Regulation Of Gap Junctions Research Articles

IL-10 and TGF- β differentially regulate gap junction formation and membrane transfer in macrophages and macrophage-like cells

Abstract In macrophages, the expression and regulation of the gap junction protein connexin43 (Cx43) is poorly understood. Here we demonstrate that inflammatory and anti-inflammatory mediators differentially regulate gap junction plaque formation in primary bone marrow-derived macrophages (BMDMs) and the macrophage-like cell line RAW264.7. RAW264.7 macrophage-like cells were transfected with GFP-tagged Cx43 to visualize Cx43 trafficking. LPS, an inflammatory mediator, is a potent inducer of Cx43 in BMDMs and RAW264-Cx43-GFP but demonstrates limited trafficking into gap junction plaques. Interestingly, the further addition of IL-10, an anti-inflammatory cytokine, resulted in no difference in protein expression but significantly increased gap junction plaque formation. In contrast to IL-10, the addition of TGF-β (beta) decreased Cx43 protein levels and prevented gap junction plaque formation. We utilized a dye transfer assay to assess gap junction intracellular communication. We could not demonstrate any channel-associated transfer of an intracellular dye, calcein. Uniquely, macrophages were observed transferring endosome-like particles containing membrane between cells as visualized with the membrane-bound dye DiI. This appeared to be through long tube-like structures, possibly tunneling nanotubes. This effect was enhanced with IL-10 treatment and decreased with TGF-β. These results suggest that IL-10 and TGF-β differentially regulate Cx43 plaque formation and that this corresponds to changes in the transfer of cellular components in a gap junction channel-independent way. Overall these results suggest a complex and novel regulation of Cx43 gap junction formation in macrophages and macrophage-like cells.

Dynamic UTR Usage Regulates Alternative Translation to Modulate Gap Junction Formation during Stress and Aging

SUMMARYConnexin43 (Cx43; gene name GJA1) is the most ubiquitously expressed gap junction protein, and understanding of its regulation largely falls under transcription and post-translational modification. In addition to Cx43, Gja1 mRNA encodes internally translated isoforms regulating gap junction formation, whose expression is modulated by TGF-β. Here, using RLM-RACE, we identify distinct Gja1 transcripts differing only in 5′ UTR length, of which two are upregulated during TGF-β exposure and hypoxia. Introduction of these transcripts into Gja1−/− cells phenocopies the response of Gja1 to TGF-β with reduced internal translation initiation. Inhibiting pathways downstream of TGF-β selectively regulates levels of Gja1 transcript isoforms and translation products. Reporter assays reveal enhanced translation of full-length Cx43 from shorter Gja1 5′ UTR isoforms. We also observe a correlation among UTR selection, translation, and reduced gap junction formation in aged heart tissue. These data elucidate a relationship between transcript isoform expression and translation initiation regulating intercellular communication.

Growth and development of the corpus luteum

The mammalian corpus luteum, which plays a central role in the reproductive process because of its production of hormones such as progesterone, is an exceptionally dynamic organ. Growth and development of the corpus luteum are extremely rapid, and even when the corpus luteum is functionally mature cellular turnover remains high. Associated with this high rate of cell turnover, the mature corpus luteum receives the greatest blood supply per unit tissue of any organ, and also exhibits a relatively high metabolic rate. Central to the growth and development of the corpus luteum, therefore, is luteal vascular growth, which appears to be regulated primarily by the angiogenic growth factors, basic fibroblast growth factor and vascular endothelial growth factor. In addition, the corpus luteum is a complex tissue composed of parenchymal (small and large steroidogenic) and nonparenchymal (for example fibroblasts, vascular smooth muscle, pericytes and endothelial) cells. Recent studies evaluating the expression, location and regulation of gap junctions in the corpus luteum indicate an important role of gap junctional intercellular communication in the coordination of function among these diverse cell types during luteal growth and development. These studies will lead to an improved understanding not only of luteal function but also of tissue growth and development in general.

