Abstract. Insulin-like growth factor binding proteins (IGFBPs) have been shown to serve as carrier proteins for the insulin-like growth factors (IGFs) and to modulate their biologic effects. Since extracellular matrix (ECM) has been shown to be a reservoir for IGF-I and IGF-U, we examined the ECM of cultured human fetal fibroblasts and found that IGFBP-5 was incorporated intact into ECM, while mostly inert proteolytic fragments were found in the medium. In contrast, two other forms of IGFBP that are secreted by these cells were either present in ECM in minimal amounts (IGFBP-3) or not detected (IGFBP-4). Likewise, when purified IGFBPs were incubated with ECM, IGFBP-5 bound preferentially. IGFBP-5 was found to bind to types III and IV collagen, laminin, and fibronectin. Increasing salt concentrations inhibited the binding of IGFBP-5 to ECM and accelerated the release of IGFBP-5 from ECM, suggesting an ionic basis for this interaction. ECM-associated IGFBP-5 had a sevenfold decrease in affinity for IGF-I compared to IGFBP-5 in solution. Furthermore, when IGFBP-5 was present in cell culture substrata, it potentiated the growth stimulatory effects of IGF-I on fibroblasts. When IGFBP-5 was present only in the medium, it was degraded to a 22-kD fragment and had no effect on IGF-I-stimulated growth. We conclude that IGFBP-5 is present in fibroblast ECM, where it is protected from degradation and can potentiate the biologic actions of IGF-I. These findings provide a molecular explanation for the association of the IGF's with the extracellular matrix, and suggest that the binding of the IGF's to matrix, via IGFBP-5, may be important in mediating the cellular growth response to these growth factors.
Insulin-like growth factor (IGF)-binding protein 1 (IGFBP-1) contains an Arg-Gly-Asp (RGD) integrin recognition sequence. In vitro mutagenesis was used to alter this RGD sequence to Trp-Gly-Asp (WGD). Migration of Chinese hamster ovary (CHO) cells expressing the wild-type protein was more than 3-fold greater in 48 hr compared with ceUs expressing the WGD mutant form of IGFBP-1. Similarly, wild-type IGFBP-1 added to the media of control CHO cells stimulated migration 2-fold compared with the WGD protein.A synthetic RGD-containing peptide, when added to the medium with wild-type IGFBP-1, blocked the effect of IGFBP-1 on cell migration. The addition of IGF-I to the culture medium had no effect on the migration of cells expressing IGFBP-1 or vector alone. Affmity chromatography of 12I-labeled CHO cel membrane proteins, using IGFBP-1 coupled to agarose, iden- These studies demonstrate that IGFBP-1 stimulates CHO cell nmgration and binds to the a4s31 integrin receptor, both by an RGD-dependent mechanism. The effect of IGFBP-1 on migration is independent of IGF-I and is probably mediated through the a43.B integrin.The insulin-like growth factor (IGF)-binding proteins (IGFBPs) are a family of homologous but distinct proteins that specifically bind IGF-I and IGF-II with high affinity and are ubiquitous in biologic fluids, tissues, and the extracellular matrix of most cell types (1). Currently six IGFBPs have been purified, cloned, and sequenced (2,3). The roles of these proteins in intercellular transport, extracellular localization, and the modulation of the actions of IGF-I and IGF-II are areas of active study. The major focus of studies published to date has been to examine the effects of the IGFBPs on modifying IGF physiology. However, few direct non-IGFmediated effects of a purified form of IGFBP on cellular functions have been reported. IGFBP-1 is a phosphorylated protein of approximately 25 kDa that is expressed in greatest amounts during fetal development. It has been reported to potentiate (4) or inhibit (5) IGF actions, depending upon the experimental conditions and degree of IGFBP-1 phosphorylation (6). IGFBP-1 contains an Arg-Gly-Asp (RGD) integrin recognition sequence (7,8) and has been shown to bind to cell surfaces (9). The specific mechanism by which IGFBP-1 or any of the other IGFBPs bind to cell surfaces has not been previously described.We undertook these studies after observing that transfected Chinese hamster ovary (CHO) cells expressing humanThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.IGFBP-1 appeared to spread and migrate on tissue culture plates more rapidly than CHO cells not expressing IGFBP-1. Cell migration is known in many cell types to involve cellular integrin receptor binding to RGD sequences in extracellular matrix proteins (10). Since IGFBP-1 binds to cell surfaces and contains an RGD sequence, we sought to determine ...
Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 1251 and immunoprecipitated with specific anti-integrin antibodies. Integrins containing aV (vitronectin receptor), a5 (fibronectin receptor), and a3 (collagen/ laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in a5-integrin subunit protein and a 25% increase in aV subunit. More importantly, ligand binding of aVf33 was increased by 2.4-fold. We therefore examined whether the function of the aVf83 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to aV.83 and not to a5j31 or other abundant integrins. The related disintegrin echistatin specifically inhibited 1251-labeled kistrin binding to cVI83, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the aiV integrin. Furthermore, an aVf83 blocking antibody inhibited SMC migration by 80%o. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and aVj33 integrin antagonists appear to be impor-
Smooth muscle cells have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the role of IGF-binding proteins (IGFBPs) in this process has not been determined. As IGFBPs are synthesized and secreted by smooth muscle cells and bind to IGFs with high affinity, this study was undertaken to determine the ability of IGFBP-1 and -2 to modulate cellular migration in response to IGF-I and -II. Confluent monolayers of porcine vascular smooth muscle cells were wounded with a razor blade. After wounding, IGF-I and -II induced increases of 159 +/- 49% (mean +/- SD) and 108 +/- 33%, respectively, above control values in the number of cells migrating over a fixed distance in 4 days. The addition of IGFBP-1 caused a 19-21% inhibition of IGF-I- or IGF-II-stimulated migration, whereas the addition of IGFBP-2 inhibited the effect of both by greater than 60%. The addition of IGFBP-2 alone had no effect, whereas IGFBP-1 addition was associated with a 42 +/- 12% increase. In contrast, [Trp221]IGFBP-1 in which the RGD sequence was changed to WGD, thus eliminating its capacity to bind to the alpha 5 beta 1 integrin, inhibited IGF-I-stimulated migration by 67 +/- 17%. An IGF analog that has a reduced affinity for IGFBP-2-stimulated migration equally well as IGF-I alone even in the presence of IGFBP-2. Likewise, the addition of insulin, which cannot bind to IGFBPs, at supraphysiological concentrations that are adequate to activate the IGF-I receptor resulted in a similar increase in migration. In summary, IGF-I and -II stimulate smooth muscle cell migration after wounding. This migratory response is modulated by IGFBPs. Both IGFBP-1 and IGFBP-2 appear to neutralize the effects of the IGFs by inhibiting their interaction with IGF receptors, but IGFBP-1 also has a direct stimulatory effect that requires an intact RGD integrin recognition sequence.
Vascular smooth muscle cells (SMC) synthesize insulin-like growth factor-I (IGF-I), which is a mitogen for this cell type in vitro. Since IGF binding proteins (IGFBP) modulate IGF bioactivity, we determined which IGFBPs were secreted by porcine SMC. Porcine SMC secreted 34,000 and 24,000 M(r) forms of IGFBPs which were identified as IGFBP-2 and IGFBP-4, respectively, by immunoblotting. Northern blot analysis showed single transcripts of 1.6 kb and 2.4 kb for IGFBP-2 and IGFBP-4, respectively. Secretion of IGFBP-2 was not regulated to a significant degree, with insulin, IGF-II, IGF-I, forskolin, and dibutyryl cyclic adenosine monophosphate (cAMP) inducing minimal changes in IGFBP-2 secretion of less than 30% by radioimmunoassay (RIA). Insulin increased (2.8 +/- 0.1-fold) the abundance of IGFBP-4 protein in conditioned media (CM) and increased IGFBP-4 mRNA levels. Growth factors for SMC such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor beta-1 (TGF beta-1) were without effect on either IGFBP-2 or -4. IGF-I treatment decreased the amount of IGFBP-4 present in CM, but a corresponding decrease in IGFBP-4 mRNA levels was not observed. In order to determine if IGFBP-4 could modulate IGF-I bioactivity, IGFBP-4 was added to pSMCs with and without IGF-I. IGF-I alone (20 ng/ml) induced a 1.6 to threefold increase in 3H-thymidine incorporation. Addition of IGFBP-4 (between 50 and 250 ng/ml) to cultures containing IGF-I (20 ng/ml) had no effect on DNA synthesis compared to that observed with IGF-I alone, while 500 ng/ml consistently caused a small decrease (15 +/- 5%; mean +/- SE). Immunoblotting of the CM obtained at the end of the 3H-thymidine assay showed a loss of intact IGFBP-4 in the cultures containing IGF-I. This corresponded with an increase in the abundance of a 16,000 M(r) immunoreactive fragment that did not bind IGF-I. Coincubation with insulin had no effect on the amount of IGFBP-4 that was converted to fragment, suggesting that the reaction was dependent upon IGF-I binding to IGFBP-4. In contrast, addition of IGFBP-4 (500 ng/ml) to human fibroblast cultures with IGF-I (20 ng/ml) almost completely inhibited the stimulatory effect of IGF-I on DNA synthesis and no increase in fragment was detected in the CM. In summary, SMC secrete IGFBP-2 and IGFBP-4, both of which have been shown to regulate IGF-mediated DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)
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