Decrease In Paracellular Permeability Research Articles

Mechanisms of Blood-Brain Barrier Protection by Microbiota-Derived Short-Chain Fatty Acids.

Impairment of the blood-brain barrier (BBB) integrity is implicated in the numerous neurological disorders associated with neuroinflammation, neurodegeneration and aging. It is now evident that short-chain fatty acids (SCFAs), mainly acetate, butyrate and propionate, produced by anaerobic bacterial fermentation of the dietary fiber in the intestine, have a key role in the communication between the gastrointestinal tract and nervous system and are critically important for the preservation of the BBB integrity under different pathological conditions. The effect of SCFAs on the improvement of the compromised BBB is mainly based on the decrease in paracellular permeability via restoration of junctional complex proteins affecting their transcription, intercellular localization or proteolytic degradation. This review is focused on the revealed and putative underlying mechanisms of the direct and indirect effects of SCFAs on the improvement of the barrier function of brain endothelial cells. We consider G-protein-coupled receptor-mediated effects of SCFAs, SCFAs-stimulated acetylation of histone and non-histone proteins via inhibition of histone deacetylases, and crosstalk of these signaling pathways with transcriptional factors NF-κB and Nrf2 as mainstream mechanisms of SCFA's effect on the preservation of the BBB integrity.

High Glucose Reduces the Paracellular Permeability of the Submandibular Gland Epithelium via the MiR-22-3p/Sp1/Claudin Pathway.

Tight junctions (TJs) play an important role in water, ion, and solute transport through the paracellular pathway of epithelial cells; however, their role in diabetes-induced salivary gland dysfunction remains unknown. Here, we found that the TJ proteins claudin-1 and claudin-3 were significantly increased in the submandibular glands (SMGs) of db/db mice and high glucose (HG)-treated human SMGs. HG decreased paracellular permeability and increased claudin-1 and claudin-3 expression in SMG-C6 cells. Knockdown of claudin-1 or claudin-3 reversed the HG-induced decrease in paracellular permeability. MiR-22-3p was significantly downregulated in diabetic SMGs and HG-treated SMG-C6 cells. A miR-22-3p mimic suppressed claudin-1 and claudin-3 expression and abolished the HG-induced increases in claudin-1 and claudin-3 levels in SMG-C6 cells, whereas a miR-22-3p inhibitor produced the opposite effects. Specificity protein-1 (Sp1) was enhanced in diabetic SMGs and HG-treated SMG-C6 cells, which promoted claudin-1 and claudin-3 transcription through binding to the corresponding promoters. A luciferase reporter assay confirmed that miR-22-3p repressed Sp1 by directly targeting the Sp1 mRNA 3′-untranslated region (3′-UTR). Consistently, the miR-22-3p mimic suppressed, whereas the miR-22-3p inhibitor enhanced, the effects of HG on Sp1 expression. Taken together, our results demonstrate a new regulatory pathway through which HG decreases the paracellular permeability of SMG cells by inhibiting miR-22-3p/Sp1-mediated claudin-1 and claudin-3 expression.

A Dual Zinc plus Arginine formulation attenuates the pathogenic properties of Porphyromonas gingivalis and protects gingival keratinocyte barrier function in an in vitro model

ABSTRACT Background and objectives Porphyromonas gingivalis, a late colonizer of the periodontal biofilm, has been strongly associated with the chronic form of periodontitis. The aim of this study was to investigate the effects of a Dual Zinc plus Arginine formulation (aqueous solution and dentifrice) on the pathogenic properties of P. gingivalis and the barrier function of an in vitro gingival epithelium model. Results The Dual Zinc plus Arginine aqueous solution and dentifrice inhibited the hemolytic and proteolytic activities of P. gingivalis. The Dual Zinc plus Arginine aqueous solution and dentifrice enhanced the barrier function of an in vitro gingival epithelium model as determined by a time-dependent increase in transepithelial electrical resistance and decrease in paracellular permeability. This was associated with an increased immunolabeling of two important tight junction proteins: zonula occludens-1 and occludin. The deleterious effects of P. gingivalis on keratinocyte barrier function as well as the ability of the bacterium to translocate through a gingival epithelium model were attenuated in the presence of either Dual Zinc plus Arginine aqueous solution or dentifrice. Conclusions The Dual Zinc plus Arginine formulation may offer benefits for patients affected by periodontal disease through its ability to attenuate the pathogenic properties of P. gingivalis and promote keratinocyte barrier function.

