Claudin-2 Gene Research Articles

Milk and Lacticaseibacillus paracasei BL23 effects on intestinal responses in a murine model of colitis.

Probiotic-containing fermented dairy foods have the potential to benefit human health, but the importance of the dairy matrix for efficacy remains unclear. We investigated the capacity of Lacticaseibacillus paracasei BL23 in phosphate-buffered saline (BL23-PBS), BL23-fermented milk (BL23-milk), and milk to modify intestinal and behavioral responses in a dextran sodium sulfate (DSS, 3% wt/vol) mouse model of colitis. Significant sex-dependent differences were found such that female mice exhibited more severe colitis, greater weight loss, and higher mortality rates. Sex differences were also found for ion transport ex vivo, colonic cytokine and tight junction gene expression, and fecal microbiota composition. Measurements of milk and BL23 effects showed BL23-PBS consumption improved weight recovery in females, whereas milk resulted in better body weight recovery in males. Occludin and Claudin-2 gene transcript levels indicated barrier function was impaired in males, but BL23-milk was still found to improve colonic ion transport in those mice. Proinflammatory and anti-inflammatory gene expression levels were increased in both male and female mice fed BL23, and to a more variable extent, milk, compared with controls. The female mouse fecal microbiota contained high proportions of Akkermansia (average of 18.1%) at baseline, and females exhibited more changes in gut microbiota composition following BL23 and milk intake. Male fecal microbiota harbored significantly more Parasutterella and less Blautia and Roseburia after DSS treatment, independent of BL23 or milk consumption. These findings show the complex interplay between dietary components and sex-dependent responses in mitigating inflammation in the digestive tract.NEW & NOTEWORTHY Sex-dependent responses to probiotic Lacticaseibacillus paracasei and milk and the potential of the dairy matrix to enhance probiotic protection against colitis in this context have not been previously explored. Female mice were more sensitive than males to colonic injury, and neither treatment effectively alleviated inflammation in both sexes. These sex-dependent responses may result from differences in the higher baseline proportions of Akkermansia in the gut microbiome of female mice.

Integrated microbiome, metabolome and transcriptome profiling reveals the beneficial effects of fish oil and Bacillus subtilis jzxj-7 on mouse gut ecosystem.

The effects of fish oil (FO) and Bacillus subtilis jzxj-7 (JZXJ-7) on the colonic physiology, bacteria, metabolites, and gene expressions were studied in C57BL/6J mice. Co-administration of FO and JZXJ-7 was more beneficial than individual supplementation, as evidenced by improved growth performance, enhanced colon crypt depth and goblet cell numbers. FO + JZXJ-7 inhibited colonic fibrosis by downregulating fibrosis marker protein expression and upregulating occludin, claudin-2 and claudin-4 gene expressions. FO + JZXJ-7 ameliorated oxidative stress and inflammation by increasing catalase, superoxide dismutase, total anti-oxidation capacity, and reducing colon tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 levels. Mechanistically, FO + JZXJ-7 modulated the colon micro-ecological environment by enriching Roseburia, Lachnospiraceae NK4B4, Faecalibaculum and Lactococcus and its derived short-chain fatty acids, and activating Ppara and Car1 mediated peroxisome proliferators-activated receptor (PPAR) and phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling. Overall, FO + JZXJ-7 may serve as a promising nutraceutical to improve health by boosting the growth of colonic beneficial bacteria, altering metabolic phenotype, and regulating gene expression.

Effect of Vitamin C and Probiotics on Broilers Performance and Claudin-2 (CLDN-2) Expression Under Heat Stress [RESEARCH NOTE

This study was conducted to investigate the effect of vitamin C and probiotic which consists of five species of beneficial bacteria and two species of fungi on performance, blood parameters, and intestinal CLDN-2 gene expression of broilers during heat stress (HS). A total of 400 broilers were randomly divided into five groups, each of which received a different treatment. The first group was considered a negative control and did not experience any HS (C-); the second group was considered a positive control (C+) and was exposed to HS; the third group received only vitamin C (250 mg/L in drinking water); the fourth group received only the recommended dose of probiotics; and the fifth group received a combination of both vitamin C and probiotics (250 mg/L in drinking water and recommended dose, respectively). The broiler’s performance, blood parameters, and intestinal CLDN-2 gene expression were assessed. The results showed that the administration of probiotics and vitamin C increased the broiler's feed intake and decreased the feed conversion ratio (p < 0.05). Heat stress increased the broiler's blood total protein, glucose, cholesterol, triglyceride, and liver enzyme concentration (p < 0.05). While the supplementation of vitamin C and probiotics decreased these parameters during HS (p < 0.05). Vitamin C administration reduced the intestinal CLDN-2 gene expression in broilers, during HS (p < 0.05). Heat stress negatively affected the broiler's performance and increased the mortality rate and intestinal CLDN-2 gene expression, whereas, the supplementation of vitamin C decreased the deleterious effect of HS in broilers.

