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. (c) 2008 Wiley-Liss, Inc.