Citric acid mitigates soybean meal induced inflammatory response and tight junction disruption by altering TLR signal transduction in the intestine of turbot, Scophthalmus maximus L

A 12-week feeding trial was conducted to investigate the effect of citric acid on the involvement of TLRs in the soybean meal induced inflammatory response and tight junction disruption in the distal intestine of juvenile turbot (Scophthalmus maximus L.). Four isonitrogenous and isolipidic practical diets were formulated: fish meal-based diet (FM); 40% fish meal protein in FM replaced with soybean meal protein (SBM); SBM + 1.5% citric acid and SBM + 3% citric acid. Compared to the FM, diet SBM significantly increased the gene expression of TLRs (TLR2, TLR3, TLR5b, TLR9, TLR21, TLR22) and MyD88, as well as TLR related molecules (NF-κB, IRF-3, p38 and JNK), which were remarkably reduced by dietary citric acid. Similarly, citric acid supplementation in SBM markedly depressed gene expression of pro-inflammatory cytokines (TNF-α and IFN-γ) and pore-forming tight junction protein Claudin-7, and enhanced gene expression of the anti-inflammatory cytokine TGF-β1 and TJ proteins related to the decrease in paracellular permeability (Claudin-3, Claudin-4, Occludin, Tricellulin and ZO-1). Compared to the SBM, the concentration of IgM and C4 in serum was significantly reduced by dietary citric acid. In brief, dietary citric acid could synchronously inhibit TLRs-dependent inflammatory response regulated by NF-κB and IRF3, as well as cause TLRs-dependent tight junction disruption modulated by p38 and JNK. Therefore, citric acid could function on mitigating soybean meal induced enteropathy in the distal intestine of juvenile turbot.

Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of human lung adenocarcinoma A549 cells

Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.

Tu1742 Human Small Intestinal Barrier Function and Tight Junction Integrity During Ischemia Reperfusion

interacts with the E-cadherin mRNA and regulates its expression posttranscriptionally, thus implicating in control of the epithelial barrier function. Methods: Caco-2 cells were used in these studies. The binding of HuR with E-Cadherin mRNA was examined by biotin pulldown assays and ribonucleoprotein/IP analysis. E-cadherin mRNA stability was examined by measuring its half-life with real-time PCR analysis, and its translation was examined by chimeric luciferaseE-cadherin 3'UTR reporter assays. HuR function was investigated by its gene silencing and overexpression. Barrier function was detected by paracellular tracer flux assay and trans epithelial electric resistant. Results: HuR bound to the E-cadherin mRNA by directly interacting with the E-cadherin 3'UTR rather than its 5'UTR and coding region. Ectopic overexpression of HuR by transient transfection increased [HuR/ E-cadherin mRNA] complex, enhanced E-cadherin translation (by~3 times), and up-regulated its protein level (by~3 times), but it failed to alter E-cadherin mRNA stability and total E-cadherin mRNA level. In contrast, silencing HuR decreased HuR/ E-cadherin mRNA association, inhibited E-cadherin translation, and down-regulated its protein abundance without affecting the stability and level of E-cadherin mRNA. Ectopic overexpression of HuR also promoted the epithelial barrier function as indicated by a decrease in paracellular permeability. Decreased E-cadherin translation by silencing HuR disrupted the barrier function and increased paracellular permeability. Conclusions: These results indicate: 1) E-cadherin mRNA is a target of HuR; 2) increased HuR/ E-cadherin mRNA association enhances E-cadherin mRNA translation; and 3) HuR plays an important role in the regulation of intestinal epithelial barrier function.

