TJP3 promotes T cell immunity escape and chemoresistance in breast cancer: a comprehensive analysis of anoikis-based prognosis prediction and drug sensitivity stratification.

BACKGROUND: Overcoming anoikis is a necessity during the metastasis and invasion of tumors. Recently, anoikis has been reported to be involved in tumor immunity and has been used to construct prognosis prediction models. However, the roles of anoikis in regulating tumor immunity and drug sensitivity in breast cancer are still not clear and therefore worth uncovering. METHODS: TCGA and GEO data are the source of gene expression profiles, which are used to identify anoikis-related-gene (ARG)-based subtypes. R4.2 is used for data analysis. RESULTS: Breast cancer is divided into three subgroups, amongst which shows prognosis differences in pan-cancer cohort, ACC, BLCA, BRCA, LUAD, MESO, PAAD, and SKCM. In breast cancer, it shows significant differences in clinical features, immune cell infiltration and drug sensitivity. Machine learning constructs prognosis prediction model, which is useful to perform chemotherapy sensitivity stratification. Following, TJP3 is identified and verified as the key ARG, up-regulation of which increases tolerance of paclitaxel-induced cell toxicity, accompanied with increased expression of caspas3 and cleaved-caspase3. In addition, Down-regulation of TJP3 weakens the cell migration, which accompanied with increased expression of E-cad and decreased expression of vimentin, twist1, zeb1, and MMP7. Furthermore, the expression level of PD-L1 is negative correlated with TJP3. CONCLUSION: ARGs-based subgroup stratification is useful to recognize chemotherapy sensitive cohort, and also is useful to predict clinical outcome. TJP3 promotes chemoresistance, tumor metastasis and potential immunotherapy escape in breast cancer.

Integrated analysis of the lncRNA-associated ceRNA network in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that worsens with age. Long non-coding RNAs (lncRNAs) dysregulation and its associated competing endogenous RNA (ceRNA) network have a potential connection with the occurrence and development of AD. A total of 358 differentially expressed genes (DEGs) were screened via RNA sequencing, including 302 differentially expressed mRNAs (DEmRNAs) and 56 differential expressed lncRNAs (DElncRNAs). Anti-sense lncRNA is the main type of DElncRNA, which plays a major role in the cis and trans regulation. The constructed ceRNA network consisted of 4 lncRNAs (NEAT1, LINC00365, FBXL19-AS1, RAI1-AS1719) and 4 microRNAs (miRNAs) (HSA-Mir-27a-3p, HSA-Mir-20b-5p, HSA-Mir-17-5p, HSA-Mir-125b-5p), and 2 mRNAs (MKNK2, F3). Functional enrichment analysis revealed that DEmRNAs are involved in related biological functions of AD. The co-expressed DEmRNAs (DNAH11, HGFAC, TJP3, TAC1, SPTSSB, SOWAHB, RGS4, ADCYAP1) of humans and mice were screened and verified by real-time quantitative polymerase chain reaction (qRT-PCR). In this study, we analyzed the expression profile of human AD-related lncRNA genes, constructed a ceRNA network, and performed functional enrichment analysis of DEmRNAs between human and mice. The obtained gene regulatory networks and target genes can be used to further analyze AD-related pathological mechanisms to optimize AD diagnosis and treatment.

Study on differentially expressed genes and participating pathways of ectopic endometrium in adenomyosis patients with different data sets.

BACKGROUND: Adenomyosis is a benign uterine disease and affected patients present with symptoms such as menorrhagia, chronic pelvic pain, abnormal uterine bleeding, and infertility. However, the specific mechanisms by which adenomyosis occurs need to be further studied. OBJECTIVE: Dataset of adenomyosis from our hospital and a public database were analyzed using bioinformatics. Corresponding differentially expressed genes (DEGs) and gene enrichment were detected to explore potential genetic adenomyosis targets. METHODS: Clinical data on adenomyosis were accessed based on the pathological specimens of patients with adenomyosis obtained from the Shengjing Hospital. R software was used to screen for DEGs, and volcano and cluster maps were drawn. Adenomyosis datasets (GSE74373) were downloaded from the GEO database. GEO2R online tool was used to screen for DEGs between adenomyosis and normal controls. Genes with P < 0.01 and |logFC| >1 were selected as DEGs. DAVID software was used for functional and pathway enrichment analyses. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on common DEGs to obtain descriptions of the genes. The online database STRING was used for interaction gene retrieval. Moreover, Cytoscape software was used to construct a protein-protein interaction (PPI) network map for common DEGs to visualize potential gene interactions and screen the hub genes. RESULTS: A total of 845 DEGs were identified in the dataset obtained from Shengjing Hospital. A total of 175 genes were downregulated, and 670 genes were upregulated. In the GSE74373 database, 1679 genes were differentially expressed, 916 genes were downregulated, and 763 genes were upregulated. A total of 40 downregulated and 148 upregulated common DEGs showed potential gene interactions. The top ten upregulated hub genes were CDH1, EPCAM, CLDN7, ESRP1, RAB25, SPINT1, PKP3, TJP3, GRHL2, and CDKN2A. CONCLUSION: Genes involved in tight junction may be key in the development of adenomyosis and may provide a potential treatment strategy for adenomyosis.

