Use of lanthanum to accurately quantify insulin-like growth factor binding to proteins on cell surfaces

Insulin-like growth factor binding proteins (IGFBPs) are found both associated with cells and in extracellular fluids. Cell-associated IGFBPs increase [¹²⁵I]-IGF binding to cell monolayers, whereas extracellular (soluble, released) IGFBPs decrease binding. In the current study, we show that either IGFBP-3 or IGFBP-5 are the major forms of IGFBP released from monolayers of human GM10 fibroblasts, T98G glioblastoma cells and forskolin-treated bovine MDBK cells. IGFBPs represent the most abundant [¹²⁵I]-IGF-I binding site on GM10 and T98G cell monolayers, but 4-17% of the total cell-associated IGFBPs are released from the cell monolayer at 8°C during their quantification. Most of the IGFBPs (> 70%) are released from MDBK cells. Quantitative estimates of [¹²⁵I]-IGF binding to the cell monolayers are altered because of the ability of the released IGFBPs to reduce the amount of radiolabeled ligand that is available to bind to the cell surface. Lanthanum (La³⁺) depresses IGFBP release from all three cell types (> 80% for GM10 and T98G cells and > 65% for MDBK cells). The effect was cation specific, noted with La³⁺ or Zn²⁺ but not with either Mn²⁺, Sr²⁺ or Se³⁺. The effect was also IGFBP specific; La³⁺ markedly depressed the release of IGFBP-3 and IGFBP-5, but had less of an effect on IGFBP-2 and IGFBP-4. Concomitant with a decrease in IGFBP-3 and IGFBP-5 release, La³⁺ caused an increase in [¹²⁵I]-IGF-I binding to cell-associated IGFBPs and type I IGF receptors. The released soluble IGFBPs have a three- to 20-fold greater affinity (K_a) for [¹²⁵I]-IGF-I compared to cell-associated IGFBPs. La³⁺ did not alter the affinity constants of cell-associated IGFBPs. In summary, we have identified a means to prevent loss of IGFBPs from cell monolayers during binding assays. This procedure will be useful in accurately quantifying the levels of IGFBPs on cell monolayers and in determining the role of cell-associated IGFBPs in controlling IGF activity. Retention of cell-associated low affinity IGFBPs may be important in controlling the size of the pericellular IGF pool and in regulating IGF-I access to the type I IGF receptor. J. Cell. Biochem. 66:256-267. © 1997 Wiley-Liss, Inc.     

Zinc partitions insulin-like growth factors (IGFs) from soluble IGF binding protein (IGFBP)-5 to the cell surface receptors of BC3H-1 muscle cells

Zinc (Zn(2+)) is a multifunctional micronutrient. The list of functions for this micronutrient expanded with the recent discovery that Zn(2+) retains insulin-like growth factors binding proteins (IGFBPs) on the surface of cultured cells, lowers the affinity of cell-associated IGFBPs, and increases the affinity of the cell surface insulin-like growth factor (IGF)-type 1 receptor (IGF-1R). However, currently there is no information concerning the effect of Zn(2+) on soluble IGFBPs. In the current study, the soluble IGFBP-5 secreted by BC(3)H-1 cells is shown to bind approximately 50% more [(125)I]-IGF-II than [(125)I]-IGF-I at pH 7.4. Zn(2+) is shown to depress the binding of both IGF-I and IGF-II to soluble secreted IGFBP-5; [(125)I]-IGF-I binding is affected more so than [(125)I]-IGF-II binding. Zn(2+) acts by lowering the affinity (K(a)) of IGFBP-5 for the IGFs. Scatchard plots are non-linear indicating the presence of high and low affinity binding sites; Zn(2+) affects only binding to the high affinity site. In contrast, Zn(2+) increases the affinity by which either [(125)I]-IGF-I or [(125)I]-R(3)-IGF-I binds to the IGF-1R, but depresses [(125)I]-IGF-II binding to the IGF-type 2 receptor (IGF-2R) on BC(3)H-1 cells. By depressing the association of the IGFs with soluble IGFBPs, Zn(2+) is shown to repartition either [(125)I]-IGF-I or [(125)I]-IGF-II from soluble IGFBP-5 onto cell surface IGF receptors. Zn(2+) was active at physiological doses depressing IGF binding to IGFBP-5 and the IGF-2R at 15-20 microM. Hence, a novel mechanism is further characterized by which the trace micronutrient Zn(2+) could regulate IGF activity.     

