Regulation of proliferation of prostate epithelial cells by 1,25-dihydroxyvitamin D3 is accompanied by an increase in insulin-like growth factor binding protein-3

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to regulate the proliferation of human prostate epithelial cell lines. Since the insulin-like growth factor (IGF) system is involved in the transformation process of epithelial cells, the following study was undertaken to determine if the IGF system, in particular IGF binding protein-3 (IGFBP-3), is altered by 1,25-(OH)2D3 in normal prostate epithelial cells as part of a mechanism for inhibition of transformation. Two cell systems were used in this study: (1) primary cultures of benign human prostate epithelial cells (PECs) and (2) an SV40-T immortalized prostate epithelial cell line (P153) that is non-tumorigenic. 1,25-(OH)2D3 was added to parallel sets of PECs and P153 cells in addition to the presence or absence of IGF-I or des(1-3)IGF-I. Treatment with 1,25-(OH)2D3 resulted in significant growth inhibition of both PECs and P153 cells. Furthermore, 1,25-(OH)2D3 inhibited IGF-induced proliferation, but this was partially reversed by high concentrations of IGF-I. Western ligand blots of condition media demonstrated a significant increase in IGFBP-3; likewise Northern blots demonstrated an increase in mRNA for IGFBP-3. Proliferation assays using an antibody designed to block the IGF-independent effects of IGFBP-3 failed to reverse the inhibitory effect of 1,25-(OH)2D3. Thus, IGFBP-3 acts in an IGF-dependent manner to inhibit cell growth of benign prostate epithelial cells.     

1,25-Dihydroxyvitamin D3 stimulates the production of insulin-like growth factor-binding proteins-2, -3 and -4 in human bone marrow stromal cells

BACKGROUND: 1,25-Dihydroxyvitamin D3 (calcitriol) inhibits proliferation and stimulates differentiation of multiple cell types, including osteoblasts. Human (h) bone marrow stromal cells (MSCs) are a homogenous non-hematopoietic population of cells present in the bone marrow and exhibit a less differentiated osteoblastic phenotype. The IGF system, including IGFs-I, and -II and IGF binding proteins (IGFBPs), plays an important role in osteoblast cell proliferation and differentiation. OBJECTIVE: To examine the pattern of expression of the IGF system in hMSCss and its regulation by calcitriol. METHODS AND RESULTS: hMSCs express mRNA of both IGFs-I, and -II and IGFBPs-1 to -6 as shown by RT-PCR and northern blot analysis. As assessed by western ligand blotting (WLB) and western immmunoblot analysis, hMSCs secrete 38-42 kDa IGFBP-3, 24-28 kDa IGFBP-4 and a 33 kDa IGFBP-2. Calcitriol (dose range 10-10 mol/l) exerted no consistent dose-dependent effects on either IGF-I or IGF-II mRNA levels. In contrast, calcitriol treatment increased steady-state mRNA levels of IGFBPs-2, -3 and -4, but had no effect on IGFBP-5 or -6. Similarly, calcitriol increased the secretion of IGFBPs-2, -3 and -4 as determined by WLB. We found no detectable basal IGFBP-3 or IGFBP-4 protease activities in the absence or presence of calcitriol treatment. CONCLUSIONS: Our results demonstrate that hMSCs expressed a distinct pattern of IGFs and IGFBPs that may be related to their stage of differentiation. The observed increase in production of IGFBPs-2, -3 and -4 by hMSCs upon treatment with calcitriol may be an important mechanism mediating the effects of calcitriol on MSC proliferation and differentiation.     

Regulation of insulin-like growth factor binding protein synthesis and secretion in a bovine epithelial cell line.

