Convergence of Integrins and EGF Receptor Signaling Via PI3K/Akt/FoxO Pathway in Early Gene Egr-1 Expression SARA CABODI, 1 VIRGINIA MORELLO, 1 ALESSIO MASI, 2,3 RICCARDO CICCHI, 3 CHIARA BROGGIO, 3,4 PAOLA DISTEFANO, 1 ELISA BRUNELLI, 5 LORENZO SILENGO, 1 FRANCESCO PAVONE, 3,6 ANNAROSA ARCANGELI, 2,3 EMILIA TURCO, 1 GUIDO TARONE, 1 LAURA MORO, 5,7 AND PAOLA DEFILIPPI 7 * 1 Centro di Biotecnologie Molecolari and Dipartimento di Genetica, Biologia e Biochimica, Universita` di Torino, Torino, Italy 2 Dipartimento di Patologia ed Oncologia Sperimentali, Universita` di Firenze, Firenze, Italy 3 European Laboratory of non Linear Spectroscopy (LENS), Universita` di Firenze, Firenze, Italy 4 Dipartimento di Fisica, Universita` di Trento, Trento, Italy 5 Dipartimento Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita` del Piemonte Orientale, Novara, Italy 6 Dipartimento di Fisica, Universita` di Firenze, Firenze, Italy 7 Centro di Biotecnologie Molecolari and Dipartimento di Genetica, Biologia e Biochimica, Universita` di Torino, Torino, Italy The early gene early growth response (Egr-1), a broadly expressed member of the zing-finger family of transcription factors, is induced in many cell types by a variety of growth and differentiation stimuli, including epidermal growth factor (EGF). Here we demonstrate that Egr-1 expression is mainly regulated by integrin-mediated adhesion. Integrin-dependent adhesion plays a dual role in Egr-1 regulation, either being sufficient ‘‘per se’’ to induce Egr-1, or required for EGF-dependent expression of Egr-1, which occurs only in adherent cells and not in cells in suspension. To dissect the molecular basis of integrin-dependent Egr-1 regulation, we show by FLIM-based FRET that in living cells beta1-integrin associates with the EGF receptor (EGFR) and that EGF further increases the extent complex formation. Interestingly, Egr-1 induction depends on integrin-dependent PI3K/Akt activation, as indicated by the decrease in Egr-1 levels in presence of the pharmacological inhibitor LY294002, the kinase-defective Akt mutant and Akt1/2 shRNAs. Indeed, upon adhesion activated Akt translocates into the nucleus and phosphorylates FoxO1, a Forkhead transcription factors. Consistently, FoxO1silencing results in Egr-1- increased levels, indicating that FoxO1 behaves as a negative regulator of Egr-1 expression. These data demonstrate that integrin/EGFR cross-talk is required for expression of Egr-1 through a novel regulatory cascade involving the activation of the PI3K/Akt/Forkhead pathway. J. Cell. Physiol. 218: 294–303, 2009. ß 2008 Wiley-Liss, Inc. Integrins are adhesive receptors formed by alpha and beta subunits, which anchor extra-cellular matrix proteins to the actin cytoskeleton. On the basis of differential expression and specific localization of the receptors, integrins also trigger multiple signaling pathways regulating cell migration, proliferation, differentiation, and survival from apoptosis (Hynes, 2002). In the last few years, increasing evidences indicate that integrins co-operate with growth factor and cytokine receptors providing that cells adhere properly to the extra-cellular matrix (Miranti and Brugge, 2002; Giancotti and Tarone, 2003; Cabodi and Defilippi, 2006). The first findings on the cross-talk between integrins and epidermal growth factor receptor (EGFR) are related to the fact that integrin-mediated adhesion triggers its activation with ligand-independent mechanisms (Miyamoto et al., 1996; Moro et al., 1998, 2002; Yamada and Even-Ram, 2002; Bill et al., 2004). Thus, in adherent cells EGFR might be a direct upstream molecule triggering integrin signal inside the cells. The number of RPTKs activated by cell–matrix adhesion indicates that this mechanism is broadly used in integrin signaling (Vuori and Ruoslahti, 1994; Giancotti and Tarone, 2003; Cabodi and Defilippi, 2006). Noteworthy is the finding that the EGFR phosphorylation in response to adhesion occurs on residues that are different from those triggered by soluble EGF (Moro et al., 2002; Boeri Erba et al., 2005, 2007), suggesting that this signaling mechanism might have specific functional consequences. For example, direct activation of Sara Cabodi and Virginia Morello contributed equally to this work. Contract grant sponsor: Italian Association for Cancer Research (AIRC). Contract grant sponsor: Association for International Cancer Research (AICR). Contract grant sponsor: EU FP7 program (Metafight). Contract grant sponsor: MUR (Ministero deIl’Universita ` e Ricerca Scientifica, cofinanziamento PRIN, fondi ex-60% and FIRB 2001). Contract grant sponsor: Special project ‘‘Oncology,’’ Compagnia San Paolo/FIRM, Torino, Italy. Contract grant sponsor: Ente Cassa di Risparmio di Firenze, Progetto ‘‘Promelab’’. *Correspondence to: Paola Defilippi, MBC Via Nizza 52, 10126 Torino, Italy. E-mail: paola.defilippi@unito.it Received 29 May 2008; Accepted 26 August 2008 Published online in Wiley InterScience (www.interscience.wiley.com.), 9 October 2008. DOI: 10.1002/jcp.21603 ORIGINAL ARTICLE 294 Journal of Journal of Cellular Physiology Cellular Physiology ß 2008 WILEY-LISS, INC.