Microcystin-LR accelerates follicular atresia in mice via JNK-mediated adherent junction damage of ovarian granulosa cells.

Microcystin-LR (MC-LR), one of aquatic environmental contaminants with reproductive toxicity produced by cyanobacterial blooms, but its toxic effects and mechanisms on the ovary are not fully understood. Here, proteomic techniques and molecular biology experiments were performed to study the potential mechanism of MC-LR-caused ovarian toxicity. Results showed that protein expression profile of ovarian granulosa cells (KK-1) was changed by 17 µg/mL MC-LR exposure. Comparing with the control group, 118 upregulated proteins as well as 97 downregulated proteins were identified in MC-LR group. Function of differentially expressed proteins was found to be enriched in pathways related to adherent junction, such as cadherin binding, cell-cell junction, cell adhesion and focal adherens. Furthermore, in vitro experiments, MC-LR significantly downregulated the expression levels of proteins associated with adherent junction (ß-catenin, N-cadherin, and α-catenin) as well as caused cytoskeletal disruption in KK-1 cells (P < 0.05), indicating that the adherent junction was damaged. Results of in vivo experiments have shown that after 14 days of acute MC-LR exposure (40 µg/kg), damaged adherent junction and an increased number of atretic follicles were observed in mouse ovaries. Moreover, MC-LR activated JNK, an upstream regulator of adherent junction proteins, in KK-1 cells and mouse ovarian tissues. In contrast, JNK inhibition alleviated MC-LR-induced adherent junction damage in vivo and in vitro, as well as the number of atretic follicles. Taken together, findings from the present study indicated that JNK is involved in MC-LR-induced granulosa cell adherent junction damage, which accelerated follicular atresia. Our study clarified a novel mechanism of MC-LR-caused ovarian toxicity, providing a theoretical foundation for protecting female reproductive health from environmental pollutants.

Autotaxin-LPA axis regulates hMSC migration by adherent junction disruption and cytoskeletal rearrangement via LPAR1/3-dependent PKC/GSK3ß/ß-catenin and PKC/Rho GTPase pathways.

Bioactive molecules and stem cell-based regenerative engineering is emerging a promising approach for regenerating tissues. Autotaxin (ATX) is a key enzyme that regulates lysophosphatidic acid (LPA) levels in biological fluids, which exerts a wide range of cellular functions. However, the biological role of ATX in human umbilical cord blood-derived mesenchymal stem cells (hMSCs) migration remains to be fully elucidated. In this study, we observed that hMSCs, which were stimulated with LPA, accelerated wound healing, and LPA increased the migration of hMSCs into a wound site in a mouse skin wound healing model. In an experiment to investigate the effect of LPA on hMSC migration, ATX and LPA increased hMSC migration in a dose-dependent manner, and LPA receptor 1/3 siRNA transfections inhibited the ATX-induced cell migration. Furthermore, LPA increased Ca(2+) influx and PKC phosphorylation, which were blocked by Gαi and Gαq knockdown as well as by Ptx pretreatment. LPA increased GSK3ß phosphorylation and ß-catenin activation. LPA induced the cytosol to nuclear translocation of ß-catenin, which was inhibited by PKC inhibitors. LPA stimulated the binding of ß-catenin on the E-box located in the promoter of the CDH-1 gene and decreased CDH-1 promoter activity. In addition, the ATX and LPA-induced increase in hMSC migration was blocked by ß-catenin siRNA transfection. LPA-induced PKC phosphorylation is also involved in Rac1 and CDC42 activation, and Rac1 and CDC42 knockdown abolished LPA-induced F-actin reorganization. In conclusion, ATX/LPA stimulates the migration of hMSCs through LPAR1/3-dependent E-cadherin reduction and cytoskeletal rearrangement via PKC/GSK3ß/ß-catenin and PKC/Rho GTPase pathways.

HN1 negatively influences the ß-catenin/E-cadherin interaction, and contributes to migration in prostate cells.