Improving effects of telmisartan on spermatogenic disorder induced by fractionated low-dose irradiation in mice.

BACKGROUND: Male infertility is a hot problem worldwide, but there are few treatments, especially male infertility caused by irradiation is difficult to treat. The aim of this study was to investigate and evaluate novel drugs for the treatment of male infertility caused by irradiation. METHODS: we randomly divided 18 male BALB/c mice into 3 groups: control, irradiated, and telmisartan. Both irradiated and telmisartan group completed whole-body 0.5 Gy five times irradiation, and the telmisartan group received intraperitoneal injection of telmisartan (1.2 mg/kg) daily on the next day after irradiation, and all groups were sampled on day 25 after irradiation. RESULTS: Sperm motility results show that total sperm motility of irradiated group was significantly lower compared with control group, and testicular HE results showed that testis in irradiated group were severely damaged. Compared with irradiated group, the total sperm motility, sperm concentration, testicular index, Johnsen score, and the seminiferous tubule layer numbers were higher in telmisartan group (P < 0.05). The immunohistochemical staining showed γ-H2AX expression is higher in telmisartan group compared with irradiated group. And the relative mRNA expression of PLZF, GFRA1, STRA8, DMRT1, SPO11, SYCP2, OVOL2, CCNA1, TJP3, RUNX2, TXNDC2 TNP1, and PRM3 in telmisartan group was all significantly higher than irradiated group (P < 0.05). CONCLUSION: In conclusion, in vivo experiments confirmed that telmisartan ameliorated the spermatogenic disorder in mice caused by fractionated low-dose irradiation via promoting spermatogenesis.

Cholesterol-rich domain formation mediated by ZO proteins is essential for tight junction formation.

Tight junctions (TJs) are cell-adhesion structures responsible for the epithelial barrier. We reported that accumulation of cholesterol at the apical junctions is required for TJ formation [K. Shigetomi, Y. Ono, T. Inai, J. Ikenouchi, J. Cell Biol. 217, 2373-2381 (2018)]. However, it is unclear how cholesterol accumulates and informs TJ formation-and whether cholesterol enrichment precedes or follows the assembly of claudins in the first place. Here, we established an epithelial cell line (claudin-null cells) that lacks TJs by knocking out claudins. Despite the lack of TJs, cholesterol normally accumulated in the vicinity of the apical junctions. Assembly of claudins at TJs is thought to require binding to zonula occludens (ZO) proteins; however, a claudin mutant that cannot bind to ZO proteins still formed TJ strands. ZO proteins were however necessary for cholesterol accumulation at the apical junctions through their effect on the junctional actomyosin cytoskeleton. We propose that ZO proteins not only function as scaffolds for claudins but also promote TJ formation of cholesterol-rich membrane domains at apical junctions.

Toll-like receptor 4 deficiency alleviates lipopolysaccharide-induced intestinal barrier dysfunction.

