Comprehensive Pan-Cancer Analysis of Connexin 43 as a Potential Biomarker and Therapeutic Target in Human Kidney Renal Clear Cell Carcinoma (KIRC).

Background and Objectives: Connexin 43 (Cx43) is involved in the transfer of small signaling molecules between neighboring cells, thereby exerting a major influence on the initiation and progression of tumorigenesis. However, there is a lack of systematic research on Cx43 expression and its predictive role in clinical diagnosis and prognosis in pan-cancer. Materials and Methods: Several biological databases were used to evaluate the expression levels of GJA1 (encoding Cx43) and its diagnostic and prognostic significance in pan-cancer. We targeted kidney renal clear cell carcinoma (KIRC) and investigated the relationship between GJA1 expression and different clinical features of KIRC patients. Then, we performed cell-based experiments to partially confirm our results and predicted several proteins that were functionally related to Cx43. Results: The expression of GJA1 has a high level of accuracy in predicting KIRC. High GJA1 expression was remarkably correlated with a favorable prognosis, and this expression was reduced in groups with poor clinical features in KIRC. Cell experiments confirmed the inhibitory effects of increased GJA1 expression on the migratory capacity of human renal cancer (RCC) cell lines, and protein-protein interaction (PPI) analysis predicted that CDH1 and CTNNB1 were closely related to Cx43. Conclusions: GJA1 could be a promising independent favorable prognostic factor for KIRC, and upregulation of GJA1 expression could inhibit the migratory capacity of renal cancer cells.

Upregulation of Connexins in the Rat Hippocampal and Cortical Neurons Following Blockade of NMDA Receptors During Postnatal Development.

BACKGROUND: Interneural gap junctional coupling represents neural development that decreases during the postnatal period. The decrease of gap junction function coincides with the main period of chemical synapse creation and increment of synaptic activity during postnatal weeks 1 to 3. METHODS: Here, we have assessed the role of chemical synapses on connexin (Cx) expression in neurons and glial cells of hippocampal and cortical neurons. We characterized the impact of NMDA receptors blockade on the expression of Cx36 and Cx43 proteins by western blot analysis in postnatal day (PND)14 and PND28. MK801 was injected subcutaneously from the first day of birth until 14 or 28 days, depending on the experimental groups. Saline was injected in the same volumes in the control group. RESULTS: Early postnatal blockade of the NMDA subtype of glutamate receptors by the non-competitive antagonist dizocilpine maleate (MK801) arrested the developmental reduction in gap junctions during the initial postnatal weeks. Expression of Cx43 declined in PND28 compared to PND14 in visual cortex (VC) neurons. Also, we found that the expression of Cx36 and Cx43 augmented in the rats' VC in PND28 following the blockade of NMDA receptors. Expression of Cx36 declined in PND28 compared to PND14 in hippocampal neurons. Also, we found that the expression of Cx36 augmented in the rats' hippocampal neurons in PND14 and PND28 following a blockade of NMDA receptors. CONCLUSION: These results suggest that the postnatal enhancement in glutamatergic synaptic activity is associated with the loss of gap junctional connections and downregulation of Cx36 and Cx43 between developing neurons and glial cells.

Estrogen Protects against Renal Ischemia-Reperfusion Injury by Regulating Th17/Treg Cell Immune Balance.

Inflammation is a critical mediator of renal ischemia-reperfusion (I/R) injury (IRI), and T lymphocytes exert a key role in the renal IRI-induced inflammation. Connexin 43 (Cx43) is related to the maintenance of T lymphocyte homeostasis. Various preclinical researches have reported that estrogen is a renoprotective agent based on its anti-inflammatory potential. The present research is aimed at studying the role of T lymphocytes activated by Cx43 in 17ß-estradiol-mediated protection against renal IRI. Female rats were classified into six groups: control rats, I/R rats, ovariectomized rats, ovariectomized I/R rats, and ovariectomized rats treated with 17ß-estradiol or gap27. Levels of serum creatinine (Scr) and blood urea nitrogen (BUN) and Paller scoring were dramatically increased in I/R rats, especially in ovariectomized rats. By contrast, these indicators were markedly decreased by administering estradiol or gap27. Immunofluorescence staining revealed that CD4+ T cells infiltrated kidney tissues in the early stage of IRI. In both peripheral blood and renal tissue, the proportion of CD3+CD4+ T cells and ratio of CD4+ to CD8+ were high in I/R rats, especially in ovariectomized rats. The proportion of CD3+CD8+ T cells was low in peripheral blood but high in renal tissues. Administration of estrogen or Gap27 reversed these effects. IL-17 levels in both serum and tissue homogenate were significantly increased in ovariectomized rats subjected to I/R but significantly decreased in estrogen or gap 27 treated rats. The opposite trend was observed for IL-10 levels. Correlation analysis demonstrated that IL-17 was correlated positively with BUN, Scr, and Paller scores, while IL-10 was negatively correlated with these indicators. Western blot showed that Cx43 expression was markedly increased in the peripheral blood T lymphocytes of I/R rats, especially ovariectomized rats. After intervention with estrogen and gap27, Cx43 expression was significantly downregulated. These findings indicate that Cx43 may participate in the regulation of Th17/Treg balance by estrogen against renal IRI.