The Madin-Darby bovine kidney cell line was used to examine regulation of insulin-like growth factor binding protein (IGFBP) synthesis by epithelial cells. Ligand and immunoblot analysis of conditioned media indicated that IGFBP-2 was the predominant IGFBP secreted by untreated cells. Treatment with forskolin decreased secretion of IGFBP-2 by 75 +/- 3% and induced the appearance of IGFBP-3 and 24,000 Mr IGFBP. Although insulin alone did not induce the appearance of either band, in the presence of forskolin it increased the IGFBP-3 and 24,000 Mr bands 4.2 +/- 1.1 and 7.3 +/- 0.9-fold, respectively, above the values for forskolin treatment alone. Exposure to forskolin resulted in a 3-fold decrease in the abundance of IGFBP-2 messenger RNA (mRNA), and a 30-fold increase in IGFBP-3 mRNA. An additional 2- to 3-fold increase in IGFBP-3 mRNA was observed when cells were treated with insulin plus forskolin. Treatment with insulin plus forskolin increased cell number 2-fold, compared to small increases (26%) observed with forskolin treatment alone. Since treatment with IGF-I or -II did not result in similar responses to those of insulin, IGF analogs with differing affinities for IGFBP and IGF type I receptor were tested. B-chain IGF-I (decreased affinity for IGFBP) increased cell number and enhanced forskolin's effects on IGFBP-3 secretion and mRNA abundance to the same extent as insulin, whereas [Leu24,1-62]IGF-I (decreased affinity for the type I IGF receptor) did not. Therefore, activation of the type I IGF receptor was required to elicit increases in cell number and IGFBP synthesis and secretion, and the actions of IGF-I and II were likely blocked by binding to the large amounts of IGFBP-2 that were secreted. These results are in direct contrast to studies with human fibroblasts in which IGF-I and [Leu24,1-62]IGF-I stimulate IGFBP-3 secretion, whereas B-chain IGF-I has only a minimal effect. The ability to differentially regulate secretion of different forms of IGFBPs by epithelial cells and the finding that regulation is distinct from that of fibroblasts may have important implications for understanding mechanisms by which IGFs and IGFBPs interact to regulate epithelial cell growth.     

Insulin and insulin-like growth factors (IGFs) stimulate production of IGF-binding proteins by ovarian granulosa cells.

Ligand blot analysis of granulosa cell (GC)-conditioned culture medium revealed several easily measurable insulin-like growth factor (IGF)-binding proteins (IGFBPs), including IGFBP-3 [40-44 kilodaltons (kDa)] and IGFBP-2 (34 kDa). In the present study, IGF-I, in a dose-dependent manner, significantly stimulated the production of these IGFBPs. Insulin, but not IGF-II, mimicked IGF-I's action on IGFBP-3 and -2 production, but was less potent. The synthetic IGF, long R3-IGF-I, which has very low affinity for IGFBPs and only slightly reduced affinity for the IGF-I (type I) receptor, had significantly greater potency in stimulating IGFBP-3 and -2 production compared to IGF-I. Des-(1-3)-IGF-I had similar effects. IGF-I, IGF-II, and the IGF-I analogs, but not insulin, also induced production of an unidentified 30-kDa IGFBP not normally detectable in these cultures. However, in the presence of epidermal growth factor (which was without independent effect on the 30-kDa IGFBP), insulin also induced this 30-kDa IGFBP. By Northern analysis the expression of IGFBP-3 mRNA was found to be significantly stimulated by IGF-I. In summary, insulin stimulated IGFBP-3 and -2 production in a manner that mimics that of IGF-I and the more potent long R3-IGF-I. However, its low potency suggested that IGFBP production is regulated via the IGF-I (type I) receptor. The much higher potency of long R3-IGF-I compared to that of IGF-I suggests that the IGFBPs themselves modulate the action of IGFs by sequestering exogenous IGFs. Thus, one cellular response to IGF stimulation is the production of IGFBPs, which, in turn, reduce or negate the biological activity of the IGFs. The effects of insulin-like peptides are exerted at least in part by increasing levels of mRNA for specific BPs.     

