatment. As illustrated in Fig. 5, (Y~-M content increased in parallel in the cells and in the medium with a very similar dose-response. This indicates that, under these conditions, there was no detectable intracellular accumulation of CY*-M, thus suggesting that the newly synthesized protein was rapidly secreted, without any evidence for an intermediary step of intracellular storage.

TGF-(3, Stimulates LY~-M mRNA Expression-The question remained whether TGF-& stimulation of (Y~-M synthesis required enhanced mRNA synthesis or could result from an increased translation of 01*-M mRNAs. To check that point, we examined whether TGF-&-induced stimulation of CQ-M production was affected in the presence of DRB, an inhibitor of RNA polymerase II (47). BAC cells were treated with DRB for 10 h in the absence or the presence of TGF-PI. When metabolically labeled proteins from the cell layers and the medium were analyzed (Fig. 6), it appeared that DRB treatment completely suppressed the TGF-P1-induced increase of (Y~-M expression. This was observed both with cells and conditioned medium. These observations suggested that the regulation of a~-M expression by TGF& was probably at the transcriptional or at the mRNA stability level. This was confirmed by a more direct experiment in which CQ-M mRNA levels were analyzed by Northern blot in BAC cells treated with TGF-&. The time course study indicated the (Y~-M mRNA was increased 4-fold after 5 h and remained elevated at least 25 h with TGF-P1 treatment (Fig. 7). As a control, glyceraldehyde-3-phosphate dehydrogenase mRNA levels remained unchanged during the same period of time, indicating the TGF-fi, was not modifying the expression of “housekeeping” genes (Fig. 7).

CY~-M Protein Secretion Is ALSO Regulated by Other Peptides Regulating Adrenocortical Cell Functions-Adrenocortical functions are under the control of growth factors such as basic FGF or insulin (proliferation), hormones such as ACTH, angiotensin II, and regulators like TGF-/3s (steroidogenesis). Thus, we investigated the effects of these peptides on (YZ-M secretion under the same conditions as those used for TGF- p1 (Fig. 8). TGF-fiz appeared as potent as TGF-P1 in stimulating az-M secretion (5-6-fold), whereas the heterodimeric molecule TGF-& was slightly less potent. Basic FGF and angiotensin II were also able to stimulate az-M secretion 2- 3-fold. On the contrary, ACTH, the major trophic hormone for adrenal cortex completely suppressed the basal production of a2-M. One could observe the presence of a slightly larger molecule in the medium of ACTH-treated, but its abundance was less than that of an-M from untreated cells (Fig. 8, lane ACTH). A more extended study is required to determine whether this larger protein is structurally related to CQ-M. We also observed that platelet-derived growth factor (10 rig/ml), acidic FGF (10 rig/ml), insulin-like growth factor-I (10 ng/ ml), Mullerian inhibiting substance (10 rig/ml), insulin (1 pg/ ml), epinepherin (10e6 M), 12-O-tetradecanoylphorbol-13-ace- tate (10e6 M), and dexamethasone (10e6 M) were without any detectable effect on (Y*-M secretion (data not shown).

DISCUSSION

In this work, we report the identification of cr2-macroglobulin (Q-M) as the major protein secreted by bovine adrenocortical (BAC) cells in primary culture. This was clearly established on the grounds of both immunological and protein


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2886 Stimulation of Adrenocortical az.M Secretion by TGF-/3,

sequencing data. This is a new and quite striking observation since az-M is usually considered as a plasma glycoprotein mainly synthesized in the liver (1, 48) and since the function of cr2-M in adrenal physiology is not obvious. (Y~-M has been shown to possess at least two functions: it is a potent protease inhibitor of broad specificity (1) and it can also act as a carrier protein for several cationic polypeptides such as platelet- derived growth factor, FGF, or TGF-fi (7-11). It is not clear at the moment whether any of these activities is implicated in adrenocortical functions. However one may recall that the adjacent tissue, the adrenal medulla, contains high levels of protease activities and that LY~-M secretion could represent an important protective mechanism for the adrenal cortex. Fur- thermore, in rats, a,-M is an important acute-phase reactant. Its concentration in plasma is elevated several hundredfold during the inflammatory response (3, 49). Glucocorticoids appear to be an important cofactor for the full induction of cuz-M production during that process. During provoked inflammation in hypophysectomized rats carrying low levels of circulating glucocorticoids, induction of olz-M is not observed, while an injection of glucocorticoids prior to experimental inflammation restores the full response (50). Abundant secretion of az-M by adrenal cortex thus emphasizes the important contribution of this tissue to the inflammatory response.

