Connexin-43 is a promising target for pulmonary hypertension due to hypoxaemic lung disease.

The mechanisms underlying pulmonary hypertension (PH) are complex and multifactorial, and involve different cell types that are interconnected through gap junctional channels. Although connexin (Cx)-43 is the most abundant gap junction protein in the heart and lungs, and critically governs intercellular signalling communication, its contribution to PH remains unknown. The focus of the present study is thus to evaluate Cx43 as a potential new target in PH.Expressions of Cx37, Cx40 and Cx43 were studied in lung specimens from patients with idiopathic pulmonary arterial hypertension (IPAH) or PH associated with chronic hypoxaemic lung diseases (chronic hypoxia-induced pulmonary hypertension (CH-PH)). Heterozygous Cx43 knockdown CD1 (Cx43+/-) and wild-type littermate (Cx43+/+) mice at 12 weeks of age were randomly divided into two groups, one of which was maintained in room air and the other exposed to hypoxia (10% oxygen) for 3 weeks. We evaluated pulmonary haemodynamics, remodelling processes in cardiac tissues and pulmonary arteries (PAs), lung inflammation and PA vasoreactivity.Cx43 levels were increased in PAs from CH-PH patients and decreased in PAs from IPAH patients; however, no difference in Cx37 or Cx40 levels was noted. Upon hypoxia treatment, the Cx43+/- mice were partially protected against CH-PH when compared to Cx43+/+ mice, with reduced pulmonary arterial muscularisation and inflammatory infiltration. Interestingly, the adaptive changes in cardiac remodelling in Cx43+/- mice were not affected. PA contraction due to endothelin-1 (ET-1) was increased in Cx43+/- mice under normoxic and hypoxic conditions.Taken together, these results indicate that targeting Cx43 may have beneficial therapeutic effects in PH without affecting compensatory cardiac hypertrophy.

Connexins, important players in the dissemination of prostate cancer cells.

Over the past 50years, increasing experimental evidences have established that connexins (Cxs) and gap junctional intercellular communication (GJIC) ensure an important role in both the onset and development of cancerous processes. In the present review, we focus on the impact of Cxs and GJIC during the development of prostate cancer (PCa), from the primary growth mainly localized in acinar glands and ducts to the distant metastasis mainly concentrated in bone. As observed in several other types of solid tumours, Cxs and especially Cx43 exhibit an ambivalent role with a tumour suppressor effect in the early stages and, conversely, a rather pro-tumoural profile for most of invasion and dissemination steps to secondary sites. We report here the current knowledge on the function of Cxs during PCa cells migration, cytoskeletal dynamics, proteinases activities and the cross talk with the surrounding stromal cells in the microenvironment of the tumour and the bones. In addition, we discuss the role of Cxs in the bone tropism even if the prostate model is rarely used to study the complete sequence of cancer dissemination compared to breast cancer or melanoma. Even if not yet fully understood, these recent findings on Cxs provide new insights into their molecular mechanisms associated with progression and bone targeted behaviour of PCa. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

An update on minding the gap in cancer.

This article is a report of the "International Colloquium on Gap junctions: 50Years of Impact on Cancer" that was held 8-9 September 2016, at the Amphitheater "Pôle Biologie Santé" of the University of Poitiers (Poitiers, France). The colloquium was organized by M Mesnil (Université 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.

Pannexin-1 in Human Lymphatic Endothelial Cells Regulates Lymphangiogenesis.

The molecular mechanisms governing the formation of lymphatic vasculature are not yet well understood. Pannexins are transmembrane proteins that form channels which allow for diffusion of ions and small molecules (<1 kDa) between the extracellular space and the cytosol. The expression and function of pannexins in blood vessels have been studied in the last few decades. Meanwhile, no studies have been conducted to evaluate the role of pannexins during human lymphatic vessel formation. Here we show, using primary human dermal lymphatic endothelial cells (HDLECs), pharmacological tools (probenecid, Brilliant Blue FCF, mimetic peptides [10Panx]) and siRNA-mediated knockdown that Pannexin-1 is necessary for capillary tube formation on Matrigel and for VEGF-C-induced invasion. These results newly identify Pannexin-1 as a protein highly expressed in HDLECs and its requirement during in vitro lymphangiogenesis.

Extracellular HSP27 mediates angiogenesis through Toll-like receptor 3.

The heat-shock protein 27 (HSP27) is up-regulated in tumor cells and released in their microenvironment. Here, we show that extracellular HSP27 has a proangiogenic effect evidenced on chick chorioallantoic membrane. To explore this effect, we test the recombinant human protein (rhHSP27) at physiopathological doses (0.1-10 µg/ml) onto human microvascular endothelial cells (HMECs) grown as monolayers or spheroids. When added onto HMECs, rhHSP27 dose-dependently accelerates cell migration (with a peak at 5 µg/ml) and favors spheroid sprouting within 12-24 h. rhHSP27 increases VEGF gene transcription and promotes secretion of VEGF-activating VEGF receptor type 2. Increased VEGF transcription is related to NF-κB activation in 30 min. All of these effects are initiated by rhHSP27 interaction with Toll-like receptor 3 (TLR3). Such an interaction can be detected by immunoprecipitation but does not seem to be direct, as we failed to detect an interaction between rhHSP27 and monomeric TLR3 by SPR analysis. rhHSP27 is rapidly internalized with a pool of TLR3 to the endosomal compartment (within 15-30 min), which is required for NF-κB activation in a cytosolic Ca(2+)-dependent manner. The HSP27/TLR3 interaction induces NF-κB activation, leading to VEGF-mediated cell migration and angiogenesis. Such a pathway provides alternative targets for antiangiogenic cancer therapy.

Down-regulation of Connexin43 expression reveals the involvement of caveolin-1 containing lipid rafts in human U251 glioblastoma cell invasion.

Glioblastoma cells are characterized by high proliferation and invasive capacities. Tumor development has been associated with a decrease of gap-junctional intercellular communication, but the concrete involvement of gap junction proteins, connexins, remains elusive since they are also suspected to promote cell invasion. In order to better understand how connexins control the glioma cell phenotype, we studied the consequences of inhibiting the intrinsic expression of the major astrocytic connexin, Connexin43, in human U251 glioblastoma cells by the shRNA strategy. The induced down-regulation of Cx43 expression has various effects on the U251 cells such as increased clonogenicity, angiogenesis and decreased adhesion on specific extracellular matrix proteins. We demonstrate that the invasion capacity measured in vitro and ex vivo correlates with Cx43 expression level. For the first time in a cancer cell context, our work demonstrates that Cx43 cofractionates, colocalizes and coimmunoprecipitates with a lipid raft marker, caveolin-1 and that this interaction is inversely correlated to the level of Cx43. This localization of Cx43 in these lipid raft microdomains regulates both homo- and heterocellular gap junctional communications (respectively between U251 cells, or between U251 cells and astrocytes). Moreover, the adhesive and invasive capacities are not dependent, in our model, on Cav-1 expression level. Our results tend to show that heterocellular gap junctional communication between cancer and stroma cells may affect the behavior of the tumor cells. Altogether, our data demonstrate that Cx43 controls the tumor phenotype of glioblastoma U251 cells and in particular, invasion capacity, through its localization in lipid rafts containing Cav-1.

Correction: Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells.

[This corrects the article DOI: 10.18632/oncotarget.3522.].

ZO-1 is involved in trophoblastic cell differentiation in human placenta.

