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Exosomes are naturally secreted nanovesicles that have emerged as a promising therapeutic nanodelivery platform, due to their specific composition and biological properties. However, challenges like considerable complexity, low isolation yield, drug payload, and potential safety concerns substantially reduce their pharmaceutical acceptability. Given that the nano-bio-interface is a crucial factor for nanocarrier behavior and function, modification of synthetic nanoparticles with the intrinsic hallmarks of exosomes' membrane to create exosome mimetics could allow for siRNA delivery in a safer and more efficient manner. Herein, connexin 43 (Cx43)-embedded, exosome-mimicking lipid bilayers coated chitosan nanoparticles (Cx43/L/CS NPs) were constructed by using cell-free (CF) synthesis systems with plasmids encoding Cx43 in the presence of lipid-coated CS NPs (L/CS NPs). The integration of de novo synthesized Cx43 into the lipid bilayers of L/CS NPs occurred cotranslationally during one-pot reaction and, more importantly, the integrated Cx43 was functionally active in transport. In addition to considerably lower cytotoxicity (

Objective: To develop a method to distinguish atypical meningiomas (AMs) with malignant progression (MP) from primary AMs without a clinical history., Methods: The clinical, radiologic, and pathologic data of 33 previously Simpson grade I resected (if any) as well as no radiotherapy treated intracranial AMs between January 2008 and December 2015 were reviewed. Immunohistochemical staining for connexin 43 (Cx43) and Ki-67 was performed. Descriptive analysis and univariate and multivariate logistic regression analyses were used to explore independent predictors of MP. A multivariable logistic model was developed to estimate the risk of MP, and its diagnostic value was determined from a receiver operating characteristic curve., Results: There were 11 AMs (33.3%) with histopathologically confirmed MP from benign meningiomas. The other 22 (66.7%) were initially diagnosed AMs with no histopathologically confirmed MP during a median 60.5 months (range, 42-126 months) of follow-up. Univariate and multivariate logistic analyses showed that irregular tumor shape (P = 0.010) and low Cx43 expression (P = 0.010) were independent predictors of the presence of MP, and the predicted probability was calculated by the following formula: P = 1/[1+exp.{1.218-(3.202×Shape)+(3.814×Cx43)}]. P > 0.5 for an irregularly shaped (score 1) AM with low Cx43 expression (score 0) indicated a high probability of MP. The sensitivity, specificity, positive predictive value, negative predictive value, and overall predictive accuracy were 63.6, 95.6, 87.5, 84.0, and 84.8%, respectively., Conclusions: Low Cx43 expression and irregular tumor shape were independent predictors of the presence of MP. The relevant logistic regression model was found to be effective in distinguishing MP-AMs from primary AMs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

It is accepted that alteration of connexin43 (Cx43) expression in glomeruli is a common pathological response in several forms of kidney diseases. To date, however the change of the Cx43 expression in obesity-related glomerulopathy (ORG) has not been reported. In this study, the alteration of Cx43 expression in the glomeruli of rat with ORG was defined. Five-week-old rats were fed with high-fat diet for 18weeks to establish the ORG model, then the histological change of glomeruli, the foot process effacement of podocyte, the markers for podocyte injury (nephrin,podocin and WT1) and Cx43 expression in glomeruli were examined respectively. The results demonstrated metabolic disorder, hyperinsulinemia, systemic inflammation and microalbuminuria in ORG rats. There was significant hypertrophy, glomerular expansion and inflammatory cell infiltration in the kidney of ORG rats compared to the control group. Significant foot process effacement of the podocyte in the glomeruli, nephrin loss and density reduction were shown in the ORG rats, and Cx43 expression was significant upregulated in glomeruli of ORG rats compared to the control group. The results indicate the correlation of overexpressed Cx43 with the obesity related renal inflammation and suggest that Cx43 might be a potential target in the development of obesity related glomerulopathy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: In this study, we hypothesized that CSCs mediated the expression of Cx43 after transplantation post MI via the ANG II/AT1R/TGF-beta1 signaling pathway., Methods: Myocardial infarction (MI) was induced in twenty male Sprague-Dawley rats. The rats were randomized into two groups and were then received the injection of 5 × 10(6) CSCs labeled with PKH26 in phosphate buffer solution (PBS) or equal PBS alone into the infarct anterior ventricular free wall two weeks after MI. Six weeks later, relevant signaling molecules involved were all examined., Results: In the CSCs group, an increased expression of Cx43 could be observed in different zones of the left ventricle (P<0.01). There was a significant reduction of the angiotensin II (ANG II) level in plasma and different regions of the left ventricular cardiac tissues (P<0.05; P<0.01). The angiotensin II type I receptor (AT1R) was decreased accompanied with an enhanced expression of angiotensin II type II receptor (AT2R) (P<0.01). Transforming growth factor beta-1(TGF-beta1) was downregulated (P<0.01). The expression of mothers against decapentaplegic homolog (SMAD) proteins including SMAD2 and SMAD3 was attenuated whereas SMAD7 was elevated (P<0.01, P<0.01, P<0.05). In addition, the expression of mitogen-activated protein kinases (MAPKs) including extracellular kinases 1/2 (ERK1/2) and p38 was also found to be reduced (P<0.01)., Conclusion: CSCs transplantation could enhance the level of Cx43 after MI. They might function through intervening the ANGII/AT1R/TGF-beta1 signaling pathway to regulate the expression of Cx43., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Decreased expression of connexins has been associated with cancer, but the underlying mechanisms are poorly understood. We have previously shown that a 5 day exposure to hexachlorobenzene (HCB) resulted in decreased connexins expression in hepatocytes 45 days later, and that this down-regulation was linked to activation of Akt through the ILK pathway. Because HCB promotes cancer in both the liver and breast, the present study aimed to determine if the mechanisms are similar in both tissues. MCF-12A breast cells were thus transfected with vectors coding for either Akt or a constitutively active form of Akt. In those cells, activation of Akt was correlated with decreased Cx43 levels. Female rats were then exposed to HCB by gavage either following the same protocol used previously for the liver or through a chronic exposure. While no changes were observed after the 5 days exposure protocol, chronic exposure to HCB resulted in increased Akt levels and decreased Cx43 levels in breast cells. In vitro, Akt was activated in MCF-12A cells exposed to HCB either for 7 days or chronically, but no changes were observed in junctional proteins. Together, these results suggested that, while activation of Akt can decrease Cx43 expression in breast cells in vitro, other mechanisms are involved during HCB exposure, leading to a decrease in Cx43 levels in a model- and duration-dependent manner. Finally, we showed that HCB effects are tissue specific, as we did not observe the same results in breast and liver tissues., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Coordinated contraction of the heart is essential for survival and is regulated by the cardiac conduction system. Contraction of ventricular myocytes is controlled by the terminal part of the conduction system known as the Purkinje fiber network. Lineage analyses in chickens and mice have established that the Purkinje fibers of the peripheral ventricular conduction system arise from working myocytes during cardiac development. It has been proposed, based primarily on gain-of-function studies, that Endothelin signaling is responsible for myocyte-to-Purkinje fiber transdifferentiation during avian heart development. However, the role of Endothelin signaling in mammalian conduction system development is less clear, and the development of the cardiac conduction system in mice lacking Endothelin signaling has not been previously addressed. Here, we assessed the specification of the cardiac conduction system in mouse embryos lacking all Endothelin signaling. We found that mouse embryos that were homozygous null for both ednra and ednrb, the genes encoding the two Endothelin receptors in mice, were born at predicted Mendelian frequency and had normal specification of the cardiac conduction system and apparently normal electrocardiograms with normal QRS intervals. In addition, we found that ednra expression within the heart was restricted to the myocardium while ednrb expression in the heart was restricted to the endocardium and coronary endothelium. By establishing that ednra and ednrb are expressed in distinct compartments within the developing mammalian heart and that Endothelin signaling is dispensable for specification and function of the cardiac conduction system, this work has important implications for our understanding of mammalian cardiac development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Previously, connexin 43-modified skeletal myoblasts (MbCx) were shown to reduce the pro-arrhythmic effect during the regeneration of heart tissue in an animal model of infarction. To increase the relevance to clinical implementation, in this study, we introduced connexin 43 into human myoblasts using a highly safe non-viral vector and demonstrated that their transplantation had a positive effect on the function of the injured heart., Methods and Results: Myoblasts were efficiently transfected with a pCiNeo-GJA1 plasmid (65.72%). qPCR analysis revealed over 32-fold higher expression of the connexin 43 gene in the MbCx cell population compared to 'native' controls. The susceptibility of the myoblasts to oxidative stress conditions (p<0.001) and the fusion index (p<0.01) were increased in the MbCx cells. Additionally, we observed changes in the MYOG and MYH2 gene expression levels in the GJA1-modified myoblasts. Finally, we observed a significant improvement in the post-infarction echocardiographic parameters after intervention using MbCx cells compared with non-transfected myoblasts (MbWt) and the control (0.9% NaCl), wherein a significant decrease in the left ventricular area change in the short axis (SAX AC%) was observed at the two-month follow-up (p<0.05 and p<0.01, respectively)., Conclusions: We demonstrated the positive effect of connexin 43 overexpression on the biology and function of human skeletal myoblasts in the context of their potential clinical applications. Our preclinical studies using a mouse infarction model indicated the positive effect of MbCx implantation on the function of the injured heart., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Doxorubicin (dox) is an effective chemotherapeutic agent that leads to cardiotoxicity. An alternative treatment for dox-cardiotoxicity is autologous mesenchymal stem cells (MSCs) transplantation. It remains unclear if dox has deleterious effects on MSCs from subjects under chemotherapy, therefore this study aimed to evaluate dox in vivo toxicological effects on ex vivo cultured MSCs, inferring whether autologous transplantation may be an alternative treatment in patients who are exposed to the drug. Wistar rats received either dox or saline. Following treatments, animals were sacrificed and bone marrow MSCs were isolated, characterized for cell surface markers and assessed according to their viability, alkaline phosphatase production, and proliferation kinetics. Moreover, MSCs were primed to cardiac differentiation and troponin T and connexin 43 expressions were evaluated. Compared to control, undifferentiated MSCs from dox group kept the pattern for surface marker and had similar viability results. In contrast, they showed lower alkaline phosphatase production, proliferation rate, and connexin 43 expression. Primed MSCs from dox group showed lower troponin T levels. It was demonstrated a toxic effect of dox in host MSCs. This result renders the possibility of autologous MSCs transplantation to treat dox-cardiotoxicity, which could be a non-suitable option for subjects receiving such antineoplastic agent., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: Although it is well known that 3,4-methylenedioxymethamphetamine (MDMA) can cause various cardiovascular abnormalities and even sudden death from cardiac arrhythmia, whether it has any effect on myocardial gap junctions, which might be one of the targets mediating MDMA-induced cardiotoxicity, remains unclear., Objective: To test the hypothesis that MDMA may affect the myocardial gap junction protein connexin43 (Cx43) and induce cardiac dysrhythmia., Method: (1) In vivo study: adult rats were treated with a single dose MDMA administration (20mg/kg, i.p.). Electrocardiogram detection and immunohistochemical analysis were performed to evaluate cardiac function and expression of Cx43, respectively; (2) in vitro study: cultured ventricular myocytes of neonatal rats were treated with MDMA (10, 100, 1000μmol/L) for 1h. Western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) were performed to investigate the total Cx43 mRNA expression. Immunofluorescent analysis was used to evaluate the amount of junctional Cx43. The phosphorylation status of Cx43 at site Ser368 and intracellular Ca(2+) oscillation were also studied., Results: Obvious changes in electrocardiographic patterns were found in rats following MDMA administration. They were characterized by prolonged QRS duration associated with increased amplitude of QRS complex. The heart rates in treated rats were significantly decreased compared to the rats in the control group. The immunohistochemical findings revealed a significant decrease in Cx43 expression. The in vitro study also showed a marked decline in total Cx43 protein associated with reduction of Cx43 mRNA, whereas the phosphorylated Cx43 at Ser368 was increased. Decrease of junctional Cx43 was found correlated with reduction in N-cadherin induced by high concentration of MDMA. Additionally, confocal microscopy findings revealed alteration of intracellular calcium oscillation patterns characterized by high frequency and increasing influx Ca(2+)., Conclusions: MDMA reduces expression of cardiac gap junction protein Cx43. The increase of phosphorylation status of Cx43 at Ser368 induced by MDMA is attributed, at least in part, to the Ca(2+)-dependent regulation of protein kinase C (PKC) activity. Our findings provide first evidence of MDMA-mediated changes in those cardiac gap junctions that may underlie MDMA-induced cardiac arrhythmia., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