Fermentation Supernatant of Elderly Feces with Inulin and Partially Hydrolyzed Guar Gum Maintains the Barrier of Inflammation-Induced Caco-2/HT29-MTX-E12 Co-Cultured Cells.

Intestinal barrier function declines with aging. We evaluated the effect of dietary fibers and indigestible oligosaccharides on intestinal barrier function by altering the microbiota of the elderly. The feces were anaerobically cultured with indigestible dextrin, inulin, partially hydrolyzed guar gum (PHGG), lactulose, raffinose, or alginate, and the fermented supernatant was added to inflammation-induced Caco-2/HT29-MTX-E12 co-cultured cells. Our data showed that inulin- and PHGG-derived supernatants exerted a protective effect on the intestinal barrier. The protective effect was significantly positively correlated with total short-chain fatty acids (SCFAs) and butyric acid production in the supernatant and negatively correlated with the claudin-2 (CLDN2) gene expression in the cultured cells. Furthermore, we showed that the CLDN2 levels are regulated by butyric acid. Thus, inulin and PHGG can change the intestinal environment of the elderly and maintain the intestinal barrier by accelerating the production of SCFAs and modifying the expression levels of barrier function-related genes.

Claudins in kidney health and disease.

Claudins are strategically located to exert their physiologic actions along with the nephron segments from the glomerulus. Claudin-1 is normally located in the Bowman’s capsule, but its overexpression can reach the podocytes and lead to albuminuria. In the proximal tubule (PT), claudin-2 forms paracellular channels selective for water, Na+, K+, and Ca2+. Claudin-2 gene mutations are associated with hypercalciuria and kidney stones. Claudin-10 has two splice variants, -10a and -10b; Claudin-10a acts as an anion-selective channel in the PT, and claudin-10b functions as a cation-selective pore in the thick ascending limb (TAL). Claudin-16 and claudin-19 mediate paracellular transport of Na+, Ca2+, and Mg2+ in the TAL, where the expression of claudin-3/16/19 and claudin-10b are mutually exclusive. The claudin-16 or -19 mutation causes familial hypomagnesemia with hypercalciuria and nephrocalcinosis. Claudin-14 polymorphisms have been linked to increased risk of hypercalciuria. Claudin-10b mutations produce HELIX syndrome, which encompasses hypohidrosis, electrolyte imbalance, lacrimal gland dysfunction, ichthyosis, and xerostomia. Hypercalciuria and magnesuria in metabolic acidosis are related to downregulation of PT and TAL claudins. In the TAL, stimulation of calcium-sensing receptors upregulates claudin-14 and negatively acts on the claudin-16/19 complex. Claudin-3 acts as a general barrier to ions in the collecting duct. If this barrier is disturbed, urine acidification might be impaired. Claudin-7 forms a nonselective paracellular channel facilitating Cl– and Na+ reabsorption in the collecting ducts. Claudin-4 and -8 serve as anion channels and mediate paracellular Cl– transport; their upregulation may contribute to pseudohypoaldosteronism II and salt-sensitive hypertension.

Role of claudins in idiopathic hypercalciuria and renal lithiasis.

Paracellular transport in the kidney is mediated by a family of proteins located in the tight junctions called claudins which confers its ionic selectivity. Claudin-2 is highly expressed in the proximal tubule and descending limb of Henle and mediate paracellular reabsorption of sodium and calcium cations. In the thick ascending limb of Henle (TALH) calcium is reabsorbed by a paracellular channel formed by Claudin-16 and-19. Claudin-16 mediates cationic permeability while Claudin-19 increases the cationic selectivity of Claudin-16 by blocking anionic permeability. On the other hand, Claudin 14, that is also located in TALH, inhibits the paracellular permeability of Claudin-16 to calcium. Recent wide genomic association analysis studies have detected four common synonymous variants (genetic polymorphisms of a single nucleotide, SNPs) at the locus of Claudin-14 gene that were significantly associated with the presence of renal lithiasis. Another study of wide genomic association and nephrolithiasis was carried out in the general population but including chromosome X, where claudin-2 gene is located. They detected nine SNPs that had a significant association with renal lithiasis risk. A greater knowledge of the paracellular pathway controlled by claudins and its regulation will allow us to develop future new treatments for idiopathic hypercalciuria and renal lithiasis.

