KLF5 promotes the ossification process of ligamentum flavum by transcriptionally activating CX43.

BACKGROUND: Ossification of ligamentum flavum (OLF) is a prevalent degenerative spinal disease, typically causing severe neurological dysfunction. Kruppel-like factor 5 (KLF5) plays an essential role in the regulation of skeletal development. However, the mechanism KLF5 plays in OLF remains unclear, necessitating further investigative studies. METHODS: qRT-PCR, immunofluorescent staining and western blot were used to measure the expression of KLF5. Alkaline Phosphatase (ALP) staining, Alizarin red staining (ARS), and the expression of Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and osteocalcin (OCN) were used to evaluate the osteogenic differentiation. Luciferase activity assay and ChIP-PCR were performed to investigate the molecular mechanisms. RESULTS: KLF5 was significantly upregulated in OLF fibroblasts in contrast to normal ligamentum flavum (LF) fibroblasts. Silencing KLF5 diminished osteogenic markers and mineralized nodules, while its overexpression had the opposite effect, confirming KLF5's role in promoting ossification. Moreover, KLF5 promotes the ossification of LF by activating the transcription of Connexin 43 (CX43), and overexpressing CX43 could reverse the suppressive impact of KLF5 knockdown on OLF fibroblasts' osteogenesis. CONCLUSION: KLF5 promotes the OLF by transcriptionally activating CX43. This finding contributes significantly to our understanding of OLF and may provide new therapeutic targets.

Semaglutide attenuates pathological electrophysiological remodeling in diabetic cardiomyopathy via restoring Cx43 expression.

BACKGROUND: Semaglutide is a relatively new anti-hyperglycemic agent that was shown to carry cardioprotective potentials. However, the exact effects of semaglutide on diabetic cardiomyopathy (DCM) and their underlining mechanism remain unclear. This study aimed to evaluate the effects of semaglutide on myocardium injury and cardiac function in DCM mice and its potential mechanisms, with emphasis on its effects on Cx43 and electrophysiological remodeling. METHODS: C57BL/6 mice were randomly divided into four groups: control group, semaglutide group, diabetes group, and diabetes + semaglutide treatment group. Type 1 diabetes were induced by intraperitoneal injection of streptozotocin. Mice in the semaglutide intervention group were injected subcutaneously with semaglutide (0.15 mg/kg) every week for 8 weeks. The blood glucose, cardiac function, oxidative stress markers, apoptosis, expression of Sirt1, AMPK, Cx43, and electrocardiogram of mice in each group were evaluated. RESULTS: Treatment with semaglutide alleviated glucose metabolism disorders and improved cardiac dysfunction in diabetic mice. In addition, semaglutide ameliorated the increase in oxidative stress and apoptosis in diabetic heart. Sirt1/AMPK pathway was activated after semaglutide treatment. Furthermore, diabetic mice showed reduced expression of Cx43 in the myocardium, accompanied by changes in electrocardiogram, including significantly prolonged RR, QRS, QT and QTc interval. Semaglutide treatment restored Cx43 expression and reversed the above-mentioned ECG abnormalities. CONCLUSIONS: Our research results showed that semaglutide protected against oxidative stress and apoptosis in diabetic heart, thereby improving cardiac function and electrophysiological remodelling in DCM mice, which may attribute to activation of Sirt1/AMPK pathway and restore of Cx43 expression.

Involvement of oxidative stress-AMPK-Cx43-NLRP3 pathway in extracellular matrix remodeling of gastric smooth muscle cells in rats with diabetic gastroparesis.

