Expression of a gap junction protein, connexin43, in a large panel of human gliomas: new insights.

Precise diagnosis of low and high grades of brain tumors permits determining therapeutical strategies. So far, diagnosis and prognosis of gliomas were based on histological and genetic criteria which need being completed by a panel of molecular markers. Highly distributed in brain, gap junction proteins, connexins, could be considered as markers of glioma progression as previous studies indicated that expression of a connexin type, connexin43 (Cx43), is inversely correlated to tumor grading. However, this assumption was weakened by the low number of glioma samples used. Taking advantage of tissue microarray technique, we pursued this analysis by studying in situ expression of Cx43 on 85 samples (37 grade IV, 18 grade III, 24 grade II, and 6 grades II to III). Our analysis confirmed the global diminution of Cx43 expression in glioblastomas that was observed in previous studies. However, this analysis brought new insights such as the following ones. First, the high number of samples permitted to show that more than 60% of glioblastomas still express Cx43. Second, no gradual decrease in Cx43 expression was observed between grades II and III, but Cx43 appeared to be a marker distinguishing oligodendrocytic and astrocytic grade III tumors. Third, independently from tumor grade, a Cx43 nuclear staining was detected in areas where leukocytes are present. In conclusion, our study emphasizes the importance of in situ immunohistochemical approaches by giving more precise insights in the subcellular localization of Cx43. It also emphasizes the necessity to carry out such analysis on a wide range of samples to circumvent the high glioma heterogeneity.

Pseudomonas aeruginosa-induced apoptosis in airway epithelial cells is mediated by gap junctional communication in a JNK-dependent manner.

Chronic infection and inflammation of the airways is a hallmark of cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The response of the CF airway epithelium to the opportunistic pathogen Pseudomonas aeruginosa is characterized by altered inflammation and apoptosis. In this study, we examined innate immune recognition and epithelial responses at the level of the gap junction protein connexin43 (Cx43) in polarized human airway epithelial cells upon infection by PAO1. We report that PAO1 activates cell surface receptors to elicit an intracellular signaling cascade leading to enhancement of gap junctional communication. Expression of Cx43 involved an opposite regulation exerted by JNK and p38 MAPKs. PAO1-induced apoptosis was increased in the presence of a JNK inhibitor, but latter effect was prevented by lentiviral expression of a Cx43-specific short hairpin RNA. Moreover, we found that JNK activity was upregulated by pharmacological inhibition of CFTR in Calu-3 cells, whereas correction of a CF airway cell line (CF15 cells) by adenoviral expression of CFTR reduced the activation of this MAPK. Interestingly, CFTR inhibition in Calu-3 cells was associated with decreased Cx43 expression and reduced apoptosis. These results indicate that Cx43 expression is a component of the response of airway epithelial cells to innate immune activation by regulating the survival/apoptosis balance. Defective CFTR could alter this equilibrium with deleterious consequences on the CF epithelial response to P. aeruginosa.

Paroxetine use during pregnancy and perinatal outcomes including types of cardiac malformations in Quebec and France: a short communication.

BACKGROUND: Antidepressants, more specifically SSRIs, represent one example of a widely prescribed class of medications in pregnant women for which less than adequate pregnancy data have been available since the first drug in this class was marketed 20 years ago. Moreover, findings from studies performed after 2005, when health governmental authorities issued warnings regarding first trimester exposure to paroxetine and the risk of cardiac malformations, may be the result of detection bias if physicians were investigating more their pregnant patients that used paroxetine than the others. OBJECTIVES: To estimate the prevalence of 1) paroxetine use during pregnancy, and 2) diagnosed cardiac malformations in the Quebec and France populations. METHODS: Two distinct pregnancy databases were used for this ecologic study: the Quebec Pregnancy Registry and the French EFEMERIS database. RESULTS: In Quebec, among the 109,344 eligible pregnancies, 1,612 (1.5%) were exposed to paroxetine. Prevalence of paroxetine use during pregnancy increased from 0.7% to 1.2% between 1998 and 2003, simultaneously to the increase of the prevalence of cardiac malformation diagnoses. In France, among 40,317 eligible pregnancies, 173 (0.4%) were exposed to paroxetine; between 2004 and 2008 the number of paroxetine fillings and cardiac malformation diagnoses remained constant. CONCLUSIONS: Despite differences in the Quebec and French healthcare systems, increase in paroxetine prevalence use during pregnancy was already consistent with an increase in the prevalence of cardiac malformations, even before the warning on the risk of cardiac malformations in newborns in 2005-2006, limiting the possibility of detection bias.