Relationship between Eimeria tenella associated-early clinical signs and molecular changes in the intestinal barrier function

The major clinical signs of coccidiosis in chickens due to Eimeria parasite are diarrhea and bloody feces. Previous studies showed that the impairment of the intestinal epithelial barrier and the elevation of the intestinal permeability are causes of clinical signs associated with coccidia challenges. Nevertheless, the information about molecular changes of the epithelial barrier at the early stage of the infection with a specific Eimeria species has not been mentioned. Hence, this study aims to elucidate the temporal relationships between epithelial barrier conditions and clinical signs in chickens infected with Eimeria tenella over the time from the earliest stages of infection.White Leghorn chickens were inoculated with 1 × 104 oocysts of E. tenella. Thereafter the chickens were monitored for their daily clinical signs through observation, and between 5 dpi to 10 dpi, feces were collected for oocysts counting. Chickens were then administrated with fluorescein isothiocyanate-dextran (FITC-d) for gastrointestinal permeability test and tissues were collected each day for histopathological observation and total RNA extraction. Finally, the mRNA expression levels of the tight and adherens junction genes and cytokine genes were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR).In this study, clinical signs such as diarrhea and bloody feces were observed concurrently from 3 to 8 dpi. Histopathology changes such as severe inflammation, hemorrhage, and epithelial desquamation were identified in the cecum specimens. The FITC-d level in the E. tenella-infected group was significantly higher than in the control group. In the infected group, the expression of claudin-2 gene was also upregulated, whereas the expressions of claudin-3 and E-cadherin genes were decreased as compared to the control group. These results implied that clinical signs of avian coccidiosis were associated with the intestinal barrier disruption via changes in expression levels of claudins and E-cadherin at the intestine.

Tight junction protein claudin-2 promotes cell entry of Bombyx mori cypovirus.

Claudin-2 is a major component of tight junctions (TJs), which play an important role in reovirus entry into host cells. The Bombyx mori cytoplasmic polyhedosis virus (BmCPV) relates to the cypovirus strain of the reovirus family. So far, the role of claudin-2 in the process of BmCPV infection is not known. In the present study, it was observed that increasing expression of the claudin-2 gene (CLDN2) may concomitantly elevate BmCPV infection. Contrarily, knockdown of CLDN2 expression by siRNAs can reduce BmCPV infection. Similarly, antibody-based blockage of claudin-2 could also decrease BmCPV cell entry. These results suggest that claudin-2 can promote BmCPV infection in vitro. Moreover, immunofluorescence (IF) assays showed that claudin-2 can interact with BmCPV during viral infection. Specifically, co-immunoprecipitation experiments indicated that claudin-2 binds the BmCPV VP7 (instead of VP3 proteins). The interaction between VP7 and claudin-2 was further confirmed by bimolecular fluorescence complementation (BIFC). Altogether, our results suggest that BmCPV cell entry can be promoted upon interaction of VP7 with claudin-2. These findings provide new mechanistic insights related to BmCPV infection. KEY POINTS: •Claudin-2 could promote BmCPV infection of cells. •Claudin-2 interacted with BmCPV during BmCPV infection. •Claudin-2 could interact with BmCPV VP7 protein, but not with VP3 proteins.

Maternal dietary linoleic acid altered intestinal barrier function in domestic pigeons (Columba livia).

Linoleic acid (LA) is predominantly essential for poultry. Poultry lacking LA show retarded growth and reduced disease resistance. Intestinal barrier function plays an important role in pigeon squab growth, whereas research on the effects of LA on intestinal health in altrices is scant. Considering that squabs are fed by their parents, the study aimed to explore the effects of maternal dietary LA on intestinal morphology, tight junction proteins, immune cytokines and microbial flora in squabs. A completely randomised design with a control group, 1 % LA supplementation group, 2 % LA supplementation group and 4 % LA supplementation group was used. Six squabs from each treatment were randomly sampled at 21 d post-hatching. The results indicated that LA supplementation improved intestinal morphology, as reflected by increased villus height, villus area and the ratio of villi to crypts. Also, 1 % LA supplementation elevated the density of goblet cells in the intestine and strengthened tight junctions by up-regulating claudin-3 and occludin gene expression but down-regulating claudin-2 gene expression. Moreover, 1 % LA supplementation reduced the secretion of proinflammatory cytokines and partly increased anti-inflammatory cytokines. The intestinal microbial diversity in the 1 % LA supplementation group was higher than that in the other groups. As beneficial bacteria, Butyrivibrio was the biomarker of 1 % LA supplementation. However, excessive (4 %) LA supplementation led to adverse impacts on intestinal immunity and microbiota. In conclusion, maternal dietary LA might alter intestinal barrier function in pigeon squabs in a dose-dependent manner. Supplementation with 1 % LA was suggested in parental pigeons.

