Role Of Connexin43 Research Articles

Expression of connexin43 in oral submucous fibrosis, oral epithelial dysplasia and oral squamous cell carcinoma: A cross-sectional immunohistochemical study

Introduction: Connexins are specialized gap junction (GJ) proteins that are necessary for the maintenance of cellular homeostasis. The loss of GJ leads to the loss of cellular cohesion, promoting tumor progression. Not much is known about the role of connexin43 (Cx43) in oral submucous fibrosis (OSF), oral epithelial dysplasia (OED), and in OSF that had transformed into oral squamous cell carcinoma (OSCC). Materials and methods: A total of 39 tissue samples was grouped into group I: Normal oral mucosa (NC) (n = 6), group II: OSF (n = 14), group III: OED (n = 12), and group IV: OSCC with a history of OSF (n = 7). Sections were stained with Cx43 antibody and analyzed for staining intensity in the basal, suprabasal cell layers, and connective tissue. The Chi-square test was used to assess significant association and Kappa statistics between the variable and interobserver variability. Results: Cx43 exhibited varying levels of expression in the cell membrane of suprabasal cell layer across all the groups. The difference in expression was statistically significant (P = 0.033). In OSF, there was a moderate presence of Cx43, whereas NC, dysplasia, and OSCC with OSF showed no detectable Cx43 expression. Notably, OSF displayed higher Cx43 expression compared to OED. Conversely, OSCC with OSF demonstrated reduced expression of Cx43 in epithelial tumor islands that had invaded the connective tissue (P = 0.033). Conclusion: Cx43 expression was significantly decreased as there was a progression in OED severity and reduced in OSCC with a history of OSF.

Connexin 43 regulates astrocyte dysfunction and cognitive deficits in early life stress-treated mice.

Early life stress such as maternal separation (MS), is a major risk factor for developing psychiatric disorders in adulthood. Connexin 43 (CX43), the main type of connexins expressed in astrocytes, has been indicated to participate in depression disorders. Nevertheless, the role of CX43 in MS-induced cognitive impairment and astrocyte dysfunction is unclear. Neonatal C57BL/6 mice were exposed to MS to mimic early life stress. Adeno-associated virus carrying CX43 was inoculated into mice for CX43 overexpression. Sucrose preference test, forced swim test and Morris water maze were performed for evaluating depression-like behaviors and spatial learning and memory of mice in adulthood. Real time quantitative polymerase chain reaction was conducted to detect CX43 mRNA expression in mouse brain. Immunofluorescence staining and western blotting were used for measuring expression levels of astrocytic markers in murine hippocampal dentate gyrus. The results showed that overexpressing CX43 attenuated MS exposure-induced depression-like behaviors and decrease in spatial learning and memory in mice. Upregulating CX43 alleviated MS exposure-induced downregulation of astrocytic markers. Collectively, CX43 overexpression attenuates cognitive deficits and astrocyte dysfunction in mice exposed to MS.

Epidemiological Characteristics and Role of Connexin-43 in Patients of Syndactyly Attending a Tertiary Care Center in Odisha, India.

Syndactyly is one of the most common hereditary limb malformations. Yet, epidemiological data in our state is not reported. The current study aims to understand the Connexin-43 expression in such patients. A retrospective cohort study was done in the Department of Plastic surgery and Anatomy of All India Institute of Medical Sciences (AIIMS), Bhubaneswar. The study duration was three years, between 2019 and 2022. The total number of cases was 49 and included patients diagnosed with Syndactyly seeking surgical intervention. The demographic details and the personal and disease history were collected, analyzed, and interpreted. Immunohistochemistry study using Connexin-43. Out of the 49 patients, 26 (53.1%) were male, and 23 (46.9%) were female. Thirty nine (79.6%) had syndactyly, and 10 (20.4%) were diagnosed with syndactyly associated with another syndrome. Both complete and incomplete syndactyly was found. Strong membranous positivity of Connexin-43was found in the keratinocytes of the stratum spinosum layer of the epidermis, while the stratum granulosum and stratum basale layer revealed negative staining. Syndactyly is mostly non-familial, sporadic with male preponderance affecting unilaterally and in incomplete form in our geographical location. We found an overt expression of Connexin-43 in these patients' stratum basale.