The gap junction protein Cx43 is involved in the bone-targeted metastatic behaviour of human prostate cancer cells.

For decades, cancer was associated with gap-junction defects. However, more recently it appeared that the gap junction proteins (connexins) could be re-expressed and participate to cancer cell dissemination during the late stages of tumor progression. Since primary tumors of prostate cancer (PCa) are known to be connexin deficient, it was interesting to verify whether their bone-targeted metastatic behaviour could be influenced by the re-expression of the connexin type (connexin43) which is originally present in prostate tissue and highly expressed in bone where it participates to the differentiation of osteoblastic cells. Thus, we investigated the effect of the increased Cx43 expression, by retroviral infection, on the metastatic behaviour of two well-characterized cell lines (PC-3 and LNCaP) representing different stages of PCa progression. It appeared that Cx43 differently behaved in those cell lines and induced different phenotypes. In LNCaP, Cx43 was functional, localized at the plasma membrane and its high expression was correlated with a more aggressive phenotype both in vitro and in vivo. In particular, those Cx43-expressing LNCaP cells exhibited a high incidence of osteolytic metastases generated by bone xenografts in mice. Interestingly, LNCaP cells were also able to decrease the proliferation of cocultured osteoblastic cells. In contrast, the increased expression of Cx43 in PC-3 cells led to an unfunctional, cytoplasmic localization of the protein and was correlated with a reduction of proliferation, adhesion and invasion of the cells. In conclusion, the localization and the functionality of Cx43 may govern the ability of PCa cells to metastasize in bones.

Opposing roles of connexin43 in glioma progression.

Despite the tremendous amount of data over the last 40years, lack of gap junctional intercellular communication (GJIC) or altered expression of gap junction proteins is still a lesser known 'hallmark' of cancer. Expression of astrocytic gap junction protein, connexin43 (Cx43), is often reduced in astrocytomas, the most common neoplasia of the central nervous system (CNS) in adults. Supported by a number of evidences, the global decrease of Cx43 expression appears to be advantageous for the growth of glioma cells. Although the mechanisms by which Cx43 regulates the expression levels of proteins involved in cell growth is unclear, there are evidences to suggest that it might be independent of their channel forming properties. In this regard, the carboxyl tail of Cx43 may have the ability to control the translocation of transcription factor regulators into the nucleus. However, this putative tumor suppressor effect of Cx43 is counterbalanced by its capacity to enhance the migration of glioma cells out of the tumor core through mechanisms that seems to implicate its carboxyl tail, possibly by interacting with the actin cytoskeleton. This ambivalence between the tumor suppressor effect and promotion of cell migration may partly be explained by the heterogeneous expression of Cx43 in the glioma core especially at the malignant glioblastoma stage; some tumor cells would be expected to migrate (Cx43 expressing cells) and others to proliferate (non-expressing Cx43 cells). Moreover, the involvement of Cx43 in glioma progression seems to be more complex since, in addition, GJIC may increase their resistance to apoptosis and Cx43 may also affect cell homeostasis in a paracrine fashion via hemichannel action. In conclusion, Cx43 appears to be involved at different levels of the glioma progression by acting on cell growth regulation, promotion of cell migration and resistance to apoptosis. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Altered expression of the gap junction protein connexin43 is associated with papillary thyroid carcinomas when compared with other noncancer pathologies of the thyroid.