Claudin-2 deficiency associates with hypercalciuria in mice and human kidney stone disease.

The major risk factor for kidney stone disease is idiopathic hypercalciuria. Recent evidence implicates a role for defective calcium reabsorption in the renal proximal tubule. We hypothesized that claudin-2, a paracellular cation channel protein, mediates proximal tubule calcium reabsorption. We found that claudin-2-null mice have hypercalciuria due to a primary defect in renal tubule calcium transport and papillary nephrocalcinosis that resembles the intratubular plugs in kidney stone formers. Our findings suggest that a proximal tubule defect in calcium reabsorption predisposes to papillary calcification, providing support for the vas washdown hypothesis. Claudin-2-null mice were also found to have increased net intestinal calcium absorption, but reduced paracellular calcium permeability in the colon, suggesting that this was due to reduced intestinal calcium secretion. Common genetic variants in the claudin-2 gene were associated with decreased tissue expression of claudin-2 and increased risk of kidney stones in 2 large population-based studies. Finally, we describe a family in which males with a rare missense variant in claudin-2 have marked hypercalciuria and kidney stone disease. Our findings indicate that claudin-2 is a key regulator of calcium excretion and a potential target for therapies to prevent kidney stones.

Alanyl-glutamine protects the intestinal barrier function in trained rats against the impact of acute exhaustive exercise.

Strenuous exercise triggers deleterious effects on the intestinal epithelium, but their mechanisms are still uncertain. Here, we investigated whether a prolonged training and an additional exhaustive training protocol alter intestinal permeability and the putative effect of alanyl-glutamine (AG) pretreatment in this condition. Rats were allocated into 5 different groups: 1) sedentary; 2 and 3) trained (50 min per day, 5 days per week for 12 weeks) with or without 6 weeks oral (1.5 g/kg) AG supplementation; 4 and 5) trained and subjected to an additional exhaustive test protocol with or without oral AG supplementation. Venous blood samples were collected to determine gasometrical indices at the end of the 12-week protocol or after exhaustive test. Lactate and glucose levels were determined before, during, and after the exhaustive test. Ileum tissue collected after all experimental procedures was used for gene expression analysis of Zonula occludens 1 (ZO-1), occludin, claudin-2, and oligopeptide transporter 1 (PepT-1). Intestinal permeability was assessed by urinary lactulose/mannitol test collected after the 12-week protocol or the exhaustive test. The exhaustive test decreased pH and base excess and increased pCO2. Training sessions delayed exhaustion time and reduced the changes in blood glucose and lactate levels. Trained rats exhibited upregulation of PEPT-1, ZO-1, and occludin mRNA, which were partially protected by AG. Exhaustive exercise induced intestinal paracellular leakage associated with the upregulation of claudin-2, a phenomenon protected by AG treatment. Thus, AG partially prevented intestinal training adaptations but also blocked paracellular leakage during exhaustive exercise involving claudin-2 and occludin gene expression.

Lactobacillus helveticus R0052 alleviates liver injury by modulating gut microbiome and metabolome in D-galactosamine-treated rats.

The liver is an important digestive gland, and acute liver failure results in high mortality. Probiotics are considered potential adjuvant therapies for liver disease. This study aimed to investigate the beneficial effects of Lactobacillus helveticus R0052 on acute liver injury and the underlying mechanisms. Sprague-Dawley rats were gavaged with L. helveticus R0052 suspensions (3 × 109 CFU) for 1 week. Subsequently, acute liver injury was induced by intraperitoneal D-galactosamine injection on the eighth day. After 24 h, samples (blood, liver, ileum, faeces) were collected and assessed for histological injury, inflammation, intestinal barrier, gut microbiome and metabolome. L. helveticus R0052 alleviated aminotransferase, bilirubin and total bile acid elevation and histological hepatic injuries. Additionally, L. helveticus R0052 exhibited anti-inflammatory properties by downregulating Toll-like receptors, tumour necrosis factor-α and nuclear factor-κb transcription in liver samples and decreasing proinflammatory cytokine plasma concentrations. Additionally, L. helveticus R0052 ameliorated intestinal abnormalities and regulated Toll-like receptors, claudin2 and mucin3 gene transcription in the intestine. These effects were associated with gut microbiome and metabolome modulation by L. helveticus R0052. Probiotic pretreatment enriched Lactobacillus and Bacteroides and depleted Flavonifractor and Acetatifactor in the gut microbiome. Meanwhile, L. helveticus R0052 improved carbohydrate and fatty acid metabolism and reduced lithocholic acid levels. These results indicate that L. helveticus R0052 is promising for alleviating acute liver injury and provide new insights regarding the correlations among the microbiome, the metabolome, the intestinal barrier and liver disease.

