Studies on regulation of insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-4 production in human bone cells.

Previous studies have shown that the actions of IGF-II in bone are determined not only by its concentration, but also by the concentration of IGFBP-4 as well as other IGFBPs. In this study, we sought to determine by Western ligand blotting the effects of growth hormone, IGF-I and IGF-II on the production of IGFBP-3 and IGFBP-4 in TE89 human osteosarcoma cells and in untransformed normal human bone cells derived from rib. Human growth hormone at 10 micrograms/l decreased the amount of IGFBP-4 but had no effect on the IGFBP-3 level in the conditioned medium of low density cultures of TE89 cells and human bone cells derived from rib. Human growth hormone had no effect on IGFBP-3 or IGFBP-4 levels in the conditioned medium of high density human bone cell cultures. IGF-I and IGF-II, which increased human bone cell proliferation, decreased the level of IGFBP-4 (30% of control at 100 micrograms/l IGF-I and IGF-II) but increased the level of IGFBP-3 (3-10 fold at 100 micrograms/l IGF-I and IGF-II) after 48 h of treatment in the conditioned medium of both low and high density TE89 cell cultures. Similar changes in IGFBP-3 and IGFBP-4 levels were also seen in the conditioned medium of human bone cells derived from rib after treatment with IGF-I and IGF-II. Studies to determine the underlying molecular mechanisms by which IGF-II decreased the amount of IGFBP-4 in the conditioned medium revealed that IGF-II decreased the IGFBP-4 mRNA abundance and increased the IGFBP-3 mRNA abundance in human bone cells. Based on the above findings, we conclude that the production of both IGFBP-3 and IGFBP-4 is regulated in bone cells and that local and systemic agents may modulate the responsiveness of bone cells to IGFs by regulated secretion of IGFBP-3 and IGFBP-4.     

Insulin-like growth factor binding protein-4 proteolytic degradation in ovine preovulatory follicles: studies of underlying mechanisms.

The regulation of insulin-like growth factor binding protein (IGFBP)-4 proteolytic degradation by insulin-like growth factors (IGFs) has been largely studied in vitro, but not in vivo. The aim of this study was to investigate the involvement of IGFs, IGFBP-2, IGFBP-3, and IGFBP-3 proteolytic fragments in the regulation of IGFBP-4 proteolytic activity in ovine ovarian follicles. Follicular fluid from preovulatory follicles contains proteolytic activity degrading exogenous IGFBP-4. The addition of an excess of IGF-I enhanced IGFBP-4 proteolytic degradation, whereas addition of IGFBP-2 or -3 or monoclonal antibodies against IGF-I and -II dose dependently inhibited IGFBP-4 proteolytic degradation. IGF-I and IGF-II, but not LongR3-IGF-I, reversed this inhibition in a dose-dependent manner. C-terminal, but not N-terminal, proteolytic fragments derived from IGFBP-3 (aa 161-264), as well as heparin-binding domain-containing peptides derived from the C-terminal domain of IGFBP-3 and -5 also induced the inhibition of IGFBP-4 proteolytic degradation. Other heparin-binding domain-containing peptides derived from the connective tissue growth factor (CTGF) and from proteins not related to IGFBP, heparan/heparin interacting protein (HIP) and vitronectin, but not from p36 subunit of annexin II tetramer, inhibited IGFBP-4 degradation. Furthermore, IGFBP-3, mutated on its heparin-binding domain, was not able to inhibit IGFBP-4 proteolytic degradation. So, in ovine preovulatory follicles, IGFBP-4 proteolytic degradation both 1) depends on IGFs, and 2) is inhibited by IGFBP-3 via its C-terminal heparin-binding domain as well as by heparin-binding domain containing peptides. These data suggest that in early atretic follicles, the increase in IGFBP-2 participates in the decrease in IGFBP-4 degradation. In late atretic follicles, the increase in the levels of C-terminal IGFBP-3 proteolytic fragments, generated by IGFBP-3 degradation, as well as the increase in IGFBP-5 expression would strengthen the inhibition of IGFBP-4 degradation. This inhibition might be partly mediated by direct interaction of IGFBP-4 proteinase(s) and heparin-binding domain within the C-terminal region from IGFBP-3 and -5.     