Bioinformatics analysis for heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) represents approximately half of heart failure but little is known about the underlying mechanisms. Dahl salt‐sensitive (Dahl‐SS) rats were fed a high‐salt diet (8% NaCl) from 7 weeks of age to induce HFpEF and controls were fed a normal‐salt diet (0.3% NaCl). Echocardiograms and electrocardiography (ECG) was performed to assess cardiac function. Diastolic dysfunction and preserved ejection fraction along with signs of heart failure was showed and QTc interval on ECG was prolonged (p<0.001) at 14 weeks of age. Hearts were harvested for gene array and label‐free quantitative proteomics. Bioinformatics analysis using DAVID website, GSEA software and BinGO plugin in Cytoscape environment. GO analysis showed microtubule binding and calcium dependent phospholipid binding protein were significant downregulated while oxidative stress response protein were upregulated (p<0.05, q<0.05). GSEA results indicated that gap junction, focal adhesion and actin cytoskeleton regulation gene sets were concentrated in upregulated transcripts (p<0.01, q<0.25) while cardiac muscle contraction and oxidative phosphorylation pathway were downregulated in proteomics (p<0.001, q<0.001). Western blot and PCR experiments showed significant changes in the expression of Dynamin‐1, Integrin‐α9 and MFF, which had important functions including cell stiffness regulation. Our study provides valuable information on the molecular functions and regulatory network of HFpEF, as well as new perspectives on the relationship between diastolic dysfunction and calcium stimulate, cytoskeleton stiffness and oxidative stress.Support or Funding InformationShenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of cardiac myocyte cohesion and gap junctions via desmosomal adhesion.

Mutations in desmosomal proteins can induce arrhythmogenic cardiomyopathy with life-threatening arrhythmia. Previous data demonstrated adrenergic signalling to be important to regulate desmosomal cohesion in cardiac myocytes. Here, we investigated how signalling pathways including adrenergic signalling, PKC and SERCA regulate desmosomal adhesion and how this controls gap junctions (GJs) in cardiac myocytes. Immunostaining, Western blot, dissociation assay and multi-electrode array were applied in HL-1 cardiac myocytes to evaluate localization, expression and function of desmosomal and GJ components. cAMP levels were determined by ELISA. Activation of PKC by PMA or adrenergic signalling increased cell cohesion and desmoglein-2 and desmoplakin localization at cell-cell junctions, whereas tryptophan (Trp) treatment to inhibit cadherin binding or inhibition of SERCA by thapsigargin reduced cell cohesion, while cAMP elevation rescued this effect. Despite no changes in protein expression, accumulation of GJ protein connexin-43 was detectable at cell-cell contacts in parallel to increased cohesion. Disruption of cell cohesion by Trp, PMA or thapsigargin impaired conduction of excitation comparable to GJ inhibition. cAMP elevation was effective to improve arrhythmia after Trp treatment. Weakened cell cohesion by Trp or depletion of desmoglein-2 or plakoglobin blocked signalling via the β1-adrenergic receptor. Moreover, silencing of desmosomal proteins increased arrhythmia and reduced conduction velocity, which were rescued by cAMP elevation. These data demonstrate the interplay of GJs, desmosomes and the β1-adrenergic receptor with regulation of their function by cell cohesion, adrenergic and PKC signalling or SERCA inhibition. These results support the identification of new targets to treat arrhythmogenic cardiomyopathy.

Regulation of Connexin32 by ephrin receptors and T-cell protein-tyrosine phosphatase

Gap junctions are intercellular conduits that permit the passage of ions, small metabolites, and signaling molecules between cells. Connexin32 (Cx32) is a major gap junction protein in the liver and brain. Phosphorylation is integral to regulating connexin assembly, degradation, and electrical and metabolic coupling, as well as to interactions with molecular partners. Cx32 contains two intracellular tyrosine residues, and tyrosine phosphorylation of Cx32 has been detected after activation of the epidermal growth factor receptor; however, the specific tyrosine residue and the functional implication of this phosphorylation remain unknown. To address the limited available information on Cx32 regulation by tyrosine kinases, here we used the Cx32 C-terminal (CT) domain in an in vitro kinase-screening assay, which identified ephrin (Eph) receptor family members as tyrosine kinases that phosphorylate Cx32. We found that EphB1 and EphA1 phosphorylate the Cx32CT domain residue Tyr243 Unlike for Cx43, the tyrosine phosphorylation of the Cx32CT increased gap junction intercellular communication. We also demonstrated that T-cell protein-tyrosine phosphatase dephosphorylates pTyr243 The data presented above along with additional examples throughout the literature of gap junction regulation by kinases, indicate that one cannot extrapolate the effect of a kinase on one connexin to another.