Abstract P449: Connexin43 As A Therapeutic For Arrhythmogenic Right Ventricular Cardiomyopathy

Limited efforts have been focused on the interventions which could therapeutically alter arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C), a fatal cardiac disease of the desmosomal (mechanical) cell-cell junction. The desmosome is a critical target for intervention as mutations in desmosomal genes underlie 40-50% of ARVD/C populations and its dysregulation is associated with severe cardiac electrical and structural alterations, which facilitate myocardial failure, arrhythmias and premature death in these populations. Cardiomyocyte reduction of the predominant ventricular gap junction protein connexin43 is a molecular alteration that underlies desmosomal deficits and arrhythmias in ARVD/C. However, the role of connexin43 in structural alterations associated with ARVD/C remains unclear. We intervened with connexin43 reduction in human and mouse models of ARVD/C via connexin43 restoration strategies, which revealed beneficial effects in both ARVD/C models. We show ARVD/C human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes that recapitulate desmosomal structural defects and reveal connexin43 diminution alterations that are reflective of disease found in donor ARVD/C hearts. Connexin43 restoration was sufficient to rescue cardiac physiological deficits and increase desmosomal gene expressions in ARVD/C hiPSC derived cardiomyocytes, encompassing structural alterations. In vivo studies exploiting a mouse model of ARVD/C harboring severe desmosomal structural alterations revealed that cardiac connexin43 restoration was sufficient to prolong lifespan and restore cardiac desmosomal proteins. Herein, we provide evidence for non-canonical functions for connexin43, classically associated with electrical function, in the mechanical modulation of junctions. Our findings have broad implications in exploiting connexin43 as a therapeutic in advanced diseases associated with cardiac structural defects.

The role of connexin43 in neuropathic pain induced by spinal cord injury.

Neuropathic pain is caused by the damage or dysfunction of the nervous system. In many neuropathic pain models, there is an increase in the number of gap junction (GJ) channels, especially the upregulation of the expression of connexin43 (Cx43), leading to the secretion of various types of cytokines and involvement in the formation of neuropathic pain. GJs are widely distributed in mammalian organs and tissues, and Cx43 is the most abundant connexin (Cx) in mammals. Astrocytes are the most abundant glial cell type in the central nervous system (CNS), which mainly express Cx43. More importantly, GJs play an important role in regulating cell metabolism, signaling, and function. Many existing literatures showed that Cx43 plays an important role in the nervous system, especially in the CNS under normal and pathological conditions. However, many internal mechanisms have not yet been thoroughly explored. In this review, we summarized the current understanding of the role and association of Cx and pannexin channels in neuropathic pain, especially after spinal cord injury, as well as some of our own insights and thoughts which suggest that Cx43 may become an emerging therapeutic target for future neuropathic pain, bringing new hope to patients.

The Role of Connexin-43 in the Inflammatory Process: A New Potential Therapy to Influence Keratitis.

The studies outlined in this review highlight the relationship between inflammatory signaling molecules and connexin-43 (Cx43). Gap junction (GJ) channels and hemichannels (HCs) participate in the metabolic activity between intra- and extracellular space. Some ions and small molecules are exchanged from cell to cell or cell to extracellular space to affect the process of inflammation via GJ. We analyzed the effects of signaling molecules, such as innate immunity messengers, transcription factors, LPS, cytokine, inflammatory chemokines, and MMPs, on Cx43 expression during the inflammatory process. At the same time, we found that these signaling molecules play a critical role in the pathogenesis of keratitis. Thus, we assessed the function of Cx43 during inflammatory corneal disease. Corneal healing plays an essential role in the late stage of keratitis. We found that Cx43 is involved in wound healing. Studies have shown that the decrease of Cx43 can decrease the time of healing. We also report several Cx43 mimic peptides which can inhibit the activity of Cx43 Hc to mediate the releasing of adenosine triphosphate (ATP), which may in turn influence the inflammatory process.

