PKC REGULATION OF MICROFILAMENT NETWORK ORGANIZATION IN KERATINOCYTESDEFINED BY A PHARMACOLOGICAL STUDY WITH PKC ACTIVATORS AND INHIBITORS

The modulation by PKC activators and inhibitors of adhesion, spreading , migration, actin cytoskeleton organization, and focal complex format ion in keratinocytes attaching to type I collagen was studied. Two act in microfilament networks, stress fibers and cortical actin, could be distinguished on the basis of cellular distribution and opposite regul ation by growth factors, tyrosine kinase inhibitors, and PKC activator s. Stress fiber formation was stimulated by growth factors and by PMA (100 ng/ml) and these stimulations were blocked by tyrosine kinase inh ibitors (0.3 mM genistein and 1 mu M herbimycin A). By contrast, the c ortical network occurred in quiescent cells, was unaffected by tyrosin e kinase inhibitors, and was broken down after PKC activation by PMA. Spreading, migration, and actin polymerization were completely blocked while adhesion efficacy was significantly decreased by three specific PKC inhibitors. Half-inhibition of migration was obtained with 0.025, 1, and 3 mu M concentrations of calphostin C, chelerytrine chloride, and D-erythrosphingosine, respectively, which are concentrations close to those known to inhibit the PKC kinase function in vitro. Paxillin clustering, which was observed even in the presence of tyrosine kinase inhibitors, disappeared only when actin polymerization was completely impaired, i.e., in cells treated with PKC inhibitors or with both tyr osine kinase inhibitors and PMA, which indicated that focal complex fo rmation was highly dependent on microfilament reorganization. The anal ysis of these data underscores a major regulation function of PKC in t he molecular events involved in growth factor and adhesion-dependent r egulation of microfilament dynamics. (C) 1997 Academic Press.