Multivalent cations and ligand affinity of the type 1 insulin-like growth factor receptor on P2A2-LISN muscle cells

Mouse P₂A₂-LISN myoblasts are transfected cells that overexpress the human type 1 insulin-like growth factor (IGF) receptor. Because the type 1 IGF receptor is the major binding site for both IGF-I and IGF-II, this cell line is an excellent model to determine the effect of multivalent cations on ligand binding specifically to this type of receptor. Competitive binding assays were performed to characterize IGF binding and Scatchard analysis to quantify affinity (K_a). ¹²⁵I-IGF-I, ¹²⁵I-IGF-II, and ¹²⁵I-R³-IGF-I bind only to the type 1 IGF receptor on these cells. Zn²⁺ increased binding of the three ligands to the type 1 IGF receptor by 17 to 35%. Cd²⁺ significantly increased binding of ¹²⁵I-IGF-I, although by only 8%. La³⁺ and Cr³⁺ did not effect binding. Au³⁺ decreased IGF binding by approximately 56%. Scatchard analysis produced nonlinear concave-down plots yielding binding constants for high and low affinity sites. Zn²⁺ increased the strength of only the high affinity sites. Au³⁺ decreased the affinity of both high and low affinity sites. Zn²⁺ increased binding with a half-maximal effect between 40 μM and 60 μM. Half-maximal dose of Au³⁺ was > 130 μM. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) and only these cations were found to affect receptor binding indicating similar mechanisms of action. Thus, multivalent cations may alter IGF binding to cell surface receptors ultimately controlling growth. Physiologically this may be especially important for the growth promoting effects of Zn²⁺. J. Cell. Physiol. 176:392–401, 1998. © 1998 Wiley-Liss, Inc.     

Insulin-like growth factor (IGF) binding to human fibroblast and glioblastoma cells: the modulating effect of cell released IGF binding proteins (IGFBPs)

The cell surface of human fibroblasts contains not only type I IGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF-I-cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125I]-IGF-I binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]-IGF-I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]-IGF-I added) since they have a higher affinity than type I IGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF-I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]-IGF-I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]-IGF-I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125I]-IGF-I that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]-IGF-I ([QAYL]-IGF-I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF-I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF-I and the [QAYL]-IGF-I compete equally with the [125I]-[QAYL]-IGF-I for binding to cell surfaces, whereas unlabeled [QAYL]-IGF-I is greater than 25-fold less potent compared to IGF-I in competing with [125I]-IGF-I for cell surface binding. Specific binding of [125I]-[QAYL]-IGF-I to GM10 and 1690 cell surfaces is less than 20% of [125I]-IGF-I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]-IGF-I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.     

Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R(3) -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were performed to distinguish [(125) I]IGF-I binding to IGF-I receptors and IGFBPs. Specific [(125) I]IGF-I binding in brain regions and the posterior pituitary was completely displaced by DES(1-3)IGF-I and R(3) -IGF-I, indicating binding to IGF-I receptors. In contrast, [(125) I]IGF-I binding in the anterior pituitary was not displaced by DES(1-3)IGF-I and R(3) -IGF-I, suggesting binding to an IGF-binding site that is different from the IGF-I receptor. Binding affinity of IGF-I to this site was about 10-fold lower than for the IGF-I receptor. Using western immunoblotting we were also unable to detect IGF-I receptors in human anterior pituitary. Instead, western immunoblotting and immunoprecipitation experiments showed a 150-kDa IGFBP-3-acid labile subunit (ALS) complex in the anterior pituitary and not in the posterior pituitary and other brain regions. RT-PCR experiments showed the expression of ALS mRNA in human anterior pituitary indicating that the anterior pituitary synthesizes ALS. In the brain regions and posterior pituitary, IGFBP-3 was easily washed away during pre-incubation procedures as used in the [(125) I]IGF-I binding experiments. In contrast, the IGFBP-3 complex in the anterior pituitary could not be removed by these washing procedures. Our results indicate that the human anterior pituitary contains a not previously described tightly cell membrane-bound 150-kDa IGFBP-3-ALS complex that is absent in brain and posterior pituitary.     

Serum Factors Inhibit Melanoma Cell Surface Expression of Type I and Type II IGF Receptors

Abstract

We have identified one class of IGF-I-binding sites and two classes of IGF-II-binding sites at the surface of the melanoma cell line IGR39. By means of affinity labeling with ¹²⁵I-IGF-I, a 290–300 kDa form was characterized. Using ¹²⁵I-IGF-II, a 270 kDa polypeptide was labeled, corresponding to the type II IGF receptor. In the two serials of experiments, the order of potency in inhibiting 125I-IGF-I or ¹²⁵I-IGF-II labeling of IGF-related peptides and αIR3, an antibody directed against type I receptor α subunit, was the same as in competition experiments. When IGR39 cells were cultured in a serum-free medium, the number of both high affinity IGF-II and IGF-I binding sites was increased, by 8-and 5-fold respectively, without any significant change in K_d values. In both culture conditions, we found IGFBP-2, -3, -4 and a 30 kDa form which Mr was consistent with IGFBP-5 or -6. Except for IGFBP-2, the amount of secreted IGFBPs was modified depending on culture conditions: in conditioned medium from cells cultured with 10% FCS, the amount of IGFBP-3 or -4 was higher, and the amount of the 30 kDa IGFBP was lower when compared to conditioned medium from cells cultured in serum-free medium.     

Characterization of insulin-like growth factor (IGF) binding proteins and their role in modulating IGF-I action in BHK cells.

We have found that over one-half of the total cell surface 125I-insulin-like growth factor I (IGF-I) binding to BHK cells represents binding to IGF binding proteins (IGFBPs) rather than to the IGF-I receptor. In addition to a number of secreted IGFBPs, we have now characterized two cell-associated IGFBPs with unique characteristics. The cell-associated IGFBPs have molecular weights of 30,000 (30K) and 25,000 (25K), as determined by the Western ligand blot technique. IGFBP-30K is located at the cell surface and can be readily labeled by affinity cross-linking with 125I-IGF-I. Surface expression of IGFBP-30K increases 5.4 +/- 1.2-fold (n = 11) with serum starvation. This induction is fully evident by 4 h, plateauing by 24 h, and is completely inhibitable by cycloheximide. The fasting-induced increase in IGFBP-30K is inhibited by IGF-I and by des-IGF-I and, to a lesser extent, by insulin. Unlike cell-associated IGFBP-30K, secretion of IGFBP was stimulated (6.8 +/- 0.5-fold, n = 2) by IGF-I, whereas IGFBP secretion was inhibited 54% by insulin. These results demonstrate coordinate regulation of IGFBP by serum starvation and IGF-I, such that at low concentrations of IGF-I, cell surface binding protein increases whereas binding protein secretion decreases. At high concentrations of IGF-I, IGFBP secretion increases and cell surface IGF-I receptor, as well as IGFBP, decreases. Taken together, these regulatory events regulate the availability of IGF-I for biologic signalling.     