BACKGROUND: The intestinal tract is considered the body's "engine" and the most impacted organ during sepsis. In this study, we explored toll-like receptor 4 (TLR4) functions in sepsis-induced intestinal barrier dysfunction. METHODS: Wild-type and TLR4-knockout (KO) mice were used to establish a sepsis-induced dysfunctional intestinal barrier model via the intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). Hematoxylin and eosin staining, Transmission electron microscope, enzyme linked immunosorbent assay, western blot, quantitative real-time polymerase chain reaction, TdT-mediated dUTP nick end labeling staining, 16 S rRNA gene sequencing were used to explore differences in inflammatory cytokines, apoptosis, tight junction (TJ) protein expression, and intestinal flora diversity between groups. RESULTS: TLR4-deficiency reduced procalcitonin and C-reactive protein to prevent sepsis, and also inhibited inflammatory response by decreasing interleukin (IL)- 1ß, IL-6 and tumor necrosis factor-α levels. Also, BAX/Bcl2 and cleaved-caspase 3 expressions were decreased in TLR4-KO mice to suppress the intestinal mucosal cell apoptosis. TJ proteins, including zonula occludens protein, Occludin and Claudin-5 were significantly increased and intestinal fatty acid binding protein, myosin light chain and myosin light chain kinase were reduced in TLR4-KO mice. Additionally, 16 S rRNA gene sequencing indicated that TLR4-deficiency improved flora diversity and altered normal and abnormal bacterial proportions. CONCLUSIONS: TLR4 deficiency alleviated LPS-induced intestinal barrier dysfunction by reducing inflammatory responses and apoptosis, impairing intestinal damage, and regulating intestinal flora disturbance.

Exosomal miRNA-328-3p targets ZO-3 and inhibits porcine epidemic diarrhea virus proliferation.

As essential transfer carriers for cell-to-cell communication and genetic material, exosomes carry microRNAs that participate in the regulation of various biological processes. MicroRNAs are a type of single-stranded noncoding RNA that bind to specific target gene mRNAs to degrade or inhibit their translation, thereby regulating target gene expression. Although it is known that a variety of microRNAs are involved in the viral infection process, there are few reports on specific microRNAs involved in porcine epidemic diarrhea virus (PEDV) infection. In this study, we isolated and identified exosomes in PEDV-infected Vero E6 cells. Using transcriptomics technology, we found that miRNA-328-3p was significantly downregulated in exosomes following PEDV infection. Moreover, exosomal miRNA-328-3p inhibited infection by PEDV by targeting and inhibiting tight junction protein 3 (TJP-3/ZO-3) in recipient cells. Our findings provide evidence that, after infecting cells, PEDV downregulates expression of miRNA-328-3p, and the resulting reduced inhibition of the target protein ZO-3 helps to enhance PEDV infection. These results provide new insight for understanding the regulatory mechanism of PEDV infection.

Dietary supplementation of ß-conglycinin, with or without sodium butyrate on the growth, immune response and intestinal health of hybrid grouper.

We investigated the effects of low and high doses of ß-conglycinin and the ameliorative effects of sodium butyrate (based on high-dose ß-conglycinin) on the growth performance, serum immunity, distal intestinal histopathology, and gene, protein expression related to intestinal health in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). The results revealed that the instantaneous growth rate (IGR) of grouper significantly increased, decreased, and increased in the low-dose ß-conglycinin (bL), high-level ß-conglycinin (bH) and high-level ß-conglycinin plus sodium butyrate (bH-NaB), respectively. The feed coefficient ratio (FCR) was significantly increased in the bH and bH-NaB, serum levels of IFN-γ, IL-1ß, and TNF-α were upregulated in the bH. The intestinal diameter/fold height ratio was significantly increased in the bH. Furthermore, there were increases in nitric oxide (NO), total nitric oxide synthase (total NOS), and peroxynitrite anion (ONOO-) in the bH, and decreases in total NOS and ONOO- in the bH-NaB. In the distal intestine, IL-1ß and TGF-ß1 mRNA levels were downregulated and upregulated, respective in the bL. The mRNA levels of TNF-α and IL-6 were upregulated in the bH, and downregulated in the bH-NaB, respectively. Occludin, claudin3 and ZO-3 mRNA levels were upregulated in the bL, downregulated in the bH and then upregulated in the bH-NaB. No significant differences were observed in the mRNA levels of IFN-γ and jam4. And the p-PI3K p85Tyr458/total PI3K p85 value was significantly increased in the bH and then decreased in the bH-NaB, and the total Akt value was significantly increased in the bH. These indicate ß-conglycinin has a regulatory effect on serum immunity and affect distal intestinal development by modulating distal intestinal injury-related parameters. Within the distal intestinal tract, low- and high-dose ß-conglycinin differentially affect immune responses and tight junctions in the distal intestine, which eventually manifests as a reduction in growth performance. Supplementing feed with sodium butyrate might represent an effective approach for enhancing serum immunity, and protects the intestines from damage caused by high-dose ß-conglycinin.

