G-00345-2005.R1 Kuemmerle-1 Occupation of αVβ3 integrin by endogenous ligands modulates IGF-I receptor activation and proliferation of human intestinal smooth muscle

We have previously shown that endogenous Insulin-like growth factor-I (IGF-I) regulates growth of human intestinal smooth muscle cells by stimulating proliferation and inhibiting apoptosis. In active Crohn’s disease, expression of IGF-I and the αVβ3 integrin receptor ligands, fibronectin and vitronectin are increased. The aim of the present study was to determine whether occupation of the αVβ3 receptor influences IGF-I receptor tyrosine kinase activation and function in human intestinal smooth muscle cells. In untreated cells, IGF-I elicited time-dependent tyrosine phosphorylation of its cognate receptor that was maximal within 2 min and sustained for 30 min. In the presence of the αVβ3 ligand, fibronectin, IGF-I stimulated IGF-I receptor activation was augmented. Conversely, in the presence of the αVβ3 specific disintegrin, echistatin, IGF-I-stimulated IGF-I receptor tyrosine kinase phosphorylation was inhibited. IGF-I-stimulated IGF-I receptor activation was accompanied by recruitment of the adapter protein, IRS-1, activation of Erk1/2, p70S6 kinase and proliferation. These effects were augmented by fibronectin and attenuated by echistatin. IGF-I also elicited time-dependent recruitment of protein tyrosine phosphatase SHP-2 that coincided with dephosphorylation of the tyrosine phosphorylated IGF-I receptor tyrosine kinase. The αVβ3 disintegrin, echistatin, accelerated the rate of SHP-2 recruitment and deactivation of the IGF-I receptor tyrosine kinase. The results show that occupancy of the αVβ3 integrin receptor modulates IGF-I-induced IGF-I receptor activation and function in human intestinal muscle cells. We hypothesis that the concomitant increases in the expression of αVβ3 ligands and of IGF-I in active Crohn’s disease may contribute to muscle hyperplasia and G-00345-2005.R1 Kuemmerle-3 stricture formation by acting in concert to augment IGF-I stimulated IGF-I receptor tyrosine kinase activity and IGF-I mediated muscle cell growth. G-00345-2005.R1 Kuemmerle-4 INTRODUCTION Integrins are a family of transmembrane proteins comprised of 18 α-subunits and 8 β-subunits which heterodimerize to form the 24 known integrin pairs (9). A wide variety of integrin ligands have been described that bind to specific integrins and together participate in outside-in and inside-out integrin signaling (17). While integrin expression, in general, is widespread throughout the body, the expression of specific integrins and their preferred ligands is tissue specific. This confers one level of specificity to an otherwise ubiquitously expressed family of proteins and is fundamental to their ability to regulate cellular events. Smooth muscle of visceral and vascular types express the α1β1, α5β1, αVβ3, α2β1, α3β1, α5β1, αVβ1, αVβ5, and α1β6 integrins (28). Unlike receptors with intrinsic tyrosine kinase-containing activity, in response to their ligands, integrin receptors undergo a conformational change that changes their activation state (5). Phosphorylation of cytoplasmic domains of integrin β-subunits results in the association of signaling proteins (25; 36). Experimental evidence in cells that were detached and reattached to extracellular matrix proteins has shown that integrins can modulate growth-factor stimulated receptor activation and thereby regulate growth-factor stimulated cell adhesion and cell motility (38). Clemmons et al have shown that in porcine vascular smooth muscle, αVβ3 integrin (the cognate vitronectin receptor), modulates IGF-I-stimulated smooth muscle motility and proliferation in stably attached cells in vitro without being subjected to detachment and reattachment (7; 37). The αVβ3 integrin also has been shown to play a key role in the regulation of IGF-I-mediated vascular smooth muscle growth during atheroma formation in vivo (29). The muscle hyperplasia that accompanies atheroma development G-00345-2005.R1 Kuemmerle-5 and contributes to arterial narrowing can be prevented by a neutralizing antibody to αVβ3 integrin (29). Integrin and IGF-I modulated uterine smooth muscle growth has also been implicated as a factor in leiomyoma formation (4; 33). In cultured porcine vascular smooth muscle cells, three transmembrane proteins: the αVβ3 integrin, tyrosine phosphatase SHP-2 substrate 1 (SHPS-1) and the IGF-I receptor tyrosine kinase jointly regulate cellular responses to IGF-I (8). Acting in concert these three proteins regulate the recruitment of the SH2 domain-containing protein tyrosine phosphatase, SHP-2, to the IGF-I receptor tyrosine kinase (27). Interplay between the αVβ3 integrin and growth factor receptors is a common theme and its modulation of PDGF, VEGF, and insulin signaling has been demonstrated (32). The EGF receptor has been shown to interact directly with α5β1, α6β4, and αVβ1 integrins (13). The activation of PDGF, FGF, insulin and EGF receptor tyrosine kinases, in addition to the IGF-I receptor tyrosine kinase, are modulated by the protein tyrosine phosphatase SHP-2. Patients with active Crohn’s Disease have elevated plasma levels of fibronectin, a ligand of αVβ3 integrin (2). In regions of inflammation and stricturing, the numbers of mast cells resident in the muscularis propria are higher than in the muscle layer of uninflamed intestine. Although immunoreactive laminin co-localized with markers of tissue mast cells, immunoreactive fibronectin or vitronectin did not (14). Neither the expression of αVβ3 integrin and of its ligands, fibronectin and vitronectin, by intestinal smooth muscle, nor the role of this system in regulating muscle cells proliferation in response to IGF-I have been examined. G-00345-2005.R1 Kuemmerle-6 This paper shows that human intestinal smooth muscle cells express the αV and β3 integrin subunits and the αVβ3 integrin ligands, vitronectin and fibronectin in vivo. Expression of all four remains constant when intestinal smooth muscle cells are cultured. IGF-I stimulates time-dependent phosphorylation of the IGF-I receptor tyrosine kinase, recruitment and phosphorylation of insulin receptor substrate-1 (IRS-1), and the downstream kinases Erk1/2 and p70S6 kinase phosphorylation which jointly stimulate muscle cell proliferation (20). SH2-domain containing protein tyrosine phosphatase, SHP-2, is recruited in a time-dependent fashion to the activated IGF-I receptor tyrosine kinase. SHP-2 recruitment coincides in time with dephosphorylation of the IGF-I receptor tyrosine kinase. The αVβ3 integrin ligand, fibronectin, augments IGF-I-stimulated IGF-I receptor tyrosine kinase phosphorylation, Erk1/2 and p70S6 kinase phosphorylation and proliferation. In contrast, the αVβ3 selective antagonist, echistatin, diminished IGF-I stimulated IGF-I receptor phosphorylation, recruitment and phosphorylation of IRS-1, phosphorylation of Erk1/2 and p70S6 kinase and muscle cell proliferation. The rate of IGF-I stimulated SHP-2 recruitment to the IGF-I receptor in the presence of echistatin was accelerated. The results implied that activation state of αVβ3 integrin is regulated by endogenous integrin ligands and modulates IGF-I stimulated receptor activation, signaling pathways and proliferation in human intestinal smooth muscle cells. The potential clinical significance of this mechanism has relevance in the setting of chronic intestinal inflammation when levels of the αVβ3 integrin ligands, vitronectin and fibronectin, are increased and could act in concert with the observed increased IGF-I levels that augment intestinal muscle cell proliferation and inhibition of apoptosis leading to hyperplasia of the muscularis propria and stricture formation. G-00345-2005.R1 Kuemmerle-7 METHODS Culture of Smooth Muscle Cells Isolated from Normal Human Jejunum. Muscle cells were isolated and cultured from the circular muscle layer of human jejunum as described previously (19; 20). Briefly, 4-5 cm segments of normal intestine were obtained from patients undergoing surgery according to a protocol approved by the VCU Institutional Review Board. After opening the segments along the mesenteric border, the mucosa was dissected away and the remaining muscle layer cut into 2X2 cm strips. Slices were obtained separately from the circular layer using a Stadie-Riggs tissue slicer. The slices were incubated overnight at 37°C in 20 ml of Dulbecco’s modification of Eagle’s medium plus 10% fetal bovine serum (DMEM-10) containing