Gap Junctional Communication Research Articles

Fabrication and characterization of a thick, viable bi-layered stem cell-derived surrogate for future myocardial tissue regeneration

Cardiac tissue surrogates show promise for restoring mechanical and electrical function in infarcted left ventricular (LV) myocardium. For these cardiac surrogates to be useful in vivo, they are required to support synchronous and forceful contraction over the infarcted region. These design requirements necessitate a thickness sufficient to produce a useful contractile force, an area large enough to cover an infarcted region, and prevascularization to overcome diffusion limitations. Attempts to meet these requirements have been hampered by diffusion limits of oxygen and nutrients (100–200 µm) leading to necrotic regions. This study demonstrates a novel layer-by-layer (LbL) fabrication method used to produce tissue surrogates that meet these requirements and mimic normal myocardium in form and function. Thick (1.5–2 mm) LbL cardiac tissues created from human induced pluripotent stem cell-derived cardiomyocytes and endothelial cells were assessed, in vitro, over a 4-week period for viability (<5.6 ± 1.4% nectrotic cells), cell morphology, viscoelastic properties and functionality. Viscoelastic properties of the cardiac surrogates were determined via stress relaxation response modeling and compared to native murine LV tissue. Viscoelastic characterization showed that the generalized Maxwell model of order 4 described the samples well (0.7 < R 2 < 0.98). Functional performance assessment showed enhanced t-tubule network development, gap junction communication as well as conduction velocity (16.9 ± 2.3 cm s−1). These results demonstrate that LbL fabrication can be utilized successfully in creating complex, functional cardiac surrogates for potential therapeutic applications.

AS602801 sensitizes glioma cells to temozolomide and vincristine by blocking gap junction communication between glioma cells and astrocytes.

Previous studies showed that the chemotherapeutic effect of temozolomide (TMZ) and vincristine (VCR) against glioma might be blunted by the co‐culture with astrocytes, and connexin‐43 (CX43) was thought to play a vital role in the communication between glioma cells and astrocytes. In this study, we aimed to investigate the combined chemotherapeutic effect of AS602801 and TMZ/ VCR in glioma cells both. Dye transfer assay was used to evaluate the gap junction activity between U251 cells and astrocytes. Western blot and immunohistochemistry were carried out to analyse the expression of p‐JNK, CX43 and CASP‐3 proteins treated under different conditions. AS602801 significantly suppressed the gap junction activity between U251 cells and astrocytes. The expression of p‐JNK and CX43 was remarkably inhibited by AS602801. TMZ/VCR‐induced apoptosis of glioma cells was effectively enhanced by AS602801 treatment. Accordingly, the inhibitory role of TMZ/VCR in the expression of p‐JNK, CX43 and CASP‐3 in glioma cells was notably restored by AS602801. Furthermore, in a glioma cell xenograft, AS602801 showed an apparent capability to enhance TMZ/VCR‐induced tumour cell apoptosis through altering the expression of p‐JNK, CX43 and CASP‐3. The findings of this study demonstrated that the co‐culture of glioma cells with astrocytes blunted the tumour killing effect of TMZ and VCR. AS602801 down‐regulated CX43 expression by inhibiting JNK. And AS602801 also sensitized glioma cells to TMZ/VCR by blocking the gap junction communication between glioma cells and astrocytes via down‐regulating CX43, indicating its potential role as a novel adjuvant chemotherapeutic agent in the treatment of glioma.

Ethanol Disrupts Hormone-Induced Calcium Signaling in Liver.