Another new observation in this report is that adrenocortical (Y~-M secretion was activated by TGF-Pi and TGF-&. TGF-/3, has been reported previously to activate the production of two protease inhibitors: plasminogen activator inhibitor-l and tissue metalloproteinase inhibitor (21-23). In both cases, the regulation appeared to be at the transcriptional level (21,23). It has been reported that TGF&-induced tissue metalloproteinase inhibitor was rapidly deposited in the growth substratum of the cells, thus suggesting that these effects of TGF-P1 contributed mainly to a stabilization of the extracellular matrix (22). The negative control exerted by TGF-P1 on several protease genes, including collagenase and transin/stromelysin, would also favor this mechanism. In adrenocortical cells, TGF-& has been shown to be a potent inducer of several extracellular matrix proteins including fibronectin.3 The stimulation of the secretion of the protease inhibitor az-M could contribute as well to an enhanced accumulation of cell substratum which could in turn regulate adrenocortical functions “from the outside” as suggested by Gospodarowicz et al. (52). Such a possibility is currently under study in our laboratory.

We and others previously reported that TGF-/3i was a potent inhibitor of adrenocortical steroidogenesis (16, 17,53- 55). TGF-PI acts at several levels on these cells (16, 17, 54, 55), an important one being the transcriptional regulation of steroid 17a-hydroxylase.’ This microsomial enzyme is an essential constituent of the corticosteroid biosynthetic pathway, regulating the ratio between cortisol and corticosterone secretions. In addition, we have demonstrated that BAC cells possess high affinity TGF-fi receptors whose expression is under hormonal control (19). In recent experiments, we observed that adrenocortical cells express the TGF-& gene and secrete TGF-P1 into the culture medium under a latent form. TGF-& thus appears as a possible regulator of adrenocortical differentiated functions, acting through an autocrine loop. In this respect, control of the latency of the secreted form of TGF-@, may be a key regulatory step in this loop. In serum, LY~-M has been reported to form an inactive complex with TGF-P1 (11). In adrenocortical cells which secrete both TGF- @I and az-M, there is a good possibility that (Y~-M participates

‘D. L. Shi, C. Savona, E. M. Chambaz, and J. J. Feige, manuscript in preparation.

in the inactive TGF-& latent complex. If so, activation of (Ye- M secretion by TGF-PI would represent a feedback regulation of TGF-pl activity. The participation of (Y*-M in the latent TGF-PI complex secreted by BAC cells is under current scrutiny.

The observation that ACTH inhibits (Y*-M secretion adds another possibility of regulation of TGF& activity in this system. It is not known at this moment whether other types of TGF-P are secreted by BAC cells besides TGF-Pi. It is of interest to notice, however, that TGF+ stimulates aa-M secretion as well as TGF-&.

Taken together, these results suggest that cuz-M may play an important role in conjunction with hormones and growth factors for the homeostatic regulation of adrenocortical ste- roidogenic functions.

The transcriptional regulation of the LY~-M gene by TGF-PI is suggested by the Northern blot analysis of (Y*-M mRNA levels in TGF-&-treated BAC cells. However nuclear run-off experiments will be necessary to confirm that the regulation of olz-M expression is at the gene transcription level as suggested by the experiments with DRB, rather than at the mRNA stability level. If TGF-/3, regulates (Y~-M message synthesis, then it would be very interesting to determine which is the transcriptional activator acting on this particular gene. It has been reported that TGF-& directly activates transcription of the mouse type I collagen gene and that the effect is mediated by a nuclear factor l-binding site located on the a2(1) collagen promoter (26). A consensus sequence for the nuclear factor 1 responsive element has also been detected in genomic sequences upstream of the coding sequence for TGF-/$-precursor cDNA (51, 56). It would be interesting to determine whether TGF-P1 exerts its effects on the ap-M gene through a similar mechanism. Another possibility would be that TGF+-induced (Y*-M gene activation is mediated through interleukin 6 (IL6). In the inflammatory response, IL6 has been shown to be the major contributor to (Y*-M acute-phase response (18) and an ILG-responsive element has been identified in the rat (Y*-M gene promoter.4 When the bovine (YZ-M genomic sequence becomes available, it will be possible to investigate the presence of nuclear factor l- or ILG-responsive elements that could mediate the TGF-P- stimulated cuz-M gene expression observed in bovine adrenocortical cells.

Acknowledgments-We are indebted to C. Blanc-Brude and I. Gaillard for their helpful preparation of adrenocortical cells. The excellent secretarial heln of S. Lidv is gratefully acknowledged. We wish to thank Dr. G. Fey at the Department of ~mmunologyScripps Clinic, La Jolla, CA, for helpful discussions and providing us with the rat ar-M cDNA probe.

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D L Shi, C Savona, J Gagnon, C Cochet, E M Chambaz and J J Feige2-macroglobulin by cultured bovine adrenocortical cells.

Transforming growth factor-beta stimulates the expression of alpha

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