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tight junction [keywords]
- Genetic Ablation of Afadin Causes Mislocalization and Deformation of Paneth Cells in the Mouse Small Intestinal Epithelium. [JOURNAL ARTICLE]
- PLoS One 2014; 9(10):e110549.
Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using afadin conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in afadin-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the afadin-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.
- Targeted 25-Hydroxyvitamin D3 1α-Hydroxylase Adoptive Gene Therapy Ameliorates DSS-induced Colitis without Causing Hypercalcemia in Mice. [JOURNAL ARTICLE]
- Mol Ther 2014 Oct 20.
Systemic 1,25(OH)2D3 treatment ameliorating murine IBD could not be applied to patients because of hypercalcemia. We tested the hypothesis that increasing 1,25(OH)2D3 synthesis locally by targeting delivery of the 1α-hydroxylase gene (CYP27B1) to the inflamed bowel would ameliorate IBD without causing hypercalcemia. Our targeting strategy is the use of CD11b(+)/Gr1(+) monocytes as the cell vehicle and a macrophage-specific promoter (Mac1) to control CYP27B1 expression. The CD11b(+)/Gr1(+) monocytes migrated initially to inflamed colon and some healthy tissues in DSS colitis mice; however, only the migration of monocytes to the inflamed colon was sustained. Adoptive transfer of Gr1(+) monocytes did not cause hepatic injury. Infusion of Mac1-CYP27B1-modified monocytes increased body weight gain, survival, and colon length, and expedited mucosal regeneration. Expression of pathogenic Th17 and Th1 cytokines (IL-17a and IFN-γ) was decreased, while expression of protective Th2 cytokines (IL-5 and IL-13) was increased, by the treatment. This therapy also enhanced tight junction gene expression in the colon. No hypercalcemia occurred following this therapy. In conclusion, we have for the first time obtained proof-of-principle evidence for a novel monocyte-based adoptive CYP27B1 gene therapy using a mouse IBD model. This strategy could be developed into a novel therapy for IBD and other autoimmune diseases.Molecular Therapy (2014); doi:10.1038/mt.2014.201.
- Matrix Metalloproteinase-3 Promotes Early Blood-Spinal Cord Barrier Disruption and Hemorrhage and Impairs Long-Term Neurological Recovery after Spinal Cord Injury. [JOURNAL ARTICLE]
- Am J Pathol 2014 Oct 1.
After spinal cord injury (SCI), blood-spinal cord barrier (BSCB) disruption by matrix metalloproteinases (MMPs) leads to BSCB permeability and blood cell infiltration, contributing to permanent neurological disability. Herein, we report that MMP-3 plays a critical role in BSCB disruption after SCI in mice. MMP-3 was induced in infiltrated neutrophils and blood vessels after SCI, and NF-κB as a transcription factor was involved in MMP-3 expression. BSCB permeability and blood cell infiltration after injury were more reduced in Mmp3 knockout (KO) mice than in wild-type (WT) mice, which was significantly inhibited by Mmp3 siRNA or a general inhibitor of MMPs, N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. The level of tight junction proteins, such as occludin and zonula occludens-1, which decreased after SCI, was also higher in Mmp3 KO than in WT mice. Exogenously, MMP-3 injection into the normal spinal cord also induced BSCB permeability. Furthermore, MMP-9 activation after injury was mediated by MMP-3 activation. Finally, improved functional recovery was observed in Mmp3 KO mice compared with WT mice after injury. These results demonstrated the role of MMP-3 in BSCB disruption after SCI for the first time and suggest that the regulation of MMP-3 can be considered a therapeutic target to inhibit BSCB disruption and hemorrhage, and thereby enhance functional recovery after acute SCI.
- The Effect of Claudin-5 Overexpression on the Interactions of Claudin-1 and -2 and Barrier Function in Retinal Cells. [JOURNAL ARTICLE]
- Curr Mol Med 2014 Oct 15.
Claudin-5, one of the dominant tight junctions (TJs) proteins, plays an important role in maintaining the barrier function in the blood brain and retinal barrier. This study aimed to investigate the effect of claudin-5 overexpression on the interactions of claudin-1 and -2 and barrier functions in primary cultured human retinal pigment epithelium cells (HRPECs) and human retina endothelial cells (HRECs). Lentivirus was used to mediate the overexpression of claudin-5 in retinal cells. Significantly increased mRNA and protein levels of claudin-5 were detected in the transfection group. After the transfected cells grew on the transwell membrane for three weeks, a stable monolayer cell barrier model was established in vitro. The claudins expressions analysis showed that overexpressed claudin-5 significantly increased the expression of claudin-1, while it decreased the expression of claudin-2 in both mRNA and protein level. Co-IP experiments and barrier function assay revealed that claudin-5 overexpression promoted the interactions of claudin-1 and claudin-2 and enhanced the barrier function of retinal cells. Intriguingly, the exogenous expression of claudin-5 induced new interaction pattern between claudin-5 and claudin-1 or -2 in HRPECs, which do not have endogenous claudin-5 expression. In addition, claudin-5 overexpression decreased cell mobility and the sprouting capability of vessel tube formation in vitro. This study demonstrated that claudin-5 has a positive regulation in the formation of retinal barrier. Claudin elements and their interactions can be modulated and that such dynamic properties are important for the functions of TJs, ranging from the regulation of retinal barrier integrity to junction-associated signaling mechanisms.