Receptor-coupled phospholipase C (PLC) is an important target for the actions of ethanol. In the ex vivo perfused rat liver, concentrations of ethanol >100 mM were required to induce a rise in cytosolic calcium (Ca2+) suggesting that these responses may only occur after binge ethanol consumption. Conversely, pharmacologically achievable concentrations of ethanol (≤30 mM) decreased the frequency and magnitude of hormone-stimulated cytosolic and nuclear Ca2+ oscillations and the parallel translocation of protein kinase C-β to the membrane. Ethanol also inhibited gap junction communication resulting in the loss of coordinated and spatially organized intercellular Ca2+ waves in hepatic lobules. Increasing the hormone concentration overcame the effects of ethanol on the frequency of Ca2+ oscillations and amplitude of the individual Ca2+ transients; however, the Ca2+ responses in the intact liver remained disorganized at the intercellular level, suggesting that gap junctions were still inhibited. Pretreating hepatocytes with an alcohol dehydrogenase inhibitor suppressed the effects of ethanol on hormone-induced Ca2+ increases, whereas inhibiting aldehyde dehydrogenase potentiated the inhibitory actions of ethanol, suggesting that acetaldehyde is the underlying mediator. Acute ethanol intoxication inhibited the rate of rise and the magnitude of hormone-stimulated production of inositol 1,4,5-trisphosphate (IP3), but had no effect on the size of Ca2+ spikes induced by photolysis of caged IP3. These findings suggest that ethanol inhibits PLC activity, but does not affect IP3 receptor function. We propose that by suppressing hormone-stimulated PLC activity, ethanol interferes with the dynamic modulation of [IP3] that is required to generate large, amplitude Ca2+ oscillations.

Cytoproliferative effect of low dose alpha radiation in human lung cancer cells is associated with connexin 43, caveolin-1, and survivin pathway

Purpose High LET including alpha radiation-based approaches have been proved as a promising mode for cancer therapy owing to their biophysical and radiobiological advantages compared to photon beams. Studies pertaining to effect of α-radiation on cancer cells are limited to cytotoxic high doses. Materials and methods In this study, human lung adenocarcinoma (A549) cells were α-irradiated using 241Am α-irradiator and effects of low dose of alpha radiation on these cells was studied under in vitro and in vivo conditions. Results Clonogenic and other assays showed increased cellular proliferation at lower doses (1.36 and 6.8 cGy) but killing at higher doses (13.6–54.4 cGy). Further studies at low dose of alpha (1.36 cGy) showed increased TGF-β1 in the conditioned medium (CM) at early time point (24 h) but CM replacement did not affect the clonogenic survival. In these cells, increased phosphorylation of connexin 43 was correlated with decrease in gap-junction communication observed by dye transfer co-culture experiment. A decrease in caveolin-1 but increase in survivin expression was observed in low dose α-irradiated cells. An increase in cyclinD1 and decrease in Bcl-2, the target proteins of survivin, was observed in these cells. Low dose α-irradiated cancer cells transplanted in SCID mice showed significantly higher tumor volume, which was accompanied with an increased fraction of mitotic and PCNA/Ki67 positive cells in these tumor tissues. Conclusions Taken together, our results suggest an increase in proliferation and tumor volume at in vitro and in vivo levels, respectively, when A549 cells were irradiated with low dose of α-radiation. These findings may be relevant for a better understanding of radiobiological processes during high LET-based cancer radiotherapy.

Connexin Gap Junctions and Hemichannels Link Oxidative Stress to Skeletal Physiology and Pathology.

The goal of this review is to provide an overview of the impact and underlying mechanism of oxidative stress on connexin channel function, and their roles in skeletal aging, estrogen deficiency, and glucocorticoid excess associated bone loss. Connexin hemichannel opening is increased under oxidative stress conditions, which confers a cell protective role against oxidative stress-induced cell death. Oxidative stress acts as a key contributor to aging, estrogen deficiency, and glucocorticoid excess-induced osteoporosis and impairs osteocytic network and connexin gap junction communication. This paper reviews the current knowledge for the role of oxidative stress and connexin channels in the pathogenesis of osteoporosis and physiological and pathological responses of connexin channels to oxidative stress. Oxidative stress decreases osteocyte viability and impairs the balance of anabolic and catabolic responses. Connexin 43 (Cx43) channels play a critical role in bone remodeling, mechanotransduction, and survival of osteocytes. Under oxidative stress conditions, there is a consistent reduction of Cx43 expression, while the opening of Cx43 hemichannels protects osteocytes against cell injury caused by oxidative stress.

Hydrogen sulfide as a mediator of endothelium-dependent relaxation evoked by Moringa oleifera leaf extract in mesenteric arterial beds isolated from L-NAME hypertensive rats.