penicillin 200U/ml, streptomycin 200 μg/ml, gentamycin 100μg/ml and amphotericin B 2 μg/ml to which was added 0.0375% collagenase (type II), and 0.1% soybean trypsin inhibitor. Muscle cells dispersed from the circular layer were harvested by filtration through 500 μm Nitex mesh and centrifugation at 150 g for 5 minutes. Cells were resuspended and washed twice by centrifugation at 150g for 5 min. After resuspension in DMEM-10 containing the same antibiotics, the cells were plated at a concentration of 5x10 cells per ml as determined by counting in a hemocytometer. Cultures were incubated in a 10% CO2 environment at 37°C. DMEM-10 medium was replaced every three days until the cells reached confluence. Primary cultures of muscle cells were passaged upon reaching confluence and used in first passage. We have previously shown that these cells express a phenotype characteristic of intestinal smooth muscle as determined by immunostaining for smooth muscle markers (clone HM 19/2, 5 μg/ml) and expression of γ-enteric actin (34). G-00345-2005.R1 Kuemmerle-8 Epithelial cells, endothelial cells, neurons and interstitial cells of Cajal are not detected in these cultures. Immunoprecipitation of IGF-I receptor. IGF-I receptors were immunoprecipitated as previously described (21). Briefly, confluent smooth muscle cells were rendered quiescent by incubation in serum-free DMEM for 24 h. Cells were incubated with the αVβ3 integrin ligand, fibronectin (5 μg/cm cultureware surface area), the αVβ3 selective disintegrin, echistatin (100 nM) or vehicle for the final 12 h (7). Cells were then stimulated with 100 nM IGF-I for periods of time from 0 – 30 min. The reaction was terminated by washing twice with ice-cold PBS. Cell lysates were prepared in an immunoprecipitation buffer consisting of (in mM): 50 Tris-HCl (pH 7.5), 150 NaCl, 50 NaF, 1 Na orthovanadate, 1 dithiothreitol, 1 phenylmethylsulfonyl fluoride and 0.5% NP40 to which was added 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 μg/ml aprotinin. The resulting lysates were clarified by centrifugation at 14,000g for 10 min at 4°C. The lysates were pre-cleared by incubation with Protein A agarose beads for 1 h at 4°C. Samples containing equal amounts of protein (1 mg) were incubated for 2 h at 4°C with 2 μg of rabbit anti-IGF-IR β-subunit. The incubation continued overnight at 4°C after the addition of 10 μl of Protein A agarose beads. The immune complex-agarose beads were washed three times with ice-cold immunoprecipitation buffer and twice with ice-cold kinase assay buffer. After washing the immune complex-agarose beads were resuspended in 25 μl sample buffer, boiled for 5 min, and then loaded onto a 7.5% polyacrylamide gel and the proteins separated by SDS-PAGE. Tyrosine phosphorylated G-00345-2005.R1 Kuemmerle-9 IGF-IR and co-immunoprecipated IRS-1, or the protein tyrosine phosphatase, SHP-2, were identified by immunoblot analysis as described below. Western Blot Analysis. Analysis of proteins and phosphorylated proteins was performed by Western blot analysis using standard methods (18; 20-22). Briefly, confluent muscle cells were rendered quiescent by incubation for 24 h in serum-free medium. The cells were stimulated with recombinant human IGF-I for periods of time from 0 – 30 min. The reaction was terminated by two rapid washes in ice-cold PBS. Sample buffer was added to cells to lyse the cells or added to immunoprecipitated proteins. After boiling for 5 min, samples adjusted to contain equal amounts of total protein prior to immunoprecipitation or samples adjusted to contain equal amounts of total protein after being directly lysed were separated with SDS-PAGE under denaturing conditions. After the proteins were electrotransferred to nitrocellulose, the membranes were incubated overnight with specific antibodies recognizing the proteins of interest (dilution): αV (1:500), β3 (1:500), fibronectin (1:1000), vitronectin (1:1000), phosphotyrosine (1:1000), SHP-2 (1:1000), IRS-1 (1:1000), phospho-IRS-1(Tyr632) (1:1000), Erk1/2 (1:1000), phospho-Erk1/2(Thr202/Tyr204) (1:2000), p70S6 kinase (1:1000) or phospho-p70S6 kinase(Ser389) (1:1000). When appropriate, nitrocellulose membranes were stripped and reblotted to determine levels of total (phosphorylated plus