To determine if adrenergic hormones play a critical role in the functional development of the cardiac pacemaking and conduction system, we employed a mouse model where adrenergic hormone production was blocked due to targeted disruption of the dopamine β-hydroxylase (Dbh) gene. Immunofluorescent histochemical evaluation of the major gap junction protein, connexin 43, revealed that its expression was substantially decreased in adrenergic-deficient (Dbh-/-) relative to adrenergic-competent (Dbh+/+ and Dbh+/-) mouse hearts at embryonic day 10.5 (E10.5), whereas pacemaker and structural protein staining appeared similar. To evaluate cardiac electrical conduction in these hearts, we cultured them on microelectrode arrays (8×8, 200 μm apart). Our results show a significant slowing of atrioventricular conduction in adrenergic-deficient hearts compared to controls (31.4±6.4 vs. 15.4±1.7 ms, respectively, p<0.05). To determine if the absence of adrenergic hormones affected heart rate and rhythm, mouse hearts from adrenergic-competent and deficient embryos were cultured ex vivo at E10.5, and heart rates were measured before and after challenge with the β-adrenergic receptor agonist, isoproterenol (0.5 μM). On average, all hearts showed increased heart rate responses following isoproterenol challenge, but a significant (p<0.05) 225% increase in the arrhythmic index (AI) was observed only in adrenergic-deficient hearts. These results show that adrenergic hormones may influence heart development by stimulating connexin 43 expression, facilitating atrioventricular conduction, and helping to maintain cardiac rhythm during a critical phase of embryonic development., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Reduced expression of connexin43 (Cx43) and sodium channel (Nav1.5) and increased expression of collagen (fibrosis) are important determinants of impulse conduction in the heart., Objective: To study the importance and interaction of these factors at very low Cx43 expression, inducible Cx43 knockout mice with and without inducible ventricular tachycardia (VT) were compared through electrophysiology and immunohistochemistry., Methods: Cx43(CreER(T)/fl) mice were induced with tamoxifen and killed after 2 weeks. Epicardial activation mapping was performed on Langendorff-perfused hearts, and arrhythmia vulnerability was tested. Mice were divided into arrhythmogenic (VT+; n = 13) and nonarrhythmogenic (VT-; n = 10) animals, and heart tissue was analyzed for Cx43, Nav1.5, and fibrosis., Results: VT+ mice had decreased Cx43 expression with increased global, but not local, heterogeneity of Cx43 than did VT- mice. Nav1.5-immunoreactive protein expression was lower in VT+ than in VT- mice, specifically at sites devoid of Cx43. Levels of fibrosis were similar between VT- and VT+ mice. QRS duration was increased and epicardial activation was more dispersed in VT+ mice than in VT- mice. The effective refractory period was similar between the 2 groups. Premature stimulation resulted in a more severe conduction slowing in VT+ than in VT- hearts in the right ventricle. Separate patch-clamp experiments in isolated rat ventricular myocytes confirmed that the loss of Cx43 expression correlated with the decreased sodium current amplitude., Conclusions: Global heterogeneity in Cx43 expression and concomitant heterogeneous downregulation of sodium-channel protein expression and sodium current leads to slowed and dispersed conduction, which sensitizes the heart for ventricular arrhythmias., (Copyright © 2012 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Connexin 43 (Cx43) gap junctions play an important role in development and differentiation of endometrium and placenta. Therefore, aberrant expression of Cx43 could result in altered physiological and pathological processes leading to recurrent early pregnancy loss (REPL). To investigate if expression of Cx43 is effected in REPL patients as compared to controls, Cx43 expression was compared in the chorionic villi and uterine decidua samples of 15 REPL patients and 15 induced abortion subjects as controls. Cx43 levels were evaluated using semi-quantitative RT-PCR, quantitative Real-time PCR and by western blot. Cx43 expression at mRNA and protein level was significantly decreased in both chorionic villi and decidua (p < 0.05) of REPL patients. Reduced expression of Cx43 transcript and protein in fetal chorionic villi and maternal decidua indicate the key role of Cx43 in REPL risk., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

