Georges Pointis

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.

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 Durands 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.

New cellular mechanisms of gap junction degradation and recycling

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.

Three-dimensional analysis of connexin43 gap junction in the ex vivo rat seminiferous tubules: Short-term effects of hormonal effectors

Cx43 gap junctions are essential for proliferation, differentiation, and apoptosis of germ cells during spermatogenesis. However, only few and indirect observations have been reported on the distribution of Cx43, the predominant Cx within the seminiferous tubules. In thepresent study, we developed an innovative method that allows visualization of the three‐ dimensional localization of Cx43 associated with gap junctions and their functionality in isolated spermatogenic stage‐specific seminiferous tubules. Cx43 gap junctions were present between myoid cells, between Sertoli cells, and between Sertoli and germ cells. Cx43 levels and coupling were stage‐dependent with higher values at stages VI–VIII of spermatogenesis and markedly reduced at stages IX–X. Short‐term exposure of seminiferous tubule fragments at stages VI–VIII and of the 42GPA9 Sertoli cell line transfected with a Cx43‐GFP vector, to FSH, cAMP, DHT, and 17β‐E2 significantly altered Cx43 distribution as well as gap junction coupling. These observations highlight a nongenomic effect of these testicular effectors on Cx43 gap junction. Microsc. Res. Tech. 2009.

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. Developmental Dynamics 239:1113–1123, 2010.

Heteromeric connexin 43/connexin 33 complex endocytosis: A connexin phosphorylation independent mechanism

The role of gap junctions in proliferation, differentiation and apoptosis has been recently highlighted. Nevertheless, the molecular mechanisms that control these physiological events by acting on gap junction channels are still unknown. We have recently demonstrated that heteromeric gap junction plaques composed by Cx43 and Cx33 are unstable at the cell boundary and are rapidly internalized by endocytosis. In the present study, we analyze the phosphorylation status of Cx43 in homomeric (Cx43/Cx43) and heteromeric (Cx33/Cx43) complexes and their association with the tyrosine kinase c-Src. Our data show that c-Src interaction and P2 phosphorylation of Cx43, which are essential for homomeric Cx43 complex endocytosis, were altered in the heteromeric Cx33/Cx43 complex: lack of association between Cx33 and activated c-Src and disappearance of the P2 phosphorylated Cx43 isoform. The present findings demonstrate that the interaction of Cx33 with Cx43 within a same heteromeric complex may conduce to channel instability through alteration of the phosphorylation status of Cx43 independently of the control of the c-Src kinase. The data described here emphasize a new mechanism of Cx43 internalization Src kinase-independent.

The anti-mitotic drug griseofulvin induces apoptosis of human germ cell tumor cells through a connexin 43-dependent molecular mechanism

Griseofulvin, a widely used antifungal antimitotic drug has been proposed as an anti-tumoral treatment by way of in vitro experiments. Recently, in vivo demonstration of griseofulvin efficacy against multiple myeloma in mice argues for its potential as therapeutics for cancer. Nevertheless, the molecular mechanisms by which griseofulvin disrupts cancerous cell progression are far from being understood. In the present study, we found that griseofulvin inhibits human germ cell tumor cell growth through activation of mitochondrial caspase pathway (caspase 9 and 3) leading to the activation of apoptosis rather than an alteration of cell proliferation. Strikingly, we demonstrated that griseofulvin triggered the expression level of connexin 43 (mRNA and protein), a well described tumor-suppressor gene, known to participate in apoptosis regulation. Consistently, together with microtubule instability, a mechanism classically associated with cell death in response to griseofulvin, we observed a disruption of connexin 43/tubulin association concomitant of an enhanced translocation of connexin 43, or an immunoreactive fragment of the protein, from the cytoplasm to the nucleus. Finally, by using siRNA approaches we demonstrated the requirement of connexin 43 in the apoptotic induction of griseofulvin on our tumor cell model. Altogether, these results described a new molecular mechanism connexin 43-dependent targeted by griseofulvin leading to apoptosis of human germ cell tumor cells.

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.

Regenerating I messenger RNA and protein expression in the failing human testis: a potential molecular prognostic marker of seminoma

Infertility has been stated as a risk factor for testicular cancer; but currently, there is no prognostic indicator of tumor development from the pathologic testis with impaired spermatogenesis. Regenerating proteins are expressed in many human tissues including the testis, and their role in carcinogenesis has been well documented. In the present work, regenerating I messenger RNA and protein expression and cellular protein localization were studied in testicular biopsies of patients with normal (obstructive azoospermia) or impaired spermatogenesis (nonobstructive azoospermia) and in seminoma testis by quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunofluorescence analyses. No significant differences in regenerating I transcripts were reported between the 3 groups studied. However, regenerating I protein was highly expressed in pure seminoma and in placental-like alkaline phosphatase-positive seminiferous tubules with in situ carcinoma. Regenerating I protein levels measured by Western blotting increased from the placental-like alkaline phosphatase-negative distal region of the seminoma to the pure placental-like alkaline phosphatase-positive tumoral region. Importantly, although cells localized in seminiferous tubules of obstructive azoospermic patients with normal spermatogenesis were very slightly labeled, persisting germ, Sertoli, and myoid cells and fibrous tissues were strongly regenerating I positive in seminiferous tubules of nonobstructive azoospermia. These results suggest the possibility to use regenerating I as a prognostic marker of tumoral development in the infertile testis.

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.