Expression of intercellular junctions during preimplantation development of the human embryo

A total of 74 human embryos were stained with gap junction proteinspecific anti-peptide antibodies and antibodies to the desmosomalprotein desmoplakin to reveal the expression pattern of intercellularjunctions during preimplantation development. Prior to implantation,the human embryo expresses predominantly connexin (Cx43)-containinggap junctions. Gap junctions were first detected in apposingcell membranes at the 4-cell stage and became increasingly organizedas development proceeded. In normal blastocysts, trophectoderm(TE) cells were linked by dense arrays of gap junctions whileinner cell mass (ICM) cells were linked by small, punctate gapjunctions. Gap junctions containing Cx32 or Cx26 were observedoccasionally in the TE of late blastocysts. Desmosomes appearedbetween outer cells prior to cavitation and were retained inthe TE, but not in the ICM. Levels of gap junction protein expressionwere variable in morphologically normal embryos at the samestage, suggesting that a normal appearance may not be a reliableindicator of future viability. Morphologically normal embryosoften possessed multinucleate, apoptotic and decompacting cells.They could show either extensive, disorganized over-expressionor reduced expression of gap junction protein. The results fitthe view that only embryos destined to survive display an organizedpattern of intercellular junctions. blastocyst/connexin/desmosome/gap junction/immunocytochemistry     

Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions

Numerous gap junctions exist between cumulus cells and betweencumulus cells and between cumulus cells and the oocyte. Theymay play a role in the regulation of both follicular developmentand oocyte status. We used primary cultures of human granulosacells to study the molecular nature and functionality of thesegap junctions. As shown by a cinemicrographic technique, duringthe first 3 days of culture, cells flattened and extended inseveral directions by means of cytoplasmic extensions. An ultrastructuralstudy showed the presence of both intercellular and annulargap junctions after 48 h of culture. As revealed by immunodetectionanalyses, connexin 43 was present. An analysis using a functionalprocedure, the gap fluorescence recovery after photobleaching(FRAP) method, indicated that: (i) diffusional communicationexisted among granulosa cells; (ii) the communication was delayedby treatment with 1-heptanol, a well-documented inhibitor ofgap junction permeability; and (iii) permeability was up-regulatedby incubation with 8-Br-cAMP, an analogue of cyclic AMP. Thedetection of connexin 43 and functional gap junctions in networksof cytoplasmic extensions indicated junction formation amongcells during culture. In conclusion, our results show that humangranulosa cells in culture exhibited functional gap junctions.Connexin 43 was present and the permeability of the gap Junctionswas up-regulated by cyclic AMP, an important modulator of humangranulosa cell function. connexin 43/cyclic AMP/FRAP method/gap junction/human granulosa-lutein cell     

Neoplastic reversal of human ovarian carcinoma cells transfected with connexin43

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

Gap junctions--major structures promoting intercellular communication

Gap junctions provide humoral and electric communication between the cells, thus contributing to their morpho-functional cooperation. Gap junction is formed by multiple intercellular channels, each of them being made by two closed hemichannels--connexons, that are oligomeric transmembrane proteins built by 6 subunits, belonging to connexin family. Permeability and electric conductivity of gap junction channels is determined by molecular peculiarities of connexins, their capacity for phosphorilation and by some extra- and intracellular factors. According to the current data, gap junctions in both cell cultures and tissues are dynamic structures with a short half-life period. Main mechanisms responsible for gap junction assembly and destruction have been discovered. These mechanisms were shown to depend upon peculiarities of differential genome activity and to be controlled by extra- and intracellular factors. The data on the gap junctions in the nervous system, heart and epidermis are presented.     

Quantitative immunoconfocal analysis of human myometrial gap junction connexin43 in relation to steroid hormone concentrations at term labour

The aim of this study was to quantify gap junction expression in the human myometrium in relation to progesterone and oestradiol concentrations, and to establish whether oxytocin-resistant dystocia is due to an abnormality in gap junction expression. Three groups of patients were investigated: (i) before labour (at term), (ii) normal labour and (iii) oxytocin-resistant dystocia (eight patients per group). For each patient, the concentrations of oestradiol and progesterone in maternal blood and in myometrial tissue were measured, and the number and area of immunostained connexin43 gap junctions per unit volume of tissue determined by quantitative analysis of digital images obtained by confocal microscopy. No significant difference in connexin43 gap junction content was observed between the three patient groups. When all groups were pooled, there was a significant positive correlation (P < 0.05) between the quantity of immunolabelled gap junctions and the oestradiol:progesterone ratio, but there was no significant difference in this correlation between the groups. Gap junction immunolabelling was not correlated with the progesterone or oestradiol concentration in the maternal blood or the myometrium. These data suggest that in human myometrium: (i) dystocia is not due to a reduced level of immunodetectable connexin43 gap junctions, (ii) onset of labour is not associated with a sudden increase in immunodetectable gap junction protein and (iii) gap junctions can be expressed in the presence of high progesterone concentrations.      