Tight Junction ZO Proteins Maintain Tissue Fluidity, Ensuring Efficient Collective Cell Migration.

Tight junctions (TJs) are essential components of epithelial tissues connecting neighboring cells to provide protective barriers. While their general function to seal compartments is well understood, their role in collective cell migration is largely unexplored. Here, the importance of the TJ zonula occludens (ZO) proteins ZO1 and ZO2 for epithelial migration is investigated employing video microscopy in conjunction with velocimetry, segmentation, cell tracking, and atomic force microscopy/spectroscopy. The results indicate that ZO proteins are necessary for fast and coherent migration. In particular, ZO1 and 2 loss (dKD) induces actomyosin remodeling away from the central cortex towards the periphery of individual cells, resulting in altered viscoelastic properties. A tug-of-war emerges between two subpopulations of cells with distinct morphological and mechanical properties: 1) smaller and highly contractile cells with an outward bulging apical membrane, and 2) larger, flattened cells, which, due to tensile stress, display a higher proliferation rate. In response, the cell density increases, leading to crowding-induced jamming and more small cells over time. Co-cultures comprising wildtype and dKD cells migrate inefficiently due to phase separation based on differences in contractility rather than differential adhesion. This study shows that ZO proteins are necessary for efficient collective cell migration by maintaining tissue fluidity and controlling proliferation.

LRRC19 Promotes Permeability of the Gut Epithelial Barrier Through Degrading PKC-ζ and PKCι/λ to Reduce Expression of ZO1, ZO3, and Occludin.

BACKGROUND: A dysfunctional gut epithelial barrier allows the augmented permeation of endotoxins, luminal antigens, and bacteria into the bloodstream, causing disease. The maintenance of gut epithelial barrier integrity may be regulated by multiple factors. Herein we analyze the role of leucine-rich repeat-containing protein 19 (LRRC19) in regulating the permeability of the gut epithelial barrier. METHODS: We utilized Lrrc19 knockout (KO) mice and clinical samples through transmission electron, intestinal permeability assay, Western blot, and immunofluorescence staining to characterize the role of LRRC19 in the permeability of the gut epithelial barrier. RESULTS: We found that LRRC19, which is expressed in gut epithelial cells, impairs gut barrier function. Transmission electron micrographs revealed a tighter junction and narrower gaps in the colon epithelium cells in LRRC19 KO mice. There were lower levels of serum lipopolysaccharide and 4 kDa-fluorescein isothiocyanate-dextran after gavage in LRRC19 KO mice than in wild-type mice. We found that LRRC19 could reduce the expression of zonula occludens (ZO)-1, ZO-3, and occludin in the colonic epithelial cells. The decreased expression of ZO-1, ZO-3, and occludin was dependent on degrading protein kinase C (PKC) ζ and PKCι/λ through K48 ubiquitination by LRRC19. The expression of LRRC19 was also negatively correlated with ZO-1, ZO-3, occludin, PKCζ, and PKCι/λ in human colorectal cancers. CONCLUSIONS: The protein LRRC19 can promote the permeability of the gut epithelial barrier through degrading PKC ζ and PKCι/λ to reduce the expression of ZO-1, ZO-3, and occludin.

Long non­coding RNA NEAT1 promotes ovarian cancer cell invasion and migration by interacting with miR­1321 and regulating tight junction protein 3 expression.

Previous studies have reported that long non­coding RNAs (lncRNAs) have a significant role in the metastasis of tumors, including ovarian cancer (OC). The aim of the present study was to demonstrate the function and working mechanism of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in OC. The expressions of NEAT1 in OC were measured by reverse transcription­quantitativePCR (RT­qPCR). The effects of NEAT1 on cell proliferation, invasion, migration and epithelial­mesenchymal transition (EMT) were detected by Cell Counting Kit­8, transwell and wound healing assays, and western blotting. Dual­luciferase reporter assays were performed to confirm the correlated between NEAT and miR­1321, miR­1321 and TJP3. The effect of NEAT1 on miR­1321 and TJP3 was confirmed by RT­qPCR and western blotting. Elevated expression of NEAT1 was observed in OC cell lines, and NEAT1 expression was found to be positively related to the expression of tight junction protein 3 (TJP3), which is important in cancer development. Moreover, the present results indicated that NEAT1 and TJP3 expression levels were negatively correlated with microRNA (miR)­1321 expression in OC. Knockdown of NEAT1 attenuated the migration and invasion of OC cells, as well as increased miR­1321 expression and in turn led to the reduction of TJP3. Thus, the present study demonstrated that NEAT1 regulates TJP3 expression by sponging miR­1321 and enhances the epithelial­mesenchymal transition, invasion and migration of OC cells. Overall, the present study identified the function and mechanism of NEAT1 in OC, suggesting that NEAT1 may be a promising therapeutic target for OC metastasis.

