ABSTRACT
Intestinal epithelial cells (IEC) play a role in mucosal inflammatory responses by producing important chemokines like CXCL8 when stimulated by TNF-α. Previously, we found that IEC cell lines required the Rho-associated kinase, ROCK, for CXCL8 responses after IL-1 stimulation. This study extends these findings by showing that inhibiting ROCK suppressed TNF-α-induced CXCL8 secretion by Caco-2 and DLD1 colonic epithelial cell lines and CXCL8 mRNA levels in Caco-2 cells. RNAi knockdown experiments indicated that the inhibitory effect was mediated by ROCK2, and not ROCK1. Inhibiting ROCK had no effect on TNF-stimulated IκBα phosphorylation and degradation or p38 MAPK phosphorylation indicating that ROCK plays no role in these signaling pathways. However, inhibiting ROCK suppressed TNF-induced phosphorylation of the p54 JNK isoform and phosphorylation of the upstream MKK4 kinase. These results suggest that ROCK is required for CXCL8 responses by TNF-stimulated IEC by affecting intracellular signaling through MKK4 and JNK.
Subject(s)
Inflammation/immunology , Interleukin-8/immunology , Intestines/immunology , JNK Mitogen-Activated Protein Kinases/immunology , Signal Transduction/immunology , Tumor Necrosis Factor-alpha/immunology , rho-Associated Kinases/immunology , Amides/pharmacology , Blotting, Western , Caco-2 Cells , Enzyme Inhibitors/pharmacology , Epithelial Cells/immunology , Humans , Interleukin-8/genetics , Interleukin-8/metabolism , Intestines/cytology , MAP Kinase Kinase 4/immunology , Phosphorylation/immunology , Pyridines/pharmacology , RNA Interference/immunology , RNA, Messenger/chemistry , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , rho-Associated Kinases/antagonists & inhibitorsABSTRACT
Intestinal epithelial cells (IEC) play a role in mucosal inflammation by producing pro-inflammatory chemokines that may initiate or amplify local responses. IL-1 is a potent activator of IEC and its receptor localizes to focal adhesions. Since the Rho-associated kinase, ROCK, also localizes to focal adhesions, we examined the role of ROCK in IL-1-induced chemokine responses in IEC cell lines. Suppressing ROCK with the Y27632 inhibitor suppressed IL-1-stimulated Caco-2 cell CXCL8/IL-8 and IEC-6 cell CCL2/MCP-1 secretion and mRNA levels. ROCK inhibition also suppressed IL-1-induced JNK phosphorylation in both cell lines, but high levels of the inhibitor had no significant effect on IL-1-stimulated Caco-2 IκBα phosphorylation and degradation or IKK phosphorylation and kinase activity. Therefore, ROCK may exert an effect on IL-1-stimulated JNK signaling to AP-1 activation, with little effect on IKK/IκBα signaling, defining a potentially important mechanism for regulating IL-1 signaling in IEC that may be essential for optimal cytokine responses.
