Involvement of gap junctional communication and connexin expression in trophoblast differentiation of the human placenta

Gap junctional intercellular communication (GJIC) permits coordinated cellular activities during development and differentiation processes, and its dysfunction or mutation of connexin genes have been implicated in pathologies. In the human placenta, two distinct differentiation pathways of cytotrophoblastic cell coexist leading to a double model: fusion phenotype (villous trophoblast) and proliferative/invasive phenotype (extravillous trophoblast). This review focuses on current knowledge on the connexin expression and the implication of GJIC in trophoblastic differentiation. Experimental evidence obtained in human placenta demonstrates the involvement of connexin 43-gap junctions in the trophoblastic fusion process and of a connexin switch during the spatially and temporally controlled proliferation/invasion process.     

Gap junctional intercellular communication capacity by gap-FRAP technique: a comparative study

Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Connexins 43 (Cx43) are the most widely expressed gap junction proteins. Cellular localization of phosphorylated Cx43 has been implicated in the capacity of gap junctional intercellular communication (GJIC). To follow the functionality of GJIC of different cell types, in monolayer cultures, characterized by different patterns of phosphorylated Cx43, we used a fluorescence recovery after photobleaching (FRAP) technique, and compared two tracers, 5(6)-carboxyfluorescein diacetate (CFDA) and calcein acetoxymethylester (AM). The GJIC capacity was quantified by estimating fluorescence redistribution parameters. The functionality of GJIC was in relation with the staining localization of phosphorylated Cx43 to the cell-cell contact areas, corresponding to gap junctions between contacting cells. GJIC involvement in fluorescence restitution after photobleaching was checked by a gap junction channel inhibition assay. We demonstrated that the choice of the dye did not significantly influence the fluorescence recovery percentages despite a cell line-dependent CFDA release, whereas it had an important impact on fluorescence kinetic profiles. This study reinforces the interest of the gap-FRAP approach to quantify modifications in the functionality of gap junctions and, above all, argues about the limits of CFDA for 3-D future approaches.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

The connexin 43 C-terminus: A tail of many tales

Connexins are chordate gap junction channel proteins that, by enabling direct communication between the cytosols of adjacent cells, create a unique cell signalling network. Gap junctional intercellular communication (GJIC) has important roles in controlling cell growth and differentiation and in tissue development and homeostasis. Moreover, several non-canonical connexin functions unrelated to GJIC have been discovered. Of the 21 members of the human connexin family, connexin 43 (Cx43) is the most widely expressed and studied. The long cytosolic C-terminus (CT) of Cx43 is subject to extensive post-translational modifications that modulate its intracellular trafficking and gap junction channel gating. Moreover, the Cx43 CT contains multiple domains involved in protein interactions that permit crosstalk between Cx43 and cytoskeletal and regulatory proteins. These domains endow Cx43 with the capacity to affect cell growth and differentiation independently of GJIC. Here, we review the current understanding of the regulation and unique functions of the Cx43 CT, both as an essential component of full-length Cx43 and as an independent signalling hub. We highlight the complex regulatory and signalling networks controlled by the Cx43 CT, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. We discuss these data in relation to the recent discovery of the direct translation of specific truncated forms of Cx43. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Involvement of gap junctions in placental functions and development

Connexin (Cx) expression and gap junctional intercellular communication (GJIC) are involved in development and differentiation processes. Mediating exchanges between mother and fetus, the placenta is formed when fetal membranes are apposed or even fusing or destroying the uterine mucosa. Therefore, an extraordinary variability of placental structures is observed throughout the mammalian species. This variability affect mainly, the maternofetal blood flow interrelationships, the kind and number of tissue layers separating maternal and fetal bloods, the trophoblast invasiveness and the formation of a syncytium (syncytiotrophoblast). Here, the expression, the localisation and the possible role of Cx and GJIC in placental functions and development are discussed. In rodents, gene knock out in mice have vastly improved our understanding of the role of Cx genes in mouse placental development: Cx26 in transplacental uptake of glucose, Cx31 in the proliferative process of trophoblastic cells and Cx45 in placental vascularisation. In human, it appears that Cx43 allows a GJIC required for the fusion process of cytotrophoblastic cells leading to the formation of the syncytiotrophoblast, the site of the numerous placental functions. On other hands, Cx40 plays a critical role in the switch from a proliferative to an invasive phenotype of the trophoblastic cells invading the endometrium. Owing to the striking diversity of Cx expression in placental structures, we must be careful when extrapolating findings from one species to another.     