Serum levels of insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 to -6 and their relationship to bone metabolism in osteoporosis patients

BACKGROUND: Insulin-like growth factor (IGF) system components are important regulators of bone formation. Alterations of individual IGF system components have been described in osteoporosis (OP) patients; however, no study has addressed changes in free IGF-I and in all six IGF binding proteins (IGFBPs). METHODS: A cross-sectional study was performed in 45 OP patients and 100 healthy matched controls. Serum levels of free and total insulin-like growth factor I (IGF-I), IGFBP-1 through -6, intact parathyroid hormone (PTH), 25-OH-vitamin D(3) (25OHD(3)), 1,25-(OH)(2)-vitamin D(3) (1,25-(OH)(2)D(3)), osteocalcin (OSC), bone alkaline phosphatase (B-ALP), and carboxyterminal propeptide of type-I procollagen (PICP) were measured with specific assays. Bone mineral density (BMD) of the lumbar spine was determined by dual-energy X-ray absorptiometry (DEXA). RESULTS: Compared with age- and sex-matched control subjects, OP patients showed a 73% decrease in free IGF-I, a 29% decrease in total IGF-I, a 10% decrease in IGFBP-3, and a 52% decrease in IGFBP-5 levels; they had higher levels of IGFBP-1 (4.1-fold), IGFBP-2 (1.8-fold), IGFBP-4 (1.3-fold), and IGFBP-6 (2.1-fold). Alterations in IGF system components were most evident in 13 OP patients with vertebral fractures in the past 4 years compared to patients without fractures. In OP patients with fractures, the ratio between IGFBP-4 and IGFBP-5 was increased whereas levels of OSC were decreased. CONCLUSIONS: Our data provide strong indirect evidence for a functional connection between circulating IGF system components and bone metabolism and the susceptibility to fractures in OP patients.     

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.     

Production of insulin-like growth factor binding proteins (IGFBPs) by porcine granulosa cells: identification of IGFBP-2 and -3 and regulation by hormones and growth factors.

Using ligand blotting and immunoprecipitation we have characterized the insulin like growth factor binding proteins (IGFBPs) produced by cultured porcine granulosa cells. Ligand blot analysis of granulosa cell conditioned medium revealed 5 bands of IGF binding activity with apparent molecular sizes of 44, 40, 34, 29, and 22 kilodaltons (kDa). The 40-44 kDa bands of granulosa- conditioned medium were identified by immunoprecipitation with an antibody to porcine IGFBP-3, the acid-stable subunit of the 150 kDa GH-dependent serum IGFBP complex. The 34 kDa band was immunoprecipitated by an antibody to the rat IGFBP-2, the major IGFBP found in fetal rat serum and in BRL-3A cell cultures. To date we have been unable to immunoprecipitate the 29 and 22 kDa bands with any of the antibodies tested including a panel of monoclonal antibodies to human IGFBP-1, the amniotic fluid IGFBP. The pattern of secretion varied with size of the follicles from which granulosa cells were obtained and the culture conditions. With cells from small (2-4 mm) follicles, short term cultures secreted mainly IGFBP-3 and IGFBP-2, while IGFBP-2 and the 29 and 22 kDa bands were pronounced in longer term cultures. In short term culture, granulosa cells from medium sized (4-6 mm) porcine follicles produced IGFBPs in substantially greater amounts than did those from small (1-3 mm) follicles, but exhibited comparable band patterns. The production of IGFBPs was inhibited by cycloheximide. IGFBP production by granulosa cells in culture was regulated by hormones and growth factors. The most striking effects were the inhibition of IGFBP-3 secretion by transforming growth factor beta and FSH. In contrast, IGFBP-3 levels were enhanced by epidermal growth factor. The IGFBPs produced by cultured porcine granulosa cells are identical in size and immunoreactivity to those previously found in porcine follicular fluid. Thus, follicular cells may be the source of follicular fluid IGFBPs. The IGFBPs may be important modulators of the IGF autocrine/paracrine system in the ovary.     

