Fibronectin (FN) is known to transduce signal(s) to rescue cells from detachment-induced apoptosis (anoikis) through an integrin-mediated survival pathway. However, the functions of individual FN domains have not been studied in detail. In the present study we investigated whether the interaction of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8h. Nuclear condensation indicated apototic cell death. This was due to the decreased activity/stability of focal adhesion kinase (pp125FAK) in the absence of HepII domain. A peptide in the HepII domain [peptide V, WQPPRARI (single-letter amino acid codes)], which has previously been implicated in cytoskeletal organization, rescued apoptotic changes. Consistently, pp125FAK phosphorylation was increased, and both cleavage of pp125FAK and activation of caspase 3 on FN120 were partly blocked by peptide V. Thus the interaction of the cell-binding domain with integrin has a major role in cell survival but is itself not sufficient for cell survival. One or more additional survival signals come from the HepII domain to regulate pp125FAK activity/stability.
Abbreviations used: Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin; DAPI, 4,6-diamidino-2-phenylindole; ECM, extracellular matrix; ERK, extracellular signal-regulated protein kinase; FN, fibronectin; FN120, fibronectin fragment lacking C-terminal heparin-binding domain; HepII, C-terminal heparin-binding domain; HUVEC, human umbilical-vein endothelial cells; MAP, mitogen-activated protein; PKC, protein kinase C; pp125FAK, focal adhesion kinase; RGD, Arg-Gly-Asp; REF, rat embryo fibroblast; SFM, serum-free medium.