0013-7227/91/1282-1057$03.00/0 Endocrinology Copyright© 1991 by The Endocrine Society
Vol. 128, No. 2 Printed in U.S.A.
Mitogens Regulate the Production of Insulin-Like Growth Factor-Binding Protein by Swiss 3T3 Cells A. N. CORPS AND K. D. BROWN Department of Biochemistry, Institute of Animal Physiology and Genetics Research, Babraham Hall, Cambridge, CB2 4AT United Kingdom
Production of this IGFBP by Swiss 3T3 cells was stimulated by 50-150% by the mitogens bombesin, vasopressin, plateletderived growth factor, epidermal growth factor, and 12-O-tetradecanoylphorbol 13-acetate and also by IGF-I. The increased production of IGFBP was first detected after 4-6 h of incubation and was then maintained for 48-72 h. Agents that elevate intracellular cAMP and the glucocorticoid dexamethasone reduced IGFBP output. In cells in which protein kinase-C had been down-modulated, the stimulation of IGFBP output by 12O-tetradecanoylphorbol 13-acetate was abolished, but the stimulation induced by the other mitogens was not prevented. Thus, the production of IGFBP by Swiss 3T3 cells can be regulated by a number of different signalling pathways. (Endocrinology 128: 1057-1064, 1991)
ABSTRACT. Quiescent Swiss 3T3 cells can be stimulated to reenter the cell cycle by various mitogens used in synergistic combinations with insulin-like growth factors (IGFs). The cells constitutively secrete an IGF-binding protein (IGFBP), which can modulate the interaction of IGFs with their receptors and could, therefore, alter cellular responsiveness to IGFs. We have now characterized the IGFBP secreted by Swiss 3T3 cells and tested whether its secretion is regulated by heterologous mitogens. Ligand blotting using [125I]IGF-I revealed a major IGFBP of 40,000 mol wt, and treatment of the cells with tunicamycin reduced the mol wt of this protein to about 32,000. mRNA from Swiss 3T3 cells hybridized to a 32P-labeled oligonucleotide (50mer) complementary to rat IGFBP-3. Taken together, these results indicate that the principal IGFBP secreted by Swiss 3T3 cells is probably the iV-glycosylated IGFBP-3.
T
HE INSULIN-like growth factors (IGFs) are polypeptide mitogens that are important in both preand postnatal growth (1, 2). Although present at high concentrations in the circulation and extravascular fluids, they are mostly complexed with specific binding proteins (1, 3, 4). These IGF-binding proteins (IGFBPs) may be classified into at least three groups according to their relative binding affinities for IGF-I and -II (5), and three separate genes coding for IGFBP have been identified by cDNA sequencing (6-10). The majority of circulating IGF is carried in a GH-dependent complex of about 150,000 mol wt (Mr), the binding subunit of which is termed IGFBP-3 (see Ref. 11 for a review of the terminology currently used). Two other IGFBPs, identified from various sources, form complexes of about 40,000 Mr and have been termed IGFBP-1 and IGFBP2. The role(s) of IGFBPs in vivo is not clear, but they modulate the interaction of IGFs with their cellular receptors in vitro. This effect is generally inhibitory, such that IGF derivatives with reduced affinity for the IGFBPs have greater biological potency (12-14); however, some researchers have reported the enhancement of IGF actions by IGFBPs (14-16), indicating that dif-
ferent effects may be observed depending on the conditions of the experiment (14). IGFBPs are also secreted by various cell types in culture (17, 18). As observed in vivo, the production of IGFBPs in culture may be regulated by hormones such as insulin, IGF, and GH (see Discussion); different IGFBPs show different patterns of regulation by these agents. Less is known about the effects of other growth factors and mitogenic hormones on the secretion of IGFBPs. We have shown previously that an IGFBP is secreted by Swiss mouse embryo-derived 3T3 cells (19). This cell system has been widely used to study the control of cell growth by mitogens that stimulate different intracellular signalling pathways, including protein kinase-C and cAMP (20). Most of these mitogens induce substantial synergistic stimulation of the cells when added together with IGF-I or insulin (19, 20). It was, therefore, of interest to determine whether any of the mitogens affected the production of IGFBP. We show here that the IGFBP secreted by Swiss 3T3 cells is likely to be IGFBP-3 and that its secretion is regulated by various mitogens.
Received July 26,1990. Address requests for reprints to: Dr. A. N. Corps, Department of Biochemistry, Institute of Animal Physiology and Genetics Research, Babraham Hall, Cambridge, CB2 4AT United Kingdom.
Materials
Materials and Methods Dulbecco's Modified Eagle's Medium (DMEM), newborn calf serum, antibiotics, and trypsin were obtained from Flow Lab-
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MITOGENS REGULATE IGFBP PRODUCTION
oratories (Irvine, Strathclyde, United Kingdom). Recombinant [Thr69]IGF-I, platelet-derived growth factor (PDGF), 125I, [732 P]ATP, and Rainbow Markers for gel electrophoresis were obtained from Amersham International (Amersham, Bucks, United Kingdom). IGF-I was iodinated by the soluble lactoperoxidase method (21), and the [125I]IGF-I (specific radioactivity, 900-1300 Ci/mmol in different preparations) was purified by gel filtration on Sephadex G-50 (Pharmacia, Uppsala, Sweden), as described previously (19). BSA, activated charcoal, tunicamycin, insulin, 12-O-tetradecanoylphorbol 13-acetate (TPA), and dexamethasone were obtained from Sigma (Poole, Dorset, United Kingdom). Bombesin, vasopressin, forskolin, and isobutylmethylxanthine (IBMX) were obtained from Bachem (Saffron Walden, United Kingdom), Cambridge Research Biochemicals (Harston, Cambridge, United Kingdom), Calbiochem, and Aldrich (Gillingham, Dorset, United Kingdom), respectively. Epidermal growth factor (EGF) was purified from mouse submaxillary glands as described previously (22). An oligonucleotide probe complementary to 50 nucleotides of the rat IGFBP-3 mRNA sequence coding for Ser166 to Thr182 (nucleotides 634-683 and 611-660 of the cDNA clones described in Refs. 23 and 24, respectively) was prepared using a Biosearch 8750 four-channel DNA synthesizer (Biosearch, San Rafael, CA). The probe was purified by denaturing polyacrylamide gel electrophoresis in the presence of 7 M urea and by ion exchange chromatography. It was labeled using T4 polynucleotide kinase and purified using a NENSORB 20 column, according to the manufacturer's instructions (DuPont, Wilmington, DE). A cDNA probe against rat IGFBP-2 (10) was kindly provided by Dr. J. Schwander (Basel, Switzerland). Supernatants from Swiss 3T3 cells Stock cultures of Swiss 3T3 cells were maintained and passaged as described previously (25). For experimental use, cells were seeded into 24-well cluster trays or 3.5-cm dishes (Nunc, Gibco, Uxbridge, Middlesex, United Kingdom) and incubated for 6-8 days, by which time they were confluent and quiescent. Cells in which protein kinase-C had been down-modulated were obtained by adding 300 nM TPA to the culture medium for the last 48 h before the experiment (26). Cell numbers at the beginning and end of the experimental incubations were determined using a Coulter counter (Coulter Electronics, Hialeah, FL). Serum-free supernatants were obtained from cultures of Swiss 3T3 cells by a protocol based on that described previously (19). Cells were rinsed twice with serum-free DMEM, incubated for 2-4 h at 37 C, and rinsed again to remove any binding proteins that may have been absorbed from the serum. The cells were then incubated at 37 C, with or without added mitogens, in 0.5 ml DMEM/well of a 24-well plate and 1 or 1.5 ml DMEM/3.5-cm dish. Cells in which protein kinase-C had been down-modulated also received 100 nM TPA to maintain the down-modulation. At the end of the incubation, the supernatant medium was collected, centrifuged to remove cellular debris, and stored frozen until required for analysis. In each experiment, duplicate or triplicate dishes or wells were run for each treatment, and the supernatants for each treatment were
