Serum half-life and biological activity of mutants of human insulin-like growth factor I which do not bind to serum binding proteins

We have characterized the biological properties of two mutants of human insulin-like growth factor I (IGF-I) which, as we have shown previously, have normal affinity for the type I IGF receptor, but drastically reduced affinity for the acid-stable components of human serum binding proteins. [Phe-1,Val1,Asn2,Gln3,His4,Ser8,His9,Glu12 ,Tyr15,Leu16]IGF I (B-chain mutant) and [Gln3,Ala4,Tyr15,Leu16]IGF I have 1000 and 500 times lower affinity than IGF-I for the native 150K binding protein in adult rat serum. Like IGF-I, these two peptides migrate as monomers during size exclusion chromatography on TSK 125. [125I]IGF-I, [125I]B-chain mutant, and [125I] [Gln3,Ala4,Tyr15,Leu16]IGF-I have in vivo serum half-lives of 100, 27.5, and 26.9 min, respectively, after iv injection. These data suggest that serum binding protein-bound peptide is cleared from the serum more slowly than free peptide. The tissue distributions of [125I]IGF-I and [125I]B-chain mutant are similar 10 min after dosing, with more than 80% of the tissue-sequestered intact radioactive peptides in the kidney. Despite decreased serum half-lives, the B-chain mutant and [Gln3,Ala4,Tyr15,Leu16]IGF-I are, respectively, 4 times and twice as active as IGF-I in stimulating the incorporation of [14C]glucose into glycogen in rat diaphragm in vivo. This effect of IGF-I is thought to be mediated by the type 1 IGF receptor in muscle, since the same doses of peptide that stimulated glycogen synthesis more than 30-fold did not stimulate the incorporation of [14C]glucose into total lipid in adipose tissue, an effect known to be mediated by the insulin receptor. These data support the hypothesis that serum- or tissue-derived binding proteins impair the ability of IGF-I to exert its effects through the type 1 IGF receptor in vivo.     

Potentiation of insulin-like growth factor (IGF) action by IGF-binding protein-3: studies of underlying mechanism.

In this study we investigated the mechanism(s) by which insulin-like growth factor-binding protein-3 (IGFBP-3) potentiates IGF-I action in cultured bovine fibroblasts. Preincubation of cells with glycosylated or nonglycosylated recombinant human IGFBP-3 enhanced responsiveness to IGF-I in a time-dependent manner. A preincubation period of at least 24 h with IGFBP-3 was required to see a significant effect. Pretreatment with IGFBP-3 for 72 h resulted in a 2- to 4-fold augmentation of IGF-I-stimulated [3H]aminoisobutyric acid uptake; IGFBP-3 had no effect on basal [3H]aminoisobutyric acid uptake. During the preincubation period, exogenous IGFBP-3 associated with the fibroblast surface and exhibited time-dependent processing to lower mol wt forms that retained the ability to bind radiolabeled IGF-I. Initial surface adherence (preincubation time of 24 h or less) was readily reversible. However, IGFBP-3, once processed, appeared to be closely associated with the cell. After 72 h of exposure to bovine fibroblasts, cell-associated IGFBP-3 had a 10-fold lower affinity for IGF-I compared to intact IGFBP-3 in solution. In addition, incubation of bovine fibroblasts with IGFBP-3 had modifying effects on type I IGF receptor-mediated signalling because 1) the bioeffectiveness of [Gln3,Ala4,Tyr15,Leu16]IGF-I and insulin, IGF-I receptor activators with little or no affinity for IGFBP-3, was potentiated by preincubation with IGFBP-3; and 2) fibroblast responsiveness to IGF-I analogs with different affinities for the type I IGF receptor was enhanced in direct relation to the ability of the peptide to bind to the receptor. There was no evidence for an increase in receptor number or affinity as a result of IGFBP-3 treatment. These data suggest that IGFBP-3 potentiation of IGF-I action in bovine fibroblasts may involve changes in IGFBP-3 and type I IGF receptor responsiveness. Thus, cell-associated IGFBP-3 may provide a mechanism for optimal presentation of IGF-I to its receptor as well as a means to heighten receptor reactivity to IGF-I and related peptides.     

