Metformin suppresses NFE2L1 pathway activation to inhibit gap junction beta protein expression in NSCLC.

OBJECTIVE: Non-small-cell lung cancer (NSCLC) is a deadly form of cancer that exhibits extensive intercellular communication which contributed to chemoradiotherapy resistance. Recent evidence suggests that arrange of key proteins are involved in lung cancer progression, including gap junction proteins (GJPs). METHODS AND RESULTS: In this study, we examined the expression patterns of GJPs in NSCLC, uncovering that both gap junction protein, beta 2 (GJB2) and gap junction protein, beta 2 (GJB3) are increased in LUAD and LUSC. We observed a correlation between the upregulation of GJB2, GJB3 in clinical samples and a worse prognosis in patients with NSCLC. By examining the mechanics, we additionally discovered that nuclear factor erythroid-2-related factor 1 (NFE2L1) had the capability to enhance the expression of connexin26 and connexin 31 in the NSCLC cell line A549. In addition, the use of metformin was discovered to cause significant downregulation of gap junction protein, betas (GJBs) by limiting the presence of NFE2L1 in the cytoplasm. CONCLUSION: This emphasizes the potential of targeting GJBs as a viable treatment approach for NSCLC patients receiving metformin.

Does astrocyte gap junction protein expression differ during development in absence epileptic rats?

Intercellular communication via gap junctions (GJs) has a wide variety of complex and essential functions in the CNS. In the present developmental study, we aimed to quantify the number of astrocytic GJs protein connexin 30 (Cx30) of genetic model of absence epilepsy rats from Strasbourg (GAERS) at postnatal P10, P30, and P60 days in the epileptic focal areas involved in the cortico-thalamic circuit. We compared the results with Wistar rats using immunohistochemistry and western blotting. The number of Cx30 immunopositive astrocytes per unit area were quantified for the somatosensory cortex (SSCx), ventrobasal (VB), and lateral geniculate (LGN) thalamic nuclei of the two strains and Cx30 western blot was applied to the tissue samples from the same regions. Both immunohistochemical and western blot results revealed the presence of Cx30 in all regions studied at P10 in both Wistar and GAERS animals. The SSCx, VB, and LGN of Wistar animals showed progressive increase in the number of Cx30 immunopositive labeled astrocytes from P10 to P30 and reached a peak at P30; then a significant decline was observed from P30 to P60 for the SSCx and VB. However, in GAERS Cx30 immunopositive labeled astrocytes showed a progressive increase from P10 to P60 for all brain regions studied. The immunohistochemical data highly corresponded with western blotting results. We conclude that the developmental disproportional expression of Cx30 in the epileptic focal areas in GAERS may be related to the onset of absence seizures or may be related to the neurogenesis of absence epilepsy.

L-carnitine suppresses cisplatin-induced renal injury in rats: impact on cytoskeleton proteins expression.

We designed this work to examine the curative role of L-carnitine (LCAR) in a rat model of cisplatin (CDDP)-induced kidney injury. We induced kidney injury in rats by a single intraperitoneal injection of 5 mg/kg of CDDP. Fifteen days post injection, rats were orally supplemented with 354 mg/kg of LCAR for another 15 days. Kidney tissues were subjected to histo-biochemical analysis along with mRNA gene expression quantification for cytoskeleton proteins encoding genes (vimentin, nestin, and connexin 43) by real-time reverse transcription polymerase chain reaction. LCAR reversed CDDP-induced renal structural and functional impairments. LCAR significantly declined serum urea and creatinine concentrations, restored oxidant/antioxidant balance, reversed inflammation, and antagonized caspase 3-mediated apoptotic cell death in renal tissues. Moreover, LCAR effectively down-regulated cytoskeleton proteins mRNA levels, reflecting amelioration of CDDP-provoked podocyte injury. We concluded that LCAR has a favorable therapeutic utility against CDDP-induced kidney injury.