Endo • 1991 Voll28«No2
pooled for analysis; consistent results were obtained in two to seven experiments with each treatment. Ligand blotting Samples of supernatant were electrophoresed under nonreducing conditions, using a 4% (wt/vol) acrylamide stacking gel and 10% or 12.5% (wt/vol) separating gels (27), and the proteins were then transferred from the gel to nitrocellulose using a LKB Novablot apparatus (LKB, Rockville, MD). The nitrocellulose was preblocked, incubated with [125I]IGF-I at 4 C, and washed at least four times, as described by Hossenlopp et al. (28). It was then dried and exposed to preflashed Fuji RX film for between 5-12 days at —70 C with intensifying screens. The blots were quantified both by scanning the autoradiographs using a Joyce-Loebl Chromoscan (Gateshead, UK) and by cutting out the radioactive bands and counting them using a Packard Minaxi 7-counter (Packard, Downers Grove, IL); samples of supernatants from mitogen-treated cells were compared against serial 2-fold dilutions of control cell supernatants. The labeling of the bands described was blocked by the inclusion of unlabeled IGF-I in the incubation of the blot with [125I]IGF-I. However, it was not affected by incubation of the supernatant samples with unlabeled IGF-I before electrophoresis, indicating that the presence of unlabeled IGF in the supernatant should not affect the results obtained by this method. Charcoal adsorption assay for IGF-binding activity IGF-binding activity in fluids and cell culture supernatants has previously been estimated using assays in which the remaining free [125I] IGF-I is adsorbed onto charcoal and, thus, separated from protein-bound [125I]IGF-I. We have used a charcoal adsorption assay based on those described previously (29, 30). Samples of supernatant were incubated with [125I]IGFI (0.1 ng) for 30 min at 37 C in a final volume of 0.5 ml 50 mM Tris-HCl (pH 7.4) containing 5 mg/ml BSA and 1 mM NaN3. The tubes were then placed on ice for 30 min, and 1 ml activated charcoal (10 mg/ml) plus protamine sulfate (0.2 mg/ml) was then added. After a further 10 min at 4 C, the tubes were centrifuged (2 min at 10,000 X g in a microcentrifuge), and 750 /xl of the supernatant containing protein-bound [126I] IGF-I were removed for counting in a 7-counter. Samples of DMEM that had not been exposed to cells were assayed in parallel, and the values obtained were subtracted from those of the supernatant samples to determine specific IGF-binding activity. No IGFbinding activity was detected if protamine sulfate was omitted from the assay. Control experiments showed that a constant proportion (>75%) of the [125I]IGF-I was adsorbed to the charcoal over a 20-fold range around the concentration of [125I]IGFI used in the assay, and that the mitogens used did not affect the adsorption of [125I]IGF-I to the charcoal. As found by Conover et al. (30), the binding of [126I]IGF-I to proteins in the supernatant samples increased approximately linearly with increasing concentration of supernatant, until about 20% of the total [125I]IGF-I was specifically bound. Samples were, therefore, compared at concentrations on the linear range, as determined by using two or three different concentrations of each supernatant in duplicate or triplicate. The results
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MITOGENS REGULATE IGFBP PRODUCTION are presented either as the percentage of [125I]IGF-I specifically bound or a percentage of the binding activity in control supernatants. Northern blotting of RNA from Swiss 3T3 cells Total RNA from Swiss 3T3 cells was isolated, as previously described (31), and stored at -70 C before use. Ten ng of RNA per lane were run on an agarose-formaldehyde gel, blotted onto Hybond N, and hybridized to the 32P-labeled oligonucleotide probe overnight at 65 C; all methods were essentially as previously described (32). Blots were washed in four changes of 250 ml 2 x SSC (300 mM NaCl-30 mM sodium citrate) with 0.1% (wt/vol) sodium dodecyl sulfate (SDS) at 65 C for 15 min/wash and exposed to preflashed film at -70 C using intensifying screens.
Results In the absence of added mitogens, Swiss 3T3 cells in serum-free medium secreted IGF-binding activity at a constant rate for at least 72 h, as determined by the charcoal adsorption assay for free [125I]IGF-I (see below). Ligand blotting using [125I]IGF-I revealed a major IGFBP of 40,000 Mr (Fig. 1), consistent with our previous demonstration of a cross-linked complex of 48,000 Mr (19). The electrophoretic mobility of this protein corresponded to that of the lower component of an IGFBP doublet (Mr, 40,000-45,000) observed in adult mouse serum (Fig. 1). The addition of tunicamycin, which inhibits N-linked protein glycosylation, caused a reduction in the Mr of the labeled band, from 40,000 to about 1
2
3
4
5
6
7
8
32,000 in both control and bombesin-stimulated cells (Fig. 1). A small decrease in the intensity of the band probably reflected a general reduction of cellular protein secretion resulting from 18-h incubation of the cells with tunicamycin (Corps, A. N., unpublished data). A second [125I]IGF-I-labeled band of 23,000 Mr was also revealed by the ligand-blotting procedure (Fig. 1). The amount of this component in control cell supernatants (~10% of the major component) is probably too low to have been detected by cross-linking in our previous study (19). Alternatively, it might be occupied by endogenously produced IGF, thus reducing the sensitivity of the crosslinking reaction. The size of the major IGFBP band detected by ligand blotting together with the effect of tunicamycin suggested that the protein might be IGFBP-3. We, therefore, probed Northern blots of RNA isolated from Swiss 3T3 cells using an oligonucleotide complementary to 50 bases of rat IGFBP-3 mRNA. A single band was obtained (Fig. 2), the size of which was estimated from several similar blots to be 2.6 kilobases, consistent with the IGFBP-3 transcript observed in various rat tissues (23, 24). On the same blots, there was no hybridization to RNA isolated from a rat epithelial cell line which we have found to secrete a different IGFBP from that produced by Swiss 3T3 cells (Fig. 2). When the blots were reprobed using a cDNA against rat IGFBP-2, no hybridization to Swiss 3T3 cell RNA was observed, whereas strong hybridization was obtained to a somewhat smaller band in the RNA from the epithelial cell line (data not shown); this 1 2
9
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FIG. 1. Ligand blot showing IGFBPs in supernatants from Swiss 3T3 cells treated with bombesin and tunicamycin. Cells were incubated for 18 h at 37 C, and supernatants were collected, electrophoresed, blotted, and probed with [125I]IGF-I, as described in Materials and Methods. Lanes 1-4, Control cells; lanes 5-8, cells treated with 10 nM bombesin. Tunicamycin was present at the following concentrations (nanograms per ml): lanes 1 and 5, 0; lanes 2 and 6, 10; lanes 3 and 7, 100; and lanes 4 and 8, 1000. Lane 9 contained 5 fil serum from an adult female mouse. The positions of Mr markers (X1O~3) are shown.
FIG. 2. Northern blot of Swiss 3T3 cell RNA probed for IGFBP-3. Total RNA (10 jug/lane) was electrophoresed, blotted, and probed with a 32P-labeled oligonucleotide (50-mer) complementary to IGFBP-3, as described in Materials and Methods. Lane 1, Cells incubated for 6 h in serum-free medium; lane 2, cells incubated for 6 h in serum-free medium containing 30 nM TPA; lane 3, cells not changed to serumfree medium. Lane 4 contains RNA from a rat intestinal epithelial cell line which secretes a different IGFBP from that secreted by Swiss 3T3 cells. The positions of 18S and 28S rRNA are shown.
