Integrin-mediated Adhesion of Endothelial Cells Induces JAK2 and STAT5A Activation: Role in the Control of c-fos Gene Expression

Abstract

Integrin-mediated adhesion induces several signaling pathways leading to regulation of gene transcription, control of cell cycle entry and survival from apoptosis. Here we investigate the involvement of the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in integrin-mediated signaling. Plating primary human endothelial cells from umbilical cord and the human endothelial cell line ECV304 on matrix proteins or on antibody to ␤1-or ␣v-integrin subunits induces transient tyrosine phosphorylation of JAK2 and STAT5A. Consistent with a role for the JAK/STAT pathway in regulation of gene transcription, adhesion to matrix proteins leads to the formation of STAT5A-containing complexes with the serum-inducible element of c-fos promoter. Stable expression of a dominant negative form of STAT5A in NIH3T3 cells reduces fibronectin-induced c-fos mRNA expression, indicating the involvement of STAT5A in integrin-mediated c-fos transcription. Thus these data present a new integrin-dependent signaling mechanism involving the JAK/STAT pathway in response to cell-matrix interaction. INTRODUCTIONEndothelial cell adhesion to extracellular matrix is mediated by the integrin family of adhesive receptors, glycoprotein heterodimers that are formed by ␣ and ␤ subunits (Hynes, 1992;Defilippi et al., 1997). Several lines of evidence indicate that integrin-mediated adhesion stimulates signaling pathways leading to actin cytoskeleton organization, cell motility, regulation of cell growth, and control of cell differentiation (for review, see Clark and Brugge, 1995;Schwartz et al., 1995). Integrin-induced signaling has been reported to affect gene expression (Dike and Farmer, 1988;Haskill et al., 1991;Chen et al., 1992;Rana et al., 1994;Schmidhauser et al., 1994;Fan et al., 1995;Tremble et al., 1995;Dike and Ingber, 1996). Among the early growth response genes encoding for transcriptional activators, adhesion-dependent induction of cfos gene transcription has been described in monocytes (Haskill et al., 1991), in endothelial cells (Dike and Ingber, 1996;Wary et al., 1996), and in fibroblasts (Tremble et al., 1995;Wary et al., 1996) and proposed as a mediator of cell cycle progression controlled by cell adhesion.The signaling pathways leading to integrin-dependent early growth response gene transcription are not completely defined. A role of activated Erk1/Erk2 MAP kinases in coupling integrins to gene expression has been recently proposed (Chen et al., 1992;Morino et al., 1995;Zhu and Assoian, 1995;Wary et al., 1996). Different signaling pathways, including activation of Shc (Wary et al., 1996), fyn kinase (Wary et al., 1998), phosphinositide 3 kinase (King et al., 1997), Raf (Howe and Juliano, 1998) (for review, see Assoian, 1997;Giancotti, 1997;, and EGF receptor (Moro et al., 1998) have been proposed to trigger adhesiondependent Erk1/Erk2 MAP kinase activation. Upon activation, Erk1 and Erk2 MAP kinases translocate to the nucleus and phosphorylate their specific Elk1 and SAP1 substrates, that together with...

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“…After 18 h, supernatants were collected and divided into two aliquots: one was stored at À801C for the assessment of MMP-2 gelatinolytic activity and the other was used  40 concentrated for VEGF protein detection by Western blot analysis. After cell lysis, the protein content was determined by the Bradford technique and samples were normalized by appropriate dilution with medium and then processed as previously described (Brizzi et al, 1999b). Unstimulated SMC or EC were lysed in DIM buffer for immunoprecipitation experiments and processed as described (Brizzi et al, 1999b).…”

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“…32 Northern blot analysis was performed as previously described. 29 Filters were hybridized to P random-labeled DNA probes corresponding to p21 waf and ␤-actin and were processed as previously described. 29 …”