ERK and miRNA-1 target Cx43 expression and phosphorylation to modulate the vascular protective effect of angiotensin II

Aims and methodsWe previously reported that angiotensin II (AngII) restores the vascular reactivity diminished by hemorrhagic shock. In this study, we investigated whether the beneficial effects of AngII are related to regulation of gap junctions (GJs) and connexin43 (Cx43), and the implication of MAPK signaling and microRNA (miR-1) in this process. Key findingsOur results show that after hemorrhagic shock or hypoxia, the blockade of GJs or knockdown of Cx43 inhibits the AngII-induced increase in vascular reactivity of superior mesenteric arteries and the contractile response of vascular smooth muscle cells (VSMCs). AngII treatment increases Cx43 expression and phosphorylation at Ser262, and restores gap-junctional communication (GJIC) between VSMCs after hypoxia. The AngII-induced up-regulation of Cx43 expression and phosphorylation is blocked in cells transduced with ERK-siRNA, but is not blocked in cells transduced with p38-siRNA. miR-1 levels are elevated after hypoxia; AngII treatment reverses the up-regulation of miR-1, while ERK-siRNA abolishes that effect of AngII. In hypoxic cells, transfection of a miR-1 mimic into VSMCs decreases Cx43 expression and VSMC reactivity, whereas a miR-1 inhibitor increases both. Also in hypoxic cells, miR-1 eliminates the restoration effects of AngII on Cx43 expression and VSMC reactivity. SignificanceAngII provides protection of vascular function through the restoration of the expression and phosphorylation of Cx43 and its mediated GJIC in VSMCs. It is ERK that mediates the AngII-induced phosphorylation of Cx43 at Ser262. Additionally, miR-1 is involved in this process, and AngII may exert its protective effect partially by inhibiting miR-1 elevation via ERK signaling.

G protein-coupled receptor 30 mediates meiosis resumption and gap junction communications downregulation in goat cumulus-oocyte complexes by 17β-estradiol

Estrogen plays a critical role in the regulation of gap junctions between oocytes and granulosa cells in mammalian ovaries. G protein-coupled receptor 30 (GPR30) was identified as a membrane estrogen receptor, mediating rapid, nongenomic signaling events that might be responsible for the regulation of oocyte meiosis resumption and gap junction intercellular communications (GJICs). The present study aimed to determine the expression and localization of GPR30 and its role in oocyte meiotic progression and GJICs in goat cumulus-oocyte complexes (COCs). Immunofluorescence experiments revealed that GPR30 was primarily located in the plasma membrane of cumulus cells and oocytes in goats. 17β-estradiol could promote oocyte meiotic progression, which was blocked by G15 (a selective GPR30 antagonist) but not ICI182780 (a nuclear estrogen receptor inhibitor) in the early stage of in vitro culture. The effect of 17β-estradiol on the GJICs was quantified by lucifer yellow (LY) microinjection and calcein-AM fluorescent dye diffusion. 17β-estradiol treatment of goat COCs resulted in rapid downregulation of GJICs. The transfer of calcein from cumulus cells to oocytes could be significantly inhibited by carbenoxolone (a known gap junction blocker), 17β-estradiol or G1 (a GPR30 agonist), and this inhibition could be reversed by G15 but not ICI182780, indicating that GPR30 mediates the effect of 17β-estradiol on the rapid downregulation of GJICs. 17β-estradiol also stimulated the serine 368 phosphorylation of connexin 43 (Cx43) when COCs were in vitro cultured for 4 h, 6 h, and 8 h. More importantly, 17β-estradiol or G1 could separately promote the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and Cx43 significantly when COCs were cultured for 4 h. Furthermore, both ERK1/2 and Cx43 phosphorylation could be inhibited by G15 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or by the ERK1/2 inhibitor PD98059, indicating that EGFR-ERK1/2 signaling was involved in these events. These results supported the hypothesis that GPR30 mediated 17β-estradiol-stimulated meiotic resumption and GJIC reduction in goat COCs. Thus, the present study provides novel insights into elucidating the mechanisms for steroid hormone action in the regulation of oocyte maturation.

Abstract 16931: Alternate UTR Usage Regulates Connexin43 mRNA Translation Limiting Gap Junction Formation During Stress

Introduction: Connexin43 (Cx43, Gja1 ) gap junctions enable electrical coupling of cardiomyocytes necessary for heart function. Pathological disruption of Cx43 expression or localization results in the arrhythmias of sudden cardiac death. Investigation of Cx43 gap junction biology has focused on transcription, vesicular trafficking, and post translational modifications. The importance of translational regulation however is highlighted by the recent discovery that Cx43 mRNA undergoes internal translation initiation to yield truncated protein isoforms, including the predominant GJA1-20k isoform capable of regulating gap junction formation. We have found that TGF-β signaling, a hallmark of cardiac stressors including hypertrophy, suppresses GJA1-20k expression to limit gap junction formation. The mechanisms regulating Cx43 mRNA internal translation in response to cellular stress are unknown but are critical in harnessing this biology for therapeutic restoration of electrical coupling in diseased hearts. We hypothesize that altered Gja1 -5’UTR usage promotes translation of full length Cx43 over internal translation products resulting in reduced gap junction formation during stress. Methods: Rapid amplification of cDNA ends was used to identify Gja1- 5’UTR isoforms following TGF-β exposure or hypoxia, and Western blotting was used to monitor Gja1 translation. Identified Gja1 -5’UTR variants were transfected into Cx43 knockout cells to assess translational potential. Luciferase assays assessed potential IRES activity within Gja1 mRNA in relation to specific 5’UTR isoforms. Results: TGF-β or hypoxia significantly increase translation of full length Cx43 over Gja1-20k correlating with an increase in truncated Gja1 -5’UTRs. Sequenced UTR isoforms map to genome wide transcription start sites and transfection of Gja1 transcripts with truncated 5’UTRs is sufficient to reduce Gja1-20k translation, phenocopying stress models. Conclusions: Alteration of Gja1 -5’UTR composition represents a powerful mechanism connecting alterations in transcription to gene-specific translational regulation to fine tune intercellular communication during stress.

Allicin attenuates calcium oxalate crystal deposition in the rat kidney by regulating gap junction function.

Renal calculus is a global common urological disease that is closely related to crystal adhesion and renal tubular epithelial cell impairment. Gap junctions (GJs) and their components (connexins and Cxs) are involved in various pathophysiology processes, but their roles in renal calculi progression are not well defined. Our previous RNA microarray analysis suggests that GJs are one of the key predicted pathways involved in the renal calcium oxalate (CaOx) crystal rat model. In the current study, we found that the Cx43 and Cx32 expression and the GJ function decreased significantly after stimulation with CaOx or sodium oxalate (NaOx) in NRK-52E, MDCK, and HK-2 cells, and Cx43 expression also decreased in renal tissues in renal CaOx crystal model rats. Inhibition of Cx43 in NRK-52E cells by small interference RNA significantly increased the CD44 and androgen receptor expression, and the adhesion between CaOx crystals and cells, which were consistent with the function of GJ inhibitors. On the other hand, after GJ function and Cx43 expression were increased by allicin, diallyl disulfide, or diallyl trisulfide, the impairment of NRK-52E cells by NaOx or other GJ inhibitors and the adhesion between CaOx crystals and renal cells decreased significantly. Furthermore, allicin also increased Cx43 expression and inhibited crystal deposition in rat kidneys. Taken together, our results provide a basis that GJs and Cx43 may participate in renal CaOx stone progression and that allicin, together with its analogues, could be potential drugs for renal calculus precaution.

Buffalo oocyte-secreted factors promote cumulus cells apoptosis and the rate of cGMP production but not steroidogenesis.

