0013-7227/91/1283-1425$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 128, No. 3 Printed in U.S.A.

Transforming Growth Factor-/? Stimulates Production of Insulin-Like Growth Factor-Binding Protein-3 by Human Skin Fibroblasts* JANET L. MARTIN AND ROBERT C. BAXTER Department of Medicine, University of Sydney, New South Wales 2006; and the Department of Endocrinology, Royal Prince Alfred Hospital, Camperdown, New South Wales 2050, Australia

ABSTRACT. Human neonatal fibroblasts in monolayer culture produce insulin-like growth factor-binding protein-3 (IGFBP-3), the IGF-binding subunit of the circulating 140-kDa IGFBP complex. We now report that transforming growth factor-/? (TGF/3) is a potent stimulator of IGFBP-3 production by fibroblasts. After 72-h incubation with 1 ng/ml TGF/3, the levels of IGFBP-3 in conditioned medium were increased 5.8 ± 1.2fold (mean ± SE; n = 9). Half-maximal stimulation of IGFBP-3 production was seen at 0.4 ± 0.05 ng/ml TGF/3 (n = 4). Coincubation of fibroblasts with TGFfl and either IGF-I or IGF-II at 50 ng/ml enhanced IGFBP-3 production 1.5- to 2-fold compared to TGF/3 alone. As previously reported, fetal calf serum (FCS) stimulated IGFBP-3 production 5- to 6-fold; 1 ng/ml TGF/3 increased the stimulated production of IGFBP-3 by FCS a

T

HE INSULIN-like growth factors IGF-I and IGFII are anabolic and mitogenic peptides of 7.5 kDa mol mass which circulate in association with specific carrier or binding proteins (IGFBPs). Three immunologically distinct classes of IGFBPs have been characterized, and the complete primary structures predicted from the cloned cDNAs for each. Known as IGFBP-1, IGFBP-2, and IGFBP-3, these proteins differ in molecular size, and degree and type of glycosylation (1, 2). IGFBP-1 and IGFBP-3 have similar high affinity for both IGF-I and IGF-II, while IGFBP-2 shows a marked preference for binding to IGF-II (1). Each of these three classes of IGFBPs has been shown to modulate some aspect of IGF action in a variety of cell types in vitro. Insulin-like activity in fat cells and incorporation of sulfate into cartilage were shown to be inhibited by impure IGFBP preparations from human plasma (3, 4). IGFBP-2 purified from the conditioned medium of rat liver-derived BRL-3A cell line inhibits Received July 30,1990. Address all correspondence and requests for reprints to: Janet Martin, Department of Medicine, University of Sydney, New South Wales 2006, Australia. * This work was supported by the National Health and Medical Research Council of Australia.

further 2.5- to 3.5-fold. Acidification of FCS before addition enhanced the stimulation of IGFBP-3 compared to that caused by untreated FCS, but decreased further potentiation by TGF/3. This effect of acidified FCS was reversed by a neutralizing antibody to TGF/3. Similarly, the stimulation of IGFBP-3 levels by human serum or conditioned serum-free fibroblast medium was significantly increased by acidification of serum or medium before addition and was reversed by TGF/3 antibody. These observations are consistent with acid-mediated activation of latent TGF/3 added in serum or secreted by fibroblasts. Since IGFBP-3 is known to regulate IGF activity in fibroblasts, these results raise the possibility that TGF/3 may modulate IGF actions in these cells by stimulating the production of IGFBP-3. (Endocrinology 128: 1425-1433, 1991)