Expression of connexin-43 in surgical resections of primary tumor and lymph node metastases of squamous cell carcinoma and adenocarcinoma of the lung: a retrospective study.

Background: Connexins are transmembrane proteins forming gap junctions between the cells, which allow intercellular communication. Significance of gap junctions and connexins in lung carcinoma is not yet understood. The objective of the study was to investigate immunohistochemical expression and the localization of connexin-43 (Cx43) in primary lung carcinoma and its lymphatic metastases. Methods: Surgical specimens of excised tumors from 88 patients (45 men and 43 women, 61.9 ± 7.4 years) with lung carcinoma (52 adenocarcinoma (AC), 36 squamous cell carcinoma (SqCC)) who were operated on at the University Hospital "Bezanijska Kosa" in a five-year period (2012-2016) were used. We conducted immunohistochemical staining for Cx43 and measured the degree of expression (percentage of positive cells and staining intensity) as well as localization of Cx43 in primary tumor and in lymphatic metastases. Results: Immunohistochemical analysis of the primary tumors revealed that SqCC showed significantly higher percentage of tumor cells expressing Cx43 as well as higher staining intensity than AC (p < 0.001). Almost 70% of samples with SqCC showed high Cx43 expression, whereas AC showed no expression in more than 50% of cases. Localization of Cx43 expression was most often cytoplasmic (AC and SqCC) and combined membranous and cytoplasmic (SqCC) with very rare instances of nuclear localization (AC). Almost the same pattern in distribution, intensity, and localization of Cx43 expression was observed in the lymph node metastases; however, almost a third of AC cases changed the pattern of Cx43 expression in the metastasis compared to primary tumor. Conclusion: The results of this study showed that lung carcinomas express Cx43 in more than 65% of cases and that it was aberrantly localized (not membranous localization). We highlighted that SqCC expressed Cx43 more than did AC, both in primary tumor and lymphatic metastases. Further research is needed to establish whether Cx43 could be used as a prognostic biomarker in lung carcinoma.

The Relationship between the Connexin 32 and Connexin 43 Genes and the Pretreatment Stage and Short-term Follow-up of Patients with Acute Myeloid Leukemia.

Background: Connexins (Cxs) are gap junction proteins involved in the communication between acute myeloid leukemia (AML) and stromal cells. They consist of intercellular channels termed "connexions", which can cause uncontrolled cell proliferation if dysregulated. This study aimed to evaluate the expression levels of the Cx32 and Cx43 genes and their correlations with other prognostic markers in patients with AML. Methods: This cross sectional study was performed on peripheral blood samples from 60 newly diagnosed patients with AML and 40 healthy control subjects at Kasr Alainy School of Medicine, Cairo University, from June 2016 to December 2017. The quantitative real-time polymerase chain reaction (qRT-PCR) test was used to examine the relative expression level of Cx43 and Cx32 genes in the patients and the control subjects. The Chi square test or the Fisher exact test was employed to examine the relationship between qualitative variables, while the independent t test or the Mann-Whitney test was employed for quantitative data. All the tests were two-tailed, and a P value of less than 0.05 was considered significant. Results: Among the patients with AML, 65% had a high Cx32 expression level, whereas 63.3% had a low Cx43 expression level. There was a statistically significant difference in the fold change values of Cx32 and Cx43 expression between the patient group and the control group (P=0.009 vs P=0.013, respectively). There was a remarkable association between both Cxs and CD34 and HLA-DR cells. Conclusion: Cx expression in samples may add to the diagnostic workup of AML. Although we found a negative correlation between Cx43 expression and the peripheral blood blast percentage, the response after the first induction of chemotherapy showed no significant relationship with Cx43 and Cx32.