Aqueous extract of Moringa oleifera leaves (MOE) is a potent inducer of endothelium-dependent relaxation of mesenteric resistance arteries of rats induced to be hypertensive using Nω-nitro-L-arginine methyl ester (L-NAME). Hydrogen sulfide (H2S) has been shown to participate in endothelium-dependent relaxation of small resistance arteries. Therefore, this study aimed to investigate whether endothelial H2S-dependent signaling plays a role in the vasorelaxation in response to MOE. Mesenteric arterial beds isolated from L-NAME hypertensive rats were set up in an exvivo perfusion system for measurement of vasoreactivity. All experiments were performed in the presence of the nitric oxide synthase inhibitor, L-NAME (100µM) and the cyclooxygenase inhibitor, indomethacin (10µM) to prevent the formation of nitric oxide and prostanoids, respectively. In the presence of the nitric oxide synthase inhibitor, L-NAME and the cyclooxygenase inhibitor, indomethacin, the endothelium-dependent vasorelaxation induced by MOE (0.001-3mg) was completely inhibited by DL-propargylglycine (100µM), which inhibits the H2Sgenerating enzyme, cystathionine γ-lyase. This H2Sdependent response was reduced by the KATP channel blocker; glibenclamide (10µM), the KCa channel blocker; tetraethylammonium (1µM), and the myo-endothelial gap-junctional uncoupler; 18α-glycyrrhetinic acid (10µM). In contrast, the muscarinic receptor antagonist, atropine (100µM), did not affect the response to MOE. The results may suggest that H2S is the likely mediator of endothelium-dependent relaxation in response to MOE in mesenteric arterial beds of L-NAME-induced hypertensive rats. MOE-induced H2S-dependent vasorelaxation involves activation of KATP and KCa channels and requires myo-endothelial gap-junctional communication.

The CAR group of Ig cell adhesion proteins-Regulators of gap junctions?

Members of the CAR group of Ig-like type I transmembrane proteins mediate homotypic cell adhesion, share a common overall extracellular domain structure and are closely related at the amino acid sequence level. CAR proteins are often found at tight junctions and interact with intracellular scaffolding proteins, suggesting that they might modulate tight junction assembly or function. However, impairment of tight junction integrity has not been reported in mouse knockout models or zebrafish mutants of CAR members. In contrast, in the same knockout models deficits in gap junction communication were detected in several organ systems, including the atrioventricular node of the heart, smooth muscle cells of the intestine and the ureter and in Sertoli cells of the testes. Possible interactions between BT-IgSF and connexin41.8 on the disturbed pattern of pigment stripes found in zebrafish mutants and between ESAM and connexin43 during hematopoiesis in the mouse are also discussed. On the basis of the combined data and phenotypic similarities between CAR member mutants and connexin mutants I hypothesize that they primarily play a role in the organization of gap junction communication. Also see the video abstract here: https://youtu.be/i0yq2KhuDAE.

Exosomes and exosomal microRNA in non-targeted radiation bystander and abscopal effects in the central nervous system.

Localized cranial radiotherapy is a dominant treatment for brain cancers. After being subjected to radiation, the central nervous system (CNS) exhibits targeted effects as well as non-targeted radiation bystander effects (RIBE) and abscopal effects (RIAE). Radiation-induced targeted effects in the CNS include autophagy and various changes in tumor cells due to radiation sensitivity, which can be regulated by microRNAs. Non-targeted radiation effects are mainly induced by gap junctional communication between cells, exosomes containing microRNAs can be transduced by intracellular endocytosis to regulate RIBE and RIAE. In this review, we discuss the involvement of microRNAs in radiation-induced targeted effects, as well as exosomes and/or exosomal microRNAs in non-targeted radiation effects in the CNS. As a target pathway, we also discuss the Akt pathway which is regulated by microRNAs, exosomes, and/or exosomal microRNAs in radiation-induced targeted effects and RIBE in CNS tumor cells. As the CNS-derived exosomes can cross the blood-brain-barrier (BBB) into the bloodstream and be isolated from peripheral blood, exosomes and exosomal microRNAs can emerge as promising minimally invasive biomarkers and therapeutic targets for radiation-induced targeted and non-targeted effects in the CNS.

