The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes.

Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.

Preserving Endothelial Integrity in Human Saphenous Veins during Preparation for Coronary Bypass Surgery.

INTRODUCTION: While multiple factors influence coronary artery bypass graft (CABG) success rates, preserving saphenous vein endothelium during surgery may improve patency. Standard preparations include saphenous vein preparation in heparinized saline (saline) which can result in endothelial loss and damage. Here, we investigated the impact of preparing saphenous graft vessels in heparinized patient blood (blood) versus saline. METHODS: Saphenous vein tissues from a total of 23 patients undergoing CABG were split into 2 groups (1) saline and (2) heparinized patient blood. Excess tissue was fixed for analysis immediately following surgery. Level of endothelial coverage, oxidative stress marker 4-hydroxynonenal (4HNE), and oxidative stress protective marker nuclear factor erythroid 2-related factor 2 (NRF2) were evaluated. RESULTS: In saline patient veins, histological analysis revealed a limited luminal layer, suggesting a loss of endothelial cells (ECs). Immunofluorescent staining of EC markers vascular endothelial cadherin (VE-cadherin) and endothelial nitric oxide identified a significant improvement in EC coverage in the blood versus saline groups. Although both treatment groups expressed 4HNE to similar levels, EC blood samples expressed higher levels of NRF2. CONCLUSION: Our data indicate that use of heparinized patient blood helps preserve the endothelium and promotes vein graft health. This has the potential to improve long-term outcomes in patients.

Connexin 43 across the Vasculature: Gap Junctions and Beyond.

Connexin 43 (Cx43) is essential to the function of the vasculature. Cx43 proteins form gap junctions that allow for the exchange of ions and molecules between vascular cells to facilitate cell-to-cell signaling and coordinate vasomotor activity. Cx43 also has intracellular signaling functions that influence vascular cell proliferation and migration. Cx43 is expressed in all vascular cell types, although its expression and function vary by vessel size and location. This includes expression in vascular smooth muscle cells (vSMC), endothelial cells (EC), and pericytes. Cx43 is thought to coordinate homocellular signaling within EC and vSMC. Cx43 gap junctions also function as conduits between different cell types (heterocellular signaling), between EC and vSMC at the myoendothelial junction, and between pericyte and EC in capillaries. Alterations in Cx43 expression, localization, and post-translational modification have been identified in vascular disease states, including atherosclerosis, hypertension, and diabetes. In this review, we discuss the current understanding of Cx43 localization and function in healthy and diseased blood vessels across all vascular beds.

Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium.

The proinflammatory cytokine IL-1ß is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1ß expression and release are tightly controlled by changes in intracellular Ca2+ ([Ca2+]i), which has been associated with ATP release and purinergic signaling. Despite this, the mechanisms that regulate these changes have not been identified. The pannexin 1 (Panx1) channels have canonically been implicated in ATP release, especially during inflammation. We examined Panx1 in human umbilical vein endothelial cells following treatment with the proinflammatory cytokine TNF-α. Analysis by whole transcriptome sequencing and immunoblot identified a dramatic increase in Panx1 mRNA and protein expression that is regulated in an NF-κB-dependent manner. Furthermore, genetic inhibition of Panx1 reduced the expression and release of IL-1ß. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL-1ß expression was not altered after direct ATP stimulation in human umbilical vein endothelial cells. Because Panx1 forms a large pore channel, we hypothesized it may permit Ca2+ diffusion into the cell to regulate IL-1ß. High-throughput flow cytometric analysis demonstrated that TNF-α treatments lead to elevated [Ca2+]i, corresponding with Panx1 membrane localization. Genetic or pharmacological inhibition of Panx1 reduced TNF-α-associated increases in [Ca2+]i, blocked phosphorylation of the NF-κB-p65 protein, and reduced IL-1ß transcription. Taken together, the data in our study provide the first evidence, to our knowledge, that [Ca2+]i regulation via the Panx1 channel induces a feed-forward effect on NF-κB to regulate IL-1ß synthesis and release in endothelium during inflammation.

Local Peroxynitrite Impairs Endothelial Transient Receptor Potential Vanilloid 4 Channels and Elevates Blood Pressure in Obesity.

