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Originally published as MBC in Press, 10.1091/mbc.E02-10-0667 on May 18, 2003

Vol. 14, Issue 9, 3699-3715, September 2003

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{alpha}4{beta}1 Integrin/Ligand Interaction Inhibits {alpha}5{beta}1-induced Stress Fibers and Focal Adhesions via Down-Regulation of RhoA and Induces Melanoma Cell Migration

José V. Moyano, Alfredo Maqueda, Benito Casanova, and Angeles Garcia-Pardo *

Departamento de Inmunología, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas, 28006 Madrid, Spain

Submitted October 18, 2002; Revised April 21, 2003; Accepted April 22, 2003
Monitoring Editor: Mark Ginsberg

We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7–10 fragment. Coimmobilization of FNIII4–5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7–10, or addition of soluble FNIII4–5 to cells preattached to FNIII7–10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7–10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7–10+FNIII4–5. Furthermore, addition of alpha4beta1 ligands to FNIII7–10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E02–10–0667. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E02-10-0667.

Online version of the article contains video material. Online version is available at www.molbiolcell.org.

* Corresponding author. e-mail address: agarciapardo{at}cib.csic.es.




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