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)     

Localization of the IGF binding domain and evaluation of the role of cysteine residues in IGF binding in IGF binding protein-4

Our previous findings suggest that binding of IGF binding protein-4 (IGFBP-4) to IGFs is essential for the inhibitory effect of IGFBP-4 on the activity of IGFs, both in vitro and in vivo. Therefore, understanding the structural determinants of IGF binding in IGFBP-4 is important to the general understanding of the biology of the IGF system. This study sought to further localize the IGF binding domain and to evaluate the role of Cys residues in IGF binding. Our data revealed that full-length IGFBP-4 peptides lacking the residues Leu(72)-Ser(91) or Leu(72)-His(74) or Gly(75)-Ser(91) failed to bind to IGF-I or IGF-II, whereas deletion of the residue Leu(72) or residues Met(80)-Ser(91) led to a 2- to 3-fold reduction in IGF-I and IGF-II binding activity. The IGF-I and IGF-II binding activities were dramatically reduced by the single mutation, Cys9/Arg (>25-fold), and to a lesser degree, by the single mutation, Cys12/Arg (the first N-terminal Cys residue was designated Cys1). The mutation Cys17/Ser or Cys18/Tyr or Cys20/Ser each resulted in a similar but moderate ( approximately 5-fold) reduction in IGF-II binding activity. The IGF-I binding activity was also dramatically reduced by the mutation Cys18/Tyr, and to a lesser extent, by the mutation Cys17/Ser or Cys20/Ser. These data suggest: 1) the IGF-I and IGF-II binding domain in IGFBP-4 involves a hydrophobic motif (Leu(72)-Met(80)) located in the distal part of the conserved N-terminal region, and 2) the N-terminal Cys residues (Cys9 and Cys12) are more critical than the C-terminal Cys residues (Cys17 and Cys20) in affecting the IGF-I and IGF-II binding. Based on these data, we speculate that the structural determinants of IGF-I and IGF-II binding in IGFBP-4 are very similar, if not identical.     

The role of cell surface attachment and proteolysis in the insulin-like growth factor (IGF)-independent effects of IGF-binding protein-3 on apoptosis in breast epithelial cells.

We have recently reported that insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can significantly increase ceramide-induced apoptosis in an Hs578T breast carcinoma cell line in an IGF-independent manner. It was observed in that study that IGFBP-3 added to the cultures was proteolytically modified, generating a specific pattern of fragmentation. We have also previously reported that almost all of the IGFBP-3 outside the circulation in extravascular fluids is in a fragmented form, apparently due to the activity of a cation-dependent serine protease. The aim of this study was to investigate the role of proteolysis in the IGFBP-3 enhancement of C2-induced apoptosis. In this study we confirmed that preincubation of Hs578T cells with IGFBP-3 enhances the apoptotic effect of the ceramide analog C2. The presence of IGF-I completely inhibited the enhancement effect, apparently by inhibiting cell surface association and proteolytic modification. The presence of a serine protease inhibitor [4-(2-aminoethyl)benesulfonyl fluoride] completely inhibited the enhancement effect of IGFBP-3, and Western immunoblotting of conditioned medium and cell surface-associated IGFBP-3 revealed that proteolytic fragmentation of the IGFBP-3 was reduced. In addition, fragments from the incubation of IGFBP-3 with plasmin were able to enhance the susceptibility of Hs578T cells to C2. The effect of these fragments could, however, also be reduced by 4-(2-aminoethyl)benesulfonyl fluoride despite the fact that IGFBP-3 was already fragmented. This suggests additional roles for serine proteases in the IGFBP-3 effect on C2-induced apoptosis in addition to the cleavage of the binding protein.     

Secretion of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 by intestinal epithelial (IEC-6) cells: implications for autocrine growth regulation.

