JOURNAL OF CELLULAR PHYSIOLOGY 153 266-276 119921

Transforming Growth Factor p, and Adrenocorticotropin Differentially Regulate the Synthesis of Adrenocortical Cell Heparan Sulfate Proteoglycans and Their Binding of Basic Fibroblast Growth Factor ZHIWEN JIANG,CATHERINE SAVONA, EDMOND M. CHAMBAZ, AND JEAN-JACQUESFEICE' I N S t R M Unif6 244, DBMS/KKCE,Ccntrc d'ftiidcq Nuc/&rrcc, 85X, r-3804 I Crenoble Crdrx, France Adrenocortical differentiated functions are under the control of both endocrine hormones such as ACTH and local factors such as transforming growth factor P (TGFP)or basic fibroblast growth factor (bFGF). Gesides their regulatory actions on the synthesis of corticosteroids, these IWO classes of factors JISO exert some important effects on the cellular environment. We have examined here the regulation by ACTH and TGFP of adrenocortical cell proteoglycan synthesis and secretion. Under basal conditions, adrenocortical cells synthesized and secreted several species of sulfated proteoglycans, 80"h of them being recovered in solution in the culture medium. When analyzed by ion exchange chromdtography, the cell extracts and the media from cells metabolically labeled with 35S-5ulfate were found to contdin two dnd three species of radioactive sulfated proteoglycans, respectively. All species were proteoheparan-sulfates. Treatment of adrenocortical cells with TGFP, or ACTH resulted in a significant increase of the incorporation of J'S into both secreted and cell-associated proteoglycans. ACTH stimulatcd more than three times the amount of sccrctcd proteoglycans eluting from DEAETrisacryl as peak B, whereas TGFB preferentially increased the amount of peak C. No important modification of the size of the synthesized proteoglycan5 wds observed. The subpopulation of heparan sulfate proleoglycans capable to bind bFLF was a150 largely increawd after ACTH or TGFP treatment and paralleled the variation in overall proteoheparan sulfate synthesis. Thus those effects of TGFB and ACTH on proteoglycan synthesis may participate in an increased ability of adrenocortical cells to bind and respond to bFCF. (D1932 \,Z!iIey-Liss. Inc.

The adrenal cortex is a morphologically and functionally zonate gland. Cells from the external zone (zona glomerulosa) secrete primarily aldosterone under the main control of angiotensin 11, whereas cells from the inner zones (zonae fasciculata and reticularis) secrete primarily cortisol and corticosterone under the main control of adrenocorticotropin (ACTH) (Vinson and Kenyon, 1978).Primary cultures of bovine adrenocortical cells (BAC cells) from the zona fasciculata-reticularis have been a model system widely used for the study of proliferative and differentiated activities (steroidogenesis) of this tissue, both functions being under the control of exogenous factors such as hormones and growth factors (Feige and Baird, 1991). The pituitary hormone ACTH is the major endocrine factor acutely stimulating the differentiated functions of cultured adrenocortical cells (i.e.,increase in the synthesis of corticosteroids). It is well known to act through a CAMP-mediated pathway (Saez et al., 1981). It also rapidly induces a variety of effects apparently unrelated to steroidogenesis such as changes of cell mor(C

1992 WILEY-LISS, INC

phology (Rainey et al., 1983) or stimulation of polyamine uptake and biosynthesis (Feige et al., 1986a).It increases the expression of most of the specific corticosteroidogenic enzymes (Simpson and Waterman, 1988). In addition, ACTH induces a n increase in the synthesis of extracellular matrix components (Turley, 1980; Feige et al., 1982). Recently, we and others reported that the multifunctional cytokine transforming growth factor PI ITGFP,) is a potent inhibitor of basal as well as ACTH-stimulated adrenocortical cell cortisol production (Feige et al., 1986b; Hotta and Baird, 1986). TGFf3, was found t o be abundantly distributed in the different zones of the adrenal cortex but absent from the adrenal medulla (Keramidas et al., 1991) and, alReceived January 6.1992; accepted May 18, 1992.

"To whom reprint requestsicorrespondence should he addressed. Zhiwen Jiang's present address is Department of Pharmacology, Medical College of Bengbu, 233001 Bengbu, Province of Anhui, Popular Republic of China.

