αvβ3 Integrins and Pyk2 Mediate Insulin-Like Growth Factor I Activation of Src and Mitogen-Activated Protein Kinase in 3T3-L1 Cells

Abstract

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.

Transforming Growth Factor β1 Induces αvβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway

“…Our data suggest through a process of inside-out signaling whereby activated TGF␤RI or TGF␤RII directly interacts with the ␤3 cytoplasmic domain, which in turn induces a conformational change in the integrin resulting in activation of the downstream SFK (4,37). A similar process of inside-out activation of ␣v␤3 integrin by ligation of insulin-like growth factor I receptor in the murine preadipocyte 3T3-L1 cell line has been described recently (17).…”

“…TGF␤1-induced ␤3 Integrin Expression Is Dependent on ␣v␤3 Activation-To evaluate the role of integrin activation on enhanced ␣v␤3 expression, we treated adherent fibroblasts with the disintegrin echistatin, which has been shown to inhibit the activation of RGD-binding integrins in several cell types (17,21,22). Echistatin had no effect on cell attachment but dose-dependently impaired the ability of fibroblasts to spread A, fibroblasts transiently transfected with Smad7 cDNA or transfection reagent alone were stimulated with TGF␤1 (10 ng/ml) for 24 h, and ␣v␤3 integrin cell surface expression was analyzed by flow cytometry as described under "Experimental Procedures."…”

“…Previous studies have also identified several molecules as inducers of ␣v integrin expression in various tissue culture systems (12)(13)(14). It was also reported that growth factors are able to activate integrins and that this activation provided an additional mechanism for a growth factor to induce a broad spectrum of cellular responses (15)(16)(17). Recently we demonstrated that TGF␤1 not only synergistically interacts with ␣v␤3 integrins but also induces their gene transcription in human * This work was supported by the British Columbia Lung Association, the Canadian Institutes of Health Research, The Wolfe and Gita Churg Foundation, and the National Health and Medical Research Council of Australia.…”

Insulin-like growth factor-I promotes migration in human androgen-independent prostate cancer cells via the αvβ3 integrin and PI3-K/Akt signaling

“…Our observations are in line with recent studies on nonmalignant cells; the ·vß3 integrin dimer has been shown to be required for IGF-I to stimulate the proliferation and migration of smooth muscle cells (42), extravillous trophoblast cells (43), and preadipocyte 3T3-L1 cells (44). In smooth muscle cells, the intracellular proteins (DOK1, SHP-2) that are recruited to the ß3 subunit of the dimer following IGF-IR stimulation, have also been identified (45)(46)(47).…”

Mechanotransduction and Osteogenesis

“…IGF-1 activity depends totally on the presence of integrins, and Akt activation rate is not altered by mechanical stimulation in the presence of IGF-1 (Desbois- Mouthon et al, 2001;Kapur et al, 2005;Nomura & Takano-Yamamoto, 2000;Sakata et al, 2004;Sekimoto et al, 2005;Sikavitsas et al, 2001).…”