Trophoblastic cell-cell fusion is an essential event required during human placental development. Several membrane proteins have been described to be directly involved in this process, including connexin 43 (Cx43), syncytin 1 (Herv-W env), and syncytin 2 (Herv-FRD env glycoprotein). Recently, zona occludens (ZO) proteins (peripheral membrane proteins associated with tight junctions, adherens junctions, and gap junctions) were shown to be involved in mouse placental development. Moreover, zona occludens 1 (ZO-1) was localized mainly at the intercellular boundaries between human trophoblastic cells. Therefore the role of ZO-1 in the dynamic process of human trophoblastic cell-cell fusion was investigated using primary trophoblastic cells in culture. In vitro as in situ, ZO-1 was localized mainly at the intercellular boundaries between trophoblastic cells where its expression substantially decreased during differentiation and during fusion. At the same time, Cx43 was localized at the interface of trophoblastic cells and its expression increased during differentiation. To determine a functional role for ZO-1 during trophoblast differentiation, small interfering RNA (siRNA) was used to knock down ZO-1 expression. Cytotrophoblasts treated with ZO-1 siRNA fused poorly, but interestingly, decreased Cx43 expression without altering the functionality of trophoblastic cell-cell communication as measured by relative permeability time constant determined using gap-FRAP experiments. Because kinetics of Cx43 and ZO-1 proteins show a mirror image, a potential association of these two proteins was investigated. By using coimmunoprecipitation experiments, a physical interaction between ZO-1 and Cx43 was demonstrated. These results demonstrate that a decrease in ZO-1 expression reduces human trophoblast cell-cell fusion and differentiation.

Effect of endothelin-1 on osteoblastic differentiation is modified by the level of connexin43: comparative study on calvarial osteoblastic cells isolated from Cx43+/- and Cx43+/+ mice.

Bone is a dynamic tissue that undergoes a precise remodeling process involving resorptive osteoclastic cells and bone-forming osteoblastic (OB) cells. The functional imbalance of either of these cell types can lead to severe skeletal diseases. The proliferation and differentiation of OB cells play a major role in bone development and turnover. These cellular processes are coordinated by connexin43 (Cx43)-based gap-junctional intercellular communication (GJIC) and by soluble factors such as endothelin-1 (ET-1). We have used the Cx43 heterozygous (Cx43(+/-)) murine model to study the possible cross-talk between Cx43 and ET-1 in cultured calvarial OB cells. On microcomputed tomographic analysis of 3-day-old pups, Cx43(+/-) mice showed hypomineralized calvaria in comparison with their Cx43(+/+) littermates. Characterization of cultured OB cells clearly demonstrated the effect of the partial deletion of the Cx43 gene on its expression, on GJIC, and subsequently on OB differentiation. In this model, ET-1 (10(-8) M) lost its mitogenic action in Cx43(+/-) OB cells compared with Cx43(+/+) cells. Moreover, a correlation between the inhibition of cell differentiation by ET-1 and the decreased amount and function of Cx43 was found in Cx43(+/+) OB cells but not in their Cx43(+/-) counterparts. Thus, as Cx43 is linked to OB differentiation, our data indicate that this mitogenic ET-1 peptide has pronounced effects on fully differentiated OB cells. With respect to roles in mechanotransduction and OB differentiation, Cx43 might modulate osteoblastic sensitivity to soluble factors.

Cx43 Present at the Leading Edge Membrane Governs Promigratory Effects of Osteoblast-Conditioned Medium on Human Prostate Cancer Cells in the Context of Bone Metastasis.

Among the different interacting molecules implicated in bone metastases, connexin43 (Cx43) may increase sensitivity of prostate cancer (PCa) cells to bone microenvironment, as suggested by our in silico and human tissue samples analyses that revealed increased level of Cx43 expression with PCa progression and a Cx43 specific expression in bone secondary sites. The goal of the present study was to understand how Cx43 influences PCa cells sensitivity and aggressiveness to bone microenvironment. By means of Cx43-overexpressing PCa cell lines, we revealed a Cx43-dependent promigratory effect of osteoblastic conditioned media (ObCM). This effect on directional migration relied on the presence of Cx43 at the plasma membrane and not on gap junctional intercellular communication and hemichannel functions. ObCM stimulation induced Rac1 activation and Cx43 interaction with cortactin in protrusions of migrating PCa cells. Finally, by transfecting two different truncated forms of Cx43 in LNCaP cells, we determined that the carboxy terminal (CT) part of Cx43 is crucial for the responsiveness of PCa cells to ObCM. Our study demonstrates that Cx43 level and its membrane localization modulate the phenotypic response of PCa cells to osteoblastic microenvironment and that its CT domain plays a pivotal role.

Development of a new model of reconstituted mouse epidermis and characterization of its response to proinflammatory cytokines.

