TGF-β1 alters esophageal epithelial barrier function by attenuation of claudin-7 in eosinophilic esophagitis

Barrier dysfunction has been implicated in the pathophysiology of eosinophilic esophagitis (EoE). TGF-β1, a potent pleiotropic molecule, is increased in EoE, however, no study has evaluated its influence on esophageal epithelial barrier. We hypothesized that TGF-β1 regulates barrier dysfunction in EoE. We aimed to determine the role of TGF-β1 in epithelial barrier in models of EoE. To examine the impact of TGF-β1 on esophageal barrier, immortalized human esophageal epithelial (EPC2-hTERT) cells were exposed to TGF-β1 during the 3-dimensional air liquid interface (3D-ALI) model in vitro. TGF-β1 exposure diminished EPC2-hTERT barrier function as measured by transepithelial electrical resistance (TEER) and 3kDa FITC dextran paracellular flux (FITC Flux) and H&E assessment revealed prominent cellular separation. In analysis of epithelial barrier molecules, TGF-β1 led to the specific reduction in expression of the tight-junction molecule, claudin-7 and this was prevented by TGF-β receptor I inhibitor. shRNA mediated claudin-7 knockdown diminished epithelial barrier function, while claudin-7 overexpression resulted in protection from TGF-β1-mediated barrier dysfunction. In analysis of pediatric EoE biopsies claudin-7 expression was decreased, altered localization was observed by immunofluorescence analysis and the TGF-β1 downstream transcription factor phosphorylated SMAD2/3 (pSMAD2/3) was increased. Our data suggest that TGF-β1 participates in esophageal epithelial barrier dysfunction through claudin-7 dysregulation

HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity

Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA–mediated depletion of the HIF1β in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1β-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1β-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis