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Concept: Tight junction protein 1

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Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization.

Concepts: Liver, Paracetamol, Hepatocyte, Tight junction protein 1

28

Tight junctions (TJs) are dynamic cellular structures that are critical for compartmentalizing environments within tissues and regulating transport of small molecules, ions, and fluids. Phosphorylation-dependent binding of the transmembrane protein occludin to the structural organizing protein ZO-1 contributes to the regulation of barrier properties; however, the details of their interaction are controversial. Using small angle X-ray scattering (SAXS), NMR chemical shift perturbation, cross-saturation, in vitro binding, and site-directed mutagenesis experiments. we define the interface between the ZO-1 PDZ3-SH3-U5-GuK (PSG) and occludin coiled-coil (CC) domains. The interface is comprised of basic residues in PSG and an acidic region in CC. Complex formation is blocked by a peptide (REESEEYM) that corresponds to CC residues 468-475 and includes a previously uncharacterized phosphosite, with the phosphorylated version having a larger effect. Furthermore, mutation of E470 and E472 reduces cell border localization of occludin. Together, these results localize the interaction to an acidic region in CC and a predominantly basic helix V within the ZO-1 GuK domain. This model has important implications for the phosphorylation-dependent regulation of the occludin:ZO-1 complex.

Concepts: DNA, Protein, Mutation, Cell membrane, Chemical shift, Tight junction, Tight junction protein 1, Occludin

24

Epithelial-mesenchymal transition (EMT) is considered to play a critical role in cancer progression and metastasis. However, the impact of EMT on the prognosis of hepatocellular carcinoma (HCC) is still elusive. In this study, we examined the relationship between the expression of EMT markers and recurrence-free survival (RFS) and overall survival (OS) in HCC patients after hepatic resection.

Concepts: DNA, Cancer, Oncology, Lung cancer, Play, The Expression of the Emotions in Man and Animals, Tight junction protein 1, John Coltrane

5

Non-alcoholic steatohepatitis (NASH) is an epidemic metabolic disease with limited therapeutic strategies. Cumulative data support the pivotal role of gut microbiota in NASH. Here, we investigated the hypothesis regarding whether fecal microbiota transplantation (FMT) is effective in attenuating high-fat diet (HFD)-induced steatohepatitis in mice. Mice were randomized into control, HFD and HFD + FMT groups. After an 8-week HFD, FMT treatment was initiated and carried out for 8 weeks. The gut microbiota structure, butyrate concentrations of the cecal content, liver pathology and intrahepatic lipid and cytokines were examined. Our results showed that after FMT, the gut microbiota disturbance was corrected in HFD-fed mice with elevated abundances of the beneficial bacteria Christensenellaceae and Lactobacillus. FMT also increased butyrate concentrations of the cecal content and the intestinal tight junction protein ZO-1, resulting in relief of endotoxima in HFD-fed mice. Steatohepatitis was alleviated after FMT, as indicated by a significant decrease in intrahepatic lipid accumulation (reduced Oli-red staining, decreased intrahepatic triglyceride and cholesterol), intrahepatic pro-inflammatory cytokines, and the NAS score. Accordingly, intrahepatic IFN-γ and IL-17 were decreased, but Foxp3, IL-4 and IL-22 were increased after FMT intervention. These data indicate that FMT attenuated HFD-induced steatohepatitis in mice via a beneficial effect on the gut microbiota.