Ultraviolet A exposure induces reversible disruption of gap junction intercellular communication in lens epithelial cells

Gap junction intercellular communication (GJIC) is essential for the proper function of many organs including the lens. Disruption of GJIC can cause lens metabolic disorder and can induce cataracts. The purpose of this study was to investigate the signal transduction pathways involved in GJIC disruption following ultraviolet A (UVA) exposure in lens epithelial cells. Following exposure of human lens epithelial cells to UVA, connexin 43 (Cx43), the main component of gap junctions, was down-regulated at both the mRNA and protein levels. Furthermore, we observed that UVA exposure can increase protein kinase C activity and stimulate reactive oxygen species generation and lipid peroxidation. Using scrape load dye transfer technique, we found that the GJIC is compromised by UVA exposure. In addition, we demonstrated that UVA-induced modulation of GJIC was associated with p38 mitogen-activated protein kinase activation. More importantly, at non-lethal doses (10 J/cm2), the UVA-induced GJIC disruption and the consequent alterations were reversible. Collectively, our data revealed a new signaling pathway in GJIC disruption following UVA exposure, suggesting that UVA-compromised gap junction activity may sensitize human lens to photoaging and cataract formation.     

Novel GJA1 mutations in patients with oculo-dento-digital dysplasia (ODDD)

Oculo-dento-digital dysplasia (ODDD) is an autosomal dominant disorder characterized by developmental anomalies of the face, the eyes, the limbs and the teeth. Patients with ODDD usually present with complete syndactyly of the fourth and fifth fingers (type III syndactyly), ocular changes, abnormalities of primary and permanent dentition and specific craniofacial malformations. Mutations in GJA1, a gene that encodes the gap junction protein connexin 43, are responsible for ODDD. Gap junctions are assemblies of intercellular channels that allow exchange of various ions and signaling molecules between cells. In this way, gap junctions play an important regulatory role in a variety of physiologic and developmental processes. We identified three novel and one previously described GJA1 mutation in two large ODDD families and two sporadic ODDD cases.     

The role of myocardial gap junctions in electrical conduction and arrhythmogenesis.

Electrical activation of the heart requires cell-cell transfer of current via gap junctions, arrays of densely packed protein channels that permit intercellular passage of ions and small molecules. Because current transfer occurs only at gap junctions, the spatial distribution and biophysical properties of gap junction channels are important determinants of the conduction properties of cardiac muscle. Gap junction channels are composed of members of a multigene family of proteins called connexins. As a general rule, individual cells express multiple connexins, which creates the potential for considerable functional diversity in gap junction channels. Although gap junction channels are relatively nonselective in their permeability to ions and small molecules, cardiac myocytes actively adjust their level of coupling by multiple mechanisms including changes in connexin expression, regulation of connexin trafficking and turnover, and modulation of channel properties. In advanced stages of heart disease, connexin expression and intercellular coupling are diminished, and gap junction channels become redistributed. These changes have been strongly implicated in the pathogenesis of lethal ventricular arrhythmias. Ongoing studies in genetically engineered mice are revealing insights into the role of individual gap junction channel proteins in normal cardiac function and arrhythmogenesis.     

Connexin expression and functional analysis of gap junctional communication in mouse embryonic stem cells

Gap junctional intercellular communication (GJIC) has been suggested to be necessary for cellular proliferation and differentiation. We wanted to investigate the function of GJIC in mouse embryonic stem (ES) cells using pharmacological inhibitors or a genetic approach to inhibit the expression of connexins, that is, the subunit proteins of gap junction channels. For this purpose, we have analyzed all known connexin genes in mouse ES cells but found only three of them, Cx31, Cx43, and Cx45, to be expressed as proteins. We have demonstrated by coimmunoprecipitation that Cx31 and Cx43, as well as Cx43 and Cx45, probably form heteromeric gap junction channels, whereas Cx31 and Cx45 do not. The pharmacological inhibitors reduced GJIC between ES cells to approximately 3% and initiated apoptosis, suggesting an antiapoptotic effect of GJIC. In contrast to these results, reduction of GJIC to approximately 5% by decreased expression of Cx31 or Cx45 via RNA interference in homozygous Cx43-deficient ES cells did not lead to apoptosis. Additional studies suggested that apoptotic death of ES cells and adult stem cells reported in the literature is likely due to a cytotoxic side effect of the inhibitors and not due to a decrease of GJIC. Using the connexin expression pattern in mouse ES cells, as determined in this study, multiple connexin-deficient ES cells can now be genetically engineered in which the level of GJIC is further decreased, to clarify whether the differentiation of ES cells is qualitatively or quantitatively compromised.     