A role of connexin 43 on the drug-induced liver, kidney, and gastrointestinal tract toxicity with associated signaling pathways.

Drug-induced organ toxicity/injury, especially in the liver, kidney, and gastrointestinal tract, is a systematic disorder that causes oxidative stress formation and inflammation resulting in cell death and organ failure. Current therapies target reactive oxygen species (ROS) scavenging and inhibit inflammatory factors in organ injury to restore the functions and temporary relief. Organ cell function and tissue homeostasis are maintained through gap junction intercellular communication, regulating connexin hemichannels. Mis-regulation of such connexin, especially connexin (Cx) 43, affects a comprehensive process, including cell differentiation, inflammation, and cell death. Aim to describe knowledge about the importance of connexin role and insights therapeutic targeting. Cx43 misregulation has been implicated in recent decades in various diseases. Moreover, in recent years there is increasing evidence that Cx43 is involved in the toxicity process, including hepatic, renal, and gastrointestinal disorders. Cx43 has the potential to initiate the immune system to cause cell death, which has been activated in the acceleration of apoptosis, necroptosis, and autophagy signaling pathway. So far, therapies targeting Cx43 have been under inspection and are subjected to clinical trial phases. This review elucidates the role of Cx43 in drug-induced vital organ injury, and recent reports compromise its function in the major signaling pathways.

Differential Action of Connexin Hemichannel and Pannexin Channel Therapeutics for Potential Treatment of Retinal Diseases.

Dysregulation of retinal function in the early stages of light-induced retinal degeneration involves pannexins and connexins. These two types of proteins may contribute to channels that release ATP, leading to activation of the inflammasome pathway, spread of inflammation and retinal dysfunction. However, the effect of pannexin channel block alone or block of both pannexin channels and connexin hemichannels in parallel on retinal activity in vivo is unknown. In this study, the pannexin channel blocker probenecid and the connexin hemichannel blocker tonabersat were used in the light-damaged rat retina. Retinal function was evaluated using electroretinography (ERG), retinal structure was analyzed using optical coherence tomography (OCT) imaging and the tissue response to light-induced injury was assessed immunohistochemically with antibodies against glial fibrillary acidic protein (GFAP), Ionized calcium binding adaptor molecule 1 (Iba-1) and Connexin43 (Cx43). Probenecid did not further enhance the therapeutic effect of connexin hemichannel block in this model, but on its own improved activity of certain inner retina neurons. The therapeutic benefit of blocking connexin hemichannels was further evaluated by comparing these data against results from our previously published studies that also used the light-damaged rat retina model. The analysis showed that treatment with tonabersat alone was better than probenecid alone at restoring retinal function in the light-damaged retina model. The results assist in the interpretation of the differential action of connexin hemichannel and pannexin channel therapeutics for potential treatment of retinal diseases.

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

PURPOSE: High LET including alpha radiation-based approaches have been proved as a promising mode for cancer therapy owing to their biophysical and radiobiological advantages compared to photon beams. Studies pertaining to effect of α-radiation on cancer cells are limited to cytotoxic high doses. MATERIALS AND METHODS: In this study, human lung adenocarcinoma (A549) cells were α-irradiated using 241Am α-irradiator and effects of low dose of alpha radiation on these cells was studied under in vitro and in vivo conditions. RESULTS: Clonogenic and other assays showed increased cellular proliferation at lower doses (1.36 and 6.8 cGy) but killing at higher doses (13.6-54.4 cGy). Further studies at low dose of alpha (1.36 cGy) showed increased TGF-ß1 in the conditioned medium (CM) at early time point (24 h) but CM replacement did not affect the clonogenic survival. In these cells, increased phosphorylation of connexin 43 was correlated with decrease in gap-junction communication observed by dye transfer co-culture experiment. A decrease in caveolin-1 but increase in survivin expression was observed in low dose α-irradiated cells. An increase in cyclinD1 and decrease in Bcl-2, the target proteins of survivin, was observed in these cells. Low dose α-irradiated cancer cells transplanted in SCID mice showed significantly higher tumor volume, which was accompanied with an increased fraction of mitotic and PCNA/Ki67 positive cells in these tumor tissues. CONCLUSIONS: Taken together, our results suggest an increase in proliferation and tumor volume at in vitro and in vivo levels, respectively, when A549 cells were irradiated with low dose of α-radiation. These findings may be relevant for a better understanding of radiobiological processes during high LET-based cancer radiotherapy.