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MITOGENS REGULATE IGFBP PRODUCTION
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was found to correspond to the IGFBP-2 band observed in RNA from rat brain or testis (as in Ref. 10). The IGFBP-3 band was present in RNA from Swiss 3T3 cells incubated under various conditions, including cells not changed from depleted medium and cells changed to fresh serum-free medium with or without added TPA (Fig. 2). In the experiment shown in Fig. 2, a slightly stronger hybridization was obtained in the RNA from control cells in serum-free medium than in the other treatments. However, no consistent effects were observed between the different treatments in other experiments, indicating that any changes at these times are small and require more quantitative methods of analysis. The intensity of the major IGFBP band detected by ligand blotting was enhanced in supernatants from cells incubated with the mitogens PDGF, bombesin, vasopressin, and TPA and to a slightly lesser extent by EGF (Fig. 3, A and B). A similar stimulation was also observed in response to IGF-I, but insulin had no effect (Fig. 3). This is consistent with different responses being mediated through the insulin and type I IGF receptors; we have shown previously that insulin acts primarily through the insulin receptor on these cells at the concentrations used in this study (19). The addition of IBMX plus isoprena1
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line, which raises intracellular cAMP levels, substantially reduced the output of IGFBP (Fig. 3, A and C), but this inhibition did not prevent stimulation by IGF-I (Fig. 3C). Dexamethasone also inhibited IGFBP output (Fig. 3A). With the exception of supernatants containing added IGF-I (which competes for the binding protein), the charcoal adsorption assay for separating proteinbound and free [125I]IGF-I gave results for the effects of mitogens that agreed with those from ligand blotting and indicated that the stimulation of IGFBP output was generally about 100-150% (Table 1). The magnitude of these stimulatory effects (Fig. 3 and Table 1) could not be attributed to increased cell numbers; none of the treatments with single mitogens induced increases of more than 30% in the number of cells, reflecting the requirement of these cells for synergistic combinations of mitogens (19, 20). Additionally, the inhibition of IGFBP output by cAMP-elevating agents occurred in cultures in which there was a small (~20%) increase in cell numbers compared with control cultures, such that the inhibition of output per cell was more marked than is apparent from the data presented. The stimulation of the output of IGF-binding activity was not an immediate response and could not be detected in a 2-h incubation with mitogens. The earliest effect was noted 4 h after the addition of TPA, while stimulation by bombesin or PDGF was first observed at 6 h (Fig. 4), which is considerably earlier than any stimulation of DNA synthesis or cell number. Subsequently, the stimulation of IGFBP output was maintained at an approximately constant rate for up to 72 h (Fig. 5). This applied to most of the mitogens tested, including TPA at a TABLE 1. Effect of mitoijens on IGF-binding activity in supernatants from Swiss 3T3 cells
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FlG. 3. Scans of ligand blots of IGFBPs in supernatants from mitogenstimulated Swiss 3T3 cells. Cells were incubated with mitogens for 48 h (A) or 24 h (B and C) at 37 C, and supernatants were collected, electrophoresed, blotted, and probed with [125I]IGF-I, as described in Materials and Methods. The autoradiographic scans shown are from three separate experiments in which mitogens were added at the following concentrations: con, control; ins, 200 nM insulin; IGF, 1.2 nM IGF-I; EGF, 3 nM; bom, 10 nM bombesin; dex, 100 nM dexamethasone; IBMX/ip, 50 /xM IBMX plus 25 nM isoprenaline; TPA, 10 nM; vp, 100 nM vasopressin; PDGF, 400 pM.
Control Bombesin (10 nM) Vasopressin (100 nM) PDGF (400 pM) TPA (10 nM) EGF (3 nM) IBMX (50 fiM) + isoprenaline (25 I*M) or + forskolin (100 fiM) Dexamethasone (100 nM) Insulin (200 nM)
[126I]IGF-I bound (% of control) 100 243 ± 8 (7) 226 ± 14 (4) 318 ± 74 (3) 235 ± 30 (4) 153 ± 14 (3) 79 ± 9 (4) 61(2) 87(2)
Cells were incubated with mitogens for 24 or 48 h (in different experiments) at 37 C, and [126I]IGF-I-binding activity in the supernatants was assayed by the charcoal adsorption assay, as described in Materials and Methods. The specific binding was expressed as a percentage of that in the control cell supernatant, and the results shown are the mean ± SEM of the number of experiments given in parentheses. Incubation of the cells with IBMX, isoprenaline, or forskolin individually did not affect the amount of [125I]IGF-I-binding activity in the supernatant.
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MITOGENS REGULATE IGFBP PRODUCTION
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IGFBP activity, but the subsequent 24-h supernatant had activity similar to that from control cells (Figs. 5 and 6A) and the re-addition of TPA did not increase the activity (Fig. 6); effective down-modulation had, therefore, occurred. This pretreatment with TPA induced an bombesin
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FIG. 4. Early time course of stimulated IGFBP production by Swiss 3T3 cells. Cells were incubated at 37 C for the times shown, and the [125I]IGF-I-binding activity (percentage of total counts per min specifically bound by 100 fA supernatants) was determined by the charcoal adsorption assay, as described in Materials and Methods. O, Control cells; • , 10 nM bombesin; A, 10 nM TPA; • , 400 pM PDGF. The means of two or three experiments with each treatment are shown; each experiment consisted of pooled supernatant from two or three dishes per treatment, which were assayed in duplicate or triplicate.
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FIG. 5. Persistence of mitogen effects on IGFBP production by Swiss 3T3 cells. Cells were incubated with mitogens for 48 h at 37 C, and the supernatants were collected. The cells were then washed and incubated with the same mitogens for a further 24 h, after which the supernatants were collected. Twenty-five microliters of the 0- to 48-h supernatants (•) or 50 /xl of the 48- to 72-h supernatants (H) were assayed by the charcoal adsorption assay, as described in Materials and Methods.
concentration (10 nM) that stimulates, but does not down-modulate, protein kinase-C. When high concentrations of TPA were used to down-modulate protein kinase-C, the initial 48-h supernatant showed stimulated
Q.
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con bom vp EGF PDGF TPA IGF FIG. 6. Effects of protein kinase-C depletion on IGFBP production by Swiss 3T3 cells. Control cells (•) or protein kinase-C-depleted cells (H) were incubated with mitogens for 24 h at 37 C. Supernatants were assayed by the charcoal adsorption assay (A; percentage of total counts per min specifically bound) or scans of ligand blots (B; percentage of activity in appropriate control supernatant), as described in Materials and Methods. The mitogens used were added at the following concentrations: con, control; bom, 10 nM bombesin; vp, 100 nM vasopressin; EGF, 3 nM; PDGF, 400 pM; TPA, 10 nM; IGF-I, 1.2 nM. The results in A are the mean ± SEM of triplicate determinations from a single experiment. All mitogen treatments caused significant stimulation relative to the appropriate controls (P = 0.001-0.05, by t test), except for TPA in protein kinase-C-depleted cells. Additionally, there were signficant differences (P = 0.01-0.05) between control and protein kinase-C-depleted cells treated with bombesin, vasopressin, EGF, and TPA. The results in B are the means from two or three separate experiments with each mitogen.
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MITOGENS REGULATE IGFBP PRODUCTION
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increase in cell number (10-50% in different experiments), and any subsequent increase in cell number due to incubation with mitogens was smaller than that in nonpretreated cells. However, the down-modulation of protein kinase-C did not prevent the stimulation of IGFBP output by the other mitogens tested, assayed by either charcoal adsorption (Fig. 6A) or ligand blotting (Fig. 6B). The stimulation induced by bombesin or vasopressin was slightly reduced in some experiments (as shown in Fig. 6A), whereas the stimulation induced by EGF or PDGF was sometimes enhanced (as shown for EGF in Fig. 6A), but these effects were not statistically significant in every experiment. The stimulation induced by IGF-I was not affected by the down-modulation of protein kinase-C (Fig. 6B). Discussion Swiss 3T3 cells secrete an IGFBP that is similar in size to IGFBP-3, the binding component of the high Mr complex in adult mouse serum (33, 34). In addition, the cells express a mRNA that hybridizes to an oligonucleotide probe specific for IGFBP-3. The secreted IGFBP is iV-glycosylated, as indicated by the effect of tunicamycin, which is consistent with it being IGFBP-3 (23, 24) and not IGFBP-1 or IGFBP-2. Swiss 3T3 cells do not contain detectable levels of mRNA coding for IGFBP-2, and the requirement for protamine sulfate in the charcoal adsorption assay to detect IGF-binding activity is consistent with the cells secreting IGFBP-3 rather than IGFBP-1 (30). Taken together, these results indicate that the major IGFBP secreted by Swiss 3T3 cells is IGFBP-3, although this remains to be confirmed by sequence analysis. In this context, we note that the partial sequence analysis of a growth inhibitor (IDF-45) secreted by Swiss 3T3 cells (35) shows only 1 amino acid change from the first 12 amino acids predicted for rat IGFBP-3 (23, 24) and is, thus, likely to be equivalent to the IGFBP described here. The secretion of IGFBP by Swiss 3T3 cells is maintained at a constant rate over long periods of culture in serum-free medium, and RNA coding for IGFBP-3 is present in cells quiesced in serum-depleted medium as well as in cells incubated in serum-free medium with or without added mitogens. Taken together with the slow onset of the effects of mitogens, this suggests that secretion occurs via a constitutive pathway, with little storage for stimulated secretion. Indeed, a comparison of serial dilutions of culture supernatants and cell extracts, subjected to ligand blotting as described above, indicated that the intracellular content of IGFBP is sufficient to maintain secretion for only about 15 min (Corps, A. N., unpublished data). This agrees with results obtained for IGFBP-2 output by BRL-3A cells and rat embryo fibro-