IGF binding, DNA synthesis, and glucose transport in chick embryo fibroblasts (5), while IGFBP-1 inhibits IGF-I binding and stimulation of aminoisobutyrate uptake in choriocarcinoma cells (6). An IGFBP purified from human bone cell-conditioned medium inhibits both IGF-stimulated and serum-stimulated bone cell proliferation (7). Interestingly, work from two laboratories has demonstrated that proteins purified on the basis of their inhibitory action on cell growth or function have sequence homology with IGFBP-3 (8, 9). Although the majority of reported effects of IGFBPs on IGF action are inhibitory, stimulation of IGF activity by IGFBPs has also been documented. When coincubated with IGF-I, a pure preparation of IGFBP-1 was found to potentiate IGF-I-stimulated DNA synthesis in porcine aortic smooth muscle cells as well as human, mouse, and chick embryo fibroblasts (10). IGFBP-3 has been shown to enhance IGF-stimulated DNA synthesis in human fibroblasts (11) and aminoisobutyrate uptake in cultured bovine fibroblasts (12) only when the cells were preincubated with IGFBP-3 before stimulation with IGF-I; both studies showed that coincubation of IGF-I with IGFBP-3 resulted in inhibition of IGF action. Blum and co-workers (13) have reported that in baby hamster kidney fibroblasts, IGF-I stimulates thymidine incorpo-

1425

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:59 For personal use only. No other uses without permission. . All rights reserved.

1426

TGF0 STIMULATES IGFBP-3 PRODUCTION

ration only if first complexed with IGFBP-3. Although one recent report has suggested that inhibition of granulosa cell steroidogenesis by IGFBP-3 is not due solely to sequestration of IGFs (14), most of the effects of IGFBPs on cell function appear to be through association with IGFs and modulation of growth factor activity. IGFBPs are synthesized by many types of cells in culture; for example, IGFBP-1 is secreted by the human hepatoma cell line HEP-G2 (15) and granulosa cells (16), IGFBP-2 is produced by the rat liver-derived BRL-3A cell (17) and the bovine kidney cell-line MDBK (18), while IGFBP-3 is secreted by human fibroblasts (19), vascular endothelial cells (20), and mouse 3T3 cells (21). Many of these cells also produce and/or are responsive to IGF-I or IGF-II in vitro; thus, the binding proteins have the potential to be important autocrine or paracrine regulators of IGF action. We have shown previously that human fibroblasts synthesize IGFBP-3, and that the production of this protein is increased in response to epidermal growth factor and a factor(s) present in fetal calf serum (FCS) (19). In this paper we demonstrate that transforming growth factor-/?l (TGF/3) is a potent stimulator of IGFBP-3 production by fibroblasts and show that both serum and culture medium conditioned by fibroblasts contain latent TGF/3, which on activation is able to stimulate IGFBP-3 production.

Materials and Methods Materials Human TGF^l and TGF/31 -neutralizing antibody (rabbit immunoglobulin G-antiporcine platelet TGF0) were purchased from British Biotechnology (Oxford, United Kingdom). Human IGF-I and IGF-II (22) and IGFBP-3 (23) were purified from Cohn fraction IV as previously described. BSA was obtained from Sigma Chemical Co. (St Louis, MO). FCS, cell culture media, and chemicals were purchased from Cytosystems (North Ryde, New South Wales, Australia). Plasticware for cell cultures were purchased from Nunc (Roskilde, Denmark). Electrophoresis consumables and mol wt standards were obtained from Pharmacia (Uppsala, Sweden). Autoradiographic film (Hyperfilm-MP) was purchased from Amersham (Bucks, United Kingdom). Tracers Radioiodinated IGF-I and IGF-II were prepared and purified to specific activities of 200 Ci/g, as previously reported (22, 24). [125I]IGF-I cross-linked to IGFBP-3 for RIA of IGFBP-3 was prepared as previously described (25).