Tu1744 The Relationship Between Common IBD-Associated Risk Alleles and Intestinal Permeability in a Cohort of Healthy First Degree Relatives of Individuals With Crohn's Disease

interacts with the E-cadherin mRNA and regulates its expression posttranscriptionally, thus implicating in control of the epithelial barrier function. Methods: Caco-2 cells were used in these studies. The binding of HuR with E-Cadherin mRNA was examined by biotin pulldown assays and ribonucleoprotein/IP analysis. E-cadherin mRNA stability was examined by measuring its half-life with real-time PCR analysis, and its translation was examined by chimeric luciferaseE-cadherin 3'UTR reporter assays. HuR function was investigated by its gene silencing and overexpression. Barrier function was detected by paracellular tracer flux assay and trans epithelial electric resistant. Results: HuR bound to the E-cadherin mRNA by directly interacting with the E-cadherin 3'UTR rather than its 5'UTR and coding region. Ectopic overexpression of HuR by transient transfection increased [HuR/ E-cadherin mRNA] complex, enhanced E-cadherin translation (by~3 times), and up-regulated its protein level (by~3 times), but it failed to alter E-cadherin mRNA stability and total E-cadherin mRNA level. In contrast, silencing HuR decreased HuR/ E-cadherin mRNA association, inhibited E-cadherin translation, and down-regulated its protein abundance without affecting the stability and level of E-cadherin mRNA. Ectopic overexpression of HuR also promoted the epithelial barrier function as indicated by a decrease in paracellular permeability. Decreased E-cadherin translation by silencing HuR disrupted the barrier function and increased paracellular permeability. Conclusions: These results indicate: 1) E-cadherin mRNA is a target of HuR; 2) increased HuR/ E-cadherin mRNA association enhances E-cadherin mRNA translation; and 3) HuR plays an important role in the regulation of intestinal epithelial barrier function.

Knockout of Ste20-Like Proline/Alanine-Rich Kinase (SPAK) Attenuates Intestinal Inflammation in Mice

Inflammatory bowel diseases are characterized by epithelial barrier disruption and alterations in immune regulation. Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but the underlying mechanisms need to be defined. Herein, SPAK knockout (KO) C57BL/6 mice exhibited significant increases in intestinal transepithelial resistance, a marked decrease in paracellular permeability to fluorescence isothiocyanate-dextran, and altered apical side tight junction sodium ion selectivity, compared with wild-type mice. Furthermore, the expression of junction protein, claudin-2, decreased. In contrast, expressions of occludin, E-cadherin, β-catenin, and claudin-5 increased significantly, whereas no obvious change of claudin-1, claudin-4, zonula occludens protein 1, and zonula occludens protein 2 expressions was observed. In murine models of colitis induced by dextran sulfate sodium and trinitrobenzene sulfuric acid, KO mice were more tolerant than wild-type mice, as demonstrated by colonoscopy features, histological characteristics, and myeloperoxidase activities. Consistent with these findings, KO mice showed increased IL-10 levels and decreased proinflammatory cytokine secretion, ameliorated bacterial translocation on treatment with dextran sulfate sodium, and regulation of with no lysine (WNK) kinase activity. Together, these features may reduce epithelial permeability. In conclusion, SPAK deficiency increases intestinal innate immune homeostasis, which is important for control or attenuation of pathological responses in inflammatory boweldiseases.