The objectives of this study were to investigate the effect of buffalo oocyte-secreted factors (OSFs) on cumulus cells (CCs) functions, apoptosis and cGMP generation, and whether the direct contact between oocyte and CCs is essential for oocyte-mediated regulation of CCs functions. Buffalo CCs were cultured during IVM within three groups: (a) intact cumulus-oocyte complexes (COCs), (b) CCs cocultured with denuded oocytes (DOs) (CCs+DOs) and (c) CCs monolayer cultured alone (CCsM). After 24hr of IVM, CCs were harvested for evaluation of the relative mRNA abundance of the genes encoding gap junction (GJA1), glycolysis (PFKP and LDHA), apoptosis (CASPASE-3 and BCL-2) and steroidogenesis (ER-β and PGR) by QRT-PCR, and CASPASE-3 proteins, using western blot. Intracellular cGMP content was also assessed by ELISA. Results showed that the relative abundance of LDHA, PFKP and BCL-2 significantly increased (p<0.05) in COCs, whereas GJA1 and CASPASE-3 exhibited lower expression (p<0.05) compared to CCs+DOs and CCsM groups. However, the expression levels of CASPASE-3, both mRNA and protein, were significantly (p<0.05) downregulated in CCs+DOs compared to CCsM. There was no significant difference in the expression level of PGR and ER-β between the groups. The intracellular content of cGMP was notably (p<0.05) higher in COCs compared to CCs+DOs and CCsM groups. In conclusion, this study demonstrated, for the first time, that buffalo OSFs protect CCs against apoptosis and stimulate their cGMP production; however, the regulation of cumulus glycolysis and gap junction is confined to those in close contact with the oocyte. Neither OSFs from COCs nor those from DOs have any effect on CCs steroidogenesis.

Gap Junctions in the Nervous System: Probing Functional Connections Using New Imaging Approaches.

Gap junctions are channels that physically connect adjacent cells, mediating the rapid exchange of small molecules, and playing an essential role in a wide range of physiological processes in nearly every system in the body, including the nervous system. Thus, altered function of gap junctions has been linked with a plethora of diseases and pathological conditions. Being able to measure and characterize the distribution, function, and regulation of gap junctions in intact tissue is therefore essential for understanding the physiological and pathophysiological roles that gap junctions play. In recent decades, several robust in vitro and in vivo methods have been developed for detecting and characterizing gap junctions. Here, we review the currently available methods with respect to invasiveness, signal-to-noise ratio, temporal resolution and others, highlighting the recently developed chemical tracers and hybrid imaging systems that use novel chemical compounds and/or genetically encoded enzymes, transporters, channels, and fluorescent proteins in order to map gap junctions. Finally, we discuss possible avenues for further improving existing techniques in order to achieve highly sensitive, cell type-specific, non-invasive measures of in vivo gap junction function with high throughput and high spatiotemporal resolution.

Connexin 43 K63-polyubiquitylation on lysines 264 and 303 regulates gap junction internalization

Gap junctions (GJs) assembled from connexin (Cx) proteins allow direct cell-cell communication. While phosphorylation is known to regulate multiple GJ functions, much less is known about the role of ubiquitin in these processes. Using ubiquitylation-type-specific antibodies and Cx43 lysine-to-arginine mutants we show that ∼8% of a GJ, localized in central plaque domains, is K63-polyubiquitylated on K264 and K303. Levels and localization of ubiquitylation correlated well with: (1) the short turnover rate of Cxs and GJs; (2) removal of older channels from the plaque center; and (3) the fact that not all Cxs in an internalizing GJ channel need to be ubiquitylated. Connexins mutated at these two sites assembled significantly larger GJs, exhibited much longer protein half-lives and were internalization impaired. Interestingly, these ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitylation is triggered by phosphorylation. Phospho-specific anti-Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282 and 255, which are well-known regulatory PKC and MAPK sites. Together, these novel findings suggest that the internalizing portion of channels in a GJ is K63-polyubiquitylated, ubiquitylation is critical for GJ internalization and that phosphorylation induces Cx K63-polyubiquitylation.This article has an associated First Person interview with the first author of the paper.

First person – Rachel Margraf

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Rachel Margraf is co-first author on ‘Connexin 43 K63-polyubiquitylation on lysines 264 and 303 regulates gap junction internalization’, published in Journal of Cell Science. Rachel recently graduated from the lab of Dr Matthias Falk at Lehigh University, Bethlehem, USA, where she studied molecular mechanisms of gap junction turnover, including phosphorylation and ubiquitylation.