Mutations in the NSD1 gene in patients with Sotos syndrome associate with endocrine and paracrine alterations in the IGF system

OBJECTIVE: To investigate the effect of nuclear receptor Su-var, 3-9, enhancer of zeste, trithorax (SET) domain-containing protein 1 (NSD1) gene alteration in patients with Sotos syndrome on plasma IGFs and IGF-binding proteins (IGFBPs), as well as on the IGF/IGFBP system activity at the tissue level. DESIGN: Twenty-nine patients suspected of Sotos syndrome were divided into two groups: patients with heterozygous deletions or mutations in the NSD1 gene (NSD1(+/-)) (n=11) and subjects without (NSD1(+/+)) (n=18). Plasma samples (n=29) and skin fibroblasts (n=23) were obtained. The results of both groups were compared and related to reference values. METHODS: IGF-I, IGF-II, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-6 levels were determined by RIAs. The mitogenic response of fibroblasts to IGFs was investigated by [methyl-(3)H]thymidine incorporation. IGFBP-3 levels in the culture media were measured by RIA. IGFBP-3 mRNA expression was determined by real time RT-PCR. RESULTS: NSD1(+/-) patients showed significantly altered levels of IGF-I (mean-1.2 SDS), IGF-II (-1.2), IGFBP-3 (-1.7), IGFBP-4 (-0.4), IGFBP-2 (+0.8) and IGFBP-6 (+1.5). The NSD1(+/+) patients did not differ from the reference, with the exception of the mean IGFBP-3 level (-1.3). Basal proliferation and mitogenic response to IGFs was diminished in NSD1(+/-) fibroblasts compared with NSD1(+/+) (basal, P=0.02; IGF-I, P<0.001; IGF-II, P=0.02). Compared with control fibroblasts, only the mitogenic response was diminished (basal, P=0.07; IGF-I, P=0.04; IGF-II, P=0.04). A trend of higher IGFBP-3 secretion after IGF-I stimulation (P=0.09) and 3.5-5 times higher mRNA expression of IGFBP-3 in basal conditions was found in NSD1(+/-) fibroblasts in comparison to controls. CONCLUSIONS: NSD1(+/-) patients show endocrine and paracrine changes in the IGF system. These changes may contribute to the abnormal growth pattern.     

Low serum levels of free and total insulin-like growth factor I (IGF-I) in patients with anorexia nervosa are not associated with increased IGF-binding protein-3 proteolysis

Patients with anorexia nervosa (AN) are GH resistant, with elevated GH levels and low serum levels of total insulin-like growth factor I (IGF-I). IGF-I action is modulated by IGF-binding proteins (IGFBPs), and a variety of catabolic states has been characterized by the presence of increased IGFBP-3 proteolysis. The present study was performed to examine the levels of free IGFs in AN and to clarify whether AN is associated with increased IGFBP-3 proteolytic activity. In 24 patients and 10 age-matched controls, the fasting serum concentrations of free IGF-I and -II were measured using ultrafiltration by centrifugation. In addition, GH, GH-binding protein, total IGFs, IGFBP-1 to -4, and IGFBP-3 proteolytic activity were measured. The IGFBPs were measured by both immunoassays and Western ligand blotting. Twelve of the patients were restudied 3 months after a minor increase in body mass index. In AN, the levels of GH-binding protein, free and total IGF-I, free IGF-II, and IGFBP-3 were significantly reduced; total IGF-II, IGFBP-2, and IGFBP-4 levels were unchanged; and IGFBP-1 was increased. No increased IGFBP-3 proteolytic activity could be detected in AN. In conclusion, the mechanisms responsible for the adaption of the GH-IGF-IGFBP axis in AN may be different from other catabolic conditions, because the low levels of free and total IGF-I in AN are not associated with increased IGFBP-3 proteolysis.     

Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 release IGF-binding protein-3 from human fibroblasts by different mechanisms.