The development of three-dimensional models of reconstituted mouse epidermis (RME) has been hampered by the difficulty to maintain murine primary keratinocyte cultures and to achieve a complete epidermal stratification. In this study, a new protocol is proposed for the rapid and convenient generation of RME, which reproduces accurately the architecture of a normal mouse epidermis. During RME morphogenesis, the expression of differentiation markers such as keratins, loricrin, filaggrin, E-cadherin and connexins was followed, showing that RME structure at day 5 was similar to those of a normal mouse epidermis, with the acquisition of the natural barrier function. It was also demonstrated that RME responded to skin-relevant proinflammatory cytokines by increasing the expression of antimicrobial peptides and chemokines, and inhibiting epidermal differentiation markers, as in the human system. This new model of RME is therefore suitable to investigate mouse epidermis physiology further and opens new perspectives to generate reconstituted epidermis from transgenic mice.

Modulation of the inwardly rectifying potassium channel Kir4.1 by the pro-invasive miR-5096 in glioblastoma cells.

Inwardly rectifying potassium channels (Kir), and especially the barium-sensitive Kir4.1 encoded by KCNJ10, are key regulators of glial functions. A lower expression or mislocation of Kir4.1 is detected in human brain tumors. MicroRNAs participate in the regulation of ionic channels and associated neurologic disorders. Here, we analyze effects of miR-5096 on the Kir4.1 expression and function in two glioblastoma cell lines, U87 and U251. Using whole-cell patch-clamp and western-blot analysis, we show that cell loading with miR-5096 decreases the Kir4.1 protein level and associated K+ current. Cell treatment with barium, a Kir4.1 blocker, or cell loading of miR-5096 both increase the outgrowth of filopodia in glioma cells, as observed by time-lapse microscopy. Knocking-down Kir4.1 expression by siRNA transfection similarly increased both filopodia formation and invasiveness of glioma cells as observed in Boyden chamber assay. MiR-5096 also promotes the release of extracellular vesicles by which it increases its own transfer to surrounding cells, in a Kir4.1-dependent manner in U251 but not in U87. Altogether, our results validate Kir4.1 as a miR-5096 target to promote invasion of glioblastoma cells. Our data highlight the complexity of microRNA effects and the role of K+ channels in cancer.

Transfer of functional microRNAs between glioblastoma and microvascular endothelial cells through gap junctions.

Extensive invasion and angiogenesis are hallmark features of malignant glioblastomas. Here, we co-cultured U87 human glioblastoma cells and human microvascular endothelial cells (HMEC) to demonstrate the exchange of microRNAs that initially involve the formation of gap junction communications between the two cell types. The functional inhibition of gap junctions by carbenoxolone blocks the transfer of the anti-tumor miR-145-5p from HMEC to U87, and the transfer of the pro-invasive miR-5096 from U87 to HMEC. These two microRNAs exert opposite effects on angiogenesis in vitro. MiR-5096 was observed to promote HMEC tubulogenesis, initially by increasing Cx43 expression and the formation of heterocellular gap junctions, and secondarily through a gap-junction independent pathway. Our results highlight the importance of microRNA exchanges between tumor and endothelial cells that in part involves the formation of functional gap junctions between the two cell types.

Expression of a gap junction protein, connexin43, in a large panel of human gliomas: new insights.

Precise diagnosis of low and high grades of brain tumors permits determining therapeutical strategies. So far, diagnosis and prognosis of gliomas were based on histological and genetic criteria which need being completed by a panel of molecular markers. Highly distributed in brain, gap junction proteins, connexins, could be considered as markers of glioma progression as previous studies indicated that expression of a connexin type, connexin43 (Cx43), is inversely correlated to tumor grading. However, this assumption was weakened by the low number of glioma samples used. Taking advantage of tissue microarray technique, we pursued this analysis by studying in situ expression of Cx43 on 85 samples (37 grade IV, 18 grade III, 24 grade II, and 6 grades II to III). Our analysis confirmed the global diminution of Cx43 expression in glioblastomas that was observed in previous studies. However, this analysis brought new insights such as the following ones. First, the high number of samples permitted to show that more than 60% of glioblastomas still express Cx43. Second, no gradual decrease in Cx43 expression was observed between grades II and III, but Cx43 appeared to be a marker distinguishing oligodendrocytic and astrocytic grade III tumors. Third, independently from tumor grade, a Cx43 nuclear staining was detected in areas where leukocytes are present. In conclusion, our study emphasizes the importance of in situ immunohistochemical approaches by giving more precise insights in the subcellular localization of Cx43. It also emphasizes the necessity to carry out such analysis on a wide range of samples to circumvent the high glioma heterogeneity.