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)     

Regulation of insulin-like growth factor (IGF)-binding protein expression by growth factors and cytokines alters IGF-mediated proliferation of postnatal lung fibroblasts

Postnatal day 5 is the beginning of septation and the peak of postnatal fibroblast proliferation. The author and colleagues studied fibroblasts from this developmental time period to determine factors that regulate cell proliferation. Exposure of cells to insulin-like growth factor (IGF)-I for 48 hours increased cell number whereas exposure to epithelial growth factor (EGF), platelet-derived growth factor (PDGF)-BB, fibroblast growth factor (FGF)-7, FGF-2, tumor necrosis factor-alpha (TNF-alpha), or interleukin (L)-1beta did not alter cell number. Long[R3]IGF-I (a synthetic IGF analog with reduced affinity for IGF-binding proteins [IGFBPs]) was more potent than IGF-I, with half-maximal stimulation at a dose of 0.6 nM for long[R3]IGF-I compared to 1.5 nM for IGF-I, suggesting that IGFBPs in the conditioned medium (CM) inhibit IGF activity. Addition of exogenous IGFBP-3 inhibited the IGF-stimulated increase in cell number. Addition of IGFBP-4 did not alter IGF activity because IGF-I stimulated proteolysis of IGFBP-4. The expression of mRNA for PAPP-A (a known IGFBP-4 protease) suggests that the clearance of IGFBP-4 is mediated by pregnancy-associated plasma protein (PAPP)-A. Exposure of cells to TNF-alpha or IL-1beta increased IGFBP-3 mRNA abundance and IGFBP-3 protein in CM. PDGF-BB and IL-1beta increased IGFBP-4 protein abundance and PDGF-BB and dibutyryl cAMP increased IGFBP-4 mRNA. The increase in CM IGFBP-3 following TNF-alpha exposure blocked IGF-mediated cell proliferation, suggesting that the growth factor- and cytokine-mediated changes in IGFBP abundance regulate postnatal fibroblast cell proliferation.     

Inhibition of human fibroblast insulin-like growth factors binding protein (IGFBP) production by IGFBP-3.

Human neonatal fibroblasts in monolayer culture 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 [125I]IGF-II ligand blotting, human neonatal fibroblasts secrete IGFBP-3, an IGFBP of 29-31 kDa, and a 22-24 kDa IGFBP after treatment with 50 ng/ml IGF-I. When IGF-I treatment was carried out in the presence of increasing concentrations (50-1000 ng/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 ng/ml) IGF-I, IGFBP-3 had approximately 20-fold reduced potency in inhibiting 29-31 kDa IGFBP. When endogenous production of IGFBP-3 was increased by treatment with transforming growth factor-beta 1 (TGF beta 1), there was complete inhibition of 29-31 kDa IGFBP, while at high IGF-I concentrations TGF beta 1 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.     

The insulin-like growth factor system in the GT1-7 GnRH neuronal cell line

Evidence suggests that insulin-like growth factors (IGFs; IGF-I and IGF-II) are involved in the regulation of reproductive function including the development of the gonadotropin-releasing hormone (GnRH) neuronal system and the modulation of GnRH secretory activities. To further characterize the regulatory role of the IGF system on GnRH neuronal function, we have examined the gene expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-binding proteins (IGFBPs) in a GnRH neuronal cell line (GT1-7 cells). The relative effects of IGFs and insulin on GnRH secretion by these cells was also investigated. RT-PCR analysis demonstrated IGF-I, IGF-II and IGF-IR mRNAs in GT1-7 cells. The mRNAs for IGFBP-2, -3, -4, -5 and -6 but not IGFBP-1 were also detected. Immunoreactive protein bands for IGFBP-2, -4 and -5 but not for other IGFBPs were demonstrated by Western blot with IGFBP-5 appearing to be the most abundant IGFBP secreted by GT1-7 cells. IGFBP-5 production by GT1-7 cells was stimulated by both IGF-I and IGF-II in a dose-dependent manner with approximately equal potency, whereas insulin caused no significant effect. GnRH secretion by GT1-7 cells treated with IGF-I or IGF-II but not insulin showed an increase (80-100%) at 2 h of treatment followed by a decrease (46%) at 6 h that continued up to 24 h. We conclude that the expression of IGFs, IGF-IR and IGFBPs and their interactions in the regulation of GnRH secretion by GT1-7 cells as demonstrated by our study provide a basis for an autocrine regulatory role for the IGF system in GnRH neuronal secretory activities.     