Previously, it has been reported that HN1 is involved in cytoplasmic retention and degradation of androgen receptor in an AKT dependent manner. As HN1 is a hormone inducible gene, and has been shown that it is upregulated in various cancers, we studied the importance of HN1 function in ß-catenin signaling in prostate cancer cell line, PC-3 and mammary cancer cell line MDA-MB231. Here, we demonstrated that HN1 physically associates with GSK3ß/ß-catenin destruction complex and abundantly localizes to cytoplasm, especially when the GSK3ß is phosphorylated on S9 residue. Further, ectopic HN1 expression results an increase in the ß-catenin degradation leading to loss of E-cadherin interaction, concurrently contributing to actin re-organization, colony formation and migration in cancer cell lines. Thus, we report that HN1 is an essential factor for ß-catenin turnover and signaling, augments cell growth and migration in prostate cancer cells.

Cigarette Smoke Mediates Nasal Epithelial Barrier Dysfunction via TNF-α.

BACKGROUND: Extensive data suggest that exposure to cigarette smoke can induce pulmonary epithelial barrier dysfunction. However, the effects of cigarette smoke on the nasal epithelial barrier are still unclear. Here, we investigated the consequence and mechanism of cigarette smoke on the nasal epithelial barrier. METHODS: Sprague Dawley rats were exposed to cigarette smoke for 3 or 6 months, and changes in inflammatory markers and nasal barrier function were evaluated. Moreover, underlying mechanisms were explored. Finally, normal human bronchial epithelial cells were cultured with or without tumor necrosis factor-alpha (TNF-α) in vitro, and the levels of continuity and tight junction-associated proteins were measured. RESULTS: In vivo experiments showed that the nasal mucosal barrier function of rats exposed to cigarette smoke was disturbed. Indeed, proteins associated with tight junctions were decreased, and the levels of inflammatory factors, such as IL-8, IL-6, and TNF-α, were dramatically increased in comparison to those of control animals. In vitro, TNF-α was shown to disrupt the continuity of proteins associated with tight junctions and to downregulate the expression of these proteins in bronchial epithelial cells. CONCLUSIONS: We found that cigarette smoke disrupted the nasal mucosal barrier, and the extent of the damage was correlated with the duration of cigarette smoke exposure. We showed that TNF-α can disrupt the continuity and attenuate the expression of tight junction proteins in human bronchial epithelial cells. Therefore, cigarette smoke may induce nasal epithelial barrier dysfunction through TNF-α.

An Antioxidant Supplement Function Exploration: Rescue of Intestinal Structure Injury by Mannan Oligosaccharides after Aeromonas hydrophila Infection in Grass Carp (Ctenopharyngodon idella).

Mannan oligosaccharides (MOS) are a type of functional oligosaccharide which have received increased attention because of their beneficial effects on fish intestinal health. However, intestinal structural integrity is a necessary prerequisite for intestinal health. This study focused on exploring the protective effects of dietary MOS supplementation on the grass carp's (Ctenopharyngodon idella) intestinal structural integrity (including tight junction (TJ) and adherent junction (AJ)) and its related signalling molecule mechanism. A total of 540 grass carp (215.85 ± 0.30 g) were fed six diets containing graded levels of dietary MOS supplementation (0, 200, 400, 600, 800 and 1000 mg/kg) for 60 days. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila for 14 days. We used ELISA, spectrophotometry, transmission electron microscope, immunohistochemistry, qRT-PCR and Western blotting to determine the effect of dietary MOS supplementation on intestinal structural integrity and antioxidant capacity. The results revealed that dietary MOS supplementation protected the microvillus of the intestine; reduced serum diamine oxidase and d-lactate levels (p < 0.05); enhanced intestinal total antioxidant capacity (p < 0.01); up-regulated most intestinal TJ and AJ mRNA levels; and decreased GTP-RhoA protein levels (p < 0.01). In addition, we also found several interesting results suggesting that MOS supplementation has no effects on ZO-2 and Claudin-15b. Overall, these findings suggested that dietary MOS supplementation could protect intestinal ultrastructure, reduce intestinal mucosal permeability and maintain intestinal structural integrity via inhibiting MLCK and RhoA/ROCK signalling pathways.

