Colon Epithelial Cell Tgf Beta Signaling Modulates The Expression Of Tight Junction Proteins And Barrier Function In Mice

Defective barrier function is a predisposing factor in inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Although TGF beta signaling defects have been associated with IBD and CAC, few studies have examined the relationship between TGF beta and intestinal barrier function. Here, we examine the role of TGF beta signaling via SMAD4 in modulation of colon barrier function. The Smad4 gene was conditionally deleted in the intestines of adult mice and intestinal permeability assessed using an in vivo 4 kDa 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 transepithelial resistance (TER). In silico analyses of human IBD and colon cancer databases were performed. 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 CIdn8/Claudin 8, and decreases in Cldn3, Cldn4, and C/dn7/Claudin 7 with intestinal SMAD4 loss in vivo without changes in Claudin protein localization. TGF beta 1/BMP2 treatment of polarized SMAD4+ colonoids increases TER. Cldn2, Cldn4, Cldn7, and CIdn8 are regulated by canonical TGF beta signaling, and TGF beta-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 with healthy controls. Canonical TGF beta signaling modulates the expression of tight junction proteins and barrier function in mouse colon.NEW & NOTEWORTHY We demonstrate that canonical TGF beta family signaling modulates the expression of critical tight junction proteins in colon epithelial cells, and that expression of these tight junction proteins is associated with maintenance of colon epithelial barrier function in mice.