To evaluate sex differences in protein expression in the heart, we performed Western blot studies on a subset of Heart Rhythm Determinant (HRD) proteins. We examined key components of a variety of types of mechanical and electrical junctions including, connexin43, plakophilin-2, N-cadherin and plakoglobin, ankyrin-2 and actin. We describe novel findings in sex differences in cardiac protein expression and membrane localization. For most proteins examined, sex differences were significantly more pronounced in the membrane compartment than in overall expression. These studies extend our previous findings in microarray studies to demonstrate that sex differences in gene expression are likely to confer distinct functional properties on male and female myocardium., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Acute hypoxia at postnatal day (P) 10 is an accepted model of human neonatal hypoxia which results, among other consequences, in increased hippocampal excitability. Hypoxic-ischemic injury, which mimics stroke, has been shown to result in changes in connexins (Cxs), however, changes in Cxs have not been studied in the P10 hypoxia model. The aim of this study was to investigate changes in the hippocampal expression of three different connexins at consecutive developmental stages after acute hypoxia at P10 (10min and 30min after reoxygenation, P11, P14, P17, P29, and P45) as compared to sham manipulated pups. After acute hypoxia at P10, Cx30 protein levels were increased at 30min after reoxygenation, at P11 and at P14, and then returned to control levels. Cx36 protein levels transiently decreased at P11 after acute hypoxia then returned to control levels. Cx43 protein levels did not change at any of the time points. Although changes in mRNA expression were observed during development for Cx30 only, acute hypoxia did not result in changes in mRNA expression of all these Cxs when compared to age matched controls suggesting that acute hypoxia induced posttranslational changes in protein expression., (Copyright © 2009 Elsevier B.V. All rights reserved.)

Dental pulp stem cells (DPSCs) are of interest to researchers and clinicians due to their ability to differentiate into various tissue types and potential uses in cell-mediated therapies and tissue engineering. Currently DPSCs are cryopreserved in suspension using Me(2)SO. However, preservation as two and three dimensional constructs, along with the elimination of toxic Me(2)SO, may be required. It was shown that intracellular ice formation (IIF), lethal to cells in suspensions, may be innocuous in cell monolayers due to ice propagation between cells through gap junctions that results in improved post-thaw recovery. We hypothesized that innocuous IIF protects confluent DPSC monolayers against injury during cryopreservation. The objective was to examine the effects of IIF on post-thaw viability of both confluent monolayers and suspensions of DPSCs. Confluent DPSC monolayers were assessed for the expression of gap junction protein Connexin-43. IIF was induced on the cryostage and in the methanol bath at different subzero temperatures. Membrane integrity and colony-forming ability were assessed post-thaw. Confluent DPSC monolayers expressed Connexin-43. In cell suspensions, 85.9+/-1.7% of cells were damaged after 100% IIF. In cell monolayers, after 100% IIF, only 25.5+/-5.5% and 14.8+/-3.3% of cells were damaged on the cryostage and in the methanol bath respectively. However, DPSC monolayers exposed to 100% IIF showed no colony-forming ability. We conclude that confluent monolayers of DPSCs express the gap junction-forming protein Connexin-43 and upon IIF retain membrane integrity, however lose the ability to proliferate., ((c) 2010 Elsevier Inc. All rights reserved.)

Intercellular communication is an important tool used by the cells to effectively regulate concerted responses. Hepatic stellate cells (HSCs) communicate to each other through functional gap junctions composed of connexin 43 (Cx43) proteins. We show that exogenous human TGF-beta1 (hTGF-beta1), a pro-fibrotic stimulus, decreases Cx43 mRNA and protein in a rat HSC cell line and primary HSCs. Furthermore, hTGF-beta1 increases the phosphorylation of Cx43 at serine 368. These effects lead to a decrease in the gap junction intercellular communication between the HSCs, as shown by gap-FRAP analysis. We also observe the binding of Snai1, from the nuclear extract of HSCs, to a Snai1 consensus sequence in the Cx43 promoter. In the same context, Snai1 siRNA transfection results in an up-regulation of Cx43 suggesting that TGF-beta1 may regulate Cx43 via Snai1. In addition, we demonstrate that the knockdown of Cx43 by siRNA transfection results in a slower proliferation of HSCs. These findings illuminate a new effect of TGF-beta1 in HSCs, namely the regulation of intercellular communication by affecting the expression level and the phosphorylation state of Cx43 through Snai1 signaling.

Carcinoma in situ (CIS) represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap junctional proteins, connexin 43 (Cx43) represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and germ cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated, that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development.

Ochratoxin A (OTA) is one of the most potent renal carcinogens studied to date, but the mechanism of tumor formation by ochratoxin A remains largely unknown. Cell adhesion and cell-cell communication participate in the regulation of signaling pathways involved in cell proliferation and growth control and it is therefore not surprising that modulation of cell-cell signaling has been implicated in cancer development. Several nephrotoxicants and renal carcinogens have been shown to alter cell-cell signaling by interference with gap junction intercell communication (GJIC) and/or cell adhesion, and the aim of this study was to determine if disruption of cell-cell interactions occurs in kidney epithelial cells in response to OTA treatment. MDCK cells were treated with OTA (0-50 microM) for up to 24h and gap junction function was analyzed using the scrape-load/dye transfer assay. In addition, expression and intracellular localization of C x 43, E-cadherin and beta-catenin were determined by immunoblot and immunofluorescence analysis. A clear decrease in the distance of dye transfer was evident following treatment with OTA at concentrations/incubation times which did not affect cell viability. Consistent with the functional inhibition of GJIC, treatment with OTA resulted in a dose-dependent decrease in C x 43 expression. In contrast to C x 43, OTA did not alter total amount of the adherens junction proteins E-cadherin and beta-catenin. Moreover, Western blot analysis of Triton X-100 soluble and insoluble protein fractions did not indicate translocation of cell adhesion molecules from the membrane to the cytoplasm. However, a approximately 78 kDa fragment of beta-catenin was detected in the detergent soluble fraction, indicating proteolytic cleavage of beta-catenin. Immunofluorescence analysis also revealed changes in the pattern of both beta-catenin and E-cadherin labeling, suggesting that OTA may alter cell-adhesion. Taken together, these data support the hypothesis that disruption of cell-cell signaling may contribute to OTA toxicity and carcinogenicity.