Human neonatal fibroblasts in monolayer culture secrete insulin-like growth factor-binding proteins (IGFBPs), which may modulate IGF action. To examine whether an increase in extracellular concentrations of IGFBPs in response to IGF-I is due to the release of cell-associated IGFBPs, we measured secreted and cell-associated IGFBP-3 immunologically in fibroblast monolayers treated with IGF-I and IGF analogs with altered affinities for the IGF receptors and IGFBPs. IGFBP-3 in medium conditioned by fibroblasts treated with IGF-I was significantly increased (P < 0.05) compared with that in medium from untreated cultures; concomitantly, cell-associated IGFBP-3 was significantly decreased (P < 0.05). [Ser24]IGF-I (reduced affinity for IGF receptors) also increased secreted IGFBP-3 and decreased cell-associated IGFBP-3. In contrast, IGFBP-3 concentrations in medium conditioned by fibroblasts treated with B-chain IGF-I (reduced affinity for IGFBPs) were not significantly increased, and cell-associated IGFBP-3 was unchanged. Heparin, which releases proteins attached to cell surface proteoglycans, increased medium concentrations of IGFBP-3 and decreased IGFBP-3 binding to fibroblasts. An IGFBP of 29-31 kilodaltons (kDa) showed a pattern of regulation similar to that of IGFBP-3, while a third IGFBP, of 24 kDa, was decreased in IGF-I- and [Ser24]IGF-I-conditioned medium and unchanged by B-chain IGF-I and heparin. Preincubation with transforming growth factor-beta 1 (TGF beta 1), which stimulates fibroblast IGFBP-3 production, or human serum-derived IGFBP-3 did not increase cell-associated IGFBP-3. Analysis of total RNA isolated from fibroblasts revealed that IGFBP-3 mRNA was increased by TGF beta 1, but not by IGF-I. These data suggest that IGFs and TGF beta 1 release fibroblast IGFBPs by distinct mechanisms: IGFs by binding and subsequent release of cell-associated IGFBP-3 and 29- to 31-kDa IGFBP, and TGF beta 1 by increased de novo synthesis of IGFBP-3.     

Control of insulin-like growth factor binding protein-5 protease synthesis and secretion by human fibroblasts and porcine aortic smooth muscle cells

IGF binding protein-5 (IGFBP-5) is an important trophic factor for controlling the actions of IGF-I in human dermal fibroblasts and porcine aortic smooth muscle cells. When IGFBP-5 is associated with extracellular matrix, it acts to enhance the cell growth response to IGF-I. The amount of IGFBP-5 within the extracellular matrix is related in part to the amount that is present in conditioned medium, which is related to its rate of synthesis and degradation. A serine protease that degrades IGFBP-5 is present in the conditioned medium of both of these cell types. Because the IGFBP-5 protease activity that is secreted by fibroblasts has been shown to be due to the complement components C1r and C1s, these studies were undertaken to determine whether smooth muscle cells also secreted these proteases and to identify some of the factors that regulate their secretion by both cell types. Both smooth muscle cells and human fibroblasts were shown to release C1r and C1s into conditioned medium. Both C1r and C1s were detected as activated forms, as determined by SDS-PAGE using reducing conditions. The addition of increasing concentrations of either IL-1beta or TNFalpha resulted in increased synthesis of C1s by fibroblasts and smooth muscle cells, and they each increased C1r release. TNFalpha (50 ng/ml) and IL-1beta (20 ng/ml) resulted in maximum stimulation of release of both proteases. In contrast dexamethasone (10(-7) M) had no effect on C1s release and stimulated C1r release only by smooth muscle cells. To determine the physiological significance of this increase in C1r and C1s, the amount of IGFBP-5 protease activity that was present in conditioned medium was determined before and after exposure to TNFalpha, IL-beta, and dexamethasone. All three compounds resulted in an increase in the amount of IGFBP-5 proteolytic activity. Dexamethasone inhibited the release of C(1) inhibitor from fibroblasts, and this contributed to the net increase in proteolytic activity. TNFalpha inhibited the smooth muscle cell DNA synthesis response to IGF-I, but the effect of IGF-I was partially restored by the addition of C1 inhibitor. In conclusion, both C1r and C1s are released by cultured fibroblasts, and the release of each into fibroblast or porcine smooth muscle cells medium is stimulated by TNFalpha and IL-1beta. This increase results in a net increase in IGFBP-5 proteolysis, which has the potential to modify IGF-I and IGFBP-5 actions.     

The induction of a specific protease for insulin-like growth factor binding protein-3 in the circulation during severe illness.