This study aimed to investigate the changes in oxidative stress, adenosine monophosphate-activated protein kinase (AMPK), connexin43 (Cx43), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) expression, and extracellular matrix (ECM) in the gastric smooth muscle tissues of rats with diabetic gastroparesis (DGP) and high glucose-cultured gastric smooth muscle cells, determine the existence of oxidative stress-AMPK-Cx43-NLRP3 pathway under high glucose condition, and the involvement of this pathway in ECM remodeling in DGP rats. The results showed that with increasing duration of diabetes, oxidation stress levels gradually increased, the AMPK activity decreased first and then increased, NLRP3, CX43 expression, and membrane/cytoplasm (M/C) ratio of Cx43 expression were increased in the gastric smooth muscle tissues of diabetic rats. Changes in ECM of gastric smooth muscle cells was observed in DGP rats. The DGP group showed higher collagen type I content, increased expression of Caspase-1, transforming growth factor-beta 3 (TGF-ß3), and matrix metalloproteinase-2 (MMP-2), decreased tissue inhibitor of metalloproteinases-1 (TIMP-1) expression, and higher interleukin-1beta (IL-1ß) content when compared with the control group. For gastric smooth muscle cells cultured under higher glucose, the MMP-2 and TGF-ß3 expression were decreased, TGF-ß1 and TIMP-1 expression were increased, the IL-1ßcontent was decreased in cells after inhibition of NLRP3 expression; the NLRP3 and Caspase-1expression were decreased, and adenosine triphosphate content was lower after inhibition of Cx43; the expression of NLRP3, Caspase-1, P2X7, and the M/C ratio of CX43 expression were decreased in cells after inhibition of AMPK and oxidative stress, the phospho-AMPK expression was also decreased after suppressing oxidative stress. Our findings suggest that high glucose induced the activation of the AMPK-Cx43-NLRP3 pathway through oxidative stress, and this pathway was involved in the ECM remodeling of gastric smooth muscles in DGP rats by regulating the biological functions of TGF-ß3, TGF-ß1, MMP-2, and TIMP-1.

NGF increases Connexin-43 expression and function in pulmonary arterial smooth muscle cells to induce pulmonary artery hyperreactivity.

AIMS: Pulmonary hypertension (PH) is characterised by an increase in pulmonary arterial pressure, ultimately leading to right ventricular failure and death. We have previously shown that nerve growth factor (NGF) plays a critical role in PH. Our objectives here were to determine whether NGF controls Connexin-43 (Cx43) expression and function in the pulmonary arterial smooth muscle, and whether this mechanism contributes to NGF-induced pulmonary artery hyperreactivity. METHODS AND RESULTS: NGF activates its TrkA receptor to increase Cx43 expression, phosphorylation, and localization at the plasma membrane in human pulmonary arterial smooth muscle cells, thus leading to enhanced activity of Cx43-dependent GAP junctions as shown by Lucifer Yellow dye assay transfer and fluorescence recovery after photobleaching -FRAP- experiments. Using both in vitro pharmacological and in vivo SiRNA approaches, we demonstrate that NGF-dependent increase in Cx43 expression and activity in the rat pulmonary circulation causes pulmonary artery hyperreactivity. We also show that, in a rat model of PH induced by chronic hypoxia, in vivo blockade of NGF or of its TrkA receptor significantly reduces Cx43 increased pulmonary arterial expression induced by chronic hypoxia and displays preventive effects on pulmonary arterial pressure increase and right heart hypertrophy. CONCLUSIONS: Modulation of Cx43 by NGF in pulmonary arterial smooth muscle cells contributes to NGF-induced alterations of pulmonary artery reactivity. Since NGF and its TrkA receptor play a role in vivo in Cx43 increased expression in PH induced by chronic hypoxia, these NGF/Cx43-dependent mechanisms may therefore play a significant role in human PH pathophysiology.

Purification, Reconstitution, and Functional Analysis of Connexin Hemichannels.

Connexins are the proteins that form the gap junction channels that are essential for cell-to-cell communication. These channels are formed by head-to-head docking of hemichannels (each from one of two adjacent cells). Free "undocked" hemichannels at the plasma membrane are mostly closed, although they are still important under physiological conditions. However, abnormal and sustained increase in hemichannel activity due to connexin mutations or acquired conditions can produce or contribute to cell damage. For example, mutations of Cx26, a connexin isoform, can increase hemichannel activity and cause deafness. Studies using purified isolated systems under well-controlled conditions are essential for a full understanding of molecular mechanisms of hemichannel function under normal conditions and in disease, and here, we present methodology for the expression, purification, and functional analysis of hemichannels formed by Cx26.

Evaluation of Connexin Hemichannel Activity In Vivo.