Abstract PD9-08: Breast cancer liver metastases vascularize by vessel co-option, not angiogenesis, and have a desert immune phenotype: A histopathological and gene expression study

Abstract Phase 3 trials of bevacizumab combined with chemotherapy in metastatic breast cancer have consistently failed to demonstrate a survival benefit for the addition of bevacizumab. When cancers metastasize to highly vascular organs (including the liver), they can utilize vessel co-option, instead of angiogenesis, as a mechanism to obtain a vascular supply (1). We have repeatedly shown by histopathological analyses that almost all (95%, 2 cohorts) breast cancer liver metastases utilize vessel co-option instead of angiogenesis to vascularize (2,3). The prevalence of vessel co-option in breast cancer could explain, at least in part, why anti-angiogenic therapy has been a disappointing therapeutic approach in metastatic breast cancer. Animal models of non-angiogenic liver and lung metastases also displayed resistance to anti-VEGF treatment (3,4). We have now undertaken a gene expression study (mRNA sequencing) of targeted samples at the tumor-liver interface to discover gene expression patterns and signaling pathways that are associated with non-angiogenic growth of metastatic cancer in the liver (n = 70). A network to detect biological themes of non-angiogenic growth was built by gene set enrichment analysis. Key components of this network are: cancer cell motility and invasion, epithelial-to-mesenchymal transition, stemness and proliferation. This contrasts with the network of angiogenic liver metastases of which the most important components are inflammation and ECM remodeling. Semi-automated image analyses of CD8-immunostained section of liver metastases confirms that non-angiogenic liver metastases have a significantly lower density of CD8-positive cytotoxic T-lymphocytes at the tumor-liver interface when compared with angiogenic liver metastases (300 cells/mm2 and 1000 cells/mm2, respectively (p<0.0001)). In addition, a clear CXCL13-driven B-cell gene expression signature is associated with angiogenic growth of liver mets but is absent in non-angiogenic growth of breast cancer liver metastases. Gene expression patterns that may be play a role in vessel co-option are the up-regulation of LAMA3, LAMB3, LAMC2, coding for the 3 subunits of laminin-5, and of ITGA3, ITGB1, ITGA6 and ITGB4, coding for both integrin-receptors of laminin-5. This supports the concept of 'adhesive' vessel co-option during which cancer cells use the basement membrane of the co-opted blood vessels as a soil (5). In addition, the claudin-2 gene (CLDN2) is significantly overexpressed in non-angiogenic liver metastases which is consistent with earlier reports on the role of claudin-2 during breast cancer metastasis to the liver (6). In conclusion, we provide evidence, based on morphology and gene expression, for the almost exclusive non-angiogenic growth of breast cancer liver metastases. In addition, non-angiogenic breast cancer liver metastases are characterized by a desert immune phenotype. Both observations can have an impact on the treatment strategy of patients with metastatic breast cancer.

Association of claudin2 and PRSS1‐PRSS2 polymorphisms with idiopathic recurrent acute and chronic pancreatitis: A case–control study from India

Gene polymorphisms, including those recently described in the claudin2 gene, have been implicated in recurrent acute (RAP) and chronic pancreatitis (CP). In India, RAP and CP have been associated with SPINK1 polymorphism. In this study, we evaluated the association of claudin2 and PRSS1-PRSS2 polymorphisms with idiopathic RAP and CP. We included 101 prospectively followed patients with documented idiopathic RAP (IRAP) and 96 patients who presented with idiopathic chronic pancreatitis (ICP) without previous history of AP. Controls were 156 unrelated individuals undergoing master health check or with non-specific symptoms. All the samples were genotyped for the SNPs rs7057398 in the claudin2 (CLDN2) gene and rs10273639 in the PRSS1 gene on Realtime polymerase chain reaction platform. Clinical data pertaining to patient and disease characteristics were recorded. Claudin2 and PRSS1 polymorphisms were seen in a significantly higher proportion of female patients (P = 0.01 and 0.039, respectively). Thirty-three (32.7%) patients with IRAP developed features of early CP during follow-up (mean [95% confidence interval, CI] duration of 11.3 [8.9-13.7] months). Female patients with claudin2 (rs7057398) CC genotype were at significantly higher risk for IRAP (odds ratio [OR] [95% CI] 6.75 [1.82-23.67]; P = 0.004) and progression from IRAP to CP (OR [95% CI] 7.05 [1.51-33.01]; P = 0.007). CT genotype of PRSS1 (rs10273639) was associated IRAP (OR [95% CI] 2.59 [1.1-6.13]; P = 0.030), and both CT and CC genotypes with ICP in women (OR [95% CI] 2.86 [1.12-7.31]; P = 0.033 and 3.73 [1.03-13.59]; P = 0.048, respectively). In this study, we have demonstrated the association of claudin2 (rs7057398) polymorphism with IRAP and progression of IRAP to CP, and PRSS1 (rs10273639) polymorphism with IRAP and ICP.

