IL-1alpha-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCzeta-ROS pathway.
OBJECTIVE
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.
Di Mari
John F
JF
Department of Internal Medicine, 9.138 MRB, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1064, USA. jdimari@utmb.edu
Mifflin
Randy C
RC
Adegboyega
Patrick A
PA
Saada
Jamal I
JI
Powell
Don W
DW
D000975:Antioxidants / Q000494:pharmacology; D002478:Cells, Cultured; D003106:Colon / Q000201:enzymology*; D051546:Cyclooxygenase 2; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000201:enzymology*; D007527:Isoenzymes / Q000096:biosynthesis*; D008565:Membrane Proteins; D016328:NF-kappa B / Q000502:physiology; D009861:Onium Compounds / Q000494:pharmacology; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D034741:RNA, Small Interfering / Q000502:physiology; D017382:Reactive Oxygen Species; D000099221:Protein Kinase C zeta
D000975:Antioxidants; D051546:Cyclooxygenase 2; D007375:Interleukin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D016328:NF-kappa B; D009861:Onium Compounds; D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D000099221:Protein Kinase C zeta; C007517:diphenyleneiodonium; C496540:PTGS2 protein, human