Conjugated activation of myocardial-specific transcription of Gja5 by a pair of Nkx2-5-Shox2 co-responsive elements.

The sinoatrial node (SAN) is the primary pacemaker in the heart. During cardiogenesis, Shox2 and Nkx2-5 are co-expressed in the junction domain of the SAN and regulate pacemaker cell fate through a Shox2-Nkx2-5 antagonism. Cx40 is a marker of working myocardium and an Nkx2-5 transcriptional output antagonized by Shox2, but the underlying regulatory mechanisms remain elusive. Here we characterized a bona fide myocardial-specific Gja5 (coding gene of Cx40) distal enhancer consisting of a pair of Nkx2-5 and Shox2 co-bound elements in the regulatory region of Gja5. Transgenic reporter assays revealed that neither element alone, but the conjugation of both elements together, drives myocardial-specific transcription. Genetic analyses confirmed that the activation of this enhancer depends on Nkx2-5 but is inhibited by Shox2 in vivo, and its presence is essential for Gja5 expression in the myocardium but not the endothelial cells of the heart. Furthermore, chromatin conformation analysis showed an Nkx2-5-dependent loop formation between these two elements and the Gja5 promoter in vivo, indicating that Nkx2-5 bridges the conjugated activation of this enhancer by pairing the two elements to the Gja5 promoter.

PUFAs supplementation affects the renal expression of pannexin 1 and connexins in diabetic kidney of rats.

In diabetic nephropathy (DN), intercellular communication is disrupted. Connexins (Cx) have a crucial role in that process. Dietary ratios and supplementation with polyunsaturated fatty acids (PUFAs) can alleviate diabetic complications and cause alterations in Cx levels. Although pannexins (Panx) share similarities with members of the Cx family, their function in diabetic nephropathy has still not been fully determined. We studied the influence of PUFA supplementation on the immunoexpression of Px1 and Cx family members in diabetic kidneys of rats. Four groups of rats in experimental DM1 model were supplemented with different dietary n-6/n-3 ratios; ≈7 in control (C) and diabetic groups (STZ), ≈ 60 in the STZ + N6 group and ≈ 1 (containing 16% EPA and 19% DHA) in the STZ + N3 group. Immunoexpression of Cx40, Cx43, Cx45 and Panx1 was evaluated in the renal tissue of diabetic rats using immunohistochemistry. Diabetes significantly decreased the protein expression of Cx40 and Cx43 and increased Panx1 protein expression in the renal cortex (p < 0.05-p < 0.01). There was a significant impact of diet on Cx and Panx1 immunoexpression. Dietary supplementation with a high n-6/n-3 ratio downregulated the protein expression of Cx45 and Panx1 in diabetic rats (p < 0.05-p < 0.01), while Cx43 immunoexpression was increased in diabetic rats fed with high and low n-6/n-3 ratios (p < 0.01-p < 0.001). Hyperglycaemic conditions in DN interfere with cell-to-cell communication and disturb the connection between cells and their immediate environment due to variations in connexin and pannexin immunoexpression. These variations can be regulated by PUFA dietary intake, suggesting their beneficial effect and possible therapeutic option.

Targeting gap junctional intercellular communication by hepatocarcinogenic compounds.

Gap junctions in liver, as in other organs, play a critical role in tissue homeostasis. Inherently, these cellular constituents are major targets for systemic toxicity and diseases, including cancer. This review provides an overview of chemicals that compromise liver gap junctions, in particular biological toxins, organic solvents, pesticides, pharmaceuticals, peroxides, metals and phthalates. The focus in this review is placed upon the mechanistic scenarios that underlie these adverse effects. Further, the potential use of gap junctional activity as an in vitro biomarker to identify non-genotoxic hepatocarcinogenic chemicals is discussed.

Knockout of P2rx7 purinergic receptor attenuates cyst growth in a rat model of ARPKD.

The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK.P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK.P2rx7+ littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.

Transcriptional and post-translational regulation of pannexins.