BACKGROUND: Impaired endothelium-dependent vasodilation is a hallmark of obesity-induced hypertension. The recognition that Ca2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca2+ signaling is compromised during obesity, but the underlying abnormality is unknown. In this regard, transient receptor potential vanilloid 4 (TRPV4) ion channels are a major Ca2+ influx pathway in endothelial cells, and regulatory protein AKAP150 (A-kinase anchoring protein 150) enhances the activity of TRPV4 channels. METHODS: We used endothelium-specific knockout mice and high-fat diet-fed mice to assess the role of endothelial AKAP150-TRPV4 signaling in blood pressure regulation under normal and obese conditions. We further determined the role of peroxynitrite, an oxidant molecule generated from the reaction between nitric oxide and superoxide radicals, in impairing endothelial AKAP150-TRPV4 signaling in obesity and assessed the effectiveness of peroxynitrite inhibition in rescuing endothelial AKAP150-TRPV4 signaling in obesity. The clinical relevance of our findings was evaluated in arteries from nonobese and obese individuals. RESULTS: We show that Ca2+ influx through TRPV4 channels at myoendothelial projections to smooth muscle cells decreases resting blood pressure in nonobese mice, a response that is diminished in obese mice. Counterintuitively, release of the vasodilator molecule nitric oxide attenuated endothelial TRPV4 channel activity and vasodilation in obese animals. Increased activities of inducible nitric oxide synthase and NADPH oxidase 1 enzymes at myoendothelial projections in obese mice generated higher levels of nitric oxide and superoxide radicals, resulting in increased local peroxynitrite formation and subsequent oxidation of the regulatory protein AKAP150 at cysteine 36, to impair AKAP150-TRPV4 channel signaling at myoendothelial projections. Strategies that lowered peroxynitrite levels prevented cysteine 36 oxidation of AKAP150 and rescued endothelial AKAP150-TRPV4 signaling, vasodilation, and blood pressure in obesity. Peroxynitrite-dependent impairment of endothelial TRPV4 channel activity and vasodilation was also observed in the arteries from obese patients. CONCLUSIONS: These data suggest that a spatially restricted impairment of endothelial TRPV4 channels contributes to obesity-induced hypertension and imply that inhibiting peroxynitrite might represent a strategy for normalizing endothelial TRPV4 channel activity, vasodilation, and blood pressure in obesity.

Mechanisms of Connexin Regulating Peptides.

Gap junctions (GJ) and connexins play integral roles in cellular physiology and have been found to be involved in multiple pathophysiological states from cancer to cardiovascular disease. Studies over the last 60 years have demonstrated the utility of altering GJ signaling pathways in experimental models, which has led to them being attractive targets for therapeutic intervention. A number of different mechanisms have been proposed to regulate GJ signaling, including channel blocking, enhancing channel open state, and disrupting protein-protein interactions. The primary mechanism for this has been through the design of numerous peptides as therapeutics, that are either currently in early development or are in various stages of clinical trials. Despite over 25 years of research into connexin targeting peptides, the overall mechanisms of action are still poorly understood. In this overview, we discuss published connexin targeting peptides, their reported mechanisms of action, and the potential for these molecules in the treatment of disease.

Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane.

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.

Pannexin 1 channels in renin-expressing cells influence renin secretion and blood pressure homeostasis.

Kidney function and blood pressure homeostasis are regulated by purinergic signaling mechanisms. These autocrine/paracrine signaling pathways are initiated by the release of cellular ATP, which influences kidney hemodynamics and steady-state renin secretion from juxtaglomerular cells. However, the mechanism responsible for ATP release that supports tonic inputs to juxtaglomerular cells and regulates renin secretion remains unclear. Pannexin 1 (Panx1) channels localize to both afferent arterioles and juxtaglomerular cells and provide a transmembrane conduit for ATP release and ion permeability in the kidney and the vasculature. We hypothesized that Panx1 channels in renin-expressing cells regulate renin secretion in vivo. Using a renin cell-specific Panx1 knockout model, we found that male Panx1 deficient mice exhibiting a heightened activation of the renin-angiotensin-aldosterone system have markedly increased plasma renin and aldosterone concentrations, and elevated mean arterial pressure with altered peripheral hemodynamics. Following ovariectomy, female mice mirrored the male phenotype. Furthermore, constitutive Panx1 channel activity was observed in As4.1 renin-secreting cells, whereby Panx1 knockdown reduced extracellular ATP accumulation, lowered basal intracellular calcium concentrations and recapitulated a hyper-secretory renin phenotype. Moreover, in response to stress stimuli that lower blood pressure, Panx1-deficient mice exhibited aberrant "renin recruitment" as evidenced by reactivation of renin expression in pre-glomerular arteriolar smooth muscle cells. Thus, renin-cell Panx1 channels suppress renin secretion and influence adaptive renin responses when blood pressure homeostasis is threatened.

