0013.7227/92/1313-1568$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
INHIBITION PRODUCTION
Vol. 131, No. 3
Printed in U.S.A.
Society
OF HUMAN FIBROBLAST BY IGFBP-3.
Janet L Martin and Robert Department of Endocrinology,
INSULIN-LIKE
C Baxter. Royal
Prince
Alfred
GROWTH
Hospital,
FACTOR
Camperdown,
BINDING
NSW
20.50,
PROTEIN
(IGFBP)
Australia.
ABSTRACT secrete a number of insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, which may alter paracrine or autocrine IGF activity. Studies in vitro have demonstrated that exogenous IGFBP-3 can both inhibit and potentiate IGF action in these cells; however, it is not known to what extent there is regulatory interaction between the IGFBPs. In this study we report that exogenous and endogenous IGFBP-3 inhibit production of an IGF inducible IGFBP. When analyzed by SDS-PAGE and [tsI]IGF-II ligand blotting, human neonatal fihroblasts secrete IGFBP-3, an IGFBP of 29-31 kDa, and a 22-24 kDa IGFBP after treatment with 50 rig/ml IGF-I. When IGF-I treatment was carried out in the presence of increasing concentrations (50-IO00 @ml) of pure human serum-derived IGFBP-3, there was a dose-dependent decrease in the 29-31 kDa protein. In the presence of excess (250 @ml) IGFI, IGFBP-3 had -20-fold reduced potency in inhibiting 29-31 kDa IGFBP. When endogenous production of IGFBP-3 was increased by treatment with transforming growth factor-p1 (TGFPt), there was complete inhibition of 29-31 kDa IGFBP, while at high IGF-I concentrations TGFpt had 2 to 3-fold reduced potency. These results demonstrate that fibroblast IGFBP production can be altered by exogenous and endogenous IGFBP-3, and suggest the existence of regulatory interactions between fibroblast IGFBPs. Human
neonatal
fibroblasts
in monolayer
culture
INTRODUCTION
MATERIALS and METHODS Materials. IGF-I, IGF-II and IGFBP-3 were purified from human Cohn fraction IV as previously described (I 1.12). Transforming growth factor-pt (TGFpt) from human platelets was purchased from British Biotech (Oxford, England). Tracers (IGF-I and IGF-II) were radioiodinated using Na1251 and chloramine T; IGFBP-3 was affinity-labelled with [IZSI]IGF-I as previously described (13). Cell culture reagents and plasticware were purchased from Cytosystems (North Ryde, NSW, Australia), Corning (Corning, NY) and Nunc (Roskilde, Denmark). Electophoresis reagents were purchased from BioRad (Richmond, CA) and BDH (Poole, England), and molecular weight markers were from Pharmacia (Uppsala, Sweden). Hybond-C Extra nitrocellulose and autoradiographic film (Hyperfilm MP) was obtained from Amersham (Bucks, UK). All other reagents were of analytical grade. Cell cultur& Human neonatal fibroblasts in monolayer culture were maintained as previously described (14). Stimulation experiments were performed on confluent monolayers of cells in 24-place multiwells after a 48-h serum free period. Test substances were added in triplicate in 0.5 ml medium (a 4:l mixture of DMEM: Ham’s F-12 supplemented with HEPES (20 mM), glutamine (2 mM), penicillin (0.06 @L), streptomycin sulphate (0.1 g/L) and bovine serum albumin (BSA, 0.5 @). After stimulation for 48h, media were collected and stored frozen until analysis by RIA or electmphoresis and ligand blotting. Assavs, Conditioned media were assayed for IGFBP-3 by RIA as previously reported (13). SDS-nolva&lamide & electrw lip-, Conditioned media were concenuated 5-fold by ultrafiltration as previously described (14). Samples and molecular weight markers were prepared for electrophoresis by addition of concenuated sample buffer (15) and boiling for 5 min without reducing agent. Proteins were fractionated on 12 % SDSpolyacrylamide gels overnight at 80 V, then transferred electrophoretically to Hybond-C extra supported niuocellulose. IGFBPs were detected by ligand blotting with [t25I]IGF-II, essentially as previously described (16). In brief, nitrocellulose sheets were incubated for 3 h at 37 C in his-buffered saline (TBS, 10 mM uis, 150 mM NaCI, pH 7.4) containing 10 gjL BSA, then overnight at 4 C in - 1 x 106 cpm [tuI]IGF-II in TBS containing 10 g/L BSA and 0.05% Nonidet P-40. Blots were then rinsed three times in cold TBS buffer, once in TBS buffer containing 0.05% Nonidet P-40, and a further three times in detergent-free TBS. Hyperfilm-MP autoradiographic film (Amersham, Bucks, UK) was exposed to dried blots for I - 3 days at -70 C, then developed using standard procedures.