To identify the factors regulating the proliferation of intestinal epithelium, we examined the effects of various growth factors on [3H] thymidine incorporation into the DNA of IEC-6 cells, an intestinal epithelial cell line derived from rat jejunal crypts. Insulin-like growth factor-I (IGF-I), IGF-II, and insulin stimulated the DNA and protein synthesis of IEC-6 cells in serum-free medium supplemented with transferrin, dexamethasone, and BSA (basal medium). Concentration-response experiments demonstrated that IGF-I is approximately 10 times more potent than IGF-II or insulin in producing 2- to 3-fold stimulations of DNA and protein synthesis by IEC-6 cells. In addition, IEC-6 cells proliferated slowly in the basal medium without any added growth factors. Analysis of medium conditioned by IEC-6 cells by gel filtration chromatography, RIA, HPLC, and N-terminal sequencing revealed that IEC-6 cells synthesize and secrete mature, 7,500 mo wt (M(r)) IGF-II as well as high M(r) forms of IGF-II. In addition, ligand blot, immunoblot, and N-terminal sequence analyses showed that IEC-6 cells produce the 34,000 M(r) IGF-binding protein-2 (IGFBP-2). To determine if IGFBP-2 modulates IGF responses in IEC-6 cells, the IGF-I analogs, Des-(1-3)-IGF-I and [Gln3,Ala4,Tyr15,Leu16]IGF-I, both of which have a reduced affinity for IGFBPs, were tested for their effects on IEC-6 cell proliferation. Both analogs exhibited 10-fold greater potency than IGF-I, presumably because endogenously secreted IGFBPs depress IGF-I binding to cell surface receptors. Finally, purified IGFBP-2 attenuated the DNA synthesis of IEC-6 cells in a dose-dependent manner. We conclude that IGFBP-2 secreted by intestinal epithelial cells is capable of limiting the mitogenic activity of both exogenous and endogenous IGFs by blocking the association of the growth factors with cell surface binding sites. These results further suggest that the growth of IEC-6 cells is modulated by autocrine mechanisms involving IGF-II and IGFBP-2.     

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.     

Basic fibroblast growth factor induces proteolysis of secreted and cell membrane-associated insulin-like growth factor binding protein-2 in human neuroblastoma cells.

Insulin-like growth factor (IGF) action in the brain is modulated by IGF-binding proteins (IGFBPs) whose abundance can be altered by other locally expressed growth factors. However, the mechanisms involved are unclear. We here employed the neuroblastoma cell line SK-N-MC as a model to define the mechanisms involved in modulation of IGFBPs in neuronal cells. Western ligand blotting analysis and immunoprecipitation of conditioned media (CM) from SK-N-MC cells showed that in these cells, as in the brain, the most abundantly expressed IGFBP was IGFBP-2. However, IGFBP-2 was barely detectable in CM from cells treated with basic fibroblast growth factor (bFGF) without a change in IGFBP-2 messenger RNA (mRNA) abundance. These CM contained specific IGFBP-2 proteolytic activity, resulting in two IGFBP-2 fragments of 14 and 22 kDa. The activity was inhibited by EDTA/phenylmethylsulfonyl fluoride or aprotinin. Competitive binding studies indicated that IGFBP-2 fragments had reduced binding affinity for IGF-I. bFGF induced IGFBP-3 mRNA and protein. Affinity cross-linking of [125I]IGF-I to neuroblastoma cell membranes followed by immunoprecipitation revealed a approximately 38 kDa [125I]IGF-I/IGFBP-2 complex. Cell surface-associated IGFBP-2 was also susceptible to bFGF-induced proteolysis, with the appearance of a single cross-linked 21-kDa complex with low affinity for IGF-I. These findings indicate that intact IGFBP-2 and the 14-kDa, but not the 22-kDa fragment, bind to the cell surface. Our data suggest that induction of IGFBP-2 proteolysis on neuronal cell surface is a novel mechanism whereby IGF availability is modulated by the local growth factor bFGF.     