Endo • 1991 Voll28'No2
blasts in pulse-chase experiments using [35S]methionine (36). The output of IGFBP by Swiss 3T3 cells is stimulated by IGF-I and a number of heterologous mitogens, including bombesin, vasopressin, PDGF, EGF, and TPA. In contrast, mitogens that elevate intracellular cAMP levels have a small inhibitory effect. The good agreement between the results obtained using the charcoal adsorption assay and ligand blotting suggest that none of the effects can be attributed to actions on the output of IGFs rather than the output of IGFBPs. Comparison with previous studies suggests that there are both similarities and differences in the pattern of regulation of IGFBP output in different systems;. For example, IGF-I has been reported to stimulate the production of IGFBP-1, IGFBP3, and uncharacterized IGF-binding activity both in vivo and in culture (35, 37-39). Additionally, stimulation by TPA has been reported both in the present study and for the output of IGFBP-1 by human granulosa luteal cells (40). However, the inhibition by dexamethasone and lack of effect of insulin in the present study contrast with the effects of these hormones on IGFBP-1 production (30, 38, 41). Similarly, whereas EGF has been shown to stimulate the production of IGFBP-3 by human skin fibroblasts (42) as well as by Swiss 3T3 cells (this study), other researchers have not detected effects of growth factors such as PDGF and EGF on IGFBP production (30, 38). These differences could reflect the presence or absence of receptors in the different cells or the coupling of alternative signalling pathways to the control of expression of different IGFBPs. At present it is not possible to deduce a common signalling pathway for the stimulation by diverse growth factors of IGFBP production by Swiss 3T3 cells; indeed, it is possible that some growth factors activate transcription, while others stimulate translation of the mRNA. The stimulation induced by TPA indicates that protein kinase-C can mediate the enhancement of IGFBP production. However, after down-modulation of protein kinase-C, the increases in IGFBP output induced by mitogens other than TPA are not eliminated, indicating that other signalling pathways mediate these effects. Recent analysis of the promoter region of the human IGFBP-3 gene revealed the presence of a DNA sequence element similar to that known to bind the transcription factor AP-2 (43). Sequence elements known to bind other transcriptional regulators were not evident, and the researchers suggested that the AP-2 site might mediate the stimulation of IGFBP-3 production by phorbol esters. It remains to be determined whether this or other, as yet unidentified, control elements are involved in the signalling pathways activated by different mitogens in Swiss 3T3 cells. A major question that arises from the present work is
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MITOGENS REGULATE IGFBP PRODUCTION the role of the IGFBP in the mitogenic or metabolic stimulation of Swiss 3T3 cells by IGFs. In previous experiments we showed that conditioned medium from Swiss 3T3 cells inhibited [125I]IGF-I binding to the type I IGF receptors on these cells, presumably due to the presence of the IGFBP (19). Furthermore, the supernatants generated in this study also inhibited [125I]IGF-I binding to cellular receptors to an extent consistent with their IGFBP content assayed by charcoal adsorption (Corps, A. N., unpublished data). It might, therefore, be expected that the binding protein would have a negative influence on cell stimulation, and the purification of IGFBP-3 as an inhibitor of 3T3 cell proliferation (35) would support this view. However, as noted in the Introduction, there is conflicting evidence from other systems as to whether endogenously produced IGFBPs released at the cell surface are inhibitory or stimulatory (12-16). The finding that c AMP-elevating mitogens reduce the output of IGFBP indicates that stimulation of the latter, as seen in response to other growth factors, is not necessary for comitogenic action. Nevertheless, since the reduction of IGFBP output by cAMP-elevating agents does not prevent a stimulation of output by IGF-I, we cannot deduce from these experiments whether the IGFBP is actually required for or modulates the contribution of IGFs to mitogenesis. The use of antisense RNA to prevent the production of IGFBP, or antibodies that block the binding site of the binding protein for IGF, may resolve this question.
8.
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15. 16.
Acknowledgments
17.
We thank John Pascall, Chris Littlewood, and Miranda Gomperts for advice and assistance, and Sue Porter, Linda Notton, and Dianne Styles for preparing the manuscript.
18.
References
19.
1. Sara VR, Hall K 1990 Insulin-like growth factors and their binding proteins. Physiol Rev 70:591-614 2. Gluckman PD 1986 The role of pituitary hormones, growth factors and insulin in the regulation of fetal growth. In: Clarke JR (ed) Oxford Reviews of Reproductive Biology. Clarendon Press, Oxford, vol 8:1-60 3. Ooi GT, Herington AC 1988 The biological and structural characterization of specific serum binding proteins for the insulin-like growth factors. J Endocrinol 118:7-18 4. Baxter RC, Martin JL 1989 Binding proteins for the insulin-like growth factors: structure, regulation and function. Prog Growth Factor Res 1:49-68 5. Forbes B, Szabo L, Baxter RC, Ballard FJ, Wallace JC 1988 Classification of the insulin-like growth factor binding proteins into three distinct categories according to their binding specificities. Biochem Biophys Res Commun 157:196-202 6. Brinkman A, Groffen C, Kortleve DJ, Geurts van Kessel A, Drop SLS 1988 Isolation and characterization of a cDNA encoding the low molecular weight insulin-like growth factor binding protein (IBP-1). EMBO J 7:2417-2423 7. Lee Y-L, Hintz RL, James PM, Lee PDK, Shively JE, Powell DR 1988 Insulin-like growth factor (IGF) binding protein complementary deoxyribonucleic acid from human HEP G2 hepatoma cells:
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predicted protein sequence suggests an IGF binding domain different from those of the IGF-I and IGF-II receptors. Mol Endocrinol 2:404-411 Wood WI, Cachianes G, Henzel WJ, Winslow GA, Spencer SA, Hellmiss R, Martin JL, Baxter RC 1988 Cloning and expression of the GH dependent IGF binding protein. Mol Endocrinol 2:11761185 Binkert C, Landwehr J, Mary J-L, Schwander J, Heinrich G 1989 Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2). EMBO J 8:2497-2502 Margot JB, Binkert C, Mary J-L, Landwehr J, Heinrich G, Schwander J 1989 A low molecular weight insulin-like growth factor binding protein from rat: cDNA cloning and tissue distribution of its messenger RNA. Mol Endocrinol 3:1053-1062 Ballard FJ, Baxter RC, Binoux M, Clemmons DR, Drop SLS, Hall K, Hintz RL, Rechler MM, Rutanen EM, Schwander JC 1990 Report on the nomenclature of the IGF-binding proteins. J Clin Endocrinol Metab 70:817-818 Ross M, Francis GL, Szabo L, Wallace JC, Ballard FJ 1989 Insulinlike growth factor (IGF)-binding proteins inhibit the biological activities of IGF-I and IGF-2 but not des-(l-3)-IGF-I. Biochem J 258:267-272 Cascieri MA, Hayes NS, Bayne ML 1989 Characterization of the increased biological potency in BALB/C 3T3 cells of two analogs of human insulin-like growth factor I which have reduced affinity for the 28 K cell-derived binding protein. J Cell Physiol 139:181188 Clemmons DR, Cascieri MA, Camacho-Hubner C, McCusker RH, Bayne ML 1990 Discrete alterations of the insulin-like growth factor I molecule which alter its affinity for insulin-like growth factor-binding proteins result in changes in bioactivity. J Biol Chem 265:12210-12216 Elgin RG, Busby Jr WH, Clemmons DR 1987 An insulin-like growth factor (IGF) binding protein enhances the biologic response to IGF-I. Proc Natl Acad Sci USA 84:3254-3258 Blum WF, Jenne EW, Reppin F, Kietzmann K, Ranke MB, Bierich JR 1989 Insulin-like growth factor I (IGF-I)-binding