Stimulation of protein kinase C activity by tumor necrosis factor-alpha in bovine bronchial epithelial cells

Bronchial epithelial cell migration, attachment, and proliferation are important processes in response to airway injury. We have shown that tumor necrosis factor (TNF)-alpha stimulates the migration of bovine bronchial epithelial cells (BBEC) in vitro. We hypothesized that protein kinase C (PKC) may be one of the intracellular signaling mediators of TNF-alpha in BBEC. In this study, we have identified multiple PKC isoforms in BBEC and measured total cellular PKC activity. Polyclonal antibodies to the PKC-alpha, -beta(2), -delta, and -epsilon isoforms recognized protein bands around 80-90 kDa. BBEC primary cultures treated with either 500 U/ml TNF-alpha for 2-4 h or 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate for 15 min resulted in three-to fivefold increases in PKC activity in the particulate fractions of crude cell lysates. This activity was inhibited by 1 mu M calphostin C or 10 mu M H-7. Similarly, TNF-alpha-stimulated BBEC migration was reduced at least twofold in the presence of H-7 or calphostin C. These studies suggest that the activation of PKC is necessary for TNF-alpha-stimulated BBEC migration.