G protein dependent alterations in [125I]iodocyanopindolol and±cyanopindolol binding at 5-HT1B binding sites in rat brain membranes

Several manipulations that affect G protein/receptor coupling also alter the binding of [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP)±cyanopindolol (±CYP) to rat brain 5-HT_1B binding sites in radiologand binding assays. Inclusion of 5 mM MgSO₄ in these assays results in a small but significant increase in the affinity of [¹²⁵I]ICYP (fromK _D=0.046 nM toK _D=0.037 nM). In contrast, 100 M Gpp(NH)p, GTP, or GDP reduce [¹²⁵I]ICYP affinity (K _D=0.056 nM with GTP) while ATP and GMP are less effective.±CYP affinity for 5-HT_1B sites labeled by [³H]dihydroergotamine ([³H]DE) also displays a small but significant reduction (from K_i=1.4 nM to K_i=3.5nM) by the inclusion of 100 M GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT_1B specific binding of [¹²⁵I]ICYP. The NEM induced reduction in [¹²⁵I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [¹²⁵I]ICYP to 5-HT_1B sites is dependent on G proteins. The effects of Mg²⁺ ion, guanine nucleotides, NEM and G protein reconstitution on [¹²⁵I]ICYP and ±CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT_1B sites. The relatively slight stabilization of [¹²⁵I]ICYP and ±CYP binding conferred by G protein/5-HT_1B receptor interaction may reflect the molecular events underlying previous observations that these compounds are partial 5-HT_1B agoinists.     

Insulin-like growth factors and binding proteins in the fetal rat: Alterations during maternal starvation and effects in fetal brain cell culture

Maternal malnutrition adversely affects fetal body and brain growth during late gestation. We utilized a fetal brain cell culture model to examine whether alternations in circulating factors may contribute to reduce brain growth during maternal starvation; we then used specific immunoassay and western blotting techniques, and purified peptides to investigate the potential role that altered levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) may play in impaired growth during maternal nutritional restriction.Fetal, body, liver, and brain weight were reduced after 72 hr maternal starvation, and plasma from starved fetuses were less potent than fed fetal plasma in stimulating brain cell growth. Circulating levels of IGF-I were reduced in starved compared to fed fetuses, while levels of IGF-II were similar in both groups. In contrast, [¹²⁵I]-IGF-I binding assay demonstrated an increase in the availability of plasma IGFBPs following starvation. Western ligand blotting and densitometry indicated that levels of 32 Kd IGFBPs were 2-fold higher in starved compared to fed fetal plasma. Immunoblotting and immunoprecipitation with antiserum against rat IGFBP-1 confirmed that heightened levels of immunoreactive IGFBP-1 accounted for the increase in 32 Kd IGFBPs in starved plasma. Levels of 34 Kd BPs, representing IGFBP-2, were unaffected by starvation. Reconstitution experiments in cell culture showed that IGF-I promoted fetal brain cell growth, and that when they were supplemented with IGF-I, the growth promoting activity of starved fetal plasma was restored to fed levels. These changes were measured using MTT to assess mitochondrial reductase activity. Conversely, addition of physiological amounts of rat IGFBP-1 inhibited the effects of fed fetal plasma on brain cell growth, and bioactivity was reduced even further with higher concentrations of IGFBP-1. Based on these results, we conclude that reciprocal changes in circulating levels of IGFBP-1 (increased) and IGF-I (decreased) may combine to reduce the availability of IGF-I to this tissue and limit fetal brain cell growth when maternal nutrition is impaired.     

Characterization of the inhibition of DNA synthesis in proliferating mink lung epithelial cells by insulin-like growth factor binding protein-3

