Intestinal Epithelial Cell-Specific Deletion of PLD2 Alleviates DSS-Induced Colitis by Regulating Occludin

Abstract

Ulcerative colitis is a multi-factorial disease involving a dysregulated immune response. Disruptions to the intestinal epithelial barrier and translocation of bacteria, resulting in inflammation, are common in colitis. The mechanisms underlying epithelial barrier dysfunction or regulation of tight junction proteins during disease progression of colitis have not been clearly elucidated. Increase in phospholipase D (PLD) activity is associated with disease severity in colitis animal models. However, the role of PLD2 in the maintenance of intestinal barrier integrity remains elusive. We have generated intestinalspecific Pld2 knockout mice (Pld2 IEC-KO) to investigate the mechanism of intestinal epithelial PLD2 in colitis. We show that the knockout of Pld2 confers protection against dextran sodium sulphate (DSS)-induced colitis in mice. Treatment with DSS induced the expression of PLD2 and downregulated occludin in colon epithelial cells. PLD2 was shown to mediate phosphorylation of occludin and induce its proteasomal degradation in a c-Src kinase-dependent pathway. Additionally, we have shown that treatment with an inhibitor of PLD2 can rescue mice from DSS-induced colitis. To our knowledge, this is the first report showing that PLD2 is pivotal in the regulation of the integrity of epithelial tight junctions and occludin turn over, thereby implicating it in the pathogenesis of colitis.Ulcerative colitis (UC) is a multi-factorial disease with genetic, immunological, environmental, and diet-related factors contributing to its etiology 1, 2 . Incidence of UC is increasing worldwide, and current research is focused on elucidating the origin and mechanism of disease initiation and perpetuation. A chronically dysregulated response, mediated by the host's immune cells against normal gut microbiota, results in severe inflammation and is the hallmark of UC 3 . Genome-wide association studies have revealed several genes associated with UC. These studies have highlighted the role of immune cell-and intestinal barrier-associated genes in the development of UC; however, convincing evidence, enabling the understanding of the complex aetiology of UC, is still lacking 4 .Maintenance of proper barrier integrity is an essential function of the epithelial layer. Disruptions in the intestinal epithelial monolayer lead to bacterial translocation across the membrane and subsequent inflammation 5 . Patients with UC show an increased intestinal permeability 6, 7 . Tight junction proteins are a major class of proteins responsible for junctional sealing and selective transport of molecules across the epithelial barrier 4,8 . Altered or compromised expression of epithelial tight junction proteins is a key factor in the pathogenesis of the disease 9, 10 . However, the mechanisms underlying epithelial barrier dysfunction or regulation of epithelial tight junction proteins during disease progression remain unexplored.

Role of Csk in intestinal epithelial barrier function and protection against colitis

“…Moreover, we previously showed that deficiency of Csk in IECs resulted in the elevated activation of the Src family tyrosine kinases c-Src, c-Yes, and Fyn in IECs [12]. In addition, it has been demonstrated that c-Src is implicated in the pathogenesis of DSS-induced colitis [25,27]. It is thus likely that intestinal epithelial Csk modulates the intestinal epithelial barrier in the colonic epithelium by controlling the c-Src-mediated degradation of occludin.…”

“…c-Src was shown to mediate the tyrosine phosphorylation of occludin in cultured IECs upon DSS exposure [25,26]. The DSS-induced tyrosine phosphorylation of occludin caused its proteasome-dependent degradation and increased paracellular permeability in cultured IECs [25]. Moreover, we previously showed that deficiency of Csk in IECs resulted in the elevated activation of the Src family tyrosine kinases c-Src, c-Yes, and Fyn in IECs [12].…”

“…By contrast, the abundance of occludin in the colon was significantly reduced for DSS-treated Csk CKO mice. Given that the reduction in occludin was observed in the colon of patients with IBDs [23] and is thought to contribute to the development of DSS-induced colitis [25], Csk in IECs likely participates in the maintenance of the intestinal epithelial barrier in part through the regulation of the abundance of occludin in the colon after DSS-induced colonic injury.…”

“…The molecular mechanism by which Csk in IECs modulates the expression of occludin in the DSS-treated colon remains to be fully elucidated. It was demonstrated that treatment of mice with DSS in drinking water induced tyrosine phosphorylation of occludin in the colonic epithelial cells [25,26]. c-Src was shown to mediate the tyrosine phosphorylation of occludin in cultured IECs upon DSS exposure [25,26].…”

“…It was demonstrated that treatment of mice with DSS in drinking water induced tyrosine phosphorylation of occludin in the colonic epithelial cells [25,26]. c-Src was shown to mediate the tyrosine phosphorylation of occludin in cultured IECs upon DSS exposure [25,26]. The DSS-induced tyrosine phosphorylation of occludin caused its proteasome-dependent degradation and increased paracellular permeability in cultured IECs [25].…”