Fast Generation of Functional Subtype Astrocytes from Human Pluripotent Stem Cells.

SummaryDifferentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4–7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.

Abstract 1090: The role of connexin43 in prostate cancer motility

Abstract Prostate cancer (PC) is one of the most common male cancers in United States. PC lethality is due to metastatic growth, when cancer cells move from the primary tumor site to a secondary site. A key step in metastsis is the acquisition of motility. Connexins (Cx) play a major role in cell-cell communication and growing evidence demonstrates their contribution to cell motility. Cx43 is up-regulated in cultured PC3 metastatic prostate cancer cells. To understand the role of Cx43 in migration we asked whether silencing Cx43 would decrease PC3 cell migration. Using cells in which short interfering RNA (sh-RNA) silenced Cx43 mRNA, we showed that silencing Cx43 decreased motility of sh-Cx43 PC3 cells in Boyden chamber assays. To confirm these results in another biological system, we performed cellular wound healing assays. Silencing Cx43 decreased motility of PC3 cells reducing movement to wounded area, leaving more area open in the Cx43 inhibited cell lines. The wound healing assay showed that knockdown Cx43 altered directional movement, velocity and distance. These results supported those of our Boyden assays showing that loss of Cx43 decreases the migratory ability of PC3. Conversely, the overexpression of Cx43 in LNCaP cells, non-migratory prostate cancer cells that lack Cx43 expression, increased their motility. A comparison of cell lines engineered to overexpress or to silence Cx43 confirms the importance of Cx43 in the motility of prostate cancer cells in vitro. Although the mechanism whereby Cx43 regulates motility is still unclear, our preliminary data revealed that Cx43 co-immunoprecipitated with N-cadherin, suggesting that they form a complex in PC3 cells. Cell fractionation studies of PC3 control cells showed that N-cadherin was located in the cell membrane and not detected free in the cytoplasm, but in sh-Cx43 PC3 cells that lack Cx43, the distribution of N-cadherin protein was altered and significant levels of N-cadherin were also detected in cytoplasmic fractions. Whether changes in N-cadherin distribution is a direct or indirect result of loss of Cx43 in sh-Cx43 PC3 cells, is unclear. However, modulation of Cx43 levels in prostate cancer cells does alter motility and N-cadherin distribution. These results indicate that Cx43 and N-cadherin are in close association in PC3 cells and their interaction may play an important role in prostate cancer cell motility. A better understanding of the requirements of migratory PC cells may lead to more effective targeting of key steps in PC progression to lethal metastatic disease. Citation Format: Abdulaziz A. Aloliqi. The role of connexin43 in prostate cancer motility [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 1090.

CX43 is essential for optimal cGAS function during cytosolic DNA-sensing

Abstract During intracellular Chlamydia infection, cytosolic DNA is sensed by cyclic GMP-AMP synthase (cGAS) that catalyzes the synthesis of cGAMP from ATP and GTP. cGAMP is a key mediator of IFN beta expression during DNA sensing and can also transfer to adjacent uninfected cells through intercellular connexin gap-junctions to induce intrinsic immunity. In this study, we investigated the role of gap junction protein connexin 43 (CX43) during Chlamydia infection. Using RNA-in situ hybridization, we discovered a distinct intracellular contribution of CX43 in IFN beta expression in the infected cells and cells transfected with DNA, a function distinct from its intercellular gap-junction role affecting IFN beta expression in adjacent uninfected cell. CX43 is essential for IFN beta expression during DNA-sensing upstream of STING activation, but not required during RNA-sensing. CX43 co-localized with transfected DNA and on the chlamydial inclusion membrane with cGAS. CX43-depleted cells showed significantly reduced cGAMP production during DNA-sensing. CX43 forms hemichannels on intracellular membrane, which open and close to allow nucleotide transfer. Blocking this transport across CX43 hemichannel reduced IFN beta expression during infection and DNA-sensing, without altering CX43 localization. These results uncover a novel role of CX43 in cGAMP synthesis by cGAS during cytosolic DNA-sensing.

The role of connexin43 in hemorrhagic transformation after thrombolysis in vivo and in vitro

Thrombolysis with recombinant tissue plasminogen activator (rtPA) is the most effective drug treatment for acute ischemic stroke within 4.5h after symptom onset. However, the use of rtPA may increase the risk of hemorrhagic transformation (HT), particularly when it is administered after the first 4.5h. However, no effective treatments are available to reduce the HT risk. Disruption of the blood–brain barrier (BBB) is central to the genesis of HT. Connexin43 (Cx43)-mediated gap junction intercellular communication (GJIC) has been demonstrated to regulate the integrity of the BBB in ischemia. We investigated the effect of Cx43 on BBB permeability during rtPA-induced HT. Spontaneously hypertensive rats (SHRs) underwent a 1.5-h middle cerebral artery occlusion and were treated with rtPA at 4.5h. The rats were sacrificed at 24h, and their brains were evaluated for BBB permeability and the expression of tight junction (TJ) proteins and Cx43. We examined whether the effects were Cx43 dependent using multiple Cx43 inhibitors. Phosphorylated Cx43 (p-Cx43) but not total Cx43 protein expression was increased after rtPA treatment. Delayed rtPA administration induced significant HT and BBB disruption. These effects were attenuated by inhibitors that blocked GJIC and Cx43 phosphorylation and expression but not Cx43 redistribution. Additionally, rtPA administration upregulated p-Cx43 expression in hypoxia/reoxygenation (H/R)-exposed brain endothelial cells. These effects were suppressed by the phosphatidylinositol 3′-kinase (PI3K) inhibitor LY294002 and the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor U0126. We suggest that rtPA-associated hemorrhage due to an alteration in the integrity of the BBB is highly associated with an increase in p-Cx43 resulting from the activation of the PI3K and ERK pathways.

Expression of Cx43 and Pax3 proteins in the human placental villi and decidua during early pregnancy.

To study the expression and roles of Connexin43 (Cx43) and Paired-box3 (Pax3) proteins in the human Chorionic villi and decidua during early pregnancy, Cx43 and Pax3 protein expression in the human placental villi and decidua at 5-7 weeks of early pregnancy was studied by immunohistochemistry (SABC). Integrated optical density (IOD) of Cx43 and Pax3 protein expression was obtained by using special image analysis software. Cx43 and Pax3 proteins were expressed in all trophoblastic cells, cells of villi central axis and decidual cells. IOD of Cx43 and Pax3 proteins expressed in the syncytiotrophoblast decreased from 5 to 7 weeks, while Cx43 and Pax3 expressed in cytotrophoblast cells and decidual cells showed increased IOD during the same period. The differences between any two groups were statistically significant (P<0.05). Cx43 and Pax3 proteins were expressed during differentiation and development of placental villous cells in early phases of pregnancy. We propose that Cx43 and Pax3 may participate in endometrial decidualization and the regulation of trophoblastic invasion and differentiation. It is likely that Cx43 and Pax3 play critical roles in cell proliferation and differentiation during the processes of chorionic villi initiation/development and placental morphogenesis.

Shifting Paradigms on the Role of Connexin43 in the Skeletal Response to Mechanical Load

