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

John F. Di Mari, Randy C. Mifflin, Patrick A. Adegboyega, Jamal I. Saada, Don W. Powell

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

Abstract

Background & Aims: Intestinal myofibroblasts (IMFs) express cyclooxygenase 2 (COX-2) early on in polyp progression and respond to pro-inflammatory cytokines. Interleukin (IL)-1α induces COX-2 expression in IMF via mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and nuclear factor κ B (NF-κB)-dependent pathways. Because NF-κB activity can be mediated by PKC activation and reactive oxygen species (ROS) generation, we examined the relationship of these pathways to IL-1α-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-1α increased PKC α, δ, and ζ 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-1α increased ROS levels 2-fold within 15 minutes and this increase was inhibited by 10 μmol/L bisindoly-lymaleimide I (BIS), a pan-specific PKC inhibitor that also inhibits COX-2 expression. Chelerythrine chloride (CC) (0.5 μmol/L) inhibited classic and novel PKC activity, but not PKCζ, and enhanced IL-1α-mediated ROS generation 4.0-fold and COX-2 expression 1.8-fold. The use of a PKCζ pseudosubstrate prevented IL-1 from increasing ROS greater than control levels and abolished IL-1α-induced COX-2 expression. Small inhibitory RNA (siRNA) for PKCζ confirmed its role in COX-2 expression. Antioxidants inhibited ROS generation and diminished IL-1α-induced COX-2 expression by 80%, without affecting PKC activation. Neither the PKC inhibitors nor the antioxidants prevented NF-κB-mediated transcription as determined by reporter gene analysis. Conclusions: PKCζ and threshold ROS generation are critical for IL-1α-induced COX-2 expression and act concomitantly with NF-κB translocation in IMF.

Original languageEnglish (US)
Pages (from-to)1855-1865
Number of pages11
JournalGastroenterology
Volume124
Issue number7
DOIs
StatePublished - Jul 1 2003

ASJC Scopus subject areas

  • Hepatology
  • Gastroenterology

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