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• W2024707576 abstract "Insulin-like growth factor (IGF)-I is a pleiotropic hormone that regulates vascular smooth muscle cell (VSMC) migration, proliferation, apoptosis, and differentiation. These actions are mediated by the IGF-I receptor. How activation of the same receptor by the same ligand leads to these diverse cellular responses is not well understood. Here we describe a novel mechanism specifying VSMC responses to IGF-I stimulation, distinctive for the pivotal roles of local IGF-binding proteins (IGFBPs). The role of local IGFBPs was indicated by comparing the activities of IGF-I and des-1–3-IGF-I, an IGF-I analog with reduced binding affinity to IGFBPs. Compared with IGF-I, des-1–3-IGF-I was more potent in stimulating DNA synthesis but much less potent in inducing directed migration of VSMCs. When the effects of individual IGFBPs were tested, IGFBP-2 and IGFBP-4 were found to inhibit IGF-I-stimulated DNA synthesis and migration. IGFBP-5 had an inhibitory effect on IGF-I-stimulated DNA synthesis, but it strongly potentiated IGF-I-induced VSMC migration. By using a non-IGF-binding IGFBP-5 mutant and an IGF-I-neutralizing antibody, it was demonstrated that IGFBP-5 also stimulates VSMC migration in an IGF-independent manner. This effect of IGFBP-5 was inhibited by soluble heparin and by treating cells with heparinase. Mutation of the heparin-binding motif of IGFBP-5 reduced its migration promoting activity. These findings suggest that local IGFBPs are important determinants of cellular responses to IGF-I stimulation, and a key player in this paradigm is IGFBP-5. IGFBP-5 not only modulates IGF-I actions, but it also stimulates cell migration by interacting with cell-surface heparan sulfate proteoglycans. Insulin-like growth factor (IGF)-I is a pleiotropic hormone that regulates vascular smooth muscle cell (VSMC) migration, proliferation, apoptosis, and differentiation. These actions are mediated by the IGF-I receptor. How activation of the same receptor by the same ligand leads to these diverse cellular responses is not well understood. Here we describe a novel mechanism specifying VSMC responses to IGF-I stimulation, distinctive for the pivotal roles of local IGF-binding proteins (IGFBPs). The role of local IGFBPs was indicated by comparing the activities of IGF-I and des-1–3-IGF-I, an IGF-I analog with reduced binding affinity to IGFBPs. Compared with IGF-I, des-1–3-IGF-I was more potent in stimulating DNA synthesis but much less potent in inducing directed migration of VSMCs. When the effects of individual IGFBPs were tested, IGFBP-2 and IGFBP-4 were found to inhibit IGF-I-stimulated DNA synthesis and migration. IGFBP-5 had an inhibitory effect on IGF-I-stimulated DNA synthesis, but it strongly potentiated IGF-I-induced VSMC migration. By using a non-IGF-binding IGFBP-5 mutant and an IGF-I-neutralizing antibody, it was demonstrated that IGFBP-5 also stimulates VSMC migration in an IGF-independent manner. This effect of IGFBP-5 was inhibited by soluble heparin and by treating cells with heparinase. Mutation of the heparin-binding motif of IGFBP-5 reduced its migration promoting activity. These findings suggest that local IGFBPs are important determinants of cellular responses to IGF-I stimulation, and a key player in this paradigm is IGFBP-5. IGFBP-5 not only modulates IGF-I actions, but it also stimulates cell migration by interacting with cell-surface heparan sulfate proteoglycans. Insulin-like growth factors (IGFs), 1The abbreviations used are: IGF, insulin-like growth factor; IGFBPs, IGF-binding proteins; VSMC, vascular smooth muscle cell; IGF-IR, IGF-I receptor; DMEM, Dulbecco's minimum essential medium; FBS, fetal bovine serum; SFM, serum-free