Multiple and complex influences of connexins and pannexins on cell death.

Cell death is a fundamental process for organogenesis, immunity and cell renewal. During the last decades a broad range of molecular tools were identified as important players for several different cell death pathways (apoptosis, pyroptosis, necrosis, autosis…). Aside from these direct regulators of cell death programs, several lines of evidence proposed connexins and pannexins as potent effectors of cell death. In the present review we discussed the potential roles played by connexins, pannexins and innexins in the different cell death programs at different scales from gap junction intercellular communication to protein-protein interactions. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

New cellular mechanisms of gap junction degradation and recycling.

BACKGROUND INFORMATION: Connexins (Cxs), the constitutive proteins of gap junctions, are key actors of many physiological processes. Therefore, alterations of Cx expression and degradation lead to the development of physiopathological disorders. Because of the formation of a double membrane vesicle termed annular gap junction (AGJ), gap junction degradation is a unique physiological process for which many cellular aspects remain unclear. RESULTS: By using a combination of time-lapse fluorescence microscopy and high-resolution transmission electron microscopy, we evidenced new specific cellular events concerning gap junction degradation and recycling. Indeed, by time lapse video microscopy we demonstrated, for the first time to our knowledge, that an entire AGJ can be fully recycled back to the plasma membrane. Moreover, we dissected the degradative processes of gap junction by electron microscopy approaches. Interestingly, in addition to canonical autophagy and heterophagy pathways, previously described, we discovered that both pathways could sometimes intermingle. Strikingly, our results also highlighted a new lysosome-based autophagy pathway that could play a pivotal role in common autophagy degradation. CONCLUSIONS: The present investigation reveals that AGJ degradation is a more complex process that it was previously thought. First, a complete recycling of the gap junction plaque after its internalisation could occur. Second, the degradation of this peculiar double membrane structure is possible through autophagy, heterophagy, hetero-autophagy or by lysosomal-based autophagy. Altogether, this work underlines novel aspects of gap junction degradation that could be extended to other cell biology processes.

Physiological roles of connexins and pannexins in reproductive organs.

Reproductive organs are complex and well-structured tissues essential to perpetuate the species. In mammals, the male and female reproductive organs vary on their organization, morphology and function. Connectivity between cells in such tissues plays pivotal roles in organogenesis and tissue functions through the regulation of cellular proliferation, migration, differentiation and apoptosis. Connexins and pannexins can be seen as major regulators of these physiological processes. In the present review, we assembled several lines of evidence demonstrating that these two families of proteins are essential for male and female reproduction.

Molecular connexin partner remodeling orchestrates connexin traffic: from physiology to pathophysiology.

Connexins, through gap junctional intercellular communication, are known to regulate many physiological functions involved in developmental processes such as cell proliferation, differentiation, migration and apoptosis. Strikingly, alterations of connexin expression and trafficking are often, if not always, associated with human developmental diseases and carcinogenesis. In this respect, disrupted trafficking dynamics and aberrant intracytoplasmic localization of connexins are considered as typical features of functionality failure leading to the pathological state. Recent findings demonstrate that interactions of connexins with numerous protein partners, which take place throughout connexin trafficking, are essential for gap junction formation, membranous stabilization and degradation. In the present study, we give an overview of the physiological molecular machinery and of the specific interactions between connexins and their partners, which are involved in connexin trafficking, and we highlight their changes in pathological situations.

Connexin 43 a check-point component of cell proliferation implicated in a wide range of human testis diseases.