Neocortical in vivo focal and spreading potassium responses and the influence of astrocytic gap junctional coupling

Raised extracellular potassium ion (K+) concentration is associated with several disorders including migraine, stroke, neurotrauma and epilepsy. K+ spatial buffering is a well-known mechanism for extracellular K+ regulation/distribution. Astrocytic gap junction-mediated buffering is a controversial candidate for K+ spatial buffering. To further investigate the existence of a K+ spatial buffering and to assess the involvement of astrocytic gap junctional coupling in K+ redistribution, we hypothesized that neocortical K+ and concomitant spreading depolarization (SD)-like responses are controlled by powerful local K+ buffering mechanisms and that K+ buffering/redistribution occurs partially through gap junctional coupling. Herein, we show, in vivo, that a threshold amount of focally applied KCl is required to trigger local and/or distal K+ responses, accompanied by a SD-like response. This observation indicates the presence of powerful local K+ buffering which mediates a rapid return of extracellular K+ to the baseline. Application of gap junctional blockers, carbenoxolone and Gap27, partially modulated the amplitude and shape of the K+ response and noticeably decreased the velocity of the spreading K+ and SD-like responses. Opening of gap junctions by trimethylamine, slightly decreased the amplitude of the K+ response and markedly increased the velocity of redistribution of K+ and SD-like events. We conclude that spreading K+ responses reflect powerful local K+ buffering mechanisms which are partially modulated by gap junctional communication. Gap junctional coupling mainly affected the velocity of the K+ and SD-like responses.

Mechanisms of Estradiol-induced EGF-like Factor Expression and Oocyte Maturation via G Protein-coupled Estrogen Receptor.

Estrogen is an important modulator of reproductive activity through nuclear receptors and G protein-coupled estrogen receptor (GPER). Here, we observed that both estradiol and the GPER-specific agonist G1 rapidly induced cyclic adenosine monophosphate (cAMP) production in cumulus cells, leading to transient stimulation of phosphorylated cAMP response element binding protein (CREB), which was conducive to the transcription of epidermal growth factor (EGF)-like factors, amphiregulin, epiregulin, and betacellulin. Inhibition of GPER by G15 significantly reduced estradiol-induced CREB phosphorylation and EGF-like factor gene expression. Consistently, the silencing of GPER expression in cultured cumulus cells abrogated the estradiol-induced CREB phosphorylation and EGF-like factor transcription. In addition, the increase in EGF-like factor expression in the cumulus cells is associated with EGF receptor (EFGR) tyrosine kinase phosphorylation and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. Furthermore, we demonstrated that GPER-mediated phosphorylation of EGFR and ERK1/2 was involved in reduced gap junction communication, cumulus expansion, increased oocyte mitochondrial activity and first polar body extrusion. Overall, our study identified a novel function for estrogen in regulating EGFR activation via GPER in cumulus cells during oocyte maturation.

Modifying gap junction communication in cancer therapy

AimDrug delivery is crucial for therapeutic efficacy and gap junction communication channels (GJIC) facilitate movement within the tumour. Pro-drug activation, a modality of cancer therapy leads to Ganciclovir triphosphate (GCV-TP) incorporation into newly synthesized DNA resulting in cell death. The objective was to enhance, with Histone deacetylase inhibitors (HDACi) and All Trans Retinoic Acid (ATRA), GJIC, crucial for drug delivery, and with combination, abrogate the observed detrimental effect of Dexamethasone (DXM). MethodsCell lines (NT8E, and HeLa) were pre-treated with Valproic Acid (VPA) (1 mM), 4 Phenyl Butyrate (4PB) (2 mM), ATRA (10 μM) and Dexamethasone (1 μM). Protein quantitated with the Bicinchoninic (BCA) assay for cell lysates, membrane and soluble fractions was assessed with Western blotting for Connexins (43, 26 and 32) and E-Cadherin. A qRT-PCR was done for CX 43-GJA1, CX 26-GJB2, CX 32-GJB1 and E-Cadherin, and normalized with Glyceraldehyde Phosphate dehydrogenase (GAPDH). Further, localization of Connexins (CX) and E-Cadherin, GJIC competence, pre-clinical in-vitro studies and the mechanism of cell death were evaluated. ResultsThere was no toxicity or change in growth patterns observed with the drugs. In both the cell lines CX 43 localized to the membrane whereas CX 32 and CX 26 were present but not membrane bound. E-Cadherin was present on the membrane in NT8E and completely absent in HeLa cells. Effects of HDACi, DXM and ATRA were seen on the expression of Connexins and E-Cadherin in both the cell lines. NT8E and HeLa cell lines showed enhanced GJIC with 4PB [30 %], VPA [36 %] and ATRA [54 %] with a 60 % increase in cytotoxicity and an abrogation of Dexamethasone inhibition on combination with VPA or ATRA. ConclusionAn enhancement of GJIC function by HDACi and ATRA increased cytotoxicity and could be effective in the presence of Dexamethasone, when combined with ATRA or VPA.