Endothelial calreticulin deletion impairs endothelial function in aged mice.

Discrete calcium signals within the vascular endothelium decrease with age and contribute to impaired endothelial-dependent vasodilation. Calreticulin (Calr), a multifunctional calcium binding protein and endoplasmic reticulum (ER) chaperone, can mediate calcium signals and vascular function within the endothelial cells (ECs) of small resistance arteries. We found Calr protein expression significantly decreases with age in mesenteric arteries and examined the functional role of EC Calr in vasodilation and calcium mobilization in the context of aging. Third-order mesenteric arteries from mice with or without EC Calr knockdown were examined for calcium signals and constriction to phenylephrine (PE) or vasodilation to carbachol (CCh) after 75 wk of age. PE constriction in aged mice with or without EC Calr was unchanged. However, calcium signals and vasodilation to endothelial-dependent agonist carbachol were significantly impaired in aged EC Calr knockdown mice. Ex vivo incubation of arteries with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) significantly improved vasodilation in mice lacking EC Calr. Our data suggests diminished vascular Calr expression with age can contribute to the detrimental effects of aging on endothelial calcium regulation and vasodilation.NEW & NOTEWORTHY Calreticulin (Calr) is responsible for key physiological processes in endoplasmic reticulum, especially in aging tissue. In particular, endothelial Calr is crucial to vascular function. In this study, we deleted Calr from the endothelium and aged the mice up to 75 wk to examine changes in vascular function. We found two key differences: 1) calcium events in endothelium were severely diminished after muscarinic stimulation, which 2) corresponded with a dramatic decrease in muscarinic vasodilation. Remarkably, we were able to rescue the effect of Calr deletion on endothelial-dependent vasodilatory function using tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum stress that is currently in clinical trials.

Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

Pannexin1 as mediator of inflammation and cell death.

Pannexins form channels at the plasma membrane surface that establish a pathway for communication between the cytosol of individual cells and their extracellular environment. By doing so, pannexin signaling dictates several physiological functions, but equally underlies a number of pathological processes. Indeed, pannexin channels drive inflammation by assisting in the activation of inflammasomes, the release of pro-inflammatory cytokines, and the activation and migration of leukocytes. Furthermore, these cellular pores facilitate cell death, including apoptosis, pyroptosis and autophagy. The present paper reviews the roles of pannexin channels in inflammation and cell death. In a first part, a state-of-the-art overview of pannexin channel structure, regulation and function is provided. In a second part, the mechanisms behind their involvement in inflammation and cell death are discussed.

Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.

Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe(3+) state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe(2+) state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells.

The Connexin Mimetic Peptide Gap27 and Cx43-Knockdown Reveal Differential Roles for Connexin43 in Wound Closure Events in Skin Model Systems.