Gap junction channels link cytoplasms of adjacent cells. Connexins, their constitutive proteins, are essential in cell homeostasis and are implicated in numerous physiological processes. Spermatogenesis is a sophisticated model of germ cell proliferation, differentiation, survival, and apoptosis, in which a connexin isotype, connexin 43, plays a crucial role as evidenced by genomic approaches based on gene deletion. The balance between cell proliferation/differentiation/apoptosis is a prerequisite for maintaining levels of spermatozoa essential for fertility and for limiting anarchic cell proliferation, a major risk of testis tumor. The present review highlights the emerging role of connexins in testis pathogenesis, focusing specifically on two intimately interconnected human testicular diseases (azoospermia with impaired spermatogenesis and testicular germ cell tumors), whose incidence increased during the last decades. This work proposes connexin 43 as a potential cancer diagnostic and prognostic marker, as well as a promising therapeutic target for testicular diseases.

Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model.

Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12days. Exposure to low concentrations of chromium (10µg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood-testis barrier dynamic is postulated.

Cooperative cell-cell actin network remodeling to perform Gap junction endocytosis.

BACKGROUND: The endocytosis of Gap junction plaques (GJP) requires cytoskeletal forces to internalize such large membranous structures. Actin, which partners the connexin proteins constituting Gap junctions and is located close to Annular Gap Junctions (AGJ), could be actively involved in this physiological process. RESULTS: Electron Microscopy and Light Microscopy images, associated with time-lapse analysis and 3D reconstruction, used at high resolution and enhanced using ImageJ based software analysis, revealed that: i) actin cables, originating from Donor cells, insert on the edge of GJP and contribute to their invagination, giving rise to AGJ, whereas actin cables on the Acceptor cell side of the plaque are not modified; ii) actin cables from the Donor cell are continuous with the actin network present over the entire GJP surface. These actin cables fuse at a single point distant from the plaque, which then detaches itself from the membrane, condensing to form an actin mass during the final internalization process; iii) the Acceptor cell participates in the last step of the endocytic invagination process by forming an annular actin structure known as an actin ring. CONCLUSIONS: Together, these data suggest that the endocytosis of GJP is an example of a unique cooperative mechanism between the Donor (the traction of its actin cables) and the Acceptor cells (forming the actin ring).

RéSUMé: CONTEXTE: L'endocytose des plaques de jonctions communicantes ou jonctions gap (GJP) nécessite les forces du cytosquelette pour internaliser ces grandes structures membranaires. L'actine, partenaire des connexines, proteins constitutives des jonctions gap (Gj), localisée proche des jonctions gap annulaires (GJA), pourrait être impliquée dans ce processus physiologique. RéSULTATS: L' imagerie par microscopie optique et électronique, associées avec des analyses vidéo et des reconstructions en relief/3D, examinées à haute résolution et améliorées après traitement par des logiciels développés sous ImageJ, montrent que: i) des câbles d'actine, originaires des cellules donneuses, s'insèrent sur le bord des plaques jonctionnelles et facilitent leur invagination pour former les GJA tandis que les câbles d'actine des cellules receveuses ne sont pas modifies; ii) les câbles d'actine des cellules donneuses sont en continuité avec le réseau d'actine qui couvre la totalité de la surface de la plaque. De plus, ces câbles fusionnent en un point unique, à distance de la plaque, qui se détache de la région membranaire pour former une masse d'actine à la fin du processus d'endocytose; iii) la cellule receveuse participe à l'étape ultime du processus d'endocytose de la plaque en formant un anneau d'actine. CONCLUSIONS: L'ensemble de nos résultats montrent que l'endocytose des plaques jonctionnelles est un exemple de coopération unique entre la cellule donneuse (grâce à la traction des câbles d'actine) et la cellule receveuse (anneau d'actine).

Connexin 33 impairs gap junction functionality by accelerating connexin 43 gap junction plaque endocytosis.

Connexin 33 (Cx33) is a testis-specific gap junction protein. We previously reported that Cx33 exerts dominant-negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild-type Sertoli cells transfected with Cx33-DsRed2 and Cx43-green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi-channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens-1 (ZO-1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell-cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO-1 specifically interacted with P(1) phosphorylated and P(0) unphosphorylated isoforms of Cx43, and the ZO-1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO-1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant-negative effect on gap junction plaque.

