Gap junctional communication promotes apoptosis in a connexin-type-dependent manner

Gap junctions (GJs) have been described to modulate cell death and survival. It still remains unclear whether this effect requires functional GJ channels or depends on channel-independent effects of connexins (Cx), the constituents of GJs. Therefore, we analysed the apoptotic response to streptonigrin (SN, intrinsic apoptotic pathway) or to α-Fas (extrinsic apoptotic pathway) in HeLa cells expressing Cx43 as compared with empty vector-transfected (CTL) cells. Apoptosis assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining was significantly higher in HeLa-Cx43 compared with HeLa-CTL cells. Moreover, the cleavage of caspase-7 or Parp occurred earlier in HeLa-Cx43 than in HeLa-CTL cells. Comparative analysis of the effect of two further (endothelial) Cx (Cx37 and Cx40) on apoptosis revealed that apoptosis was highest in HeLa-Cx43 and lowest in HeLa-Cx37 cells, and correlated with the GJ permeability (assessed by spreading of a GJ-permeable dye and locally induced Ca(2+) signals). Pharmacologic inhibition of GJ formation in HeLa-Cx43 cells reduced apoptosis significantly. The role of GJ communication was further analysed by the expression of truncated Cx43 proteins with and without channel-forming capacity. Activation of caspases was higher in cells expressing the channel-building part (HeLa-Cx43NT-GFP) than in cells expressing the channel-incompetent C-terminal part of Cx43 (HeLa-Cx43CT-GFP) only. A hemichannel-dependent release and, hence, paracrine effect of proapoptotic signals could be excluded since the addition of a peptide (Pep)-blocking Cx43-dependent hemichannels (but not GJs) did not reduce apoptosis in HeLa-Cx43 cells. Treatment with SN resulted in a significant higher increase of the intracellular free Ca(2+) concentration in HeLa-Cx43 and HeLa-Cx43NT-GFP cells compared with HeLa-CTL or HeLa-Cx43CT-GFP cells, suggesting that Ca(2+) or a Ca(2+)-releasing agent could play a signalling role. Blocking of inositol triphosphate receptors reduced the SN-induced Ca(2+) increase as well as the increase in apoptosis. Our observations suggest that Cx43 and Cx40 but not Cx37 promote apoptosis via gap junctional transfer of pro-apoptotic signals between cells

Gap Junction Mediated Intercellular Metabolite Transfer in the Cochlea Is Compromised in Connexin30 Null Mice

Connexin26 (Cx26) and connexin30 (Cx30) are two major protein subunits that co-assemble to form gap junctions (GJs) in the cochlea. Mutations in either one of them are the major cause of non-syndromic prelingual deafness in humans. Because the mechanisms of cochlear pathogenesis caused by Cx mutations are unclear, we investigated effects of Cx30 null mutation on GJ-mediated ionic and metabolic coupling in the cochlea of mice. A novel flattened cochlear preparation was used to directly assess intercellular coupling in the sensory epithelium of the cochlea. Double-electrode patch clamp recordings revealed that the absence of Cx30 did not significantly change GJ conductance among the cochlear supporting cells. The preserved electrical coupling is consistent with immunolabeling data showing extensive Cx26 GJs in the cochlea of the mutant mice. In contrast, dye diffusion assays showed that the rate and extent of intercellular transfer of multiple fluorescent dyes (including a non-metabolizable D-glucose analogue, 2-NBDG) among cochlear supporting cells were severely reduced in Cx30 null mice. Since the sensory epithelium in the cochlea is an avascular organ, GJ-facilitated intercellular transfer of nutrient and signaling molecules may play essential roles in cellular homeostasis. To test this possibility, NBDG was used as a tracer to study the contribution of GJs in transporting glucose into the cochlear sensory epithelium when delivered systemically. NBDG uptake in cochlear supporting cells was significantly reduced in Cx30 null mice. The decrease was also observed with GJ blockers or glucose competition, supporting the specificity of our tests. These data indicate that GJs facilitate efficient uptake of glucose in the supporting cells. This study provides the first direct experimental evidence showing that the transfer of metabolically-important molecules in cochlear supporting cells is dependent on the normal function of GJs, thereby suggesting a novel pathogenesis process in the cochlea for Cx-mutation-linked deafness

Nitric oxide and cyclic nucleotides: Their roles in junction dynamics and spermatogenesis