Melatonin decreases duodenal epithelial paracellular permeability via a nicotinic receptor–dependent pathway in rats in vivo

Intestinal epithelial intercellular tight junctions (TJs) provide a rate-limiting barrier restricting passive transepithelial movement of solutes. TJs are highly dynamic areas, and their permeability is changed in response to various stimuli. Defects in the intestinal epithelial TJ barrier may contribute to intestinal inflammation or leaky gut. The gastrointestinal tract may be the largest extrapineal source of endogenous melatonin. Melatonin released from the duodenal mucosa is a potent stimulant of duodenal mucosal bicarbonate secretion (DBS). The aim of this study was to elucidate the role of melatonin in regulating duodenal mucosal barrier functions, including mucosal permeability, DBS, net fluid flux, and duodenal motor activity, in the living animal. Rats were anesthetized with thiobarbiturate, and a ~30-mm segment of the proximal duodenum with an intact blood supply was perfused in situ. Melatonin and the selective melatonin receptor antagonist luzindole were perfused luminally or given intravenously. Effects on permeability (blood-to-lumen clearance of (51)Cr-EDTA), DBS, mucosal net fluid flux, and duodenal motility were monitored. Luminal melatonin caused a rapid decrease in paracellular permeability and an increase in DBS, but had no effect on duodenal motor activity or net fluid flux. Luzindole did not influence any of the basal parameters studied, but significantly inhibited the effects of melatonin. The nonselective and noncompetitive nicotinic acetylcholine receptor antagonist mecamylamine abolished the effect of melatonin on duodenal permeability and reduced that on DBS. In conclusion, these findings provide evidence that melatonin significantly decreases duodenal mucosal paracellular permeability and increases DBS. The data support the important role of melatonin in the neurohumoral regulation of duodenal mucosal barrier.

Indole-3-carbinol에 의한 OVCAR-3 인체 난소암세포의 침윤 억제

In the present study, we investigated the effect of indole-3-carbinol (I3C), a natural compound present in vegetables, on the cell migration and invasion of OVCAR-3 ovarian cancer cells. Our results indicated that I3C inhibited the proliferation of OVCAR-3 cells, a process which was associated with inhibition of cell motility as determined by wound healing experiments and cell invasion studies. I3C treatment increased the tightness of the tight junctions (TJs), which was demonstrated by an increase in transepithelial electrical resistance and a decrease in paracellular permeability. The RT-PCR and immunoblotting results indicated that I3C repressed the levels of claudin-3 as well as claudin-4, proteins that comprise a major part of TJs and play a key role in the control and selectivity of paracellular transport. Furthermore, the activities of matrix metalloproteinase (MMP)-2 and MMP-9 were also decreased by treatment with I3C, which was connected with the down-regulation of their mRNAs and protein expression. The results suggest that I3C may be expected to inhibit cancer cell metastasis and invasion by restoring TJs and decreasing MMP activity in ovarian cancer cell line OVCAR-3.

Ligation of CD24 expressed by oral epithelial cells induces kinase dependent decrease in paracellular permeability mediated by tight junction proteins

In previous studies we demonstrated uniform strong expression of CD24 in the epithelial attachment to the tooth and in the migrating epithelium of the periodontitis lesion. Titers of serum antibodies auto-reactive with CD24 peptide correlated with reduced severity of periodontal disease. In the present study an epithelial culture model with close correspondence for expression patterns for tight junction components in periodontal epithelia was used. Ligation of CD24 expressed by oral epithelial cells with an anti-CD24 antibody induced formation of tight junctions and live-cell imaging confirmed that paracellular diffusion of fluorochrome-labeled dextran was reduced. Expression of mRNA and protein for zona occludens-1, -2, junction adhesion molecule-A (JAM-A), occludin and claudins-1, -4, -8, -15, -18 was significantly increased following ligation of CD24 but only claudins-4 and -15, JAM-A, occludin and zona occludens-1 and -2 were increased at cell contacts. This change in expression patterns reflected that observed between the epithelium of the periodontal lesion and that of the healthy gingival attachment. In the model system, response profiles to kinase inhibitors indicated a key role of c-Src kinase activation in the development of diffusion-limiting tight junction complexes. Activation was confirmed by demonstrating concomitant phosphorylation of the kinase. Pre-incubation with antibodies against JAM-A and claudin-15 prevented barrier-enhancing effects of anti-CD24 antibodies while pre-incubation with antibody to claudin-4 was partially effective. It is concluded that antibodies to CD24 facilitate expression and location of JAM-A, claudins-4 and -15 that mediate enhanced epithelial barrier function in a protective response against bacterial products.