In this study, we examined the interaction of the osteoblast which forms bone and sulfated hyaluronan (SHya). For the purpose of the creation of a new functional polysaccharide, we introduced a sulfate group in hyaluronan (Hya) of high molecular weight, and SHya of high molecular weight could be obtained for the first time. When rat calvarial osteoblast (rOB) cells were cultured with a high concentration of SHya, they formed aggregated spheroids after 4h and the spheroids grew to about 200microm after 24h. We examined the expression of cell adhesion molecules in order to clarify the mechanism of aggregate formation. The N-cadherin (N-cad) and Connexin43 (Cx43) expression level of rOB cells cultured with SHya remarkably increased after 2h. A difference in the expression of Integrin beta1 (Intbeta1) could not be observed between the SHya addition and control group. The alkaline phosphatase (ALPase) activity of rOB cells cultured with SHya after 8h was significantly enhanced in comparison with control. Therefore, the sulfate group of SHya seems to enhance expression of cell adhesion protein such as N-cad and Cx43, resulting in aggregate formation and further remarkable induction of the ALPase activity of rOB cells.

Several studies have demonstrated that connexin 43 (Cx43) mediates signals important for osteoblast function and osteogenesis. The role of gap junctional communication in bone resorption is less clear. We have investigated the expression of Cx43 mRNA in osteoclasts and bone resorption cultures and furthermore, the functional importance of gap junctional communication in bone resorption. RT-PCR analysis demonstrated Cx43 mRNA expression in mouse bone marrow cultures and in osteoclasts microisolated from the marrow cultures. Cx43 mRNA was also expressed in bone resorption cultures with osteoclasts and osteoblasts/stromal cells incubated for 48h on devitalized bone slices. An up-regulation of Cx43 mRNA was detected in parathyroid (PTH)-stimulated (0.1 nM) bone resorption. Two inhibitors of gap junction communication, 18alpha-glycyrrhetinic acid (30 microM) and oleamide (100 microM), significantly inhibited PTH- and 1,25-(OH)(2)D(3)-stimulated osteoclastic pit formation. In conclusion, our data indicate a functional role for gap junction communication in bone resorption.

Okadaic acid (OA) is a phycotoxin produced by dinoflagellates. It accumulates in the digestive tracts of shellfish causing diarrhetic shellfish poisoning (DSP) in consumers. OA is a tumour promoter, and an inhibitor of both protein phosphatases and protein synthesis. OA induces DNA adducts, suggesting it may be carcinogenic. Since the Ames test without S(9) was negative, but a mutagenesis test was positive in mammalian cells, the question as to whether its molecular mechanism is genotoxic or epigenetic became unavoidable. Therefore, experiments were performed to search for epigenetic effects, since evidence for DNA-adduct formation using the gamma-(32)P-ATP post-labelling method was not obtained. We found that OA is a potent inducer of lipid peroxidation in human intestinal cells (Caco-2) at low concentrations (0.75-7.5 ng/ml versus IC50 of 15 ng/ml) with increased rates of 8-OH-dG and m(5)dC formation causing CG to AT transversion mutations and gene deregulation, respectively. The transcription and translation of connexin 43-specific mRNA were inhibited, and 3H-uridine incorporation in RNA was concomitantly increased. Consequently gap junction intracellular communication (GJIC) was inhibited, making possible cellular anarchic proliferation. Higher OA concentrations also disorganized the cellular cytoskeleton, since both actin and tubulin formations were impaired. Our results suggest that OA may induce tumours via an epigenetic mechanism.

Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of focal segmental glomerulosclerosis. However, it is poorly understood how podocytes respond to injury. In this study, glomerular expression of connexin43 (Cx43) gap junction protein was examined at both protein and transcript levels in an experimental model of podocyte injury, puromycin aminonucleoside (PAN) nephrosis. A striking increase in the number of immunoreactive dots with anti-Cx43 antibody was demonstrated along the glomerular capillary wall in the early to nephrotic stage of PAN nephrosis. The conspicuous change was not detected in the other areas including the mesangium and Bowman's capsule. Immunoelectron microscopy showed that the immunogold particles for Cx43 along the capillary wall were localized predominantly at the cell-cell contact sites of podocytes. Consistently, Western blotting and ribonuclease protection assay revealed a distinct increase of Cx43 protein, phosphorylation, and transcript in glomeruli during PAN nephrosis. The changes were detected by 6 hours after PAN injection. These findings indicate that the increase of Cx43 expression is one of the earliest responses that have ever been reported in podocyte injury. To show the presence of functional gap junctional intercellular communication (GJIC) in podocytes, GJIC was assessed in podocytes in the primary culture by transfer of fluorescent dye, Lucifer yellow, after a single-cell microinjection. Diffusion of the dye into adjacent cells was observed frequently in the cultured podocytes, but scarcely in cultured parietal epithelial cells of Bowman's capsule, which was compatible with their Cx43 staining. Thus, it is concluded that Cx43-mediated GJIC is present between podocytes, suggesting that podocytes may respond to injury as an integrated epithelium on a glomerulus rather than individually as a separate cell.