283 Impact of PRSS1 and CLDN2 Variants in Alcoholic Versus Non-Alcoholic Chronic Pancreatitis

Background & Aims: Chronic pancreatitis (CP) is an inflammatory destructive disorder with a complex pathogenesis. In non-alcoholic chronic pancreatitis (NACP) several genetic risk factors have been identified. A genome wide association study reported that PRSS1 (rs10273639; cationic trypsinogen) and CLDN2 (rs7057398 and rs12688220; Claudin 2) variants contribute to CP susceptibility or resistance. Here, we aimed to refine these findings in a large European cohort. Methods: In total, 3,002 alcohol-related (ACP) and NACP patients and 4,523 controls were enrolled. In addition we included 1,560 German patients with alcoholic liver cirrhosis (ALC) or alcohol dependence (AD) for comparison with German ACP patients. We used a logistic regression model to examine genotype-phenotype relationships. Results: The association with ACP was strongest for PRSS1 SNP (OR 0.63, 95% CI [0.58-0.69]). In male ACP patients both CLDN2 SNPs (OR 1.56, [1.45-1.69] and OR 1.51, [1.39-1.63]) associated with the disease. In female ACP patients CLDN2 SNPs were similarly associated (OR 1.51, [1.25-1.84] and OR 1.36, [1.16-1.59)], while the impact was lower. Similar results were obtained for the comparison of German ACP to German ALC and AD patients. In the overall NACP population, the association of the PRSS1 SNP was marginal (OR 0.82, [0.72-0.94]), while only CLDN2 rs12688220 SNP associated with NACP in males (OR 1.52, [1.13-2.06]). Conclusions: Our results implicate that PRSS1 and CLDN2 gene variants are associated with CP and strongly impact ACP, but only marginally affect NACP.

Interleukin-6 Modulation of Intestinal Epithelial Tight Junction Permeability Is Mediated by JNK Pathway Activation of Claudin-2 Gene

Defective intestinal epithelial tight junction (TJ) barrier has been shown to be a pathogenic factor in the development of intestinal inflammation. Interleukin-6 (IL-6) is a pleiotropic, pro-inflammatory cytokine which plays an important role in promoting inflammatory response in the gut and in the systemic circulation. Despite its key role in mediating variety inflammatory response, the effect of IL-6 on intestinal epithelial barrier remains unclear. The purpose of this study was to investigate the effect of IL-6 on intestinal epithelial TJ barrier and to delineate the intracellular mechanisms involved using in-vitro (filter-grown Caco-2 monolayers) and in-vivo model (mouse intestinal perfusion) systems. Our results indicated that IL-6 causes a site-selective increase in Caco-2 intestinal epithelia TJ permeability, causing an increase in flux of small-sized molecules having molecular radius <4 Å. The size-selective increase in Caco-2 TJ permeability was regulated by protein-specific increase in claudin-2 expression. The IL-6 increase in TJ permeability required activation of JNK signaling cascade. The JNK pathway activation of AP-1 resulted in AP-1 binding to its binding sequence on the claudin-2 promoter region, leading to promoter activation and subsequent increase in claudin-2 gene transcription and protein synthesis and TJ permeability. Our in-vivo mouse perfusion showed that IL-6 modulation of mouse intestinal permeability was also mediated by AP-1 dependent increase in claudin-2 expression. In conclusion, our studies show for the first time that the IL-6 modulation of intestinal TJ permeability was regulated by JNK activation of AP-1 and AP-1 activation of claudin-2 gene.

Genetics of acute and chronic pancreatitis: An update.