DMEM; BrdUrd, 5-bromo-2-deoxyuridine; WT, wild type; GAGs, glycosaminoglycans; BSA, bovine serum albumin; HS, heparan sulfate. including IGF-I and IGF-II, are peptide growth factors structurally related to proinsulin. IGFs are chemoattractants, survival factors, and mitogens for vascular smooth muscle cells (VSMCs) (1Clemmons D.R. Diabetes Rev. 1997; 5: 353-364Google Scholar, 2Bayes-Genis A. Conover C.A. Schwartz R.S. Circ. Res. 2000; 86: 125-130Crossref PubMed Scopus (385) Google Scholar, 3Zaina S. Pettersson L. Ahrén B. Brånén L. Hassan A.B. Lindhol M. Mattsson R. Thyberg J. Nilsson J. J. Biol. Chem. 2002; 277: 4505-4511Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 4Bornfeldt K.E. Raines E.W. Nakano T. Graves L.M. Krebs E.G. Ross R. J. Clin. Investig. 1994; 93: 1266-1274Crossref PubMed Scopus (382) Google Scholar, 5Wang J.W. Niu W. Nikiforov Y. Naito S. Chernausek S. Witte D. LeRoith D. Strauch A. Fagin J.A. J. Clin. Investig. 1997; 100: 1425-1439Crossref PubMed Scopus (169) Google Scholar, 6Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 16836-16842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 7Bai H.Z. Pollman M.J. Inishi Y. Gibbons G.H. Circ. Res. 1999; 85: 229-237Crossref PubMed Scopus (93) Google Scholar, 8Duan C. Bauchat J.R. Hsieh T. Circ. Res. 2000; 86: 15-23Crossref PubMed Scopus (150) Google Scholar, 9Patel V.A. Zhang Q.J. Siddle K. Soos M.A. Goddard M. Weissberg P.L. Bennett M.R. Circ. Res. 2001; 88: 895-902Crossref PubMed Scopus (99) Google Scholar). In addition, IGFs have been shown to regulate VSMC differentiation, glucose uptake, protein synthesis, and contractility (10Sowers J.R. Hypertension. 1997; 29: 691-699Crossref PubMed Scopus (168) Google Scholar). These diverse actions of IGFs are mediated through the IGF-I receptor (IGF-IR) and its downstream signaling transducers, including mitogen-activated protein kinases, phosphatidylinositol 3-kinase, and protein kinase C (7Bai H.Z. Pollman M.J. Inishi Y. Gibbons G.H. Circ. Res. 1999; 85: 229-237Crossref PubMed Scopus (93) Google Scholar, 8Duan C. Bauchat J.R. Hsieh T. Circ. Res. 2000; 86: 15-23Crossref PubMed Scopus (150) Google Scholar, 11Imai Y. Clemmons D.R. Endocrinology. 1999; 140: 4228-4235Crossref PubMed Google Scholar, 12Yano K. Bauchat J.R. Limatta M. Clemmons D.R. Duan C. Endocrinology. 1999; 140: 4622-4632Crossref PubMed Google Scholar, 13Duan C. Mol. Cell. Endocrinol. 2003; 206: 75-83Crossref PubMed Scopus (24) Google Scholar). However, it remains poorly understood how activation of the same receptor by the same ligand(s) leads to these diverse, sometimes even mutually exclusive, cellular responses. It is now understood that most, if not all, IGFs in the extra-cellular environment are bound to specific, high affinity IGF-binding proteins (IGFBPs). IGFBPs are a family of secreted proteins that specifically bind IGF-I and IGF-II with affinities that are equal to or greater than those of the IGF-IR. Six distinct IGFBPs, designated as IGFBP-1–6, have been isolated and characterized from a variety of vertebrate species, ranging from humans to teleost fish (14Baxter R.C. Am. J. Physiol. 2000; 278: E967-E976Crossref PubMed Google Scholar, 15Clemmons D.R. Endocr. Rev. 2001; 22: 800-817Crossref PubMed Scopus (122) Google Scholar, 16Duan C Ding J. Li Q. Tsai W. Pozios K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15274-15279Crossref PubMed Scopus (121) Google Scholar, 17Maures T. Duan C. Endocrinology. 2002; 143: 2722-2731Crossref PubMed Scopus (77) Google Scholar). The circulating IGF·IGFBP complexes prolong the half-lives of IGFs and inhibit the potential hypoglycemic effects of high concentrations of circulating IGFs. Locally expressed IGFBPs are thought to provide a means of localizing IGFs to specific cells and regulating their local availability and biological activity (14Baxter R.C. Am. J. Physiol. 