ARACHIDONATE INITIATED PROTEIN-KINASE-C ACTIVATION REGULATES HELA-CELL SPREADING ON A GELATIN SUBSTRATE BY INDUCING F-ACTIN FORMATION AND EXOCYTOTIC UP-REGULATION OF BETA-1 INTEGRIN

HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-in duced activation of phospholipase A2 (PLA2) to produce arachidonic aci d (AA) and metabolism of AA by lipoxyginase (LOX). The present study e xamines how PKC activation affects the actin-and microtubule-based cyt oskeletal machinery to facilitate HeLa cell spreading on gelatin. Cell spreading on gelatin is contingent on PKC induction of both actin pol ymerization and microtubule-facilitated exocytosis, which is based on the following observations. There is an increase in the relative conte nt of filamentous (F)-actin during HeLa cell spreading, and treating H eLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoy l phorbol 13-acetate (TPA) further increases the relative content of F -actin and the rate and extent to which the cells spread. Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F-actin content. The increased F-actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F-actin were similar to that for PKC activation. In addition, PKC epsilon, which is the PKC isoform most involved in re gulating HeLa cell spreading in response to AA production, is more rap idly translocated to the membrane in response to TPA treatment than is the increase in F-actin. Blocking the activities of either PLA2 or LO X inhibited F-actin formation and cell spreading, both of which were r eversed by TPA treatment. This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F-actin formation and cell spreading. PKC ap pears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activate d PKC was associated not only with the basolateral plasma membrane dom ain contacting the culture dish but also with the apical plama membran e domain exposed to the culture medium and with an intracellular membr ane fraction. In addition to the facilitation of F-actin formation, ac tivation of PKC induces the exocytotic upregulation of beta 1 integrin s from an intracellular domain to the cell surface, possibly in a micr otubule-dependent manner because the upregulation is inhibited by Noco dazole. The results support the concept that cell-attachment-induced A A production and its metabolism by LOX results in the activation of PK C, which has a dual role in regulating the cytoskeletal machinery duri ng HeLa cell spreading. One is through the formation of F-actin that i nduces the structural reorganization of the cells from round to spread , and the other is the exocytotic upregulation of collagen receptors t o the cell surface to enhance cell spreading. (C) 1997 Wiley-Liss, Inc .