Progress made in identifying the genetic susceptibility underlying acute and chronic pancreatitis has benefitted the clinicians in understanding the pathogenesis of the disease in a better way. The identification of mutations in cationic trypsinogen gene (PRSS1 gene; functional gain mutations) and serine protease inhibitor kazal type 1 (SPINK1 gene; functional loss mutations) and other potential susceptibility factors in genes that play an important role in the pancreatic secretory functions or response to inflammation during pancreatic injury has changed the current concepts and understanding of a complex multifactorial disease like pancreatitis. An individual's susceptibility to the disease is governed by genetic factors in combination with environmental factors. Candidate gene and genetic linkage studies have identified polymorphisms in cationic trypsinogen (PRSS1), SPINK1, cystic fibrosis trans-membrane conductance regulator (CFTR), Chymotrypsinogen C (CTRC), Cathepsin B (CTSB) and calcium sensing receptor (CASR). Individuals with polymorphisms in the mentioned genes and other as yet identified genes are at an enhanced risk for the disease. Recently, polymorphisms in genes other than those involved in "intra-pancreatic trypsin regulatory mechanism" namely Claudin-2 (CLDN2) and Carboxypeptidase A1 (CPA1) gene have also been identified for their association with pancreatitis. With ever growing number of studies trying to identify the genetic susceptibility in the form of single nucleotide polymorphisms, this review is an attempt to compile the available information on the topic.

Ephrin-A1 inhibits NSCLC tumor growth via induction of Cdx-2 a tumor suppressor gene

BackgroundTumor formation is a complex process which involves constitutive activation of oncogenes and suppression of tumor suppressor genes. Receptor EphA2 and its ligand ephrin-A1 form an important cell communication system with its functional role in cell-cell interaction and tumor growth. Loss of cell-cell adhesion is central to the cellular transformation and acquisition of metastatic potential. Claudins, the integrated tight junction (TJ) cell-cell adhesion proteins located on the apico-lateral portion of epithelial cells, functions in maintaining cell polarity. There is extensive evidence implicating Eph receptors and ephrins in malignancy, but the mechanisms how these molecular players affect TJ proteins and regulate tumor growth are not clear. In the present study we hypothesized that EphA2 signaling modulates claudin-2 gene expression via induction of cdx-2, a tumor suppressor gene in NSCLC cells.MethodsThe expression of EphA2, claudin-2 was determined in various NSCLC cell lines by using real-time quantitative polymerase chain reaction and Western blot analysis. The claudin-2 expression was also analyzed by immunofluorescence analysis. EphA2 and erk1/erk2 phosphorylation in ephrin-A1 activated cells was evaluated by Western blot analysis. The cell proliferation and tumor colony formation were determined by WST-1 and 3-D matrigel assays respectively.ResultsNSCLC cells over expressed receptor EphA2 and claudin-2. Ephrin-A1 treatment significantly down regulated the claudin-2 and EphA2 expression in NSCLC cells. The transient transfection of cells with vector containing ephrin-A1 construct (pcDNA-EFNA1) decreased the expression of claudin-2, EphA2 when compared to empty vector. In addition ephrin-A1 activation increased cdx-2 expression in A549 cells. In contrast over-expression of EphA2 with plasmid pcDNA-EphA2 up regulated claudin-2 mRNA expression and decreased cdx-2 expression. The transient transfection of cells with vector containing cdx-2 construct (pcMV-cdx-2) decreased the expression of claudin-2 in A549 cells. Moreover, silencing the expression of receptor EphA2 by siRNA significantly reduced claudin-2 expression and decreased cell proliferation and tumor formation. Furthermore, silencing cdx-2 gene expression before ephrin-A1 treatment increased claudin-2 expression along with increased cell proliferation and tumor growth in A549 cells.ConclusionsOur study suggests that EphA2 signaling up-regulates the expression of the TJ-protein claudin-2 that plays an important role in promoting cell proliferation and tumor growth in NSCLC cells. We conclude that receptor EphA2 activation by ephrin-A1 induces tumor suppressor gene cdx-2 expression which attenuates cell proliferation, tumor growth and thus may be a promising therapeutic target against NSCLC.

991 RNA-Binding Protein CUGBP1 Represses Translation of Junctional Adhesion Molecule-1 and Modulates Epithelial Barrier Function