2000; 278: E967-E976Crossref PubMed Google Scholar, 15Clemmons D.R. Endocr. Rev. 2001; 22: 800-817Crossref PubMed Scopus (122) Google Scholar). Previous studies in porcine, bovine, rat, and mouse indicate that mammalian VSMCs secrete IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 (6Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 16836-16842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 9Patel V.A. Zhang Q.J. Siddle K. Soos M.A. Goddard M. Weissberg P.L. Bennett M.R. Circ. Res. 2001; 88: 895-902Crossref PubMed Scopus (99) Google Scholar, 18Giannella-Neto D. Kamyar A. Sharifi B. Pirola C.J. Kupfer J. Rosenfeld R.G. Forrester J.S. Fagin J.A. Circ. Res. 1992; 71: 646-656Crossref PubMed Scopus (60) Google Scholar, 19Cohick W. Gockerman A. Clemmons D.R. J. Cell. Physiol. 1993; 157: 52-60Crossref PubMed Scopus (79) Google Scholar, 20Cohick W. Gockerman A. Clemmons D.R. J. Cell. Physiol. 1995; 164: 187-196Crossref PubMed Scopus (25) Google Scholar, 21Kamyar A. Pirola C.J. Wang H.M. Sharifi B. Mohan S. Forrester J.S. Fagin J.A. Circ. Res. 1994; 74: 576-585Crossref PubMed Scopus (52) Google Scholar, 22Duan C. Hawes S.B. Prevette T. Clemmons D.R. J. Biol. Chem. 1996; 272: 4280-4288Abstract Full Text Full Text PDF Scopus (84) Google Scholar, 23Anwar A. Zahid A.A. Scheidegger K.J. Brink M. Delafontaine P. Circulation. 2002; 105: 1220-1225Crossref PubMed Scopus (90) Google Scholar). These IGFBPs, when added exogenously to cultured VSMCs in combination with IGF-I, can either inhibit or potentiate IGF-I-induced DNA synthesis and migration (6Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 16836-16842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 19Cohick W. Gockerman A. Clemmons D.R. J. Cell. Physiol. 1993; 157: 52-60Crossref PubMed Scopus (79) Google Scholar, 21Kamyar A. Pirola C.J. Wang H.M. Sharifi B. Mohan S. Forrester J.S. Fagin J.A. Circ. Res. 1994; 74: 576-585Crossref PubMed Scopus (52) Google Scholar, 24Gockerman A. Prevette T. Jones J.I. Clemmons D.R. Endocrinology. 1995; 136: 4168-4173Crossref PubMed Google Scholar, 25Rees C. Clemmons D.R. J. Cell. Biochem. 1998; 71: 375-381Crossref PubMed Scopus (20) Google Scholar, 26Duan C. J. Endocrinol. 2002; 175: 41-54Crossref PubMed Scopus (127) Google Scholar). Recent studies have also revealed that the abundance of these local IGFBPs vary significantly under different pathophysiological conditions and different cellular contexts. For example, higher levels of IGFBP-5 were found in atherosclerotic plaques compared with tissue from the normal vessel wall (27Zheng B. Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 8994-9000Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In vitro, synthetic VSMCs maintained on fibronectin synthesize and release more IGFBP-5 compared with differentiated contractile cells grown on laminin and type IV collagen (27Zheng B. Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 8994-9000Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Likewise, the biosynthesis and secretion of IGFBP-5 by porcine VSMCs decreased by 18-fold when cell density increases from 30 to 100% confluence, whereas the concentrations of IGFBP-4 increased up to 15-fold (6Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 16836-16842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Because different IGFBPs have different biological effects (14Baxter R.C. Am. J. Physiol. 2000; 278: E967-E976Crossref PubMed Google Scholar, 15Clemmons D.R. Endocr. Rev. 2001; 22: 800-817Crossref PubMed Scopus (122) Google Scholar), and because the availability of IGFBPs is differentially regulated by the cellular context, we hypothesized that locally produced IGFBPs may be important in determining specific responses to the IGF signal. The goals of this study were to determine whether IGFBPs regulate the chemotactic and mitogenic responses of VSMCs to IGF stimulation, and to elucidate the individual role(s) and underlying mechanisms of local IGFBPs in regulating VSMC migration and proliferation. Materials—All chemicals and reagents were purchased from Fisher unless noted otherwise. The neutralizing IGF-I antibody (Sm1.2) was a gift from Dr. Judson VanWyk at the University of North Carolina, Chapel Hill. The rainbow molecular weight marker and [3H]thymidine were obtained from ICN Biomedicals Inc. (Irvine, CA). Human IGF-I, des-1–3-IGF-I, IGFBP-2, and IGFBP-4 were purchased from GroPep (Adelaide, Australia). Recombinant human IGFBP-5 was purchased from IBT-Immunological and Biochemical Testsystems GmbH (Reutlingen, Germany). The non-IGF-binding human IGFBP-5 mutant and heparin-binding deficient human IGFBP-5 mutant (K202A/R206N/R207A) were prepared as described previously (28Imai Y. Moralez A. Andag U. Clarke J.B. Busby Jr., W.H. Clemmons D.R. J. Biol. Chem. 2000; 275: 18188-18194Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 29Arai T. Clarke J. Parker A.J. Busby W.H. Nam T.J. Clemmons D.R. J. Biol. Chem. 1996; 271: 6099-6106Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Heparitinase (heparinase III) was purchased from Calbiochem. Chondroitinase, heparin, and various chondroitin sulfates were purchased from Sigma. Fetal bovine serum (FBS), Dulbecco's minimum essential medium (DMEM), penicillin/streptomycin, and trypsin were purchased from Invitrogen. Cell Culture and Transfection—Porcine VSMCs were isolated from thoracic aorta of 3-week-old piglets (22Duan C. Hawes S.B. Prevette T. Clemmons D.R. J. Biol. Chem. 1996; 272: 4280-4288Abstract Full Text Full Text PDF Scopus (84) Google Scholar). The cells were grown in 10-cm dishes (Falcon, BD Biosciences) in DMEM supplemented with glutamine (4 mm), penicillin (100 units/ml), streptomycin (100 μg/ml), and 10% FBS. The medium was changed every 4th day until the cells became confluent. Before stimulation experiments, medium was changed to serum-free DMEM (SFM) for 20–24 h. This SFM was then replaced with fresh SFM plus the indicated growth factors for various times. Cultured porcine VSMCs were transfected with the pMEP4/IGFBP-5 construct or empty pMEP4 following a procedure published previously (30Parker A. Rees C. Clarke J. Busby Jr., W.H. Clemmons D.R. Mol. Biol. Cell. 1998; 9: 2383-2392Crossref PubMed Scopus (70) Google Scholar). The transfected cells were trypsinized, plated on three 10-cm plates, and grown in DMEM plus 10% FBS with 120 μg/ml hygromycin B. Medium was analyzed for secretion of IGFBP-5 as described below. Western Immunoblot, Immunoprecipitation, and Ligand Blot Analysis—In order to identify the forms of IGFBPs secreted by VSMCs, conditioned media were concentrated 20 times through a Centricon-10 microconcentrator (Amicon) by centrifugation (22Duan C. Hawes S.B. Prevette T. Clemmons D.R. J. Biol. Chem. 1996; 272: 4280-4288Abstract Full Text Full Text PDF Scopus (84) Google Scholar). The proteins were separated by 12.5% SDS-PAGE under nonreducing conditions, and they were transferred to filters (Immobilon P, 0.45-μm pore size, Millipore Corp., Bedford, MA) and subjected to immunoblot and ligand blot analysis following standard procedures (6Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 16836-16842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Biological Assays—The effects of IGF-I and/or IGFBPs on VSMC DNA synthesis and growth was examined by direct cell counting, [3H]thymidine incorporation, and 5-bromo-2-deoxyuridine (BrdUrd) labeling assays as reported previously (8Duan C. Bauchat J.R. Hsieh T. Circ. Res. 2000; 86: 15-23Crossref PubMed Scopus (150) Google Scholar). The effects of IGF-I, IGFBPs, and selected soluble glycosaminoglycans (GAGs) on VSMC migration were investigated using the trans-well migration assay and gold particle cell motility assay. The trans-well migration assay measures the directional movements of VSMCs across a porous membrane, as described previously (8Duan C. Bauchat J.R. Hsieh T. Circ. Res. 2000; 86: 15-23Crossref PubMed Scopus (150) Google Scholar). The gold particle cell motility assay measures cell motility in the absence of a concentration gradient (31Alberch-Buehler G. Cell. 1977; 11: 395-404Abstract Full Text PDF PubMed Scopus (384) Google Scholar, 32Cheng H.-L. Steinway M. Russell J.W. Feldman E.L. J. Biol. Chem. 2000; 275: 27197-27204Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, a uniform carpet of gold particles was prepared on BSA-coated glass coverslips placed in 6-well plates. Colloidal gold-coated coverslips were rinsed and then seeded with 1 × 104 VSMCs in 2 ml of DMEM or DMEM + 100 ng/ml IGF-I. After 6 h, cells were fixed with 3.5% glutaraldehyde, air-dried, and mounted on glass slides. The gold particle-free clear zone surrounding the cells was photographed, and VSMC motility was represented as the average area of at least 60 pericellular clear zones, quantitated using Scion Image software (Frederick, MD). Removal of Cell-surface GAGs by GAG Lyase Treatment—Enzymatic treatment with GAG lyases was performed following published methods (33Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). Briefly, VSMCs cells were incubated with the GAG lyases in 1× phosphate-buffered saline containing 0.1% BSA, 0.2% gelatin, and 0.1% glucose for 40 min at 37 °C in a CO2 incubator. Cells were then washed 3 times with 1× phosphate-buffered saline, trypsinized, washed again with SFM, and used for migration assays. Statistical Analysis—Values are means ± S.E. Differences among groups were analyzed by one-way analysis of variance followed by Fisher's protected least significance difference test using StatView (Abacus Concept, Berkeley, CA). Significance was accepted at p < 0.05. The Local Repertoire of IGFBPs Is Critical in Determining Whether VSMCs Migrate or Proliferate in Response to IGF-I Stimulation—To test the hypothesis that the local repertoire of IGFBPs may affect the cellular responses to IGF-I stimulation, we examined the effects of IGF-I and des-1–3-IGF-I on VSMC growth and migration. Des-1–3-IGF-I is an IGF-I analog that binds to IGF-IR with normal affinity but has greatly reduced affinity for IGFBPs (34Francis G.L. Ross M. Ballard F.J. Milner S.J. Senn C. McNeil K.A. Wallace J.C. King R. Wells J.R.E. J. Mol. Endocrinol. 1992; 8: 213-223Crossref PubMed Scopus (167) Google Scholar). As shown in Fig. 1A, both IGF-I and des-1–3-IGF-I stimulated [3H]thymidine incorporation in a dose-dependent fashion. Compared with IGF-I, however, des-1–3-IGF-I was more potent at concentrations ranging from 1 to 20 ng/ml. At 10 ng/ml, des-1–3-IGF-I and IGF-I induced a 362.6 ± 10 and a 221.3 ± 15.7% increase over the control, respectively. The difference between these two groups was statistically significant (p < 0.05, n = 4). Similarly, 20 ng/ml of des-1–3-IGF-I caused a 444.2 ± 37.7% increase which is significantly higher than the 304.7 ± 31.4% increase induced by IGF-I at the same concentration (p < 0.05, n = 4). When the peptide concentration was increased to 50 ng/ml and higher, the difference between the two peptides disappeared, indicating saturation of endogenous IGFBPs by excess IGF-I added. When the chemotactic activities of IGF-I and des-1–3-IGF-I were examined, an opposite relationship was observed, i.e. IGF-I was a much stronger