non-phosphorylated) protein. Bands of interest were visualized with enhanced chemiluminescence using a FluoChem 8800 (Alpha Innotech, San Leandro, CA) and the resulting digital images quantifies using AlphaEaseFC version 3.1.2 software. G-00345-2005.R1 Kuemmerle-10 [H]Thymidine Incorporation Assay. Proliferation of smooth muscle cells in culture was measured by the incorporation of [H]thymidine as described previously (19; 22). Briefly, the cells were washed free of serum and incubated for 24 h in serum-free DMEM in the presence or absence of various test agents. During the final 4 h of this incubation period, 1 μCi/ml [H]thymidine was added to the medium. [H]Thymidine incorporation into the perchloric acid extractable pool was used as a measure of DNA synthesis. Measurement of Protein Content. The protein content of cell lysates was measured using the BioRad DC protein assay kit according to manufacturer’s direction. Samples were adjusted in order to provide aliquots of equal protein content prior to in vitro kinase assay or Western blot analysis. Statistical analysis. Values given represent the mean ± SE of n experiments where n represents the number of experiments on cells derived from separate primary cultures. Statistical significance was tested by Student’s t-test for either paired or unpaired data as was appropriate. Densitometric values for protein bands of phosphorylated signaling intermediates were reported in arbitrary units above background values after normalization to total protein levels. Bands of interest were visualized with enhanced chemiluminescence using a FluoChem 8800 (Alpha Innotech, San Leandro, CA) and the resulting digital images quantified using AlphaEaseFC version 3.1.2 software. Materials. Recombinant human IGF-I was obtained from Austral Biologicals (San Ramon, CA), collagenase and soybean trypsin inhibitor were obtained from Worthington G-00345-2005.R1 Kuemmerle-11 Biochemical Inc (Freehold, NJ); HEPES was obtained from Research Organics (Cleveland, OH); Dulbecco’s modification of Eagles medium and Hank’s balanced salt solution were obtained from Mediatech Inc (Herndon, VA); fetal bovine serum was obtained from Summit Biotechnologies (Fort Collins, CO); rabbit polyclonal antibodies to p-IRS-1(Tyr632), IRS-1, and p70S6 kinase, mouse monoclonal antibodies to phosphotyrosine (p-Tyr100), p-Erk1/2(Thr202/Tyr204), and Erk1/2, anti-rabbit IgGHRP, and anti-mouse-HRP were obtained from Cell Signaling Technology (Beverly, MA); rabbit polyclonal antibody to p-p70S6 kinase(Ser389) was obtained from Upstate Biotechnology (Lake Placid, NY); rabbit polyclonal antibody to β3 was obtained from Biosource International (Camarillo, CA); human fibronectin and rabbit polyclonal antibody to αV were obtained from Becton-Dickinson (Franklin Lakes, NJ); Western blotting materials and DC protein assay kit were obtained from BioRad Laboratories (Hercules, CA); mouse-rat monoclonal antibody to smooth muscle (HM19/2) from Biogenesis (Sandown, NH); and plastic cultureware was obtained from Corning (Corning, NY). Echistatin and all other chemicals were obtained from Sigma (St Louis, MO). G-00345-2005.R1 Kuemmerle-12 RESULTS Expression of αV and β3 integrin subunits in fresh and cultured muscle cells. The expression of the αV and β3 integrin subunits was examined in human intestinal circular smooth muscle in vivo and their continued expression confirmed in cultured intestinal smooth muscle cells in vitro. Cell lysates were prepared from fresh and cultured smooth muscle cells and adjusted to contain equal concentrations of total protein. Western blot analysis of these cell lysates using an antibody selective for the αV integrin subunit identified a protein of 125 kDa size, corresponding to the know size of the αV integrin subunit (Figure 1A). Western blot analysis of cell lysates from fresh and cultured using a selective β3 integrin subunit antibody identified a protein of 105 kDa size, corresponding to the know size of the β3 integrin subunit (Figure 1B). Similar levels of integrin subunit expression were present in fresh muscle cells compared to cultured muscle cells based on total cell protein. Expression of αVβ3 integrin ligands in fresh and cultured muscle cells. Two important ligands of the αVβ3 