protein complex is a better mitogen than free IGF-I. Endocrinology 125:766772 Nissley SP, Rechler MM 1984 Insulin-like growth factors: biosynthesis, receptors and carrier proteins. In: Li CH (ed) Hormonal Proteins and Peptides. Academic Press, New York, vol 12:127-203 Yang YW-H, Brown AL, Orlowski CC, Graham DE, Tseng LY-H, Romanus JA, Rechler MM 1990 Identification of rat cell lines that preferentially express insulin-like growth factor binding proteins rIGFBP-1,2 or 3. Mol Endocrinol 4:29-38 Corps AN, Brown KD 1988 Ligand-receptor interactions involved in the stimulation of Swiss 3T3 fibroblasts by insulin-like growth factors and insulin. Biochem J 252:119-125 Rozengurt E 1986 Early signals in the mitogenic response. Science 234:161-166 Thorell JI, Johansson BG 1971 Enzymatic iodination of polypeptide with 125I to high specific activity. Biochim Biophys Acta 251:363-369 Savage CR, Cohen S 1972 Epidermal growth factor and a new derivative. Rapid isolation procedures and biological and chemical characterisation. J Biol Chem 247:7609-7611 Shimasaki S, Koba A, Mercado M, Shimonaka M, Ling N 1989 Complementary DNA structure of the high molecular weight rat insulin-like growth factor binding protein (IGF-BP3) and tissue distribution of its mRNA. Biochem Biophys Res Commun 165:907912 Albiston AL, Herington AC 1990 Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. Biochem Biophys Res Commun 166:892-897 Brown KD, Blakeley DM 1983 Inhibition of the binding of 125Ilabelled epidermal growth factor to mouse cells by a mitogen in goat mammary secretions. Biochem J 21:465-472 Brown KD, Blakeley DM, Hamon MH, Laurie MS, Corps AN 1987 Protein kinase C-mediated negative-feedback inhibition of unstim-
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MITOGENS REGULATE IGFBP PRODUCTION
ulated and bombesin-stimulated polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells. Biochem J 245:631-639 Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M 1986 Analysis of serum insulin-like growth factor binding proteins using Western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138-143 Martin JL, Baxter RC 1986 Insulin-like growth factor-binding protein from human plasma. J Biol Chem 261:8754-8760 Conover CA, Liu F, Powell D, Rosenfeld RG, Hintz RL 1989 Insulin-like growth factor binding proteins from cultured human fibroblasts. J Clin Invest 83:852-859 Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159 Pascall JC, Saunders J, Blakeley DM, Laurie MS, Brown KD 1989 Tissue-specific effects of castration and ovariectomy on murine epidermal growth factor and its mRNA. J Endocrinol 121:501-506 Zapf J, Born W, Chang J-Y, James P, Froesch ER, Fischer JA 1988 Isolation and NH2-terminal amino acid sequences of rat serum carrier proteins for insulin-like growth factors. Biochem Biophys Res Commun 156:1187-1194 Zapf J, Hauri C, Waldvogel M, Futo E, Hsler H, Binz K, Guler HP, Schmid C, Froesch ER 1989 Recombinant human insulin-like growth factor I induces its own specific carrier protein in hypophysectomized and diabetic rats. Proc Natl Acad Sci USA 86:38133817 Blat C, Bohlen P, Villaudy J, Chatelain G, Golde A, Harel L 1989 Isolation and amino-terminal sequence of a novel cellular growth
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inhibitor (inhibitory diffusible factor 45) secreted by 3T3 fibroblasts. J Biol Chem 254:6021-6024 Romanus JA, Yang YW-H, Nissley SP, Rechler MM 1987 Biosynthesis of the low molecular weight carrier protein for insulin-like growth factors in rat liver and fibroblasts. Endocrinology 121:10411050 McCusker RH, Clemmons DR 1988 Insulin-like growth factor binding protein secretion by muscle cells: effect of cellular differentiation and proliferation. J Cell Physiol 137:505-512 Hill DJ, Camacho-Hubner C, Rashid P, Strain AJ, Clemmons DR 1989 Insulin-like growth factor (IGF)-binding protein release by human fetal fibroblasits: dependency on cell density and IGF peptides. J Endocrinol l!J2:87-98 Schmid C, Zapf J, Froesch ER 1989 Production of carrier proteins for insulin-like growth factors (IGFs) by rat osteoblastic cells. FEBS Lett 244:328-332 Jalkanen J, Suikkari A, Koistinen R, Btzow R, Ritvos O, Seppl M, Ranta T 1989 Regulation of insulin-like growth factor-binding protein-1 production in human granulosa-luteal cells. J Clin Endocrinol Metab 69:1174-1179 Suikkari A-M, Koivisto VA, Koistinen R, Seppala M, Yki-Jarvinen H 1989 Dose-response characteristics for suppression of low molecular weight plasma insulin-like growth factor-binding protein by insulin. J Clin Endocrinol Metab 68:135-139 Martin JL, Baxter RC 1988 Insulin-like growth factor-binding proteins (IGF-BPs) produced by human skin fibroblasts: immunological relationship to other human IGF-BPs. Endocrinology 123:1907-1915 Cubbage ML, Suwanichkul A, Powell DR 1990 Insulin-like growth factor binding protein-3. Organisation of the human chromosomal gene and demonstration of promoter activity. J Biol Chem 265:12642-12649
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Vol. 128, No. 2 Printed in U.S.A.
Mitogens Regulate the Production of Insulin-Like Growth Factor-Binding Protein by Swiss 3T3 Cells A. N. CORPS AND K. D. BROWN Department of Biochemistry, Institute of Animal Physiology and Genetics Research, Babraham Hall, Cambridge, CB2 4AT United Kingdom
Production of this IGFBP by Swiss 3T3 cells was stimulated by 50-150% by the mitogens bombesin, vasopressin, plateletderived growth factor, epidermal growth factor, and 12-O-tetradecanoylphorbol 13-acetate and also by IGF-I. The increased production of IGFBP was first detected after 4-6 h of incubation and was then maintained for 48-72 h. Agents that elevate intracellular cAMP and the glucocorticoid dexamethasone reduced IGFBP output. In cells in which protein kinase-C had been down-modulated, the stimulation of IGFBP output by 12O-tetradecanoylphorbol 13-acetate was abolished, but the stimulation induced by the other mitogens was not prevented. Thus, the production of IGFBP by Swiss 3T3 cells can be regulated by a number of different signalling pathways. (Endocrinology 128: 1057-1064, 1991)
ABSTRACT. Quiescent Swiss 3T3 cells can be stimulated to reenter the cell cycle by various mitogens used in synergistic combinations with insulin-like growth factors (IGFs). The cells constitutively secrete an IGF-binding protein (IGFBP), which can modulate the interaction of IGFs with their receptors and could, therefore, alter cellular responsiveness to IGFs. We have now characterized the IGFBP secreted by Swiss 3T3 cells and tested whether its secretion is regulated by heterologous mitogens. Ligand blotting using [125I]IGF-I revealed a major IGFBP of 40,000 mol wt, and treatment of the cells with tunicamycin reduced the mol wt of this protein to about 32,000. mRNA from Swiss 3T3 cells hybridized to a 32P-labeled oligonucleotide (50mer) complementary to rat IGFBP-3. Taken together, these results indicate that the principal IGFBP secreted by Swiss 3T3 cells is probably the iV-glycosylated IGFBP-3.
T
HE INSULIN-like growth factors (IGFs) are polypeptide mitogens that are important in both preand postnatal growth (1, 2). Although present at high concentrations in the circulation and extravascular fluids, they are mostly complexed with specific binding proteins (1, 3, 4). These IGF-binding proteins (IGFBPs) may be classified into at least three groups according to their relative binding affinities for IGF-I and -II (5), and three separate genes coding for IGFBP have been identified by cDNA sequencing (6-10). The majority of circulating IGF is carried in a GH-dependent complex of about 150,000 mol wt (Mr), the binding subunit of which is termed IGFBP-3 (see Ref. 11 for a review of the terminology currently used). Two other IGFBPs, identified from various sources, form complexes of about 40,000 Mr and have been termed IGFBP-1 and IGFBP2. The role(s) of IGFBPs in vivo is not clear, but they modulate the interaction of IGFs with their cellular receptors in vitro. This effect is generally inhibitory, such that IGF derivatives with reduced affinity for the IGFBPs have greater biological potency (12-14); however, some researchers have reported the enhancement of IGF actions by IGFBPs (14-16), indicating that dif-
ferent effects may be observed depending on the conditions of the experiment (14). IGFBPs are also secreted by various cell types in culture (17, 18). As observed in vivo, the production of IGFBPs in culture may be regulated by hormones such as insulin, IGF, and GH (see Discussion); different IGFBPs show different patterns of regulation by these agents. Less is known about the effects of other growth factors and mitogenic hormones on the secretion of IGFBPs. We have shown previously that an IGFBP is secreted by Swiss mouse embryo-derived 3T3 cells (19). This cell system has been widely used to study the control of cell growth by mitogens that stimulate different intracellular signalling pathways, including protein kinase-C and cAMP (20). Most of these mitogens induce substantial synergistic stimulation of the cells when added together with IGF-I or insulin (19, 20). It was, therefore, of interest to determine whether any of the mitogens affected the production of IGFBP. We show here that the IGFBP secreted by Swiss 3T3 cells is likely to be IGFBP-3 and that its secretion is regulated by various mitogens.