The insulin-like growth factors (IGF-I and IGF-II) are almost completely bound in the circulation to specific binding proteins (IGFBPs). These IGFBPs appear to play a pivotal role in maintaining circulating levels and modulating the delivery of the IGFs to the tissues. A large proportion of the circulating IGFs are bound with high affinity to one of the binding proteins. IGFBP-3. The mechanism by which these IGFs are transferred from the circulatory pool to the tissue receptors is at present unclear. Recent studies in late pregnancy have demonstrated the presence of specific proteases which may modify the IGFBPs such that their affinities for the IGFs are reduced. In this paper, we have demonstrated the presence of a heat-sensitive cation-dependent proteolytic enzyme specific for IGFBP-3 in the serum of five severely ill patients. The activity of this protease was found to vary in these patients, becoming more apparent during fasting than when studied after commencement of parenteral nutrition, indicating that one of the influencing factors in the activity of this protease is the nutritional intake of the patient. Age- and sex-matched healthy adults were also studied in a similar protocol, but no proteolytic modification of any of the IGFBPs was found in any of the samples examined. As the levels of both IGF-I and IGF-II were found to be low in the patients, the presence of a circulatory protease suggests that this may be an adaptive response to increase the bioavailability of the IGFs and possibly to improve the nitrogen retention and counter the catabolic state in severe illness.     

Pregnancy-associated plasma protein-a is the insulin-like growth factor binding protein-4 protease secreted by human ovarian granulosa cells and is a marker of dominant follicle selection and the corpus luteum

Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and IGFBP proteases are important in ovarian function. IGFs stimulate granulosa steroidogenesis, an effect that is inhibited by IGFBP-4 and augmented by IGFBP-4 proteolysis. We have recently identified the IGFBP-4 protease in human ovarian follicular fluid (FF) as pregnancy-associated plasma protein-A (PAPP-A). In the current study, we identify the IGFBP-4 protease secreted by cultured human ovarian granulosa cells as PAPP-A, based on specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with PAPP-A polyclonal antibodies and immunorecognition by PAPP-A monoclonal antibodies in ELISA. PAPP-A was barely detectable in conditioned media (CM) from granulosa derived from /=9 mm, coincident with dominant follicle selection, and by luteinizing granulosa. PAPP-A levels in CM from the latter did not change in response to IGF-II or hCG (100 ng/mL). A naturally occurring inhibitor of PAPP-A, proform of eosinophil major basic protein (proMBP), was detected by ELISA in estrogen-dominant follicular fluid FF, but not in CM from granulosa or luteinizing granulosa cells treated with IGF-II (0-200 ng/mL), FSH (0-100 ng/mL) or hCG (0-100 ng/mL), suggesting an alternative source (other than granulosa) for proMBP, compared to PAPP-A. The data demonstrate granulosa cells as a source of PAPP-A in human ovary and suggest that PAPP-A is a marker of ovarian follicle selection and corpus luteum formation. In addition the data suggest complex regulation of this system in human ovary.     

Pregnancy-associated plasma protein-A accounts for the insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) proteolytic activity in human pregnancy serum and enhances the mitogenic activity of IGF by degrading IGFBP-4 in vitro

Pregnancy-associated plasma protein-A (PAPP-A) has been identified as the insulin-like growth factor (IGF)-dependent IGF-binding protein-4 (IGFBP-4) protease produced by human fibroblasts. Recently, we found that serum proteases induced during human pregnancy cleaved IGFBP-4 in both an IGF-II-dependent and an IGF-II-independent fashion. This study sought to determine whether PAPP-A is the predominant IGFBP-4 protease in human pregnancy serum (PS) and to assess the in vitro role of serum PAPP-A. Immunoprecipitation with PAPP-A antibody effectively depleted PAPP-A from the PS and completely abolished both IGF-II-dependent and IGF-II-independent IGFBP-4 proteolytic activity in PS. Direct addition of PAPP-A antibody to PS completely blocked IGFBP-4 proteolysis and partially blocked IGFBP-5 proteolysis, but had no effect on IGFBP-3 proteolysis. To evaluate the role of serum PAPP-A, we tested whether PAPP-A in PS modulated the inhibitory activity of IGFBP-4 on IGF-II-induced cell proliferation in human osteosarcoma MG63 cells. The wild-type IGFBP-4 (WTBP-4; 200 ng/mL) failed to inhibit proliferation of the cells treated with PS (0.1% or 0.3%) alone or in combination with IGF-II (40 ng/mL), whereas the inhibitory effect of WTBP-4 was observed in the cells treated with nonpregnancy serum alone or in combination with IGF-II (P < 0.05). In contrast to WTBP-4, a protease-resistant IGFBP-4 was able to inhibit proliferation of the cells treated with PS alone or in combination with IGF-II (P < 0.05). In the presence of PAPP-A neutralizing antibody, the inhibitory effect of WTBP-4 on proliferation of the cells treated with IGF-II and PS was restored. In summary, these data demonstrate 1) that PAPP-A represents the predominant IGFBP-4 protease in PS; 2) that PAPP-A may in part contribute to IGFBP-5, but not IGFBP-3, proteolytic activity in PS; and 3) that PAPP-A enhances the bioactivity of IGFs in vitro by degrading IGFBP-4.     