The insulin-like growth factor binding proteins (IGFBPs) are believed to play an important role in modulating the biological activity of IGF-I and IGF-II in cell growth and metabolism. Studies in vitro have demonstrated that purified preparations of IGFBP-1, IGFBP-2 and IGFBP-3 inhibit IGF action in a variety of cell types (1.2.3). presumably by sequestration of the growth factor before receptor interaction is possible. Purified preparations of IGFBPs have been shown to potentiate IGF actions by as yet unidentified mechanisms: pure IGFBP-3 enhances IGF-I activity in human and bovine fibroblasts in monolayer culture (3,4,5), while a potentiating form of IGFBP-1 has been isolated from human amniotic fluid (6.7). These findings suggest that IGFBPs produced by cells may profoundly affect paracrine or autccrine actions of the IGFs. In spite of widespread interest in the physiological role of IGFBPs, the regulation of production of these proteins by IGFtarget cells is still poorly understood. Transforming growth factor-gt (TGF~I) is a potent stimulator of IGFBP-3 in human neonatal fibroblast cultures (8). and the secretion of IGFBP-3 and other IGFBPs by these cells is altered in response to IGFs (9,lO). However, the possibility of regulatory interaction between different IGFBPs has not yet been examined. We have addressed this question, and found that IGFBP-3 markedly inhibits secretion of an IGF-inducible IGFBP by human fibroblasts.
ACKNOWLEDGEMENT
This work was supported by the National Research Council of Australia.
Received
in
Iowa
City,
June
15,
Health
and Medical
1992.
1568
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:04 For personal use only. No other uses without permission. . All rights reserved.
RAPID
COMMUNICATIONS
RESULTS and DISCUSSION We have previously demonstrated that treatment of human fibroblast cultures with IGF-I results in 8- to g-fold increased secretion of an IGFBP of 29-31 kDa, detected by SDS-PAGE and ligand blotting with [125I]IGF-II (9). IGFBP-3 is known to inhibit a number of IGF-I-stimulated events in fibroblasts (3,4,10); therefore, we examined the effect of addition of exogenous IGFBP-3 on production1 of fibroblast IGFBPs. Fibroblasts were incubated with IGF-I (50 @ml) in the presence of increasing concentrations (SO, 200 and 1000 &ml) of pure IGFBP3. Conditioned media were then analyzed by SDS-PAGE and ligand blotting. In the absence of exogenous IGFBP-3, IGFI-treated fibroblasts secreted three main IGF-binding species: IGFBP-3 doublet of -50 kDa, the 29-31 kDa doublet IGFBP, and a third IGFBP of 22-24 kDa (Figure la). As the concentration of exogenous IGFBP-3 in the medium was increased, the 29-31 kDa IGFBP disappeared, so that at a concentration of 200 rig/ml added IGFBP-3, the 29-31 kDa IGFBP was no longer detectable by ligand blotting. These results were consistent with IGFBP-3 inhibiting 29-31 kDa IGFBP production by fibroblasts. An apparent increase in the level of the 22-24 kDa IGFBP accompanied the decrease in the 29-31 kDa IGFBP in some experiments; however, this response was quite variable, suggesting that production of this IGFBP is subject to regulatory factors not yet known. b
67-
67-c
,
1569