Novel GJA1 mutations in patients with oculo-dento-digital dysplasia (ODDD)

Oculo-dento-digital dysplasia (ODDD) is an autosomal dominant disorder characterized by developmental anomalies of the face, the eyes, the limbs and the teeth. Patients with ODDD usually present with complete syndactyly of the fourth and fifth fingers (type III syndactyly), ocular changes, abnormalities of primary and permanent dentition and specific craniofacial malformations. Mutations in GJA1, a gene that encodes the gap junction protein connexin 43, are responsible for ODDD. Gap junctions are assemblies of intercellular channels that allow exchange of various ions and signaling molecules between cells. In this way, gap junctions play an important regulatory role in a variety of physiologic and developmental processes. We identified three novel and one previously described GJA1 mutation in two large ODDD families and two sporadic ODDD cases.     

Gap junction intercellular communication: a review of a potential platform to modulate craniofacial tissue engineering

Defects in craniofacial tissues, resulting from trauma, congenital abnormalities, oncologic resection or progressive deforming diseases, may result in aesthetic deformity, pain and reduced function. Restoring the structure, function and aesthetics of craniofacial tissues represents a substantial clinical problem in need of new solutions. More biologically-interactive biomaterials could potentially improve the treatment of craniofacial defects, and an understanding of developmental processes may help identify strategies and materials that can be used in tissue engineering. One such strategy that can potentially advance tissue engineering is cell-cell communication. Gap junction intercellular communication is the most direct way of achieving such signaling. Gap junction communication through connexin-mediated junctions, in particular connexin 43 (Cx43), plays a major role bone development. Given the important role of Cx43 in controlling development and differentiation, especially in bone cells, controlling the expression of Cx43 may provide control over cell-to-cell communication and may help overcome some of the challenges in craniofacial tissue engineering. Following a review of gap junctions in bone cells, the ability to enhance cell-cell communication and osteogenic differentiation via control of gap junctions is discussed, as is the potential utility of this approach in craniofacial tissue engineering.     

Cell-cell communication in heterocellular cultures of alveolar epithelial cells

The mammalian alveolar epithelium is composed of alveolar type I (AT1) and alveolar type II (AT2) cells that together coordinate tissue function. We used a heterocellular culture model of AT1 and AT2 cells to determine pathways for intercellular signaling between these two phenotypes. Gap junction protein (connexin) profiles of AT1 and AT2 cells in heterocellular cultures were similar to those seen in rat lung alveolar sections. Dye coupling studies revealed functional gap junctions between and among each cell phenotype. Localized mechanical stimulation resulted in propagated changes of intracellular Ca2+ to AT1 or AT2 cells independent of the stimulated cell phenotype. Ca2+ communication that originated after AT1 cell stimulation was inhibited by gap junction blockers, but not by an inhibitor of extracellular nucleotide signaling (apyrase). Conversely, Ca2+ communication after stimulation of AT2 cells was not significantly reduced by gap junction inhibitors. However, apyrase significantly reduced Ca2+ communication from AT2 to AT1 cells, but not from AT2 to AT2 cells. In conclusion, AT1 and AT2 cells have unique connexin profiles that allow for functional coupling and distinct intercellular pathways for coordination of Ca2+ signaling.     

Gap junctions and connexins: potential contributors to the immunological synapse

Gap junctional communication is a widespread mechanism for metabolic coupling of adjoining cells. In the immune system, evidence has built up showing that lymphocytes possess the protein building blocks of gap junctions, the connexins. The most widespread is connexin 43, but connexin 40 is also present in secondary lymphoid organs. Inhibitors of gap junctional communication, especially the highly specific connexin mimetic peptides, have been shown to decrease the secretion of immunoglobulins and cytokines by T and B lymphocyte cocultures, indicating that connexins may play a fundamental role in lymphocyte physiology. Traditionally, connexins function when assembled into gap junction-intercellular channels. However, the possibility is now arising that gap junction hemichannels, previously viewed as plasma membrane precursors of gap junctions, are also involved in the release from cells of small metabolites, e.g., adenosine 5'-triphosphate and nicotinamide adenine dinucleotide(+), and this opens up a second, possible paracrine function for connexins detected in lymphocytes. The increasing structural and functional evidence points to a potential role that lymphocyte gap junctional intercellular communication may play within the complex signaling components of the immunological synapse.     