Sheep insulin-like growth factors I and II: sequences, activities and assays

This report describes the purification, sequences, and activities of insulin-like growth factors (IGFs) from adult and fetal sheep plasma. IGF-1 from adult sheep is identical to human and bovine IGF-I, except for substitution in the sheep of Ala at residue 66 for Pro in the human and bovine polypeptides. IGF-II from adult sheep differs from bovine IGF-II also by a single amino acid, with residue 62 being Ala in ovine and Thr in bovine IGF-2. The first 10 amino-terminal residues of fetal sheep plasma IGF-I and 92% of the amino acids of fetal IGF-II were identified and found to be the same as those of the corresponding IGFs isolated from adult sheep. Ovine IGF-I was virtually equipotent with human IGF-I in growth-related bioassays and in a RIA for human and bovine IGF-I and inhibited the binding of radiolabeled human IGF-I to type I IGF receptors and to a pure IGF-binding protein. Ovine and bovine IGF-II were also found to be similar to each other in biological and immunochemical activities, and in their binding to type I and II IGF receptors and IGF-binding protein. As observed with human and bovine IGF-I and IGF-II, ovine IGF-I bound slightly better to type I IGF receptors than ovine IGF-II, but bound very poorly to type II IGF receptors. This study shows that IGFs from sheep are very similar to those of human and bovine in structure and activity and defines sensitive radioligand assays specific for ovine IGF-I and ovine IGF-II.     

Testosterone and insulin-like growth factor (IGF) I interact in controlling IGF-binding protein production in androgen-responsive foreskin fibroblasts

The growth of the male external genitalia is primarily regulated by androgens. However, human genital fibroblast growth is also stimulated by insulin-like growth factor (IGF) I. In this study, we report that IGF-binding protein (IGFBP) production in human foreskin fibroblasts is regulated by androgens and IGF-I. Human foreskin fibroblasts secrete IGFBP-3, IGFBP-4, and IGFBP-5. IGF-I increased the abundance of both intact IGFBP-3 and -5 in the culture medium. Testosterone increased IGFBP-3, and the combination of IGF-I and testosterone had an additive effect. Following its secretion, IGFBP-5 was degraded, but the effect of IGF-I on IGFBP-5 peptide abundance in conditioned media did not seem to be due to inhibition of proteolysis. Testosterone had no effect on IGFBP-5 degradation. Intact IGFBP-4 was decreased by IGF-I, and the combination resulted in a similar reduction. The mechanism seemed to be decreased synthesis, since IGFBP-4 messenger RNA was also decreased. The increase in IGFBP-5 synthesis was associated with an increase in the abundance of intact IGFBP-5 in the extracellular matrix. The combination of testosterone and IGF-I resulted in a synergistic stimulation of total protein synthesis by the fibroblast cultures, suggesting that a maximum anabolic response requires both hormones. These observations suggest that combined exposure to androgen and IGF-I altered the abundance of some forms of IGFBPs and that the IGFBPs that are regulated may play a role in modulating the effects of IGF-I on the anabolic response.     

A new radioimmunoassay to measure rat insulin-like growth factor binding protein-4 (IGFBP-4) in serum, wound fluid and conditioned media.