We examined the effect of 2',5'-dihydroxychalcone on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). The result showed that expression of Cx43 is rapidly reduced by 2',5'-dihydroxychalcone in a dose-dependent manner, Concomitantly, the communication function, determined by fluorescence recovery after photobleaching (FRAP), is decreased. We further investigated whether the mitogen-activated protein (MAP) kinase and the degradation pathway of gap junctions are involved in these processes. Although the change of Cx43 is not affected by the level of fetal calf serum (FCS) used in the medium, activation of MAP kinase varies, depending on the FCS level. At a low level (0.5%), the chalcone inhibits the activation, like PD98059, a specific inhibitor of MAP kinase kinase. However, at a high level (20%), MAP kinase is activated. On the other hand, the chalcone's down-regulating effect on Cx43, while is totally blocked by protease inhibitors leupeptin and N-acetyl-leucyl-norleucinal (ALLN), persists in the presence of PD98059, We concluded that 2',5'-dihydroxychalcone down-regulates Cx43 expression and gap-junctional communication in the HUVEC via enhancement of the proteolysis pathway, and this compound possesses dual effects on MAP kinase activation.

Gap junctional intercellular communication and expression of gap junction proteins (connexins) are decreased frequently in neoplastic cells including human ovarian carcinoma cells. In order to test the hypothesis that these changes contribute to the neoplastic phenotype of ovarian carcinoma cells, we transfected human ovarian carcinoma SKOV-3 cells with connexin43. Stable, connexin43-expressing transfectants were characterized for cell proliferation in vitro in normal, low-serum, and serum-free culture medium, for tumorigenicity in nude mice, and for sensitivity to adriamycin in vitro. Transfected clones expressed higher levels of connexin43 and gap junctional intercellular communication, reduced proliferation and greater dependence upon serum for growth in vitro, decreased tumor formation, increased sensitivity to adriamycin, and reduced expression of p-glycoprotein. These data suggest that gap junctional intercellular communication and/or connexin43 expression suppresses the neoplastic phenotype of ovarian carcinoma cells and their downregulation is involved in neoplastic transformation of ovarian epithelial cells. The increased sensitivity to adriamycin and elevated expression of p-glycoprotein by the transfected cells also suggest that gap junctional intercellular communication and connexin43 expression are involved in drug sensitivity and might be manipulated to enhance the clinical response.

Objective: Our purpose was to test the hypothesis that nitric oxide signals regulate the expression of the gap-junction protein connexin 43 in primary uterine myocytes from pregnant women at term., Study Design: Northern analysis and immunoblotting were used to determine the expression of connexin 43 in myocytes cultured in the presence of the nitric oxide donors S -nitroso-N -acetyl-penicillamine (SNAP) (100 micromol/L) and (Z)-1-[2-(2-aminoethyl)-N -(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate diethylenetriamine (NOC-18) (100 micromol/L). We also tested the effect of the NO stimulants 8-bromo-cyclic adenosine 3',5'-monophosphate (cAMP) (100 micromol/L) and 8-bromo-cyclic guanosine 3',5'-monophosphate (cGMP) (200 micromol/L), and the nitric oxide synthase inhibitors N -nitro-L-arginine methyl ester (NAME) (100 micromol/L) and L -N (1-iminoethyl)lysine (NIL) (50 micromol/L)., Results: Nitric oxide and 8-bromo-cAMP reduced the level of connexin 43 expression, and 8-bromo-cGMP had no effect. In contrast, NIL, but not NAME, increased the levels of connexin 43 protein without affecting the level of connexin 43 messenger RNA. With immunoblotting, expression of inducible nitric oxide synthase was not detected in these cells., Conclusion: Nitric oxide down-regulates the expression of connexin 43 in cultured human myocytes. We speculate that this effect may decrease the efficacy of intermyocyte signaling and thus contribute to uterine quiescence during human pregnancy.

To study the mechanism(s) underlying the proliferation of heterogeneous cell populations within a solid tumour, the NBT-II rat bladder carcinoma system was used. It has been first investigated whether the different cell populations are coupled through gap junctions (GJIC). Cells overexpressing the Cx43 were generated to test for any tumour suppressive activity in vivo. To determine whether GJIC is essential for tumour proliferation and the establishment of a cooperative community effect, NBT-II cells that are incompetent for cell coupling were generated. The data report that (i) carcinoma cells expressing or not FGF-1 are coupled through GJIC in vitro and in coculture and express the gap junction protein Cx43, (ii) overexpression of Cx43 in these cells does not affect their in vitro coupling capacities and in vivo tumourigenic growth properties, (iii) inhibition of GJIC through antisense strategy has no in vivo obvious consequence on the tumour growth properties of the carcinoma, and (iv) the community effect between two carcinoma cell populations does not critically involve cell coupling through gap junctions.

Developmental regulation of growth promoting activities in mammary secretions of pregnant Awassi ewes was defined, and growth factors contained in these secretions were partially purified and characterised. Mammary secretions from pregnant ewes enhanced fibroblast cell (AKR-2B) and mammary cell (CID-9 cell strain) proliferation to levels comparable to that induced by 10% Foetal calf serum. Major milk proteins in mammary secretions collected from pregnant ewes one month prior to lambing up to one week after lambing, were resolved by SDS-PAGE, while gelatinases were resolved by zymography. Gelatinase activity was noted prior to P134 and decreased thereafter to reach a minimum during lactation. This decrease was concomitant with the onset of casein production. It is during this critical developmental period that highest growth promoting activity in mammary secretions was detected. Secretions with highest growth promoting activity were fractionated by ion exchange and gel filtration chromatography. Two heat-resistant, trypsin/chymotrypsin sensitive, growth-promoting activities were characterised. The first, designated ovine mammary derived growth factor-1 (oMDGF-1), had around a 30 kDa molecular weight and eluted at 0.65 M NaCl gradient on cation ion exchange chromatography. The second, oMDGF-2, eluted under gel filtration conditions at a molecular weight of 50 kDa and 150 kDa. oMDGF-1 induced changes in Connexin 43, but not in beta-casein mRNA expression by CID-9 mammary cells. In conclusion, growth factor activities in ewe mammary secretions peak during gestation at a period that overlaps maximal gelatinase expression and precedes milk protein synthesis. The factors modulate mammary cell function and may play a role in mammary gland development.