Connexin hemichannels (Cx HCs) are hexameric structures at the cell plasma membrane, whose function as membrane transport proteins allows for the passive flow of small hydrophilic molecules and ions (≤1 kDa) between the cytosol and the extracellular environment. Activation of Cx HCs is highly dependent on pathological conditions. HC activity provokes changes in the microenvironment, inducing the dissemination of signaling molecules in both an autocrine and paracrine manner. Given the elicitation of a variety of signaling pathways, and assortment of Cx species and dispersion throughout the body, Cx HCs have been implicated in a range of processes such as cell proliferation, differentiation, cell death, and tissue modeling and remodeling. While studying the expression and localization of Cx HCs can be done using traditional laboratory techniques, such as immunoblot analysis, measuring the functionality/activity of the HCs requires a more explicit methodology and is essential for determining Cx-mediated physiological changes. The study of Cx HC function/activity has focused mainly on in vitro measurements through electrophysiological characterization or, more commonly, using HC-permeable dye uptake studies. Here, we describe the use of dye uptake to measure Cx HC activity in vivo using mechanically stimulated osteocytic Cx43 HCs with Evans blue dye as our model.

A Modified Tridecapeptide Probe for Imaging Cell Junction.

Cell junctions, which are typically associated with dynamic cytoskeletons, are essential for a wide range of cellular activities, including cell migration, cell communication, barrier function and signal transduction. Observing cell junctions in real-time can help us understand the mechanisms by which they regulate these cellular activities. This study examined the binding capacity of a modified tridecapeptide from Connexin 43 (Cx43) to the cell junction protein zonula occludens-1 (ZO-1). The goal was to create a fluorescent peptide that can label cell junctions. A cell-penetrating peptide was linked to the modified tridecapeptide. The heterotrimeric peptide molecule was then synthesized. The binding of the modified tridecapeptide was tested using pulldown and immunoprecipitation assays. The ability of the peptide to label cell junctions was assessed by adding it to fixed or live Caco-2 cells. The testing assays revealed that the Cx43-derived peptide can bind to ZO-1. Additionally, the peptide was able to label cell junctions of fixed cells, although no obvious cell junction labeling was observed clearly in live cells, probably due to the inadequate affinity. These findings suggest that labeling cell junctions using a peptide-based strategy is feasible. Further efforts to improve its affinity are warranted in the future.

Remazolam affects the phenotype and function of astrocytes to improve traumatic brain injury by regulating the Cx43.

PURPOSE: To explore the mechanism by which Remazolam affects the phenotype and function of astrocytes to improve traumatic brain injury (TBI). METHODS: The oxygen -glucose deprivation/recovery (OGD/R) cell model was constructed to simulate the pathological state of astrocytes in a TBI environment. The viability of astrocytes was measured by CCK-8, and the cytoskeleton changes were observed by Phalloidin- TRITC staining. The expressions of differentiation markers, Cx43 and phosphorylated Cx43 (P-Cx43) of A1/A2 astrocytes were detected by Western blot, and the complement C3 and S100A10 of A1/A2 astrocytes were detected by ELISA. The TBI rat model was established. The water content of brain tissue was measured by dry-wet specific gravity method, the pathological morphology of brain tissue in cortical injury area was observed by HE staining method, ROS was detected by fluorescence quantitative method, Cx43 expression was detected by immunohistochemistry method, and the differentiation markers of A1/A2 astrocytes were detected by immunofluorescence. RESULTS: In the TBI environment, astrocytes showed decreased cell viability, blurred skeleton, and increased expression of Cx43. In TBI rats, the water content of brain tissue increased, the brain tissue in the cortex injury area was seriously damaged, ROS and Cx43 expression were significantly increased, and mainly distributed in A2 astrocytes. Remazolam can reverse the above results after the intervention. CONCLUSION: Remazolam affects the phenotype and function of astrocytes to improve TBI via regulating Cx43, and plays a role in protecting the neurological function of TBI rats.

Connexin 43 in the function and homeostasis of osteocytes: a narrative review.