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility.

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, beta-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO(-/-)) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO(-/-) as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO(-/-) testis. Occludin, N-cadherin and beta-catenin levels were enhanced in SCCx43KO(-/-) mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.

Differential time course of FSH/FSH receptor complex endocytosis within sertoli and germ cells during rat testis development.

Follicle-stimulating hormone (FSH) is required for initiation and maintenance of spermatogenesis, a dynamic process of cell proliferation and maturation. By using FSH-gold particles and pulse-chase experiments, we analyzed the kinetics of FSH endocytosis in Sertoli and germ cells during development. Ultrastructural time-dependent analysis demonstrates that FSH was first located on plasma membrane, before being accumulated within the endosomal compartment, in the early endosomes, identified by morphological criteria and Rab-5 colocalization. Thereafter, FSH-gold was routed to the degradation pathway. The FSH endocytosis kinetic was similar in Sertoli cells, spermatogonia and spermatocytes. However, quantitative analysis of gold particles revealed differences in the dynamic of FSH accumulation in the endosomes between immature and mature rats. This age-dependent kinetic of FSH endocytosis, mostly detectable by ultrastructural analysis associated with quantitative data, argues for a potential new regulatory mechanism of the FSH signalling pathway that could occur during maturation of testicular cells.

[European regulation REACH and the assessment of testicular toxicity]. / Analyse de la spermatogenèse ex vivo : application à l'analyse de la toxicité testiculaire.

Several studies suggest that exposure to environmental pollutants is partly responsible for testicular pathologies that have considerably increased over the last decades (cryptorchidism, hypospadias, cancer, decrease in the number of ejaculated spermatozoa). However, the cellular and molecular mechanisms involved in this reprotoxicity remain mostly unknown. One of the challenges of the european regulation REACH is to improve the knowledge on the chemical, toxic and ecotoxic properties of substances used in everyday life. As for the testicular toxicity, the few in vivo models used are not always the most appropriate for mechanistic studies. Our laboratory has developed and validated on a physiological point of view, coculture systems of germ cells in bicameral chambers, which reproduce a blood-testis barrier, allowing the determination of the mechanisms responsible for the toxicity of organic or mineral compounds on spermatogenesis, while reducing greatly the number of animals required.

Effects of a mixture of low doses of atrazine and benzo[a]pyrene on the rat seminiferous epithelium either during or after the establishment of the blood-testis barrier in the rat seminiferous tubule culture model.

Atrazine (ATZ), a widely used agricultural pesticide and benzo[a]pyrene (BaP), a ubiquitous environmental human carcinogen can induce alterations of spermatogenesis. In the present study, we showed first that our seminiferous tubule culture model, in bicameral chambers, allowed the settlement of the blood-testis barrier (BTB) in 8-day-old male rat cultures and the differentiation of spermatogonia into round spermatids.The effect of a mixture of 1 µg/L of ATZ and 1 µg/L of BaP was then investigated either during or after the establishment of the BTB by using 8- or 20-22-day-old rats. Cultures were performed over a 3-week period. Our results show that claudin-11 and connexin 43 two proteins of the BTB, were impaired by the mixture which also reduced the number of round spermatids (the direct precursors of spermatozoa), by targeting the middle to late pachytene spermatocytes. These effects were observed in 8- and 20-22-day -old rat seminiferous tubule cultures. However, the decrease of the number of round spermatids was faster and more marked in the 8-day- than in the 20-22-day -old rat seminiferous tubule cultures. Our study emphasizes the possible influence of the age of an individual on the effect of (a) toxicant(s) on spermatogenesis.

Accelerated internalization of junctional membrane proteins (connexin 43, N-cadherin and ZO-1) within endocytic vacuoles: an early event of DDT carcinogenicity.