Insulin-like growth factor I (IGF-I) and long R(3)IGF-I differently affect development and messenger ribonucleic acid abundance for IGF-binding proteins and type I IGF receptors in in vitro produced bovine embryos

The insulin-like growth factor (IGF) system is a complex network, including ligands (IGF-I and -II), binding proteins (IGFBP-1 to -6), and receptors, of which the type I IGF receptor (IGF-I-R) is important for transmission of most biological effects of IGFs. As IGFs are secreted in large amounts by the female reproductive tract, it has been hypothesized that maternal IGFs may affect embryonic growth and differentiation in a fine-tuned manner, involving modulation of IGF effects by embryonic IGFBP and IGF-I-R expression. To address this point, we cultured in vitro produced bovine embryos in a chemically defined culture system in the presence (100 ng/ml) of recombinant human IGF-I, long R(3)IGF-I (LR(3)), or without IGF supplementation (control). The affinity of LR(3) to IGFBPs measured by competition assays and Western ligand blots is at least 3 orders of magnitude lower than that of IGF-I. LR(3) was most efficient in stimulating early embryonic cleavage, whereas further development was most potently supported by IGF-I. Total cell numbers of blastocysts were highest in the presence of LR(3) (105 +/- 4), followed by IGF-I (96 +/- 5), and the control group (91 +/- 3; P < 0.05). Differential cell staining of blastocysts revealed that these differences were mainly represented by trophectoderm cell numbers. Analysis of messenger RNA (mRNA) expression for IGFBPs and IGF-I-R was performed by RT-real-time PCR, using expression of the nonregulated housekeeping gene glyceraldehyde-3-phosphate dehydrogenase for normalization. Embryonic IGFBP-2 mRNA levels in the LR(3) treatment group were 1.7-fold (P < 0.001) and 2.8-fold (P < 0.001) higher than those in the IGF-I and control groups, respectively. IGFBP-5 mRNA levels were about 2-fold (P < 0.001) elevated in both IGF treatment groups, with slightly (P < 0.05) higher levels in IGF-I- than in LR(3)-treated embryos. Similarly, IGFBP-3 mRNA abundance was increased (P < 0.05) in embryos from the IGF-I vs. the LR(3) culture system. IGF-I-R mRNA levels were reduced by IGF-I (80% of control; P < 0.01), but increased by LR(3) (1.3-fold vs. control; P < 0.001). These data show that the affinity for IGFBPs of IGF peptides is relevant for their effects on preimplantation embryos and affects different parameters, i.e. development, cell numbers, and mRNA expression for components of the IGF system, in different directions.     

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.     

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.     

Increased activity of insulin-like growth factor (IGF) in osteoblastic cells in the presence of growth hormone (GH): positive correlation with the presence of the GH-induced IGF-binding protein BP-3

Insulin-like growth factors (IGFs) are bound in the circulation to specific binding proteins (BP). The predominant BP is a GH-dependent glycosylated protein of 42-49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BP-3), whereas nonglycosylated GH-independent IGFBPs of 32 kDa and less are minor constituents. Primary cultures of rat osteoblastic cells constitutively produce IGFBP species of 32 kDa, while GH induces the accumulation of BP-3. To examine whether BP-3 could regulate the biological activity of IGF-I on osteoblasts, we compared the effects of recombinant native human IGF-I (hIGF-I) on primary cultures of osteoblasts in the presence and absence of GH. hIGF-I stimulated cell replication and alpha 1(I) collagen gene expression in a dose-dependent manner, and these effects were potentiated by the presence of GH, which increased the accumulation of BP-3. To further examine this correlation, we compared the effects of two IGF-I peptides on the osteoblastic cell line PyMS, which constitutively produces BP-3, to those in RCT-3 cells, which do not secrete this IGFBP. Using hIGF-I and [Gln3,Ala4,Tyr15,Leu16]IGF-I ([QAYL]IGF-I), a mutated IGF-I with reduced affinity to IGFBPs, we found that at equimolar concentrations hIGF-I produced higher stimulation than [QAYL]IGF-I on [3H]thymidine incorporation, cell replication, and collagen gene expression in PyMS cells. In contrast, both IGF-I peptides had similar potency in RCT-3 cells. Hence, these data show that the accumulation of BP-3 correlates with enhanced hIGF-I activity on osteoblastic cells, suggesting that BPs may act locally to augment the effects of IGF-I in bone.     