Insulin-like growth factor binding protein-3 (IGFBP-3) can inhibit cell growth by directly interacting with cells, as well as by forming complexes with IGF-I and IGF-II that prevent their growth-promoting activity. The present study examines the mechanism of inhibition of DNA synthesis by IGFBP-3 in CCL64 mink lung epithelial cells. DNA synthesis was measured by the incorporation of 5-bromo-2'-deoxyuridine, using an immunocolorimetric assay. Recombinant human IGFBP-3 (rh[N109D,N172D]IGFBP-3) inhibited DNA synthesis in proliferating and quiescent CCL64 cells. Inhibition was abolished by co-incubation of IGFBP-3 with a 20% molar excess of Leu(60)-IGF-I, a biologically inactive IGF-I analogue that binds to IGFBP-3 but not to IGF-I receptors. DNA synthesis was not inhibited by incubation with a preformed 1:1 molar complex of Leu(60)-IGF-I and IGFBP-3, indicating that only free IGFBP-3 inhibits CCL64 DNA synthesis. Inhibition by IGFBP-3 is not due to the formation of biologically inactive complexes with free IGF, since endogenous IGFs could not be detected in CCL64 conditioned media; any IGFs that might have been present could only have existed in inactive complexes, since endogenous IGFBPs were present in excess; and biologically active IGFs were not displaced from endogenous IGFBP complexes by Leu(60)-IGF-I. After incubation with CCL64 cells, (125)I-IGFBP-3 was covalently cross-linked to a major thick similar400-kDa complex. This complex co-migrated with a complex formed after incubation with (125)I-labeled transforming growth factor-beta (TGF-beta) that has been designated the type V TGF-beta receptor. (125)I-IGFBP-3 binding to the thick similar400-kDa receptor was inhibited by co-incubation with unlabeled IGF-I or Leu(60)-IGF-I. The ability of Leu(60)-IGF-I to decrease both the inhibition of DNA synthesis by IGFBP-3 and IGFBP-3 binding to the thick similar400-kDa receptor is consistent with the hypothesis that the thick similar400-kDa IGFBP-3 receptor mediates the inhibition of CCL64 DNA synthesis by IGFBP-3.     

Consequences of the presence of the Booroola F gene on the intraovarian insulin-like growth factor system and terminal follicular maturation in Mérinos d'Arles ewes

In sheep, the presence of the Booroola F gene has several important consequences for ovarian function. This study investigated the consequences of the presence of the F gene for the insulin-like growth factor (IGF) system in the ewe ovary. Studies were undertaken in ovaries from F+ and ++ Mérinos d'Arles ewes to determine 1) the levels of type I IGF receptors and IGF binding proteins (IGFBPs) in follicular cells by quantitative autoradiography of [(125)]-IGF-I binding sites on ovarian sections; 2) the pattern of intrafollicular IGFBPs, by Western-ligand blotting on follicular fluids; and 3) the effects of IGF-I and FSH on proliferation and differentiation of granulosa cells in vitro, assessed by progesterone secretion and cytochrome P450 side-chain cleavage (P450(scc)) expression. The amounts of type I IGF receptors were similar in F+ and ++ follicular cells; however, at the same follicular size, F+ healthy follicles contained lower concentrations of IGFBPs smaller than 40 kDa (particularly IGFBP-2) than ++ healthy follicles. In vitro, in basal conditions as well as in IGF-I- or FSH-stimulated conditions (or both), granulosa cells from F+ follicles had a lower proliferative activity, secreted higher amounts of progesterone, and expressed higher levels of P450(scc) than granulosa cells from ++ follicles of the same size. When F+ and ++ preovulatory follicles were compared at the end of the follicular phase, IGFBPs <40 kDa concentrations were slightly higher, and responsiveness of granulosa cells to FSH in vitro was lower in F+ than in ++ follicles, suggesting that terminal maturation of F+ follicles, although precocious, was less complete than it was in ++ follicles. The early decrease in intrafollicular IGFBPs <40 kDa concentrations observed in F+ antral follicles, which likely leads to an early increase in IGF bioavailability, may at least partly account for the increased ovulation rate that characterizes F-carrier ewes.     