Stability of cell-to-cell interactions and integrity of junctional membrane proteins are essential for biological processes including cancer prevention. The present study shows that DDT, a non-genomic carcinogen used at a non-cytotoxic dose (1 microM), rapidly disrupted the cell-cell contacts and concomitantly induced the formation of cytoplasmic vacuoles close to the plasma membrane in the SerW3 Sertoli cell line. High-resolution deconvolution microscopy reveals that this vacuolization process was clathrin-dependent since a hyperosmotic media (0.2 M sucrose) blocked rhodamine-dextran endocytosis. In response to DDT, junctional proteins such as Cx43, N-Cadherin and ZO-1 were internalized and present in vacuoles. In Cx43-GFP transfected cells, time lapse videomicroscopy demonstrates that DDT rapidly enhanced fragmentation of the gap junction plaques and abolished the gap junction coupling without major modification of Cx43 phosphorylation status. Repeated exposure to DDT resulted in chronic gap junction coupling injury. The present results demonstrate that one of the early effect of DDT is to interfere with the plasma membrane and to perturb its function, specifically its ability to establish cell-cell junctions that are essential for tissue homeostasis and control of cell proliferation and differentiation. Such an alteration may play a specific role during carcinogenesis.

Modified expression of cytoplasmic isocitrate dehydrogenase electrophoretic isoforms in seminal plasma of men with sertoli-cell-only syndrome and seminoma.

Two isoforms of human cytoplasmic isocitrate dehydrogenase (IDPc) of close molecular weights and different isoelectric points were identified in human seminal plasma (SP) by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). These two isoforms were detected in the normospermic men SP and their expressions were markedly altered in patients with testicular seminoma, the most frequent testicular germ cell cancer (TGCC): increase of the more acidic spot and decrease of the more basic one. Since oligospermia has been considered as a high risk pathological condition for developing a testicular cancer, the two IDPc isoforms were analyzed in SP of a group of secretory azoospermic patients. In this group the two spots displayed similar variations of expression to those observed in testicular seminoma. These results propose IDPc as a promising SP biomarker of testicular seminoma. Whether IDPc alteration in secretory azoospermia is predictive of testicular seminoma remains to be elucidated.

Effects of low doses of carbendazim or iprodione either separately or in mixture on the pubertal rat seminiferous epithelium: An ex vivo study.

It has been shown that non-cytotoxic doses of Carbendazim (CBZ), a broad-spectrum benzimidazole fungicide, possess endocrine-disrupting (androgen-like) actions, ex vivo, on the pubertal rat seminiferous epithelium. Iprodione (IPR), a dicarboximide fungicide, is also known to be an endocrine-disrupter (anti-androgen). The effect of a mixture of these two pesticides was investigated in the validated rat seminiferous tubule culture model. Cultures were performed in the absence or presence of CBZ 50nM or IPR 50nM either alone or in mixture (Mix), over a 3-week period. Mix exerted a dramatic effect on two proteins (Connexin 43 and Claudin-11) of the blood-testis barrier and possessed similar effects to IPR on some germ cell populations. The presence of IPR together with CBZ (Mix) cancelled the effect of CBZ on the increase of the androgen-dependent TP1 and TP2 mRNAs and on the decrease of ERα, ERß mRNAs. Nevertheless, CBZ alone or IPR alone or Mix induced toxicity on spermatogenesis resulting in a decrease of round spermatids (the precursors of spermatozoa). These results strongly suggest that, even at these low concentrations, the effects of IPR and of CBZ are not solely dependent on their respective anti-androgenic and androgen-like effects and should involve several mechanisms of action.

Gap junctional communication in the male reproductive system.