integrin, the cognate vitronectin receptor, are vitronectin and fibronectin. The expression of vitronectin and of fibronectin in human intestinal smooth muscle cells in vivo and in cultured smooth muscle in vitro was therefore examined. Western blot analysis of cell lysates prepared from fresh tissue and from cultured cells using a fibronectin specific antibody identified a protein of 90 kDa size, similar to the predicted size of fibronectin and coinciding with authentic fibronectin (Figure 2A). Western blot analysis of cell lysates prepared from fresh tissue and from cultured cells using a vitronectin specific antibody identified a protein of 60 kDa size, G-00345-2005.R1 Kuemmerle-13 similar to the predicted size of vitronectin and coinciding with authentic vitronectin (Figure 2B). Similar levels of fibronectin and vitronectin were present in fresh muscle cells compared to cultured muscle cells Role of αVβ3 integrin in IGF-IR activation. The effect of αVβ3 integrin occupancy, by its endogenous ligands, on IGF-I stimulated IGF-IR activation was examined in cultured human intestinal smooth muscle cells. Muscle cells were rendered quiescent by incubation in serum-free DMEM for 24 h. During the final 12 h of the incubation the cells were exposed to either the αVβ3 integrin ligand, fibronectin (5 μg/cm cultureware surface area) or the αVβ3 integrin specific antagonist, echistatin (100 nM). Echistatin, a selective αVβ3 antagonist, binds to the cytoplasmic portion of the β3 subunit of αVβ3 integrin and disrupts its function. At the end of the 24 h, cells were stimulated with a maximally effective concentration of IGF-I (100 nM) for periods of 0 – 30 min. Immunoprecipitation and immunoblot analysis of samples containing equal amounts of protein showed that IGF-I elicited phosphorylation of the IGF-I receptor that was rapid, attained a maximum of 341 ± 46% above basal within 2 min and within 30 min IGF-I receptor phosphorylation declined to 190 ± 35% above basal, i.e. IGF-I receptor dephosphorylation had occurred (Figure 3). In the presence of fibronectin, IGF-I-induced IGF-I receptor phosphorylation was increased at all time periods. The results implied that the extent of αVβ3 integrin occupancy influences the extent of IGF-I receptor activation. In the presence of echistatin, IGF-I stimulated IGF-IR phosphorylation at 2 min was inhibited by 79 ± 6% and was abolished at longer time periods (Figure 3). The ability of the αVβ3 receptor antagonist, echistatin, to alter the kinetics of IGF-I stimulated IGFG-00345-2005.R1 Kuemmerle-14 IR receptor phosphorylation implied that endogenous αVβ3 integrin activity contributes to the regulation of extent and duration of IGF-IR phosphorylation. The SH2 domaincontaining tyrosine phosphatase, SHP-2, is recruited in other cell types to the activated IGF-I receptor tyrosine kinase and regulates is activity by mediating receptor tyrosine kinase dephosphorylation (1; 27). Recruitment of tyrosine phosphatase SHP-2 to the activated IGF-I receptor. The time-course of SHP-2 recruitment to the activated IGF-I receptor was examined in quiescent muscle cells and in cells treated with the αVβ3 integrin antagonist, echistatin. Incubation of quiescent muscle cells with 100 nM IGF-I for periods of 0 – 30 min elicited time-dependent association of SHP-2 with the IGF-I activated IGF-I receptor tyrosine kinase (Figure 4). The association was gradual and attained a maximum of 200 ± 30% above basal after 30 min. In cells treated with the αVβ3 integrin antagonist, the timecourse of SHP-2 association was accelerated. In the presence of echistatin, maximal SHP-2 association with the IGF-I receptor of 255 ± 27% above basal occurred within 5 min and had declined to basal levels after 30 min. The recruitment of SHP-2 to the activated IGF-I receptor, in both naïve and echistatin treated cells, coincided in time with IGF-I receptor dephosphorylation suggesting that the SHP-2 phosphatase may be responsible for inactivation of the IGF-I receptor that follows IGF-I stimulation. The ability of the αVβ3 antagonist, echistatin, to alter the kinetics of IGF-I stimulated IGF-I receptor phosphorylation supported the hypothesis that endogenous αVβ3 integrin ligands regulated the extent and duration of