REGULAI'ION OF ADRENOCORTlCAL bFGF-BJNDING HSPGS

267

though it is secreted under a latent form in the culture purified from human platelets was purchased from R & medium, it has been proposed to act as a n autocrine D Systems (Minneapolis, MN). Human recombinant factor in the long-range control of adrenocortical ste- basic FGF was a generous gift from Dr. L. Cousens roidogenesis (Feige et al., 1991). Two main targets of (Chiron Co., Emeryville, CA). Pronase, chondroitinase TGFP, action on adrenocortical cells were identified: ABC, and standard glycosaminoglycans were purthe low-density lipoprotein receptors whose expression chased from Sigma (St Louis, MO). Heparitinase from is reduced (Hotta and Baird, 1987) and the biosynthetic Flauobacteriurn heparinurn was obtained from Seikacytochrome P-450, 7cr (steroid 17a hydroxylase) whose gaku Kogyo Co. (Tokyo, Japan). Rabbit polyclonal antimRNA and protein levels are greatly decreased follow- bFGF antibodies (Antiserum 773) were a generous gift ing TGFP, treatment (Feige et al., 1987; Perrin et al., from Dr. A. Baird (Whittier Institute, La Jolla, CAI. 1991). TGFP, was also observed to stimulate the pro- Protein molecular weight standards, Sephadex G50 duction of fibronectin (Shi et al., 1990a; Williams and fine, Sepharose CLGB, and protein A-Sepharose were Allen-Hoffmann, 1990) and a,-macroglobulin (Shi et purchased from Pharmacia (Uppsala, Sweden). "S-sulal., 1990b) in BAC cells, thus contributing to a n in- fate (1.7 GBqimmol), Amplify, and MP-pmax autoradiography films were purchased from Amersham (Buckcreased accumulation of extracellular matrix. Acidic and basic fibroblast growth factors (FGFs) are inghamshire, England). Ultrogel ACA 44 and DEAEwell-characterized mitogens for adrenocortical cells Trisacryl were from IBF Biotechnics (Villeneuve-la (Esch et al., 1985). Corneal endothelial cell-derived ex- Garenne, France). Cell culture media and sera were tracellular matrices also stimulate adrenocortical cell purchased from Boehringer Mannheim (Meylan, proliferation but these effects could be explained by France). All other reagents were of the highest purity FGF associated t o these matrices (Gospodarowicz et al., grade available. 1980). Indeed, FGFs present a strong affinity for Cell culture and 35S-sulfatelabeling heparan sulfate proteoglycans (HSPG) and are abundantly distributed in HSPG-rich basal membranes Bovine adrenocortical cells (from the fasciculata-rethroughout the organism (Gonzalez et al., 1990; Vlo- ticularis zone) were prepared weekly from fresh adredavsky et al., 1987). nal glands collected a t the local slaughterhouse, accordProteoglycans associate in the extracellular matrices ing t o a previously described protocol (Duperray and with collagen fibers, fibronectin, and other adhesion Chambaz, 1980). After their plating in 10 cm diameter proteins to constitute a macromolecular network. Al- culture dishes ( 4 x lo6 cells/75 cm2 dish), cells were though the biological function of proteoglycans is still grown a t 37°C in Ham's F12 medium supplemented poorly understood, the notion that they play a n impor- with 10% horse serum and 2.5% foetal calf serum, untant role as modulators of growth factor functions has der a n airiCO, (955) atmosphere. For metabolic labelemerged recently (Ruoslahti, 1989; Massague, 1991; ing with "S-sulfate, cells were preincubated for 2 h in Ruoslahti and Yamaguchi, 1991). Members of both the sulfate-free DMEM supplemented with 6% dialyzed FGF and the TGFp families associate with proteogly- foetal calf serum, and then incubated for 24 h in the cans at the cell surface and in the extracellular matri- same medium containing 1mgiml BSA and 0.2 mCiiml ces (Gonzalez et al., 1990; Vlodavsky et al., 1987; Sa- "S-sulfatejn the presence or absence of 2 ngiml TGFP, vona et al., 1991; Kiefer et al., 1990; Flanders et al., or 3 x lo-' M ACTH. At the end of the incubation pe1989; Cheifetz et al., 1988). Release of growth factors riod, the medium was collected and the cell layer was from these reservoir-proteoglycans may result from rinsed twice with cold PBS and extracted in 10 mM proteolysis of the proteoglycan core proteins or from Tris-HC1 (pH 7.41, 1M urea, 0.5% Triton X-100, 1mM partial degradation of the heparan sulfate polyosidic DTT, 10 mM EDTA, 2 mM PMSF (extraction buffer) for chains (Saksela and Rifkin, 1990; Ishai-Michaeli et al., 1 h on ice. The cell extracts were then centrifuged for 1990). Binding of bFGF to heparan sulfate proteogly- 10 min a t 15,000g and the supernatants were stored a t cans may also be a prerequisite for binding to its high- -80°C for further analysis. We measured the uptake of affinity tyrosine kinase receptors. Recent studies have "S-sulfate by control, TGFp,-treated, and ACTHcharacterized such a function of proteoglycans in the treated cells under these same labeling conditions. The presentation of FGF t,o its signal transducing receptors values were not more than 10% different from each (Yayon et al., 1991;Rapraeger et al., 1991). other under these three conditions, ruling out the possiWe reported previously that adrenocortical cells in bility that variations in the amounts of "S-labeled molprimary culture synthesize heparan sulfate proteogly- ecules could be due to variations in the transport of cans and that ACTH stimulates this synthesis (Feige et sulfate into the cells and therefore in the specific actival., 1982). Given the recent interest devoted to proteo- ity of intracellular "S-sulfate. glycans a s modulators of growth factor function, we Quantitation of "S-sulfate incorporation extended this initial study and investigated the regulainto proteoglycans tion of proteoglycan synthesis by ACTH and TGFB in cultured adrenocortical cells. Furthermore, we examSeparation of 35S-labeled macromolecules from free ined whether changes in proteoglycan synthesis re- "S-sulfate was performed using the centrifugationsulted in changes in their FGF-binding capacity. accelerated gel filtration technique described by Penefsky (1977). Disposable 1ml syringes were filled with MATERIALS AND METHODS Sephadex G50 fine preequilibrated with 50 mM sodium Materials acetate (pH 6.0) 0.15 M NaCI, 8 M urea, 0.5% Triton Synthetic pl-24 adrenocorticotropin (Synacthen)was X-100. The gel was first centrifuged for 2 min a t 800g. obtained from Ciba-Geigy (Basel, Switzerland).TGFP, Then 0.1 ml samples (medium or cell extracts from "S-