Metabolic co-dependence of the oocyte and cumulus cells: essential role in determining oocyte developmental competence.

Within the antral follicle, the oocyte is reliant on metabolic support from its surrounding somatic cells. Metabolism plays a critical role in oocyte developmental competence (oocyte quality). In the last decade, there has been significant progress in understanding the metabolism of the cumulus-oocyte complex (COC) during its final stages of growth and maturation in the follicle. Certain metabolic conditions (e.g. obesity) or ART (e.g. IVM) perturb COC metabolism, providing insights into metabolic regulation of oocyte quality. This review provides an update on the progress made in our understanding of COC metabolism, and the metabolic conditions that influence both meiotic and developmental competence of the oocyte. The PubMed database was used to search for peer-reviewed original and review articles. Searches were performed adopting the main terms 'oocyte metabolism', 'cumulus cell metabolism', 'oocyte maturation', 'oocyte mitochondria', 'oocyte metabolism', 'oocyte developmental competence' and 'oocyte IVM'. Metabolism is a major determinant of oocyte quality. Glucose is an essential requirement for both meiotic and cytoplasmic maturation of the COC. Glucose is the driver of cumulus cell metabolism and is essential for energy production, extracellular matrix formation and supply of pyruvate to the oocyte for ATP production. Mitochondria are the primary source of ATP production within the oocyte. Recent advances in real-time live cell imaging reveal dynamic fluctuations in ATP demand throughout oocyte maturation. Cumulus cells have been shown to play a central role in maintaining adequate oocyte ATP levels by providing metabolic support through gap junctional communication. New insights have highlighted the importance of oocyte lipid metabolism for oocyte oxidative phosphorylation for ATP production, meiotic progression and developmental competence. Within the last decade, several new strategies for improving the developmental competence of oocytes undergoing IVM have emerged, including modulation of cyclic nucleotides, the addition of precursors for the antioxidant glutathione or endogenous maturation mediators such as epidermal growth factor-like peptides and growth differentiation factor 9/bone morphogenetic protein 15. These IVM additives positively alter COC metabolic endpoints commonly associated with oocyte competence. There remain significant challenges in the study of COC metabolism. Owing to the paucity in non-invasive or in situ techniques to assess metabolism, most work to date has used in vitro or ex vivo models. Additionally, the difficulty of measuring oocyte and cumulus cell metabolism separately while still in a complex has led to the frequent use of denuded oocytes, the results from which should be interpreted with caution since the oocyte and cumulus cell compartments are metabolically interdependent, and oocytes do not naturally exist in a naked state until after fertilization. There are emerging tools, including live fluorescence imaging and photonics probes, which may provide ways to measure the dynamic nature of metabolism in a single oocyte, potentially while in situ. There is an association between oocyte metabolism and oocyte developmental competence. Advancing our understanding of basic cellular and biochemical mechanisms regulating oocyte metabolism may identify new avenues to augment oocyte quality and assess developmental potential in assisted reproduction.

A PKA/AKAP signaling complex is essential for regulation of ventricular cardiomyocytes Cx43 gap junction communication