Clinicopathological Bird's-Eye View of Left Atrial Myocardial Fibrosis in 121 Patients With Persistent Atrial Fibrillation: Developing Architecture and Main Cellular Players.

BACKGROUND: Scientific research on atrial fibrosis in atrial fibrillation (AF) has mainly focused on quantitative or molecular features. The purpose of this study was to perform a clinicoarchitectural/structural investigation of fibrosis to provide one key to understanding the electrophysiological/clinical aspects of AF. METHODS: We characterized the fibrosis (amount, architecture, cellular components, and ultrastructure) in left atrial biopsies from 121 patients with persistent/long-lasting persistent AF (group 1; 59 males; 60±11 years; 91 mitral disease-related AF, 30 nonmitral disease-related AF) and from 39 patients in sinus rhythm with mitral valve regurgitation (group 2; 32 males; 59±12 years). Ten autopsy hearts served as controls. RESULTS: Qualitatively, the fibrosis exhibited the same characteristics in all cases and displayed particular architectural scenarios (which we arbitrarily subdivided into 4 stages) ranging from isolated foci to confluent sclerotic areas. The percentage of fibrosis was larger and at a more advanced stage in group 1 versus group 2 and, within group 1, in patients with rheumatic disease versus nonrheumatic cases. In patients with AF with mitral disease and no rheumatic disease, the percentage of fibrosis and the fibrosis stages correlated with both left atrial volume index and AF duration. The fibrotic areas mainly consisted of type I collagen with only a minor cellular component (especially fibroblasts/myofibroblasts; average value range 69-150 cells/mm2, depending on the areas in AF biopsies). A few fibrocytes-circulating and bone marrow-derived mesenchymal cells-were also detectable. The fibrosis-entrapped cardiomyocytes showed sarcolemmal damage and connexin 43 redistribution/internalization. CONCLUSIONS: Atrial fibrosis is an evolving and inhomogeneous histological/architectural change that progresses through different stages ranging from isolated foci to confluent sclerotic zones which-seemingly-constrain impulse conduction across restricted regions of electrotonically coupled cardiomyocytes. The fibrotic areas mainly consist of type I collagen extracellular matrix and, only to a lesser extent, mesenchymal cells.

Connexin43 expression in bone marrow derived cells contributes to the electrophysiological properties of cardiac scar tissue.

Cardiac pathologies associated with arrhythmic activity are often accompanied by inflammation. The contribution of inflammatory cells to the electrophysiological properties of injured myocardium is unknown. Myocardial scar cell types and intercellular contacts were analyzed using a three-dimensional reconstruction from serial blockface scanning electron microscopy data. Three distinct cell populations were identified: inflammatory, fibroblastic and endocardial cells. While individual fibroblastic cells interface with a greater number of cells, inflammatory cells have the largest contact area suggesting a role in establishing intercellular electrical connections in scar tissue. Optical mapping was used to study the electrophysiological properties of scars in fetal liver chimeric mice generated using connexin43 knockout donors (bmpKO). Voltage changes were elicited in response to applied current pulses. Isopotential maps showed a steeper pattern of decay with distance from the electrode in scars compared with uninjured regions, suggesting reduced electrical coupling. The tissue decay constant, defined as the distance voltage reaches 37% of the amplitude at the edge of the scar, was 0.48 ± 0.04 mm (n = 11) in the scar of the bmpCTL group and decreased 37.5% in the bmpKO group (n = 10). Together these data demonstrate inflammatory cells significantly contribute to scar electrophysiology through coupling mediated at least partially by connexin43 expression.

Impact of left ventricular assist device therapy on the cardiac proteome and metabolome composition in ischemic cardiomyopathy.