268

JlANG ET AL

sulfate labeled cells) were loaded on the top of the dried gel and the syringes were centriguged again for 3 min a t 8OOg. The radiolabeled macromolecules present in the eluates were then quantitated by liquid scintillation counting.

Separation of proteoglycans b y ion-exchange c h r o m a t o g r a p h y The protocol was adapted from those described by Yanagishita et al. (1987) and Rasmussen and Rapraeger (1988). DEAE-Trisacryl columns (3 ml) were equilibrated in 50 mM sodium acetate (pH 4.5), 0.15 M NaC1, 8 M urea, 1%Triton X-100. After application of the samples, the columns were washed with 3 bed volumes of equilibration buffer and the elution was performed with a linear gradient of 0.15-1.0 M NaCl in the same buffer (10 bed volumes). Then 1 ml fractions were collected and the radioactivity present in each fraction was determined by liquid scintillation counting. Size fractionation of proteoglycans b y gel filtration The radioactive peak fractions from the DEAESephacryl column were pooled, precipitated at 20°C in ethanol containing 1.3%potassium acetate, and resuspended in water. Aliquots were eventually treated with pronase and nitrous acid, as described below. Untreated and treated samples were loaded onto 1 0 m l columns 10.7 cm2 x 14 cm) of Sepharose CL6B or Ultrogel ACA44 equilibrated in 50 mM Tris-HCI (pH 8.0) 0.1 M NaCI, 1%SDS. The elution was performed in the same buffer a t a flow rate of 6 ml/h. Then 0.25 ml fractions were collected and their radioactivity content was determined by scintillation counting. The columns were calibrated by running independently the following MW standards under the same conditions: ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa), chymot,rypsinogen A (25 kDa), ribonuclease A (13.7 kDa), and ACTH (4.6 kDa). ~

TABLE 1. Effcct of ACTH and TGFp on the biosynthesis of fiulfated macromolecules' 'SO, Medium

incorporated (cpm x 10 Increase

Control ACTH TGFp

2 17 + 0 15 4 19 0 14 3.93 i 0 09 +

5,

Cell extract

-

+ 93CZ +SO%

Inrrpnw

0 49 t 0 01 0 96 ? 0 09 0 72 I 0 03

-

+96% +478

'Bovine adrenucortical cclls (8 x 10' celld75 cm2 plate) were labeled wilh "S-sulfate 10.25 mCiJmll for 24 h a t 37°C in the absence (control)or prescnre of either 3 x M ACTH or 2 n g h l TGFp, At the end of thc incubation. the media were collected and the cells were exti-actrd in I ml of urea-containing extraction buffer. As descrihrd in %aterials and Methods, 0.1 ml aliquot%of bolh Ihe medium nnd the cell extract were suhjected to centrifugation-acceleratcd gel filtration through Sephader G50 minicolumns. The radioactivity present in the cxcluded fraction was quantitated hy scintillation counting. Thr w l u e s represent (he radioactivity present in the total volume of medium and in Ihe total cell extract corresponding to cellb from one plate. Each value 1s the mean ? SD of duplicate determinations.

serum (Gonzalez et al., 1990) preadsorbed on protein A-Sepharose were added and the incubation was performed under rotary shaking for 1h at 4°C. The suspension was centrifuged and the beads were washed three times with cold PBS. The immune complexes were then dissociated by boiling in Laemmli's sample buffer (Laemmli, 1970) and analyzed by SDS-PAGE on 5-15% polyacrylamide continuous gradients. The immunoprecipitated complexes were then visualized by fluorograPhY. Fluorography Visualization of %-labeled molecules separated on SDS-polyacrylamide gel was done by fluorography. Either the swelled gel was soaked for 30 min in Amplify (Amersham, Buchinghamshire, England), dried, and exposed to X-0 Mat films (Kodak, Rochester, NJ) or the dried gel w a s exposed directly to MP-P max films (Amersham).