Mutations in the human GJB3 gene that codes for Connexin31 (Cx31), a protein subunit of gap junction channels, have recently been reported to cause deafness and the skin disorder erythrokeratodermia variabilis. To study the function of this gene in mice, we generated animals with targeted replacement of the Cx31 gene (Gjb3) by a lacZ reporter gene. Although homozygous Cx31-deficient adult mice (Gjb3(-/-)) were found among the offspring of heterozygous Cx31-deficient parents (Gjb3(+/-)), 60% of the animals expected according to Mendelian inheritance were lost between ED 10.5 and 13.5. Placentas of Gjb3(-/-) embryos at ED 9.5 were smaller than controls as a result of severely reduced labyrinth and spongiotrophoblast size. From ED 10.5 onward, placentas of surviving Gjb3(-/-) embryos recovered progressively and reached normal size and morphology by ED 18.5. This corresponds to a time period in which another connexin isoform, Connexin43, is upregulated in spongiotrophoblast cells of Cx31-deficient and control placentas. No morphological or functional defects of skin or inner ear were observed in surviving adult Gjb3(-/-) mice. We conclude that Cx31 is essential for early placentation but can be compensated for by other connexins in the embryo proper and adult mouse., (Copyright 2001 Academic Press.)

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.

We used transgenic mice in which the promoter sequence for connexin 43 linked to a lacZ reporter was expressed in neural crest but not myocardial cells to document the pattern of cardiac neural crest cells in the caudal pharyngeal arches and cardiac outflow tract. Expression of lacZ was strikingly similar to that of cardiac neural crest cells in quail-chick chimeras. By using this transgenic mouse line to compare cardiac neural crest involvement in cardiac outflow septation and aortic arch artery development in mouse and chick, we were able to note differences and similarities in their cardiovascular development. Similar to neural crest cells in the chick, lacZ-positive cells formed a sheath around the persisting aortic arch arteries, comprised the aorticopulmonary septation complex, were located at the site of final fusion of the conal cushions, and populated the cardiac ganglia. In quail-chick chimeras generated for this study, neural crest cells entered the outflow tract by two pathways, submyocardially and subendocardially. In the mouse only the subendocardial population of lacZ-positive cells could be seen as the cells entered the outflow tract. In addition lacZ-positive cells completely surrounded the aortic sac prior to septation, while in the chick, neural crest cells were scattered around the aortic sac with the bulk of cells distributed in the bridging portion of the aorticopulmonary septation complex. In the chick, submyocardial populations of neural crest cells assembled on opposite sides of the aortic sac and entered the conotruncal ridges. Even though the aortic sac in the mouse was initially surrounded by lacZ-positive cells, the two outflow vessels that resulted from its septation showed differential lacZ expression. The ascending aorta was invested by lacZ-positive cells while the pulmonary trunk was devoid of lacZ staining. In the chick, both of these vessels were invested by neural crest cells, but the cells arrived secondarily by displacement from the aortic arch arteries during vessel elongation. This may indicate a difference in derivation of the pulmonary trunk in the mouse or a difference in distribution of cardiac neural crest cells. An independent mouse neural crest marker is needed to confirm whether the differences are indeed due to species differences in cardiovascular and/or neural crest development. Nevertheless, with the differences noted, we believe that this mouse model faithfully represents the location of cardiac neural crest cells. The similarities in location of lacZ-expressing cells in the mouse to that of cardiac neural crest cells in the chick suggest that this mouse is a good model for studying mammalian cardiac neural crest and that the mammalian cardiac neural crest performs functions similar to those shown for chick., (Copyright 1999 Academic Press.)

During chick limb development the gap junction protein Connexin-43 (Cx43) is expressed in discrete spatially restricted domains in the apical ectodermal ridge (AER) and mesenchyme of the zone of polarising activity. Antisense oligonucleotides (ODNs) were used to investigate the role of Connexin-43 (Cx43) in the development of the chick limb bud. We have used unmodified ODNs in Pluronic F-127 gel, which is liquid at low temperature but sets at room temperature and so remains situated at the point of application. As a mild surfactant, the gel increases antisense ODN penetration and supplies ODNs to the embryo continually for 12-18 h. We have shown a strong decrease in Cx43 protein expression after application of specific antisense oligonucleotides but the abundance of a closely related protein, Connexin-32 (Cx32), was not affected. Application of antisense Cx43 ODNs at stages 8-15 HH before limb outgrowth resulted in dramatic limb phenotypes. About 40% of treated embryos exhibited defects such as truncation of the limb bud, fragmentation into two or more domains, or complete splitting of the limb bud into two or three branches. Molecular analysis of antisense treated embryos failed to detect Shh or Bmp-2 in anterior structures and suggested that extra lobes seen in nicked and split limbs were not a result of establishment of new signalling centres as found after the application of FGF to the flank. However, examination of markers for the AER showed a number of abnormalities. In severely truncated specimens we were unable to detect the expression of either Fgf-4 or Fgf-8. In both nicked and split limbs the expression of these genes was discontinuous. Down-regulation of Cx43 after the antisense application could be comparable to AER removal and results in distal truncation of the limb bud. Taken together these data suggest the existence of a feedback loop between the FGFs and signalling mediated by Cx43., (Copyright 1999 Academic Press.)