IGF-I receptor activity via SHP-2 recruitment and receptor tyrosine kinase dephosphorylation. G-00345-2005.R1 Kuemmerle-15 Role of αVβ3 in recruitment of IRS-1 to the IGF-IR. The involvement of αVβ3 integrin on downstream signaling emanating from the activated IGF-I receptor tyrosine kinase was also investigated. IRS-1 is a principle substrate of the activated IGF-I receptor. Accordingly, the ability of IGF-I to stimulate the recruitment of IRS-1 to the activated IGF-I receptor and the role of the αVβ3 integrin in regulating this process was examined using a co-immunoprecipitation approach. Quiescent muscle cells or muscle cells incubated with echistatin were treated with 100 nM IGF-I for periods of time from 0 – 30 min. IGF-I elicited a prompt association of IRS-1 with the IGF-I receptor that was maximal within 2 min, 272 ± 16% above basal, and that was sustained at lower levels for up to 30 min (Figure 5). The 2 min peak in association of IRS-1 and the IGF-I receptor corresponded to the time of maximal IGF-I receptor phosphorylation stimulated by IGF-I. In the presence of echistatin, the recruitment of IRS-1 to the activated IGF-I receptor at the 2 min maximum was inhibited by 87 ± 8% (Figure 5). Role of αVβ3 in IGF-I stimulated IRS-1 phosphorylation. The IRS-1 protein possesses approximately 30 potential tyrosine, serine and threonine phosphorylation sites. Following the recruitment of IRS-1 to the activated IGF-I receptor, IRS-1 contains numerous potential tyrosine phosphorylation sites, including Tyr-612 and Tyr-632, that result in its activation and its ability to activate its downstream kinases, p85-PI 3-kinase and Grb2, which lead to p70S6 kinase and Erk1/2 activation, respectively (15). Subsequent phosphorylation of IRS-1 on serine residues cause its inactivation (11; 12; G-00345-2005.R1 Kuemmerle-16 31). Since IRS-1(Tyr632) is specifically phosphorylated by the activated IGF-I receptor this was used as a measure of IGF-I receptor mediated IRS-1 activation. Incubation of quiescent muscle cells for periods of time from 0 to 30 min with 100 nM IGF-I elicited time-dependent IRS-1(Tyr632) phosphorylation (Figure 6). IGF-Iinduced IRS-1 phosphorylation was prompt, attaining a maximum of 290 ± 26% above basal within 2 min, was sustained for 10 min before declining after 30 min. In cells treated with the αVβ3 integrin antagonist, echistatin, the ability of IGF-I to elicit IRS1(Tyr632) phosphorylation at 2 min was inhibited by 85 ± 9% and abolished at longer time points (Figure 6). Role of αVβ3 integrin in IGF-I stimulated Erk1/2 phosphorylation. As noted above, we have previously shown that endogenous IGF-I stimulates muscle cell growth by activation of distinct signaling pathways downstream of the IGF-I receptor that lead to Erk1/2 and p70S6 kinase activation (20; 22). The role of αVβ3 integrin in modulating IGF-I stimulated Erk1/2 activation was examined in the presence of the αVβ3 disintegrin, echistatin and in the presence of the αVβ3 integrin ligand, fibronectin. Incubation of quiescent muscle cells with 100 nM IGF-I for time periods of 0 to 30 min caused phosphorylation of Erk1/2(Thr202/Tyr204) that was maximal within 10 min, and declined after 30 min (Figure 7). In the presence of fibronectin (5 μg/cm cultureware surface area), IGF-I-stimulated maximal Erk1/2(Thr204/Tyr204) phosphorylation was significantly increased and phosphorylation was sustained at maximal levels for 30 minutes (Figure 7). In contrast, in the presence of echistatin, IGF-I stimulated Erk1/2(Thr202/Tyr204) phosphorylation was significantly inhibited at all time G-00345-2005.R1 Kuemmerle-17 points measured (Figure 7). These results implied that the endogenous αVβ3 ligands produced by intestinal muscle cells, vitronectin and fibronectin (Figure 1), modulate the effects of IGF-I on Erk1/2 activation. Role of αVβ3 integrin in IGF-I stimulated p70S6 kinase phosphorylation. The role of αVβ3 integrin on IGF-I stimulated p70S6 kinase phosphorylation was also examined. p70S6 kinase(Ser389) phosphorylation was used as a measure of activation since of the multiple residues phosphorylated during p70S6 kinase activation, Ser389 phosphorylation is most closely correlated with its kinase activity (22; 22). As shown previously, incubation