concentrations of TGFpi (Figure lb). Concomitant with the increase in IGFBP-3, the 29-31 kDa IGFBP disappeared. The effect was noticeable at a concentration of 0.1 @ml TGFPl, and complete inhibition was apparent at 0.3 rig/ml TGFpl. The equivalent concentrations of immunoreactive IGFBP-3 in these media were 159.5k7.3 and 315.Okl6.8 rig/ml (mean&SE), respectively. These results suggested that increased endogenous production of IGFBP-3 inhibits production of IGFBPs. Although some inhibitory effects of IGFBP-3 on IGF action occur through sequestration of the growth factor, there are several reports of inhibitory actions occurring in the absence of added IGFs (6,7). While our results were consistent with the former mechanism of action, they did not preclude the possibility that IGFBP-3 or TGFPi had intrinsic inhibitory activity distinct from IGF binding-mediated inhibition. To distinguish between these possibilities, the effects of exogenous IGFBP-3 at concentrations up to 1000 &ml, and TGFPi to 3.0 @ml, were determined in the presence of 250 rig/ml IGF-I. At this level of IGF-I, the IGFBP-3 concentration would be insufficient to fully neutralize IGF bioactivity, and intrinsic IGFBP-3 or TGFplinhibitory activity would be reflected by persisting inhibition of 29-31 kDa IGFBP. Figure 2 shows the results of these studies. Addition of 50 or 200 rig/ml IGFBP-3 (final medium. concentrations of 107.3fl.8 and 235f9.5 n&ml IGFBP-3, respectively, by RIA) had no effect on the concentration of 29-31 kDa IGFBP in the medium, even though similar concentrations inhibited 29-31 kDa IGFBP in the presence of 50 rig/ml IGF-I (see Figure la). A slight inhibition of 29-31 kDa IGFBP was apparent at 1000 rig/ml IGFBP-3, suggesting that IGF-I activity was beginning to be affected by this concentration of IGFBP-3.
ix
3--' 43' / 0'
67-c
20*
0
50
IGFBP-3
200 1000 (w/ml)-
w. Inhibition of 29-31 kDa IGFBP by IGFBP-3 and TGFpl at low IGF-I concentrations. Media was conditioned by human fibroblasts in the presence of 50 rig/ml IGF-I, and the indicated concentrations of IGFBP-3 (panel a) or TGFPl (panel b). IGFBPs were analyzed by 12% SDS-PAGE, and IGF-II ligand blotting. Although IGFBP-3 purified from human serum is similar in size and IGF-binding characteristics to fibroblast IGFBP-3 (14), the ratio of the glycosylation forms appears different for the two (9,17). To determine whether increased endogenous production of IGFBP-3 resulted in the same inhibition of 29-31 kDa IGFBP, IGF-I-treated fibroblasts were exposed to TGFPl, which potently stimulates IGFBP-3 production by fibroblasts (8). Consistent with results obtained by RIA (not shown), ligand blot analysis of these media showed increased IGFBP-3 in response to increasing
20-9
0
10
50
IGFBP-3
200 1ooa (rig/ml)
Figure 2. Inhibition of 29-31 kDa IGFBP by IGFBP-3 and TGFpl in the presence of 250 rig/ml IGF-I. Media were analyzed as described for Figure 1. 1 In this report, a change in the “production” of a particular IGFBP refers to a change in the abundance of that protein in the conditioned medium as detected by SDS-PAGE and ligand blotting. Such changes may reflect a variety of events, including altered de nova synthesis, secretion or proteolysis of specific IGFBPs. Changes in production per se by measurement of mRNA have not been demonstrated.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:04 For personal use only. No other uses without permission. . All rights reserved.
