Colon epithelial cell TGFβ signaling modulates the expression of tight junction proteins and barrier function in mice.

BACKGROUND: Defective barrier function is a predisposing factor in inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). While TGFβ signaling defects have been associated with IBD and CAC, few studies have examined the relationship between TGFβ and intestinal barrier function. Here, we examine the role of TGFβ signaling via SMAD4 in modulation of colon barrier function.

METHODS: The Smad4 gene was conditionally deleted in the intestines of adult mice and intestinal permeability assessed using an in vivo 4kD FITC-Dextran (FD4) permeability assay. Mouse colon was isolated for gene expression (RNA-sequencing), western blot, and immunofluorescence analysis. In vitro colon organoid culture was utilized to assess junction-related gene expression by qPCR and trans-epithelial resistance (TER). In silico analyses of human IBD and colon cancer databases were performed.

RESULTS: Mice lacking intestinal expression of Smad4 demonstrate increased colonic permeability to FD4 without gross mucosal damage. mRNA/protein expression analyses demonstrate significant increases in Cldn2/Claudin 2 and Cldn8/Claudin 8, and decreases in Cldn3, Cldn4, and Cldn7/Claudin 7 with intestinal SMAD4 loss in vivo without changes in Claudin protein localization. TGFb1/BMP2 treatment of polarized SMAD4+ colonoids increases TER. Cldn2, Cldn4, Cldn7, and Cldn8 are regulated by canonical TGFβ signaling, and TGFβ-dependent regulation of these genes is dependent on nascent RNA transcription (Cldn2, Cldn4, Cldn8) but not nascent protein translation (Cldn4, Cldn8). Human IBD/colon cancer specimens demonstrate decreased SMAD4, CLDN4, CLDN7, and CLDN8 and increased CLDN2 compared to healthy controls.

CONCLUSION: Canonical TGFβ signaling modulates the expression of tight junction proteins and barrier function in mouse colon.