Insulin-like growth factor binding protein-4 (IGFBP-4) is, like the other five IGFBPs, a critical regulator of the activity of insulin-like growth factor-I (IGF-I) and IGF-II. Because of the possible role of IGFBP-4 in multiple systems including reproduction, pregnancy, bone formation and wound healing, we developed a radioimmunoassay (RIA) to measure the concentration of IGFBP-4 in the serum, extracellular fluid and conditioned media of cells of rodents so that these animal models could be better exploited. Rat IGFBP-4 was expressed in Escherichia coli and purified to homogeneity. The recombinant material was used to raise a rabbit polyclonal antibody against rat IGFBP-4 and for iodination and standards. The RIA developed was sensitive to less than 1 ng/mL of rat IGFBP-4 and IGF-I did not interfere. There was no cross-reactivity with other rat IGFBPs on immuno-Western analysis of serum and wound fluid. IGFBP-4 from mouse serum did cross-react in our assay; however, serum from horse, pig or human showed no cross-reaction. Human IGFBP-1 and IGFBP-3 showed a very weak cross-reactivity. Serum IGFBP-4 levels showed no gender difference but did reveal a significant 66% increase in older rats. During the course of wound healing, which is IGF-I dependent, IGFBP-4 showed no changes. In conclusion an RIA for rat IGFBP-4 has been developed that specifically measures the concentration of IGFBP-4 in the sera, extracellular fluid and conditioned media of rodents. With this assay, the role of IGFBP-4 and IGFs in growth, development and the function of multiple systems can be further investigated using rodent models.     

An autocrine/paracrine role for insulin-like growth factors in the regulation of human placental growth

Tissue derived from preterm (9-19 weeks gestation) and term (38-41 weeks gestation) human placentae were examined for their ability to synthesize and secrete insulin-like growth factors (IGFs) in organ culture. IGF-I was measured by a specific RIA, and IGF-II by a rat placental membrane radioreceptor assay. First, explants of placental tissue were maintained in organ culture. These explants secreted immunoreactive IGF-I (IR-IGF-I). There were no differences in the IR-IGF-I content of media conditioned by term and preterm placentae under these conditions. The similarity of this material to authentic human IGF-I was supported by parallel displacement in a specific RIA and coelution during Sephadex G-50 gel filtration. Second, monolayer cultures of fibroblasts from normal human preterm placentae (15-19 weeks gestation) were established. Confluent monolayers of these fibroblasts secreted IR-IGF-I (3-10 pg/10(5) cells X 40 h). IR-IGF-I secretion was reversibly inhibited by 5.3 microM cycloheximide, suggesting that the IR-IGF-I was the result of de novo protein synthesis. IR-IGF-I secretion was stimulated 5-fold by platelet-derived growth factor (0.6 U/ml). The response of monolayers of placental fibroblasts to IGF-I also was tested. IGF-I stimulated alpha-[3H]aminoisobutyric acid transport in these fibroblasts, with half-maximal stimulation occurring at 2-3 ng/ml. Stimulation of alpha-[3H]aminoisobutyric acid uptake by IGF-I correlated with specific binding of [125I]iodo-IGF-I. Half-maximal inhibition of [125I]iodo-IGF-I binding occurred at 2-3 ng/ml IGF-I. Placental tissue also secreted IGF-II-like activity, as measured by radioreceptor assay. Media conditioned by placental explants contained 15-20 ng/mg protein X 48 h, and media conditioned by placental fibroblasts contained 3-7 ng/10(5) cells X 40 h IGF-II determined by radioreceptor assay. These data support the hypothesis that the human placenta produces IGFs (IGF-II and/or IGF-I) that act locally to regulate placental growth.     

Transforming growth factor-beta stimulates production of insulin-like growth factor-binding protein-3 by human skin fibroblasts.

Human neonatal fibroblasts in monolayer culture produce insulin-like growth factor-binding protein-3 (IGFBP-3), the IGF-binding subunit of the circulating 140-kDa IGFBP complex. We now report that transforming growth factor-beta (TGF beta) is a potent stimulator of IGFBP-3 production by fibroblasts. After 72-h incubation with 1 ng/ml TGF beta, the levels of IGFBP-3 in conditioned medium were increased 5.8 +/- 1.2-fold (mean +/- SE; n = 9). Half-maximal stimulation of IGFBP-3 production was seen at 0.4 +/- 0.05 ng/ml TGF beta (n = 4). Coincubation of fibroblasts with TGF beta and either IGF-I or IGF-II at 50 ng/ml enhanced IGFBP-3 production 1.5- to 2-fold compared to TGF beta alone. As previously reported, fetal calf serum (FCS) stimulated IGFBP-3 production 5- to 6-fold; 1 ng/ml TGF beta increased the stimulated production of IGFBP-3 by FCS a further 2.5- to 3.5-fold. Acidification of FCS before addition enhanced the stimulation of IGFBP-3 compared to that caused by untreated FCS, but decreased further potentiation by TGF beta. This effect of acidified FCS was reversed by a neutralizing antibody to TGF beta. Similarly, the stimulation of IGFBP-3 levels by human serum or conditioned serum-free fibroblast medium was significantly increased by acidification of serum or medium before addition and was reversed by TGF beta antibody. These observations are consistent with acid-mediated activation of latent TGF beta added in serum or secreted by fibroblasts. Since IGFBP-3 is known to regulate IGF activity in fibroblasts, these results raise the possibility that TGF beta may modulate IGF actions in these cells by stimulating the production of IGFBP-3.     

Insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) concentration and stimulate IGFBP-3 independently of IGF receptors in human fibroblasts and epidermal cells.

Recent studies have provided a consensus that insulin-like growth factor-I (IGF-I) stimulates IGF-binding protein-3 (IGFBP-3) in vivo and in vitro. While it also appears well established that IGFBP-1 is inversely related to insulin concentrations, evidence regarding regulation of other IGFBP is inconclusive. Using immunoprecipitation and Western ligand blot, we have characterized the IGFBPs released into conditioned medium (CM) by cells from the adult human fibroblast cell line N3652 and the human epidermal squamous cell carcinoma line SCL-1. N3652 cells expressed IGFBP-3, IGFBP-2, a 24-kilodalton (kDa) IGFBP presumed to be IGFBP-4, and IGFBPs at 30 and 28 kDa. SCL-1 expressed IGFBP-3 and a putative IGFBP-4, with intermediate bands at 34 and 30 kDa. As determined by ligand blot of CM from confluent cells 72 h after the addition of peptides to serum-free medium, IGF-I and IGF-II potently stimulated IGFBP-3 in both cell lines, but otherwise IGFBP regulation in the two cells diverged. In N3652 cells, IGFBP-3 concentrations in CM increased to 700% and 800% of basal levels in the presence of IGF-I and IGF-II (at 100 ng/ml; n = 5 experiments), respectively. IGFBP-3 was not affected by insulin up to 10 micrograms/ml. In contrast, IGFBP-4 levels were diminished 54% and 73% by 100 ng/ml IGF-I and IGF-II, respectively, with no response to insulin. In SCL-1 cells, IGF-I and IGF-II were virtually identical in stimulating a mean 200% increase in IGFBP-3 (n = 5 experiments). Insulin was less potent, but caused a significant stimulation of IGFBP-3 levels. IGF-I, IGF-II, and insulin all stimulated an approximately 50% increase in IGFBP-4 concentrations. To test the hypothesis that IGF-induced alterations in IGFBP-3 and IGFBP-4 concentrations were regulated via the type 1 IGF receptor, we attempted to block IGFBP changes with type 1 IGF receptor antibody alpha IR-3 and to induce IGFBP changes with an IGF-II analog, [Leu27]IGF-II, with little affinity for the type 1 receptor. alpha IR-3 failed to block either the IGF-induced rise in IGFBP-3 in each cell line or the decline in IGFBP-4 in N3652 CM. [Leu27]IGF-II was as potent as IGF-II or IGF-I in inducing changes in IGFBP-3 and IGFBP-4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization and partial purification of the insulin-like growth factor (IGF)-dependent IGF binding protein-4-specific protease from human fibroblast conditioned media.

Insulin-like growth factors (IGFs) are one of the most potent stimulators of cell growth. IGFs are modulated by six high affinity binding proteins (IGFBPs) which are, in turn, regulated through post-translational modifications such as proteolysis. In the conditioned media of human fibroblasts, IGFBP-4 is cleaved by an apparently novel IGFBP-4-specific protease that requires IGF for functional activity. We have used several biochemical manipulations, including size exclusion chromatography, native gel electrophoresis, chaotropic salt precipitation, hydrophobic interaction chromatography, ion exchange chromatography, isoelectric focusing, lectin affinity chromatography, and metal chelating affinity chromatography to both characterize and partially purify the IGF-dependent IGFBP-4 protease. Our results indicate that this protease is a highly glycosylated, Zn+2 binding metalloprotease with a native molecular weight greater than 200 kDa.     