In the epidermis, remodelling of Connexin43 is a key event in wound closure. However, controversy between the role of connexin channel and non-channel functions exist. We compared the impact of SiRNA targeted to Connexin43 and the connexin mimetic peptide Gap27 on scrape wound closure rates and hemichannel signalling in adult keratinocytes (AK) and fibroblasts sourced from juvenile foreskin (JFF), human neonatal fibroblasts (HNDF) and adult dermal tissue (ADF). The impact of these agents, following 24 h exposure, on GJA1 (encoding Connexin43), Ki67 and TGF-ß1 gene expression, and Connexin43 and pSmad3 protein expression levels, were examined by qPCR and Western Blot respectively. In all cell types Gap27 (100-100 µM) attenuated hemichannel activity. In AK and JFF cells, Gap27 (100 nM-100 µM) enhanced scrape wound closure rates by ~50% but did not influence movement in HNDF or ADF cells. In both JF and AK cells, exposure to Gap27 for 24 h reduced the level of Cx43 protein expression but did not affect the level in ADF and HNDF cells. Connexin43-SiRNA enhanced scrape wound closure in all the cell types under investigation. In HDNF and ADF, Connexin43-SiRNA enhanced cell proliferation rates, with enhanced proliferation also observed following exposure of HDNF to Gap27. By contrast, in JFF and AK cells no changes in proliferation occurred. In JFF cells, Connexin43-SiRNA enhanced TGF-ß1 levels and in JFF and ADF cells both Connexin43-SiRNA and Gap27 enhanced pSmad3 protein expression levels. We conclude that Connexin43 signalling plays an important role in cell migration in keratinocytes and foreskin derived fibroblasts, however, different pathways are evoked and in dermal derived adult and neonatal fibroblasts, inhibition of Connexin43 signalling plays a more significant role in regulating cell proliferation than cell migration.

Regulation of cellular communication by signaling microdomains in the blood vessel wall.

It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function.

The connexin 43/ZO-1 complex regulates cerebral endothelial F-actin architecture and migration.

Endothelial cell migration is a fundamental process during angiogenesis and, therefore, a point of intervention for therapeutic strategies aimed at controlling pathologies involving blood vessel growth. We sought to determine the role of the gap junction protein connexin 43 (Cx43) in key features of angiogenesis in the central nervous system. We used an in vitro model to test the hypothesis that a complex of interacting proteins, including Cx43 and zonula occludens-1 (ZO-1), regulates the migratory behavior of cerebral endothelium. With knockdown and overexpression experiments, we demonstrate that the rate of healing following scrape-wounding of endothelium is regulated by the level of Cx43 protein expression. The effects on cell motility and proliferation were independent of gap junction communication as cells were sensitive to altered Cx43 expression in single plated cells. Coupling of Cx43/ZO-1 critically regulates this process as demonstrated with the use of a Cx43 α-carboxy terminus 1 peptide mimetic (αCT1) and overexpression of a mutant ZO-1 with the Cx43-binding PDZ2 domain deleted. Disrupting the Cx43/ZO-1 complex with these treatments resulted in collapse of the organized F-actin cytoskeleton and the appearance of actin nodes. Preincubation with the myosin 2 inhibitors blebbistatin or Y-27632 disrupted the Cx43/ZO-1 complex and inhibited cell spreading at the leading edge of migration. Cells studied individually in time-lapse open field locomotion assays wandered less when Cx43/ZO-1 interaction was disrupted without significant change in speed, suggesting that faster wound healing is a product of linearized migration. In contrast to the breakdown of F-actin architecture, microtubule architecture was not obviously affected by treatments. This study provides new insight into the fundamental regulatory mechanisms of cerebral endothelial cell locomotion. Cx43 tethers the F-actin cytoskeleton through a ZO-1 linker and supports cell spreading and exploration during locomotion. Here, we demonstrate that releasing this actin-coupled tether shifts the balance of directional migration control to a more linear movement that enhances the rate of wound healing.

ß-Adrenergic modulation of myocardial conduction velocity: Connexins vs. sodium current.

The heart is capable of rapid changes in cardiac output: these are caused in large part by changes in the activity of the autonomic nervous system that alter heart rate, force and time course of contraction. While studies of autonomic control have focussed on heart rate and contractile mechanisms, fewer studies have considered the influence of electrical propagation across the chamber. Conduction velocity (CV) of the action potential (AP) is an important variable, which ensures efficient pumping action of the heart and, along with AP duration, is a determinant of the electrical stability of the myocardium. CV depends on multiple factors, including tissue excitability and intercellular resistance: the latter is controlled by the number and arrangement of gap junctions (GJs) linking adjacent cardiac cells. Whole heart studies (in vivo and in vitro) report variable effects of sympathetic nervous system stimulation on ventricular CV, a major complication in interpretation being the accompanying increase in heart rate. At the cellular level, changes in cardiomyocyte electrophysiology, mediated via ß-adrenoreceptor (ß-AR) activation, alter the AP shape and amplitude but the influence of these effects on the CV is unclear. Alternatively, CV changes may occur via altered GJ conductance, but despite detailed knowledge of the underlying channel protein (connexin), little consensus exists on the extent and time course of the change in GJ conductance induced by AR activation. This review will examine the literature on the modulation of ventricular AP conduction velocity by ß-AR activation in a range of physiological preparations and highlight unresolved issues.

