Mindin is upregulated during colitis and may activate NF-kappaB in a TLR-9 mediated manner

Abstract

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.

Figure 1

Mindin mRNA expression is upregulated during acute intestinal inflammation. A: Histological analysis of normal tissue and of acute inflammation specimen after 6 d of DSS. HE staining of colonic sections (magnification 40 ×); B: Quantitative mRNA expression of mindin in study mice (left graph, n = 3 of each group, triplicate samples from each mouse, right graph combines the data from each group); C: Relative mRNA expression of NF-κB p65 in study groups. Bars represent mean ± SD; ^bP < 0.01 vs day 0.

Figure 2

Mindin mRNA expression is upregulated by CpG-ODN stimulation. About 1 × 10⁵ RAW 264.7 cells were cultured in 12-well plates and stimulated with cytokines of mouse recombinant tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), toll-like receptor (TLR) ligands of Pam3-CSK4, peptidoglycans (PGN), lipopolysaccharide (LPS), flagellin and CpG-ODN 1585 (B) for 8 h, then cells were harvested for RNA isolation and relative mRNA expression of mindin was analyzed using quantitative real time polymerase chain reaction (RT-PCR). In each group, bars represent mean ± SD; ^bP < 0.01 vs unstimulated cells.

Figure 3

Mindin induces nuclear factor (NF)-κB promoter activation in a TLR-9 mediated manner. A: Mindin mRNA expression in mouse multiple organs, HPRT expression as a total RNA control; B: HEK293 cells were transfected with pCMV-Flag (lane at left side), pCMV-Mindin-Flag clone 1 and clone 2 for 24 h and Western blotting analysis was performed. RAW 264.7 (C) and CMT93 (D) cells were transfected with pNF-κB-Luc (firefly luciferase), pRL-0 vector and pCMV-mindin-Flag or pCMV-Flag control vectors. Twelve hours after transfection, cells were stimulated with the different TLR ligands of Pam3-CSK4, PGN, LPS, flagellin and CpG-ODN 1585 for an additional 12 h, then luciferase assays were performed. NS: Not significant. ^bP < 0.01 vs control.