The divergent effect of insulin-like growth factor binding protein (IGFBP) - 1 on IGF-induced Steroidogenesis in bovine adrenocortical cells is not due to its phosphorylation status.

In previous studies we have shown that insulin-like growth factor (IGF)-II stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I. The steroidogenic effect of both, IGF-I and IGF-II, is mediated through the IGF-I receptor and modified by locally produced IGF binding proteins. Further studies demonstrate that bovine adrenocortical cells do synthesize IGFBP-1 to -4 and that their secretion is regulated differentially by IGFs and ACTH. Since IGFBP-1 selectively is induced 3- to 4- fold by ACTH, the aim of the following studies was to evaluate the effect of IGFBP-1 on IGF-induced cortisol secretion from bovine adrenocortical cells in primary culture. Coincubation of bovine adrenocortical cells with purified IGFBP-1 (30 nM) induced a time--and dose-dependent potentiating effect (38+/-2%) on IGF-I-stimulated (6.5 nM) cortisol-secretion, wheras the IGF-II induced steroidogenic effect was inhibited (40+/-3%) by IGFBP-1. Similar results were found when bovine adrenocortical cells were preincubated with IGFBP-1 before stimulation experiments with IGFs were performed. In order to evaluate the influence of different phosphorylation states of IGFBP-1 on the steroidogenic effect of IGF-I and IGF-II and on the affinity to IGFs, a highly phosphorylated (pIGFBP-1) and a dephosphorylated isoform (dIGFBP-1) of IGFBP-1 have been separated by anion exchange chromatography for further incubation experiments and binding studies. No different effects on IGF-I or IGF-II-induced steroidogenesis were observed when a highly phosphorylated IGFBP-1 isoform was used, compared to the dephosphorylated IGFBP-1 isoforms. In binding studies IGFBP-1 did show high affinity binding for IGF-I with a calculated association constant (K (a)) of 2,17 x 10 (9) M (-1). Similar association constants were calculated for dIGFBP-1 and pIGFBP-1 (1,93 x 10 (9) M (-1) and 2,67 x 10 (9) M (-1) respectively) and no difference in binding capacity was observed when IGF-II was used as ligand. IN CONCLUSION, these results demonstrate that in bovine adrenocortical cells IGFBP-1 time- and dose-dependently inhibits the steroidogenic effect of IGF-II and stimulates the effect of IGF-I. The observed modulatory effect of IGFBP-1 is independent of the mode of incubation and its phosphorylation status. The previously observed stronger steroidogenic potency of IGF-II in bovine adrenocortical cells therefore can not be explained by an interaction with IGFBP-1. The mechanisms by which IGFBP-1 divergently regulates the steroidogenic effect of IGF-I and IGF-II remain unclear at present and further investigation is necessary to elucidate the mechanisms by which IGFBP-1 modulates IGF action in the adult adrenal gland.     

Post-transcriptional and post-translational regulation of insulin-like growth factor binding protein-3 and -4 by insulin-like growth factor-I in uterine myometrial cells.

Involution of the uterus induced by oestrogen depletion is associated with a decrease in uterine insulin-like growth factor (IGF)-I and an increase in IGF binding protein (IGFBP) gene expression. We examined the effects of IGF-I on primary uterine myometrial cell proliferation, and on IGFBP-3 and IGFBP-4 gene expression. IGF-I enhanced DNA synthesis in these cells. In conditioned media, IGF-I increased IGFBP-3 accumulation by release of cell associated IGFBP-3. A low dose of IGF-I increased IGFBP-4 accumulation, and a high dose caused IGFBP-4 to disappear. In cell-free conditioned media IGF-I protected IGFBP-3 and enhanced IGFBP-4 proteolysis. Co-incubation of [(125)I]-IGFBP-4 with cell-free conditioned media cleaved IGFBP-4 into 18 and 12 kDa fragments. Northern blot analysis indicated that IGF-I increased IGFBP-4 mRNA accumulation by stabilizing the mRNA while IGFBP-3 gene expression was slightly decreased. The results demonstrate that IGF-I regulates IGFBP-4 post-trancriptionally and post-translationally, whereas IGFBP-3 is only affected post-translationally. By enhancing IGFBP-4 proteolysis, increasing cell-associated IGFBP-3 and stabilizing IGFBP-3, IGF-I may initiate a mitogenic response.