Characterization of insulin-like growth factor binding proteins (IGFBP) and regulation of IGFBP-4 in bone marrow stromal cells

Summary. Bone marrow stromal cells synthesize and secrete insulin-like growth factor (IGF)-I and IGF-binding proteins (IGFBP). IGFBPs may modulate the action of IGF-I or IGF-II on haemopoiesis. However, the specific IGFBPs produced by various stromal cell types have not been identified. We examined six different stromal phenotypes for IGFBP protein and IGFBP-1 to -6 mRNA expression. [¹²⁵I]IGF-I ligand blot analysis of conditioned medium demonstrate different patterns of IGFBP secretion by each cell type. The most prominent IGFBPs were 24 and 29 kD species, consistent with IGFBP4 and IGFBP5, respectively. RNase protection assays demonstrate that, overall, stromal cells express IGFBP-2 to -6 mRNAs, with IGFBP4, IGFBP5 and IGFBP6 mRNAs predominating. Since agents that modulate cAMP levels may influence haemopoiesis via the release of stromal-derived cytokines, we determined the effect of forskolin, a cAMP agonist, on IGFBP4 expression in TC-1 cells. Forskolin (10 ⁵ M) up-regulated IGFBP4 mRNA and protein secretion in a time-dependent manner. These findings suggest that IGFBP-4, -5 and -6 released by stromal cells may be key modulators of the haemopoietic response to IGFs. Release of IGFBP4 by agents that increase cAMP may be an important mechanism involved in regulating IGF bioavailability in the marrow microenvironment.     

Identification and characterization of insulin-like growth factor (IGF)-binding protein expression and secretion by adult human adrenocortical cells: differential regulation by IGFs and adrenocorticotropin

In previous studies we have shown that IGF-II stimulates basal as well as ACTH-induced cortisol secretion from adult human adrenocortical cells more potently than IGF-I, and that both IGFs predominantly stimulate androgen biosynthesis. The steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we identified and characterized IGFBP synthesis in normal adult human adrenocortical cells in primary culture, and investigated the effect of ACTH and recombinant human IGF-I and -II on the regulation of IGFBP expression and secretion. Using RT-PCR, we identified the mRNA of all six high-affinity IGFBPs, in both adrenocortical tissue and monolayer cell cultures of adrenocortical cells. Using Western ligand and immunoblotting and two-dimensional Western ligand blotting we confirmed the secretion of IGFBP-1, -2, -3, -4 and -5 by adrenocortical cells in primary culture. The quantification of IGFBPs indicated that IGFBP-3 accounts for almost half the binding activity in conditioned medium of unstimulated cells (47%), followed by IGFBP-4 (20%), IGFBP-5 (15%), IGFBP-2 (12%) and IGFBP-1 (6%). After treatment with ACTH, the abundance of IGFBP-1 was upregulated significantly 2.6-fold, while IGFBP-3 was induced only slightly (1.3-fold). IGFBP-2, -4 and -5 remained unchanged. In contrast, IGF-I and -II (6.5 nM) predominantly induced the abundance of IGFBP-5 (2- and 1.6-fold respectively) and IGFBP-3 (2- and 1.7-fold respectively), while IGFBP-1, -2 and -4 were unaltered. The induction of IGFBP-1 and -5 by ACTH and IGFs, respectively, was paralleled by an increase in the amount of IGFBP-1 and -5 mRNA in these cells. In conclusion, all six high-affinity IGFBPs are expressed in the adult human adrenal gland, and the presence of at least five high-affinity IGFBPs has been demonstrated in conditioned medium of adult human adrenocortical cells. Furthermore, the expression and secretion of IGFBP-1 is upregulated by ACTH, whereas IGFBP-5 is induced by IGF-I and -II. Together with earlier findings, these results suggest that IGFBPs play an important modulatory role in the regulation of the differentiated adrenocortical function.     