- Intestinal microbial variation may predict early acute rejection after liver transplantation in rats. [Journal Article]
- Transplantation 2014 Oct 27; 98(8):844-52.
Acute rejection (AR) remains a life-threatening complication after orthotopic liver transplantation (OLT) and there are few available diagnostic biomarkers clinically for AR. This study aims to identify intestinal microbial profile and explore potential application of microbial profile as a biomarker for AR after OLT.The OLT models in rats were established. Hepatic graft histology, ultrastructure, function, and intestinal barrier function were tested. Ileocecal contents were collected for intestinal microbial analysis.Hepatic graft suffered from the ischemia-reperfusion (I/R) injury on day 1, initial AR on day 3, and severe AR on day 7 after OLT. Real-time quantitative polymerase chain reaction results showed that genus Faecalibacterium prausnitzii and Lactobacillus were decreased, whereas Clostridium bolteae was increased during AR. Notably, cluster analysis of denaturing gradient gel electrophoresis (DGGE) profiles showed the 7AR and 3AR groups clustered together with 73.4% similarity, suggesting that intestinal microbiota was more sensitive than hepatic function in responding to AR. Microbial diversity and species richness were decreased during AR. Phylogenetic tree analysis showed that most of the decreased key bacteria belonged to phylum Firmicutes, whereas increased key bacteria belonged to phylum Bacteroidetes. Moreover, intestinal microvilli loss and tight junction damage were noted, and intestinal barrier dysfunction during AR presented a decrease of fecal secretory immunoglobulin A (sIgA) and increase of blood bacteremia, endotoxin, and tumor necrosis factor-α.We dynamically detail intestinal microbial characterization and find a high sensitivity of microbial change during AR after OLT, suggesting that intestinal microbial variation may predict AR in early phase and become an assistant therapeutic target to improve rejection after OLT.
- Non-canonical PAR3 activation by factor Xa identifies a novel pathway for Tie2 activation and stabilization of vascular integrity. [JOURNAL ARTICLE]
- Blood 2014 Oct 15.
Endothelial barrier protective effects of activated protein C (APC) require the endothelial protein C receptor (EPCR), protease activated receptor 1 (PAR1), and PAR3. In contrast, PAR1 and PAR3 activation by thrombin results in barrier disruption. Non-canonical PAR1 and PAR3 activation by APC versus canonical activation by thrombin provide an explanation for the functional selectivity of these proteases. Here we found that FXa activated PAR1 at canonical Arg41 similar to thrombin but cleaved PAR3 at non-canonical Arg41 similar to APC. This unique PAR1-PAR3 activation profile permitted the identification of non-canonical PAR3 activation as a novel activation pathway for barrier protective Tie2. APC, FXa, and the non-canonical PAR3 tethered-ligand peptide induced prolonged activation of Tie2, whereas thrombin and the canonical PAR3 tethered-ligand peptide did not. Tie2 activation by FXa required PAR3 and EPCR. FXa and the non-canonical PAR3 tethered-ligand peptide induced Tie2- and PAR3-dependent upregulation of ZO-1, translocation of ZO-1 to cell-cell borders, and the formation of typical ZO-1 honeycomb patterns that are indicative of tight junction stabilization. These data provide intriguing novel insights into the diversification of functional selectivity of proteases signaling achievable by canonical and non-canonical PAR activation, such as the activation of vascular-protective Tie2 by non-canonical PAR3 activation.
- Increased permeability of the epithelium of middle ear cholesteatoma. [JOURNAL ARTICLE]
- Clin Otolaryngol 2014 Oct 16.
We investigated the electrical impedance of and the expressions of tight junction molecules in the cholesteatoma epithelium to provide supporting evidence for the acid lysis theory of bone resorption in middle ear cholesteatoma.Study subjects were patients with primary acquired middle ear cholesteatoma and those with non-cholesteatomatous chronic otitis media who underwent tympanomastoidectomy. The electrical impedance of the cholesteatoma epithelium was measured during tympanomastoidectomy by loading alternating currents of 320 Hz and 30.7 kHz. The expressions of tricellulin (MARVELD2), claudin-1 (CLDN1), and claudin-3 (CLDN3) were examined by fluorescence immunohistochemistry and quantitative reverse transcription-polymerase chain reaction.The electrical impedance of the cholesteatoma epithelium was significantly lower than that of the postauricular skin and external auditory canal skin at both 320 Hz and 30.7 kHz. Immunoreactivity for MARVELD2, CLDN1, and CLDN3 was localized mainly in the granular layer, and to lesser degree, in the horny and spinous layers in both the cholesteatoma tissue and postauricular skin. Fluorescence intensity was moderate for MARVELD2, weak for CLDN1, and strong for CLDN3. The expressions of MARVELD2, CLDN1, and CLDN3 mRNA were significantly lower in the cholesteatoma tissue than in the postauricular skin.These results indicate the increased permeability of the cholesteatoma epithelium, and suggest that this change is, at least partially, dependent on the decrease in the expressions of the tight junction molecules. This evidence supports the acid lysis hypothesis of bone resorption in cholesteatoma. This article is protected by copyright. All rights reserved.
