Jean-Claude Hervé

Reversible Interruption of Gap Junctional Communication by Testosterone Propionate in Cultured Sertoli Cells and Cardiac Myocytes

Abstract. A direct cell-to-cell exchange of ions and molecules occurs through specialized membrane channels built by the interaction of two half channels, termed connexons, contributed by each of the two adjacent cells. The electrical and diffusional couplings have been investigated by monitoring respectively the cell-to-cell conductance and the fluorescence recovery after photobleaching, in Sertoli and cardiac cells of young rat. In both cell types, a rapid impairment of the intercellular coupling has been observed in the presence of testosterone propionate. This interruption of the cell-to-cell communication through gap junction channels was dose-dependent, observed in the concentration range 1 to 25 μm and was progressively reversed after withdrawing the testosterone ester. Pretreatment with cyproterone acetate, an antiandrogen which blocks the nuclear testosterone receptor by binding, did not prevent the uncoupling action of the androgen ester. This observation, together with the rapid time course of the uncoupling and recoupling, and the rather high effective concentration (micromolar) of the steroid compound, suggests a nongenomic mechanism of action. The uncoupling concentrations were very similar to those of other steroid compounds known to interrupt gap junctional communication. The uncoupling could result from a direct interaction of the steroid with the proteolipidic structure of the membrane, that might alter the conformation of the gap junction channels and their functional state.

Contraceptive gossypol blocks cell-to-cell communication in human and rat cells

Gossypol (a polycyclic lipophilic agent naturally present in cottonseed, known as a potent non-steroid antifertility agent and a non-specific enzyme inhibitor) irreversibly impaired the intercellular communication between homologous pairs of various cultured cells, from man or rat, involved (Sertoli or trophoblastic cells) or not involved (ventricular myocytes) in steroidogenesis, in a dose-dependent manner. In serum-free assays, a rapid junctional uncoupling occurred in non-cytotoxic conditions. At 5 microM (approximately twice the peak plasma concentration measured in human patients during chronic administration), gap junctional communication was interrupted within 4 to 10 min, without concomitant rise in the intracellular Ca2+ concentration. The latter importantly increased when gossypol treatment was prolonged (cytotoxic effect). The short term uncoupling effect of gossypol was prevented by serum proteins, but long-lasting treatments (48 h) with moderate concentrations (3 microM) elicited junctional uncoupling and impeded the in vitro differentiation of human trophoblasts.

REVERSIBLE INHIBITION OF GAP JUNCTIONAL COMMUNICATION BY TAMOXIFEN IN CULTURED CARDIAC MYOCYTES

Abstractu2002Gap junction channels provide a cell-to-cell conduction pathway for direct exchange of ions and small molecules. The intercellular diffusion of a fluorescent dye, quantified in cardiac myocytes from neonatal rats by monitoring the fluorescence recovery after photobleaching, was found to be interrupted after short-term exposure (15 min) to tamoxifen, an anti-oestrogen drug often used in the treatment of human breast cancer. This diffusional uncoupling was dose dependent, occurred in the concentration range 3–25 μM and reversed after tamoxifen withdrawal. Some possible mechanisms of junctional channel closure have been examined. The cytosolic calcium concentration, examined using the fluorescent indicator Indo-1, did not vary during the short-term action of tamoxifen. A second anti-oestrogen agent (clomiphene) was able to impair gap junctional communication, whereas a third (nafoxidine) had no effect. Protein-kinase-C-inhibitor properties of tamoxifen did not seem to be involved in its uncoupling action. The characteristics of tamoxifen’s action (i.e. channel inhibition delay, active concentration range, reversibility, etc.) were very similar to the previously observed effects of several other lipophilic compounds (e.g. 17β-oestradiol, etc.) on junctional channels, and to recently reported effects of tamoxifen on voltage-gated calcium currents.

