Key role of PKC and Ca in EGF protection of microtubules and intestinal barrier against oxidants

Banan, A., J. Z. Fields, Y. Zhang, and A. Keshavarzian. Key role of PKC and Ca21 in EGF protection of microtubules and intestinal barrier against oxidants. Am J Physiol Gastrointest Liver Physiol 280: G828–G843, 2001.—Using monolayers of human intestinal (Caco-2) cells, we showed that growth factors (GFs) protect microtubules and barrier integrity against oxidative injury. Studies in nongastrointestinal cell models suggest that protein kinase C (PKC) signaling is key in GF-induced effects and that cytosolic calcium concentration ([Ca]i) is essential in cell integrity. We hypothesized that GF protection involves activating PKC and maintaining normal [Ca]i. Monolayers were pretreated with epidermal growth factor (EGF) or PKC or Ca21 modulators before exposure to oxidants (H2O2 or HOCl). Oxidants disrupted microtubules and barrier integrity, and EGF protected from this damage. EGF caused rapid distribution of PKC-a, PKC-bI, and PKC-z isoforms to cell membranes, enhancing PKC activity of membrane fractions while reducing PKC activity of cytosolic fractions. EGF enhanced 45Ca21 efflux and prevented oxidant-induced (sustained) rises in [Ca]i. PKC inhibitors abolished and PKC activators mimicked EGF protection. Oxidant damage was mimicked by and potentiated by a Ca21 ionophore (A-23187), exacerbated by high-Ca21 media, and prevented by calcium removal or chelation or by Ca21 channel antagonists. PKC activators mimicked EGF on both 45Ca21 efflux and [Ca]i. Membrane Ca21-ATPase pump inhibitors prevented protection by EGF or PKC activators. In conclusion, EGF protection of microtubules and the intestinal epithelial barrier requires activation of PKC signal transduction and normalization of [Ca]i.