chemoattractant (Fig. 1B). At 20 ng/ml, IGF-I caused 833 ± 182% increase over the control group (p < 0.01, n = 3), whereas des-1–3-IGF-I only caused a 450 ± 113% increase over the control group (p < 0.05, n = 3). The difference between the IGF-I and des-1–3-IGF-I groups at this concentration was also statistically significant (p < 0.05). At 50 ng/ml, des-1–3-IGF-I induced migration was 441.7 ± 71.2%. This value was significantly lower than that of the IGF-I group (1183 ± 176.4%, p < 0.01). Similarly, IGF-I at a higher concentration (100 ng/ml) caused a significantly greater increase in VSMC migration than that of des-1–3-IGF-I (1216.7 ± 154.3% versus 208.3 ± 109.3%, p < 0.05). These data suggest that the endogenous IGFBPs strongly promote VSMC migration toward IGF-I, but they inhibit IGF-I-induced DNA synthesis. The distinct dose-response curves of IGF-I and des-1–3-IGF-I on cell migration are also suggestive of different mechanisms underlying their actions. Different IGFBPs Exhibit Different Biological Effects in Regulating Mitogenic and Chemotactic Actions of IGF-I—Cultured porcine VSMCs synthesize and secrete IGFBP-2, IGFBP-4, and IGFBP-5 (22Duan C. Hawes S.B. Prevette T. Clemmons D.R. J. Biol. Chem. 1996; 272: 4280-4288Abstract Full Text Full Text PDF Scopus (84) Google Scholar). To determine the individual effects of these IGFBPs, we tested the ability of each IGFBP to modulate the mitogenic and chemotactic actions of IGF-I. IGFBP-2 and IGFBP-4, when added together with IGF-I at an equal molar concentration, significantly inhibited IGF-I-stimulated BrdUrd incorporation (Fig. 2A). Neither IGFBP-2 nor IGFBP-4 alone caused any significant change. In migration assays, IGF-I (50 ng/ml) alone induced a 642 ± 181% increase (p < 0.05) (Fig. 2B). The addition of IGFBP-2, at an equal molar concentration (200 ng/ml), reduced IGF-I-induced migration to 133 ± 28% of the control; this was significantly less than that observed in the IGF-I alone group (p < 0.01). IGFBP-4 (150 ng/ml) had a similar, but more modest, inhibitory effect; it caused a 52% reduction in IGF-I-induced migration (p < 0.05). When either IGFBP-2 or IGFBP-4 was added in the absence of IGF-I, no significant change was observed (Fig. 2B). When added with IGF-I, IGFBP-5 caused a statistically insignificant 21% increase in IGF-I-induced DNA synthesis (Fig. 2A). In contrast, addition of IGFBP-5 together with IGF-I at the same concentrations resulted in a 1393 ± 372% increase in directed migration toward IGF-I (Fig. 2B). This value was significantly higher than that of the IGF-I alone group (642 ± 181%, p < 0.05). Intriguingly, addition of IGFBP-5 alone resulted in a statistically significant increase as compared with the control group (216 ± 32%, p < 0.05). IGFBP-5 alone had no effect on DNA synthesis in these cells. To examine further the effect of IGFBP-5, cells were subjected to different concentrations of IGFBP-5 in the presence and absence of IGF-I (20 ng/ml). As shown in Fig. 2C, addition of IGFBP-5 stimulated VSMC migration in a dose-dependent fashion with or without IGF-I. These results suggest that IGFBP-2 and IGFBP-4 act as inhibitors of IGF-I actions, whereas IGFBP-5 potentiates IGF-induced VSMC migration. The data also suggest that IGFBP-5 may alter the VSMC motility by itself. Expression of IGFBP-5 in VSMCs Increases the Basal and the IGF-I Induced Cell Migration but Decreases Cell Growth— Earlier studies showed that porcine VSMCs secrete a protease that specifically degrades IGFBP-5, but this protease does not cleave IGFBP-2 or IGFBP-4 (22Duan C. Hawes S.B. Prevette T. Clemmons D.R. J. Biol. Chem. 1996; 272: 4280-4288Abstract Full Text Full Text PDF Scopus (84) Google Scholar, 35Busby Jr., W.H. Nam T.