RAPID
1570
COMMUNICATIONS
In contrast, equivalent concentrations of IGFBP-3 in the medium of TGFgt-stimulated fibroblasts (Figure 2b), effected by 0.3 rig/ml and 1 rig/ml TGFgt (103.2f6.6 @ml and 263.3f4.0 @ml, respectively) were accompanied by a marked decrease in 29-31 kDa IGFBP. To quantify these differences, autoradiographs from three experiments comparing the effects of IGFBP-3 and TGFgl in the presence of low and high IGF concentrations were scanned with a densitometer (Figure 3). In the presence of 50 &ml IGF-I, half-maximal inhibition of 29-31 kDa IGFBP was apparent with SO-60 rig/ml IGFBP-3; at 250 @ml IGF-I, IGFBP-3 was much less potent, with half-maximal inhibition requiring greater than 1000 rig/ml IGFBP-3 (Figure 3a). The potency of TGFgl was also lower in the presence of high IGF-I concentrations: the EDSO for 50 rig/ml and 250 &ml IGF-I were 0.05-0.06 rig/ml and 0.1-0.2 n&ml TGFpl, respectively (Figure 3b). These results suggest that whereas addition of pure IGFBP-3 inhibits 29-31 kDa IGFBP through binding of IGF-I, inhibition of this protein by TGFgl probably occurs via two distinct events: through sequestration of IGF by increased endogenous IGFBP-3 production, and by action independent of IGFBP-3. a Inhibition
by
10
100
IGFBP-3
100
Inhibition
0.1
by TGFP
1
TGFP (rig/ml) IGFBP-3 (nglml) Figure Densitometric analysis of autoradiographs. Ligand blots similar to those shown in Figs 1 and 2 were scanned, and the OD of the 29-31 kDa peak for IGFBP-3 or TGFgt-treated samples expressed as a percentage of the peak height in the presence of 50 @ml IGF-I ( ) or 250 @ml IGF-I ( ) without added IGFBP-3 or TGFpt.
Addition of exogenous IGFBP-3 to cultures of human or bovine fibroblasts has been shown to inhibit IGF-stimulated DNA synthesis when added with the growth factor, and potentiate IGF action if cells are preincubated with the binding protein prior to exposure to IGF-I (3,5). The mechanism by which such augmentation of IGF bioactivity occurs is not known. It has recently been suggested that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I may be involved in this potentiation (17). It is possible, however, that changes to endogenous IGFBP production occurring as a result of addition of exogenous IGFBP-3, may be important in the determining whether subsequent IGF action is inhibitory or stimulatory. Resolving such questions will prove important in achieving a full understanding of the role of IGFs and their binding proteins in cell growth.
Endo l 1992 Voll31. No 3
REFERENCES Ritvos 0, Ranta T, Jalkanen J, Suikkari A-M, Voutilainen R, Bohn H, Rutanen E-M 1988 Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells. Endocrinology 122: 2150-2157 2. Knauer DJ, Smith GL 1980 Inhibition of biological activity of multiplication-stimulating activity by binding to its carrier protein. Proc Natl Acad Sci USA 77: 7252-7256 De Mellow JSM. Baxter RC 1988 Growth hormone3. dependent insulin-like growth factor (IGF) binding protein both inhibits and potentiates IGF-I-stimulated DNA synthesis in human &in fibroblasts. Biochem Biophys Res Commun 156: 199-204 4. Blum WF. Jenne EW. ReDDin F. Kietzmann K, Ranke MB, Bier&h JR 1989 Insul&ke growth factor I (IGF-I)bindine orotein comnlex is a better mitogen than free IGF-I. Endoc%ology 125:?66-772 5. Conover CA, Ronk M, Lombana F, Powell DR 1990 Structural and biological characterization of bovine insulinlike growth factor binding protein-3. Endocrinology 127: 2795-2803 6. Busby WH Jr, Klapper DG, Clemmons DR 1988 Purification of a 31 OOO-Dalton insulin-like growth factor binding protein from human amniotic fluid. J Biol Chem 263: 14203-14210 7. Busby WI-I Jr, Hossenlopp P, Binoux M, Clemmons DR 1989 Purified preparations of the amniotic fluid-derived insulin-like growth factor-bidning protein contain multimeric fotms that are biologically _ active. Endocrinology-125: 