Functional gap junctions in thymic epithelial cells are formed by connexin 43

A multiparametric study was carried out to investigate the presence and possible role of communicating junctions in the thymus, particularly in the thymic epithelium, the major component of the thymic microenvironment. The presence of direct cell-cell communication mediated by gap junctions was demonstrated in human and murine thymic epithelial cells (TEC) by means of in situ and in vitro immunohistochemical labeling as well as in vitro fluorochrome injection and double whole-cell patch clamp experiments. Moreover, both immuno- and Northern blot studies revealed that the gap junction protein connexin 43 and its mRNA were present in TEC. Importantly, we showed that thymic endocrine activity, as ascertained by thymulin production, could be specifically downmodulated in vitro by a gap junction inhibitor, octanol. We also investigated the existence of gap junctions between TEC and thymocytes. In thymic nurse cells we were able to detect cell-cell communication, although only a minor percentage of epithelial/thymocyte pairs were coupled in a given moment. In contrast, intercellular communication was not detected between cultured phagocytic cells of the thymic reticulum and the respective rosetting thymocytes. We suggest that gap junctions formed by connexin 43 may represent a novel (and rather cell type-specific) pathway for intrathymic cellular communication, including TEC/TEC as well as possible TEC/thymocyte interactions.     

Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell-cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.     

Gap junctions in health and disease

Gap junctions are communicating junctions that consist of hexameric proteins called connexins and mediate the exchange of low molecular weight metabolites and ions between cells in contact. It has long been hypothesized that gap junctional intercellular communication plays a crucial role in the maintenance of homeostasis, morphogenesis, cell differentiation, and growth control in multicellular organisms. Recent discoveries of human genetic disorders associated with mutations in connexin genes and experimental data on connexin knockout mice have provided direct evidence of this. Connexin 32 mutations cause X-linked Charcot-Marie-Tooth disease, an inherited peripheral demyelinating neuropathy. Connexin 26 mutations have been found in hereditary nonsyndromic sensorineural deafness. Connexin 43 knockout mice die shortly after delivery because of cardiac malformation. Connexin 32 knockout mice show high incidences of spontaneous and chemically induced liver tumors, and develop a late-onset progressive peripheral neuropathy analogous to human Charcot-Marie-Tooth disease. Female connexin 37 knockout mice are infertile as the result of abnormalities in ovarian follicular growth, control of luteinization, and oocyte maturation. Connexin 46 knockout mice develop nuclear cataracts. Further identification of connexin mutations in other human diseases and generation of mice with modified connexin genes will aid our understanding of the biology of gap junctions.     

Plasma membrane channels formed by connexins: their regulation and functions

Members of the connexin gene family are integral membrane proteins that form hexamers called connexons. Most cells express two or more connexins. Open connexons found at the nonjunctional plasma membrane connect the cell interior with the extracellular milieu. They have been implicated in physiological functions including paracrine intercellular signaling and in induction of cell death under pathological conditions. Gap junction channels are formed by docking of two connexons and are found at cell-cell appositions. Gap junction channels are responsible for direct intercellular transfer of ions and small molecules including propagation of inositol trisphosphate-dependent calcium waves. They are involved in coordinating the electrical and metabolic responses of heterogeneous cells. New approaches have expanded our knowledge of channel structure and connexin biochemistry (e.g., protein trafficking/assembly, phosphorylation, and interactions with other connexins or other proteins). The physiological role of gap junctions in several tissues has been elucidated by the discovery of mutant connexins associated with genetic diseases and by the generation of mice with targeted ablation of specific connexin genes. The observed phenotypes range from specific tissue dysfunction to embryonic lethality.     

Gap junction diseases of the skin

Gap junctions are intercellular channels that allow the passage of water, ions, and small molecules. They are involved in quick, short-range messaging between cells and are found in skin, nervous tissue, heart, and muscle. An increasing number of hereditary skin disorders appear to be caused by mutations in one of the genes coding for the constituent proteins of gap junctions, known as connexins. In this review, the currently known connexin disorders that feature skin abnormalities are described: keratitis-ichthyosis deafness syndrome, erythrokeratoderma variabilis, Vohwinkel's syndrome, and a novel disorder called hypotrichosis-deafness syndrome. What is known about the pathogenesis of these disorders is discussed and related to gap junction physiology.     

In vivo and in vitro expression of connexin 43 in human teeth

Gap junctions are composed of transmembrane proteins belonging to the connexin family. These proteins permit the exchange of mall regulatory molecules directly between cells for the control of growth, development and differentiation. Although the presence of gap junctions in teeth has been already evidenced, the involved connexins have not yet been identified in human species. Here, we examined the distribution of connexin 43 (Cx43) in embryonic and permanent intact and carious human teeth. During tooth development, Cx43 localized both in epithelial and mesenchymal dental cells, correlated with cytodifferentiation gradients. In adult intact teeth, Cx43 was distributed in odontoblast processes. While Cx43 expression was downregulated in mature intact teeth, Cx43 appeared to be upregulated in odontoblasts facing carious lesions. In cultured pulp cells, Cx43 expression was related to the formation of mineralized nodules. These results indicate that Cx43 expression is developmentally regulated in human dental tissues, and suggest that Cx43 may participate in the processes of dentin formation and pathology.