Gap junctions (GJs) are membrane-spanning channels that allow for the movement of small molecules across cell membranes. Connexin43 (Cx43) is the predominant GJ protein in bone. In vitro studies suggest that gap junctional intercellular communication (GJIC) sensitizes bone cells to mechanical signals. Additionally, mechanical signals detected by osteocytes are communicated to osteoblasts via GJIC, and osteocytic Cx43 hemichannels release anabolic factors, such as PGE2 and ATP, in response to mechanical load. These findings and others have led to near consensus among researchers in the field that GJIC, hemichannels or connexins facilitate the anabolic response of bone to mechanical load and, in their absence, bone would be less sensitive to load. However, recent in vivo evidence suggests the opposite is true. Studies from our laboratory and others demonstrate that Cx43-deficient mice have an increased anabolic response to mechanical load and are protected against the catabolic effects of mechanical unloading. These developments suggest a paradigm shift in our understanding of connexins, GJIC, and mechanotransduction in bone. That is, inhibiting bone cell Cx43 expression or GJIC has a beneficial effect on bone's response to its mechanical environment, preserving bone during unloading and enhancing its formation during loading. Here, we review literature in support of this hypothesis and suggest a mechanism by which Cx43, through interaction with WNT/β-catenin signaling, moderates both arms of bone remodeling.

Proteomic and metabolomic responses to connexin43 silencing in primary hepatocyte cultures

Freshly established cultures of primary hepatocytes progressively adopt a foetal-like phenotype and display increased production of connexin43. The latter is a multifaceted cellular entity with variable subcellular locations, including the mitochondrial compartment. Cx43 forms hemichannels and gap junctions that are involved in a plethora of physiological and pathological processes, such as apoptosis. The present study was conducted with the goal of shedding more light onto the role of connexin43 in primary hepatocyte cultures. Connexin43 expression was suppressed by means of RNA interference technology, and the overall outcome of this treatment on the hepatocellular proteome and metabolome was investigated using tandem mass tag-based differential protein profiling and (1)H NMR spectroscopy, respectively. Global protein profiling revealed a number of targets of the connexin43 knock-down procedure, including mitochondrial proteins (heat shock protein 60, glucose-regulated protein 75, thiosulphate sulphurtransferase and adenosine triphosphate synthase) and detoxifying enzymes (glutathione S-transferase μ 2 and cytochrome P450 2C70). At the metabolomic level, connexin43 silencing caused no overt changes, though there was some evidence for a subtle increase in intracellular glycine quantities. Collectively, these data could further substantiate the established existence of a mitochondrial connexin pool and could be reconciled with the previously reported involvement of connexin43 signalling in spontaneously occurring apoptosis in primary hepatocyte cultures.

Role of connexin-43 in protective PI3K-Akt-GSK-3β signaling in cardiomyocytes

Sarcolemmal connexin-43 (Cx43) and mitochondrial Cx43 play distinct roles: formation of gap junctions and production of reactive oxygen species (ROS) for redox signaling. In this study, we examined the hypothesis that Cx43 contributes to activation of a major cytoprotective signal pathway, phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3β (GSK-3β) signaling, in cardiomyocytes. A δ-opioid receptor agonist {[d-Ala(2),d-Leu(5)]enkephalin acetate (DADLE)}, endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) induced phosphorylation of Akt and GSK-3β in H9c2 cardiomyocytes. Reduction of Cx43 protein to 20% of the normal level by Cx43 small interfering RNA abolished phosphorylation of Akt and GSK-3β induced by DADLE or ET-1 but not that induced by IGF-1. DADLE and IGF-1 protected H9c2 cells from necrosis after treatment with H(2)O(2) or antimycin A. The protection by DADLE or ET-1, but not that by IGF-1, was lost by reduction of Cx43 protein expression. In contrast to Akt and GSK-3β, PKC-ε, ERK and p38 mitogen-activated protein kinase were phosphorylated by ET-1 in Cx43-knocked-down cells. Like diazoxide, an activator of the mitochondrial ATP-sensitive K(+) channel, DADLE and ET-1 induced significant ROS production in mitochondria, although such an effect was not observed for IGF-1. Cx43 knockdown did not attenuate the mitochondrial ROS production by DADLE or ET-1. Cx43 was coimmunoprecipitated with the β-subunit of G protein (Gβ), and knockdown of Gβ mimicked the effect of Cx43 knockdown on ET-1-induced phosphorylation of Akt and GSK-3β. These results suggest that Cx43 contributes to activation of class I(B) PI3K in PI3K-Akt-GSK-3β signaling possibly as a cofactor of Gβ in cardiomyocytes.

Altered expression of connexin43 and its possible role in endothelin - 1 - induced contraction in rabbit basilar artery

Objective To investigate the role of connexin43 in ET- 1 -induced contraction in rabbit basilar artery.Methods The ET - 1 - induced contraction without or with carbenoxolone was studied with an isometric tension system.The expression of connexin43 protein in ET - t stimulated basilar arteries was studied with Western blot.Scrape/scratch method was used to analyze the function of gap junction in cultured rabbit cerebrovascular smooth muscle ceils.Results ET - 1 produced a concentration - dependent contraction.Carbenoxolone inhibited ET- 1 induced contraction.The connexin43 protein level was increased in ET- 1stimulated basilar arteries.Carbenoxolone decreased the connexin43 protein level increased by ET - 1.Cells treated with ET - 1 appeared positive communication and the dye transfer was increased in a time - dependent fashion.Carbenoxolone suppressed the ET - 1 - induced increasement of dye transfer.Conclusions The enhancement of gap junction intercellular communication is activated by ET - 1 via modulating the expression of connexin43,and plays an important role in the pathogenesis of cerebral vasospasm.Inhibition of vascular GJIC as a means of reducing cerebral vasospasm after SAH may have therapeutic advantage. Key words: Connexin43 ; Gap junction ; ET - 1 ; Cerebral vasospasm

Role of connexin43 and ATP in long-range bystander radiation damage and oncogenesis in vivo

Ionizing radiation is a genotoxic agent and human carcinogen. Recent work has questioned long-held dogmas by showing that cancer-associated genetic alterations occur in cells and tissues not directly exposed to radiation, questioning the robustness of the current system of radiation risk assessment. In vitro, diverse mechanisms involving secreted soluble factors, gap junction intercellular communication (GJIC) and oxidative metabolism are proposed to mediate these indirect effects. In vivo, the mechanisms behind long-range 'bystander' responses remain largely unknown. Here, we investigate the role of GJIC in propagating radiation stress signals in vivo, and in mediating radiation-associated bystander tumorigenesis in mouse central nervous system using a mouse model in which intercellular communication is downregulated by targeted deletion of the connexin43 (Cx43) gene. We show that GJIC is critical for transmission of oncogenic radiation damage to the non-targeted cerebellum, and that a mechanism involving adenosine triphosphate release and upregulation of Cx43, the major GJIC constituent, regulates transduction of oncogenic damage to unirradiated tissues in vivo. Our data provide a novel hypothesis for transduction of distant bystander effects and suggest that the highly branched nervous system, similar to the vascular network, has an important role.

Effect of pravastatin on ventricular arrhythmias in infarcted rats: role of connexin43

Epidemiologic studies showed that men treated with statins appear to have a lower incidence of sudden death than men without statins. However, the specific factor for this remained disappointingly elusive. We assessed whether pravastatin enhanced connexin43 expression after myocardial infarction through attenuation of endothelin-1. Twenty-four hours after ligation of the anterior descending artery, male Wistar rats were randomized to vehicle, pravastatin, mevalonate, bosentan, or a combination of pravastatin and mevalonate or pravastatin and bosentan for 4 wk. Myocardial endothelin-1 levels were significantly elevated in vehicle-treated rats at the border zone compared with sham-operated rats. Myocardial connexin43 expression at the border zone was significantly decreased in vehicle-treated infarcted rats compared with sham-operated rats. Attenuated connexin43 expression was blunted after administration of pravastatin, as assessed by immunofluorescence analysis, Western blotting, and real-time quantitative RT-PCR of connexin43. Bosentan enhanced connexin43 amount in infarcted rats and did not have additional beneficial effects on pravastatin-treated rats. Arrhythmic scores during programmed stimulation in vehicle-treated rats were significantly higher than scores in those treated with pravastatin. In contrast, the beneficial effects of pravastatin-induced connexin43 were abolished by the addition of mevalonate and a protein kinase C inducer. In addition, the amount of connexin43 showed significant increase after addition of bisindolylmaleimide, implicating that protein kinase C is a relevant target in endothelin-1-mediated connexin43 expression. Thus chronic use of pravastatin after infarction, resulting in enhanced connexin43 amount by attenuation of mevalonate-dependent endothelin-1 through a protein kinase C-dependent pathway, may attenuate the arrhythmogenic response to programmed electrical stimulation.