Paracellular transport of insulin-like growth factor-I (IGF-I) across human umbilical vein endothelial cell monolayers

Insulin-like growth factors (IGFs) are well defined mitogens and growth promoters, which are found in blood associated with high affinity IGF binding proteins (IGFBPs). In vivo, the endothelium is potentially the primary site of uptake of IGFs or IGF-IGFBP complexes from blood for transport to the extravascular space. However, the pathway and mechanisms by which IGFs cross the endothelial cell barrier are not known. The presence of high affinity receptors for IGF-I and IGF-II on human umbilical vein endothelial (HUVE) cells was demonstrated by (i) radio-receptor assays using both IGF-I and IGF-II and (ii) affinity label cross-linking studies. In addition, Western ligand blotting and immunoblotting revealed that IGFBP-2, -3, and -4 are secreted into serum-free media conditioned by confluent HUVE cell monolayers. To study transendothelial migration of IGF-I, HUVE cells were grown on microporous membranes in a bichamber system. When compared with membranes without cells, HUVE monolayers restricted the passage of ¹²⁵I-IGF-I and [³H]inulin, whereas the control Madin Darby canine kidney (MDCK) cell line virtually excluded all passage of these molecules. Transport of ¹²⁵I-IGF-I across HUVE cell monolayers was not significantly different to that of [³H]inulin, a paracellular probe. Moreover, ¹²⁵I-IGF-I transport was not inhibited by either excess unlabelled IGF-I or a monoclonal antibody to the type I IGF receptor at a concentration shown to inhibit ¹²⁵I-IGF-I binding to HUVE cell monolayers. Our findings show that the movement of free IGF-I across HUVE cell monolayers occurs via a paracellular route and not by a receptor-mediated, transcellular pathway. J. Cell. Physiol. 170:290–298, 1997. © 1997 Wiley-Liss, Inc.     

Matrix metalloproteinase-7 degrades all insulin-like growth factor binding proteins and facilitates insulin-like growth factor bioavailability

Proteolytic modification of insulin-like growth factor binding proteins (IGFBPs) plays an important physiological role in regulating insulin-like growth factor (IGF) bioavailability. Recently, we demonstrated that matrix metalloproteinase-7 (MMP-7)/Matrilysin produced by various cancer cells catalyzes the proteolysis of IGFBP-3 in vitro and regulates IGF bioavailability, resulting in an anti-apoptotic effect against anchorage-independent culture. In the present study, we investigated whether MMP-7 contributes to proteolysis of the other five IGFBPs, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6, and whether this results in phosphorylation of the IGF type 1 receptor (IGF-1R). MMP-7 cleaved all six IGFBPs, resulting in IGF-mediated IGF-1R phosphorylation, which was inhibited by EDTA treatment. These results suggest that MMP-7 derived from cancer cells can regulate IGF bioavailability in the microenvironment surrounding the tumor, where various kinds of IGF/IGFBP complexes are found, thereby favoring cancer cell growth and survival during the processes of invasion and metastasis.     

Expression of IGF-binding protein-1 phosphoisoforms in fasted rat skin and its role in regulation of collagen biosynthesis

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen and glycosaminoglycan (GAG) biosynthesis in tissues. IGF-I activity is modulated by a family of IGF-binding proteins (IGFBPs) with different IGF-I binding affinities. At least IGFBP-1 and IGFBP-2 are known as inhibitors of IGF functions. Some IGFBPs (IGFBP-1, IGFBP-3 and IGFBP-5) may undergo phosphorylation that dramatically increase their affinity for IGF. During fasting of animals there is a significant decrease of the collagen and GAG content of the skin, accompanied by a reduction of plasma IGF-I levels. However, in previous studies we showed that in the skin of fasted rats IGF-I as well as IGFBP-1 and IGFBP-2 expressions were not different, compared to control rat skin, although collagen content was significantly decreased. In the present study we show that fasted rat skin contains similar amounts of IGF-I, IGFBP-3 and IGFBP-1, although extract from fasted rat skin induced inhibition of collagen biosynthesis in cultured fibroblasts, compared to control rat skin extract. Western immunoblot analysis of control and fasted rat skin extracts, using anti-phosphoserine antibodies for immunoprecipitated IGFBP-1 and IGFBP-3, revealed that both proteins are present in phosphorylated form. Although no differences were found in the expression of phosphorylated IGFBP-3 between control and fasted rat skins, that of phosphorylated IGFBP-1 in fasted rat skin extract was higher than in control one. We suggest that there is an increased level of IGFBP-1 phosphoisoform in fasted rat skin, associated with increased affinity for IGF-I. The increase of phosphorylated IGFBP-1 in fasted rat skin tissue may augment IGF-I binding affinity for IGF and decrease its bioavailability for receptor interaction. This mechanism may prevent IGF-I dependent stimulation of fibroblasts to produce extracellular matrix components. The specific expression of IGFBPs and their phosphoisoforms in tissues may play an important role in regulation of IGF-I action during physiologic and pathologic responses.     

