IL-1α-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCζ-ROS pathway

Background & Aims: Intestinal myofibroblasts (IMFs) express cyclooxygenase 2 (COX-2) early on in polyp progression and respond to pro-inflammatory cytokines. Interleukin (IL-1alpha induces COX-2 expression in IMF via mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and nuclear factor kappa B (NF-kappaB)-dependent pathways. Because NF-kappaB activity can be mediated by PKC activation and reactive oxygen species (ROS) generation, we examined the relationship of these pathways to IL-1alpha-induced COX-2 expression. Methods: The effects of specific PKC inhibitors and antioxidants on PKC activation, ROS generation, and COX-2 expression were studied. Results: Immunoprecipitation/kinase (IPK) analysis showed that IL-1alpha increased PKC ot, 8, and activity 4.5-, 3.1-, and 2.6-fold, respectively, within 5 minutes. Single-cell fluorescence microscopy of 2',7-dichlorofluorescin diacetate (DCF)-loaded cells showed that IL-1alpha increased ROS levels 2-fold within 15 minutes and this increase was inhibited by 10 mumol/L bisindoly-lymaleimide I (BIS), a pan-specific PKC inhibitor that also inhibits COX-2 expression. Chelerythrine chloride (CC) (0.5 mumol/L) inhibited classic and novel PKC activity, but not PKCxi, and enhanced IL-1alpha-mediated ROS generation 4.0-fold and COX-2 expression 1.8-fold. The use of a PKC pseudosubstrate prevented IL-1 from increasing ROS greater than control levels and abolished IL-1alpha-induced COX-2 expression. Small inhibitory RNA (slRNA) for PKCt confirmed its role in COX-2 expression. Antioxidants inhibited ROS generation and diminished IL-1alpha-induced COX-2 expression by 80%, without affecting PKC activation. Neither the PKC inhibitors nor the antioxidants prevented NF-kappaB-mediated transcription as determined by reporter gene analysis. Conclusions: PKCt and threshold ROS generation are critical for IL-1alpha-induced COX-2 expression and act concomitantly with NF-kappaB translocation in IMF.