773-777 8. Martin JL, Baxter RC 1991 Transforming growth factor-p stimulates production of insulin-like growth factor-binding protein-3 by human skin fibroblasts. Endocrinology 128: 1425-1433 9. Martin JL, Baxter RC 1990 Production of an insulin-like growth factor (IGF)-inducible IGF-binding protein by human skin fibroblasts. Endocrinology 127: 781-788 10. Conover CA 1991 A uniaue receutor-indeuendent mechanism by which insulin-hke growth factor I regulates the availabilitv of insulinlike growth factor binding uroteins in normal andtransformed himan fibroblasts. J cii’n Invest 88: 1354-1361 11. Baxter RC, De Mellow JSM 1986 Measurement of insulinlike growth factor-II by radioreceptor assay using ovine placental membranes. Clin Endocrinol (Gxf) 24: 267-278 12. Martin JL, Baxter RC 1986 Insulin-like growth factorbinding protein from human plasma: p&cation and characterization. J Biol Chem 261: 8754-8760 13. Baxter RC, Martin JL 1986 Radioimmunoassay of growth hormone-dependent insulin-like growth factor binding protein in human plasma. J Clin Invest 78: 1504-1512 14. 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 15. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 16. 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 biding proteins and competitive binding studies. Analyt Biochem 154: 138-143 17. Conover CA 1991 Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: evidence for processing of cell-bound IGFBP-3. Endocrinology 129: 3259-3268 1.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:04 For personal use only. No other uses without permission. . All rights reserved.
INHIBITION PRODUCTION
Vol. 131, No. 3
Printed in U.S.A.
Society
OF HUMAN FIBROBLAST BY IGFBP-3.
Janet L Martin and Robert Department of Endocrinology,
INSULIN-LIKE
C Baxter. Royal
Prince
Alfred
GROWTH
Hospital,
FACTOR
Camperdown,
BINDING
NSW
20.50,
PROTEIN
(IGFBP)
Australia.
ABSTRACT secrete a number of insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, which may alter paracrine or autocrine IGF activity. Studies in vitro have demonstrated that exogenous IGFBP-3 can both inhibit and potentiate IGF action in these cells; however, it is not known to what extent there is regulatory interaction between the IGFBPs. In this study we report that exogenous and endogenous IGFBP-3 inhibit production of an IGF inducible IGFBP. When analyzed by SDS-PAGE and [tsI]IGF-II ligand blotting, human neonatal fihroblasts secrete IGFBP-3, an IGFBP of 29-31 kDa, and a 22-24 kDa IGFBP after treatment with 50 rig/ml IGF-I. When IGF-I treatment was carried out in the presence of increasing concentrations (50-IO00 @ml) of pure human serum-derived IGFBP-3, there was a dose-dependent decrease in the 29-31 kDa protein. In the presence of excess (250 @ml) IGFI, IGFBP-3 had -20-fold reduced potency in inhibiting 29-31 kDa IGFBP. When endogenous production of IGFBP-3 was increased by treatment with transforming growth factor-p1 (TGFPt), there was complete inhibition of 29-31 kDa IGFBP, while at high IGF-I concentrations TGFpt had 2 to 3-fold reduced potency. These results demonstrate that fibroblast IGFBP production can be altered by exogenous and endogenous IGFBP-3, and suggest the existence of regulatory interactions between fibroblast IGFBPs. Human
neonatal
fibroblasts
in monolayer
culture
INTRODUCTION
MATERIALS and METHODS Materials. IGF-I, IGF-II and IGFBP-3 were purified from human Cohn fraction IV as previously described (I 1.12). Transforming growth factor-pt (TGFpt) from human platelets was purchased from British Biotech (Oxford, England). Tracers (IGF-I and IGF-II) were radioiodinated using Na1251 and chloramine T; IGFBP-3 was affinity-labelled with [IZSI]IGF-I as previously described (13). Cell culture reagents and plasticware were purchased from Cytosystems (North Ryde, NSW, Australia), Corning (Corning, NY) and Nunc (Roskilde, Denmark). Electophoresis reagents were purchased from BioRad (Richmond, CA) and BDH (Poole, England), and molecular weight markers were from Pharmacia (Uppsala, Sweden). Hybond-C Extra nitrocellulose and autoradiographic film (Hyperfilm MP) was obtained from Amersham (Bucks, UK). All other reagents were of analytical grade. Cell cultur& Human neonatal fibroblasts in monolayer culture were maintained as previously described (14). Stimulation experiments were performed on confluent monolayers of cells in 24-place multiwells after a 48-h serum free period. Test substances were added in triplicate in 0.5 ml medium (a 4:l mixture of DMEM: Ham’s F-12 supplemented with HEPES (20 mM), glutamine (2 mM), penicillin (0.06 @L), streptomycin sulphate (0.1 g/L) and bovine serum albumin (BSA, 0.5 @). After stimulation for 48h, media were collected and stored frozen until analysis by RIA or electmphoresis and ligand blotting. Assavs, Conditioned media were assayed for IGFBP-3 by RIA as previously reported (13). SDS-nolva&lamide & electrw lip-, Conditioned media were concenuated 5-fold by ultrafiltration as previously described (14). Samples and molecular weight markers were prepared for electrophoresis by addition of concenuated sample buffer (15) and boiling for 5 min without reducing agent. Proteins were fractionated on 12 % SDSpolyacrylamide gels overnight at 80 V, then transferred electrophoretically to Hybond-C extra supported niuocellulose. IGFBPs were detected by ligand blotting with [t25I]IGF-II, essentially as previously described (16). In brief, nitrocellulose sheets were incubated for 3 h at 37 C in his-buffered saline (TBS, 10 mM uis, 150 mM NaCI, pH 7.4) containing 10 gjL BSA, then overnight at 4 C in - 1 x 106 cpm [tuI]IGF-II in TBS containing 10 g/L BSA and 0.05% Nonidet P-40. Blots were then rinsed three times in cold TBS buffer, once in TBS buffer containing 0.05% Nonidet P-40, and a further three times in detergent-free TBS. Hyperfilm-MP autoradiographic film (Amersham, Bucks, UK) was exposed to dried blots for I - 3 days at -70 C, then developed using standard procedures.
The insulin-like growth factor binding proteins (IGFBPs) are believed to play an important role in modulating the biological activity of IGF-I and IGF-II in cell growth and metabolism. Studies in vitro have demonstrated that purified preparations of IGFBP-1, IGFBP-2 and IGFBP-3 inhibit IGF action in a variety of cell types (1.2.3). presumably by sequestration of the growth factor before receptor interaction is possible. Purified preparations of IGFBPs have been shown to potentiate IGF actions by as yet unidentified mechanisms: pure IGFBP-3 enhances IGF-I activity in human and bovine fibroblasts in monolayer culture (3,4,5), while a potentiating form of IGFBP-1 has been isolated from human amniotic fluid (6.7). These findings suggest that IGFBPs produced by cells may profoundly affect paracrine or autccrine actions of the IGFs. In spite of widespread interest in the physiological role of IGFBPs, the regulation of production of these proteins by IGFtarget cells is still poorly understood. Transforming growth factor-gt (TGF~I) is a potent stimulator of IGFBP-3 in human neonatal fibroblast cultures (8). and the secretion of IGFBP-3 and other IGFBPs by these cells is altered in response to IGFs (9,lO). However, the possibility of regulatory interaction between different IGFBPs has not yet been examined. We have addressed this question, and found that IGFBP-3 markedly inhibits secretion of an IGF-inducible IGFBP by human fibroblasts.
ACKNOWLEDGEMENT
This work was supported by the National Research Council of Australia.
Received
in
Iowa
City,
June
15,
Health
and Medical
1992.
1568
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:04 For personal use only. No other uses without permission. . All rights reserved.