BACKGROUND: Gap junctions are membrane structures composed of connexins (Cx) that allow diffusion of small molecules between cells. They are involved in tissue homeostasis, and various organ dysfunctions have been associated with gap junction defects. To verify their possible involvement in thyroid pathologies, the expression of connexin43 (Cx43), the major Cx in the human thyroid, was evaluated in a variety of diseases including cancer. METHODS: There were 122 samples from various thyroid pathologies that were collected to analyze the presence of Cx43 by immunofluorescence. Through confocal microscopy, different patterns of Cx43 localization were identified as normal (membrane) or abnormal (cytoplasmic or lack of detection). The analysis of Cx43 expression was further performed by quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry in a subset of 25 papillary carcinomas and compared with nontumoral thyroid tissues. RESULTS: The presence of Cx43 was commonly altered in thyroid cancer, as abnormal Cx43 staining was detected in 94.1% of cancer, 47.4% of adenomas, 45.7% of multinodular goiter, 16.7% of Graves' disease, and 25% of thyroiditis. In papillary carcinoma samples, the deregulation of Cx43 expression was mostly the consequence of a decrease of Cx43 mRNA (68% of cases) when compared with normal tissue. When Cx43 mRNA was not downregulated (32% of cases), both loss of membrane staining and aberrant cytoplasmic distribution of the protein were observed. CONCLUSIONS: These results show that aberrations of Cx43 expression are associated with thyroid papillary carcinoma.

Drug prescribing before and during pregnancy in south west France: a retrolective study.

BACKGROUND: Several drugs that are known to exhibit teratogenic or fetotoxic risks when used during pregnancy should not be prescribed to pregnant women. However, most women of childbearing age use medications, and drug use cannot always be avoided during pregnancy, especially for women with chronic diseases for whom the benefit of treatment outweighs the potential risk of the drug for the fetus. Nevertheless, it is often possible to replace a drug with another one that has been better evaluated. OBJECTIVE: The aim of the present study was to describe the prescribing of drugs to pregnant women before and during pregnancy in order to examine whether the occurrence of pregnancy modifies drug prescribing and dispensing to women. In particular, drugs that are contraindicated or must be avoided during pregnancy, such as retinoids, ACE inhibitors, angiotensin II receptor blockers, NSAIDs and valproic acid, will be analysed. METHODS: This retrolective study used data already prospectively recorded in the database of the French Health Insurance Service. It analysed pharmacy records of women who gave birth between 1 January 2007 and 31 December 2007 in Midi-Pyrenees. Pharmacy data were analysed from 9 months before pregnancy until delivery. Drugs were classified according to the Anatomical Therapeutic Chemical code. RESULTS: The study included 23 898 women. Approximately 77% and 96% of the women received at least one prescription before and during pregnancy, respectively. The number of women who were prescribed contraindicated drugs significantly decreased with pregnancy (p < 0.0001). Most of the drugs were stopped during the 3 months before pregnancy without alternative treatment, even for chronic diseases. However, for some women, potentially dangerous prescriptions were maintained during pregnancy, and for others these drugs were dispensed for the first time during critical periods of pregnancy. CONCLUSION: Despite recommendations, some teratogenic and/or fetotoxic drugs are still prescribed and dispensed to pregnant women in France. There is a need to repeat information to sensitize health professionals and women to the harmful potential of drugs. Moreover, discontinuation of a needed treatment must be avoided. Therefore, attention must be given to ensuring that younger females and women of childbearing potential who are likely to need continued treatment in adolescence and adulthood are aware of the potential risks that some drugs may pose during pregnancy.

Approaches to study differentiation and repair of human airway epithelial cells.

One of the main functions of the airway mucosa is to maintain a mechanical barrier at the air-surface interface and to protect the respiratory tract from external injuries. Differentiation of human airway epithelial cells (hAECs) to polarized airway mucosa can be reproduced in vitro by culturing the cells on microporous membrane at the air-liquid interface. Here, we describe approaches to study differentiation as well as repair of the hAECs by using a commercially available airway cell culture model called MucilAir™.

Connexins as therapeutic targets in lung disease.

INTRODUCTION: The lung is a mechanically active system exposed to the external environment and is particularly sensitive to injury and inflammation. Studies have identified intercellular communication pathways that promote proper lung function in response to injury and disease. These pathways involve connexins (Cxs) and gap junctional intercellular communication (GJIC). AREAS COVERED IN THIS REVIEW: The functional expression of Cxs in airway epithelium and vasculature, under normal and pathological conditions, is reviewed. Inhibition of GJIC and/or silencing of Cxs have been shown to modulate the course of disease development. Cx-based channels: i) coordinate ciliary beating and fluid transport to promote clearance of particulates, ii) regulate secretion of pulmonary surfactant, in response to deep inhalation by interconnecting type I and type II alveolar epithelial cells, and iii) are key mediators of pro- and anti-inflammatory signalling by the pulmonary endothelium, in order to modulate leukocyte recruitment from the circulation. EXPERT OPINION: Cx-based channels play several central roles in promoting a regulated inflammatory response and facilitating lung repair, thus enabling the pulmonary epithelium and vasculature to behave as integrated systems. Several pathologies can disrupt the normal communication pathways required for proper lung function, including acute lung injury, asthma, cystic fibrosis, pulmonary fibrosis and cancer.

Connexin channel-dependent signaling pathways in inflammation.

Inflammation is a highly regulated process with common but also specific characteristics in each tissue affected. Recruitment of leukocytes from the blood to the injured tissue is an important early step in the inflammatory cascade. This review highlights the role of connexins (Cxs) in the regulation of both acute and chronic inflammatory processes. Cxs form gap junction channels that provide a cytoplasmic continuity between adjacent cells allowing the intercellular exchange of ions and metabolites. Their structural halves form connexons or hemichannels. Each of them consists of 6 Cx proteins and hemichannels not taking part in gap junction formation but facilitating the release of small molecules such as ATP. Based on the differential distribution of various Cxs in different tissues such as the brain, lung capillaries and large blood vessels, our aim was to analyze the specific roles of Cxs in the inflammatory process in these tissues. Three typical sites of inflammation were chosen to shed light on similarities and differences in several types of responses: (1) atherosclerosis as a model for chronic inflammation, (2) the lung as an example of acute inflammation and (3) the 'immune-privileged' environment of the brain to highlight specific reactions of the vasculature to ischemic damage and inflammation at this site.

Prostaglandin E2regulation of cystic fibrosis transmembrane conductance regulator activity and airway surface liquid volume requires gap junctional communication.

Stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by protease-activated receptors (PARs) at the basolateral membranes and by adenosine receptors (ADO-Rs) at the apical membrane maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Both pathways involve the release of prostaglandin E2 (PGE2) and the stimulation of their basolateral receptors (EP-Rs). We sought to determine whether gap junctions contribute to the coordination of these pathways for modulating CFTR activity and mucus hydration. We used RT-PCR and Western blotting to determine connexin (Cx), CD73, and EP-R expression in a Calu-3 airway epithelial cell line grown on Transwell (Corning Costar, Cambridge, MA) inserts. We used dye coupling to evaluate gap junctional intercellular communication (GJIC). We used Ussing chamber studies and X-Z confocal microscopy to monitor Cl(-) secretion and ASL volume regulation. We found that connexin 43 (Cx43)-mediated GJIC was increased either by endogenous ADO after the hydrolysis of purine nucleotides by CD73 or by the direct activation of ADO-Rs. Inhibition of phospholipase A2 and cyclooxygenase prevented ADO-dependent increases in GJIC, suggesting the involvement of PGE2. PGE2 was found to increase GJIC markedly by stimulating EP4-Rs. The modulation of ADO signaling also affected the PAR-dependent activation of CFTR. The reduction of GJIC by CD73 or Cx43 inhibition prevented PAR-evoked CFTR currents in Ussing chambers. The inhibition of GJIC resulted in a failure of PGE2 to increase ASL volume in Calu-3 cells and in primary cultures of well-differentiated human airway epithelial cells. Thus, gap junctions coordinate a signaling network comprising CFTR, ADO-Rs, PARs, and EP-Rs, and are required for ASL volume homeostasis.