Claudin-4 Levels Are Associated with Intact Alveolar Fluid Clearance in Human Lungs

The removal of edema from the air spaces is a critical function of the alveolar barrier requiring intact tight junctions. Alveolar fluid clearance contributes to graft function after transplantation and is associated with survival in patients with acute lung injury. Claudin-4 concentrations are known to increase during lung injury and the loss of claudin-4 decreases alveolar fluid clearance in mice. This study was therefore undertaken to evaluate whether differences in lung expression of the tight junction protein claudin-4 are associated with alveolar fluid clearance or clinical measures of lung function. Alveolar fluid clearance rates were measured in ex vivo perfused human lungs not used for transplantation and were compared with histological lung injury and clinical measures of lung injury in the donors. Claudin-4 staining demonstrated a positive correlation with alveolar fluid clearance (Spearman rank correlation [r(s)] = 0.71; P < 0.003); however, claudin-4 staining was not strongly associated with histological measures of lung injury. The expression of other tight junction proteins (including ZO-1) was not associated with alveolar fluid clearance or claudin-4 levels. Claudin-4 staining was lower in lungs from donors with greater impairment in respiratory physiology. These data suggest that claudin-4 may promote alveolar fluid clearance and demonstrate that the amount of claudin-4 expressed may provide specific information regarding alveolar epithelial barrier function that strengthens the link between histological changes and physiological impairment.

Cytoskeletal organization alters both epithelial barrier function and immune responses

Changing microtubule polymerization in primary airway epithelial cells alters paracellular permeability. Treatment with nocodazole, which causes microtubule depolymerization, we see a decrease in paracellular permeability. In addition, when we knockdown AQP5, a membrane water channel, we see a decrease in paracellular permeability along with destabilization of microtubules. Our data suggests that AQP5 has a novel role as a microtubule stabilizing protein. Regulation of AQP5 modulates microtubule organization and dynamically regulates barrier function. Furthermore, our data shows that these changes in paracellular permeability regulate polarized epithelial responses to known stimuli such as EGF, IFNg and Substance P, as well as epithelial ligand expression, which mediates one mechanism by which epithelial cells interact with immune cells. Knocking down AQP5 in primary airway epithelial cells significantly increases the expression of the co‐repressor molecule, B7‐H1, which functions to decrease the immune response. Similar findings occur with treatment of the cells with microtubule destabilizing agents. These data suggest that alterations in cytoskeletal organization effect both how an epithelial cell interacts with a neighboring epithelial cell, as well as how it interacts with the immune system.

Abstract P6-08-07: Claudin-20 Promotes an Aggressive Phenotype in Human Breast Cancer Cells

Abstract Background: Claudin-20 is a member of the claudin family of transmembrane proteins located in the tight junction (TJ) of cells of epithelial origin. Claudin-20 was originally described as being distributed in the TJ of intestinal epithelial cells. Due to the increasing evidence supporting the role of TJ proteins in preventing tumour cell metastatic behaviour, this study sought to evaluate the effect of claudin-20 overexpression in human breast cancer cells. Methods: Breast cancer primary tumours (n= 114) and matched background tissue (n=30) were processed for RNA extraction. RNA was reverse transcribed and quantified before analysis by Q-PCR to obtain survival curves. 42 human cancer cell lines were screened for expression of claudin-20. Invasive and non-invasive breast cancer cell lines, MDA-MB-231 (MDAwt) and MCF-7 (MCF-7wt) respectively, which exhibited negligible expression were chosen for over-expression of the claudin-20 gene (MDAexp &amp; MCF-7exp). The gene, after amplification from normal breast cDNA was cloned into a GFP-Fusion TOPO plasmid before electroporation. Expression of the gene product was confirmed by RT-PCR and fluorescent microscopy. Functional testing was carried out using TER (trans-epithelial resistance), paracellular permeability (PCP), cell invasion, cell adhesion and cell growth assays. In addition, in vivo growth was evaluated using an athymic nu/nu mice model. Results: Q-PCR data showed that high claudin-20 expression was correlated with poor survival of patients with breast cancer (p=0.022). We therefore decided to investigate the effect of claudin-20 over-expression in human breast cancer cell behaviour. Following transformation of the breast cancer cell lines MDA-MB-231 &amp; MCF-7 with a claudin-20 expression construct, the expression of the gene was demonstrated by the presence of mRNA expression and by the protein signal clearly observable at the cell-cell membrane. Claudin-20 transformed cells showed significantly increased invasion compared to WT cells (MDAexp 0.332±0.02 RU, MDAwt 0.304±0.001; p=0.05: MCF-7exp0 0.07±0.002 RU, MCF-7wt 0.05±0.0001; p=0.0014). Both MDAexp and MCF-7exp cells were significantly less adhesive than WT cells, (number of cells adhering to matrix: MDAexp 0.2±0.02 RU versus MDAwt 0.27±0.004, p=0.007; MCF-7exp 0.043±0.0005 RU versus MCF-7wt 0.052±0.0.005, p=0.05). There was no effect on growth (either in vitro or in vivo) for either cell line. Over-expression of Claudin-20 resulted in reduced TER (induced by the motogen HGF at 25 ng/ml: MDAexp change in TER at 2h -74+/−1.5 vs MDAwt at 2h -40+/−4.4, p=0.0007). Interestingly, this was not mirrored by PCP, as over-expression of claudin-20 caused a reduction in permeability (MDAexp change in PCP at 2h 182+/−5 vs MDAwt at 2h 260+/−10). Conclusion: The introduction of claudin-20 into human breast cancer cells resulted in breast cancer cells with an aggressive phenotype and reduced trans-epithelial resistance. There was no corresponding decrease in paracellular permeability, indicating that this claudin has a differential function in epithelial TJ. This provides further insight into the importance of correctly functioning TJ in preventing the progression of human breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-08-07.

인체유방암세포의 tight junction 기능 조절을 통한 genistein의 암세포 침윤 억제 효과

Tight junctions (TJs) that act as paracellular permeability barriers play an essential role in regulating the diffusion of fluid, electrolytes and macromolecules through the paracellular pathway. In this study, we investigated the correlation between the tightening of TJs, permeability and the invasive activity of genistein - a bioactive isoflavone of soybeans - in human breast carcinoma MCF-7 and MDA-MB-231 cells. The inhibitory effects of genistein on cell proliferation, motility and invasiveness were found to be associated with the increased tightness of the TJs, which was demonstrated by an increase in transepithelial electrical resistance and a decrease in paracellular permeability. Additionally, the immunoblotting results indicated that genistein repressed the levels of the proteins that comprise the major components of TJ, claudin-3 and claudin-4, which play a key role in the control and selectivity of paracellular transport. Furthermore, genistein decreased the metastasis-related gene expressions of insulin like growth factor-1 receptor and snail, while concurrently increasing that of thrombospondin-1 and E-cadherin. In addition, we demonstrated that claudins play an important role in the anti-motility and invasiveness of genistein using claudin-3 small interfering RNA. Taken together, our results indicate a possible role for genistein as an inhibitor of cancer cell invasion through the tightening of TJs, which may counteract the up-regulation of claudins. In addition, our results indicate that this may be beneficial for the inhibition of tumor metastasis.

