Abstract

Using intestinal monolayers, we showed that F-actin cytoskeletal stabilization and Ca²⁺ 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-β1 (PKC-β1) isoform is abundant in our naive (N) cells, we hypothesized that PKC-β1 is essential to EGF protection. Monolayers of N Caco-2 cells were exposed to H₂O₂ ± EGF, PKC, or Ca²⁺ modulators. Other cells were transfected to over-express PKC-β1 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 H₂O₂. In N monolayers exposed to oxidant, pretreatment with EGF or PKC activators activated PKC-β1, enhanced ⁴⁵Ca²⁺efflux, normalized Ca²⁺, decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of the actin. PKC inhibitors prevented these protective effects. Transfected cells stably over-expressing PKC-β1 (+3.1-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-β1. Antisense inhibition of PKC-β1 expression (−90%) prevented all measures of EGF protection. Inhibitors of Ca²⁺-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-β1. EGF normalizes Ca²⁺ by enhancing Ca²⁺ efflux through PKC-β1. We have identified novel biologic functions, protection of actin and Ca²⁺homeostasis, among the classical isoforms of PKC.