TGFβ Signaling as a Modulator of Epithelial Inflammation and Barrier Function in the Mouse Colon

Abstract

TGFβ signaling via SMAD4 is frequently altered in sporadic colorectal cancer (CRC), inflammatory bowel disease (IBD), and colitis-associated cancer (CAC). Little is known about how TGFβ/SMAD4 modulate inflammation or barrier function in the colon, or how these elements of homeostasis could impinge on CRC, IBD, or CAC. Here, we examine the role of TGFβ signaling via SMAD4 in modulation of colon inflammation and barrier function. The Smad4 gene was conditionally deleted in the intestines of adult mice (Smad4ΔLrig1). RNA-sequencing and flow cytometry of the colonic lamina propria as well as RNA-sequencing, western blot, and immunofluorescence analysis of the colonic epithelium was performed. Intestinal permeability was assessed using an in vivo 4kD FITC-Dextran (FITC-D) permeability assay. In vitro colon organoid (colonoid) culture was utilized to assess for TGFβ regulation of inflammation- and junction-related genes by qPCR, and colonoids were grown on transwells to assess TGFβ’s effect on transepithelial resistance (TER). Smad4ΔLrig1 mice demonstrate increased inflammatory signaling in their colon epithelium and stroma as well as increased leukocyte infiltration into their colonic lamina propria. A subset of inflammation-related genes up-regulated with SMAD4 loss in vivo, including Ccl20, were also directly regulated by canonical TGFβ signaling in vitro. Knockout of CCL20’s receptor, CCR6, from Smad4ΔLrig1 mice significantly reduced the occurrence of adenocarcinoma development after Dextran Sulfate Sodium-induced colitis (8 of 8 [100%] Smad4ΔLrig1; Ccr6+/- mice versus 2 of 11 [18%] Smad4Lrig1; Ccr6-/- mice, p<0.001). Colonic permeability to 4kD FITC-D was significantly increased in Smad4Lrig1 mice. Gene and protein expression analyses demonstrated significant alterations in tight junction gene and protein abundance with intestinal SMAD4 loss in vivo without significant changes in protein localization. Analysis of colonoids demonstrate that multiple tight junction-related genes are regulated by canonical TGFβ signaling and that TGFβ pathway stimulation increased TER in vitro. Canonical TGFβ signaling directly modulates the expression of inflammatory genes as well as critical tight junction proteins and barrier function in mouse colon. How these two phenotypes interact and effect one another remains undetermined.