Structural and functional evidence for the interaction of insulin-like growth factors (IGFs) and IGF binding proteins with vitronectin

Previous studies demonstrated that IGF-II binds directly to vitronectin (VN), whereas IGF-I binds poorly. However, binding of VN to integrins has been demonstrated to be essential for a range of IGF-I-stimulated biological effects, including IGF binding protein (IGFBP)-5 production, IGF type-1 receptor autophosphorylation, and cell migration. Thus, we hypothesized that a link between IGF-I and VN must occur and may be mediated through IGFBPs. This was tested using competitive binding assays with VN and (125)iodine-labeled IGFs in the absence and presence of IGFBPs. IGFBP-4, IGFBP-5, and nonglycosylated IGFBP-3 were shown to significantly enhance binding of IGF-I to VN, whereas IGFBP-2 and glycosylated IGFBP-3 had a smaller effect. Furthermore, binding studies with analogs indicate that glycosylation status and the heparin-binding domain of IGFBP-3 are important in this interaction. To examine the functional significance of IGFs binding to VN, cell migration in MCF7 cells was measured and found to be enhanced when VN was prebound to IGF-I in the presence of IGFBP-5. The effect required IGF:IGFBP:VN complex formation; this was demonstrated by use of a non-IGFBP-binding IGF-I analog. Together, these data indicate the importance of IGFBPs in modulating IGF-I binding to VN and that this binding has functional consequences in cells.     

Regulation of insulin-like growth factor-I and of insulin-like growth factor binding protein-1, -3 and -4 in cocultures of rat hepatocytes and Kupffer cells by interleukin-6.

BACKGROUND/AIMS: Catabolism is associated with decreased serum concentrations of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 associated with elevated IGFBP-3 protease activity and increased concentrations of IGFBP-1 and -4. The effects of the acute phase mediators interleukin (IL)-6, IL-1beta and tumor necrosis factor alpha (TNFalpha) on the biosynthesis of IGF-I and IGFBPs were studied in primary rat liver cells. METHODS: mRNA levels of IGF-I and of IGFBPs were analyzed by Northern blotting, secretion of IGFBPs by [(125)I]IGF-I ligand blotting. Proteolytic activity was measured using iodinated recombinant IGFBP-3 as the substrate. RESULTS: In hepatocytes, Kupffer cells (KC) and cocultures of hepatocytes with KC, IL-6 reduced IGF-I biosynthesis dose-dependently. IL-6 stimulated mRNA expression and protein secretion of IGFBP-1 and -4 in hepatocytes and that of IGFBP-3 in KC, respectively. In cocultures, biosynthesis of IGFBP-1, -3 and -4 was increased dose-dependently by IL-6, while the effects of IL-1beta or TNFalpha were less prominent. At neutral pH, proteolytic activity against IGFBP-3 was not detected in media of cocultures treated with IL-6. CONCLUSIONS: The alterations of IGF-I, IGFBP-1 and -4 observed in catabolism correlate with the effects of IL-6 on the biosynthesis of these components in primary rat liver cells, while a neutral IGFBP-3 protease was not detectable.     

Is there a local IGF-system in human adrenocortical cells?

