Background: Ral, a small GTPase of the Ras subfamily protein, has various biological functions such as cell proliferation, exocytosis, and actin organization through the downstream of Ras signaling pathway. Also, Ral is considered to be involved in inflammation, but its role of intestinal inflammation remains unclear. The aim of this study is to investigate the involvement of Ral activation in intestinal inflammation of experimental murine colitis using RalGAPα2, inhibitory molecule of Ral activation, knockout (KO) mice.
Methods: We assessed the difference of Ral activation in the colonic tissues under steady-state condition between RalGAPα2 KO and wild type (WT) mice using pull-down assay. To compare the phenotype of the colonic tissues under steady-state condition between RalGAPα2 KO and WT mice, we evaluated the histologic findings of their colonic tissues by hematoxylin and eosin staining and immunohistochemistry (IHC) over time. We also assessed the gene expression of inflammatory cytokines in the colonic tissues of both mice. Moreover, we examined the expression of the colonic tight junction molecules in both mice by IHC and western blotting (WB). Next, to investigate a role of Ral activation on colonic inflammation, we compared the histologic findings and the gene expression of inflammatory cytokines in the colonic tissues of murine dextran sodium sulfate (DSS)-induced colitis between RalGAPα2 KO and WT mice. Moreover, we compared the difference of intestinal epithelial permeability in DSS-induced colitis of both mice using fluorescein isothiocyanate (FITC)-dextran permeability assay.
Results: Under steady-state condition, pull-down assay showed more Ral activation in the colonic tissues of RalGAPα2 KO mice compared to WT mice. RalGAPα2 KO mice did not develop spontaneous colitis. However, IHC revealed that the number of CD11b positive cells in the colonic tissues of RalGAPα2 KO mice was significantly higher compared to WT mice. Additionally, RalGAPα2 KO mice showed significant increased gene expression of TNF-α, IL-12p40, and IL-1β compared to WT mice. Furthermore, decreased expression of ZO-1 in the colonic tissues of RalGAPα2 KO mice was found by IHC and WB. On the other hand, under inflammatory condition caused by DSS-induced colitis, RalGAPα2 KO mice showed more severe colitis and significant increased gene expression of TNF-α and IL-1β compared to WT mice. Moreover, RalGAPα2 KO mice showed more increased epithelial permeability compared to WT mice in the FITC-dextran permeability assay (1017±250 vs 405±38 ng/ml, p<0.05).
Conclusions: Our data indicate that RalGAPα2 KO mice showed more susceptible to DSS-induced colitis. Ral activation might be involved in the colonic inflammation through the impairment of intestinal barrier function.