The changes in the myocardial proteome and metabolome associated with left ventricular assist device (LVAD) therapy in patients with ischemic cardiomyopathy (ICM) are poorly characterized. We investigated the impact of mechanical unloading following LVAD therapy on the myocardial proteome and metabolome. Matched samples of 5 patients' myocardial tissue, harvested at the time of LVAD implant ("pre-LVAD") or heart transplant ("post-LVAD"), were studied by quantitative proteomics and metabolomics as well as being probed for T-tubule structure and connexin-43 distribution. Moreover, pre-LVAD proteome profiles of ICM context were bioinformatically compared to pre-LVAD proteome profiles of dilated cardiac myopathy (DCM). More than 2120 proteins were reliably identified and quantified in paired patient samples. LVAD therapy led to proteomic remodeling, including reduced levels of α-1-antichymotrypsin together with an overall decrease of immune response proteins and an increase of proteins involved in membrane biology. Metabolomics highlighted increased glucose and glucose-6-phosphate levels in the left ventricle upon LVAD therapy. Wheat germ agglutinin staining demonstrated improved T-tubule structure. Connexin-43 displayed a trend for more pronounced intercalated disc localization. In comparing pre-LVAD proteome profiles of ICM context with pre-LVAD proteome profiles of dilated cardiac myopathy (DCM), we noticed an overrepresentation in ICM of proteins associated with humoral immune response. Our findings underline an impact of LVAD therapy on left ventricular biology in ICM. The proteomic, metabolomic, and structural alterations described here are typically associated with cardiac recovery. On the molecular level, our findings indicate the possibility of cardiac remodeling under LVAD therapy in ICM.

The liver injury following ischemia and reperfusion is worse in experimental knockout heterozygote mouse model for expression of connexin 431.

PURPOSE: To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury. METHODS: We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology. RESULTS: The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group. CONCLUSIONS: This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.

The liver injury following ischemia and reperfusion is worse in experimental knockout heterozygote mouse model for expression of connexin 43

Abstract Purpose: To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury. Methods: We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology. Results: The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group. Conclusions: This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.

Immunohistochemical and Ultrastructural Features of the Seasonal Changes in the Epididymal Epithelium of Camel (Camelus dromedarius).

In order to evaluate the influence of reproductive activity on the functional role of the epididymal epithelium in the Egyptian dromedary camel, Connexin-43 (Cx-43), vascular endothelial growth factor (VEGF), and androgen receptor (AR) immunoreactivity in the epididymal epithelium and the fine structure of the principal, dark, basal, apical, and halo cells were investigated. The secretory activity of the principal cells was amplified in the breeding season, while its endocytotic function became more active in the nonbreeding season. This was evidenced by punctate strong immunoreactive signals for Cx-43, which appeared to be more intense in the apical region of these epithelial cells, and the extremely long slender stereocilia (microvilli) with multiple junctional complexes. The nonbreeding principal cells revealed granular immunoreactive signals for VEGF scattered in the apical and basal cytoplasm. Ultrastructurally, both extreme vacuolation and several multivesicular inclusion bodies were observed in their cytoplasm. Dark cell size greatly diminished in the nonbreeding season and their nuclear morphology greatly changed from oval to lobulated shape. The plasma membrane of the apical cells expressed several infoldings (microvilli) in the breeding season. However, it was almost smooth in the nonbreeding season except for a small microvillus that appeared as a bleb-like projection. In some regions, a strong dense immunoreactivity for VEGF could be recognized in the cytoplasm of the apical cells and some basal ones. Halo cells with numerous multivesicular inclusions occupying most of the cytoplasm and a lobulated eccentric nucleus were detected in the nonbreeding season. In conclusion, these findings indicate that the reproductive activity has a significant impact on the immunohistochemical and ultrastructural profiles of the epithelial cells lining the Egyptian dromedary camel epididymis.

Novel Targets for Therapy of Renal Fibrosis.

Renal fibrosis is an important component of chronic kidney disease, an incurable pathology with increasing prevalence worldwide. With a lack of available therapeutic options, end-stage renal disease is currently treated with renal replacement therapy through dialysis or transplantation. In recent years, many efforts have been made to identify novel targets for therapy of renal diseases, with special focus on the characterization of unknown mediators and pathways participating in renal fibrosis development. Using experimental models of renal disease and patient biopsies, we identified four novel mediators of renal fibrosis with potential to constitute future therapeutic targets against kidney disease: discoidin domain receptor 1, periostin, connexin 43, and cannabinoid receptor 1. The four candidates were highly upregulated in different models of renal disease and were localized at the sites of injury. Subsequent studies showed that they are centrally involved in the underlying mechanisms of renal fibrosis progression. Interestingly, inhibition of either of these proteins by different strategies, including gene deletion, antisense administration, or specific blockers, delayed the progression of renal disease and preserved renal structure and function, even when the inhibition started after initiation of the disease. This review will summarize the current findings on these candidates emphasizing on their potential to constitute future targets of therapy.

