title: Cdc42 and RhoA are differentially regulated during arachidonate-mediated HeLa cell adhesion.
abstract: Cell adhesion to extracellular matrix requires stimulation of an eicosanoid signaling pathway through the metabolism of arachidonate by 5-lipoxygenase to leukotrienes and cyclooxygenase-1/2 to prostaglandins, as well as activation of the small GTPase signaling pathway involving Cdc42 and Rho. These signaling pathways direct remodeling of the actin cytoskeleton during the adhesion process, specifically the polymerization of actin during cell spreading and the bundling of actin filaments when cells migrate. However, few studies linking these signaling pathways have been described in the literature. We have previously shown that HeLa cell adhesion to collagen requires oxidation of arachidonic acid (AA) by lipoxygenase for actin polymerization and cell spreading, and cyclooxygenase for bundling actin filaments during cell migration. We demonstrate that small GTPase activity is required for HeLa cell spreading upon gelatin, and that Cdc42 is activated while Rho is downregulated during the spreading process. Using constitutively active and dominant negative expression studies, we show that Cdc42 is required for HeLa cell spreading and migration, while activated RhoA is antagonistic towards spreading. Constitutively active RhoA promotes cell migration and increases the degree of actin bundling in HeLa cells. Further, we demonstrate that activation of either the AA oxidation pathway or the small GTPase pathway cannot rescue inhibition of spreading when the alternate pathway is blocked. Our results suggest (1) both the eicosanoid signaling pathway and small GTPase activation are required during HeLa cell adhesion, and (2) these signaling pathways converge to properly direct remodeling of the actin cytoskeleton during HeLa cell spreading and migration upon collagen.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003, USA. lroberts@bio.umass.edu
authors.forename: Louis A
authors.initials: LA
authors.lastname: Roberts
authors.forename: Honor
authors.initials: H
authors.lastname: Glenn
authors.forename: Chang S
authors.initials: CS
authors.lastname: Hahn
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Jacobson
mesh_terms: D000199:Actins / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism*; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005109:Extracellular Matrix / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015398:Signal Transduction; D020764:cdc42 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000199:Actins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C031815:toxB protein, Clostridium difficile; D016718:Arachidonic Acid; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein