Semaphorin 3A Increases FAK Phosphorylation at FocalAdhesions to Modulate MDA-MB-231 Cell Migration andSpreading on Different Substratum Concentrations

Interactions between integrin-mediated adhesions and the extracellular matrix (ECM) are important regulators of cell migrationand spreading. However, mechanisms by which extracellular ligands regulate cell migration and spreading in response to changesin substratum concentration are not well understood. Semaphorin 3A (Sema3A) has been shown to inhibit cell motility and alterintegrin signaling in various cell types. We propose that Sema3A alters focal adhesions to modulate breast carcinoma cell migrationand spreading on substrata coated with different concentrations of ECM. We demonstrate that Sema3A inhibits MDA-MB-231 cellmigration and spreading on substrata coated with high concentrations of collagen and fibronectin but enhances migration andspreading at lower concentrations of collagen and fibronectin. Sema3A increases focal adhesion kinase phosphorylation at tyrosine397 (pFAK397) at focal adhesions on all substratum concentrations of collagen and fibronectin but decreased pFAK397levels onlaminin. Rho-associated protein kinase (ROCK) inhibition blocks the Sema3A-mediated effects on cell migration, spreading, andpFAK397at focal adhesions when cultured on all concentrations of collagen. These results suggest that Sema3A shifts the optimallevel of cell-matrix adhesions to a nonoptimal ECM coating concentration, in particular collagen, to yield maximal cell migrationand spreading that may be mediated through a ROCK-dependent mechanism.