Expression of Connexin 43 (Cx43) in Benign Cutaneous Tumors With Follicular Differentiation.

INTRODUCTION: Benign cutaneous tumors with follicular differentiation are alleged to differentiate toward parts of the hair follicle. Connexin 43 (Cx43) is a gap junction protein, the tumoral role of which has been investigated in several types of tumors. OBJECTIVE: To study the pattern of expression of Cx43 in benign cutaneous tumors with follicular differentiation and to compare it with that shown by their alleged anatomical counterparts of the hair follicle. MATERIALS AND METHODS: Five cases each of trichofolliculoma, trichilemmoma, fibrofolliculoma/trichodiscoma, trichoblastoma, trichoepithelioma, pilomatrixoma, and proliferating trichilemmal tumor, 3 cases of pilar sheath acanthoma, and 1 case of tumor of the follicular infundibulum were examined. Anti-Cx43 antibody was used. RESULTS: Cx43 was expressed by all follicular tumors studied. Comparisons between trichoblastoma and trichoepithelioma and their respective normal counterparts could not be made. In 3 tumors (trichofolliculoma, pilomatrixoma, and the spectrum fibrofolliculoma/trichodiscoma), there was a parallelism between their Cx43 expression pattern and that of their alleged anatomical counterparts. In pilar sheath acanthoma, trichilemmoma, and the tumor of the follicular infundibulum, we only found partial similarities in Cx43 expression. Only the proliferating trichilemmal tumor showed a discordant pattern of expression. CONCLUSIONS: Cx43 expression is preserved in benign cutaneous tumors with follicular differentiation and the patterns of Cx43 expression in benign cutaneous tumors with follicular differentiation parallel those of their alleged anatomical counterparts in 5 types (either totally or partially). This preservation might be related to the good behavior of the entities studied.

Connexin 43 is an independent predictor of patient outcome in breast cancer patients.

PURPOSE: Gap junctions are specialized membrane structures that form channels between adjacent cells allowing cell communication. Gap junctions and specifically Connexin 43 (Cx43) are down-regulated in cancer; however, there are contrasting reports on how this effects breast cancer patient survival. This paper is the first large-scale tissue microarray analysis of Cx43 expression in breast cancer patients with an associated clinical long-term follow-up. METHODS: Using a validated TMA of 1118 primary breast cancers, coupled to a comprehensive database of clinicopathological variables, the expression levels and subcellular localisation of Cx43 was assessed by immunohistochemistry. Its impact in terms of survival, distant metastasis-free survival, and clinicopathological variables was determined. RESULTS: Patients whose tumors expressed high levels of Cx43 had significantly better survival (p < 0.001) than patients with low levels. High Cx43 expression within tumors was associated with an 18-month survival advantage. Loss of Cx43 expression was associated with markers of poor prognosis, namely large tumor size, high grade, high proliferation status, high pleomorphism, high mitosis, poor Nottingham Prognostic Index (NPI), and triple negative tumors. Cx43 expression was independent of tumor size, grade, stage and ER-status in predicting poor survival on multivariate analysis (p = 0.004). CONCLUSION: Connexin 43 (Cx43) is an independent predictor of breast cancer survival and distant metastasis-free survival. High expression of Cx43 was seen in only 13% of tumors, suggesting that drugs to increase Cx43 expression may result in prolonged patients survival.

Extracellular Matrix Elasticity Regulates Osteocyte Gap Junction Elongation: Involvement of Paxillin in Intracellular Signal Transduction.

BACKGROUND/AIMS: Osteocytes can sense and respond to extracellular stimuli, including biochemical factors throughout the cell body, dendritic processes, and cilia bending. However, further exploration is required of osteocyte function in response to substrate stiffness, an important passive mechanical cue at the interface between osteocytes and the extracellular matrix, and the deep bio-mechanism in osteocytes involving mechanosensing of cell behavior. METHODS: We fabricated silicon-based elastomer polydimethylsiloxane substrates with different stiffnesses but with the same surface topologies. We then seeded osteocytes onto the substrates to examine their responses. Methodologies used included scanning electron microscopy (SEM) for cell morphology, confocal laser scanning microscopy (CLSM) for protein distribution, western blot for protein levels, co-immunoprecipitation for protein interactions, and quantitative real-time polymerase chain reaction for gene expression. RESULTS: SEM images revealed that substrate stiffness induced a change in osteocyte morphology, and CLSM of F-actin staining revealed that substrate stiffness can alter the cytoskeleton. These results were accompanied by changes in focal adhesion capacity in osteocytes, determined via characterization of vinculin expression and distribution. Furthermore, on the exterior of the cell membrane, fibronectin was altered by substrate stiffness. The fibronectin then induced a change in paxillin on the inner membrane of the cell via protein-protein interaction through transmembrane processing. Paxillin led to changes in connexin 43 via protein-protein binding, thereby influencing osteocyte gap junction elongation. CONCLUSION: This process -from mechanosensing and mechanotransduction to cell function - not only indicates that the effects of mechanical factors on osteocytes can be directly sensed from the cell body, but also indicates the involvement of paxillin transduction.