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.     

Insulin-like growth factor binding protein-3 is secreted as a phosphoprotein by human breast cancer cells

The growth regulatory activity of the insulin-like growth factor binding proteins (IGFBPs) may be modulated by post-translational modifications such as glycosylation, limited proteolysis and phosphorylation. In this study, we have examined phosphorylation of IGFBP-3 in two breast cancer cell lines: the estrogen receptor negative (ER-ve) Hs578T cell line in which IGFBP-3 is normally expressed, and ER+ve T47D breast cancer cells transfected with IGFBP-3 cDNA (T47D(BP-3)) and therefore expressing IGFBP-3 constitutively. Metabolic labelling with [32P] orthophosphate revealed that both cell lines secreted phosphorylated IGFBP-3 similar in size to plasma IGFBP-3 phosphorylated in vitro with casein kinase II, and that IGFBP-3 phosphorylation was differentially modulated in the two cell lines. In Hs578T cells, retinoic acid (10-100 nM) increased IGFBP-3 phosphorylation to a maximum of 150% of control. IGF-I, but not [LR3]IGF-I, reduced the proportion of phosphorylated IGFBP-3 in Hs578T conditioned medium, consistent with increased release of non-phosphorylated, cell-associated IGFBP-3. By contrast, IGFBP-3 phosphorylation in T47D(BP-3) cells was not affected by retinoic acid or IGF-I, but appeared slightly increased by estradiol. Together these data indicate that phosphorylation of IGFBP-3 in breast cancer cells may be regulated by agents known to affect breast cancer cell proliferation.     

Binding properties and distribution of insulin-like growth factor binding protein-related protein 3 (IGFBP-rP3/NovH), an additional member of the IGFBP Superfamily

The protein product of the novH oncogene, a member of the CCN family, is structurally related to the insulin-like growth factor (IGF) binding proteins (IGFBPs). We have characterized aspects of structure, function, and distribution of this protein, which, as IGFBP-related protein 3 (IGFBP-rP3), is a proposed member of the IGFBP Superfamily. Affinity cross-linking experiments performed with baculovirus synthesized recombinant human IGFBP-rP3 established that rhIGFBP-rP3 binds IGF-I, IGF-II, and insulin with low affinity. Specificity of binding was shown by competitive cross-linking experiments; binding to IGF-I and -II was also demonstrated by nondenaturing Western ligand blots. Northern blot analysis indicated the presence of IGFBP-rP3 messenger RNA (mRNA) in a broad range of human tissues. Western immunoblotting studies, using a polyclonal rabbit anti-rhIGFBP-rP3 antibody, demonstrated that IGFBP-rP3 protein is synthesized in vitro by several breast and prostate cancer cell lines: Hs578T, PC3, P69, and LNCaP cells. Western immunoblotting studies of human biological fluids identified that IGFBP-rP3 was present in normal serum, pregnancy serum, serum from patients with growth hormone receptor deficiency, cerebrospinal fluid, amniotic fluid, peritoneal fluid, and follicular fluid, while IGFBP-rP3 fragments were identified in cerebrospinal fluid, amniotic fluid, and prepubertal and pubertal urine samples. Our studies demonstrate that IGFBP-rP3 exhibits IGF binding, albeit at low affinity, and IGFBP-rP3 thus merits inclusion in the IGFBP Superfamily. The low affinity IGF binding suggests that IGFBP-rP3 may act primarily independently of the IGFs. The synthesis of IGFBP-rP3 by several malignant cell lines and its presence in human biological fluids suggest that this protein possesses other interesting roles, potentially in cell growth regulation.     