Characterization and Regulation of Gap Junctions in Porcine Ciliary Epithelium.

Gap junctions provide a conduit between the intracellular fluids of the pigmented (PE) and non-pigmented (NPE) ciliary epithelial cells, and are therefore critical in the secretion of the aqueous humor (AH). However, opinions differ concerning the connexin (Cx) composition of the gap junctions. Therefore, we aimed to characterize the expression of Cx in the porcine ciliary epithelium (CE), a favorable model for humans; and determine the contribution of the highest expressed Cx to AH secretion. Freshly-harvested porcine CE cells were used. The mRNA and protein expressions of gap junctions were assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. The relative gene expressions of various Cx were determined by quantitative PCR. The gap junction permeability of isolated PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer. Using RT-PCR and WB, Cx43, Cx45, Cx47, Cx50, and Cx60 were present in porcine CE, with Cx43 being the most abundant isoform, having over 200-fold higher expression than other Cx. Cx43 was primarily localized in the PE-NPE interface and the basolateral membranes of PE cells. Knockdown of Cx43 by siRNA significantly reduced gene and protein expressions, resulting in reduction of transcellular fluid flow by 90%. Cx43 was found to be the major component of gap junctions in porcine CE. Consistent with results from a bovine model, our results support the important role of Cx43 in mediating AH secretion. This finding may shed light on the development of a novel ocular hypotensive agent.

Abstract 3402: Castration resistance transcriptome in prostate cancer revealed by single-cell RNA-seq

Abstract Fatal metastatic castration-resistant prostate cancer (mCRPC) remains without sensitive early detection biomarkers and effective therapeutic targets. Among 2.5 millions of prostate cancer patients, the majority will face a dilemma, to treat or not to treat, at a point as cancer progresses. Biomarkers for mRCPC at an early stage represent an unmet need. With early identification, clinicians could design new treatment strategies to reduce metastasis-related morbidity and to extend survival of patients. In this study, we deployed single-cell RNA-seq on prostate cancer cells (LNCaP, ABL and PC3) to determine the transcriptome in androgen independency and castration resistance of prostate cancer. We identified potential 336 androgen-independence specific genes and 2396 castration resistance specific genes in ABL and PC3 cells respectively, while only 136 genes were shared in both cells. These genes, mostly upregulated were enriched in 43 and 166 signaling pathways that implicated the complexity of the castration resistance transcriptomic systems and networks. The signaling pathways are involved in advanced and metastatic malignancies including WNT, TGFB, ITGA/B, STAT, EPH, focal adhesion, adherens junction, regulation of actin cytoskeleton, gap junction, tight junction and EMT. Malignant potencies of ~ 40 pathways were validated by in silico analysis of the RNA-seq data from the prostate cancer cohort of The Cancer Genomic Atlas (TCGA) using Kaplan-Meier disease free and survival curve analyses. The transcriptomic regulation of these genes was further validated and correlated with ATAC-seq data. In order to further verify the functions of those signaling pathways in castration resistance, 9 major signaling pathways were evaluated using small molecule inhibitors. Castration resistant prostate cancer cells showed significant defective cell proliferation, migration, invasion and sphere formation in the presence of inhibitors, whereas LNCaP and ABL cells displayed limited or non-significant changes. Interestingly, 4 small molecule inhibitors showed significant suppression on the growth of stem-like circulating tumor cells that were derived from clinical blood samples of prostate cancer patients. Our data suggest that those castration resistance specific genes and signaling pathways revealed by single-cell RNA-seq may serve as potential markers and therapeutic targets. Citation Format: Aaron M. Horning, Che-Kuang Lin, Yao Wang, Brandon Lieberman, Devalingam Mahalingam, Ming Gao, Pei Wang, Chiou-Miin Wang, Zhijie Liu, Jianhua Ruan, Michael A. Liss, Victor X. Jin, Tim H-M Huang, Chun-Liang Chen. Castration resistance transcriptome in prostate cancer revealed by single-cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3402.

