title: Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of beta 1 integrin.
abstract: HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell-attachment-induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX). The present study examines how PKC activation affects the actin- and microtubule-based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin. Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule-facilitated exocytosis, which is based on the following observations. There is an increase in the relative content of filamentous (F)-actin during HeLa cell spreading, and treating HeLa cells with PKC-activating phorbol esters such as 12-O-tetradecanoyl 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 regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F-actin. Blocking the activities of either PLA2 or LOX inhibited F-actin formation and cell spreading, both of which were reversed 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 appears to activate actin polymerization in the entire body of the cell and not just in the region of cell-substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plasma membrane domain exposed to the culture medium and with an intracellular membrane fraction. In addition to the facilitation of F-actin formation, activation of PKC induces the exocytotic upregulation of beta 1 integrins from an intracellular domain to the cell surface, possibly in a microtubule-dependent manner because the upregulation is inhibited by Nocodazole. The results support the concept that cell-attachment-induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading. One is through the formation of F-actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading.
authors.affiliation: Department of Biology, Kyungpook National University, Taegu, Korea.
authors.forename: J
authors.initials: J
authors.lastname: Chun
authors.forename: K A
authors.initials: KA
authors.lastname: Auer
authors.forename: B S
authors.initials: BS
authors.lastname: Jacobson
mesh_terms: D000199:Actins / Q000096:biosynthesis*; D016718:Arachidonic Acid / Q000494:pharmacology*; D002448:Cell Adhesion; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D003571:Cytochalasin B / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D019012:Integrin beta1 / Q000096:biosynthesis*; D007700:Kinetics; D008870:Microtubules / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D009281:Naphthalenes / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*
chemical_list: D000199:Actins; D004791:Enzyme Inhibitors; D019012:Integrin beta1; D009281:Naphthalenes; D016718:Arachidonic Acid; D003571:Cytochalasin B; D011493:Protein Kinase C; C058819:calphostin C; D015739:Nocodazole