Epithelial cells in primary ovine lens cultures express the gap junction proteins connexin43 (Cx43) and connexin49 (Cx49; a.k.a. MP70), a homologue of mouse connexin50. In contrast, lens cultures of differentiated, fiber-like cells (termed lentoid cells) express Cx49 and connexin46 (Cx46), but not Cx43. To investigate the regulation of lens cell gap junctions by protein kinase C (PKC), differentiating lens cultures were treated with the PKC activator 12-O-tetradecanoylphorbol-13-acetate (beta-TPA). Within 10 min, beta-TPA significantly inhibited the transfer of Lucifer Yellow dye between epithelial, but not lentoid, cells. This inhibition was correlated with the phosphorylation of Cx43 and was followed by the gradual disappearance of Cx43 from cell interfaces. The protein kinase inhibitor staurosporine prevented Cx43 phosphorylation and the loss of Cx43 from intercellular junctions. Following treatment of cultures with beta-TPA for 2-6 hr, Cx49 disappeared from epithelial cell interfaces, and by 24 hr of beta-TPA treatment, levels of Cx49 detected on immunoblots of purified epithelial membrane fractions had also diminished significantly. The beta-TPA-induced loss of Cx49 both from regions of epithelial cell contact and from isolated membranes was correlated with the disappearance of Cx49 mRNA. In contrast to the epithelial connexins, the lentoid connexins Cx49 and Cx46 were unaffected by even extended beta-TPA treatment. In spite of lentoid dye transfer being refractory to beta-TPA, significant levels of PKC-alpha (a beta-TPA-sensitive isoform) were detected in the lentoid cell. The response of lens gap junctions to beta-TPA depends upon the stage of differentiation and the complement of connexins expressed. The contrasting effects of beta-TPA on Cx43 and Cx49 in lens epithelial cells indicate a fundamental difference in the regulation of these connexin proteins in the developing mammalian lens., (Copyright 1997 Academic Press.)

Gap junction-mediated communication is required for normal cellular growth and differentiation. As cancer is thought to be a manifestation of the breakdown of cell-cell communication, with the concomitant loss of growth control, it would be expected that alterations in the primary structure, processing, oligomerization or trafficking of connexin (cxn) molecules would have a profound effect on the neoplastic process. Here we a present a preliminary immunohistochemical and molecular analysis of cxn 43 expression in prostatic epithelial cells from resected human tissue. Our data indicate that benign prostatic epithelial cells express cxn 43 protein, but that this expression is diminished in more advanced, anaplastic cancer cells. These data suggest that decreased connexin expression is not involved in the initiation of prostate cancer, but rather occurs during the progression of the disease.

Uterine leiomyomas, benign tumors of myometrial origin, are the most common neoplasms of the uterus and occur in 20-30% of women over 30 years of age. Despite their high prevalence, little is known about the pathophysiology of these tumors. Recently, several studies have compared leiomyoma gene expression with that of normal myometrium. In general, the major differences in leiomyoma and myometrial gene expression are found for estrogen-regulated genes, which have elevated expression in myometrium during pregnancy. The tumors appear to maintain high sensitivity to estrogen during the estrogen-dominated follicular phase of the menstrual cycle, unlike normal myometrium. The abnormal gene expression of leiomyomas suggests that they are tumors of dysregulated differentiation and resemble the myometrium of pregnancy in several aspects.

Objective: We tested the hypothesis that uterine leiomyomas are hypersensitive to estrogen as compared with autologous human myometrium., Methods: The estrogen-induced transcriptional responses of uterine leiomyoma and myometrial primary cultures were determined by transient expression assays. The relative levels of estrogen receptor (ER) in myometrial and leiomyoma tissues were determined by immunoblot., Results: Myometrial and leiomyoma primary cultures were transcriptionally responsive to the estrogen ethinyl estradiol (eE2). The partial agonist tamoxifen did not elicit a positive transcriptional response and antagonized estrogen-induced transcription in the cultured cells. The responses of hormone-treated leiomyoma cells averaged 4.5-fold higher than those in controls with no hormone (P = .0001). The myometrial cells from women in the follicular phase exhibited little if any transcriptional response to eE2, whereas myometrial cells from women in the luteal phase had a transcriptional response to eE2 averaging threefold higher than that in no-hormone controls (P = .0083). Differences in response between autologous myometrial and leiomyoma cultures were statistically significant by the two-tailed Wilcoxon paired nonparametric signed-rank test (n = 11; P = .0137). These differences were more pronounced in cultures from women in the follicular or early luteal phase. In addition, the levels of ER increased in follicular and early luteal phase myometrial tissues, which correlated well with the number of days from the last menstrual period (n = 8; r = 0.9046; P = .002). Estrogen-receptor levels in myometrial tissues decreased during the late luteal phase. Levels in leiomyoma tissues did not follow the same pattern as in the myometrium and were elevated in tissues taken from women in the follicular phase., Conclusions: Autologous leiomyoma cultures have a significantly higher response to estrogen than do matched myometrial cultures, especially if the cultures are derived from the follicular phase. The levels of ER in leiomyoma tissue from women in the follicular phase are significantly elevated.

Two distinct gap junction proteins (connexins) are expressed in rat corneal epithelium in a way which parallels cellular differentiation processes in this tissue. Connexin43 is restricted predominantly to the basal cells of the corneal epithelium and is present in significantly reduced amounts compared to the situation in the adjacent conjunctival epithelium. In contrast, a gap junction protein recognized by antibodies against MP70 which is the ovine homolog of mouse connexin50, is strongly expressed in the corneal epithelium and is present in the basal cells, wing cells and surface cells. While the functional significance of this differential expression of corneal epithelial connexins has yet to be established, the corneal epithelium is the third avascular tissue besides lens and heart valves which expresses a gap junction protein recognized by anti-MP70 antibodies.