FGF-7 Dictates Osteocyte Cell Processes Through Beta-Catenin Transduction.

It is well recognized that osteocytes communicate with each other via gap junctions and that connxin43 (Cx43) shows its great potential in gap junction for the contribution enabling transmission of small molecules and operating in an autocrine/a paracrine manner. Fibroblast growth factors (FGFs) play significant roles in new bone formation and adult bone remodeling, and FGF signaling is regulated by the precise spatiotemporal approaches. However, the influence of FGF7 on osteocyte cell processes is not well elucidated. In this study, we aimed to examine the impact of FGF7 on osteocyte cell processes by characterizing the expression of Cx43 and to reveal the underlying mechanism regulating this cell process. We first found that the mRNA level of FGF7 was higher relative to other FGF family members both in osteocytes cell line (MLO-Y4) and bone tissue. We then demonstrated that FGF7 could increase the expression of Cx43 in osteocytes and promote the cell processes in the form of gap junctions between osteocytes. This modulation was due to the FGF7-induced cytoplasmic accumulation and resultant nuclear translocation of ß-catenin. Our results could help us to further understand the importance of FGF7 on bone cell behavior and bone physiology and even pathology.

The Protective Effect of Propofol Against Ischemia-Reperfusion Injury in the Interlobar Arteries: Reduction of Abnormal Cx43 Expression as a Possible Mechanism.

BACKGROUND/AIMS: This experimental study aims to observe whether the protective effect of propofol against renal ischemia-reperfusion injury (IRI) in the rat interlobar artery occurs through altered expression of the gap junction protein connexin 43 (Cx43). METHODS: This study randomly divided male Sprague Dawley (SD) rats into an untreated control group, a sham-operated control group (sham group), an ischemia-reperfusion group (IR group), a propofol group (propofol+IR group) and a fat emulsion group (Intralipid group). The ischemia/reperfusion model was prepared through resection of the right kidney and noninvasive arterial occlusion of the left kidney. Forty-five minutes after renal ischemia-reperfusion, an automatic biochemical analyzer was employed to measure blood urea nitrogen (BUN) and serum creatinine (SCr); changes in renal tissue pathology were observed using hematoxylin and eosin (HE) staining, and the vasomotor activity of the interlobar artery was detected using a pressure mechanogram technique. The protein expression of Cx43 in renal artery cross-sections was determined through western blotting. RESULTS: The experimental study confirmed that the BUN and SCr of rats markedly increased after ischemia-reperfusion injury; additionally, we observed some coagulation necrosis and shedding of cells, some solidification of nuclear chromatin, degeneration of cytoplasmic vacuoles, high renal interstitial vascular congestion and obvious inflammatory cell infiltration, characterized by focal hemorrhages. Furthermore, the contraction activity of the renal interlobar artery greatly decreased, and the tension of the arteries in the renal lobe increased remarkably. After the gap junction blocking agents 2-APB and Gap27 were applied, the systolic velocity of blood vessels and the vascular contraction rate both decreased. In addition, the expression of Cx43 in kidney tissues increased markedly. The damage was more severe after 24 h of ischemic reperfusion than after only 4 h. However, after pretreatment with propofol, regardless of whether ischemia-reperfusion was applied for 4 h or 24 h, the previously increased expression of Cx43 decreased obviously, and all forms of renal damage were reversed. CONCLUSION: Our research suggests new ways for propofol to relieve ischemia-reperfusion injury by decreasing the abnormal expression of the gap junction protein Cx43. This study reveals a novel mechanism for the action of propofol against IRI, and we hope this finding will lead to new treatments for IRI.