ADHESION OF BOVINE AIRWAY SMOOTH-MUSCLE CELLS ACTIVATES EXTRACELLULARSIGNAL-REGULATED KINASES

Extracellular signal-regulated kinases (ERKs) phosphorylate and regula te cytoskeletal components of contractile cells and have been implicat ed in integrin-mediated adhesion. In this study, we examined the contr ibutions of adherence, cell flattening, and cytoskeletal reorganizatio n to adhesion-induced ERK activation in cultured bovine tracheal myocy tes. We found, as evidenced by a reduction in electrophoretic mobility , that adhesion to fibronectin induced phosphorylation of both p44(ERK 1) and p42(ERK2). In-gel kinase assays confirmed activation of both p4 4(ERK1) and p42(ERK2) in fibronectin-adherent cells, consistent with t he notion that ligand-integrin binding is required for adhesion-induce d ERK activation. However, ERK activation was maximal 2-4 h after plat ing, and adherence to either polystyrene or poly-L-lysine also caused ERK activation (fold increase 4 h after plating: fibronectin, 3.75 +/- 0.33; polystyrene, 3.95 +/- 0.78; poly-L-lysine, 2.14 +/- 0.36). Insp ection of myocytes following passage onto fibronectin showed near 100% adhesion and cell spreading after 4 h, whereas cells plated onto poly -L-lysine demonstrated adherence but minimal spreading. To test whethe r the cytoskeletal reorganization accompanying cell spreading is requi red for adhesion-induced ERK activation? we assessed ERK activity foll owing pretreatment with cytochalasin D, an inhibitor of actin polymeri zation. Cytochalasin inhibited both cell spreading and ERK activation following adhesion to fibronectin, but had no effect on growth factor- induced ERK activation in adherent cells. We conclude that adhesion-in duced ERK activation in bovine tracheal myocytes may occur independent ly of ligand-integrin binding and is primarily related to the cell spr eading that follows adhesion.