SAM68 IN THE INTESTINAL EPITHELIUM IS A CRITICAL REGULATOR OF INFLAMMATION AND EPITHELIAL INTEGRITY IN ULCERATIVE COLITIS

Abstract Ulcerative colitis (UC) is a chronic, idiopathic inflammatory disorder of the colon characterized by a dysregulated immune response, epithelial barrier disruption, and heightened sensitivity to microbial and inflammatory cues. Both TNFR- and TLR-mediated inflammatory signaling has been implicated in UC pathogenesis and the RNA-binding protein Sam68 (KHDRBS1) has been shown to be a critical regulator of these signaling pathways. Our previous studies demonstrated that Sam68 expression is upregulated in colonic biopsies from UC patients. We also showed that global Sam68 knockout (KO) mice display resistance to experimental colitis, associated with attenuated induction of proinflammatory genes in colon tissues and in TNF-stimulated three-dimensional colon organoids. Bone marrow transfer studies indicated that Sam68 function in hematopoietic cells is dispensable for UC pathogenesis, suggesting a potential role for Sam68 in non-hematopoietic compartments. To dissect the epithelial-specific role of Sam68, we generated intestinal epithelial cell (IEC)-specific Sam68 KO mice. Here we show that, lack of Sam68 in IECs significantly protected mice from DSS-induced acute colitis, marked by reduced weight loss, decreased expression of proinflammatory cytokines in colon tissue, and improved intestinal barrier function, as assessed by FITC-dextran permeability and endoscopy. Immunofluorescence staining showed that acute DSS treatment downregulated tight junction proteins, most notably claudin-2 in WT mice, while it remained stable in IEC-Sam68 deficient mice, suggesting that Sam68 deficiency protects against DSS-induced epithelial barrier dysfunction. In a chronic DSS model, IEC-Sam68 KO mice displayed markedly reduced mucosal inflammation and preserved epithelial integrity compared to wild-type (WT) controls. We also generated IEC-Sam68 KO mice on the TNFΔARE background that develops chronic intestinal inflammation due to overproduction of TNF. We found that Sam68 deficient TNFΔARE mice showed significantly lower intestinal inflammation compared to the Sam68-sufficient littermates. Consistent with human IBD pathology, we found that TNF stimulation increased claudin-2 gene and protein expression in colon epithelial cells, and Sam68 deficiency blocked this induction, underscoring the critical role of epithelial Sam68 in inflammatory barrier disruption. In summary, our findings identify IEC Sam68 as a central regulator of inflammatory signaling and barrier function in the intestine and suggest targeting Sam68 as a promising therapeutic strategy to treat UC.