The carboxy-terminal tail of connexin43 gap junction protein is sufficient to mediate cytoskeleton changes in human glioma cells.

Connexin43 (Cx43) is a ubiquitously expressed member of the gap junction protein family that mediates gap junction intercellular communication (GJIC) by allowing exchange of cytosolic materials. Previous studies have used Cx43 truncated at the cytoplasmic tail (C-tail) to demonstrate that the C-tail is essential to regulate cell growth and motility. Therefore, the aim of our study was to delineate the respective role of the truncated Cx43 and the C-tail in mediating Cx43-dependent signaling. A truncated Cx43 expressing the channel part of the protein (TrCx43, amino acid 1-242) and a construct encompassing only the C-tail from amino acid 243 (243Cx43) were transduced into LN18 human glioma cells. Our results showed that the ability of Cx43 to suppress growth was independent of GJIC as assessed by dye transfer, but was dependent on the presence of a rigid extracellular matrix. We further demonstrated that the C-tail alone is sufficient to promote motility. Surprisingly, Cx43 is also able to increase migration in the absence of the C-tail, suggesting the presence of at least two distinct signaling mechanisms utilized by Cx43 to affect motility. Finally, we used time-lapse imaging to examine the behavior of migrating cells and it was apparent that the C-tail was associated with a lamellipodia-based migration not observed in either mock or TrCx43 expressing LN18 cells. Our study shows for the first time that a free C-tail is sufficient to induce Cx43-dependent changes in cell morphology and that Cx43 signaling is linked to the regulation of the actin cytoskeleton.

Gap junctions and cancer: new functions for an old story.

Cancer was one of the first pathologies to be associated with gap-junction defect. Despite the evidence accumulated over the last 40-year period, the molecular involvement of gap junctions and their structural proteins (connexins) in cancer has not been elucidated. The lack of a satisfying explanation may come from the complexity of the disease, evolving through various stages during tumor progression, with cancer cells exhibiting different phenotypes. Here, the question of the involvement of gap junctions has been readdressed by considering the connexin expression/function level at different fundamental stages of carcinogenesis (cell proliferation, cell invasion, and cancer cell dissemination). By performing this analysis, it becomes clear that gap junctions are probably differently involved, depending on the stage of the cancer progression considered. In particular, the most recent data suggest that connexins may act on cell growth by controlling gene expression through a variety of processes (independent of or dependent on the gap-junctional communication capacity). During invasion, connexins have been demonstrated to enhance adherence of cancer cells to the stroma, migration, and probably their dissemination by establishing communication with the endothelial barrier. All these data present a complex picture of connexins in various functions, depending on the cell phenotype.

Defective gap junctional intercellular communication in the carcinogenic process.

Gap junctions are membrane structures made of intercellular channels which permit the diffusion from cytoplasm to cytoplasm of small hydrophilic molecules. Nearly 40 years ago, the loss of functional gap junctions has been described in cancer cells and led to the hypothesis that such type of intercellular communication is involved in the carcinogenesis process. From this time, a lot of data has been accumulated confirming that gap junctions are frequently decreased or absent in cancer cells whatever their tissue and species origins. Here, we review such data by insisting on the possible links existing between altered gap-junctional intercellular communication capacity (or the altered expression of their constitutive proteins, the connexins) and the stages of cancer progression in various cancer models. Then, we analyse particular aspects of the disturbance of connexin-mediated communication in cancer such as the cytoplasmic localization of connexins, the lack of heterologous communication between cancer cells and normal cells, the role of connexin gene mutations in cancer. In a separate part of the review, we also analyse the disturbance of gap-junctional intercellular communication during the late stages of cancer (invasion and metastasis processes).