Primary tumor- and metastasis-derived colon cancer cells differently modulate connexin expression and function in human capillary endothelial cells.

A gradual loss of functional gap junction between tumor cells has been reported with colorectal cancer (CRC) progression. Here, we explored if colon cancer cells could also affect gap junctions in blood capillary cells. Human microvascular endothelial cells (HMEC) were cultured with two CRC cell lines established from a unique patient. SW480 cells, derived from the primary tumor, migrate much faster across HMEC monolayer than SW620 cells derived from a metastatic site. The motile SW480 cells highly express and release HSP27 that increases gap junction formation with HMEC. Soluble HSP27 phosphorylates the connexin Cx43 on serine residues and induces its interaction with the oncoprotein 14-3-3, which promotes Cx43 delivery at the plasma membrane. The factors secreted by less motile SW620 cells do not affect Cx43 expression but up-regulate the expression of the connexin Cx32 through an activation of the chemokine receptor CXCR2. In turn, SW620 secreted factors induce tubulogenesis and ATP release. Altogether, cell lines derived from CRC primary tumor and metastasis differentially adapt endothelial cell functions by modulating connexin expression through released mediators.

Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells.

High levels of circulating heat shock protein 70 (HSP70) are detected in many cancers. In order to explore the effects of extracellular HSP70 on human microvascular endothelial cells (HMEC), we initially used gap-FRAP technique. Extracellular human HSP70 (rhHSP70), but not rhHSP27, blocks the gap-junction intercellular communication (GJIC) between HMEC, disrupts the structural integrity of HMEC junction plaques, and decreases connexin43 (Cx43) expression, which correlates with the phosphorylation of Cx43 serine residues. Further exploration of these effects identified a rapid transactivation of the Epidermal Growth Factor Receptor in a Toll-Like Receptor 4-dependent manner, preceding its internalization. In turn, cytosolic Ca2+ oscillations are generated. Both GJIC blockade and Ca2+ mobilization partially depend on ATP release through Cx43 and pannexin (Panx-1) channels, as demonstrated by blocking activity or expression of channels, and inactivating extracellular ATP. By monitoring dye-spreading into adjacent cells, we show that HSP70 released from human monocytes in response to macrophage colony-stimulating factor, prevents the formation of GJIC between monocytes and HMEC. Therapeutic manipulation of this pathway could be of interest in inflammatory and tumor growth.

Genomic organization and alternative transcripts of the human Connexin40 gene.

The human Cx40 gene (NT_004434.5) was sorted out from the GenBank database and as a result of a BLAST homology search, two ESTs (BE784549 from a human lung database, and BE732411 from a human placenta database) overlapping with the coding exon 2 sequence and upstream regions of the gene were identified. These ESTs correspond to two transcripts 1A and 1B, which diverge from each other in their 5' regions. The transcript 1A corresponds to the only transcript previously identified for the mouse and rat Cx40 genes; whereas the transcript 1B is a new transcript. The human Cx40 gene therefore comprises three exons: exon 1A (100 bp), exon 1B (132 bp) and coding exon 2, with the exons 1A and 1B at 14 and 1.3 kb of the exon 2, respectively. The expression of these transcripts is cell-type specific. Transcript 1A is expressed in endothelial cells. Its expression was demonstrated in human umbilical vein endothelial cells (HUVEC). Transcript 1B is expressed in placental cytotrophoblasts. Its expression was demonstrated in malignant trophoblastic cells, BeWo, JAR and JEG-3, and purified cytotrophoblasts from human first trimester placental tissues. Interestingly, both transcripts 1A and 1B are expressed in the right atrial appendages (RAA), although the cell-type expression of the two transcripts in this particular tissue has not yet been determined. Both transcripts were found to be expressed in the various heart regions investigated, where transcript 1B was found to always occur rarely in comparison with transcript 1A. Transcripts 1A and 1B are both more abundant in the atria than in the ventricles. Luciferase reporter gene assays demonstrated that two genomic regions containing the exons 1A and 1B induced a cell-type specific expression. The 1.2 kb sequence, containing the exon 1A, induced an increase of the luciferase activity in HUVEC; whereas the 1.9 kb sequence, containing the exon 1B, induces an increase of expression of the luciferase activity in BeWo cells. The DNA sequence upstream of the exon 1A contains SP1 binding sites, but no TATA- or CAAT-box; whereas the region upstream of the exon 1B is preceded by three CAAT-boxes. Thus, in contrast to the mouse and rat Cx40 genes, the human Cx40 gene organized in three exons and generates two transcripts, which are cell-type specific.