Pannexins are a 3-membered family of proteins that form large pore ion and metabolite channels in vertebrates. The impact of pannexins on vertebrate biology is intricately tied to where and when they are expressed, and how they are modified, once produced. The purpose of this review is therefore to outline our current understanding of transcriptional and post-translational regulation of pannexins. First, we briefly summarize their discovery and characteristics. Next, we describe several aspects of transcriptional regulation, including cell and tissue-specific expression, dynamic expression over development and disease, as well as new insights into the underlying molecular machinery involved. Following this, we delve into the role of post-translational modifications in the regulation of trafficking and channel properties, highlighting important work on glycosylation, phosphorylation, S-nitrosylation and proteolytic cleavage. Embedded throughout, we also highlight important knowledge gaps and avenues of future research. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Regulation of connexin signaling by the epigenetic machinery.

Connexins and their channels are involved in the control of all aspects of the cellular life cycle, ranging from cell growth to cell death, by mediating extracellular, intercellular and intracellular communication. These multifaceted aspects of connexin-related cellular signaling obviously require strict regulation. While connexin channel activity is mainly directed by posttranslational modifications, connexin expression as such is managed by classical cis/trans mechanisms. Over the past few years, it has become clear that connexin production is equally dictated by epigenetic actions. This paper provides an overview of the role of major determinants of the epigenome, including DNA methylation, histone acetylation and microRNA species, in connexin expression.

Dexamethasone-induced muscular atrophy is mediated by functional expression of connexin-based hemichannels.

Long-term treatment with high glucocorticoid doses induces skeletal muscle atrophy. However, the molecular mechanism of such atrophy remains unclear. We evaluated the possible involvement of connexin-based hemichannels (Cx HCs) in muscle atrophy induced by dexamethasone (DEX), a synthetic glucocorticoid, on control (Cx43(fl/fl)Cx45(fl/fl)) and Cx43/Cx45 expression-deficient (Cx43(fl/fl)Cx45(fl/fl):Myo-Cre) skeletal myofibers. Myofibers of Cx43(fl/fl)Cx45(fl/fl) mice treated with DEX (5h) expressed several proteins that form non-selective membrane channels (Cx39, Cx43, Cx45, Panx1, P2X7 receptor and TRPV2). After 5h DEX treatment in vivo, myofibers of Cx43(fl/fl)Cx45(fl/fl) mice showed Evans blue uptake, which was absent in myofibers of Cx43(fl/fl)Cx45(fl/fl):Myo-Cre mice. Similar results were obtained in vitro using ethidium as an HC permeability probe, and DEX-induced dye uptake in control myofibers was blocked by P2X7 receptor inhibitors. DEX also induced a significant increase in basal intracellular Ca(2+) signal and a reduction in resting membrane potential in Cx43(fl/fl)Cx45(fl/fl) myofibers, changes that were not elicited by myofibers deficient in Cx43/Cx45 expression. Moreover, treatment with DEX induced NFκB activation and increased mRNA levels of TNF-α in control but not in Cx43/Cx45 expression-deficient myofibers. Finally, a prolonged DEX treatment (7days) increased atrogin-1 and Murf-1 and reduced the cross sectional area of Cx43(fl/fl)Cx45(fl/fl) myofibers, but these parameters remained unaffected in Cx43(fl/fl)Cx45(fl/fl):Myo-Cre myofibers. Therefore, DEX-induced expression of Cx43 and Cx45 plays a critical role in early sarcolemma changes that lead to atrophy. Consequently, this side effect of chronic glucocorticoid treatment might be avoided by co-administration with a Cx HC blocker.

Gq-activated fibroblasts induce cardiomyocyte action potential prolongation and automaticity in a three-dimensional microtissue environment.

Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, Gq-mediated signaling was selectively enhanced in CFs by Gαq adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage- or Ca2+-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMPCF). Enhanced Gq signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gαq in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca2+ transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive Gq activation in CFs also depolarized RMPCF from -33 to -20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMPCF and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca2+ transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias.NEW & NOTEWORTHY In a three-dimensional microtissue model, which lowers baseline activation of cardiac fibroblasts but enables cell-cell, paracrine, and cell-extracellular matrix interactions, we demonstrate that selective cardiac fibroblast activation by enhanced Gq signaling, a pathophysiological trigger in the diseased heart, modulates cardiomyocyte electrical activity, leading to proarrhythmogenic automaticity.

The effects of polyunsaturated fatty acids and antioxidant vitamins on atrial oxidative stress, nitrotyrosine residues, and connexins following extracorporeal circulation in patients undergoing cardiac surgery.

Cardiac surgery with extracorporeal circulation is characterized by different degrees of myocardial ischemia/reperfusion, which is often associated with postoperative atrial fibrillation (POAF). We have previously shown that a novel preventive therapy based on the reinforcement of the antioxidant system using omega-3 fatty acids plus antioxidant vitamin supplementation applied to patients undergoing cardiac surgery reduces POAF occurrence. We hypothesized that oxidative stress and nitrosative stress are involved in the development of an arrhythmogenic substrate by their effect on connexins (Cx40, Cx43 and Cx45) abundance and distribution pattern. Therefore, we have assessed the effect of redox status on atrial tissue in patients undergoing cardiac surgery. Placebo/POAF and supplemented/POAF patients showed 276 and 170% higher reactive oxygen species (ROS) levels and 223 and 96% higher nitrotyrosine residues levels, respectively, compared to sinus rhythm (SR). In POAF tissue, antioxidant supplementation prevented Cx40 and Cx43 lateralization on cardiomyocyte sarcolemma, keeping them at the intercalated disks. POAF samples showed Cx40 heterogeneous distribution pattern, presenting tissue areas lacking this protein (49 and 55% lower levels in placebo/POAF and supplemented/POAF groups, respectively, compared to SR). Of note, Cx45 overexpression occurred in POAF, being 211 and 167% higher in placebo/POAF and supplemented/POAF groups, respectively, compared to SR. It is concluded that treatment with omega-3 fatty acids and antioxidant vitamins reduces oxidative and nitrosative stress and prevents Cx40/Cx43 lateralization in atrial tissue likely contributing to POAF prevention. However, it failed to fully prevent POAF occurrence because these compounds have no effects on the normalization of Cx40 down-regulation and Cx45 up-regulation, which may promote POAF.

Expression of connexins in porcine buccal pouch mucosa cells during real-time long-term cell proliferation in vitro: a primary culture approach.

In this study we investigated the expression of connexins Cx36, Cx37, Cx40, Cx43, and Cx45 mRNAs during real-time cellular proliferation in vitro. The oral mucosa cells were isolated from 80 pubertal crossbred Landrace gilts. The cells were transferred into primary in vitro culture (IVC) and cultured for 30 days. The cells were collected to RNA isolation after 7, 15 and 30 days of IVC and were checked for their real-time proliferative status using real-time cell analysis (RTCA). We found an increased expression of Cx43 mRNA after 30 days of IVC as compared to control (P<0.05). The expression level of Cx36 was significantly decreased after 30 days. The expression of Cx37, Cx40 and Cx45 mRNAs was not changed. The expression of Cx43 was statistically increased when compared to Cx40, Cx37, Cx45 and Cx36 (P<0.001, for all time periods, respectively). We confirmed the expression of selected connexins in porcine buccal mucosa cells during their long-term primary IVC, which suggests the existence of functional gap junction connections (GJCs) communication network between these cells. We also confirmed the observations of other authors that Cx43 plays a substantial role in GJC structure. However, the increased expression of Cx43 in buccal mucosa cells, accompanied with their proliferation during real-time primary culture, is presented, to our knowledge, for the first time.

The carboxyl tail of connexin32 regulates gap junction assembly in human prostate and pancreatic cancer cells.

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size.