Vitex negundo Linn. extract alleviates inflammatory aggravation and lung injury by modulating AMPK/PI3K/Akt/p38-NF-κB and TGF-ß/Smad/Bcl2/caspase/LC3 cascade and macrophages activation in murine model of OVA-LPS induced allergic asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: There is growing inclination towards developing bioactive molecule-based strategies for the management of allergic airway inflammation associated respiratory diseases. Vitex negundo Linn., also known as Nirgundi, is one such medicinal plant enriched with phytochemicals and used for inflammatory and respiratory disorders including asthma in traditional system of medicine. Preliminary studies have claimed anti-tussive and bronchodilator potential of V. negundo Linn. However, its attributes as well as molecular mechanism (s) in modulation of asthma mediated by allergic inflammation are yet to be delineated scientifically. AIM OF THE STUDY: Present study attempted to assess the effectiveness of Vitex negundo leaf extract (VNLE) in mitigation of allergen induced inflammation associated asthmatic lung damage with emphasis to delineate its molecular mechanism (s). MATERIALS AND METHODS: Allergic lung inflammation was established in Balb/c mice using Ovalbumin-lipopolysaccharide (OVA-LPS). Several allergic inflammatory parameters, histopathological changes, alveolar macrophage activation and signalling pathways were assessed to examine protective effects of VNLE. UHPLC-DAD-QTOF-ESI-IMS was used to characterize VLNE. RESULTS: VNLE administration effectively attenuated LPS-induced oxi-inflammatory stress in macrophages suggesting its anti-inflammatory potential. Further, VNLE showed protective effect in mitigating asthmatic lung damage as evident by reversal of pathological changes including inflammatory cell influx, congestion, fibrosis, bronchial thickness and alveolar collapse observed in allergen group. VNLE suppressed expressions of inflammatory Th1/Th2 cytokines, chemokines, endopeptidases (MMPs), oxidative effector enzyme (iNOS), adhesion molecules, IL-4/IFN-γ release with simultaneous enhancement in levels of IL-10, IFN-γ, MUC3 and tight junction proteins. Subsequent mechanistic investigation revealed that OVA-LPS concomitantly enhanced phosphorylation of NF-κB, PI3K, Akt and p38MAPKs and downregulated AMPK which was categorically counteracted by VNLE treatment. VNLE also suppressed OVA-LPS induced fibrosis, apoptosis, autophagy and gap junction proteins which were affirmed by reduction in TGF-ß, Smad2/3/4, Caspase9/3, Bax, LC3A/B, connexin 50, connexin 43 and enhancement in Bcl2 expression. Additionally, suppression of alveolar macrophage activation, inflammatory cells in blood and elevation of splenic CD8+T cells was demonstrated. UHPLC-DAD-QTOF-ESI-IMS revealed presence of iridoids glycoside and phenolics which might contribute these findings. CONCLUSION: These findings confer protective effect of VNLE in attenuation of allergic lung inflammation and suggest that it could be considered as valuable medicinal source for developing safe natural therapeutics for mitigation of allergic inflammation during asthma.

The Role of Connexin 43 in Lung Disease.

The term lung disease describes a broad category of disorders that impair lung function. More than 35 million Americans have a preventable chronic lung disease with high mortality rates due to limited treatment efficacy. The recent increase in patients with lung disease highlights the need to increase our understanding of mechanisms driving lung inflammation. Connexins, gap junction proteins, and more specifically connexin 43 (Cx43), are abundantly expressed in the lung and are known to play a role in lung diseases. This review focuses on the role of Cx43 in pathology associated with acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD) and asthma. Additionally, we discuss the role of Cx43 in preventing disease through the transfer of mitochondria between cells. We aim to highlight the need to better understand what cell types are expressing Cx43 and how this expression influences lung disease.

How does Helicobacter pylori cause gastric cancer through connexins: An opinion review.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium with a number of virulence factors, such as cytotoxin-associated gene A, vacuolating cytotoxin A, its pathogenicity island, and lipopolysaccharide, which cause gastrointestinal diseases. Connexins function in gap junctional homeostasis, and their downregulation is closely related to gastric carcinogenesis. Investigations into H. pylori infection and the fine-tuning of connexins in cells or tissues have been reported in previous studies. Therefore, in this review, the potential mechanisms of H. pylori-induced gastric cancer through connexins are summarized in detail.