Background and Objective: Connexin 43 (Cx43) is the main gap junction (GJ) protein and hemichannel protein in bone tissue. It is involved in the formation of hemichannels and GJs and establishes channels that can communicate directly to exchange substances and signals, affecting the structure and function of osteocytes. CX43 is very important for the normal development of bone tissue and the establishment and balance of bone reconstruction. However, the molecular mechanisms by which CX43 regulates osteoblast function and homeostasis have been less well studied, and this article provides a review of research in this area. Methods: We searched the PubMed, EMBASE, Cochrane Library, and Web of Science databases for studies published up to June 2023 using the keywords Connexin 43/Cx43 and Osteocytes. Screening of literatures according to inclusion and exclusion guidelines and summarized the results. Key Content and Findings: Osteocytes, osteoblasts, and osteoclasts all express Cx43 and form an overall network through the interaction between GJs. Cx43 is not only involved in the mechanical response of bone tissue but also in the regulation of signal transduction, which could provide new molecular markers and novel targets for the treatment of certain bone diseases. Conclusions: Cx43 is expressed in osteoblasts, osteoclasts, and osteoclasts and plays an important role in regulating the function, signal transduction, and mechanotransduction of osteocytes. This review offers a new contribution to the literature by summarizing the relationship between Cx43, a key protein of bone tissue, and osteoblasts.

Glial Cx43 hemichannels and neuronal Panx1 hemichannels and P2X7 receptors orchestrate presynaptic homeostatic plasticity.

The emerging role of glial cells in modulating neuronal excitability and synaptic strength is a growing field in neuroscience. In recent years, a pivotal role of gliotransmission in homeostatic presynaptic plasticity has been highlighted and glial-derived ATP arises as a key contributor. However, very little is known about the glial non-vesicular ATP-release pathway and how ATP participates in the modulation of synaptic strength. Here, we investigated the functional changes occurring in neurons upon chronic inactivity and the role of the purinergic signaling, connexin43 and pannexin1 hemichannels in this process. By using hippocampal dissociated cultures, we showed that blocking connexin43 and pannexin1 hemichannels decreases the amount of extracellular ATP. Moreover, Ca2+ imaging assays using Fluo-4/AM revealed that blocking connexin43, neuronal P2X7Rs and pannexin1 hemichannels decreases the amount of basal Ca2+ in neurons. A significant impairment in synaptic vesicle pool size was also evidenced under these conditions. Interestingly, rescue experiments where Panx1HCs are blocked showed that the compensatory adjustment of cytosolic Ca2+ was recovered after P2X7Rs activation, suggesting that Panx1 acts downstream P2X7Rs. These changes were accompanied by a modulation of neuronal permeability, as revealed by ethidium bromide uptake experiments. In particular, the permeability of neuronal P2X7Rs and pannexin1 hemichannels is increased upon 24 h of inactivity. Taken together, we have uncovered a role for connexin43-dependent ATP release and neuronal P2X7Rs and pannexin1 hemichannels in the adjustment of presynaptic strength by modulating neuronal permeability, the entrance of Ca2+ into neurons and the size of the recycling pool of synaptic vesicles.

Administration of USP7 inhibitor p22077 alleviates Angiotensin II (Ang II)-induced atrial fibrillation in Mice.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is an important contributor to mortality and morbidity. Ubquitin-specific protease 7 (USP7), one of the most abundant ubiquitin-specific proteases (USP), participated in many cellular events, such as cell proliferation, apoptosis, and tumourigenesis. However, its role in AF remains unknown. Here, the mice were treated with Ang II infusion to induce the AF model. Echocardiography was used to measure the atrial diameter. Electrical stimulation was programmed to measure the induction and duration of AF. The changes in atrial remodeling were measured using routine histologic analysis. Here, a significant increase in USP7 expression was observed in Ang II-stimulated atrial cardiomyocytes and atrial tissues, as well as in atrial tissues from patients with AF. The administration of p22077, the inhibitor of USP7, attenuated Ang II-induced inducibility and duration of AF, atrial dilatation, connexin dysfunction, atrial fibrosis, atrial inflammation, and atrial oxidase stress, and then inhibited the progression of AF. Mechanistically, the administration of p22077 alleviated Ang II-induced activation of TGF-ß/Smad2, NF-κB/NLRP3, NADPH oxidases (NOX2 and NOX4) signals, the up-regulation of CX43, ox-CaMKII, CaMKII, Kir2.1, and down-regulation of SERCA2a. Together, this study, for the first time, suggests that USP7 is a critical driver of AF and revealing USP7 may present a new target for atrial fibrillation therapeutic strategies.

Relationship between astrocyte damage and different levels of cerebrospinal fluid markers and prognosis in patients with subarachnoid hemorrhage.

INTRODUCTION: The aim of the study was to explore the relationship between astrocyte damage and different levels of cerebrospinal fluid markers and prognosis in patients with subarachnoid hemorrhage (SAH). MATERIAL AND METHODS: A total of 168 SAH patients diagnosed and treated in the emergency department of our hospital during the period October 2019 to February 2022 were randomly selected as the study subjects. The severity of these patients' condition was evaluated by Hunt-Hess grading and these subjects were graded as the low-level group (78 patients) and high-level group (90 patients) according to the evaluation results. The Extended Disability Status Scale (EDSS) score was employed to evaluate the astrocyte damage. The content of atypical chemokine receptor 3 (ACKR3), Connexin 43 (Cx43), oxygenated hemoglobin (HbO 2 ), and endothelin (ET) in cerebrospinal fluid was measured. The relationship between the content of ACKR3, Cx43, HbO 2 , and ET in cerebrospinal fluid with EDSS score was analyzed through Pearson correlation analysis. Multivariate logistic regression analysis was adopted to analyze the risk factors. RESULTS: ACKR3 was mainly expressed in the cytoplasm of cerebrospinal fluid monocytes, and Cx43 was mainly expressed in the cell membrane and cytoplasm. Patients in the high-level group had markedly higher expression rates of ACKR3 and Cx43 positive cells in cerebrospinal fluid than those in the low-level group ( p < 0.05). Patients in the high-level group had higher content of HbO 2 and ET in cerebrospinal fluid and EDSS score than patients in the low-level group ( p < 0.05). The content of ACKR3, Cx43, HbO 2 , and ET in cerebrospinal fluid of SAH patients was positively correlated with EDSS scores ( p < 0.05). Systolic blood pressure, Hunt-Hess grade, rebleeding, emotional control, EDSS score, ACKR3, Cx43 positive cell rate, and HbO 2 and ET expression levels were independent risk factors for the prognosis of SAH patients ( p < 0.05). CONCLUSIONS: Astrocyte damage in SAH patients was positively correlated with the content of ACKR3, Cx43, HbO 2 , and ET in cerebrospinal fluid. These indicators increased significantly with the increasing severity of the disease, and had certain value in reflecting the patient's condition. Astrocyte damage combined with cerebrospinal fluid markers had potential value in evaluating the severity and prognosis of patients.

Reuterin isolated from the probiotic Lactobacillus reuteri promotes periodontal tissue regeneration by inhibiting Cx43-mediated the intercellular transmission of endoplasmic reticulum stress.

OBJECTIVE: The present study aimed to evaluate the effects of reuterin, a bioactive isolated from the probiotic Lactobacillus reuteri (L. reuteri) on periodontal tissue regeneration, and provide a new strategy for periodontitis treatment in the future. BACKGROUND: Data discussing the present state of the field: Probiotics are essential for maintaining oral microecological balance. Our previous study confirmed that probiotic L. reuteri extracts could rescue the function of mesenchymal stem cells (MSCs) and promote soft tissue wound healing by neutralizing inflammatory Porphyromonas gingivalis-LPS. Periodontitis is a chronic inflammatory disease caused by bacteria seriously leading to tooth loss. In this study, we isolated and purified reuterin from an extract of L. reuteri to characterize from the extracts of L. reuteri to characterize its role in promoting periodontal tissue regeneration and controlling inflammation in periodontitis. METHODS: Chromatographic analysis was used to isolate and purify reuterin from an extract of L. reuteri, and HNMR was used to characterize its structure. The inflammatory cytokine TNFα was used to simulate the inflammatory environment. Periodontal ligament stem cells (PDLSCs) were treated with TNFα and reuterin after which their effects were characterized using scratch wound cell migration assays to determine the concentration of reuterin, an experimental periodontitis model in rats was used to investigate the function of reuterin in periodontal regeneration and inflammation control in vivo. Real-time PCR, dye transfer experiments, image analysis, alkaline phosphatase activity, Alizarin red staining, cell proliferation, RNA-sequencing and Western Blot assays were used to detect the function of PDLSCs. RESULTS: In vivo, local injection of reuterin promoted periodontal tissue regeneration of experimental periodontitis in rats and reduced local inflammatory response. Moreover, we found that TNFα stimulation caused endoplasmic reticulum (ER) stress in PDLSCs, which resulted in decreased osteogenic differentiation. Treatment with reuterin inhibited the ER stress state of PDLSCs caused by the inflammatory environment and restored the osteogenic differentiation and cell proliferation functions of inflammatory PDLSCs. Mechanistically, we found that reuterin restored the functions of inflammatory PDLSCs by inhibiting the intercellular transmission of ER stress mediated by Cx43 in inflammatory PDLSCs and regulated osteogenic differentiation capacity. CONCLUSION: Our findings identified reuterin isolated from extracts of the probiotic L. reuteri, which improves tissue regeneration and controls inflammation, thus providing a new therapeutic method for treating periodontitis.

Evaluation of Tyrosine Kinase Inhibitors Loaded Injectable Hydrogels for Improving Connexin43 Gap Junction Intercellular Communication.

Myocardial infarction (MI) is one of the leading causes of death in the developed world, and the loss of cardiomyocytes plays a critical role in the pathogenesis of heart failure. Implicated in this process is a decrease in gap junction intercellular communication due to remodeling of Connexin43 (Cx43). We previously identified that intraperitoneal injection of the Pyk2 inhibitor PF4618433 reduced infarct size, maintained Cx43 at the intercalated disc in left ventricle hypertrophic myocytes, and improved cardiac function in an MI animal model of heart failure. With the emergence of injectable hydrogels as a therapeutic toward the regeneration of cardiac tissue after MI, here, we provide proof of concept that the release of tyrosine kinase inhibitors from hydrogels could have beneficial effects on cardiomyocytes. We developed an injectable hydrogel consisting of thiolated hyaluronic acid and P123-maleimide micelles that can incorporate PF4618433 as well as the Src inhibitor Saracatinib and achieved sustained release (of note, Src activates Pyk2). Using neonatal rat ventricular myocytes in the presence of a phorbol ester, endothelin-1, or phenylephrine to stimulate cardiac hypertrophy, the release of PF4618433 from the hydrogel had the same ability to decrease Cx43 tyrosine phosphorylation and maintain Cx43 localization at the plasma membrane as when directly added to the growth media. Additional beneficial effects included decreases in apoptosis, the hypertrophic marker atrial natriuretic peptide (ANP), and serine kinases upregulated in hypertrophy. Finally, the presence of both PF4618433 and Saracatinib further decreased the level of ANP and apoptosis than each inhibitor alone, suggesting that a combinatorial approach may be most beneficial. These findings provide the groundwork to test if tyrosine kinase inhibitor release from hydrogels will have a beneficial effect in an animal model of MI-induced heart failure.

AS602801 treatment suppresses breast cancer metastasis to the brain by interfering with gap-junction communication by regulating Cx43 expression.

AS602801 has been reported as a potential drug candidate against brain metastasis by suppressing the gap-junction communication between lung cancer stem cells and astrocytes. In this study, we aimed to study the molecular mechanism underlying the role of AS602801 in the treatment of brain metastasis in breast cancer. We utilized female athymic BALB/c nude mice and MDA-MB-231/BT-474BR cells to establish experimental models. Polymerase chain reaction assays were performed to observe changes in the connexin 43 (Cx43) messenger RNA (mRNA) and c-Jun N-terminal kinase (JNK) mRNA levels. Dye transfer assay was used to observe the effect of AS602801 on cell-cell communication. An organotypic blood-brain barrier (BBB) model was utilized to observe the effect of AS602801 on transmigration through the BBB barrier. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and flow cytometry were performed to evaluate the proliferation and apoptosis of breast cancer cells co-cultivated with astrocytes. AS602801 inhibited the upregulation of Cx43 and JNK in brain metastasized breast cancer cells in a dose-dependent manner. Also, AS602801 significantly decreased the dye transfer rate from astrocytes to breast cancer cells, indicating the inhibitory effect of AS602801 on cell-cell communication. The transmigration ability of breast cancer cells co-cultured with astrocytes was decreased by AS602801. Furthermore, AS602801 reduced the elevated Cx43/JNK mRNA expression in the co-astrocyte group while suppressing the increased proliferation and promoting the decreased apoptosis of breast cancer cells co-cultivated with astrocytes. AS602801 also suppressed the brain metastasis of breast cancer cells and increased mouse survival. AS602801 downregulates the expressions of JNK and Cx43 to suppress the gap-junction activity. AS602801 also inhibits the communication between breast cancer cells and astrocytes, thus contributing to the treatment of brain metastasis in breast cancer.

