The β1 isoform of protein kinase C mediates the protective effects of epidermal growth factor on the dynamic assembly of F-actin cytoskeleton and normalization of calcium homeostasis in human colonic cells (original) (raw)

Abstract

Using intestinal monolayers, we showed that F-actin cytoskeletal stabilization and Ca 2ϩ normalization contribute to epidermal growth factor (EGF)-mediated protection against oxidant injury. However, the intracellular mediator responsible for these protective effects remains unknown. Since the protein kinase C-␤1 (PKC-␤1) isoform is abundant in our naive (N) cells, we hypothesized that PKC-␤1 is essential to EGF protection. Monolayers of N Caco-2 cells were exposed to H 2 O 2 Ϯ EGF, PKC, or Ca 2ϩ modulators. Other cells were transfected to over-express PKC-␤1 or to inhibit its expression and then pretreated with low or high doses of EGF or a PKC activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), before H 2 O 2 . In N monolayers exposed to oxidant, pretreatment with EGF or PKC activators activated PKC-␤1, enhanced 45 Ca 2ϩ efflux, normalized Ca 2ϩ , decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of the actin. PKC inhibitors prevented these protective effects. Transfected cells stably overexpressing PKC-␤1 (ϩ3.1-fold) but not N cell monolayers were protected from injury by even lower doses of EGF or OAG. EGF or OAG rapidly activated the over-expressed PKC-␤1. Antisense inhibition of PKC-␤1 expression (Ϫ90%) prevented all measures of EGF protection. Inhibitors of Ca 2ϩ -ATPase prevented EGF protection in N cells as well as protective synergism in transfected cells. EGF protects the assembly of the F-actin cytoskeleton in intestinal monolayers against oxidants in large part through the activation of PKC-␤1. EGF normalizes Ca 2ϩ by enhancing Ca 2ϩ efflux through PKC-␤1. We have identified novel biologic functions, protection of actin and Ca 2ϩ homeostasis, among the classical isoforms of PKC.

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  41. Address correspondence to: Dr. Ali Banan, Rush University Medical Cen- ter, Division of Digestive Diseases, 1725 W. Harrison, Suite 206, Chicago, IL 60612. E-mail: ali_banan@rush.edu