Received July 26,1990. Address requests for reprints to: Dr. A. N. Corps, Department of Biochemistry, Institute of Animal Physiology and Genetics Research, Babraham Hall, Cambridge, CB2 4AT United Kingdom.
Materials
Materials and Methods Dulbecco's Modified Eagle's Medium (DMEM), newborn calf serum, antibiotics, and trypsin were obtained from Flow Lab-
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MITOGENS REGULATE IGFBP PRODUCTION
oratories (Irvine, Strathclyde, United Kingdom). Recombinant [Thr69]IGF-I, platelet-derived growth factor (PDGF), 125I, [732 P]ATP, and Rainbow Markers for gel electrophoresis were obtained from Amersham International (Amersham, Bucks, United Kingdom). IGF-I was iodinated by the soluble lactoperoxidase method (21), and the [125I]IGF-I (specific radioactivity, 900-1300 Ci/mmol in different preparations) was purified by gel filtration on Sephadex G-50 (Pharmacia, Uppsala, Sweden), as described previously (19). BSA, activated charcoal, tunicamycin, insulin, 12-O-tetradecanoylphorbol 13-acetate (TPA), and dexamethasone were obtained from Sigma (Poole, Dorset, United Kingdom). Bombesin, vasopressin, forskolin, and isobutylmethylxanthine (IBMX) were obtained from Bachem (Saffron Walden, United Kingdom), Cambridge Research Biochemicals (Harston, Cambridge, United Kingdom), Calbiochem, and Aldrich (Gillingham, Dorset, United Kingdom), respectively. Epidermal growth factor (EGF) was purified from mouse submaxillary glands as described previously (22). An oligonucleotide probe complementary to 50 nucleotides of the rat IGFBP-3 mRNA sequence coding for Ser166 to Thr182 (nucleotides 634-683 and 611-660 of the cDNA clones described in Refs. 23 and 24, respectively) was prepared using a Biosearch 8750 four-channel DNA synthesizer (Biosearch, San Rafael, CA). The probe was purified by denaturing polyacrylamide gel electrophoresis in the presence of 7 M urea and by ion exchange chromatography. It was labeled using T4 polynucleotide kinase and purified using a NENSORB 20 column, according to the manufacturer's instructions (DuPont, Wilmington, DE). A cDNA probe against rat IGFBP-2 (10) was kindly provided by Dr. J. Schwander (Basel, Switzerland). Supernatants from Swiss 3T3 cells Stock cultures of Swiss 3T3 cells were maintained and passaged as described previously (25). For experimental use, cells were seeded into 24-well cluster trays or 3.5-cm dishes (Nunc, Gibco, Uxbridge, Middlesex, United Kingdom) and incubated for 6-8 days, by which time they were confluent and quiescent. Cells in which protein kinase-C had been down-modulated were obtained by adding 300 nM TPA to the culture medium for the last 48 h before the experiment (26). Cell numbers at the beginning and end of the experimental incubations were determined using a Coulter counter (Coulter Electronics, Hialeah, FL). Serum-free supernatants were obtained from cultures of Swiss 3T3 cells by a protocol based on that described previously (19). Cells were rinsed twice with serum-free DMEM, incubated for 2-4 h at 37 C, and rinsed again to remove any binding proteins that may have been absorbed from the serum. The cells were then incubated at 37 C, with or without added mitogens, in 0.5 ml DMEM/well of a 24-well plate and 1 or 1.5 ml DMEM/3.5-cm dish. Cells in which protein kinase-C had been down-modulated also received 100 nM TPA to maintain the down-modulation. At the end of the incubation, the supernatant medium was collected, centrifuged to remove cellular debris, and stored frozen until required for analysis. In each experiment, duplicate or triplicate dishes or wells were run for each treatment, and the supernatants for each treatment were
Endo • 1991 Voll28«No2
pooled for analysis; consistent results were obtained in two to seven experiments with each treatment. Ligand blotting Samples of supernatant were electrophoresed under nonreducing conditions, using a 4% (wt/vol) acrylamide stacking gel and 10% or 12.5% (wt/vol) separating gels (27), and the proteins were then transferred from the gel to nitrocellulose using a LKB Novablot apparatus (LKB, Rockville, MD). The nitrocellulose was preblocked, incubated with [125I]IGF-I at 4 C, and washed at least four times, as described by Hossenlopp et al. (28). It was then dried and exposed to preflashed Fuji RX film for between 5-12 days at —70 C with intensifying screens. The blots were quantified both by scanning the autoradiographs using a Joyce-Loebl Chromoscan (Gateshead, UK) and by cutting out the radioactive bands and counting them using a Packard Minaxi 7-counter (Packard, Downers Grove, IL); samples of supernatants from mitogen-treated cells were compared against serial 2-fold dilutions of control cell supernatants. The labeling of the bands described was blocked by the inclusion of unlabeled IGF-I in the incubation of the blot with [125I]IGF-I. However, it was not affected by incubation of the supernatant samples with unlabeled IGF-I before electrophoresis, indicating that the presence of unlabeled IGF in the supernatant should not affect the results obtained by this method. Charcoal adsorption assay for IGF-binding activity IGF-binding activity in fluids and cell culture supernatants has previously been estimated using assays in which the remaining free [125I] IGF-I is adsorbed onto charcoal and, thus, separated from protein-bound [125I]IGF-I. We have used a charcoal adsorption assay based on those described previously (29, 30). Samples of supernatant were incubated with [125I]IGFI (0.1 ng) for 30 min at 37 C in a final volume of 0.5 ml 50 mM Tris-HCl (pH 7.4) containing 5 mg/ml BSA and 1 mM NaN3. The tubes were then placed on ice for 30 min, and 1 ml activated charcoal (10 mg/ml) plus protamine sulfate (0.2 mg/ml) was then added. After a further 10 min at 4 C, the tubes were centrifuged (2 min at 10,000 X g in a microcentrifuge), and 750 /xl of the supernatant containing protein-bound [126I] IGF-I were removed for counting in a 7-counter. Samples of DMEM that had not been exposed to cells were assayed in parallel, and the values obtained were subtracted from those of the supernatant samples to determine specific IGF-binding activity. No IGFbinding activity was detected if protamine sulfate was omitted from the assay. Control experiments showed that a constant proportion (>75%) of the [125I]IGF-I was adsorbed to the charcoal over a 20-fold range around the concentration of [125I]IGFI used in the assay, and that the mitogens used did not affect the adsorption of [125I]IGF-I to the charcoal. As found by Conover et al. (30), the binding of [126I]IGF-I to proteins in the supernatant samples increased approximately linearly with increasing concentration of supernatant, until about 20% of the total [125I]IGF-I was specifically bound. Samples were, therefore, compared at concentrations on the linear range, as determined by using two or three different concentrations of each supernatant in duplicate or triplicate. The results
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MITOGENS REGULATE IGFBP PRODUCTION are presented either as the percentage of [125I]IGF-I specifically bound or a percentage of the binding activity in control supernatants. Northern blotting of RNA from Swiss 3T3 cells Total RNA from Swiss 3T3 cells was isolated, as previously described (31), and stored at -70 C before use. Ten ng of RNA per lane were run on an agarose-formaldehyde gel, blotted onto Hybond N, and hybridized to the 32P-labeled oligonucleotide probe overnight at 65 C; all methods were essentially as previously described (32). Blots were washed in four changes of 250 ml 2 x SSC (300 mM NaCl-30 mM sodium citrate) with 0.1% (wt/vol) sodium dodecyl sulfate (SDS) at 65 C for 15 min/wash and exposed to preflashed film at -70 C using intensifying screens.
Results In the absence of added mitogens, Swiss 3T3 cells in serum-free medium secreted IGF-binding activity at a constant rate for at least 72 h, as determined by the charcoal adsorption assay for free [125I]IGF-I (see below). Ligand blotting using [125I]IGF-I revealed a major IGFBP of 40,000 Mr (Fig. 1), consistent with our previous demonstration of a cross-linked complex of 48,000 Mr (19). The electrophoretic mobility of this protein corresponded to that of the lower component of an IGFBP doublet (Mr, 40,000-45,000) observed in adult mouse serum (Fig. 1). The addition of tunicamycin, which inhibits N-linked protein glycosylation, caused a reduction in the Mr of the labeled band, from 40,000 to about 1
2
3
4
5
6
7
8
32,000 in both control and bombesin-stimulated cells (Fig. 1). A small decrease in the intensity of the band probably reflected a general reduction of cellular protein secretion resulting from 18-h incubation of the cells with tunicamycin (Corps, A. N., unpublished data). A second [125I]IGF-I-labeled band of 23,000 Mr was also revealed by the ligand-blotting procedure (Fig. 1). The amount of this component in control cell supernatants (~10% of the major component) is probably too low to have been detected by cross-linking in our previous study (19). Alternatively, it might be occupied by endogenously produced IGF, thus reducing the sensitivity of the crosslinking reaction. The size of the major IGFBP band detected by ligand blotting together with the effect of tunicamycin suggested that the protein might be IGFBP-3. We, therefore, probed Northern blots of RNA isolated from Swiss 3T3 cells using an oligonucleotide complementary to 50 bases of rat IGFBP-3 mRNA. A single band was obtained (Fig. 2), the size of which was estimated from several similar blots to be 2.6 kilobases, consistent with the IGFBP-3 transcript observed in various rat tissues (23, 24). On the same blots, there was no hybridization to RNA isolated from a rat epithelial cell line which we have found to secrete a different IGFBP from that produced by Swiss 3T3 cells (Fig. 2). When the blots were reprobed using a cDNA against rat IGFBP-2, no hybridization to Swiss 3T3 cell RNA was observed, whereas strong hybridization was obtained to a somewhat smaller band in the RNA from the epithelial cell line (data not shown); this 1 2
9
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3
4
- 28S
-30
-
18S
-21
FIG. 1. Ligand blot showing IGFBPs in supernatants from Swiss 3T3 cells treated with bombesin and tunicamycin. Cells were incubated for 18 h at 37 C, and supernatants were collected, electrophoresed, blotted, and probed with [125I]IGF-I, as described in Materials and Methods. Lanes 1-4, Control cells; lanes 5-8, cells treated with 10 nM bombesin. Tunicamycin was present at the following concentrations (nanograms per ml): lanes 1 and 5, 0; lanes 2 and 6, 10; lanes 3 and 7, 100; and lanes 4 and 8, 1000. Lane 9 contained 5 fil serum from an adult female mouse. The positions of Mr markers (X1O~3) are shown.
FIG. 2. Northern blot of Swiss 3T3 cell RNA probed for IGFBP-3. Total RNA (10 jug/lane) was electrophoresed, blotted, and probed with a 32P-labeled oligonucleotide (50-mer) complementary to IGFBP-3, as described in Materials and Methods. Lane 1, Cells incubated for 6 h in serum-free medium; lane 2, cells incubated for 6 h in serum-free medium containing 30 nM TPA; lane 3, cells not changed to serumfree medium. Lane 4 contains RNA from a rat intestinal epithelial cell line which secretes a different IGFBP from that secreted by Swiss 3T3 cells. The positions of 18S and 28S rRNA are shown.
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MITOGENS REGULATE IGFBP PRODUCTION
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was found to correspond to the IGFBP-2 band observed in RNA from rat brain or testis (as in Ref. 10). The IGFBP-3 band was present in RNA from Swiss 3T3 cells incubated under various conditions, including cells not changed from depleted medium and cells changed to fresh serum-free medium with or without added TPA (Fig. 2). In the experiment shown in Fig. 2, a slightly stronger hybridization was obtained in the RNA from control cells in serum-free medium than in the other treatments. However, no consistent effects were observed between the different treatments in other experiments, indicating that any changes at these times are small and require more quantitative methods of analysis. The intensity of the major IGFBP band detected by ligand blotting was enhanced in supernatants from cells incubated with the mitogens PDGF, bombesin, vasopressin, and TPA and to a slightly lesser extent by EGF (Fig. 3, A and B). A similar stimulation was also observed in response to IGF-I, but insulin had no effect (Fig. 3). This is consistent with different responses being mediated through the insulin and type I IGF receptors; we have shown previously that insulin acts primarily through the insulin receptor on these cells at the concentrations used in this study (19). The addition of IBMX plus isoprena1
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line, which raises intracellular cAMP levels, substantially reduced the output of IGFBP (Fig. 3, A and C), but this inhibition did not prevent stimulation by IGF-I (Fig. 3C). Dexamethasone also inhibited IGFBP output (Fig. 3A). With the exception of supernatants containing added IGF-I (which competes for the binding protein), the charcoal adsorption assay for separating proteinbound and free [125I]IGF-I gave results for the effects of mitogens that agreed with those from ligand blotting and indicated that the stimulation of IGFBP output was generally about 100-150% (Table 1). The magnitude of these stimulatory effects (Fig. 3 and Table 1) could not be attributed to increased cell numbers; none of the treatments with single mitogens induced increases of more than 30% in the number of cells, reflecting the requirement of these cells for synergistic combinations of mitogens (19, 20). Additionally, the inhibition of IGFBP output by cAMP-elevating agents occurred in cultures in which there was a small (~20%) increase in cell numbers compared with control cultures, such that the inhibition of output per cell was more marked than is apparent from the data presented. The stimulation of the output of IGF-binding activity was not an immediate response and could not be detected in a 2-h incubation with mitogens. The earliest effect was noted 4 h after the addition of TPA, while stimulation by bombesin or PDGF was first observed at 6 h (Fig. 4), which is considerably earlier than any stimulation of DNA synthesis or cell number. Subsequently, the stimulation of IGFBP output was maintained at an approximately constant rate for up to 72 h (Fig. 5). This applied to most of the mitogens tested, including TPA at a TABLE 1. Effect of mitoijens on IGF-binding activity in supernatants from Swiss 3T3 cells
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FlG. 3. Scans of ligand blots of IGFBPs in supernatants from mitogenstimulated Swiss 3T3 cells. Cells were incubated with mitogens for 48 h (A) or 24 h (B and C) at 37 C, and supernatants were collected, electrophoresed, blotted, and probed with [125I]IGF-I, as described in Materials and Methods. The autoradiographic scans shown are from three separate experiments in which mitogens were added at the following concentrations: con, control; ins, 200 nM insulin; IGF, 1.2 nM IGF-I; EGF, 3 nM; bom, 10 nM bombesin; dex, 100 nM dexamethasone; IBMX/ip, 50 /xM IBMX plus 25 nM isoprenaline; TPA, 10 nM; vp, 100 nM vasopressin; PDGF, 400 pM.
Control Bombesin (10 nM) Vasopressin (100 nM) PDGF (400 pM) TPA (10 nM) EGF (3 nM) IBMX (50 fiM) + isoprenaline (25 I*M) or + forskolin (100 fiM) Dexamethasone (100 nM) Insulin (200 nM)
[126I]IGF-I bound (% of control) 100 243 ± 8 (7) 226 ± 14 (4) 318 ± 74 (3) 235 ± 30 (4) 153 ± 14 (3) 79 ± 9 (4) 61(2) 87(2)
Cells were incubated with mitogens for 24 or 48 h (in different experiments) at 37 C, and [126I]IGF-I-binding activity in the supernatants was assayed by the charcoal adsorption assay, as described in Materials and Methods. The specific binding was expressed as a percentage of that in the control cell supernatant, and the results shown are the mean ± SEM of the number of experiments given in parentheses. Incubation of the cells with IBMX, isoprenaline, or forskolin individually did not affect the amount of [125I]IGF-I-binding activity in the supernatant.
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MITOGENS REGULATE IGFBP PRODUCTION
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IGFBP activity, but the subsequent 24-h supernatant had activity similar to that from control cells (Figs. 5 and 6A) and the re-addition of TPA did not increase the activity (Fig. 6); effective down-modulation had, therefore, occurred. This pretreatment with TPA induced an bombesin
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FIG. 4. Early time course of stimulated IGFBP production by Swiss 3T3 cells. Cells were incubated at 37 C for the times shown, and the [125I]IGF-I-binding activity (percentage of total counts per min specifically bound by 100 fA supernatants) was determined by the charcoal adsorption assay, as described in Materials and Methods. O, Control cells; • , 10 nM bombesin; A, 10 nM TPA; • , 400 pM PDGF. The means of two or three experiments with each treatment are shown; each experiment consisted of pooled supernatant from two or three dishes per treatment, which were assayed in duplicate or triplicate.