Insulin-like growth factor binding protein-6: the "forgotten" binding protein?

Insulin-like growth factor binding protein-6 (IGFBP-6) differs from IGFBPs 1-5 in that it binds IGF-II with marked preferential affinity over IGF-I. Human and rat IGFBP-6 lack 2 and 4 N-terminal cysteines and therefore the Gly-Cys-Gly-Cys-Cys motif present in IGFBPs 1-5. IGFBP-6 is O-glycolsylated, and five serine/threonine glycosylation sites in the non-conserved mid-region of human IGFBP-6 have been identified. O-Glycosylation inhibits proteolysis of IGFBP-6 by chymotrypsin and trypsin, but has no effect on high affinity IGF binding. IGFBP-6 is a relatively specific inhibitor of IGF-II actions; it has not been shown to potentiate IGF actions. IGFBP-6 is only cell-associated to a very limited extent, if at all. IGFBP-6 is often expressed in non-proliferative, quiescent states in vitro and differentiating agents increase its expression. IGFBP-6 expression is associated with inhibition of growth of tumour cells in vitro and in vivo. Although many questions remain regarding the biological role of IGFBP-6, its major function appears to be the regulation of IGF-II actions. This could be especially significant since IGF-II has been implicated as an autocrine tumour growth factor.     

Enhanced insulin-like growth factor-I (IGF-I) cell association at reduced pH is dependent on IGF binding protein-3 (IGFBP-3) interaction

The cellular microenvironment impacts how signals are transduced by cells and plays a key role in tissue homeostasis. Although pH is generally well regulated, there are a number of situations where acidosis occurs and our work addresses how low pH impacts cell association of insulin-like growth factor-I (IGF-I) in the presence of IGF binding protein-3 (IGFBP-3). We have previously shown that IGF-I cell binding was enhanced in the presence of IGFBP-3 at low pH and now show that this binding is IGFBP-mediated as it is inhibited by Y60L-IGF-I, a mutant with reduced affinity for the IGF receptor (IGF-IR), and unaffected by insulin, which binds but not IGFBPs. Using surface plasmon resonance (SPR), we show that direct binding between IGF-I and IGFBP-3 is pH sensitive. Despite this, the key step in the process appears to be IGFBP-3 cell surface association as Long-R(3)-IGF-I, a mutant with reduced affinity for IGFBPs, shows a similar increase in cell association at pH 5.8 in the presence of IGFBP-3 but does not exhibit pH-dependent binding by SPR. Further, analysis indicates a large increase in low-affinity binding sites for IGF-I in the presence of IGFBP-3 and an elimination of IGF-I enhanced binding when a non-cell associating mutant of IGFBP-3 is added in place of IGFBP-3. That the IGFBP-3-mediated binding localizes IGF-I away from IGF-IR is suggested by triton-solubility testing and indicates additional complexities to IGF-I regulation by IGFBP-3. Identifying the pH-dependent binding partner(s) for IGFBP-3 is a necessary next step in deciphering this process.     