Binding of α-synuclein oligomers to Cx32 facilitates protein uptake and transfer in neurons and oligodendrocytes.

The intercellular transfer of alpha-synuclein (α-syn) has been implicated in the progression of Parkinson's disease (PD) and multiple system atrophy (MSA). The cellular mechanisms underlying this process are now beginning to be elucidated. In this study, we demonstrate that the gap junction protein connexin-32 (Cx32) is centrally involved in the preferential uptake of α-syn oligomeric assemblies (oα-syn) in neurons and oligodendrocytes. In vitro, we demonstrate a clear correlation between Cx32 expression and oα-syn uptake. Pharmacological and genetic strategies targeting Cx32 successfully blocked oα-syn uptake. In cellular and transgenic mice modeling PD and MSA, we observed significant upregulation of Cx32 which correlates with α-syn accumulation. Notably, we could also demonstrate a direct interaction between α-syn and Cx32 in two out of four human PD cases that was absent in all four age-matched controls. These data are suggestive of a link between Cx32 and PD pathophysiology. Collectively, our results provide compelling evidence for Cx32 as a novel target for therapeutic intervention in PD and related α-synucleinopathies.

The Role of Pannexin 3 in Bone Biology.

Cell-cell and cell-matrix communications play important roles in both cell proliferation and differentiation. Gap junction proteins mediate signaling communication by exchanging small molecules and dramatically stimulating intracellular signaling pathways to determine cell fate. Vertebrates have 2 gap junction families: pannexins (Panxs) and connexins (Cxs). Unlike Cxs, the functions of Panxs are not fully understood. In skeletal formation, Panx3 and Cx43 are the most abundantly expressed gap junction proteins from each family. Panx3 is induced in the transient stage from the proliferation and differentiation of chondrocytes and osteoprogenitor cells. Panx3 regulates both chondrocyte and osteoblast differentiation via the activation of intracellular Ca2+ signaling pathways through multiple channel activities: hemichannels, endoplasmic reticulum (ER) Ca2+ channels, and gap junctions. Moreover, Panx3 also inhibits osteoprogenitor cell proliferation and promotes cell cycle exit through the inactivation of Wnt/ß-catenin signaling and the activation of p21. Panx3-knockout (KO) mice have more severe skeletal abnormalities than those of Cx43-KO mice. A phenotypic analysis of Panx3-KO mice indicates that Panx3 regulates the terminal differentiation of chondrocytes by promoting vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP) 13. Based on the generation of Panx3-/-; Cx43-/- mice, Panx3 is upstream of Cx43 in osteogenesis. Panx3 promotes Cx43 expression by regulating Wnt/ß-catenin signaling and osterix expression. Further, although Panx3 can function in 3 ways, Cx43 cannot function through the ER Ca2+ channel, only via the hemichannels and gap junction routes. In this review, we discuss the current knowledge regarding the roles of Panx3 in skeletal formation and address the potential for new therapies in the treatment of diseases and pathologies associated with Panx3, such as osteoarthritis (OA).

The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system.

Cardiomyocytes are electrically coupled by gap junctions, defined as clusters of low-resistance multisubunit transmembrane channels composed of connexins (Cxs). The expression of Cx40, Cx43 and Cx45, which are present in cardiomyocytes, is known to be developmentally regulated. This study investigates the premise that alterations in gap junction proteins are one of the mechanisms by which teratogens may act. Specifically, those molecules known to be teratogenic in humans could cause their effects via disruption of cell-to-cell communication pathways, resulting in an inability to co-ordinate tissue development. Caffeine significantly inhibited contractile activity at concentrations above and including 1500 µm (P < 0.05), while not affecting cell viability and total protein, in the embryonic chick cardiomyocyte micromass culture system. The effects of caffeine on key cardiac gap junction protein (Cx40, Cx43 and Cx45) expression were analysed using immunocytochemistry and in-cell Western blotting. The results indicated that caffeine altered the expression pattern of Cx40, Cx43 and Cx45 at non-cytotoxic concentrations (≥2000 µm), i.e., at concentrations that did not affect total cell protein and cell viability. In addition the effects of caffeine on cardiomyocyte formation and function (contractile activity score) were correlated with modulation of Cxs (Cx40, Cx43 and Cx45) expression, at above and including 2000 µm caffeine concentrations (P < 0.05). These experiments provide evidence that embryonic chick cardiomyocyte micromass culture may be a useful in vitro method for mechanistic studies of perturbation of embryonic heart development. Copyright © 2015 John Wiley & Sons, Ltd.