RAPID
COMMUNICATIONS
RESULTS and DISCUSSION We have previously demonstrated that treatment of human fibroblast cultures with IGF-I results in 8- to g-fold increased secretion of an IGFBP of 29-31 kDa, detected by SDS-PAGE and ligand blotting with [125I]IGF-II (9). IGFBP-3 is known to inhibit a number of IGF-I-stimulated events in fibroblasts (3,4,10); therefore, we examined the effect of addition of exogenous IGFBP-3 on production1 of fibroblast IGFBPs. Fibroblasts were incubated with IGF-I (50 @ml) in the presence of increasing concentrations (SO, 200 and 1000 &ml) of pure IGFBP3. Conditioned media were then analyzed by SDS-PAGE and ligand blotting. In the absence of exogenous IGFBP-3, IGFI-treated fibroblasts secreted three main IGF-binding species: IGFBP-3 doublet of -50 kDa, the 29-31 kDa doublet IGFBP, and a third IGFBP of 22-24 kDa (Figure la). As the concentration of exogenous IGFBP-3 in the medium was increased, the 29-31 kDa IGFBP disappeared, so that at a concentration of 200 rig/ml added IGFBP-3, the 29-31 kDa IGFBP was no longer detectable by ligand blotting. These results were consistent with IGFBP-3 inhibiting 29-31 kDa IGFBP production by fibroblasts. An apparent increase in the level of the 22-24 kDa IGFBP accompanied the decrease in the 29-31 kDa IGFBP in some experiments; however, this response was quite variable, suggesting that production of this IGFBP is subject to regulatory factors not yet known. b
67-
67-c
,
1569
concentrations of TGFpi (Figure lb). Concomitant with the increase in IGFBP-3, the 29-31 kDa IGFBP disappeared. The effect was noticeable at a concentration of 0.1 @ml TGFPl, and complete inhibition was apparent at 0.3 rig/ml TGFpl. The equivalent concentrations of immunoreactive IGFBP-3 in these media were 159.5k7.3 and 315.Okl6.8 rig/ml (mean&SE), respectively. These results suggested that increased endogenous production of IGFBP-3 inhibits production of IGFBPs. Although some inhibitory effects of IGFBP-3 on IGF action occur through sequestration of the growth factor, there are several reports of inhibitory actions occurring in the absence of added IGFs (6,7). While our results were consistent with the former mechanism of action, they did not preclude the possibility that IGFBP-3 or TGFPi had intrinsic inhibitory activity distinct from IGF binding-mediated inhibition. To distinguish between these possibilities, the effects of exogenous IGFBP-3 at concentrations up to 1000 &ml, and TGFPi to 3.0 @ml, were determined in the presence of 250 rig/ml IGF-I. At this level of IGF-I, the IGFBP-3 concentration would be insufficient to fully neutralize IGF bioactivity, and intrinsic IGFBP-3 or TGFplinhibitory activity would be reflected by persisting inhibition of 29-31 kDa IGFBP. Figure 2 shows the results of these studies. Addition of 50 or 200 rig/ml IGFBP-3 (final medium. concentrations of 107.3fl.8 and 235f9.5 n&ml IGFBP-3, respectively, by RIA) had no effect on the concentration of 29-31 kDa IGFBP in the medium, even though similar concentrations inhibited 29-31 kDa IGFBP in the presence of 50 rig/ml IGF-I (see Figure la). A slight inhibition of 29-31 kDa IGFBP was apparent at 1000 rig/ml IGFBP-3, suggesting that IGF-I activity was beginning to be affected by this concentration of IGFBP-3.
