title: Cellular zinc is required for intestinal epithelial barrier maintenance via the regulation of claudin-3 and occludin expression.
abstract: Intracellular zinc is required for a variety of cell functions, but its precise roles in the maintenance of the intestinal tight junction (TJ) barrier remain unclear. The present study investigated the essential roles of intracellular zinc in the preservation of intestinal TJ integrity and the underlying molecular mechanisms. Depletion of intracellular zinc in both intestinal Caco-2 cells and mouse colons through the application of a cell-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induced a disruption of the TJ barrier, as indicated by increased FITC-labeled dextran flux and decreased transepithelial electrical resistance. The TPEN-induced TJ disruption is associated with downregulation of two TJ proteins, occludin and claudin-3. Biotinylation of cell surface proteins revealed that the zinc depletion induced the proteolysis of occludin but not claudin-3. Occludin proteolysis was sensitive to the inhibition of calpain activity, and increased calpain activity was observed in the zinc-depleted cells. Although quantitative PCR analysis and promoter reporter assay have demonstrated that the zinc depletion-induced claudin-3 downregulation occurred at transcriptional levels, a site-directed mutation in the egr1 binding site in the claudin-3 promoter sequence induced loss of both the basal promoter activity and the TPEN-induced decreases. Reduced egr1 expression by a specific siRNA also inhibited claudin-3 expression and transepithelial electrical resistance maintenance in cells. This study shows that intracellular zinc has an essential role in the maintenance of the intestinal epithelial TJ barrier through regulation of occludin proteolysis and claudin-3 transcription.
authors.affiliation: Department of Biofunctional Science and Technology, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Miyoshi
authors.affiliation: Department of Biofunctional Science and Technology, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan.
authors.forename: Soichi
authors.initials: S
authors.lastname: Tanabe
authors.affiliation: Department of Biofunctional Science and Technology, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Japan takuya@hiroshima-u.ac.jp.
authors.forename: Takuya
authors.identifier: 0000-0003-3709-543X
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D001665:Binding Sites; D018938:Caco-2 Cells; D002614:Chelating Agents / Q000494:pharmacology; D062465:Claudin-3 / Q000235:genetics / Q000378:metabolism*; D003106:Colon / Q000187:drug effects* / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000378:metabolism; D017097:Electric Impedance; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008813:Mice, Inbred ICR; D062793:Occludin / Q000378:metabolism*; D010539:Permeability; D011401:Promoter Regions, Genetic; D059748:Proteolysis; D034622:RNA Interference; D015398:Signal Transduction; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D015032:Zinc / Q000172:deficiency / Q000378:metabolism*; D019287:Zinc Sulfate / Q000378:metabolism / Q000494:pharmacology*
chemical_list: C568122:CLDN3 protein, human; D002614:Chelating Agents; D062465:Claudin-3; C568123:Cldn3 protein, mouse; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse; D019287:Zinc Sulfate; D015032:Zinc
keywords: claudin-3; intestines; occludin; tight junction; zinc