Physiological consequences of gill remodeling in goldfish (Carassius auratus) during exposure to long-term hypoxia

Goldfish (Carassius auratus) acclimated to 7 degrees C and exposed to hypoxia ( approximately 10 mmHg) for 7 days exhibited a pronounced remodeling of the gill consisting of the removal of an interlamellar cell mass (ILCM). Subsequent experiments were designed to assess the impact of gill remodeling and the associated increase in functional lamellar surface area on the distribution of branchial ionocytes and Cl(-) flux across the gill. Despite the increased functional lamellar surface area during hypoxia, there was no corresponding increase in Cl(-) loss or efflux of the extracellular marker polyethylene glycol (PEG 4000). However, when hypoxic fish were returned to normoxic water for 12 h, rates of Cl(-) and PEG efflux were markedly stimulated in keeping with an increased surface area for solute movement. Similarly, the rate of branchial Cl(-) uptake was reduced (105 +/- 22 vs. 45 +/- 8 micromol x kg(-1) x h(-1)) in normoxic and hypoxic fish, respectively, but then stimulated (345 micromol x kg(-1) x h(-1)) upon reestablishment of normoxic conditions. Hypoxia (7 days) was accompanied by a significant decrease in the total cross-sectional area of branchial ionocytes owing to a decrease in their numbers and individual sizes. Thus, despite experiencing an increase in functional lamellar surface area, hypoxic goldfish limit branchial Cl(-) loss likely by a hypoxia-mediated decrease in paracellular permeability. In normoxic fish, the ionocytes were largely confined to the outer edges of the ILCM. During hypoxia, preexisting ionocytes migrated with the shrinking ILCM, while a smaller proportion of newly differentiated cells appeared below the surface of the ILCM. The capacity to maintain a population of ionocytes in contact with the water is an appropriate strategy to retain ionoregulatory capabilities regardless of whether the lamellae are uncovered or covered.

The tight junctional protein occludin is found in the uterine epithelium of squamate reptiles

Occludin, an integral protein associated with the mammalian tight junction, has for the first time been identified in the uterus of squamate reptiles. The tight junction is made up of anastamosing strands and forms a selective barrier that regulates paracellular diffusion of solutes across uterine epithelium. Occludin exclusively labels tight junctional strands and is an excellent marker for tight junction permeability. Using western blotting and immunohistochemistry, occludin expression was examined in the uterine epithelium of five species of Australian skinks at different stages of gestation. More occludin was detected during late stage pregnancy/gravidity compared to the lower levels of occludin detected in vitellogenic and post-parturient females in three of the five species. We conclude that the paracellular permeability of the squamate uterine epithelium decreases as gestation progresses. As placental transport of ions and solutes to the embryo is highest during the last third of pregnancy in viviparous squamates, it is likely that a decrease in paracellular permeability is compensated by an upregulation of other transporting mechanisms such as histotrophy.

IL‐6 INDUCES KERATIN EXPRESSION IN INTESTINAL EPITHELIAL CELLS: POTENTIAL ROLE OF KERATIN‐8 IN IL‐6 INDUCED BARRIER FUNCTION ALTERATIONS

The regulation and function of keratin in the intestinal epithelia is largely unknown. In this study we addressed the role and regulation of K8 and K18 expression by IL-6. Caco2-BBE cell line and IL-6 null mice were used to study the effect of IL-6 on keratin expression. Keratin expression was studied by Northern blot, Western blot and confocal microscopy. Paracellular permeability was assessed by apical-to-basal transport of a FITC dextran probe (FD-4). K8 was silenced using siRNA approach. IL-6 significantly upregulated mRNA and protein levels of K8 and K18. Confocal microscopy showed a reticular pattern of intracellular keratin localized to the sub-apical region after IL-6 treatment. IL-6 also induced serine phosphorylation of K8. IL-6 decreased paracellular flux of FD-4 compared to vehicle treated monolayer. K8 silencing abolished the decrease in paracellular permeability induced by IL-6. Interestingly, IL-6−/− mice showed decreased baseline levels of K8/K18 expression. Administration of dextran sodium sulfate (DSS) significantly increased intestinal permeability in IL-6−/− mice compared to WT mice given DSS. Collectively, our data demonstrate that IL-6 regulates the colonic expression of K8 and K18 and K8/K18 mediates barrier protection by IL-6 under conditions where intestinal barrier is compromised. Thus our data uncover a novel function of these abundant cytoskeletal proteins which may have implications in intestinal disorders such as inflammatory bowel disease wherein barrier dysfunction underlies the inflammatory response.