ix
3--' 43' / 0'
67-c
20*
0
50
IGFBP-3
200 1000 (w/ml)-
w. Inhibition of 29-31 kDa IGFBP by IGFBP-3 and TGFpl at low IGF-I concentrations. Media was conditioned by human fibroblasts in the presence of 50 rig/ml IGF-I, and the indicated concentrations of IGFBP-3 (panel a) or TGFPl (panel b). IGFBPs were analyzed by 12% SDS-PAGE, and IGF-II ligand blotting. Although IGFBP-3 purified from human serum is similar in size and IGF-binding characteristics to fibroblast IGFBP-3 (14), the ratio of the glycosylation forms appears different for the two (9,17). To determine whether increased endogenous production of IGFBP-3 resulted in the same inhibition of 29-31 kDa IGFBP, IGF-I-treated fibroblasts were exposed to TGFPl, which potently stimulates IGFBP-3 production by fibroblasts (8). Consistent with results obtained by RIA (not shown), ligand blot analysis of these media showed increased IGFBP-3 in response to increasing
20-9
0
10
50
IGFBP-3
200 1ooa (rig/ml)
Figure 2. Inhibition of 29-31 kDa IGFBP by IGFBP-3 and TGFpl in the presence of 250 rig/ml IGF-I. Media were analyzed as described for Figure 1. 1 In this report, a change in the “production” of a particular IGFBP refers to a change in the abundance of that protein in the conditioned medium as detected by SDS-PAGE and ligand blotting. Such changes may reflect a variety of events, including altered de nova synthesis, secretion or proteolysis of specific IGFBPs. Changes in production per se by measurement of mRNA have not been demonstrated.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 15:04 For personal use only. No other uses without permission. . All rights reserved.
RAPID
1570
COMMUNICATIONS
In contrast, equivalent concentrations of IGFBP-3 in the medium of TGFgt-stimulated fibroblasts (Figure 2b), effected by 0.3 rig/ml and 1 rig/ml TGFgt (103.2f6.6 @ml and 263.3f4.0 @ml, respectively) were accompanied by a marked decrease in 29-31 kDa IGFBP. To quantify these differences, autoradiographs from three experiments comparing the effects of IGFBP-3 and TGFgl in the presence of low and high IGF concentrations were scanned with a densitometer (Figure 3). In the presence of 50 &ml IGF-I, half-maximal inhibition of 29-31 kDa IGFBP was apparent with SO-60 rig/ml IGFBP-3; at 250 @ml IGF-I, IGFBP-3 was much less potent, with half-maximal inhibition requiring greater than 1000 rig/ml IGFBP-3 (Figure 3a). The potency of TGFgl was also lower in the presence of high IGF-I concentrations: the EDSO for 50 rig/ml and 250 &ml IGF-I were 0.05-0.06 rig/ml and 0.1-0.2 n&ml TGFpl, respectively (Figure 3b). These results suggest that whereas addition of pure IGFBP-3 inhibits 29-31 kDa IGFBP through binding of IGF-I, inhibition of this protein by TGFgl probably occurs via two distinct events: through sequestration of IGF by increased endogenous IGFBP-3 production, and by action independent of IGFBP-3. a Inhibition
by
10
100
IGFBP-3
100
Inhibition
0.1
by TGFP
1
TGFP (rig/ml) IGFBP-3 (nglml) Figure Densitometric analysis of autoradiographs. Ligand blots similar to those shown in Figs 1 and 2 were scanned, and the OD of the 29-31 kDa peak for IGFBP-3 or TGFgt-treated samples expressed as a percentage of the peak height in the presence of 50 @ml IGF-I ( ) or 250 @ml IGF-I ( ) without added IGFBP-3 or TGFpt.
Addition of exogenous IGFBP-3 to cultures of human or bovine fibroblasts has been shown to inhibit IGF-stimulated DNA synthesis when added with the growth factor, and potentiate IGF action if cells are preincubated with the binding protein prior to exposure to IGF-I (3,5). The mechanism by which such augmentation of IGF bioactivity occurs is not known. It has recently been suggested that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I may be involved in this potentiation (17). It is possible, however, that changes to endogenous IGFBP production occurring as a result of addition of exogenous IGFBP-3, may be important in the determining whether subsequent IGF action is inhibitory or stimulatory. Resolving such questions will prove important in achieving a full understanding of the role of IGFs and their binding proteins in cell growth.
Endo l 1992 Voll31. No 3
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