A transiently expressed connexin is essential for anterior neural plate development in Ciona intestinalis.

A forward genetic screen in the ascidian Ciona intestinalis identified a mutant line (frimousse) with a profound disruption in neural plate development. In embryos with the frimousse mutation, the anteriormost neural plate cells, which are products of an FGF induction at the blastula and gastrula stages, initially express neural plate-specific genes but fail to maintain the induced state and ultimately default to epidermis. The genetic lesion in the frimousse mutant lies within a connexin gene (cx-11) that is transiently expressed in the developing neural plate in a temporal window corresponding to the period of a-lineage neural induction. Using a genetically encoded calcium indicator we observed multiple calcium transients throughout the developing neural plate in wild-type embryos, but not in mutant embryos. A series of treatments at the gastrula and neurula stages that block the calcium transients, including gap junction inhibition and calcium depletion, were also found to disrupt the development of the anterior neural plate in a similar way to the frimousse mutation. The requirement for cx-11 for anterior neural fate points to a crucial role for intercellular communication via gap junctions, probably through mediation of Ca(2+) transients, in Ciona intestinalis neural induction.

Pannexin-1 expression in developing mouse nervous system: new evidence for expression in sensory ganglia.

Pannexin1 (Panx1) is one of three members of the pannexin protein family. The expression of Panx1 mRNA has been extensively investigated from late embryonic to adult stages. In contrast, expression during early embryonic development is largely unknown. Our aim is to examine the temporal and spatial expression of Panx1 in mouse embryonic development by focusing on embryonic days (E) 9.5 to 12.5. Whole embryos are investigated in order to provide a comprehensive survey. Analyses were performed at the mRNA level by using reverse transcription plus the polymerase chain reaction and whole-mount in situ hybridization. Panx1 mRNA was detected in the heads and bodies of embryos at all developmental stages investigated (E9.5, E10.5, E11.5, E12.5). In particular, the nervous system expressed Panx1 at an early time point. Interestingly, Panx1 expression was found in afferent ganglia of the cranial nerves and spinal cord. This finding is of particular interest in the context of neuropathic pain and other Panx1-related neurological disorders. Our study shows, for the first time, that Panx1 is expressed in the central and peripheral nervous system during early developmental stages. The consequences of Panx1 deficiency or inhibition in a number of experimental paradigms might therefore be predicated on changes during early development.

Specification of the mouse cardiac conduction system in the absence of Endothelin signaling.

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.

Connexin43 reduces melanoma growth within a keratinocyte microenvironment and during tumorigenesis in vivo.

Connexins (Cx) have been identified as tumor suppressors or enhancers, a distinction that appears to be dependent on the type and stage of disease. However, the role of connexins in melanoma tumorigenesis and their status during cancer onset and progression remain controversial and unclear. Here, we show that the aggressive B16-BL6 mouse melanoma cell line expresses low basal levels of Cx26 and Cx43, rendering them gap junctional intercellular communication-deficient as elucidated by immunofluorescence, Western blotting, and dye transfer studies. Following ectopic expression of green fluorescent protein-tagged Cx26 and Cx43 in these connexin-deficient melanomas, punctate gap junction-like plaques were evident at sites of cell-cell apposition, and the incidence of dye transfer was significantly increased similar to connexin-rich keratinocytes. We found that the expression of Cx43, but not Cx26, significantly reduced cellular proliferation and anchorage-independent growth from control melanomas, whereas migration was unaffected. Additionally, melanomas expressing Cx43 displayed significantly reduced growth within the in situ-like microenvironment of keratinocytes, despite a lack of heterocellular gap junctional intercellular communication between the two cell types. Furthermore, when grown in vivo in the chicken chorioallantoic membrane, primary tumors derived from Cx43-expressing melanomas were significantly smaller than controls, whereas Cx26-expressing melanomas produced tumors similar to controls. Collectively, these results suggest that Cx43, and not Cx26, can act as a tumor suppressor during melanoma tumorigenesis.