Interleukin-6 Induces Keratin Expression in Intestinal Epithelial Cells: POTENTIAL ROLE OF KERATIN-8 IN INTERLEUKIN-6-INDUCED BARRIER FUNCTION ALTERATIONS

Keratin 8 (K8) and keratin-18 (K18) are the major intermediate filament proteins in the intestinal epithelia. The regulation and function of keratin in the intestinal epithelia is largely unknown. In this study we addressed the role and regulation of K8 and K18 expression by interleukin 6 (IL-6). Caco2-BBE cell line and IL-6 null mice were used to study the effect of IL-6 on keratin expression. Keratin expression was studied by Northern blot, Western blot, and confocal microscopy. Paracellular permeability was assessed by apical-to-basal transport of a fluorescein isothiocyanate dextran probe (FD-4). K8 was silenced using the small interfering RNA approach. IL-6 significantly up-regulated mRNA and protein levels of K8 and K18. Confocal microscopy showed a reticular pattern of intracellular keratin localized to the subapical region after IL-6 treatment. IL-6 also induced serine phosphorylation of K8. IL-6 decreased paracellular flux of FD-4 compared with vehicle-treated monolayers. K8 silencing abolished the decrease in paracellular permeability induced by IL-6. Administration of dextran sodium sulfate (DSS) significantly increased intestinal permeability in IL-6-/- mice compared with wild type mice given DSS. Collectively, our data demonstrate that IL-6 regulates the colonic expression of K8 and K18, and K8/K18 mediates barrier protection by IL-6 under conditions where intestinal barrier is compromised. Thus, our data uncover a novel function of these abundant cytoskeletal proteins, which may have implications in intestinal disorders such as inflammatory bowel disease wherein barrier dysfunction underlies the inflammatory response.

Modulation of airway epithelial barrier function in response to bacterial ligands

The barrier function of the airway epithelium provides immune and mechanical defenses against infection by inhaled pathogens. The paracellular junctions are a major potential route of bacterial invasion, thus we postulated that components of the junctional complexes: the tight, adherens and gap junctions, are directly linked to bacterial receptors such as TLR2 and respond dynamically to bacterial ligands. To test this hypothesis, we first monitored changes in transepithelial permeability using 2 μm FluoSpheres or fluoresceinated dextran. At 4 hours following stimulation with either heat killed bacteria or Pam3Cys-Ser-Lys4, a TLR2 specific agonist, there was a 40 % decrease in paracellular permeability as compared to untreated monolayers. To determine which junctional components contribute to this “tightening” of the junctions, the expression and localization of E-Cadherin, as well as the Connexins (26, 32 and 43) were examined. E-Cadherin was mobilized into a lipid raft along with TLR2 shortly after bacterial stimulation. All three Connexins (Cx) were present in the human airway cells. The cell-cell signaling function of Cx43 was clearly affected by bacterial ligands. Inhibition of Cx43 with glycyrrhetinic acid significantly decreased epithelial CXCL8 production (by 50 %, P<0.05) in response to bacteria, limiting the cell-to-cell spread of proinflammatory activation. We identified c-Src in the lipid rafts along with Cx43 and TLR2 and confirmed the participation of c-Src family kinases in regulating Cx43 activity as well as in TLR2 mediated signaling. These results suggest that TLR2-dependent recognition of bacterial ligands directly affects junction function in airway epithelial cells.