Male fertility is a highly controlled process that allows proliferation, meiosis and differentiation of male germ cells in the testis, final maturation in the epididymis and also requires functional male accessory glands: seminal vesicles, prostate and corpus cavernosum. In addition to classical endocrine and paracrine controls, mainly by gonadotropins LH and FSH and steroids, there is now strong evidence that all these processes are dependent upon the presence of homocellular or heterocellular junctions, including gap junctions and their specific connexins (Cxs), between the different cell types that structure the male reproductive tract. The present review is focused on the identification of Cxs, their distribution in the testis and in different structures of the male genital tract (epididymis, seminal vesicle, prostate, corpus cavernosum), their crucial role in the control of spermatogenesis and their implication in the function of the male accessory glands, including functional smooth muscle tone. Their potential dysfunctions in some testis (spermatogenic arrest, seminoma) and prostate (benign hyperplasia, adenocarcinoma) diseases and in the physiopathology of the human erectile function are also discussed.

Connexin43: emerging role in erectile function.

Connexins, that have their main function as part of gap junction channels, are proteins expressed in a large number of tissues such as endocrine, nervous, vascular, and muscular tissues. Gap junctions are implicated in tissue homeostasis and control of cell proliferation and differentiation. Interestingly, mutations of connexin genes have been reported in several human diseases (peripheral neuropathies, cardiovascular and dermatological diseases, hereditary cataract, and deafness) and altered expression of connexins have been associated with tumoral progression. Today, several lines of study argue for a critical role of gap junctions in corporal smooth muscle relaxation and erectile response. The present review highlights the emerging role of connexin43, one of these membranous proteins, in the physiology and physiopathology of human erectile function and its possible medical application.

Role of connexin-based gap junction channels in testis.

Spermatogenesis is a highly controlled process that allows proliferation and differentiation of male germ cells. This is under classical endocrine and paracrine controls. There is also evidence that gap junctions between Leydig cells, between Sertoli cells and between Sertoli and germ cells participate in the local regulation of spermatogenesis. Recent studies reveal that connexin 43 (Cx43), the predominant gap junction protein in the testis, is essential for the initiation and maintenance of spermatogenesis. In this review, we focus on the identification, distribution and control of connexins in the mammalian testis. The implication of connexin-based gap junctions in testicular physiology and in pathological disorders of spermatogenesis (spermatogenic arrest and testis cancer) is also discussed.

Sertoli cell junctional proteins as early targets for different classes of reproductive toxicants.

In the testis, Sertoli cells establish intercellular junctions that are essential for spermatogenesis. The SerW3 Sertoli cell line displays some features of native Sertoli cells. Western blot and immunofluorescence analyses showed that SerW3 Sertoli cells expressed typical components of tight (occludin and zonula occludens-1), anchoring (N-cadherin) and gap (connexin 43) junctions. Testicular toxicants (DDT, pentachlorophenol, dieldrin, dinitrobenzene, cadmium chloride, cisplatin, gossypol, bisphenol A and tert-octylphenol) affected intercellular junctions by either reducing the amount or inducing aberrant intracellular localization of these membranous proteins. Phosphodiesterase inhibitors (isobutyl methylxantine, rolipram, zaprinast, zardaverine) did not alter junctional-complex component levels but caused a rapid and reversible redistribution of these proteins to the cytoplasmic compartment. The present study showed that occludin, ZO-1, N-cadherin and specifically Cx43 could be early targets for testicular toxicants. The SerW3 cell line therefore appears as a useful in vitro model to evaluate molecules with potential anti-reproductive effects.

Connexin a check-point component of cell apoptosis in normal and physiopathological conditions.

Gap junction protein connexins (Cxs) play essential roles in cell homeostasis, growth, differentiation and death. Therefore, Cx dysfunction has been associated with many diseases and with tumor development. Cxs control cell apoptosis through different molecular mechanisms. First, gap junction channels classically facilitate the influx and flux of apoptotic signals between adjacent cells and hemichannels between the intracellular and extracellular environments. Second, recent studies demonstrate that Cx proteins, independently from their functional role through channels or hemichannels and in conjunction with their intracytoplasmic localization, may act as signaling effectors able to activate the canonical mitochondrial apoptotic pathway. In the present review, we dissected both functions of Cx in apoptosis, providing new avenues for apoptosis-mediated cancer therapy.