Human Cx50 Isoleucine177 prevents heterotypic docking and formation of functional gap junction channels with Cx43.

The human lens is an avascular organ, and its transparency is dependent on gap junction (GJ)-mediated microcirculation. Lens GJs are composed of three connexins with Cx46 and Cx50 being expressed in lens fiber cells and Cx43 and Cx50 in the epithelial cells. Impairment of GJ communication by either Cx46 or Cx50 mutations has been shown to be one of the main molecular mechanisms of congenital cataracts in mutant carrier families. The docking compatibility and formation of functional heterotypic GJs for human lens connexins have not been studied. Previous study on rodent lens connexins revealed that Cx46 can form functional heterotypic GJs with Cx50 and Cx43, but Cx50 cannot form heterotypic GJ with Cx43 due to its second extracellular (EL2) domain. To study human lens connexin docking and formation of functional heterotypic GJs, we developed a genetically engineered HEK293 cell line with endogenously expressed Cx43 and Cx45 ablated. The human lens connexins showed docking compatibility identical to those found in the rodent connexins. To reveal the structural mechanisms of the docking incompatibility between Cx50 and Cx43, we designed eight variants based on the differences between the EL2 of Cx50 and Cx46. We found that Cx50I177L is sufficient to establish heterotypic docking with Cx43 with some interesting gating properties. Our structure models indicate this residue is important for interdomain interactions within a single connexin, Cx50 I177L showed an increased interdomain interaction which might alter the docking interface structure to be compatible with Cx43.NEW & NOTEWORTHY The human lens is an avascular organ, and its transparency is partially dependent on gap junction (GJ) network composed of Cx46, Cx50, and Cx43. We found that human Cx46 can dock and form functional heterotypic GJs with Cx50 and Cx43, but Cx50 is unable to form functional heterotypic GJs with Cx43. Through mutagenesis and patch-clamp study of several designed variants, we found that Cx50 I177L was sufficient to form functional heterotypic GJs with Cx43.

Inhibition of astroglial hemichannels ameliorates infrasonic noise induced short-term learning and memory impairment.

As a kind of environmental noise, infrasonic noise has negative effects on various human organs. To date, research has shown that infrasound impairs cognitive function, especially the ability for learning and memory. Previously, we demonstrated that impaired learning and memory induced by infrasound was closely related with glia activation; however, the underlying mechanisms remain unclear. Connexin 43 hemichannels (Cx43 HCs), which are mainly expressed in hippocampal astrocytes, are activated under pathological conditions, lending support to the hypothesis that Cx43 HCs might function in the impaired learning and memory induced by infrasound. This study revealed that that blocking hippocampal Cx43 HCs or downregulating hippocampal Cx43 expression significantly alleviated impaired learning and memory induced by infrasound. We also observed that infrasound exposure led to the abundant release of glutamate and ATP through Cx43 HCs. In addition, the abundant release of glutamate and ATP depended on proinflammatory cytokines. Our finds suggested that the enhanced release of ATP and glutamate by astroglial Cx43 HCs may be involved in the learning and memory deficits caused by infrasound exposure.

Targeting Cx43 to Reduce the Severity of Pressure Ulcer Progression.

In the skin, repeated incidents of ischemia followed by reperfusion can result in the breakdown of the skin and the formation of a pressure ulcer. Here we gently applied paired magnets to the backs of mice to cause ischemia for 1.5 h and then removed them to allow reperfusion. The sterile inflammatory response generated within 4 h causes a stage 1 pressure ulcer with an elevation of the gap junction protein Cx43 in the epidermis. If this process is repeated the insult will result in a more severe stage 2 pressure ulcer with a breakdown of the epidermis 2-3 days later. After a single pinch, the elevation of Cx43 in the epidermis is associated with the inflammatory response with an increased number of neutrophils, HMGB1 (marker of necrosis) and RIP3 (responsible for necroptosis). Delivering Cx43 specific antisense oligonucleotides sub-dermally after a single insult, was able to significantly reduce the elevation of epidermal Cx43 protein expression and reduce the number of neutrophils and prevent the elevation of HMGB1 and RIP3. In a double pinch model, the Cx43 antisense treatment was able to reduce the level of inflammation, necroptosis, and the extent of tissue damage and progression to an open wound. This approach may be useful in reducing the progression of stage 1 pressure ulcers to stage 2.

