15-Deoxy-Δ^12,14-prostaglandin J₂-mediated ERK signaling inhibits gram-negative bacteria-induced RelA phosphorylation and interleukin-6 gene expression in intestinal epithelial cells through modulation of protein phosphatase 2A activity

We have previously shown that non-pathogenic Gram-negative Bacteroides vulgatus induces transient RelA phosphorylation (Ser-536), NF-κB activity, and proinflammatory gene expression in native and intestinal epithelial cell (IEC) lines. We now demonstrate that 15-deoxy-Δ^12,14- prostaglandin J₂ (15d-PGJ₂) but not prostaglandin E ₂ inhibits lipopolysaccharide (LPS) (B. vulgatus)/LPS (Escherichia coli)-induced RelA phosphorylation and interleukin-6 gene expression in the colonic epithelial cell line CMT-93. This inhibitory effect of 15d-PGJ ₂ was mediated independently of LPS-induced IκBα phosphorylation/degradation and RelA nuclear translocation as well as RelA DNA binding activity. Interestingly, although B. vulgatus induced nuclear expression of peroxisome proliferator-activated receptor γ (PPARγ) in native epithelium of monoassociated Fisher rats, PPARγ-specific knock-down in CMT-93 cells using small interference RNA failed to reverse the inhibitory effects of PPARγ agonist 15d-PGJ₂ suggesting PPARγ-independent mechanisms. In addition, 15d-PGJ₂ but not the synthetic high affinity PPARγ ligand rosiglitazone triggered ERK1/2 phosphorylation in IEC, and most importantly, MEK1 inhibitor PD98059 reversed the inhibitory effect of 15dPGJ₂ on LPS-induced RelA phosphorylation and interleukin-6 gene expression. Calyculin A, a specific phosphoserine/ phosphothreonine phosphatase inhibitor increased the basal phosphorylation of RelA and reversed the inhibitory effect of 15d-PGJ₂ on LPS-induced RelA phosphorylation. We further demonstrated in co-immunoprecipitation experiments that 15d-PGJ₂ triggered protein phosphatase 2A activity, which directly dephosphorylated RelA in LPS-stimulated CMT-93 cells. We concluded that 15d-PGJ₂ may help to control NF-κB signaling and normal intestinal homeostasis to the enteric microflora by modulating RelA phosphorylation in IEC through altered protein phosphatase 2A activity.