Background: Defective intestinal epithelial tight junction (TJ) barrier, leading to increased antigenic penetration, has been recognized as an important pathogenic mechanism contributing to the development of intestinal inflammation in Crohn's disease. Interleukin-6 (IL-6) is a pro-inflammatory cytokine which is elevated in various inflammatory diseases including Crohn's disease. Previous studies have shown that IL-6 induced increase in intestinal epithelial TJ permeability is mediated in part by an increase in claudin-2 expression. However, the cellular andmolecular mechanisms that regulate the claudin-2 gene activity and TJ permeability remains unclear. Aim: The aim of this study was to delineate the molecular mechanisms involved in IL-6 induced increase in intestinal epithelial TJ permeability. The major focus of these studies was to elucidate the AP-1 regulation of claudin-2 expression and TJ barrier. Methods: Filter-grown Caco-2 cells were used for TJ permeability studies. Various molecular and biochemical methods were used to determine claudin-2 gene activity and expression. Results: 1) IL-6 caused a time-dependent drop in Caco-2 transepithelial resistance and a size-selective increase in paracellular flux to small permeability markers ( 4 A in radius. 2) IL-6 caused a timedependent increase in claudin-2 expression, and did not affect other TJ protein expression including occludin and claudin-1, 3, 4, or 8. 3) IL-6 up-regulation of claudin-2 expression was preceded by the activation of JNK pathway. Inhibition of JNK activation by inhibitor (SP600125) or siRNA prevented the IL-6 induced increase in claudin-2 expression and TJ permeability. 4) IL-6 induced activation of JNK pathway resulted in AP-1 activation. 5) A 3 kb of claudin-2 promoter region was identified by 5' RACE, and cloned into pGL3 basic vector. 6) Minimal promoter region was determined by progressively 5' deletion. Four AP1 binding sites were identified within this region. 7) IL-6 induced an increase in claudin2 promoter activity, mRNA and protein expression. 8) SiRNA of AP-1 inhibited the IL-6 induced increase in claudin-2 promoter activity, expression, and increase in TJ permeability. Conclusion: IL-6 induced increase in Caco-2 TJ permeability was mediated by JNK pathway regulated activation of claudin-2 gene. The JNK pathway induced activation of AP-1 led to a step-wise binding of AP-1 to claudin-2 promoter and activation of promoter activity, increase in claudin-2 gene and protein expression, and claudin-2 dependent increase in TJ permeability. Thus, these studies demonstrate a novel mechanism of intestinal TJ barrier regulation.

990 IL-6 Induced Increase in Intestinal Epithelial Tight Junction Permeability is Mediated by AP-1 Induced Up-Regulation of Claudin-2 Gene Activity

Background: Defective intestinal epithelial tight junction (TJ) barrier, leading to increased antigenic penetration, has been recognized as an important pathogenic mechanism contributing to the development of intestinal inflammation in Crohn's disease. Interleukin-6 (IL-6) is a pro-inflammatory cytokine which is elevated in various inflammatory diseases including Crohn's disease. Previous studies have shown that IL-6 induced increase in intestinal epithelial TJ permeability is mediated in part by an increase in claudin-2 expression. However, the cellular andmolecular mechanisms that regulate the claudin-2 gene activity and TJ permeability remains unclear. Aim: The aim of this study was to delineate the molecular mechanisms involved in IL-6 induced increase in intestinal epithelial TJ permeability. The major focus of these studies was to elucidate the AP-1 regulation of claudin-2 expression and TJ barrier. Methods: Filter-grown Caco-2 cells were used for TJ permeability studies. Various molecular and biochemical methods were used to determine claudin-2 gene activity and expression. Results: 1) IL-6 caused a time-dependent drop in Caco-2 transepithelial resistance and a size-selective increase in paracellular flux to small permeability markers ( 4 A in radius. 2) IL-6 caused a timedependent increase in claudin-2 expression, and did not affect other TJ protein expression including occludin and claudin-1, 3, 4, or 8. 3) IL-6 up-regulation of claudin-2 expression was preceded by the activation of JNK pathway. Inhibition of JNK activation by inhibitor (SP600125) or siRNA prevented the IL-6 induced increase in claudin-2 expression and TJ permeability. 4) IL-6 induced activation of JNK pathway resulted in AP-1 activation. 5) A 3 kb of claudin-2 promoter region was identified by 5' RACE, and cloned into pGL3 basic vector. 6) Minimal promoter region was determined by progressively 5' deletion. Four AP1 binding sites were identified within this region. 7) IL-6 induced an increase in claudin2 promoter activity, mRNA and protein expression. 8) SiRNA of AP-1 inhibited the IL-6 induced increase in claudin-2 promoter activity, expression, and increase in TJ permeability. Conclusion: IL-6 induced increase in Caco-2 TJ permeability was mediated by JNK pathway regulated activation of claudin-2 gene. The JNK pathway induced activation of AP-1 led to a step-wise binding of AP-1 to claudin-2 promoter and activation of promoter activity, increase in claudin-2 gene and protein expression, and claudin-2 dependent increase in TJ permeability. Thus, these studies demonstrate a novel mechanism of intestinal TJ barrier regulation.