A galactosidase-responsive doxorubicin-folate conjugate for selective targeting of acute myelogenous leukemia blasts.

Cytarabine combined with an anthracycline or an anthracenedione represents the usual intensive induction therapy for the treatment of AML. However, this protocol induces severe side effects and treatment-related mortality due to the lack of selectivity of these cytotoxic agents. In this paper, we present the study of the first galactosidase-responsive molecular "Trojan Horse" programmed for the delivery of doxorubicin exclusively inside AML blasts over-expressing the folate receptor (FR). This targeting system allows the selective killing of AML blasts without affecting normal endothelial, cardiac or hematologic cells from healthy donors suggesting that FDC could reduce adverse events usually recorded with anthracyclines.

The gap junction protein Cx43 is involved in the bone-targeted metastatic behaviour of human prostate cancer cells.

For decades, cancer was associated with gap-junction defects. However, more recently it appeared that the gap junction proteins (connexins) could be re-expressed and participate to cancer cell dissemination during the late stages of tumor progression. Since primary tumors of prostate cancer (PCa) are known to be connexin deficient, it was interesting to verify whether their bone-targeted metastatic behaviour could be influenced by the re-expression of the connexin type (connexin43) which is originally present in prostate tissue and highly expressed in bone where it participates to the differentiation of osteoblastic cells. Thus, we investigated the effect of the increased Cx43 expression, by retroviral infection, on the metastatic behaviour of two well-characterized cell lines (PC-3 and LNCaP) representing different stages of PCa progression. It appeared that Cx43 differently behaved in those cell lines and induced different phenotypes. In LNCaP, Cx43 was functional, localized at the plasma membrane and its high expression was correlated with a more aggressive phenotype both in vitro and in vivo. In particular, those Cx43-expressing LNCaP cells exhibited a high incidence of osteolytic metastases generated by bone xenografts in mice. Interestingly, LNCaP cells were also able to decrease the proliferation of cocultured osteoblastic cells. In contrast, the increased expression of Cx43 in PC-3 cells led to an unfunctional, cytoplasmic localization of the protein and was correlated with a reduction of proliferation, adhesion and invasion of the cells. In conclusion, the localization and the functionality of Cx43 may govern the ability of PCa cells to metastasize in bones.

Gap junctions and cancer: new functions for an old story.

Cancer was one of the first pathologies to be associated with gap-junction defect. Despite the evidence accumulated over the last 40-year period, the molecular involvement of gap junctions and their structural proteins (connexins) in cancer has not been elucidated. The lack of a satisfying explanation may come from the complexity of the disease, evolving through various stages during tumor progression, with cancer cells exhibiting different phenotypes. Here, the question of the involvement of gap junctions has been readdressed by considering the connexin expression/function level at different fundamental stages of carcinogenesis (cell proliferation, cell invasion, and cancer cell dissemination). By performing this analysis, it becomes clear that gap junctions are probably differently involved, depending on the stage of the cancer progression considered. In particular, the most recent data suggest that connexins may act on cell growth by controlling gene expression through a variety of processes (independent of or dependent on the gap-junctional communication capacity). During invasion, connexins have been demonstrated to enhance adherence of cancer cells to the stroma, migration, and probably their dissemination by establishing communication with the endothelial barrier. All these data present a complex picture of connexins in various functions, depending on the cell phenotype.