Cell Biology: Tight Junctions as Biomolecular Condensates.

Two recent studies report that ZO proteins, the main scaffolding proteins of tight junctions, undergo liquid phase separation. This new concept provides understanding at the mechanistic level of how tight junctions are formed and how they participate in mechanochemical signaling in early development.

When Separation Strengthens Ties.

Phase separation underlies functional compartmentalization in living systems. Two recent studies (Beutel et al. and Schwayer et al.) show that zonula occludens (ZO) proteins of tight junctions (TJs) condense into compartments within the cytoplasm that display liquid properties. This ability to condense predicts normal TJ assembly and epithelial barrier function which are essential for vertebrate embryogenesis.

Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions.

Tight junctions are cell-adhesion complexes that seal tissues and are involved in cell polarity and signaling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands are dependent on the membrane-associated scaffolding proteins ZO1 and ZO2. To understand how zona occludens (ZO) proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane-attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight-junction proteins, including adhesion receptors, cytoskeletal adapters, and transcription factors. Our results suggest that an active-phase transition of ZO proteins into a condensed membrane-bound compartment drives claudin polymerization and coalescence of a continuous tight-junction belt.

Actin-independent trafficking of cochlear connexin 26 to non-lipid raft gap junction plaques.

Hereditary hearing loss affects about 1 per 1000 children. Mutations in GJB2, which encodes the connexin 26 protein (Cx26) involved in cochlear homeostasis, are found in about 50% of patients with autosomal recessive non-syndromic hearing loss. Deciphering the trafficking pathway of cochlear Cx26 in situ should represent an advance in understanding the pathogenic significance of many of these mutations. Connexins trafficking and delivery to lipid raft-associated gap junction plaques usually requires successively microtubule and actin networks. Here we show that cochlear Cx26 exhibits an unusual trafficking pathway. We observed that Cx26 assembly occurs in non-lipid raft membrane domains and that junctional plaques are devoid of actin and associated zonula occludens proteins. Using cytoskeleton-disrupting drugs in organotypic culture, we found that cochlear Cx26 gap junction assembly requires microtubules but not actin filaments. Altogether, our data provide an unexpected insight into Cx26 trafficking pathway and gap junction assembly in the cochlea.

Study of cross talk between phosphatases and OGA on a ZO-3-derived peptide.

O-GlcNAcylation, like phosphorylation, is a dynamic and rapid posttranslational modification which regulates many cellular processes. Phosphorylation on tyrosine and O-GlcNAcylation on nearby serine or threonine residues may modulate each other. Indeed, by using a microarray with a peptide model system based on the ZO-3 protein, extensive cross talk between O-GlcNAcylation by OGT and phosphorylation by kinases was observed. However, studying the effects of kinases and OGT without the reverse processes catalyzed by phosphatases and O-GlcNAcase (OGA) does not provide a complete picture of the cross talk. The study of the missing part showed that nearby phosphorylation affects the de-O-GlcNAcylation by OGA, but not to the same extent as it affects the O-GlcNAcylation by OGT. Both the phosphorylation and de-phosphorylation processes were only slightly affected by the presence of an O-GlcNAc residue on a nearby serine.

Heterogeneity of tissue IL-17 and tight junction proteins expression demonstrated in patients with autoimmune thyroid diseases.