Sortilin regulates blood-brain barrier integrity.

Brain homeostasis depends on the existence of the blood-brain barrier (BBB). Despite decades of research, the factors and signalling pathways for modulating and maintaining BBB integrity are not fully elucidated. Here, we characterise the expression and function of the multifunctional receptor, sortilin, in the cells of the BBB, in vivo and in vitro. We show that sortilin acts as an important regulatory protein of the BBB's tightness. In rats lacking sortilin, the BBB was leaky, which correlated well with relocated distribution of the localisation of zonula occludens-1, VE-cadherin and ß-catenin junctional proteins. Furthermore, the absence of sortilin in brain endothelial cells resulted in decreased phosphorylation of Akt signalling protein and increased the level of phospho-ERK1/2. As a putative result of MAPK/ERK pathway activity, the junctions between the brain endothelial cells were disintegrated and the integrity of the BBB became compromised. The identified barrier differences between wild-type and Sort1-/- brain endothelial cells can pave the way for a better understanding of sortilin's role in the healthy and diseased BBB.

Goose nephritic astrovirus infection increases autophagy, destroys intercellular junctions in renal tubular epithelial cells, and damages podocytes in the kidneys of infected goslings.

Goose nephritic astrovirus (GNAstV) has recently been identified, which causes kidney swelling and visceral gout in goslings. However, the pathological changes in kidney tissue due to GNAstV infection have not yet been described. In the study, fifty goslings were orally infected with GNAstV, and fifty goslings received PBS as a control. Kidney tissue was collected at different days following infection (dpi) to assess the injury. GNAstV infection reduced body weight, increased the relative weight of the kidney, and increased serum uric acid and creatinine levels. GNAstV was found within renal epithelial cells, and the viral load in the kidney peaked at 7 dpi. Pale and swollen kidney tissue was observed in infected goslings, especially at 5 and 7 dpi. GNAstV infection caused degeneration and necrosis of renal epithelial cells, structural destruction of the brush border, glycogen deposition in the glomerular mesangium, increased fibrosis, and infiltration of inflammatory cells into the renal interstitium. Moreover, swollen mitochondria, broken mitochondrial ridges, autophagosomes, and autophagolysosomes were observed under ultrahistopathological examination. GNAstV infection increased levels of LC3B, ATG5, and Beclin 1, and decreased p62, and downregulated WT1 mRNA and upregulated desmin mRNA. At early stages, GNAstV infection decreased expression of intercellular junction-related genes, including ZO-1, occludin, claudin-10, and catenin-α2. In conclusion, GNAstV infection causes renal epithelial cell autophagy, destruction of brush border and intercellular junctions, podocyte damage, and increased fibrosis, ultimately resulting in damage to the kidney.

Rosette morphology in zona glomerulosa formation and function.

How morphology informs function is a fundamental biological question. Here, we review the morphological features of the adrenal zona glomerulosa (zG), highlighting recent cellular and molecular discoveries that govern its formation. The zG consists of glomeruli enwrapped in a Laminin-ß1-enriched basement membrane (BM). Within each glomerulus, zG cells are organized as rosettes, a multicellular structure widely used throughout development to mediate epithelial remodeling, but not often found in healthy adult tissues. Rosettes arise by constriction at a common cellular contact point mediated/facilitated by adherens junctions (AJs). In mice, small, dispersed AJs first appear postnatally and enrich along the entire cell-cell contact around 10 days after birth. Subsequently, these AJ-rich contacts contract, allowing rosettes to form. Concurrently, flat sheet-like domains in the nascent zG, undergo invagination and folding, gradually giving rise to the compact round glomeruli that comprise the adult zG. How these structures impact adrenal function is discussed.