There is increasing evidence that in the fetal and postnatal development of the adrenal gland, trophic and differentiating effects of ACTH are locally modulated by a species-specific pattern of growth factors. As we have shown previously in human adult adrenocortical cells (HAC) in culture, IGF-I and, even more, IGF-II enhance the steroidogenesis and ACTH responsiveness. We now examined the secretion of IGFs and their binding proteins (IGFBP) in the medium of 12 serum free primary cultures of HAC by specific RIAs and [¹²⁵I]IGF ligand blot or by immunoblot, and their long-term regulation by ACTH. HAC secrete 0.41 and 0.91 ng IGF-I and IGF-II/5×10⁵ cells per day, respectively, and their secretion is significantly stimulated 2- and 1.6-fold, respectively, by ACTH. HAC secrete at least three IGFBPs. The 43–46 kDa and the 29 kDa proteins correspond to glycosylated and fragmented forms of IGFBP-3, and the 36 kDa protein to IGFBP-2. The most abundant protein is the 24 kDa IGFBP, with identical electrophoretic mobility to IGFBP-4. IGFBP-3, as measured by RIA, is in the range of 1 ng/day. None of the IGFBPs is significantly changed by ACTH. Thus, we have evidence for a local IGF system, and the IGF-levels are in a range compatible with a physiological auto or paracrine action on steroidogenesis.     

Basal and regulated secretion of insulin-like growth factor binding proteins in osteoblast-like cells is cell line specific.

Local secretion of insulin-like growth factor binding proteins (IGFBPs) may modulate the effects of IGF-I and IGF-II on bone cell metabolism and proliferation. Several osteoblast-derived cell lines are currently used as interchangeable models to study IGFBP production, although it is unknown whether findings in one cell line can be extrapolated to another cell line or to normal human osteoblasts. In this study, we examined by Western ligand blotting both basal and regulated secretion of IGFBPs in vitro in 1) normal human osteoblast-like (hOB) cells cultured from explants of human trabecular bone; 2) an SV40-transformed hOB (HOBIT) cell line; and 3) several human (U-2, MG-63, TE-85) and rat (ROS 17/2.8 and UMR-106-01) osteosarcoma cell lines. Constitutively, hOB and HOBIT cells produced a similar pattern of IGFBPs, while all other cell lines produced their own unique pattern of IGFBPs. The two rat cell lines differed from the human cell lines as well as from each other. The response to hormonal stimulation also varied among the cell lines. Treatment of hOB and HOBIT cells with IGF-I resulted in a 2-fold increase in medium levels of IGFBP-3; IGF-I decreased levels of 24-kilodalton (kDa) IGFBP in hOB cell-conditioned medium. In addition, IGF-I markedly increased levels of the 29/32/34 kDa IGFBP triplet in U-2 cells, but had little or no effect in the other human and rat osteosarcoma cell lines. PTH increased a 29-kDa IGFBP apparent only in UMR 106-01 cell-conditioned medium, whereas GH had no direct effect on IGFBP secretion in any of the osteoblast-like cells tested. We conclude that basal and regulated secretion of IGFBPs from osteoblast-like cells is cell-line specific. Spontaneously transformed human or rat osteoblast-like cells provide unique model systems to study features of distinct IGFBPs and their regulation; however, hOB cells and their derivatives may be more appropriate models for understanding the regulation of IGFs in human bone.     

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.     

Insulin-like growth factor binding protein production in bovine retinal endothelial cells