Concepts: Bacteria, Gut flora, Cytokine, Metabolism, Triglyceride, Bile, Lipid, Tight junction protein 1

3

Owing to the frequent incidence of blast-induced traumatic brain injury (bTBI) in recent military conflicts, there is an urgent need to develop effective therapies for bTBI-related pathologies. Blood-brain barrier (BBB) breakdown has been reported to occur after primary blast exposure, making restoration of BBB function and integrity a promising therapeutic target. We tested the hypothesis that treatment with dexamethasone (DEX) after primary blast injury potentiates recovery of an in vitro BBB model consisting of mouse brain endothelial cells (bEnd.3). DEX treatment resulted in complete recovery of transendothelial electrical resistance and hydraulic conductivity 1 day after injury, compared with 3 days for vehicle-treated injured cultures. Administration of RU486 (mifepristone) inhibited effects of DEX, confirming that barrier restoration was mediated by glucocorticoid receptor signaling. Potentiated recovery with DEX treatment was accompanied by stronger zonula occludens (ZO)-1 tight junction immunostaining and expression, suggesting that increased ZO-1 expression was a structural correlate to BBB recovery after blast. Interestingly, augmented ZO-1 protein expression was associated with specific upregulation of the α(+) isoform but not the α(-) isoform. This is the first study to provide a mechanistic basis for potentiated functional recovery of an in vitro BBB model because of glucocorticoid treatment after primary blast injury.Journal of Cerebral Blood Flow & Metabolism advance online publication, 11 March 2015; doi:10.1038/jcbfm.2015.38.

Concepts: Protein, Brain, Hormone, Traumatic brain injury, Cortisol, Cerebral blood flow, Tight junction protein 1, Occludin

3

Methamphetamine (METH) is a drug of abuse with neurotoxic and neuroinflammatory effects, which include disruption of the blood-brain barrier (BBB) and alterations of tight junction protein expression. The present study focused on the actin cytoskeletal rearrangement as a modulator of METH-induced redistribution of tight junction protein occludin in brain endothelial cells. Exposure to METH resulted in a shift of occludin localization from plasma membranes to endosomes. These changes were accompanied by activation of the actin-related protein 2/3 (Arp2/3) complex, which stimulates actin polymerization by promoting actin nucleation. In addition, METH induced coronin-1b phosphorylation, which diminishes the inhibitory effect of non-phosphorylated coronin-1b on actin nucleation. Blocking actin nucleation with CK-666, a specific inhibitor of the Arp2/3 complex, protected against METH-induced occludin internalization and increased transendothelial monocyte migration. Importantly, treatment with CK-666 attenuated a decrease in occludin levels in brain microvessels of METH-injected mice. The present findings indicate that actin cytoskeletal dynamics is detrimental to METH-induced BBB dysfunction by increasing internalization of occludin.

Concepts: Protein, Cell membrane, Neurology, Actin, Phosphorus, Arp2/3 complex, Tight junction protein 1, Lamellipodia

1

Propolis has abundant polyphenolic constituents and is used widely as a health/functional food. Here, we investigated the effects of polyphenol-rich propolis extracts (PPE) on intestinal barrier function in human intestinal epithelial Caco-2 cells, as well as in rats. In Caco-2 cells, PPE increased transepithelial electrical resistance and decreased lucifer yellow flux. PPE-treated cells showed increased expression of the tight junction (TJ) loci occludin and zona occludens (ZO)-1. Confocal microscopy showed organized expressions in proteins related to TJ assembly, i.e., occludin and ZO-1, in response to PPE. Furthermore, PPE led to the activation of AMPK, ERK1/2, p38, and Akt. Using selective inhibitors, we found that the positive effects of PPE on barrier function were abolished in cells in which AMPK and ERK1/2 signaling were inhibited. Moreover, rats fed a diet supplemented with PPE (0.3% in the diet) exhibited increased colonic epithelium ZO-1 expression. Overall, these data suggest that PPE strengthens intestinal barrier function by activating AMPK and ERK signaling and provide novel insights into the potential application of propolis for human gut health.