Gap Junctions at the Dendritic Cell-T Cell Interface Are Key Elements for Antigen-Dependent T Cell Activation

The acquired immune response begins with Ag presentation by dendritic cells (DCs) to naive T cells in a heterocellular cell-cell contact-dependent process. Although both DCs and T cells are known to express connexin43, a gap junction protein subunit, the role of connexin43 on the initiation of T cell responses remains to be elucidated. In the present work, we report the formation of gap junctions between DCs and T cells and their role on T cell activation during Ag presentation by DCs. In cocultures of DCs and T cells, Lucifer yellow microinjected into DCs is transferred to adjacent transgenic CD4(+) T cells, only if the specific antigenic peptide was present at least during the first 24 h of cocultures. This dye transfer was sensitive to gap junction blockers, such as oleamide, and small peptides containing the extracellular loop sequences of conexin. Furthermore, in this system, gap junction blockers drastically reduced T cell activation as reflected by lower proliferation, CD69 expression, and IL-2 secretion. This lower T cell activation produced by gap junction blockers was not due to a lower expression of CD80, CD86, CD40, and MHC-II on DCs. Furthermore, gap junction blocker did not affect polyclonal activation of T cell induced with anti-CD3 plus anti-CD28 Abs in the absence of DCs. These results strongly suggest that functional gap junctions assemble at the interface between DCs and T cells during Ag presentation and that they play an essential role in T cell activation.

Altered expression of connexin43 and its possible role in endothelin-1-induced contraction in rabbit basilar artery

Background and purpose: Endothelin-1 (ET-1) plays an important role in the pathogenesis of cerebral vasospasm. Gap junction participates in the pathologic processes of cerebrovascular diseases and may play an important role. The aim of this study is to investigate the role of connexin43 in ET-1-induced contraction in rabbit basilar artery.Methods: The ET-1-induced contraction without or with carbenoxolone was studied with an isometric tension system. Expression of connexin43 protein in ET-1–stimulated basilar arteries was studied with Western blot. Scrape/scratch method was used to analyse the function of gap junction in cultured rabbit cerebrovascular smooth muscle cells.Results: (1) ET-1 produced a concentration-dependent contraction. Carbenoxolone inhibited ET-1-induced contraction; (2) connexin43 protein level is increased in ET-1-stimulated basilar arteries. Carbenoxolone decreases the connexin43 protein level increased by ET-1; (3) cells treated with ET-1 appeared positive communicate and the dye transfer was increased in a time-dependent fashion; 4 carbenoxolone suppressed the ET-1-induced increases in dye transfer.Conclusion: The enhancement of gap junction intercellular communication is activated by ET-1 via modulating the expression of connexin43, and plays an important role in the pathogenesis of cerebral vasospasm. Inhibiting vascular GJIC as a means of reducing cerebral vasospasm after SAH may be of therapeutic advantage.

A Role for Connexin43 in Macrophage Phagocytosis and Host Survival after Bacterial Peritoneal Infection

The pathways that lead to the internalization of pathogens via phagocytosis remain incompletely understood. We now demonstrate a previously unrecognized role for the gap junction protein connexin43 (Cx43) in the regulation of phagocytosis by macrophages and in the host response to bacterial infection of the peritoneal cavity. Primary and cultured macrophages were found to express Cx43, which localized to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation. Remarkably, mortality was significantly increased in a mouse model of bacterial peritonitis after Cx43 inhibition and in Cx43 heterozygous mice compared with untreated and wild-type counterparts. These findings reveal a novel role for Cx43 in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and they implicate Cx43 in the regulation of the host response to microbial infection.