Retinopathy is the most frequent microangiopathic complication in diabetes. Many circulating hormones and locally produced mitogenic factors have been involved. Bovine retinal endothelial cells (BRECs) were cultured to investigate if insulin, insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and a chronic high-glucose condition could control endothelial cell growth. Specific IGF-I receptors with two binding sites with high (K_d 0.03 nmol/L) and low (K_d 1.3 nmol/L) affinity were found when analyzing families of displacement curves between IGF-I versus IGF-I and IGF-I versus insulin. However, IGFs failed to be mitogenic factors in these cells. This could be explained by an inhibitory effect due to the presence of specific IGFBPs with a molecular weight between 24 and 43 kd. Using Western blot and immunoblot analysis, Northern blot study, and specific radioimmunoassay (RIA), these IGFBPs have been identified as IGFBP-3, -2, -5, and -4. Insulin, which does not bind to IGFBPs, was a potent mitogenic factor in these cells at a high concentration (10 nmol/L), suggesting a cross-reaction to IGF-I receptor. These IGFBPs, except the 24-kd form (IGFBP-4), were modulated by both IGF-I and IGF-II, with a maximum effect at 100 and 10 nmol/L, respectively. This regulation on IGFBPs was IGF-I receptor—independent. In fact, (1) IGFBP mRNA levels were not modified after stimulation with 100 nmol/L IGF-I, (2) 100 nmol/L IGF plus an equimolar concentration of αIR3 did not affect IGFBP production, (3) Des(1–3)IGF-I had no effect on IGFBP modulation, whereas at 10 nmol/L it enhanced BREC thymidine cell incorporation, and (4) 100 nmol/L insulin, which at this concentration can cross-react with the IGF-I receptor, did not modify the IGFBP pattern. Chronic exposure (4 weeks) of BRECs to 25 mmol/L glucose had no effect on cell growth. However, after 3 weeks, we observed a decreased IGFBP detection, and addition of 100 nmol/L IGF-I did not change IGFBP levels and did not modify cell growth. Conversely, BRECs grown in regular medium for 4 weeks showed increased IGFBP production. In conclusion, we showed that conditions mimicking hyperinsulinemia, rather than high levels of IGFs, could regulate BREC growth and that the IGF-I analog, Des(1–3), even with reduced affinity for IGFBPs but in part capable of binding to IGFBP-3, significantly stimulated BRECs growth only at 10 nmol/L. IGF actions are modulated by locally produced endothelial IGFBPs, and in turn, these endothelial IGFBPs are regulated, via an IGF-I receptor—independent mechanism, by the presence of IGFs. The autoregulatory IGF system together with the direct glucose modulation of IGFBPs could contribute in diabetic subjects to the retinal endothelial cell growth and metabolism through local changes in IGF bioavailability.     

Coordinated control of fetal gastric epithelial functions by insulin-like growth factors and their binding proteins

The influence of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) on human gastric functions are unknown. This study was undertaken to evaluate the ability of fetal gastric mucosa to produce IGFBPs and to test the effects of IGF-I, IGF-II, and synthetic truncated IGFs that do not interact with IGFBPs on epithelial cell proliferation and glandular zymogenic function. Western blots, Far Western blots, and immunohistochemistry were performed to characterize the expression of IGFBP-1 to -6 and IGF-I receptor. The effects of growth factors on DNA synthesis and lipase and pepsin activities were determined in gastric explants maintained in serum-free organ culture. All gastric epithelial cells expressed the IGF-I receptor. IGFBP-2 to -6 were produced endogenously, and they were differentially localized along the foveolus-gland axis and modulated in culture. Exogenous IGF-I and IGF-II were able to reduce lipase activity without affecting pepsin, whereas they exerted different effects on cellular proliferation: IGF-I was stimulatory and IGF-II had no influence. Illustrating the complex regulatory effect that IGFBPs exert on IGF bioactivity, both truncated IGF-I and IGF-II stimulated DNA synthesis more than IGF-I. Moreover, the striking difference in mitogenic activity between truncated and native forms of IGF-II probably reflects the abundance of IGFBP-2 and IGFBP-6, two IGF-II carriers, in the foveolus/neck region, including the proliferative compartment. This study provides new evidence for the involvement of an intragastric IGF/IGFBP system in the fine regulation of epithelial cell division and also in the control of zymogen synthesis. Moreover, the specific influence of IGF-II as a mitogen appears to be tightly regulated by IGFBP isoforms preferentially associated with this growth factor and proliferative cells.