RESULTS TGFp and ACTH stimulate BAC cell proteoglycan s y n t h e s i s Enzymatic and chemical characterization In a first series of experiments, we examined the of proteoglycans effects of TGFP and ACTH on the overall synthesis of The proteoglycan nature of the "S-labeled macro- proteoglycans by bovine adrenocortical cells. After 24 h molecules was assessed by the following enzymatic or of treatment with these factors, primary cultures of chemical treatments: ii0.5 unitdml of chondroitinase these cells were metabolically labeled with "S-sulfate ABC in 50 mM Tris-HC1 (pH 8.0), 30 mM sodium ace- and the radioactivity incorporated into macromolecules tate for 2 h at 37°C; iiiO.1 unitiml of heparitinase in was separated from free 35S-sulfateb a centrifugation50 mM Tris-HCI (pH 7.0) 0.5 mM calcium acetate for accelerated gel filtration technique. 15S-labeledmacro17 h at 37°C; iiii20 units/ml of pronase in 50 mM ammo- molecules were measured both in the medium and in a n nium bicarbonate (pH 8) for 17 h a t 37°C; ivinitrous urea Triton X-100 cell extract. Results in Table 1 acid:l volume of sodium nitrite and 1 volume of acetic clearly show that 80% of the 35S-labeled material was acid added t o 1 volume of sample for 80 min at room found in the culture medium and only 20% remained temperature. Excess of nitrous acid was destroyed by cell associated. ACTH treatment almost doubled the addition of ammonium sulfamate (20 mgiml) and neu- amount of "S-labeled macromolecules present both in tralized with sodium hydroxyde. the medium and in the cell extract, whereas TGFP increased the amount of 35S-labeled macromolecules by Proteoglycan-bFGF binding a s s a y 80% in the culture medium and by 47% in the cells. Centrifugation through G50 columns equilibrated in "S-sulfate can be incorporated into sulfated proteogly10 mM Tris-HC1,0.15 M NaCl (pH 7.4) buffer was used cans (Yanagishita et al., 1987) o r in tyrosine-sulfated to free 85 kl fractions of media and 200 ~1 fractions of proteins (Huttner, 1984). In order to characterize the cell extracts from "S-sulfate-labeled cells of 35S-su1- nature of the radiolabeled macromolecules, we anafate. After incubation of the samples with 0.1 pg of lyzed the culture media from "5S-sulfate-labeled cells bFGF for 2 h at 4"C, 3 pl of anti-bFGF rabbit anti- by PAGE-SDS. Selective degradation of heparan sul-

269

REGULATION OF ADRENOCORTICAL bFGF-BINDING HSPGS

Control Pronase Nitrous Acid

-

+

- -

+

TGFB1 +

-

-

-

+

ACTH +

-

+

-

I

+

+

MW (kDa)

212

-

170

-

76

-

53

-

Fig. 1. SUS-PAGE analysis o f ”S-labeled proteoglycans secreted by adrenocortical cells. Confluent adrenocortical cells (8 x l o h cells/ 75 cm” plate) were incubated for 24 h at 37°C in 6 ml of labeling medium containing 0.2 mCi/ml J5S-sulfate.Labeling was performed in the absence (control) or in the presence o f either 3 X lO-’M ACTH or 2 ngiml TGFP,. At the end of the incubation, the media were collected

and the proteoglycans from 0.1 ml aliquots of the media were precipitated with ethanol containing 1.3% potassium acetate. The samples were treated without or with pronase or with pronase followed by nitrous acid as described in Materials and Methods. They were then analyzed by 6 8 PAGE-SDS and the radiolabeled proteoglycans were visualized by fluorography.

fates and proteins with nitrous acid and pronase, respectively, followed by electrophoretic analysis of the reaction products (Fig. 1)were performed. In every lane corresponding to untreated samples, one could detect a radioactive smear a t the top of the separation gel, migrating like a polydisperse mixture of proteoglycans, in addition to several minor well-defined protein bands. The smear was 1.7 times more intense in the TGFp condition and 1.2 times more intense in the ACTH condition than in the control condition, a s assessed by densitometric scanning of the autoradiograms. In each case, treatment of the media with pronase decreased the size of the 35S-labeled molecules from 250-300 kDa to 50-100 kDa. This indicated that the major “S-labeled macromolecules contained a protein component. Adciitional treatment with nitrous acid, a reagent that degrades heparin and heparan sulfate chains, resulted in the disappearance of labeled products. Enzymatic treatment with chondroitinase ABC, a n enzyme t h a t

degrades all three types of chondroitin sulfates, did not modify the migration of the radiolabeled species (data not shown). Taken together, these results indicate that adrenocortical cells synthesize and secrete large proteoheparan-sulfates and that TGFp highly stimulates this synthesis. There exists some discrepancy between the results in Table 1 which indicate that ACTH doubles the incorporation of 35Sinto macromolecules and those from Figure 1 where ACTH does not appear to strongly stimulate the secretion of the large proteoheparan-sulfate. However, one should observe that, under the conditions used, the SDS-PAGE analysis did not allow to detect the molecules of MW < 45 kDa. We thus analyzed the metabolically labeled proteoglycans by ion-exchange chromatography on DEAETrisacryl. The media and the cell extracts from control, TGFp-treated, and ACTH-treated “S-labeled cells were loaded onto DEAE-Trisacryl columns and the different proteoglycan species were eluted with a 0.15-

+

270

JIANG ET AL.