Oxygen Sensitivity of Placental Trophoblast Connexins 43 and 46: A Role in Preeclampsia?

Several gap junction connexins have been shown to be essential for appropriate placental development and function. It is known that the expression and distribution of connexins change in response to environmental oxygen levels. The placenta develops under various oxygen levels, beginning at a low oxygen tension of approximately 2% and increasing to a tension of 8% after the onset of the uteroplacental circulation. Moreover, it has been shown that during preeclampsia (PE) placentas are subjected to chronic hypoxia. Therefore, we investigated oxygen sensitivity of placental connexins 43 and 46. Using the trophoblast cell line Jar, we demonstrated that the expression of connexin43 increased during acute hypoxia but decreased during chronic hypoxia. Chronic hypoxia resulted in the translocation of connexin43 from the membrane to the cytoplasm and in a reduction in its communication properties. In contrast, the expression of connexin46 was down-regulated during chronic hypoxia and was translocated from perinuclear areas to the cell membrane. Hypoxia-inducible factor (HIF) knockdown showed that the translocation of connexin43 but not that of connexin46 was HIF-2α dependent and was mediated by phosphoinositide 3-kinase. The up-regulation of connexin43 in combination with the down-regulation of connexin46 was confirmed in placental explants cultivated under low oxygen and in placentas with early-onset PE. Taken together, in Jar cells, placental connexins 43 and 46 are regulated during periods of low oxygen in opposite manners. The oxygen sensing of connexins in the trophoblast may play a role in physiological and pathophysiological oxygen conditions and thus may contribute to PE.

Junctional abnormalities in human airway epithelial cells expressing F508del CFTR.

Cystic fibrosis (CF) has a profound impact on airway physiology. Accumulating evidence suggests that intercellular junctions are impaired in CF. We examined changes to CF transmembrane conductance regulator (CFTR) function, tight junctions, and gap junctions in NuLi-1 (CFTR(wt/wt)) and CuFi-5 (CFTR(ΔF508/ΔF508)) cells. Cells were studied at air-liquid interface (ALI) and compared with primary human bronchial epithelial cells. On the basis of fluorescent lectin binding, the phenotype of the NuLi-1 and CuFi-5 cells at week 8 resembled that of serous, glycoprotein-rich airway cells. After week 7, CuFi-5 cells possessed 130% of the epithelial Na(+) channel activity and 17% of the CFTR activity of NuLi-1 cells. In both cell types, expression levels of CFTR were comparable to those in primary airway epithelia. Transepithelial resistance of NuLi-1 and CuFi-5 cells stabilized during maturation in ALI culture, with significantly lower transepithelial resistance for CuFi-5 than NuLi-1 cells. We also found that F508del CFTR negatively affects gap junction function in the airway. NuLi-1 and CuFi-5 cells express the connexins Cx43 and Cx26. While both connexins were properly trafficked by NuLi-1 cells, Cx43 was mistrafficked by CuFi-5 cells. Cx43 trafficking was rescued in CuFi-5 cells treated with 4-phenylbutyric acid (4-PBA), as assessed by intracellular dye transfer. 4-PBA-treated CuFi-5 cells also exhibited an increase in forskolin-induced CFTR-mediated currents. The Cx43 trafficking defect was confirmed using IB3-1 cells and found to be corrected by 4-PBA treatment. These data support the use of NuLi-1 and CuFi-5 cells to examine the effects of F508del CFTR expression on tight junction and gap junction function in the context of serous human airway cells.

Follicle-Stimulating Hormone Increases Gap Junctional Communication Between Somatic and Germ-Line Follicular Compartments During Murine Oogenesis.