Th17 cells together with their hallmark cytokine interleukin (IL)-17 were identified as crucial contributing factors in the pathogenesis of thyroid autoimmunity. The cytokine-regulated tight junction (Tj) disruption is thought to be essential in the initiation and/or development of several diseases. Still, the role of IL-17 maintaining Tj integrity in autoimmune thyroid diseases (AITDs) has not yet been evaluated. We aimed to investigate integrity of the thyroid follicle by studying immunoexpression of cellular Tj - zonula occludens (ZO)-1 and claudin-1 proteins coupled to IL-17A and CD68 detection in AITD patients compared with controls.Thirty-five adult patients undergoing thyroidectomy and presenting 18 cases of Hashimoto thyroiditis (HT), 7 of Graves' disease (GD) as well as 10 subjects of colloid goiter without autoimmune component served as controls were enrolled in this study. An immunohistochemical analysis including IL-17A, ZO-1, claudin-1, and CD68 detection was performed in each case. The correlation of IL-17A with Tj and CD68 in patients with AITD was also analyzed.Apart from inflammatory cells, we evidenced a stronger expression level of IL17A in the thyroid follicular cells in HT patients when compared with GD or colloid goiter. A significant reduction of ZO-1 immunoreactivity was observed in the thyrocytes in HT patients, whereas no significant differences were found in claudin-1 expression in HT and GD compared with colloid goiter patients. A significantly higher number of thyroid follicles with CD68-positive cells was found in HT patients than that in patients with GD or colloid goiter. In HT patients, the expression of IL-17A in the follicular cells was positively correlated with CD68 immunopositivity, whereas no association with claudin-1 or ZO-1 expression was found. GD patients did not reveal any significant correlation of IL-17A with Tj and CD68.Strong overexpression of IL-17A observed in the thyroid epithelial cells is associated with the presence of intrafollicular CD68-positive cells in HT patients. We evidenced the changes in molecules of thyrocyte junctional complexes highlighting impairment of the thyroid follicle integrity in HT, but no association with IL-17A was found.

Soybean-derived recombinant human epidermal growth factor protects against experimental necrotizing enterocolitis.

BACKGROUND: Epidermal Growth Factor (EGF) reduces necrotizing enterocolitis (NEC). However, its high cost virtually prohibits clinical use. To reduce cost, soybean expressing human EGF was developed. Here we report effectiveness of soybean-derived EGF in experimental NEC. METHODS: Newborn rats were subjected to the NEC-inducing regimen of formula feeding and hypoxia. Formula was supplemented with extract from EGF-expressing or empty soybeans. NEC pathology was determined microscopically. Localization of tight junction proteins JAM-A and ZO-1 was examined by immunofluorescence and levels of mucosal COX-2 and iNOS mRNAs by real time PCR. RESULTS: Soybean extract amounts corresponding to 150µg/kg/day EGF caused considerable mortality, whereas those corresponding to 75µg/kg/day EGF were well tolerated. There was no significant difference in NEC scores between animals fed plain formula and formula supplemented with empty soybean extract. Soybean-EGF-supplemented formula at 75µg/kg/day EGF significantly decreased NEC, attenuated dissociation of JAM-A and ZO-1 proteins from tight junctions, and reduced intestinal expression of COX-2 and iNOS mRNAs. CONCLUSION: Supplementation with soybean-expressed EGF significantly decreased NEC in the rat model. Soybean-expressed EGF may provide an economical solution for EGF administration and prophylaxis of clinical NEC.

Essential roles of Akt/Snail pathway in microcystin-LR-induced tight junction toxicity in Sertoli cell.

Microcystin (MC)-LR is a cyclic heptapeptide that acts as a potent reproductive system toxin. However, the underlying pathways of MCLR-induced reproductive system toxicity have not been well elucidated. The blood-testis barrier is mainly constituted by tight junctions (TJs) between adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. The present study was designed to investigate changes in TJs and the underlying pathway in MC-LR-induced TJs toxicity in Sertoli cell. In our study, the transepithelial electrical resistance (TER) value was decreased in a dose dependent manner due to the markers of TJs occludin, claudin and zonula occludens-1 (ZO-1) expression decline. MC-LR is shown to induce cytotoxicity by inhibiting protein phosphatase 2A (PP2A) activity. Our results also showed that the PP2A activity presented a dose-dependent decline. Moreover, MC-LR stimulated protein expression of snail by Akt/GSK-3ß activation. The activated Akt/GSK-3ß and snail signaling pathway largely accounted for MC-LRinduced TJs toxicity, which could be partially reversed by snail siRNA interference or AKT chemical inhibitor in TM4 cells. These findings indicated that MC-LR inhibit the protein expression of TJs, and the activation of Akt/Snail signaling pathways due to PP2A inhibition is proposed to participate in this process.