Evidence for a novel insulin-like growth factor (IGF)-dependent protease regulating IGF-binding protein-4 in dermal fibroblasts.

The mechanisms by which insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) levels in cellular conditioned media are poorly understood. The effect of IGFs on IGFBP-4 levels in fibroblast conditioned media is not mediated via the type 1 or type 2 cellular IGF receptors, and the IGFs exert little or no effects on IGFBP-4 messenger RNA levels in human adult fibroblasts or in rat neuroblastoma cells. To determine whether the effects of IGFs on IGFBP-4 might be exerted through alterations in IGFBP-4 degradation, we incubated cell-free, fibroblast-conditioned media from either sheep or human dermal fibroblasts with or without IGF-I, IGF-II (each 1 microgram/ml), or insulin (10 micrograms/ml) for 72 h at 37 C. Samples were then analyzed by Western ligand blot using radiolabeled IGFs and by immunoblotting using a polyclonal antisera to human IGFBP-4. In the absence of IGFs, no apparent changes in the basal concentrations of the various IGFBPs were observed. In contrast, incubation of media with IGFs caused a 70-80% reduction in levels of both sheep and human IGFBP-4, whereas incubation with insulin was without effect. Similarly, incubation of cell-free conditioned media containing recombinant human IGFBP-4 with IGF-I caused a reduction in detectable levels of the 28K protein. The decrease in IGFBP-4 levels was accompanied by the appearance of an immunoreactive approximate 17-20K fragment that did not bind radiolabeled IGFs by ligand blot. The IGF-dependent decrease in IGFBP-4 was prevented by coincubation of the media with serine protease inhibitors, EDTA, or 1,10-phenanthrolene, suggesting that IGFs may activate an IGFBP-4 specific metallo-serine protease present in fibroblast conditioned media. Alternatively, binding of IGF-I or -II to IGFBP-4 may enhance the susceptibility of IGFBP-4 to proteolytic degradation. The demonstration that IGF-I and IGF-II can promote directly the proteolytic degradation of IGFBP-4 into fragments that do not bind IGFs provides a novel mechanism by which the IGFs may increase their own availability and/or activity in biological fluids.     

Zinc partitions IGFs from soluble IGF binding proteins (IGFBP)-5, but not soluble IGFBP-4, to myoblast IGF type 1 receptors

Zinc (Zn(2+)), a multifunctional micronutrient, was recently shown to lower the affinity of cell-associated insulin-like growth factor (IGF) binding protein (IGFBP)-3 and IGFBP-5 for both IGF-I and IGF-II, but to increase the affinity of the cell surface type 1 IGF receptor (IGF-1R) for the same two ligands. However, there is a need for data concerning the effects of Zn(2+) on soluble IGFBPs and the type 2 IGF receptor (IGF-2R). In the current work, we demonstrate that Zn(2+) affects the affinity of IGFBP-5 secreted by myoblasts but not IGFBP-4. Zn(2+), at physiological levels, depressed binding of both IGF-I and IGF-II to IGFBP-5, affecting (125)I-IGF-I more than (125)I-IGF-II. Both (125)I-IGF-I and (125)I-IGF-II bound to high and low affinity sites on IGFBP-5. Zn(2+) converted the high affinity binding sites of IGFBP-5 into low affinity binding sites. An IGF-I analog, (125)I-R(3)-IGF-I, did not bind to the soluble murine IGFBP-5. Zn(2+) also decreased the affinity of the IGF-2R on L6 myoblasts. In contrast, Zn(2+) increased IGF-I, IGF-II and R(3)-IGF-I binding to the IGF-1R by increasing ligand binding affinity on both P(2)A(2a)-LISN and L6 myoblasts. Soluble IGFBP-5 and IGFBP-4 depressed the binding of (125)I-IGF-I and (125)I-IGF-II to the IGF-1R, but did not affect binding of (125)I-R(3)-IGF-I. By depressing the association of the IGFs with soluble IGFBP-5, Zn(2+) partitioned (125)I-IGF-I and (125)I-IGF-II from soluble IGFBP-5 onto cell surface IGF-1Rs. This effect is not seen when soluble L6-derived IGFBP-4 is present in extracellular fluids. We introduce a novel mechanism by which the trace micronutrient Zn(2+) may alter IGF distribution, i.e. Zn(2+) acts to increase IGF-1R binding at the expense of IGF binding to soluble IGFBP-5 and the IGF-2R.     