MLKL regulates Cx43 ubiquitinational degradation and mediates neuronal necroptosis in ipsilateral thalamus after focal cortical infarction.

Necroptosis is known to play an important role in the pathophysiology of cerebral ischemia; however, its role in the occurrence of secondary thalamic injury after focal cerebral infarction and the mechanism about how mixed lineage kinase domain-like (MLKL) executes necroptosis in this pathophysiology are still unclear. In this study, Sprague-Dawley rats were subjected to distal branch of middle cerebral artery occlusion (dMCAO). The expression of MLKL, connexin 43 (Cx43) and Von Hippel-Lindau (VHL) in vitro and in vivo were assessed by Western blot. Bioinformatic methods were used to predict the potential binding sites where MLKL interacted with Cx43, and the ubiquitination degradation of Cx43 regulated by VHL. The interactions among MLKL, Cx43, VHL, and Ubiquitin were assessed by immunoprecipitation. Dye uptake assay were used to examine the Cx43 hemichannels. Intracellular Ca2+ concentration was measured using Fluo-4 AM. Overexpression and site-directed mutagenesis studies were used to study the mechanisms by which MLKL regulates Cx43 ubiquitinational degradation to mediate neuronal necroptosis. We found that MLKL and Cx43 were upregulated in the ventral posterolateral nucleus (VPN) of the ipsilateral thalamus after dMCAO. In the in vitro experiments MLKL and Cx43 were upregulated after TSZ-mediated necroptosis in SH-SY5Y cells. The interaction between MLKL and Cx43 inhibited the K48-linked ubiquitination of Cx43 in necroptotic SH-SY5Y cells. VHL is an E3 ubiquitin ligase for Cx43, and MLKL competes with VHL for binding to Cx43. Interaction of MLKL Ser454 with Cx43 can trigger the opening of Cx43 hemichannels, causing increased intracellular Ca2+, and cell necroptosis. This innovative study at animal models, cellular, and molecular levels is anticipated to clarify the roles of MLKL and Cx43 in thalamic damage after focal cortical infarction. Our findings may help identify novel targets for neurological recovery after cortical infarction.

Effects of a Sertoli cell-specific knockout of Connexin43 on maturation and proliferation of postnatal Sertoli cells.

Adult male Sertoli cell-specific Connexin43 knockout mice (SCCx43KO) exhibit higher Sertoli cell (SC) numbers per seminiferous tubule compared to their wild type (WT) littermates. Thus, deletion of this testicular gap junction protein seems to affect the proliferative potential and differentiation of "younger" SC. Although SC have so far mostly been characterised as postmitotic cells that cease to divide and become an adult, terminally differentiated cell population at around puberty, there is rising evidence that there exist exceptions from this for a very long time accepted paradigm. Aim of this study was to investigate postnatal SC development and to figure out underlying causes for observed higher SC numbers in adult KO mice. Therefore, the amount of SC mitotic figures was compared, resulting in slightly more and prolonged detection of SC mitotic figures in KO mice compared to WT. SC counting per tubular cross section revealed significantly different time curves, and comparing proliferation rates using Bromodesoxyuridine and Sox9 showed higher proliferation rates in 8-day old KO mice. SC proliferation was further investigated by Ki67 immunohistochemistry. SC in KO mice displayed a delayed initiation of cell-cycle-inhibitor p27Kip1 synthesis and prolonged synthesis of the phosphorylated tumour suppressor pRb and proliferation marker Ki67. Thus, the higher SC numbers in adult male SCCx43KO mice may arise due to two different reasons: Firstly, in prepubertal KO mice, the proliferation rate of SC was higher. Secondly, there were differences in their ability to cease proliferation as shown by the delayed initiation of p27Kip1 synthesis and the prolonged production of phosphorylated pRb and Ki67. Immunohistochemical results indicating a prolonged period of SC proliferation in SCCx43KO were confirmed by detection of proliferating SC in 17-days-old KO mice. In conclusion, deletion of the testicular gap junction protein Cx43 might prevent normal SC maturation and might even alter also the proliferation potential of adult SC.