Endo • 1991 Voll28«No3

fractionated at 4 C using a 1.5 X 100-cm Sephacryl S-200 HR column equilibrated with Ham's F-12 medium containing 20 mM HEPES, 1 g/liter BSA, 0.06 g/liter penicillin, and 0.1 g/ liter streptomycin sulfate, pH 7.6. Two milliliters of FCS were applied to the column and eluted in medium at 6 ml/h, and 2ml fractions were collected. Aliquots of each fraction were acidified/neutralized or treated with neutralized HCl, as described above, diluted to a final concentration of 10% by volume in medium, and sterilized through 0.22-jtun filters before use in stimulation experiments. In experiments where human serum was used in place of FCS, it was first immunodepleted of endogenous IGFBP-3 by passing diluted serum (1:1 dilution in medium) through a column containing an anti-IGFBP-3 antiserum bound to agarose, as previously described (26). Cell culture Neonatal human foreskin fibroblasts were maintained in monolayer culture in Ham's F-12 medium containing 10% FCS, 20 mM HEPES, and 2 mM L-glutamine, pH 7.6. Cultures were passaged every 7-10 days by treatment with trypsin-EDTA and used for experiments between passages 3-10. For stimulation experiments, cells were plated at a density of 2 x 104 cells/well (unless otherwise indicated) in 24-place multiwells in serumcontaining medium. After 48 h, this medium was replaced with serum-free Ham's F-12. The cells were maintained serum free for 48 h, then changed to medium containing TGF/3 or other additives. Incubations were continued for a further 24, 48, or 72 h (as indicated for individual experiments) before collection of medium for analysis. DNA was measured at the end of some experiments using the fluorescent dye H 33258 (Calbiochem, La Jolla, CA). To prepare a pool of conditioned fibroblast medium, confluent cultures of fibroblasts were washed with serum-free medium and maintained in serum free medium for 48 h. This medium was discarded, and cultures were incubated with fresh serum-free medium for a further 72 h. The conditioned medium was collected, centrifuged to remove cell debris, and stored frozen until use. Conditioned medium was diluted 1:1 with serum-free medium, adjusted to pH 7.6, and sterilized by filtration before use in stimulation experiments. Assays IGFBP-3 was measured in medium samples by RIA, as previously reported, using [125I]IGF-I cross-linked to IGFBP-3 as tracer (25) and antibody R7 at a final dilution of 1:10,000 (19). This assay is specific for IGFBP-3 from higher primate species (25). As determined by immunoblotting, only the IGFBP-3 doublet of approximately 50 kDa is detected in fibroblast-conditioned medium by antiserum R7 (27); however, due to the limited sensitivity of the immunoblotting technique, we cannot exclude the possibility that a minor proportion of immunoreactive IGFBP-3, as detected by our RIA, might be in a form smaller than 50 kDa.

Preparation of sera for stimulation experiments Acidification of FCS was achieved by the addition of 5 M HCl to a give a final pH of 2, followed by incubation at 22 C for 1 h and neutralization with 5 M NaOH. Control serum was treated with an equivalent volume of neutralized HCl. FCS was

Electrophoresis and ligand blotting Conditioned medium samples were prepared for electrophoresis and ligand blotting by acidification to 1 M acetic acid and concentration by centrifuging through a Centricon 10 mem-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 20:59 For personal use only. No other uses without permission. . All rights reserved.

TGF/3 STIMULATES IGFBP-3 PRODUCTION brane (Amicon, Danvers, MA). These concentrates were washed with a further 5 ml 1 M acetic acid and centrifuged to a final volume of 100-200 jul. Concentrates were lyophilized, and the pellets were dissolved in 50 mM sodium phosphate buffer, pH 6.5, to a final 5- or 10-fold concentration over the starting sample. Medium samples and electrophoresis mol wt standards were mixed with concentrated electrophoresis sample buffer to give final concentrations of 0.0125 M Tris, 3% sodium dodecyl sulfate (SDS), and 10% glycerol, then boiled without reducing agent for 5 min. Samples were fractionated on 12% SDS-polyacrylamide gels overnight at 100 V, and proteins were transferred to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany), as previously described (27). After electroblotting, the nitrocellulose sheet was incubated for 3 h at 37 C in 0.1 M sodium phosphate, pH 6.5, containing 3% BSA and 0.02% sodium azide. Blots were then incubated overnight at 4 C with radioiodinated IGF-I or IGF-II (1 X 106 cpm) in buffer containing 1% BSA. Nitrocellulose sheets were washed three times in cold phosphate buffer, once in buffer containing 0.05% Nonidet P40, then a further three times in phosphate buffer without detergent. Hyperfilm-MP was exposed to dried blots for 1-2 days before developing. Data analysis Experiments were performed at least three times unless otherwise stated, with values shown indicating the mean ± SE for determinations made in triplicate or quadruplicate in a single experiment. Statistical analysis was performed by analysis of variance and Duncan's multiple range test unless otherwise indicated.