- The kidney tight junction (Review). [JOURNAL ARTICLE]
- Int J Mol Med 2014 Dec; 34(6):1451-1457.
The tight junction is an important subcellular organelle which plays a vital role in epithelial barrier function. Claudin, as the integral membrane component of tight junctions, creates a paracellular transport pathway for various ions to be reabsorbed by the kidneys. This review summarizes advances in claudin structure, function and pathophysiology in kidney diseases. Different claudin species confer selective paracellular permeability to each of three major renal tubular segments: the proximal tubule, the thick ascending limb of Henle's loop and the distal nephron. Defects in claudin function can cause a wide spectrum of kidney diseases, such as hypomagnesemia, hypercalciuria, kidney stones and hypertension. Studies using transgenic mouse models with claudin mutations have recapitulated several of these renal disease phenotypes and have elucidated the underlying biological mechanisms. Modern recording approaches based upon scanning ion conductance microscopy may resolve the biophysical nature of claudin transport function and provide novel insight into tight junction architecture.
- Amelioration of hypoxia and LPS‑induced intestinal epithelial barrier dysfunction by emodin through the suppression of the NF‑κB and HIF‑1α signaling pathways. [JOURNAL ARTICLE]
- Int J Mol Med 2014 Dec; 34(6):1629-1639.
Intestinal barrier dysfunction occurs in critical illnesses and involves the inflammatory and hypoxic injury of intestinal epithelial cells. Researchers are still defining the underlying mechanisms and evaluating therapeutic strategies for restoring intestinal barrier function. The anti‑inflammatory drug, emodin, has been shown to exert a protective effect on intestinal barrier function; however, its mechanisms of action remain unknown. In this study, we investigated the protective effects of emodin on intestinal barrier function and the underlying mechanisms in intestinal epithelial cells challenged with lipopolysaccharide (LPS) and hypoxia/reoxygenation (HR). To induce barrier dysfunction, Caco‑2 monolayers were subjected to HR with or without LPS treatment. Transepithelial electrical resistance and paracellular permeability were measured to evaluate barrier function. The expression of the tight junction (TJ) proteins, zonula occludens (ZO)‑1, occludin, and claudin‑1, as well as that of hypoxia‑inducible factor (HIF)‑1α, phospho‑IκB‑α, phospho‑nuclear factor (NF)‑κB p65 and cyclooxygenase (COX)‑2 was determined by western blot analysis. The results revealed that emodin markedly attenuated the decrease in transepithelial electrical resistance and the increase in paracellular permeability in the Caco‑2 monolayers treated with LPS and subjected to HR. Emodin also markedly alleviated the damage caused by LPS and HR (manifested by a decrease in the expression of the TJ protein, ZO‑1), and inhibited the expression of HIF‑1α, IκB‑α, NF‑κB and COX‑2 in a dose‑dependent manner. In conclusion, our data suggest that emodin attenuates LPS- and HR‑induced intestinal epithelial barrier dysfunction by inhibiting the HIF‑1α and NF‑κB signaling pathways and preventing the damage caused to the TJ barrier (shown by the decrease in the expression of ZO‑1).
- Interleukin-1β induces blood-brain barrier disruption by downregulating sonic hedgehog in astrocytes. [Journal Article]
- PLoS One 2014; 9(10):e110024.
The blood-brain barrier (BBB) is composed of capillary endothelial cells, pericytes, and perivascular astrocytes, which regulate central nervous system homeostasis. Sonic hedgehog (SHH) released from astrocytes plays an important role in the maintenance of BBB integrity. BBB disruption and microglial activation are common pathological features of various neurologic diseases such as multiple sclerosis, Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Interleukin-1β (IL-1β), a major pro-inflammatory cytokine released from activated microglia, increases BBB permeability. Here we show that IL-1β abolishes the protective effect of astrocytes on BBB integrity by suppressing astrocytic SHH production. Astrocyte conditioned media, SHH, or SHH signal agonist strengthened BBB integrity by upregulating tight junction proteins, whereas SHH signal inhibitor abrogated these effects. Moreover, IL-1β increased astrocytic production of pro-inflammatory chemokines such as CCL2, CCL20, and CXCL2, which induce immune cell migration and exacerbate BBB disruption and neuroinflammation. Our findings suggest that astrocytic SHH is a potential therapeutic target that could be used to restore disrupted BBB in patients with neurologic diseases.