RAPID ONSET AND CALCIUM INDEPENDENCE OF THE GAP JUNCTION UNCOUPLING INDUCED BY HEPTANOL IN CULTURED HEART CELLS

The kinetics of the reversible interruption of gap junction communication by the aliphatic alcohol heptanol and the possible mediation of an increase of the cytosolic Ca2+ concentration have been investigated in pairs of myocytes dissociated from neonatal rat ventricles and cultured for 2–3 days. Junctional communication was estimated by measuring either the cell-to-cell electrical conductance with a double wholecell voltage-clamp method, or the rate constant of dye diffusion with the fluorescence recovery after photobleaching (gap FRAP) technique. Electrical coupling was seen to be abruptly interrupted (in less than 0.5 s) by heptanol (1–3 mm). The cytosolic Ca2+ concentration was not affected, even at a saturating heptanol concentration. Heptanol removal allowed a gradual re-opening of gap Junctional channels, as shown by the recovery curve of the cell-to-cell conductance, which is 90 % complete within 90 s. These data are consistent with a direct interaction of heptanol with channel proteins or with their lipid environment.

INFLUENCE OF THE MOLECULAR STRUCTURE OF STEROIDS ON THEIR ABILITY TO INTERRUPT GAP JUNCTIONAL COMMUNICATION

Abstract. 17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration.Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized.These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism.

Transforming growth factor β1 (TGFβ1) inhibits gap junctional communication during human trophoblast differentiation

Summary It was recently established that, during trophoblast differentiation, gap junctional communication (GJC) precedes the formation of the syncytiotrophoblast and is required for trophoblast cell fusion. Therefore, the end of cell-to-cell communication through gap junctions appears as a judicious criterion of cell fusion. Although GJC was seen to be stimulated by human Chorionic Gonadotropin (hCG), its regulation remains poorly understood. Transforming Growth Factor-β1 (TGF-β1), a multifunctional cytokine has been shown to modulate trophoblast differentiation. Therefore, the effects of TGF-β1 on intercellular dye diffusion have been investigated in cultured trophoblast of human term placenta, by means of the Fluorescence Recovery After Photobleaching (gap FRAP) technique. In parallel, trophoblast differnetiation, hCG production and human Chorionic Somatomammotropin (hCS, a specific syncytiotrophoblast hormonal product) expression were assessed. The presence of TGF-β1 (5 or 10 ng/ml) in the culture medium for two days partially inhibited syncytium formation. The percentage of coupled cells was significantly decreased (2.8 times) after two days in presence of 10 ng/ml of TGF β1. Simultaneously, hCG release in culture medium was reduced (at this concentration, to 0.65 and 0.79 after respectively two and three days). In these conditions, Western blot analysis of trophoblast cellular proteins revealed that, after two days, hCS expression was reduced by 40% compared to control. Furthermore, the stimulation of trophoblastic GJC by exogenous hCG (500 mIU/ml) was considerably reduced by simultaneous exposure to TGF-β1 (10 mg/ml). The addition of a polyclonal hCG antibody in excess decreased basal GJC. In the presence of hCG antibody, no significant additive inhibition by TGFβ1 was observed. In conclusion, TGF-β1 was found to inhibit intercellular communication and, subsequently, differentiation and concomitant placental hormone secretions.

The apical junctional complexes, roles, and dysfunctions.

In multicellular organisms, epithelial barriers, which promote organ homeostasis by restricting the flow of ions and solutes between cells, are fundamental to the physiology of organ systems. Epithelia form diffusion barriers between cellular compartments of very different fluid and solute composition through both asymmetrically distributed transcellular transport mechanisms (transcellular pathway) and also by structures that regulate the diffusion of ions and small, noncharged solutes through the paracellular pathway. At the apical end of the paracellular space, adjacent cell membranes are indeed in close apposition, a site that was termed by early anatomists as the “terminal bar.” These intercellular junctional complexes are composed of the tight junctions or zonula occludens, the adherens junctions or zonula adherens, and desmosomes or macular adherens, whereas gap junctions provide for intercellular communication. Tight junctions (TJs) form an intercellular diffusion gate regulating the passage of ions, water, and various macromolecules through the paracellular spaces, and a fence restricting the apical/basolateral diffusion of membrane proteins and lipids whereas adherens junctions and desmosomes link membrane and cytoskeletal components at discrete contact regions. The present issue of Biochimica et Biophysica Acta “Biomembranes” and the companion issue “The apical junctional complexes, composition, structure and characteristics” (Biochim. Biophys. Acta 1778 (3), 2008) were designed to summarize the main aspects of the state of the art on the characteristics, composition, structure, and roles of the apical junctional complexes as well as some of the consequences of their dysfunctions. The polarized architecture of epithelial cells and tissues is a fundamental determinant of animal anatomy and physiology, and mammalian epithelial tumors for example appear to lose polarity as they progress toward malignancy. Drosophila melanogaster is a genetically simplemodel, particularly suited to examinehowaproper epithelial architecture is intimately involved in a cells ability to control its growth. Badouel and McNeill [1] explore the links between growth and apical junction proteins in the regulation of growth control in this model. Several submembranous components of apical junctional complexes are able to influence gene expression through their nuclear shuttling or their specific binding to transcription factors. Balda and Matter [2] describehow tight junctionproteinsparticipate in the regulation of gene expression and cell proliferation, as well as how they are regulated themselves by different mechanisms involved in gene expression and cell differentiation. Tight-junction-associated signaling pathways are deregulated in cancer cells but whether these changes are a cause or consequence of transformation remains to be elucidated. The different types of intercellular junctions (tight, anchoring and gap junctions), sharing common adaptor molecules (particularly zonula occludens-1), frequently present intermingled relationships, their proteins coassemble into macromolecular complexes, and their expressions are coordinately regulated. Derangeon et al. [3] present an

Preface: The apical junctional complexes, composition, structure, and characteristics

In multicellular organisms, epithelial barriers, which promote organ homeostasis by restricting the flow of ions and solutes between cells, are fundamental to the physiology of organ systems. Epithelia form diffusion barriers between cellular compartments of very different fluid and solute composition not only through asymmetrically distributed transcellular transport mechanisms (transcellular pathway) but also by structures that regulate the diffusion of ions and small, noncharged solutes through the paracellular pathway. At the apical end of the paracellular space, adjacent cell membranes are indeed in close apposition, a site that was termed by early anatomists as the “terminal bar”. These intercellular junctional complexes are composed of the tight junctions or zonula occludens, the adherens junctions or zonula adherens, and desmosomes or macular adherens, whereas gap junctions provide for intercellular communication. Tight junctions form an intercellular diffusion gate regulating the passage of ions, water, and various macromolecules through the paracellular spaces, and a fence restricting the apical/basolateral diffusion of membrane proteins and lipids whereas adherens junctions and desmosomes link membrane and cytoskeletal components at discrete contact regions. The present issue of Biochimica et Biophysica Acta–Biomembranes and the companion issue in preparation, titled “The apical junctional complexes, roles and dysfunctions,“ are designed to summarize some of the new information on some of the characteristics, composition, and structure of the apical junctional complexes. Adherens junctions mediate adhesion between neighboring cells by linking the actin cytoskeleton of one cell to that of the next cell via transmembrane adhesion molecules and their associated proteins complexes. The core of these junctions consists of two basic adhesive units, the interactions among transmembrane glycoproteins of the classical cadherin superfamily and the catenin family members (including p120-catenin, β-catenin, and α-catenin) and the nectin/afadin complexes. Niessen and Gottardi [1] highlight the diverse and extensive molecular complexity of this structure. Desmosomes are adhesive intercellular junctions of epithelia and cardiac muscle that anchor the intermediate filament network to the plasma membrane. By functioning both as an adhesive complex and as a cell-surface attachment site for intermediate filaments, desmosomes integrate the intermediate filament cytoskeleton between cells and play an important role