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J. Biol. Chem., Vol. 280, Issue 31, 28803-28810, August 5, 2005
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From the
Departments of
Dermatology,
Biomedical Engineering, and ||Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794 and the ¶Department of Medicine, University of Wisconsin, Madison, Wisconsin 53706
Fibroblast adhesion to fibronectin (FN) induces formation of focal adhesions (FAs), structures that have significant effect on cell migration and signaling. FA formation requires actomyosin-based contractility that is regulated by Rho-dependent myosin light chain (MLC) phosphorylation. Previous studies indicated that the FN central cell-binding (and integrin-binding) domain (CBD) is insufficient for FA formation and that the major heparin-binding domain (HepII) facilitates FA formation in a Rho-dependent manner. We describe here conditions under which FN CBD alone is sufficient for FA formation in both human dermal fibroblasts and the FN-null murine fibroblasts. CBD-mediated FA formation is dependent on its surface adsorption and the adhesion activity of the cells. Attachment of FN-null fibroblasts to CBD elicits the same biphasic regulation of Rho activity as seen on intact FN, whereas adhesion to HepII alone does not activate Rho. Activation of Rho requires high levels of integrin occupancy. However, FN or CBD may induce FAs without increased activation of Rho (i.e. the basal level of GTP-Rho induces sufficient phospho-MLC for FA assembly under this condition). In contrast, adhesion to HepII alone does not sustain MLC phosphorylation. Pulse stimulation of cells on CBD or HepII with lysophosphatidic acid elevates Rho GTP loading to the same level, but the lysophosphatidic acid-stimulated MLC phosphorylation is significantly lower in cells on HepII than on CBD. Coating HepII with suboptimal concentrations of CBD induces FAs without increased activation of Rho. Therefore, FN CBD can support FA formation and generate contraction by activating Rho or by facilitating Rho downstream signaling.
Received for publication, February 7, 2005 , and in revised form, June 15, 2005.
* This work was supported by NIAMS, National Institutes of Health (NIH), Grant AR47894, the 2001 Dermatology Foundation Research Grant (to X.-D. R), and NIA, NIH, Grant AG10143 (to R. A. F. C.). This work was also supported by United States Army Grant DAMD17-01-1-0754 for the state-of-the-art instruments. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Dermatology, Life Science Bldg., Rm. 004, State University of New York at Stony Brook, Stony Brook, NY 11794-5200. Tel.: 631-632-1291; Fax: 631-632-1847; E-mail: xiang-dong.ren{at}sunysb.edu.
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