Results Treatment of neonatal human skin fibroblasts with TGF/3 resulted in increased production of immunoreactive IGFBP-3. Preliminary experiments indicated that this effect was maximal at 1 ng/ml TGF/3. As shown in Fig. 1, within 24 h of addition, 1 ng/ml TGF/3 increased IGFBP-3 production approximately 2-fold compared to that in untreated cells (P < 0.01, by t test); after 72 h, the degree of stimulation was much greater. Total DNA levels did not increase significantly over the same period in either the absence or presence of TGFjS (not shown). Thus, for the experiment illustrated in Fig. 1, TGF/3 caused a 5.34-fold stimulation after 72 h when calculated on the basis of IGFBP-3 production per well and a 5.30fold stimulation when calculated on the basis of IGFBP3 production per fxg DNA. For this reason, results were not routinely corrected for DNA content. Averaged over nine experiments, IGFBP-3 production was increased 5.8 ± 1.2-fold by TGF/3 compared to that in untreated cells (mean ± SE). Analysis of conditioned medium by SDS-polyacrylamide gel electrophoresis and ligand blotting with IGF-II tracer (Fig. lb) confirmed that the immunoreactive IGFBP-3 stimulated by TGF0 was a doublet species identical in size to IGFBP-3 purified from human plasma (23). An IGF-binding species of

1427

22 kDa was also detected in medium by ligand blotting; however, over the same time period, production of this smaller IGFBP was increased only 1.5-fold (determined by densitometric analysis of autoradiographs). As initial experiments indicated that the degree of stimulation of IGFBP-3 production varied with cell density, we examined this in more detail. Fibroblasts were plated at densities of 1, 2, 4, and 8 X 104 cells/well, changed to serum-free medium after 24 h, and stimulated with IGFs or TGF,3 for 72 h. At the end of this period, cells were counted to ensure that cell numbers were in the same ratio as at plating (not shown). Figure 2 shows the IGFBP-3 production at the four cell densities, expressed as percent stimulation per 104 cells compared to that in untreated cells. TGF/3 at 1 ng/ml showed optimal stimulation of IGFBP-3 production when cells were subconfluent (2-4 X 104 cells/well), with the degree of stimulation decreasing with high cell number. Sparsely growing cells showed no response. When added in isolation, 50 ng/ml IGF-I or IGF-II increased IGFBP-3 production at all cell densities, with the maximum effect (2.2- to 2.4fold stimulation; P < 0.01) observed at a cell density of 4 X 104 cells/well. IGF-I and IGF-II did not significantly differ in their stimulation of IGFBP-3. At cell densities of 2, 4, and 8 X 104, coincubation of fibroblasts with TGF/3 and either IGF-I or IGF-II resulted in an enhancement of TGF/3 stimulation of IGFBP-3 production; however, the lack of responsiveness to TGF/3 by cells at low density was not altered by coincubation with IGF-I or IGF-II. There was no significant difference between IGFI and IGF-II in the enhancement of the TGF/3 effect. Subsequent experiments confirmed the IGF potentiation of the TGF/3 effect; in seven experiments (at a density of 2 X 104 cells/well), 50 ng/ml IGF-I caused a 2.02 ± 0.22-fold greater stimulation of IGFBP-3 production by 1 ng/ml TGF/3 than that caused by TGF0 alone. This is characterized more fully in Fig. 3a, which shows a dose-response curve for TGF/3 over the range 0.05-5 ng/ml in the presence or absence of 50 ng/ml IGF-I, with IGFBP-3 levels determined after 72-h incubation. In the absence of IGF-I, half-maximal stimulation of IGFBP-3 was achieved at 0.40 ± 0.05 ng/ml TGF/3 (mean ± SE for four experiments). The presence of 50 ng/ml IGF-I significantly potentiated the stimulation by TGF/3, without altering the sensitivity to TGF/3. Although IGF-I alone caused little stimulation of IGFBP-3 production (Fig. 3b), an enhancement of the effect of 1 ng/ml TGF/3 was observed at IGF-I concentrations of 5 ng/ml or greater (P