MAPK phosphorylation of connexin 43 promotes binding of cyclin E and smooth muscle cell proliferation.

RATIONALE: Dedifferentiation of vascular smooth muscle cells (VSMC) leading to a proliferative cell phenotype significantly contributes to the development of atherosclerosis. Mitogen-activated protein kinase (MAPK) phosphorylation of proteins including connexin 43 (Cx43) has been associated with VSMC proliferation in atherosclerosis. OBJECTIVE: To investigate whether MAPK phosphorylation of Cx43 is directly involved in VSMC proliferation. METHODS AND RESULTS: We show in vivo that MAPK-phosphorylated Cx43 forms complexes with the cell cycle control proteins cyclin E and cyclin-dependent kinase 2 (CDK2) in carotids of apolipoprotein-E receptor null (ApoE(-/-)) mice and in C57Bl/6 mice treated with platelet-derived growth factor-BB (PDGF). We tested the involvement of Cx43 MAPK phosphorylation in vitro using constructs for full-length Cx43 (Cx43) or the Cx43 C-terminus (Cx43(CT)) and produced null phosphorylation Ser>Ala (Cx43(MK4A)/Cx43(CTMK4A)) and phospho-mimetic Ser>Asp (Cx43(MK4D)/Cx43(CTMK4D)) mutations. Coimmunoprecipitation studies in primary VSMC isolated from Cx43 wild-type (Cx43(+/+)) and Cx43 null (Cx43(-/-)) mice and analytic size exclusion studies of purified proteins identify that interactions between cyclin E and Cx43 requires Cx43 MAPK phosphorylation. We further demonstrate that Cx43 MAPK phosphorylation is required for PDGF-mediated VSMC proliferation. Finally, using a novel knock-in mouse containing Cx43-MK4A mutation, we show in vivo that interactions between Cx43 and cyclin E are lost and VSMC proliferation does not occur after treatment of carotids with PDGF and that neointima formation is significantly reduced in carotids after injury. CONCLUSIONS: We identify MAPK-phosphorylated Cx43 as a novel interacting partner of cyclin E in VSMC and show that this interaction is critical for VSMC proliferation. This novel interaction may be important in the development of atherosclerotic lesions.

Pannexin-3 stabilizes the transcription factor Bcl6 in a channel-independent manner to protect against vascular oxidative stress.

Targeted degradation regulates the activity of the transcriptional repressor Bcl6 and its ability to suppress oxidative stress and inflammation. Here, we report that abundance of endothelial Bcl6 is determined by its interaction with Golgi-localized pannexin 3 (Panx3) and that Bcl6 transcriptional activity protects against vascular oxidative stress. Consistent with data from obese, hypertensive humans, mice with an endothelial cell-specific deficiency in Panx3 had spontaneous systemic hypertension without obvious changes in channel function, as assessed by Ca2+ handling, ATP amounts, or Golgi luminal pH. Panx3 bound to Bcl6, and its absence reduced Bcl6 protein abundance, suggesting that the interaction with Panx3 stabilized Bcl6 by preventing its degradation. Panx3 deficiency was associated with increased expression of the gene encoding the H2O2-producing enzyme Nox4, which is normally repressed by Bcl6, resulting in H2O2-induced oxidative damage in the vasculature. Catalase rescued impaired vasodilation in mice lacking endothelial Panx3. Administration of a newly developed peptide to inhibit the Panx3-Bcl6 interaction recapitulated the increase in Nox4 expression and in blood pressure seen in mice with endothelial Panx3 deficiency. Panx3-Bcl6-Nox4 dysregulation occurred in obesity-related hypertension, but not when hypertension was induced in the absence of obesity. Our findings provide insight into a channel-independent role of Panx3 wherein its interaction with Bcl6 determines vascular oxidative state, particularly under the adverse conditions of obesity.