TIMP1 preserves the blood-brain barrier through interacting with CD63/integrin ß 1 complex and regulating downstream FAK/RhoA signaling.

Blood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability are hallmark features of several neurological disorders, including traumatic brain injury (TBI). However, there is no viable therapeutic strategy to rescue BBB function. Tissue inhibitor of metalloproteinase-1 (TIMP1) has been considered to be beneficial for vascular integrity, but the molecular mechanisms underlying the functions of TIMP1 remain elusive. Here, we report that TIMP1 executes a protective role on neuroprotective function via ameliorating BBB disruption in mice with experimental TBI. In human brain microvessel endothelial cells (HBMECs) exposed to hypoxia and inflammation injury, the recombinant TIMP1 (rTIMP1) treatment maintained integrity of junctional proteins and trans-endothelial tightness. Mechanistically, TIMP1 interacts with CD63/integrin ß1 complex and activates downstream FAK signaling, leading to attenuation of RhoA activation and F-actin depolymerization for endothelial cells structure stabilization. Notably, these effects depend on CD63/integrin ß1 complex, instead of the MMP-inhibitory function. Together, our results identified a novel MMP-independent function of TIMP1 in regulating endothelial barrier integrity. Therapeutic interventions targeting TIMP1 and its downstream signaling may be beneficial to protect BBB function following brain injury and neurological disorders.

Single-nucleotide polymorphisms of the adherent junction component cadherin gene are associated with leukoaraiosis.

BACKGROUND: Leukoaraiosis (LA) is one of the manifestations of cerebral small vessel disease. Blood-brain barrier (BBB) disruption plays a key role in LA. Cadherin is a component of adherent junctions (AJ), which play a crucial role in cell-cell adhesion, cell-cell recognition and homeostasis in BBB development. We hypothesized that alterations in cadherin genes might be a potential cause of BBB abnormalities that result in LA. METHODS: A total of 339 LA individuals (LA-PVWM, 183; LA-DWM 156) were enrolled, who underwent brain magnetic resonance imaging with obtainable vascular risk factors. Genotyping of cadherin single-nucleotide polymorphisms (SNPs) (rs5030625, rs1801026, and rs16260) was performed by real-time polymerase chain reaction with LightSNiP reagents (coupled primer and probe) and FastStart DNAMaster HybProbe (Roche Diagnostic, GmBH, Mannheim, Germany) on a LightCycler 2.0 instrument. RESULTS: Two SNPs, rs1801026 and rs16260, were significantly different between the control and LA groups. The combinatorial effects of the three SNPs were also significant. The haplotypes G-T-C and GA-T-A increased the development of LA-PVWM (OR = 1.76 and OR = 40.7, respectively). The haplotypes G-T-A and GA-T-A increased the development of LA-DWM (OR = 2.56 and OR = 10.48, respectively), but G-C-C decreased the development of LA-DWM (OR = 17.57). CONCLUSION: This study provides evidence for genetic polymorphisms of the AJ component cadherin gene and the association of its haplotypes with LA.

Pomegranate prevents binge alcohol-induced gut leakiness and hepatic inflammation by suppressing oxidative and nitrative stress.

