Diminished tumor necrosis factor-alpha mediated protein kinase c activity in passaged bovine bronchial epithelial cells

Epithelial cells play an important role in mediating the effects of pro-inflammatory cytokine.s, such as tumor necrosis facto r-alpha (TNF-a), in the airways. Previously, we have identified that TNF-a stimulates the migration of bovine bronchial epithelial cells (BBEC) in cultured monolayers and one pathway implicated in the transduetion of TNF-a signaling is protein kinase C (PKC). However, the signal transduetion mechanism of bronchial epithelial cell migration is not well understood and the role of protein kinase C (PKC) in these cells has not been extensively investigated. In this study, the subcellular distribution and the enzyme activity of multiple PKC isoforms was investigated in primary vs. passaged cultures of BBEC. We have identified the presence of a, ,. 2, and 6 PKC isoforms in primary cultures of BBEC by Western blot and immunocytochemisiry. PKC activity was demonstrated in subcellular fractions of primary BBEC by assaying Ca^-2-dependent PKC activity. TNF-a (5(K) U/ml) induces an increase in migration in primary cultures of BBEC and this response is correlated with the lime of increased PKC activation. Both migration and PKC activity were inhibited by 1 [4M ealphosiin C. Late passage BBEC crude homogenates fractionated by DEAEKophacel Chromatograph y demonstrated the loss of a distinct peak of PKC activity eluting at (O M NaCl, corresponding to ihi? loss of PKC β₁,β₂, und 8 immunoreuctivity. Additionally, PKC activity in response to TNF-u is decreased in long term cultures of passaged BBEC. We hypoihesi/e that this loss of PKC activity may directly relate to changes in cylokine responsiveness of bronchial epithelial cells of various cellular differentiation states- Supported by Department of Veterans Affairs Research Services.