Medium

Cell Extract

83W

c o "1

I.

I

C.nlr.1

i ] 6oM

tiO

0

10

20 F r n r l l o n Number

30

TOF8

C

A

B

M: osa

20

I0

Frmcllon

----

I

I

30

40

Number

ACTH

I

B

Fwcllon

Number

Fig. 2. Separation of the "S-labeled proteoglycans by ion-exchange chromatography on DEAE-Trisacryl. Adrenocortical cells (8 X lo6 cellsi75 cmLplatel were incubated for 24 h at 37°C in 6 ml of labeling medium containing 0.2 mCi/ml 35SS-sulfata. Labeling was performed in the absence (control)or in the presence of 3 x 10-7M ACTH or 2 ng!ml TGFB,. At the end of the incubation, the media were collected and the cells were extracted in I ml o f 8 M urea-containing buffer, as described in Materials and Methods. Free :'"S-sulfate was removed from media

and cell extracts by gel filtration through G50 columns. As described in Materials and Methods. 0.2 ml aliquots of medium and 0.1 ml aliquots of cell extracts were then analyzed by ion exchange chromatography on DEAE-Sephacryl columns. The data presented are representative of results obtained in three independent experiments. The various peaks of proteoglycans have been labeled A, B, C bv order of elution.

0.9 M NaCl gradient (Fig. 2). Three peaks ofradioactivity were detected in the profiles from the media, eluting a t NaCl concentrations of 0.15M (peak A), 0.22 M (peak B), and 0.6 M (peak C), respectively. Two radiolabeled proteoglycans corresponding to peaks A and C could be separated from cell extracts. The total radioactivity contained in each peak was quantitated and the results are represented in Table 2. They indicated that both peak A and peak C were increased by 80-90% in the extracts from ACTH-treated cells and by 67% in those from TGFP-treated cells. More differences were observed in the media from these cells. ACTH increased

by 56-59% the amounts of peak A and peak C and by 264% the amount of peak B. TGFp increased by 60% the amounts of radioactivity in peak A and peak B and by 1035%the amount in peak C. Thus ACTH appears to preferentially stimulate the synthesis andlor secretion of peak B HSPGs, whereas TGFp mainly stimulates those of peak C HSPGs. We attempted next t o characterize the molecular size of these different HSPG species. The radioactive fractions from each peak were pooled and analyzed by gel filtration on Sepharose CL 6B columns. As illustrated in Figure 3, approximate MWs of 230 kDa for peak A

REGULATION OF ADRENOCORTICAL bFGF-BINDING HSPGS

271

TABLE 2. Quantitative analysis of the distribution of the diffevent peaks of 35S-labeled proteoglycans separated on DEAE-Trisacryl' "%04 incorporated (cpm x Medium Control ACTH

TGFp

Cell extract

Peak A

Peak B

Peak C

Peak A

Peak C

2.15 3.42 (+ 59%) 3.47

5.46 19.88

4.93 7.69 ( + 56%)

0.55

3.19

(+

61%)

(+

2646)

8.75 (t 60%1

10.Y f t 103%)

1.06 (+ 93W) 0.92 (+ 67%)

5.85

83%) 5.34 ( + 67%) (+

'The lotal radioactivity present in each peak Crom the chromatogram shown in Fig. 2 was quantitated The percentages between parentheses indicate the increase in the radioactivity content of each peak in the ACTH- or TGFB,-treated cells, as compared to the control cells.

and 330 kDa for peak C could be determined, whereas peak B eluted in the total volume of the column. We thus used Ultrogel ACA 44, a support better adapted to the separation of small size proteins, to determine the MW of peak B and found i t t o be about 8 kDa (Fig. 3 ) . Treatment of each peak material with pronase resulted in every case in a shift of the elution position of the radioactivity towards lower molecular weights (Fig. 3). Sequential treatment with pronase and nitrous acid resulted in every case in the elution of the radioactive fractions within the total volume of the columns. The biochemical characteristics of the three labeled species are summarized in Table 3. All of them possess protein and heparan sulfate components and can thus be characterized as proteoheparan sulfates. Peaks A and C are large molecules (MW > 200 kDa) recovered from both the cell layer and the medium, whereas peak B is a smaller proteoglycan that is only present in the medium.

Regulation of FGF binding by adrenocortical proteoglycans Since low affinity FGF receptors are of proteoheparan sulfate nature (Moscatelli, 1987; Kiefer et al., 1990; Savona et al., 1991), we investigated the FGFbinding capacity of adrenocortical cell proteoheparansulfates. For this purpose, we designed a n assay based on the use of a polyclonal anti-bFGF antibody to immunoprecipitate complexes between exogenously added bFGF and "S-labeled proteoheparan-sulfates. Under control conditions, where bFGF was omitted or where non-immune serum was used instead of anti-bFGF antiserum, no radiolabeled proteoglycan was precipitated (Fig. 4).When the medium from 35S-sulfate-labeled cells was analyzed using the anti-bFGF antibody a large size "S-labeled proteoglycans (MW > 200 kDa) was immunoprecipitated. When the experiment was performed with the media from TGFp-treated and ACTH-treated cells, we observed that the amount of immunoprecipitated proteoglycans was increased in the ACTH condition (1.7X) a s well a s in the TGFp condition ( 1 . 9 ~(Fig. ) 4). There appears to be a correlation between the respective amounts of radioactivity found in peak C and those found in the anti-FGF immunopreciptates. A similar study was carried out with the cell extracts. The results in Figure 5 clearly indicated that antibFGF antibodies could imrnunoprecipitate both a high molecular weight 35S-labeled proteoglycan and a dis-

perse population of glycosaminoglycans. In TGFPtreated cells, the bFGF-binding proteoglycan band was more intense ( 1 . 7 ~and ) better defined than in control cells. The smear corresponding to bFGF-binding glycosaminoglycans was also more intense and its size appeared larger than in control cells. In ACTH-treated cells, the intensity of bFGF-binding proteoglycans was increased twofold but their size was not significantly modified. With the exception of the presence of bFGFbinding glycosaminoglycans, the results obtained with the cell extracts are very similar to those obtained with the media. The increase in the synthesis of large molecular weight FGF binding proteoglycans t h a t is induced by TGFp and ACTH appears to be positively correlated with the effect of these peptides on the overall synthesis of large size proteoglycans.

DISCUSSION Proteoglycans are long known to present diverse cellular functions and to participate in important biological mechanisms such as cell adhesion, control of cell morphology, and embryonic development (Fransson, 1987; Ruoslahti, 1989; Esko, 1991). Recently, the capacity to bind growth factors has emerged as a major additional property of these macromolecules (Massague, 1991; Ruoslahti and Yamaguchi, 1991; Esko, 1991).

Adrenocortical cell proteoglycans are very poorly characterized. A previous report from our laboratory indicated that these cells grown in primary cultures synthesize essentially proteoheparan-sulfates and that ACTH is a potent stimulator of this synthesis (Feige et al., 1982). We here report that transforming growth factor (TGFP,) and ACTH induce both a n increase of the synthesis of proteoheparan-sulfates and a concomitant increase in the capacity of these molecules to bind bFGF. However, careful analysis of the nature of the proteoglycans synthesized under ACTH and TGFp treatments revealed differences between the effects of these two peptides. When analyzed by ion-exchange chromatography on DEAE-Trisacryl columns, the cell extracts and the conditioned media from cells metabolically labeled with 35S-sulfate were found to contain two and three species of radioactive sulfated proteoglycans, respectively (Fig. 2). All three species were proteoheparan-sulfates since pronase treatment resulted in reduced size of these molecules and additional nitrous acid treatment totally degraded them (Fig. 3). Analysis of the size of these different proteoglycan spe-

272

JIANG ET AL. TABLE 3. Biochemical features of the three species of proteoglycans svnthesized and secreted bv bovlne adrenocortical cells’

2wO

Psak A

Sopharose

CL 6

Peak A

R C

ElutioniDEAE NaCl (MI

MW (kDa)

MW of GAG chain (kDa)

Nature

0 15 M 0 22 M 0 60 M

230 83 330

30 2.2 150

HS-PG HS-PG HY-PG

‘HS PG = hepaian sulfate-proteuglvcnn

10

20

30

40

Frmclian

I

1

-f

Peek

50

60

Num0.r

c

Scphsross CL 6 8

E

Fraction

Number

zoo0

Ultrogel ACA 44

Psak B

20

I

no

So

40

Frecrion

60

70

Numb.,

Fig. 3. Hydrodynamic size analysis of secreted heparan sulfate proteoglycans by gel-filtration chromatography. Radioactive peaks collected from the separation of radiolabeled secreted proteoglycans on DEAE-Sephacryl (Fig. 2) were precipitated with ethanol containing 1.3% potassium acetate and either untreated (3-9) treated with pronase ( 0 - 0 ) or sequentially treated with pronase and nitrous acid (0-0) as described in Materials and Methods. The samples were then analyzed by gel filtration through Sepharose CL-6B (peaks A and C) or Ultrogel ACA 44 (peak B) columns under the conditions described in Materials and Methods. Arrows indicate the elution position and MW of standard proteins used for calibration.