Insulin-like growth factor binding protein-2 mediates the inhibition of DNA synthesis by transforming growth factor-beta in mink lung epithelial cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) has been proposed to mediate the growth inhibitory effects of transforming growth factor (TGF)-beta in breast and prostate cancer cells. Both TGF-beta and exogenous IGFBP-3 inhibit DNA synthesis in Mv1 mink lung epithelial cells (CCL64). The present study asks whether IGFBPs synthesized by CCL64 cells mediate growth inhibition by TGF-beta. CCL64 cells synthesize and secrete a single 34-kDa IGFBP that was identified as IGFBP-2 by immunoprecipitation and immunodepletion. Recombinant bovine IGFBP-2 inhibited CCL64 DNA synthesis in serum-free media in an IGF-independent manner. Coincubation with Leu(60)-IGF-I, an IGF-I analog that binds to IGFBPs with higher affinity than to IGF-I receptors, decreased the inhibition by bIGFBP-2. Leu(60)-IGF-I also decreased the inhibition of CCL64 DNA synthesis by TGF-beta by up to 70%, whereas Long-R3-IGF-I, an IGF-I analog with higher affinity for IGF-I receptors than for IGFBPs, did not decrease inhibition, suggesting that the effect of Leu(60)-IGF-I resulted from its forming complexes with endogenous IGFBPs. Leu(60)-IGF-I did not decrease TGF-beta stimulation of a Smad3-dependent reporter gene. Following incubation of intact CCL64 cells with bIGFBP-2 at 0 degrees C, bIGFBP-2 was recovered in membrane fractions; membrane association was abolished by coincubation with Leu(60)-IGF-I. If exogenous and secreted IGFBP-2 must bind to CCL64 cells to inhibit DNA synthesis, Leu(60)-IGF-I might reduce the inhibition of DNA synthesis by bIGFBP-2 or TGF-beta by inhibiting the association of IGFBP-2 in the media with CCL64 cells. Since TGF-beta does not increase IGFBP-2 abundance, we propose that TGF-beta sensitizes CCL64 cells to the latent growth inhibitory activity of endogenous IGFBP-2 by potentiating an intracellular IGFBP-2 signaling pathway or by promoting the association of secreted IGFBP-2 with the plasma membrane.     

Mitogenic and antiapoptotic effects of insulin-like growth factor binding protein-6 in the human osteoblastic osteosarcoma cell line Saos-2/B-10.

Insulin-like growth factor (IGF) I is a potent mitogen for human osteosarcoma cells such as the Saos-2/B-10 cell line. IGF binding proteins (IGFBPs) prevent stimulation of DNA synthesis by IGFs. In contrast to recombinant human (rh) IGFBP-2, -3, -4, and -5, 10-100 nM rhIGFBP-6 stimulated [(3)H]thymidine incorporation into DNA and multiplication of Saos-2/B-10 cells. Upon withdrawal of serum, 30 nM IGFBP-6 also decreased apoptosis (within 4 h) and increased protein content and sodium-dependent phosphate uptake (within 24 h), but less potently than IGF I. (125)I-labeled rhIGFBP-6 did not bind to the cells, and cold IGFBP-6 did not affect (125)I-labeled IGF I binding. Production of IGF I, IGF II, and IGFBP-6 by the cells or significant degradation of rhIGFBP-6 could not be detected within 24 h of incubation. Thus, among the rhIGFBPs tested, rhIGFBP-6 is unique in stimulating osteosarcoma cell growth. Furthermore, it has an antiapoptotic effect.     

Characterization of insulin-like growth factor binding protein-1 kinases from human hepatoma cells

The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate M_r 150,000 (peak I kinase) and M_r 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC₅₀ = 2.5 and 16.5 μg/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC₅₀ = 50 μM and 100 μM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-³²P]ATP lowered the incorporation of ³²P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-³²P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and peak II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases. © 1996 Wiley-Liss, Inc.