Germ cells develop in intimate contact and communication with somatic cells of the gonad. In female mammals, oocyte development depends crucially on gap junctions that couple it to the surrounding somatic granulosa cells of the follicle, yet the mechanisms that regulate this essential intercellular communication remain incompletely understood. Follicle-stimulating hormone (FSH) drives the terminal stage of follicular development. We found that FSH increases the steady-state levels of mRNAs encoding the principal connexins that constitute gap junctions and cadherins that mediate cell attachment. This increase occurs both in granulosa cells, which express the FSH-receptor, and in oocytes, which do not. FSH also increased the number of transzonal projections that provide the sites of granulosa cell-oocyte contact. Consistent with increased connexin expression, FSH increased gap junctional communication between granulosa cells and between the oocyte and granulosa cells, and it accelerated oocyte development. These results demonstrate that FSH regulates communication between the female germ cell and its somatic microenvironment. We propose that FSH-regulated gap junctional communication ensures that differentiation processes occurring in distinct cellular compartments within the follicle are precisely coordinated to ensure production of a fertilizable egg.

Down-regulation of connexin43 and connexin32 in keratocystic odontogenic tumours: potential association with clinical features.

AIMS: The objective of this study was to explore the potential involvement of connexin43 (Cx43) and connexin32 (Cx32), two vital members of the connexin families, in the pathogenesis of keratocystic odontogenic tumours (KCOT). METHODS AND RESULTS: The expression levels of Cx43 and Cx32 in human KCOT and normal oral mucosa (OM) tissues were measured using immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). The relationship between Cx43 and Cx32 expression and markers of proliferation [proliferating cell nuclear antigen (PCNA), cyclin D1], anti-apoptosis [B cell lymphoma 2 (Bcl-2)] and autophagy [light chain 3 (LC3), Sequestosome 1 p62 (p62)] was then investigated in the KCOT samples. The results showed that Cx43 and Cx32 expression was down-regulated significantly in KCOT samples relative to OM samples. Meanwhile, the expression levels of Cx43 and Cx32 were correlated negatively with the expression levels of PCNA, cyclin D1, Bcl-2, LC3 and p62, as confirmed further by double-labelling immunofluorescence analyses. CONCLUSIONS: This study reveals for the first time that Cx43 and Cx32 are down-regulated in KCOT and suggests an association with growth regulation, anti-apoptosis and autophagy in KCOT.

Analysis of connexin expression during seizures induced by 4-aminopyridine in the rat hippocampus.

BACKGROUND: In epilepsy, seizures are generated by abnormal synchronous activity in neurons. In the rat hippocampus (HIP), epileptiform activity has been found to be associated with gap junctions (GJs). GJs are formed by the combination of two hemichannels, each composed of six connexins. At low doses, the convulsive drug 4-aminopyridine (4-AP) produces epileptiform activity without affecting glutamate levels; therefore, GJs could participate in its effect. Based on this argument, in this study, the expression of Cx 32, Cx 36 and Cx 43 protein and mRNA in the HIP of rats treated with 4-AP was evaluated. The evaluation of connexins was carried out by chemifluorescent immunoassay, semiquantitative RT-PCR and immunofluorescence to detect the amount and distribution of connexins and of cellular markers in the HIP and dentate gyrus (DG) of animals treated with NaCl and 4-AP in the right entorhinal cortex. In these animals, convulsive behavior and EEG signals were analyzed. RESULTS: The animals treated with 4-AP showed convulsive behavior and epileptiform activity 60 min after the administration. A significant increase in the protein expression of Cx 32, Cx 36 and Cx 43 was found in the HIP contralateral and ipsilateral to the site of 4-AP administration. A trend toward an increase in the mRNA of Cx 32 and Cx 43 was also found. An increase in the cellular density of Cx 32 and Cx 43 was found in the right HIP and DG, and an increase in the cellular density of oligodendrocytes in the DG and a decrease in the number of cells marked with NeuN were observed in the left HIP. CONCLUSIONS: Cx 32 and Cx 43 associated with oligodendrocytes and astrocytes had an important role in the first stages of seizures induced by 4-AP, whereas Cx36 localized to neurons could be associated with later stages. Additionally, these results contribute to our understanding of the role of connexins in acute seizures and allow us to direct our efforts to other new anticonvulsant strategies for seizure treatment.