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1
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FIG. 5. Persistence of mitogen effects on IGFBP production by Swiss 3T3 cells. Cells were incubated with mitogens for 48 h at 37 C, and the supernatants were collected. The cells were then washed and incubated with the same mitogens for a further 24 h, after which the supernatants were collected. Twenty-five microliters of the 0- to 48-h supernatants (•) or 50 /xl of the 48- to 72-h supernatants (H) were assayed by the charcoal adsorption assay, as described in Materials and Methods.
concentration (10 nM) that stimulates, but does not down-modulate, protein kinase-C. When high concentrations of TPA were used to down-modulate protein kinase-C, the initial 48-h supernatant showed stimulated
Q.
0
SII
1
1
con bom vp EGF PDGF TPA IGF FIG. 6. Effects of protein kinase-C depletion on IGFBP production by Swiss 3T3 cells. Control cells (•) or protein kinase-C-depleted cells (H) were incubated with mitogens for 24 h at 37 C. Supernatants were assayed by the charcoal adsorption assay (A; percentage of total counts per min specifically bound) or scans of ligand blots (B; percentage of activity in appropriate control supernatant), as described in Materials and Methods. The mitogens used were added at the following concentrations: con, control; bom, 10 nM bombesin; vp, 100 nM vasopressin; EGF, 3 nM; PDGF, 400 pM; TPA, 10 nM; IGF-I, 1.2 nM. The results in A are the mean ± SEM of triplicate determinations from a single experiment. All mitogen treatments caused significant stimulation relative to the appropriate controls (P = 0.001-0.05, by t test), except for TPA in protein kinase-C-depleted cells. Additionally, there were signficant differences (P = 0.01-0.05) between control and protein kinase-C-depleted cells treated with bombesin, vasopressin, EGF, and TPA. The results in B are the means from two or three separate experiments with each mitogen.
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MITOGENS REGULATE IGFBP PRODUCTION
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increase in cell number (10-50% in different experiments), and any subsequent increase in cell number due to incubation with mitogens was smaller than that in nonpretreated cells. However, the down-modulation of protein kinase-C did not prevent the stimulation of IGFBP output by the other mitogens tested, assayed by either charcoal adsorption (Fig. 6A) or ligand blotting (Fig. 6B). The stimulation induced by bombesin or vasopressin was slightly reduced in some experiments (as shown in Fig. 6A), whereas the stimulation induced by EGF or PDGF was sometimes enhanced (as shown for EGF in Fig. 6A), but these effects were not statistically significant in every experiment. The stimulation induced by IGF-I was not affected by the down-modulation of protein kinase-C (Fig. 6B). Discussion Swiss 3T3 cells secrete an IGFBP that is similar in size to IGFBP-3, the binding component of the high Mr complex in adult mouse serum (33, 34). In addition, the cells express a mRNA that hybridizes to an oligonucleotide probe specific for IGFBP-3. The secreted IGFBP is iV-glycosylated, as indicated by the effect of tunicamycin, which is consistent with it being IGFBP-3 (23, 24) and not IGFBP-1 or IGFBP-2. Swiss 3T3 cells do not contain detectable levels of mRNA coding for IGFBP-2, and the requirement for protamine sulfate in the charcoal adsorption assay to detect IGF-binding activity is consistent with the cells secreting IGFBP-3 rather than IGFBP-1 (30). Taken together, these results indicate that the major IGFBP secreted by Swiss 3T3 cells is IGFBP-3, although this remains to be confirmed by sequence analysis. In this context, we note that the partial sequence analysis of a growth inhibitor (IDF-45) secreted by Swiss 3T3 cells (35) shows only 1 amino acid change from the first 12 amino acids predicted for rat IGFBP-3 (23, 24) and is, thus, likely to be equivalent to the IGFBP described here. The secretion of IGFBP by Swiss 3T3 cells is maintained at a constant rate over long periods of culture in serum-free medium, and RNA coding for IGFBP-3 is present in cells quiesced in serum-depleted medium as well as in cells incubated in serum-free medium with or without added mitogens. Taken together with the slow onset of the effects of mitogens, this suggests that secretion occurs via a constitutive pathway, with little storage for stimulated secretion. Indeed, a comparison of serial dilutions of culture supernatants and cell extracts, subjected to ligand blotting as described above, indicated that the intracellular content of IGFBP is sufficient to maintain secretion for only about 15 min (Corps, A. N., unpublished data). This agrees with results obtained for IGFBP-2 output by BRL-3A cells and rat embryo fibro-
Endo • 1991 Voll28'No2
blasts in pulse-chase experiments using [35S]methionine (36). The output of IGFBP by Swiss 3T3 cells is stimulated by IGF-I and a number of heterologous mitogens, including bombesin, vasopressin, PDGF, EGF, and TPA. In contrast, mitogens that elevate intracellular cAMP levels have a small inhibitory effect. The good agreement between the results obtained using the charcoal adsorption assay and ligand blotting suggest that none of the effects can be attributed to actions on the output of IGFs rather than the output of IGFBPs. Comparison with previous studies suggests that there are both similarities and differences in the pattern of regulation of IGFBP output in different systems;. For example, IGF-I has been reported to stimulate the production of IGFBP-1, IGFBP3, and uncharacterized IGF-binding activity both in vivo and in culture (35, 37-39). Additionally, stimulation by TPA has been reported both in the present study and for the output of IGFBP-1 by human granulosa luteal cells (40). However, the inhibition by dexamethasone and lack of effect of insulin in the present study contrast with the effects of these hormones on IGFBP-1 production (30, 38, 41). Similarly, whereas EGF has been shown to stimulate the production of IGFBP-3 by human skin fibroblasts (42) as well as by Swiss 3T3 cells (this study), other researchers have not detected effects of growth factors such as PDGF and EGF on IGFBP production (30, 38). These differences could reflect the presence or absence of receptors in the different cells or the coupling of alternative signalling pathways to the control of expression of different IGFBPs. At present it is not possible to deduce a common signalling pathway for the stimulation by diverse growth factors of IGFBP production by Swiss 3T3 cells; indeed, it is possible that some growth factors activate transcription, while others stimulate translation of the mRNA. The stimulation induced by TPA indicates that protein kinase-C can mediate the enhancement of IGFBP production. However, after down-modulation of protein kinase-C, the increases in IGFBP output induced by mitogens other than TPA are not eliminated, indicating that other signalling pathways mediate these effects. Recent analysis of the promoter region of the human IGFBP-3 gene revealed the presence of a DNA sequence element similar to that known to bind the transcription factor AP-2 (43). Sequence elements known to bind other transcriptional regulators were not evident, and the researchers suggested that the AP-2 site might mediate the stimulation of IGFBP-3 production by phorbol esters. It remains to be determined whether this or other, as yet unidentified, control elements are involved in the signalling pathways activated by different mitogens in Swiss 3T3 cells. A major question that arises from the present work is
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MITOGENS REGULATE IGFBP PRODUCTION the role of the IGFBP in the mitogenic or metabolic stimulation of Swiss 3T3 cells by IGFs. In previous experiments we showed that conditioned medium from Swiss 3T3 cells inhibited [125I]IGF-I binding to the type I IGF receptors on these cells, presumably due to the presence of the IGFBP (19). Furthermore, the supernatants generated in this study also inhibited [125I]IGF-I binding to cellular receptors to an extent consistent with their IGFBP content assayed by charcoal adsorption (Corps, A. N., unpublished data). It might, therefore, be expected that the binding protein would have a negative influence on cell stimulation, and the purification of IGFBP-3 as an inhibitor of 3T3 cell proliferation (35) would support this view. However, as noted in the Introduction, there is conflicting evidence from other systems as to whether endogenously produced IGFBPs released at the cell surface are inhibitory or stimulatory (12-16). The finding that c AMP-elevating mitogens reduce the output of IGFBP indicates that stimulation of the latter, as seen in response to other growth factors, is not necessary for comitogenic action. Nevertheless, since the reduction of IGFBP output by cAMP-elevating agents does not prevent a stimulation of output by IGF-I, we cannot deduce from these experiments whether the IGFBP is actually required for or modulates the contribution of IGFs to mitogenesis. The use of antisense RNA to prevent the production of IGFBP, or antibodies that block the binding site of the binding protein for IGF, may resolve this question.
8.
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12.
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15. 16.
Acknowledgments
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We thank John Pascall, Chris Littlewood, and Miranda Gomperts for advice and assistance, and Sue Porter, Linda Notton, and Dianne Styles for preparing the manuscript.
18.
References
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