Cellular zinc is required for intestinal epithelial barrier maintenance via the regulation of claudin-3 and occludin expression.

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.

Miyoshi Y ，Tanabe S ，Suzuki T 《-》

Naringenin enhances intestinal barrier function through the expression and cytoskeletal association of tight junction proteins in Caco-2 cells.

We have previously reported that naringenin promotes the tight junction (TJ) integrity in intestinal Caco-2 cells. This study investigated the naringenin-mediated effect in Caco-2 cells with a particular focus on the modulation of TJ structure and claudin-4 transcriptional regulation. Naringenin (10~100 μM) dose-dependently enhanced TJ barrier integrity of Caco-2 cells, indicated by transepithelial electrical resistance and FITC-dextran flux. Immunoblot analysis showed that naringenin increased the cytoskeletal association of TJ proteins, zonula occludens-2, occludin, claudin-1, and claudin-4, simultaneously with increased occludin phosphorylation. The total expression of claudin-4 was also increased by naringenin. Quantitative RT-PCR and luciferase reporter assay revealed that naringenin transcriptionally increased the claudin-4 expression with activation of claudin-4 promoter. The mutation of the binding site of the transcriptional factor Sp1 in the claudin-4 promoter sequence and the pharmacological inhibition of Sp1 partially suppressed the naringenin-mediated activation of the claudin-4 promoter. Further, naringenin induced the heat shock protein 70 expression in the cells. Naringenin enhances barrier integrity through the assembly and expression of TJ proteins in intestinal epithelial cells. Naringenin-mediated claudin-4 expression occurs, at least partially, through Sp1-dependent transcriptional regulation. The induction of heat shock protein 70 may be also involved in the increased claudin-4 expression.

Noda S ，Tanabe S ，Suzuki T 《-》

L-Glutamine Enhances Tight Junction Integrity by Activating CaMK Kinase 2-AMP-Activated Protein Kinase Signaling in Intestinal Porcine Epithelial Cells.

The tight junctions (TJs) are essential for maintenance of the intestinal mucosal barrier integrity. Results of our recent work show that dietary l-glutamine (Gln) supplementation enhances the protein abundance of TJ proteins in the small intestine of piglets. However, the underlying mechanisms remain largely unknown. This study was conducted to test the hypothesis that Gln regulates TJ integrity through calcium/calmodulin-dependent kinase 2 (CaMKK2)-AMP-activated protein kinase (AMPK) signaling which, in turn, contributes to improved intestinal mucosal barrier function. Jejunal enterocytes isolated from a newborn pig were cultured in the presence of 0-2.0 mmol Gln/L for indicated time points. Cell proliferation, monolayer transepithelial electrical resistance (TEER), paracellular permeability, expression and distribution of TJ proteins, and phosphorylated AMPK were determined. Compared with 0 mmol Gln/L, 2.0 mmol Gln/L enhanced (P < 0.05) cell growth (by 31.9% at 48 h and 11.1% at 60 h). Cells treated with 2 mmol Gln/L increased TEER by 32.2% at 60 h, and decreased (P < 0.05) TJ permeability by 20.3-40.0% at 36-60 h. In addition, 2.0 mmol Gln/L increased (P < 0.05) the abundance of transmembrane proteins, such as occludin, claudin-4, junction adhesion molecule (JAM)-A, and the plaque proteins zonula occludens (ZO)-1, ZO-2, and ZO-3 by 1.8-6 times. In contrast, 0.5 mmol Gln/L had a moderate effect on TJ protein abundance (20.2-70.5%; P < 0.05) of occludin, claudin-3, claudin-4, JAM-A, and ZO-1. 2.0 mmol Gln/L treatment led to a greater distribution of claudin-1, claudin-4, and ZO-1 at plasma membranes compared with 0 mmol Gln/L. This effect of Gln was mediated by the activation of CaMKK2-AMPK signaling, because either depletion of calcium from the medium or the presence of an inhibitor of CaMKK2 abrogated the effect of Gln on epithelial integrity. Our findings indicate that activation of CaMKK2-AMPK signaling by Gln is associated with improved intestinal mucosal barrier function through enhancing the abundance of TJ proteins and altering their intracellular localization in intestinal porcine epithelial cells.

Wang B ，Wu Z ，Ji Y ，Sun K ，Dai Z ，Wu G ... -  《-》

Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier.

Al-Sadi R ，Khatib K ，Guo S ，Ye D ，Youssef M ，Ma T ... -  《-》

3,5,7,3',4'-Pentamethoxyflavone Enhances the Barrier Function through Transcriptional Regulation of the Tight Junction in Human Intestinal Caco-2 Cells.

Mayangsari Y ，Sugimachi N ，Xu W ，Mano C ，Tanaka Y ，Ueda O ，Sakuta T ，Suzuki Y ，Yamamoto Y ，Suzuki T ... -  《-》