Aim: To investigate the regulation of mindin expression and the signaling pathway involved during inflammation.
Methods: C57BL/6 mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 6 d to induce acute colitis, and then the colon was harvested for histological analysis or for RNA isolation. mRNA expression of mindin and nuclear factor (NF)-kappaB p65 was analyzed by quantitative real time polymerase chain reaction (RT-PCR) and mindin expression construct was confirmed by Western blotting. Mouse macrophage and intestinal epithelial lineage cells were stimulated with different cytokines and toll-like receptor (TLR) ligands, before pNF-kappaB-luciferase activity was assessed using the Dual-Luciferase reporter assay system.
Results: mRNA expression of mindin was upregulated 4.7 + or - 1.1 fold compared with the baseline during DSS-induced intestinal inflammation in the mice. Stimulation with CpG-ODN (a known TLR-9 ligand) induced 4.2 + or - 0.3 fold upregulation of mindin expression in RAW 264.7 cells. Full-length of mindin was cloned from cDNA of mouse mesenteric lymph node, then the pCMV-Mindin-Flag expression vector was established and the protein expression level was confirmed. Transfection of the mindin construct and stimulation with CpG-ODN significantly increased the NF-kappaB-luciferase activity by 2.5 + or - 0.3 and 4.5 + or - 0.5 fold in RAW264.7 and CMT93 cells, respectively (P < 0.01).
Conclusion: Mindin expression is upregulated during intestinal inflammation and may induce NF-kappaB promoter activation in a TLR-9 mediated manner.