cies by gel-filtration chromatography revealed that two of them (peaks A and C) were large molecules (MW > 200 kDa), whereas the third one (peak B) was much smaller (MW 2: 8 kDa) (Fig. 3; Table 3). Since this latter species was only observed in the conditioned

medium, it could have been generated by proteolytic cleavage of the cell-associated proteoheparan-sulfates. The differences in the affinities of these three species for DEAE-Trisacryl are likely to reflect differences in the degree of sulfation of their respective glycosaminoglycan chains. The identity of the two large proteoheparan-sulfates, present both in the culture medium and in the adrenocortical cell layer, was not established in this study but these proteoglycans appeared to possess the ability to bind bFGF. Recently, Kiefer et al. reported that syndecan, a proteoglycan bearing both heparan sulfate and chondroitin sulfate chains, represented the low affinity receptor for bFGF (Kiefer et al., 1990). Meanwhile, the expression of syndecan appears to be limited to epithelial cells and absent from mesenchymal cells (Saunders et al., 1989). It is thus possible that adrenocortical cell heparan sulfate proteoglycans are distinct from syndecan. Whether they belong to the fibroglycan (Marynen et al., 1989), the glypican (David et al., 1990), or t,he betaglycan (Andres et al., 1989) families of HSPGs will require purification and determination of the sequence of their core protein. We demonstrated previously that adrenocortical cells express type I11 TGFP receptors (Cochet et al., 1988), which have been identified as betaglycans (Andres et a]., 1989).But i t is not known whether this HSPG species is also able to bind bFGF. Since TGFp binds to the core protein of betaglycan and bFGF binds to the heparan sulfate chains, betaglycans may represent bifunctional storage receptors for these two factors. Treatment of adrenocortical cells with TGF& or ACTH resulted in a significant increase in the incorporation of 36Sinto both secreted and cell-associated proteoglycans (Table 1). ACTH appeared to stimulate more than three times the secretion of the proteoglycan eluting from DEAE-Ultrogel as peak B, whereas TGFp was observed t o preferentially increase the amount of peak C (Table 2). This is in agreement with the analysis of secreted proteoglycans by SDS-PAGE which only allows the detection of large proteoglycans (peaks A and C) and which indicated a more pronounced stimulation of the secretion of large proteoglycans by TGFP, than by ACTH (Fig. 1). Stimulation of proteoglycan synthesis by TGFp has been observed previously in several different cell types (Border et al., 1990; Rapraeger, 1989; Chen et al., 1987; Redini e t al., 1991; Bassols and Massague, 1988; Morales and Roberts, 1988). TGFp appeared to up-regulate both the transcription of the core protein (Kahari et al., 1991; Romaris et al., 1991) and the synthesis of the glycosaminoglycan chains (Rapraeger, 1989; Redini et al., 1991; Bassols and Massague, 1988).Depending on the cell type studied, TGFP induces changes of

273

REGULATION OF ADRENOCORTICAL bFGF-BINDING HSPGS

Control 773

-

bFGF

TGFBI

NRS

+ - +

-

ACTH

773

NRS

773

NRS

+

- +

- +

-

+

MW (kDa)

212 170r

-

116

-

76

-

53

-

Fig. 4. Immunoprecipitation o f bFGF-binding secreted proteoglycans with anti-bFGF antibodies. Bovine adrenocortical cells (8 10" cellsi75 cm2 plate) were labeled with %-sulfate (0.25 mCiiml) for 24 h at 37°C in the absence (control) or presence of either 3 x M ACTH or 2 ngiml TGFp,. A t the end ofthe incubation, the media were collected and subjected to centrifugation-accelerated gel filtration through Sephadex G50 mini-columns equilibrated in 8 M urea-containing buffer. Then 100 pl samples were incubated for 1 h a t 4°C in

the presence or absence of 0.1 pg of recombinant bFGF. Free and complexed bFGF were then immunoprecipitated by protein A-Sepbarose adsorbed anti-bFGF antiserum (773)as described in Materials and Methods. A mock precipitation was performed in the presence of protein A-Sepharose-adsorbed normal rabbit serum (NRS). The immunoprecipitates were analyzed by PAGE-SDS on a 6 1 5 % polyacrylamide gradient and visualized by fluorography.

the size, the composition, and/or the number of the glycosaminoglycan chains. In adrenocortical cells, the size of the proteoglycans was not modified by TGFP treatment; only their abundance was. We previously reported that TGFP stimulates the synthesis of fibronectin (Shi et al., 1990a) and that of the broad specificity protease inhibitor a,-macroglobulin (Shi e t al., 1990b) in adrenocortical cells. The stimulation of the secretion of large proteoglycans thus appears as another of the multiple coordinated effects of TGFP that contribute to a n increased accumulation of the pericellular and extracellular matrices. The observation that the synthesis of the subpopulation of HSPGs that bind bFGF is stimulated by TGFp and ACTH to the same extents as the overall synthesis of HSPGs (Figs. 4 , 5 ) raises some very interesting questions. First, it is known that HSPGs represent the low affinity 1Kd lo-' M) binding sites for bFGF in different cell types including the adrenocortical cells (Moscatelli, 1987; Savona et al., 1991). First considered as reservoirs for bFGF, HSPGs have recently been attributed a more crucial function in the presentation of bFGF to its high-affinity receptors (Rapraeger et al.,

1991; Yayon et al., 1991). In adrenocortical cells, we have observed that degradation of HSPGs by heparitinase treatment resulted in a decreased binding of bFGF to both the high-affinity and the low-affinity receptors (Savona et al., 1991). bFGF is long known to be the most potent mitogen for adrenocortical cells in culture (Esch et al., 1985). It has been purified from the adrenal gland (Gospodarowicz et al., 1986) and localized in the fasciculata-reticularislayers of the fetal adrenal cortex (Gonzalez et al., 1990). The involvement of bFGF in the ontogenic development or the pathogenic proliferation of the adrenal gland has been proposed although not demonstrated yet (Dallman, 1984-1985; Feige and Baird, 1991). TGFp does not affect the proliferation of bovine adult adrenocortical cells in primary culture (Feige et al., 1986b; Hotta and Baird, 1986), whereas ACTH is a strong inhibitor of DNA synthesis in vitro (Ramachandran and Suyama, 1975). In vivo, ACTH has opposite effects since it stimulates adrenocortical growth (Dallman, 1984-1985). The proliferative action of ACTH in vivo is thus very probably indirect and could be mediated through the bFGF pathway. In line with this hypothesis, Mesiano et al. (1990) have re-

274

JIANG ET AL.

Control bFGF:

TGFB

ACTH

- - + + - - + + - -

+ +

MW (kDa)

Fig. 5 . Immunoprecipitation of cell-associated bFGF-binding proteoglycans with anti-bFGF antibodies. Bovine adrenocortical cells (8 x 106 celld75 cmz plate) were labeled with 35S-sulfate (0.25 mCii ml) for 24 h at 37°C in the absence (control) or presence of either 3X M ACTH or 2 ngiml TGFP,. At the end ofthe incubation, the cell layer was extracted in 1 ml of urea-containing buffer as described in Materials and Methods. After gel filtration through Sephadex G50

mini-columns, 200 ~1 samples were incubated overnight a t 4°C in the presence or absence of 0.1 pg of bFGF. Free and complexed bFGF were then immunoprecipitated with protein A-Sepharose-adsorbed antibFGF antiserum (antiserum 773) as described in Materials and Methods. The immunoprecipitates were analyzed by PAGE-SDS on a 5-15% polyacrylamide gradient and visualized by fluorography.

ported that ACTH induces the expression of bFGF and IGF-I1 in human fetal adrenals. Our observations that ACTH increases the synthesis of heparan sulfate proteoglycans and that most of these HSPGs are secreted raise some interesting questions about the role of this regulation and its possible implication in the proliferative action of ACTH in vivo. It is known that heparan sulfates protect bFGF from proteolytic degradation (Saksela et al., 1988)and increase its radius of diffusion (Flaumenhaft et al., 1990). It was shown that secreted heparan sulfates are able to release bFGF bound to endothelial cell extracellular matrices (Moscatelli, 1988).Under the physiological situation, bFGF appears to be stored in the basement membranes or in the pericellular matrix of diverse tissues including the adrenal cortex and its vasculature (Gonzalez et al., 1990). Thus, an increase in the secretion of HSPGs of small size such as that observed in adrenocortical cells treated by ACTH could release matrix-bound bFGF and make it available for its highaffinity signalling receptors. In fact, this is not observed in vitro where ACTH (through c ~ strongly ~ inhibits adrenocortical cell proliferation. This proposed mechanism thus does not seem to take place in the

adrenocortical cells themselves but bFGF could act as a paracrine factor and modify the proliferation of neighbouring cell types such as endothelial cells.

ACKNOWLEDGMENTS We thank Dr. A. Baird (Whittier Institute, La Jolla, CA) for his gift of anti-bFGF antibodies. We are grateful to Claude Blanc-Brude and Isabelle Gaillard for their helpful contribution to the preparation of adrenocortical cell primary cultures. We are indebted to Sonia Lidy for the preparation of the manuscript. This research was supported by the Institut National de la Sante et de la Recherche M6dieale (INSERM U 244) and by the Commissariat a I’Energie Atomique (DSVDBMS). Z. Jiang was supported by a fellowship from INSERM. LITERATURE CITED Andres, J.L., Stanley, K., Cheifetz, S., and Massape, J. (1989) Memand soluble forms of betaglycan, a polymorphic pro~ brane-anchored ) teoglycan that binds transforming growth factor-p. J. Cell Biol.,

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