Bee venom phospholipase A2-induced phasic contractions in mouse rectum: independent roles of eicosanoid and gap junction proteins and their loss in experimental colitis.

Various events including digestion and inflammation are regulated by secreted phospholipase A2 (sPLA2) in gastrointestinal tissues, however, the role of sPLA2 on contractile activity has not been elucidated. We investigated the effect of bee venom PLA2 (bvPLA2), which is homologous to the central domain of group III sPLA2, on contractile activity in mouse rectum. The longitudinal preparations of rectum showed rhythmic phasic contractions (RPCs) with varied amplitude and high frequency. Treatment with bvPLA2 at 1 µg/ml increased amplitudes of RPCs without marked changes in frequency and basal tone. RPCs by bvPLA2 were affected neither by atropine nor by inhibition of nitric oxide synthase, and partly inhibited by dual inhibition of the cyclooxygenase and lipoxygenase pathways. Pretreatment of bvPLA2 with dithiothreitol, which inhibits the enzyme activity, partly reduced bvPLA2-induced RPCs, and arachidonic acid-increased RPCs were completely abolished by cyclooxygenase/lipoxygenase inhibition. Phasic contractions have been shown to be regulated by gap junction and to be decreased in gastrointestinal tissues with experimental colitis. Treatment with inhibitors of gap junction proteins, 50 µM 18ß-glycyrrhetinic acid and 100 µM carbenoxolone, partly and almost completely reduced bvPLA2-induced RPCs without and with the cyclooxygenase/lipoxygenase inhibitors, respectively, but not arachidonic acid-induced RPCs. In rectum from mouse having colitis, where total levels and modified forms of connexin43 increased, bvPLA2-induced RPCs were markedly decreased. Our results suggest that both arachidonic acid metabolism and gap junction proteins independently regulated the sPLA2-induced RPCs in mouse rectum. An increased expression and/or modification of connexin43 may influence sPLA2-induced RPCs in rectum with colitis.

Expression of gap junctional proteins Cx37 mRNA in cerebral arterial endothelium induced by soluble eggs antigen of Schistosome / 中国地方病学杂志

Objective To explore the effects of gap junctional(GJ)proteins in pathogenesis of cerebral schistosomiasis, through observing the expression of gap junctional proteins Cx37 mRNA in cultured cerebral arterial endothelium incubated with soluble eggs antigen(SEA). MethodsCerebral artery endothelial cells of rabbits were incubated with SEA, and the experiments were divided into control group and SEA 1 - 5 groups (SEA concentrations were 10.0％ ,5.0％ ,3.3％ ,2.5％,2.0％, respectively), reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of Cx37 mRNA and protein in cerebral artery endothelial cells of rabbits, respectively. Results Cx37 mRNA levels of control and SEA 1 - 5 groups were 0.239 ± 0.037, 0.260 ± 0.043, 0.218 ± 0.310, 0.647 ± 0.040, 0.419 ± 0.036, and 0.513 ± 0.038, respectively;SEA 3 - 5 groups were higher than control group of mRNA levels(all P＜ 0.05). Cx37 protein levels of control and SEA 1 - 5 group were 0.401 ± 0.045, 0.485 ± 0.048, 0.749 ± 0.052, 1.119 ± 0.063, 1.015 ± 0.057 and 0.605 ±0.047, respectively, of which SEA 2 - 5 groups were higher than control group(all P ＜ 0.05). ConclusionsExpression levels of Cx37 mRNA and protein in cultured cerebral artery endothelial cells incubated with SEA are higher than those of control cerebral artery endothelial cells, which suggests that the gap junction proteins may play an important role in pathogenesis of cerebral schistosomiasis through SEA and its secretion in infiltration of brain tissue and deposition in the cerebral arteries.