Cell adhesion and migration contribute to normal processes such as differentiation, embryonic development, and wound healing as well as to the progression of diseases and pathological conditions that can result from either acute or chronic exposure to environmental toxicants, such as cancer and inflammatory responses. Key mechanistic steps in these processes involve the interactions of extracellular glycoproteins--such as fibronectin, laminin, and collagens--with specific adhesive receptors, the best characterized of which are the integrins, a family of heterodimeric complexes consisting of an alpha subunit and a beta subunit. Integrins are highly regulated receptors that can exist in either an active or inactive state. Our research has focused recently on the possible role of cues from the tumor microenvironment that can regulate integrin-mediated tumor cell migration and invasion. As a model system, we are examining the ability of one selectin, P-selectin, to trigger integrin-mediated adhesion and migration of cultured human tumor cells. We focus on two closely related aspects of this project: to characterize the mechanisms of P-selectin-induced activation of integrin-mediated cell adhesion and cell migration. Arachidonic acid stimulates cell adhesion by activating alpha2-beta1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. We have investigated the interaction of cytoskeletal components with key signaling molecules that contribute to spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of beta1 integrin-containing pseudopodia whereas untreated cells displayed elongated stress fibers and fewer clusters of beta1 integrins. Vinculin and phospho-p38 both appeared to be enriched in pseudopodia and at the tips of actin filaments arachidonic acid-treated cells.Fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading and that this association can be regulated by factors in the tumor microenvironment.

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