The Notch-1 signaling pathway is required for a functional colonic barrier in WT mice

Abstract

Gut lympho-epithelial interactions occur in the epithelial layer and in the sub-epithelial space. We have recently shown that the presence of LPLs promotes mucosal barrier function in the RAG1−/− transfer model of colitis putatively due to the Notch-1 signaling pathway. This finding was corroborated in vitro as epithelial barrier function correlated with the activation of the Notch-1 signaling pathway in vitro. Moreover, the activation of Notch-1 extends throughout the epithelium in CD mucosa compared to normal or UC mucosa. To determine whether the Notch pathway is mandatory for barrier function in vivo. A specific Notch-1 or a scrambled siRNA sequence was freshly complexed with Lipofectamine 2000. Anesthetized WT mice received 2 intra-rectal injections of complexed siRNA (5 nmol in 20 μl of solution/mouse) 4 days apart. The injected mice were sacrificed one day after the last injection. Colonic tissues were either mounted into Ussing chambers to assess permeability or snap-frozen and subjected to Real-Time PCR for Notch-1, Hes-1, CDX-2, Villin, Occludin, Claudin-5, β-catenin, and E-cadherin. Notch-1 mRNA expression was decreased in the Notch-1 siRNA treated WT mice (p=0.001). Hes-1, CDX-2, β-catenin and E-cadherin mRNA expression was decreased in the Notch-1 siRNA treated WT mice (p=0.01, p=0.009, p<0.0001 and p=0.0004). These findings correlated with a decrease in villin, occludin, and claudin-5 mRNA expression (p=0.0002, p=0.0002 and p=0.002). While no differences in resistance were seen between the groups, there was a significant decrease in flux in mice that received the Notch-1 siRNA (10925 ± 3389 ng of Dextran-FITC/ml/min/cm2 vs. 2912 ± 1054 ng, p=0.01). Local knock down of Notch-1 impaired expression of the genes related to Notch-1 and to barrier function affecting epithelial integrity. Thus, the Notch-1 signaling pathway is critical for the epithelium in order to achieve differentiation and functional barrier integrity.