Spermatogenesis is a highly complicated process in which functional spermatozoa (haploid, 1n) are generated from primitive mitotic spermatogonia (diploid, 2n). This process involves the differentiation and transformation of several types of germ cells as spermatocytes and spermatids undergo meiosis and differentiation. Due to its sophistication and complexity, testis possesses intrinsic mechanisms to modulate and regulate different stages of germ cell development under the intimate and indirect cooperation with Sertoli and Leydig cells, respectively. Furthermore, developing germ cells must translocate from the basal to the apical (adluminal) compartment of the seminiferous epithelium. Thus, extensive junction restructuring must occur to assist germ cell movement. Within the seminiferous tubules, three principal types of junctions are found namely anchoring junctions, tight junctions, and gap junctions. Other less studied junctions are desmosome-like junctions and hemidesmosome junctions. With these varieties of junction types, testes are using different regulators to monitor junction turnover. Among the uncountable junction modulators, nitric oxide (NO) is a prominent candidate due to its versatility and extensive downstream network. NO is synthesized by nitric oxide synthase (NOS). Three traditional NOS, specified as endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS), and one testis-specific nNOS (TnNOS) are found in the testis. For these, eNOS and iNOS were recently shown to have putative junction regulation properties. More important, these two NOSs likely rely on the downstream soluble guanylyl cyclase/cGMP/protein kinase G signaling pathway to regulate the structural components at the tight junctions and adherens junctions in the testes. Apart from the involvement in junction regulation, NOS/NO also participates in controlling the levels of cytokines and hormones in the testes. On the other hand, NO is playing a unique role in modulating germ cell viability and development, and indirectly acting on some aspects of male infertility and testicular pathological conditions. Thus, NOS/NO bears an irreplaceable role in maintaining the homeostasis of the microenvironment in the seminiferous epithelium via its different downstream signaling pathways

Connexins in cancer: bridging the gap to the clinic

Gap junctions comprise arrays of intercellular channels formed by connexin proteins and provide for the direct communication between adjacent cells. This type of intercellular communication permits the coordination of cellular activities and plays key roles in the control of cell growth and differentiation and in the maintenance of tissue homoeostasis. After more than 50 years, deciphering the links among connexins, gap junctions and cancer, researchers are now beginning to translate this knowledge to the clinic. The emergence of new strategies for connexin targeting, combined with an improved understanding of the molecular bases underlying the dysregulation of connexins during cancer development, offers novel opportunities for clinical applications. However, different connexin isoforms have diverse channel-dependent and -independent functions that are tissue and stage specific. This can elicit both pro- and anti-tumorigenic effects that engender significant challenges in the path towards personalised medicine. Here, we review the current understanding of the role of connexins and gap junctions in cancer, with particular focus on the recent progress made in determining their prognostic and therapeutic potential

An update on minding the gap in cancer

This article is a report of the International Colloquium on Gap junctions: 50 Years of Impact on Cancer that was held 8-9 September 2016, at the Amphitheater Pole Biologie Sante of the University of Poitiers (Poitiers, France). The colloquium was organized by M Mesnil (Universite de Poitiers, Poitiers, France) and C Naus (University of British Columbia, Vancouver, Canada) to celebrate the 50th anniversary of the seminal work published in 1966 by Loewenstein and Kanno [Intercellular communication and the control of tissue growth: lack of communication between cancer cells, Nature, 116 (1966) 1248-1249] which initiated studies on the involvement of gap junctions in carcinogenesis. During the colloquium, 15 participants presented reviews or research updates in the field which are summarized below

Modulation of Rac1 Activity by ADMA/DDAH Regulates Pulmonary Endothelial Barrier Function

Endogenously produced nitric oxide synthase inhibitor, asymmetric methylarginine (ADMA) is associated with vascular dysfunction and endothelial leakage. We studied the role of ADMA, and the enzymes metabolizing it, dimethylarginine dimethylaminohydrolases (DDAH) in the regulation of endothelial barrier function in pulmonary macrovascular and microvascular cells in vitro and in lungs of genetically modified heterozygous DDAHI knockout mice in vivo. We show that ADMA increases pulmonary endothelial permeability in vitro and in in vivo and that this effect is mediated by nitric oxide (NO) acting via protein kinase G (PKG) and independent of reactive oxygen species formation. ADMA-induced remodeling of actin cytoskeleton and intercellular adherens junctions results from a decrease in PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and a subsequent down-regulation of Rac1 activity. The effects of ADMA on endothelial permeability, Rac1 activation and VASP phosphorylation are prevented by overexpression of active DDAHI and DDAHII, whereas inactive DDAH mutants have no effect. These findings demonstrate for the first time that ADMA metabolism critically determines pulmonary endothelial barrier function by modulating Rac1-mediated remodeling of the actin cytoskeleton and intercellular junctions