-J. Moralez A. Smith C. Jennings M. Clemmons D.R. J. Biol. Chem. 2000; 275: 37638-37644Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The presence of this potent IGFBP-5 protease complicates the interpretation of the data obtained with the use of exogenous IGFBP-5. To define further the role of IGFBP-5, porcine VSMCs were transfected with an IGFBP-5 expression construct, and three independent groups of cells that stably expressed the transgene (+BP5) were obtained. The three +BP5 clones secreted considerably more IGFBP-5 (250, 345, and 180% compared with the wild-type control (WT), respectively). The empty expression vector (Mock)-transfected cells had low levels of IGFBP-5, comparable with those of WT. No significant variation in the IGFBP-2 levels was detected among +BP5, Mock, and WT cells (data not shown). The growth curves of three independent clones of Mock cells were similar to that of WT (Fig. 3A). In contrast, all three +BP5 clones had significantly reduced growth rate when compared with WT and Mock throughout the period. The difference was highly significant at 288 h (p < 0.01). Next, the IGF-I-induced DNA synthesis in these cells was examined by [3H]thymidine incorporation assays. As shown in Fig. 3B, IGF-I stimulated DNA synthesis in a dose-dependent manner in all groups. However, the basal levels of [3H]thymidine incorporation in the +BP5 clones were only 26 ± 7% of those observed in WT and Mock cells, and the level remained significantly lower even in the presence of increasing concentrations of IGF-I. Together, these data indicate that overexpression of IGFBP-5 inhibits IGF-I-stimulated porcine VSMC growth. The effect of IGFBP-5 expression on cell migration is shown in Fig. 4. In the absence of IGF-I, Mock-transfected cells were similar to the WT group in the basal migration rate (4.8 ± 0.2 versus 3.7 ± 1.2 cells/well). IGF-I caused a similar degree of chemotactic response in the WT and Mock cells (Fig. 4A). In contrast, the +BP-5 cells showed significantly higher levels of migration at all the concentrations of IGF-I tested (p < 0.01). Even in the absence of IGF-I, these cells showed an 8-fold increase over the WT control (29.6 versus 3.7 cells/well, p < 0.01, n = 3). Closer observation of the response curves revealed that IGFBP-5 overexpression caused a left shift in the dose-response curve to IGF-I. The maximum response was detected at 5 ng/ml of IGF-I in the +BP5 cells, which is much lower than the maximal response concentrations seen in WT and Mock cells (50 ng/ml), suggesting that IGFBP-5 enhances the cellular responsiveness to the IGF-I stimulation. To examine further the effect of IGFBP-5, cells from +BP5, Mock, and WT groups were analyzed using the gold particle cell motility assay. As shown in Fig. 4B, WT and Mock cells showed a similar degree of motility. The +BP5 clones showed a mean increase of 264 ± 52% compared with WT cells. To determine whether the effect of IGFBP-5 on basal cell motility was IGF-dependent, an IGF-I-neutralizing antibody (Sm1.2) was added. This antibody has been shown to neutralize IGF-I actions in porcine VSMCs (6Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 16836-16842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Addition of Sm1.2 did not cause any significant change in basal or IGFBP-5-induced motility. These results suggest that overexpression of IGFBP-5 in VSMCs not only potentiates IGF-I-induced chemotaxis but also stimulates basal cell motility rate through a ligand-independent mechanism(s). IGFBP-5 Stimulates VSMC Migration via an IGF-independent Mechanism—Further evidence for IGF-independent action of IGFBP-5 came from the check board analysis. IGF-I is a chemoattractant for VSMCs. It stimulated VSMC migration when added to the lower chamber (Fig. 5A). When the IGF-I concentration gradient was eliminated by adding the same concentration of IGF-I to both lower and upper chambers, it did not stimulate VSMCs migration. In contrast, addition of IGFBP-5 to both chambers resulted in an increase that was comparable with that in the lower chamber alone (Fig. 5A), suggesting that the effect of IGFBP-5 on VSMC migration is not chemotactic. Because it is known that cultured porcine VSMCs synthesize and secrete IGF-I (6Duan C. Clemmons D.R. J. Biol. Chem. 1998; 273: 16836-16842Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), it is possible that the exogenously added IGFBP-5 influences VSMC migration by interacting with the endogenous IGF-I. To clarify this issue, migration assays were performed in the presence and absence of the IGF-I-neutralizing antibody (Sm1.2). As shown in Fig. 5B, Sm1.2 completely abolished the chemotactic action of IGF-I (50 ng/ml) without affecting the level of basal migration. The addition of Sm1.2, however, did not change the higher basal migration rate of the IGFBP-5 group. To ascertain that the action of IGFBP-5 is independent from its IGF binding, we next examined the activity of a non-IGF binding IGFBP-5 mutant. This mutant has a 1000-fold reduced affinity for IGF-I (28Imai Y. Moralez A. Andag U. Clarke J.B. Busby Jr., W.H. Clemmons D.R. J. Biol. Chem. 2000; 275: 18188-18194Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and has been used to determine IGF-I-independent effects of IGFBP-5 on cellular physiologic processes in cell types that secrete IGF-I (36Perks C.M. McCaig C. Clarke J.B. Clemmons D.R. Holly J.M.P. Biochem. Biophys. Res. Commun. 2002; 294: 995-1000Crossref PubMed Scopus (22) Google Scholar). As shown in Fig. 5C, wild-type IGFBP-5 and the non-IGF-binding IGFBP-5 mutant both caused a significant increase. The mutant caused a 565% increase (32.7 ± 4.1 versus 5.8 ± 1.4 cells/well, p < 0.01 compared with the BSA control). This value was even higher than that of the wild-type IGFBP-5 group (21.8 ± 3.9 cells/well, a 376% increase over the BSA control) although the difference was not statistically significant. Taken together, these data indicate that IGFBP-5 stimulates VSMC migration through an IGF-I-independent mechanism. IGFBP-5 Stimulates VSM Migration by Interacting with Cell-surface GAGs—Because IGFBP-5 binds to heparin and GAG-containing proteoglycans located on VSMC surface (29Arai T. Clarke J. Parker A.J. Busby W.H. Nam T.J. Clemmons D.R. J. Biol. Chem. 1996; 271: 6099-6106Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 37Arai T. Parker A.J. Busby W.H. Clemmons D.R. J. Biol. Chem. 1994; 269: 20388-20393Abstract Full Text PDF PubMed Google Scholar) and because a peptide derived from the heparin-binding motif of IGFBP-5 has been shown to stimulate mesangial cell migration (38Abrass C.K. Berfield A.K. Andress D.L. Am. J. Physiol. 1997; 273: F899-F906PubMed Google Scholar), we postulated that IGFBP-5 may regulate VSMC motility by interacting with GAGs containing proteoglycans on the cell surface. If this were correct, then one or more of the four groups of GAGs found on membrane-associated proteoglycans should act as competitive inhibitors of IGFBP-5 actions. We therefore tested the impact of the 4 major GAGs found on membranes, i.e. heparin/heparan sulfate (HS), chondroitin A, chondroitin B, and chondroitin C. Of the four GAGs, heparin at t" @default.
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• W2024707576 title "Regulation of Vascular Smooth Muscle Cell Responses to Insulin-like Growth Factor (IGF)-I by Local IGF-binding Proteins" @default.
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• W2024707576 doi "https://doi.org/10.1074/jbc.m303835200" @default.
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