In cardiomyocytes, β-adrenergic receptor (β-AR) stimulation activates protein kinase A (PKA), which increases electrical conduction velocity. Connexin-43 (Cx43) form gap junction (GJ) channels at the intercalated discs of cardiomyocytes and ensure electrical impulse propagation. This is essential for coordinated cardiac contractions. In myocardial infarction (MI), Cx43 is altered that affects GJ communication, electrical conduction leading to fatal arrhythmias. Regardless the cause, the β-AR system is activated after a MI. Considering the key role of Cx43, we investigated the functional regulation of GJ by PKA-anchored through A-Kinase Anchoring Protein (AKAP) in cardiac physiology and pathophysiology (MI). Neonatal rat ventricular cardiomyocytes were cultured with peptides that inhibit PKA or displace PKA anchoring from AKAP in basal condition or after β-AR stimulation. Cx43 phosphorylation level and GJ communication were assessed by immunoblot and GAP-Fluorescence Loss In Photobleaching experiments. A Cx43-PKA-AKAP signaling complex in cardiomyocytes and heart was investigated by immunofluorescence, immunoblot or immunoprecipitation. Proximity ligation assays were performed in cells and tissue in physiological conditions and after MI. Our experiments point to a PKA-scaffold protein required for PKA-mediated regulation of Cx43 phosphorylation and GJ communication. We show that the AKAP named ezrin forms a molecular complex with PKA and Cx43 in rat heart at the cellular and tissue levels. While PKA and ezrin expression levels remain constant, Cx43 showed a significant reduction after a MI. Interestingly, we noticed a decreased in Cx43 vicinity with ezrin at the ID of ventricular cardiomyocytes. These data suggest for a role of PKA-ezrin-Cx43 complex in the regulation of GJ communication in cardiac physiology. A deregulation of this complex could explain partly the altered electrical propagation observed after a MI.

Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9\u2009\xc5

Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid environment that is specific to the extracellular lipid leaflet. In addition, ~400 water molecules are resolved in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to the channel architecture. These results illustrate how the aqueous-lipid environment is integrated with the architectural stability, structure and function of gap junction communication channels.

High-mobility group box-1 induces mechanical pain hypersensitivity through astrocytic connexin 43 via the toll-like receptor-4/JNK signaling pathway.

The present study aimed to investigate the effects of high-mobility group box-1 (HMGB-1) on mechanical pain hypersensitivity and the underlying mechanism. Mouse primary astrocytes were isolated and treated as specified. A CCK-8 assay was used to determine cytotoxicity and a gap junctional communication assay was performed. Ethidium bromide (EtBr) uptake was used to evaluate the hemichannel activity of primary astrocytes. A mouse model of neuropathic pain was developed and paw withdrawal threshold was used to evaluate hind paw sensitivity. RT-qPCR and Western blot were used to determine mRNA and protein expression of genes, respectively. ELISA was used to measure the release of inflammatory cytokines. Treatment with HMGB-1 increased the expression of both toll-like receptor-4 (TLR-4) and connexin 43 (Cx43) in mouse primary astrocytes. HMGB-1 also promoted gap junctional intercellular communication and hemichannel function. Our results also demonstrated that HMGB-1-regulated Cx43 through the JNK signaling pathway, and Cx43 was involved in HMGB-1-mediated inflammation in astrocytes. In vivo analysis supported the idea that HMGB-1-induced mechanical hypersensitivity was associated with Cx43. We therefore conclude that HMGB-1-induced mechanical pain hypersensitivity occurs through modulating astrocytic Cx43 via the TLR-4/JNK signaling pathway.

Gap junction with MLO-A5 osteoblast-like cell line induces ALP and BSP transcription of 3T3-L1 pre-adipocyte like cell line via Hspb1 while retaining adipogenic differentiation ability.

In bone tissues, gap junctions form direct links between the cytoplasm of an osteocyte and another adjacent osteocyte or osteoblast, which underlie both bone formation and bone resorption. We have previously demonstrated that alkaline phosphatase (ALP) and bone sialoprotein (BSP), which are osteoblast markers, were induced in mesenchymal stem cells (MSCs) co-cultured with osteoblast-like cell line. However, the molecular mechanism of this process has not been fully addressed. Furthermore, few advances have been made toward elucidating the communication networks that link the status of committed cells such as (pre-) adipocytes that differentiated from MSCs as well as osteoblasts. Therefore, the objective of the present study was to investigate the mechanism underlying the communication network between pre-adipocytes and osteoblasts. We evaluated the effect of co-culture with osteoblast on the cell status of pre-adipocytes using murine osteoblast-like cell line, MLO-A5, and pre-adipocyte-like cell line, 3T3-L1, respectively. The results presented here demonstrated that osteoblasts and pre-adipocytes communicate via gap junctions, and the ensuing drastic increase in ALP and BSP transcription in co-cultured pre-adipocytes was induced, at least partly, via heat shock protein family B member 1 (Hspb1). In addition, terminal differentiation into adipocytes was suppressed in pre-adipocytes during co-culture with osteoblast without loss of adipogenic differentiation ability. Interestingly, after co-culture with osteoblasts, isolated co-cultured pre-adipocytes were able to differentiate to adipocytes as well as original pre-adipocytes. These results suggest that gap junctional communication with osteoblasts suppressed adipogenic differentiation of pre-adipocytes without loss of adipogenic differentiation ability.