Insulin-like growth factors (IGFs), IGF receptors, and IGF-binding proteins in primary cultures of prostate epithelial cells.

Insulin-like growth factors (IGFs) are potent mitogens that bind with high affinity and specificity to IGF receptors and IGF-binding proteins (IGFBPs). We studied the roles of these three groups of proteins in prostate epithelial cells (PEC) in primary culture grown under serum-free conditions. Affinity cross-linking of IGF-I and IGF-II to crude membranes prepared from PEC revealed an abundance of type 1 IGF receptors and no evidence of type 2 IGF receptors. Western ligand blots of conditioned media (CM) from PEC demonstrated the presence of two specific IGFBP bands similar to those previously demonstrated in seminal plasma, with approximate mol wt of 31 and 24 kDa. The 31-kDa band was immunoprecipitable with an antibody to IGFBP-2, and neither band could be deglycosylated with endoglycosidase-F. Northern blot analysis of poly(A)+ RNA prepared from PEC with cDNAs for hIGFBP-1, -2, and -3 documented the expression of mRNA for hIGFBP-2 only. Modifications of the serum-free conditions of PEC did not significantly alter the IGFBP profile of PEC CM. The ability of IGF-I, IGF-II, and insulin to stimulate clonal growth of PEC was examined. IGF-I stimulated PEC growth with an ED50 of 0.1 ng/mL. IGF-II and insulin, respectively, were 1 and 3 orders of magnitude less effective than IGF-I in stimulating the growth of PEC. Radioimmunoassayable IGF-I and IGF-II levels in PEC CM were below the assay detection levels. In conclusion, we suggest that IGFs are important growth stimulators of PEC in culture, that their actions are mediated through the type 1 IGF receptor, and that PEC produce hIGFBP-2 and a 24-kDa IGFBP which may modulate IGF action in these cells.     

Competition for binding to insulin-like growth factor (IGF) binding protein-2, 3, 4, and 5 by the IGFs and IGF analogs.

The insulin-like growth factors (IGF) I and II bind to IGF binding proteins (BP) with high affinity. The affinity of each of the IGFs for individual BPs and the regions of the IGF-I molecule that are required for this high affinity binding have been defined only for IGFBP-1 and IGFBP-3. The present studies have determined the affinity of several IGF analogs (prepared using in vitro mutagenesis) for pure IGFBP-2, 3, 4, and 5. The results show IGFBP-2 binds these analogs in a manner similar to IGFBP-1. For example, a mutation in the A chain region (positions 49, 50, 51) or B chain (positions 3, 4) results in greater than 20-fold reduction in affinity for either IGFBP-1 or 2. In contrast, mutations in the A chain region have minimal effect on binding to IGFBP-3, whereas substitutions at the 3, 4, 15, 16 positions of the B chain reduce IGF-I affinity by at least 50-fold. At pH 7.4, binding of the analogs to IGFBP-4 is less affected by substitutions at the B chain 3, 4 positions compared to IGFBP-1, 2, and 3, but IGFBP-4 affinity for analogs containing the A chain substitutions is greatly reduced similarly to IGFBP-1 and 2. Binding to IGFBP-5 is greatly reduced by either A or B chain substitutions and most of the mutations result in greater than 100-fold reduction in affinity. Acidic pH 6.0 was associated with increased affinity of IGFBP-4 for the A chain containing mutants. The results indicate that only IGFBP-1 and 2 have nearly identical affinity for each of these analogs, whereas IGFBP-3, 4, and 5 have similarities and significant differences. The findings suggest that different binding proteins have differential structural requirements for optimal IGF-I binding.