Mindin is upregulated during colitis and may activate NF-κB 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.