Connexin Mutants Compromise the Lens Circulation and Cause Cataracts through Biomineralization.

Gap junction-mediated intercellular communication facilitates the circulation of ions, small molecules, and metabolites in the avascular eye lens. Mutants of the lens fiber cell gap junction proteins, connexin46 (Cx46) and connexin50 (Cx50), cause cataracts in people and in mice. Studies in mouse models have begun to elucidate the mechanisms by which these mutants lead to cataracts. The expression of the dominant mutants causes severe decreases in connexin levels, reducing the gap junctional communication between lens fiber cells and compromising the lens circulation. The impairment of the lens circulation results in several changes, including the accumulation of Ca2+ in central lens regions, leading to the formation of precipitates that stain with Alizarin red. The cataract morphology and the distribution of Alizarin red-stained material are similar, suggesting that the cataracts result from biomineralization within the organ. In this review, we suggest that this may be a general process for the formation of cataracts of different etiologies.

Abstract 5997: Increased connexin 43 expression and gap junction communication correlates with invasion following reduced glucose metabolism in breast cancer cells

Abstract Dysregulation of gap junction intercellular communication (GJIC) is a common feature during cancer progression. GJIC is a means of direct cell-cell communication mediated by regulated membrane channels composed of connexin proteins. This communication is frequently lost between primary tumor cells but may be upregulated at secondary metastatic sites with stromal cells. Control of this process by cancer cells has been shown to facilitate aggressive qualities both in vitro and in vivo. During the process of metastasis, cells encounter numerous metabolic challenges that must be overcome, particularly during growth of primary tumors. In this study, we set out to evaluate if changes to cancer cell metabolism affect GJIC in breast cancer cells. To address this question, we generated a metabolic variant of the MDA-MB-231 cell line conditioned to grow in glucose-limiting conditions. These cells were grown in FBS supplemented RPMI with &amp;lt;0.130mM glucose compared to 2mM in control conditions for more than 4 weeks. Substantial cell death over this time revealed a small population of cells capable of surviving in glucose reduced conditions. Growth of these cells normalized following a period of quiescence and exhibited stable viability and proliferative capacity. Following STR validation, these cells were designated MDA-MB-231LG for their ability to grow in low glucose media. MDA-MB-231LG exhibited a larger and more rounded morphologic appearance with formation of strong cell-cell contacts as demonstrated by scanning electron microscopy in contrast to parental cells which showed a higher degree of membrane overlap. Further comparison of the two cell lines through western blot and immunofluorescence analysis of connexin 43, a major connexin expressed in breast tissue, revealed higher levels in MDA-MB-231LG and increased membrane localization. Using a double label dye transfer technique, the gap junction permeable fluorescent dye calcein showed increased movement from CM-DiI labeled donor cells into neighboring cells in MDA-MB-231LG indicating functional gap junction coupling while MDA-MB-231 had little to no dye movement. To evaluate phenotypic qualities, both cell lines were grown in Matrigel and MDA-MB-231LG displayed increased stellate morphology. Use of a Matrigel invasion chamber assay confirmed the increased invasive qualities with significantly more MDA-MB-231LG invading through the lower portion of the membrane compared to MDA-MB-231 parental cells. Our data demonstrate a clear upregulation of gap junction activity following metabolic adaptation to reduced glucose availability. It also suggests a possible connection between GJIC and invasive qualities in breast cancer cells and may represent an inducible phenotype that occurs in primary tumors when tumor growth limits blood vessel penetration and nutrient availability. Citation Format: Jennifer C. Jones, Amanda M. Miceli, Mary M. Chaudhry, Mallika A. Jai, Romel N. Pancho, Alan Lazzar, Bradley S. Taylor, Vishnupriya Bodempudi, Prarthana P. Jain, Sheeri Hanjra, Alexander E. Urban, Brian Zanotti, Ellen K. Kohlmeir, Thomas M. Bodenstine. Increased connexin 43 expression and gap junction communication correlates with invasion following reduced glucose metabolism in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5997.