Regulation of Cardiomyocyte Cohesion and Gap Junctions via Desmosomal Adhesion

Mutations in desmosomal proteins such as desmoglein‐2, desmoplakin or plakoglobin can induce arrhythmogenic cardiomyopathy, a disease leading to arrhythmia and sudden cardiac death. Previous data demonstrated adrenergic signaling to be important to regulate desmosomal cohesion in cardiomyocytes. Here, we investigated how signaling pathways including adrenergic signaling, PKC and SERCA regulate desmosomal adhesion and how desmosomal adhesion controls gap junctions in cardiomyocytes. As revealed by dissociation and immunostaining experiments in cardiomyocytes, activation of PKC by PMA or adrenergic signaling increased cell cohesion and desmoglein‐2 and desmoplakin localization at cell junctions whereas Trp‐treatment to inhibit cadherin binding or inhibition of SERCA pump by thapsigargin reduced cardiomyocyte cohesion. Cohesion was rescued by both cAMP elevation or PMA. In parallel to increased cohesion elevated phosphorylation of the gap junctions component connexin‐43 was revealed by Western blotting and immunostaining. Using a multi‐electrode array, disruption of cell cohesion by Trp impaired conduction of excitation comparable to the connexin inhibitor carbenoxolone. Elevation of cAMP levels was effective to increase conduction velocity and to improve arrhythmia under baseline conditions as well as after Trp treatment. Immunoprecipitation revealed connexin‐43 to interact with desmoglein‐2 and β1‐adrenergic receptor. Weakened cell cohesion by Trp or siRNA‐mediated depletion of desmoglein‐2 or plakoglobin were sufficient to block signaling via the β1‐adrenergic receptor. Moreover, depletion of desmosomal proteins increased arrhythmia and reduced conduction velocity, both of which was rescued by cAMP elevation. These data demonstrate gap junction and desmosomal proteins to form a complex with the β1‐adrenergic receptor. The function of this complex was regulated by cell cohesion, adrenergic and PKC signaling or inhibition of SERCA. These results support the identification of new targets to treat arrhythmogenic cardiomyopathy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phenotyping of Gap-Junctional Coupling in the Mouse Retina.

In the mammalian retina, gap junctions, made of connexin proteins, are found in all neuronal cell types and are important for the transmission of rod photoreceptor signals, spike synchronization, noise reduction, and signal averaging. There are several methods available to assess gap junctional coupling in the retina: simultaneous electrical recordings from two adjacent cells, cut-loading, and intracellular injection of gap junction-permeable tracers. Here, we focus on the latter as it allows precise targeting of the cell of interest and is suitable to assess tracer coupling in a wide variety of retinal cell types, e.g., horizontal cells, amacrine cells, and ganglion cells. Tracer coupling experiments are usually performed in the intact retina and can provide information on the extent of coupling, the identity of synaptic partners, and (when combined with immunohistochemistry or pharmacology) the underlying connexin or the regulation of gap junctions.

Inhibition of Gap Junction Elevates Glutamate Uptake in Cultured Astrocytes.

Glutamate uptake is a main function of astrocytes to keep extracellular glutamate levels low and protect neurons against glutamate-induced excitotoxicity. On the other hand, astrocyte networks formed by gap junctions, which are consisted with connexins and connecting neighboring cells, are reported to play a critical role in maintaining the homeostasis in the brain. In the present study, we examined the effects of gap junction inhibitors on the glutamate uptake activity in cultured rat cortical astrocytes. At first, we confirmed the effects of gap junction inhibitors, 1-octanol and carbenoxolone, on cell-cell communication by the scrape-loading assay using a fluorescent dye Lucifer yellow. Both of 1-octanol and carbenoxolone treatments for 20min in cultured astrocytes significantly suppressed the cell-cell communication assessed as the distance of dye-spreading. 1-octanol and carbenoxolone increased the glutamate uptake by astrocytes and glutamate aspartate transporter (GLAST) expression on the cell membrane. These results suggest that gap junction inhibitors increase the glutamate uptake activity through the increase of GLAST proteins located on the cell membrane. The regulation of gap junction in astrocytes might protect neurons against glutamate-induced excitotoxicity.

CELF1 regulates gap junction integrity contributing to dilated cardiomyopathy

Cardiovascular disease is a major contributor of adult mortality and accounts for approximately 801,000 deaths annually throughout the United States alone (1).