Concepts: Protein, Gene expression, Cell membrane, Epithelium, Skin, Tight junction, Tight junction protein 1, Occludin

1

Proteasome inhibitors have revolutionized outcomes in multiple myeloma, but they are used empirically, and primary and secondary resistance are emerging problems. We have identified TJP1 as a determinant of plasma cell proteasome inhibitor susceptibility. TJP1 suppressed expression of the catalytically active immunoproteasome subunits LMP7 and LMP2, decreased proteasome activity, and enhanced proteasome inhibitor sensitivity in vitro and in vivo. This occurred through TJP1-mediated suppression of EGFR/JAK1/STAT3 signaling, which modulated LMP7 and LMP2 levels. In the clinic, high TJP1 expression in patient myeloma cells was associated with a significantly higher likelihood of responding to bortezomib and a longer response duration, supporting the use of TJP1 as a biomarker to identify patients most likely to benefit from proteasome inhibitors.

Concepts: Multiple myeloma, Gene expression, Patient, Proteasome, Proteasome inhibitor, Bortezomib, Tight junction protein 1

1

The purposes of this study are to assess whether 7 days of oral glutamine supplementation: (1) reduces exercise induced intestinal permeability; (2) prevents the pro-inflammatory response; and (3) to determine whether these changes are associated with the up-regulation of the heat shock response. On separate occasions, eight human subjects participated in baseline testing, glutamine (GLN), and placebo (PLA) trials followed by a 60-min treadmill run. Intestinal permeability was higher in the PLA trial compared to baseline and GLN (0.0604 ± 0.047 vs. 0.0218 ±0.008 and 0.0272 ± 0.007, respectively, p<0.05). PBMC IκBα expression was higher 240-min post-ex in GLN trial compared to PLA (1.411 ± 0.523 vs. 0.9839 ± 0.343, p<0.05). In vitro (Caco-2) we measured effects of glutamine supplementation (0 mM, 4 mM, and 6 mM) on heat-induced (37° or 41.8°C) HSP70, HSF-1, and occludin expression. HSF-1 and HSP70 levels increased in 6 mM 41ºC compared to 0 mM 41ºC (1.785 ± 0.495 vs. 0.6681 ± 0.290, and 1.973 ± 0.325 vs. 1.133 ± 0.129, respectively, p<0.05). Occludin levels increased after 4 mM 41ºC and 6 mM 41ºC compared to 0 mM 41ºC (1.236 ± 0.219 and 1.849 ± 0.564 vs. 0.7434 ± 0.027, p<0.001, respectively). Glutamine supplementation prevented exercise-induced permeability, possibly through HSF-1 activation.

Concepts: Shock, In vitro fertilisation, In vitro, Heat shock protein, Tight junction protein 1, Occludin

0

Nucleic acid-based targeting of nuclear factor kappaB (NF-κB) is gaining attention as a treatment option for skin diseases like atopic dermatitis (AD). Transdermal administration improves patient quality of life because of non-invasive; however, siRNA delivery into the skin can be challenging owing to the barrier of tight junctions in the granular layer. Therefore, we aimed to develop a delivery system of siRNA for topical skin application using functional peptides. We previously reported that combined treatment with a cytoplasm-responsive stearylated-arginine-rich peptide (STR-CH2R4H2C) and a tight junction opening peptide (AT1002) showed high siRNA permeability in the skin of AD-induced and normal mice. Here, we used murine macrophage RAW264.7 cells to examine siRNA permeation and the therapeutic effect of anti-NF-κB (RelA) siRNA (siRelA) complexed with STR-CH2R4H2C and AT1002 for AD-induced mice. We showed that significantly higher siRNA cellular uptake occurs after this treatment as well as decreased TNF-α and IL-6 expression. Additionally, we showed that effective siRNA transdermal delivery occurs with the suppression of the tight junction protein ZO-1. Moreover, topical skin application of siRelA with STR-CH2R4H2C and AT1002 improved AD-like symptoms in model mice. Thus, the combined treatment of STR-CH2R4H2C and AT1002 could serve as an effective transdermal siRNA therapeutic system for AD.

Concepts: Protein, Cell membrane, Transdermal patch, Peptide, Route of administration, Peptides, Tight junction protein 1, Occludin