Cardiac β-adrenergic receptor activation mediates distinct and cell type-dependent changes in the expression and distribution of connexin 43.

Activation of the sympatho‐β‐adrenergic receptors (β‐ARs) system is a hallmark of heart failure, leading to fibrosis and arrhythmias. Connexin 43 (Cx43) is the most abundant gap junctional protein in the myocardium. Current knowledge is limited regarding Cx43 remodelling in diverse cell types in the diseased myocardium and the underlying mechanism. We studied cell type‐dependent changes in Cx43 remodelling due to β‐AR overactivation and molecular mechanisms involved. Mouse models of isoproterenol stimulation or transgenic cardiomyocyte overexpression of β2‐AR were used, which exhibited cardiac fibrosis and up‐regulated total Cx43 abundance. In both models, whereas Cx43 expression in cardiomyocytes was reduced and more laterally distributed, fibroblasts exhibited elevated Cx43 expression and enhanced gap junction communication. Mechanistically, activation of β2‐AR in fibroblasts in vitro elevated Cx43 expression, which was abolished by the β2‐antagonist ICI‐118551 or protein kinase A inhibitor H‐89, but simulated by the adenylyl cyclase activator forskolin. Our in vitro and in vivo data showed that β‐AR activation‐induced production of IL‐18 sequentially stimulated Cx43 expression in fibroblasts in a paracrine fashion. In summary, our findings demonstrate a pivotal role of β‐AR in mediating distinct and cell type‐dependent changes in the expression and distribution of Cx43, leading to pathological gap junction remodelling in the myocardium.

Soluble silica stimulates osteogenic differentiation and gap junction communication in human dental follicle cells

Several studies have indicated that dietary silicon (Si) is beneficial for bone homeostasis and skeletal health. Furthermore, Si-containing bioactive glass biomaterials have positive effects on bone regeneration when used for repair of bone defects. Si has been demonstrated to stimulate osteoblast differentiation and bone mineralisation in vitro. However, the mechanisms underlying these effects of Si are not well understood. The aim of the present study was to investigate the effects of soluble Si on osteogenic differentiation and connexin 43 (CX43) gap junction communication in cultured pluripotent cells from human dental follicles (hDFC). Neutral Red uptake assay demonstrated that 25 μg/ml of Si significantly stimulated hDFC cell proliferation. Dosages of Si above 100 μg/ml decreased cell proliferation. Alizarin Red staining showed that osteogenic induction medium (OIM) by itself and in combination with Si (25 μg/ml) significantly increased mineralisation in hDFC cultures, although Si alone had no such effect. The expression of osteoblast-related markers in hDFC was analysed with RT-qPCR. OSX, RUNX2, BMP2, ALP, OCN, BSP and CX43 genes were expressed in hDFC cultured for 1, 7, 14 and 21 days. Expression levels of BMP-2 and BSP were significantly upregulated by OIM and Si (25 μg/ml) and were also induced by Si alone. Notably, the expression levels of OCN and CX43 on Day 21 were significantly increased only in the Si group. Flow cytometric measurements revealed that Si (50 μg/ml) significantly increased CX43 protein expression and gap junction communication in hDFC. Next-generation sequencing (NGS) and bioinformatics processing were used for the identification of differentially regulated genes and pathways. The influence of OIM over the cell differentiation profile was more prominent than the influence of Si alone. However, Si in combination with OIM increased the magnitude of expression (up or down) of the differentially regulated genes. The gene for cartilage oligomeric matrix protein (COMP) was the most significantly upregulated. Genes for the regulator of G protein signalling 4 (RGS4), regulator of G protein signalling 2 (RGS2), and matrix metalloproteinases (MMPs) 1, 8, and 10 were also strongly upregulated. Our findings reveal that soluble Si stimulates Cx43 gap junction communication in hDFC and induces gene expression patterns associated with osteogenic differentiation. Taken together, the results support the conclusion that Si is beneficial for bone health.