Alcoholic liver disease (ALD) is a major chronic liver disease worldwide and can range from simple steatosis, inflammation to fibrosis/cirrhosis possibly through leaky gut and systemic endotoxemia. We investigated whether pomegranate (POM) protects against binge alcohol-induced gut leakiness, endotoxemia, and inflammatory liver damage. After POM pretreatment for 10 days, rats were exposed to 3 oral doses of binge alcohol (5 g/kg/dose) or dextrose (as control) at 12-h intervals. Binge alcohol exposure induced leaky gut with significantly elevated plasma endotoxin and inflammatory fatty liver by increasing the levels of oxidative and nitrative stress marker proteins such as ethanol-inducible CYP2E1, inducible nitric oxide synthase, and nitrated proteins in the small intestine and liver. POM pretreatment significantly reduced the alcohol-induced gut barrier dysfunction, plasma endotoxin and inflammatory liver disease by inhibiting the elevated oxidative and nitrative stress marker proteins. POM pretreatment significantly restored the levels of intestinal tight junction (TJ) proteins such as ZO-1, occludin, claudin-1, and claundin-3 markedly diminished after alcohol-exposure. In addition, the levels of gut adherent junction (AJ) proteins (e.g., ß-catenin and E-cadherin) and desmosome plakoglobin along with associated protein α-tubulin were clearly decreased in binge alcohol-exposed rats but restored to basal levels in POM-pretreated rats. Immunoprecipitation followed by immunoblot analyses revealed that intestinal claudin-1 protein was nitrated and ubiquitinated in alcohol-exposed rats, whereas these modifications were significantly blocked by POM pretreatment. These results showed for the first time that POM can prevent alcohol-induced gut leakiness and inflammatory liver injury by suppressing oxidative and nitrative stress.

The Interplay between Cofilin and Phospho-Cofilin: Its Role in Maintaining Blood Brain Barrier Integrity.

The blood brain barrier (BBB) is a continuous, non-fenestrated vessel system that tightly regulates the movement of molecules, ions, and cells between the blood and the central nervous system. Endothelial cells are the major constituents of the BBB and these cells are linked to each other through intercellular contact points composed of tight junctions, adherent junctions and gap junctions. These three types of junctions are connected to the intracellular actin cytoskeleton via various adaptor proteins. Thus, the actin cytoskeleton plays a crucial role in regulating the stability of endothelial cell contacts and vascular permeability. Shear stress, growth factors, and Wnt/ß-catenin pathway modulators contribute to maintaining endothelial cell integrity by controlling actin dynamics under homeostatic conditions. Interestingly, the downstream signaling of the aforementioned factors converges at Rac1, which mediates cortical actin stabilization, stress fiber destabilization and junctional complex stabilization by controlling subcellular cofilin dynamics. However, Rac1 is not the only modulator of cofilin activity; many other agents activated during inflammatory, ischemic, and excitotoxic conditions can disturb homeostatic cofilin dynamics and induce BBB disruption. Therefore, in this review, we discuss organization of the actin cytoskeleton in BBB endothelial cells and how interactions between the actin cytoskeleton and junctional complexes are maintained during homeostatic conditions. Furthermore, we discuss how an imbalance in subcellular cofilin dynamics can contribute to BBB disruption and highlight Rac1 as a potential target that can be exploited to preserve BBB stability.

Mono-(2-ethylhexyl) phthalate (MEHP) affects intercellular junctions of Sertoli cell: A potential role of oxidative stress.

We analyzed the potential role of oxidative stress induced by mono (2-ethylhexyl) phthalate (MEHP) in adherent cell junction protein expression of prepubertal rat Sertoli cells (SC) in vitro. Five-day SC cultures were treated with MEHP (200µM) for 24h and compared to cells in basal conditions. Western blot and immunofluorescent (IF) analyses showed that MEHP induced increase of N-cadherin and catenin expression, modifying its distribution. Concomitantly, Cx-43 expression decreased significantly and delocalization of the IF signal for tight junction proteins (occludin, claudin-11 and ZO-1) occurred. Indicative of oxidative stress, MEHP induced in SC an increase of lipoperoxides, a decrease in glutathione (GSH) levels and a concomitant increase in Glutathione S-Transferases (GST) activity. Antioxidant N-acetyl-cysteine (1mM) treatment prevented GSH decrease and N-cadherin and α-catenin up-regulation induced by MEHP. Our data suggest that oxidative stress signaling is a mechanism involved in adherent cell junctions disruption induced by MEHP in SC cultures.