IL-1alpha-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCzeta-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 alpha, delta, and zeta 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 micromol/L bisindolylymaleimide I ( BIS), a pan-specific PKC inhibitor that also inhibits COX-2 expression. Chelerythrine chloride (CC) (0.5 micromol/L) inhibited classic and novel PKC activity, but not PKCzeta, and enhanced IL-1alpha-mediated ROS generation 4.0-fold and COX-2 expression 1.8-fold. The use of a PKCzeta pseudosubstrate prevented IL-1 from increasing ROS greater than control levels and abolished IL-1alpha- induced COX-2 expression. Small inhibitory RNA (siRNA) for PKCzeta 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: PKCzeta and threshold ROS generation are critical for IL-1alpha-induced COX-2 expression and act concomitantly with NF-kappaB translocation in IMF.[1]References
- IL-1alpha-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCzeta-ROS pathway. Di Mari, J.F., Mifflin, R.C., Adegboyega, P.A., Saada, J.I., Powell, D.W. Gastroenterology (2003) [Pubmed]
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