of quiescent muscle cells with 100 nM IGF-I increased the phosphorylation of p70S6 kinase(Ser389) in a time-dependent fashion that was maximal after 30 min (Figure 8). In the presence of fibronectin (5 μg/cm cultureware surface area) IGF-I stimulated p70S6 kinase(Ser389) phosphorylation was significantly increased (Figure 8). In contrast, in the presence of the αVβ3 specific disintegrin, echistatin, IGF-I stimulated p70S6 kinase(Ser389) phosphorylation was significantly inhibited at all time points (Figure 8). Role of αVβ3 in IGF-I stimulated proliferation. We have previously shown that endogenous IGF-I stimulates human intestinal smooth muscle cells proliferation by activation of the IGF-I receptor tyrosine kinase (21). The participation of endogenous αVβ3 integrin ligands in modulating IGF-I stimulated activation of the IGF-I receptor tyrosine kinase, IRS-1, and the signaling intermediates Erk1/2 and p70S6 kinase, suggested that endogenous integrin ligands might modulate IGF-I stimulated G-00345-2005.R1 Kuemmerle-18 proliferation. This possibility was examined by measuring the proliferative response to IGF-I (100 nM) in the presence of the αVβ3 integrin antagonist, echistatin (100 nM) or the αVβ3 integrin ligand fibronectin (5 μg/cm cultureware surface area). Basal levels of [H]thymidine incorporation were augmented 50 ± 14% by fibronectin and inhibited 54 ± 11% by echistatin (Figure 9). In the presence of fibronectin, IGF-I stimulated proliferation was increased by 400 ± 30% above that elicited by 100 nM IGF-I under control conditions (440 ± 29% above basal levels). In contrast in the presence of echistatin, IGF-I stimulated proliferation was inhibited by 47 ± 10% from that observed under control conditions. These results, taken together with the finding that human intestinal smooth muscle cells produce vitronectin and fibronectin, suggest that occupancy of αVβ3 integrin by endogenous αVβ3 integrin ligands modulate IGF-I stimulated growth of human intestinal smooth muscle cells. G-00345-2005.R1 Kuemmerle-19 DISCUSSION Endogenous IGF-I regulates the growth of human intestinal smooth muscle cells by jointing stimulating proliferation and inhibiting apoptosis (18; 19). These effects are mediated by IGF-I stimulated activation of its cognate receptor tyrosine kinase. Binding of IGF-I to the IGF-I receptor tyrosine kinase results in rapid, time dependent phosphorylation of specific residues within the ATP-binding domain, the autophosphorylation domain and in distinct domains that couple to the primary substrates of the activated IGF-I receptor tyrosine kinase (11). While phosphorylation of the IGF-I receptor by IGF-I is rapid (maximal within 2 min), it is also transient and declines to suprabasal levels within 30 min. The biphasic kinetics of receptor activation and rapid deactivation suggest that dephosphorylation of the receptor might also a regulated process. The present paper shows that occupancy of the αVβ3 integrin by its endogenous ligands regulates the extent and duration of IGF-I stimulated IGF-I receptor activation by determining the rate of recruitment of SHP-2 phosphatase to the activated IGF-I receptor. We have previously shown that IGF-I is an autocrine growth factor of human intestinal smooth muscle cells (19). The present paper shows that the αV and β3 integrin subunits and the αVβ3 ligands, vitronectin and fibronectin, are expressed by these cells. The evidence supporting a role for αVβ3 integrin-dependent modulation of IGF-I stimulated IGF-I receptor activation and proliferation in human intestinal smooth muscle cells can be summarized as follows: i. IGF-I-stimulated IGF-I receptor phosphorylation is augmented by the αVβ3 integrin ligand, fibronectin, and inhibited by the αVβ3 selective disintegrin, echistatin; ii. SHP-2 phosphatase recruitment to the IGF-I receptor is G-00345-2005.R1 Kuemmerle-20 accelerated in the presence of echistatin and maximal association is correlated in time with IGF-I receptor dephosphorylation; iii. IGF-I-induced recruitment of IRS-1 to the activated IGF-I receptor and IRS-1 phosphorylation is inhibited by echistatin; iv. IGF-Iinduced activation of Erk1/2 and p70S6 kinase is augmented by fibronectin and inhibited by echistatin. v. basal and IGF-I stimulated proliferation of intestinal smooth muscle cells are inhibited by echistatin and augmented by fibronectin. In human intestinal smooth muscle cells, and porcine vascular smooth muscle cells, occupancy of the αVβ3 integrin is required for optimal activation of IGF-I receptor tyrosine kinase by IGF-I This paper and work by Clemmons et al, show that in the presence of peptide and non-peptide antagonists of the αVβ3 integrin, the duration and extent of IGF-I stimulated IGF-I receptor phosphorylation is significantly reduced and results in decreased IGF-I-stimulated IGF-I receptor tyrosine kinase activation, proliferation of intestinal and vascular muscle cells, and migration and protein synthesis in vascular muscle cells. (7; 26; 27; 29). αVβ3 integrin-dependent modulation of these events is mediated by its ability to regulate the rate of recruitment of the protein tyrosine phosphatase SHP-2, to the activated IGF-I receptor (27). When activation of αVβ3 integrin is blocked, SHP-2 is recruited more rapidly to specific phosphotyrosine motifs of the activated IGF-I receptor tyrosine kinase. SHP-2 specifically binds to phosphorylated tyrosine residues that are followed by a specific motif YXXL/I and causes dephosphorylates of these phosphotyrosine residues (6; 23). Following dephosphorylation SHP-2 looses affinity and disassociates. In addition to its role in regulating the extent and duration of IGF-I stimulated IGF-I receptor tyrosine kinase activation, protein tyrosine phosphatase SHP-2 can be G-00345-2005.R1 Kuemmerle-21 recruited to other activated growth factor receptors (PDGF, Insulin and EGF) that possess these specific phosphotyrosine containing motifs where it functions similarly to regulate the activity these receptor tyrosine kinases (3; 13). SHP-2 binds to Tyr 771 of the PDGFβ receptor, elicits receptor tyrosine kinase dephosphorylation and diminishes signaling to the Ras/Erk pathway (10). SHP-2 also binds to Tyr 992 of the EGF receptor negatively regulating ligand-dependent signaling (1). In addition to its affinity for phosphotyrosine residues in growth factor receptors, SHP-2 can also modulate signaling via interleukin receptors through their tyrosine residues. SHP-2 binds to Tyr 759 of the IL-6 signal transducing receptor subunit, gp130, and attenuates IL-6 stimulated Jak/STAT activation (24) The canonical immunoreceptor tyrosine-based inhibitory motif (I/VxYxxL), which is also found in the IL-4 receptor, is a target of protein tyrosine phosphatase SHP-2 and modulates IL-4 stimulated proliferation (16). The fundamental role of IGF-I in regulating smooth muscle growth and development throughout the body, and in the intestine in particular, is highlighted by investigations using mutant mice under-expressing or over-expressing IGF-I. Mice overexpressing IGF-I exhibit hyperplasia of the intestinal smooth muscle layers (30). Mice with a Cre-LoxP targeted disruption of hepatic IGF-I production have greatly diminished levels of circulating IGF-I, yet their intestinal muscle layers develop normally (35). These results imply that the mechanisms regulating IGF-I stimulated activation of its cognate receptor in intestinal smooth muscle are autocrine and/or paracrine in nature and are important determinates of smooth muscle growth. The potential clinical significance of the mechanisms investigated in this paper occurs in the setting of chronic intestinal inflammation. Associated with chronic G-00345-2005.R1 Kuemmerle-22 intestinal inflammation of Crohn’s Disease are increased levels of the αVβ3 integrin ligands vitronectin and fibronectin (2). Endogenous expression of IGF-I by inflamed intestinal muscle is also significantly increased (39). The concomitant increased activation of αVβ3 integrin and IGF-I receptor tyrosine kinase of human intestinal smooth muscle can result in increased activation of IGF-I-stimulated signaling pathways leading to increased muscle cell proliferation, Erk1/2 and p70S6 kinase (22), and inhibition of apoptosis, GSK-3β (18). The result of these events that jointly stimulate intestinal smooth muscle cells proliferation and inhibition of apoptosis could lead to the muscle hyperplasia and stricture formation in patients with Crohn’s Disease. G-00345-2005.R1 Kuemmerle-23