Expression of connexins 26, 32 and 43 in the human colon--an immunohistochemical study

Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions (GJs), which consist of proteins called connexins (Cxs). GJIC plays a critical role in tissue development and differentiation and is important in maintenance of tissue homeostasis. The purpose of the study was to evaluate the expression of Cx26, Cx32 and Cx43 in the human colon. Surgical specimens were obtained from patients who underwent surgical resection of colorectal tumours. Tissue samples (50 cases) were collected from normal colon, at the maximum distance from the tumor. Using antibodies for Cx26, Cx32 and Cx43, immunohistochemical detection was made. In epithelial cells, strong Cx26 immunoreactivity was found, whereas Cx32 and Cx43 were sparsely distributed. Strong Cx43 immunostaining in muscularis mucosae was observed. In the circular layer of muscularis externa, expression of Cx43 and Cx26 was seen, but only in the portion closest to the submucosa. No immunoreactivity was found in the longitudinal muscle layer. Small vessels stained positively only for Cx43. Furthermore, there was no difference in staining between samples derived from various sections of the colon. This study showed immunohistochemically for the first time the expression of Cx26 in human colon mucosa.     

A Neuroprotective Role for Gap Junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

A neuroprotective role for gap junctions

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein, connexin43 (Cx43), and provide a substrate for formation of a functional syncytium implicated in the process of spatial buffering in the CNS. Thus gap junctional communication may be neuroprotective following a CNS insult that entails glutamate cytotoxicity (i.e. ischemia). We have shown that blocking gap junctions during a glutamate insult to co-cultures of astrocytes and neurons results in increased neuronal injury. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild type and Cx43 heterozygote null mice following focal ischemia. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared to wild type. At the cellular level, a significant increase in TUNEL positive cells was observed in the penumbral region of the Cx43 heterozygote mice. These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection following ischemic injury. These findings support the hypothesis that gap junctions play a neuroprotective role against glutamate cytotoxicity.     

Gap junctions and hemichannels in signal transmission, function and development of bone

Gap junctional intercellular communication (GJIC) mediated by connexins, in particular connexin 43 (Cx43), plays important roles in regulating signal transmission among different bone cells and thereby regulates development, differentiation, modeling and remodeling of the bone. GJIC regulates osteoblast formation, differentiation, survival and apoptosis. Osteoclast formation and resorptive ability are also reported to be modulated by GJIC. Furthermore, osteocytes utilize GJIC to coordinate bone remodeling in response to anabolic factors and mechanical loading. Apart from gap junctions, connexins also form hemichannels, which are localized on the cell surface and function independently of the gap junction channels. Both these channels mediate the transfer of molecules smaller than 1.2kDa including small ions, metabolites, ATP, prostaglandin and IP(3). The biological importance of the communication mediated by connexin-forming channels in bone development is revealed by the low bone mass and osteoblast dysfunction in the Cx43-null mice and the skeletal malformations observed in occulodentodigital dysplasia (ODDD) caused by mutations in the Cx43 gene. The current review summarizes the role of gap junctions and hemichannels in regulating signaling, function and development of bone cells. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Involvement of tyrosine kinase in the hypoxia/reoxygenation-induced gap junctional intercellular communication abnormality in cultured human umbilical vein endothelial cells

Vascular endothelial cells (EC), communicating with one another across gap junctions, are usually made dysfunctional by hypoxia and reoxygenation (H/R); however, very limited information exists regarding the effects of H/R on the endothelial gap junctions. We investigated whether H/R interferes with endothelial gap junctional intercellular communication (GJIC). After human umbilical vein EC had grown to confluence, they were exposed to hypoxia (pO₂ < 0.1%) for 12–16 h and then returned to normal atmospheric conditions for reoxygenation. At 0-, 2-, 4-, 6-h reoxygenation, GJIC was detected by means of a fluorescence recovery after a photobleaching technique. The results demonstrated that a GJIC reduction (about 20% less than that under normoxia) was induced after 2 h of reoxygenation; after 4 h of reoxygenation, it began to recover (to about 10% less than that under normoxia); and after 6 h of reoxygenation, GJIC was restored to the normal level. Calphostin C (1 × 10⁻⁷ mol/l), a specific protein kinase C inhibitor, partially inhibited the reduction in GJIC (resulting in a level about 10% less than that under normoxia), whereas the tyrosine kinase inhibitor genistein (10 µmol/L) completely blocked the reduction in GJIC. Vanadate (1.5 mmol/l), a tyrosine phosphatase inhibitor, amplified the inhibitory effect of H/R on GJIC (to about 40% less than that under normoxia). Immunofluorescence and immunoprecipitation showed that 2-h reoxygenation significantly stimulated tyrosine protein phosphorylation, and this phosphorylation event was obviously enhanced by vanadate. The results of Western blotting showed that the gap junctional protein connexin 43 (Cx43) was phosphorylated by H/R; moreover, immunoprecipitation demonstrated that 2-h reoxygenation induced a prominent increase of tyrosine phosphorylation of Cx43 compared with that under normoxia. These data indicate that H/R induces a transient endothelial GJIC dysfunction through the activation of tyrosine kinase and phosphorylation of tyrosine residues of Cx43. J. Cell. Physiol. 180:305–313, 1999. © 1999 Wiley-Liss, Inc.     

Correlation between expression of cadherin and gap junctional communication in human lung carcinoma cells

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Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells

Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions.     

Gap junction-mediated intercellular communication between dendritic cells (DCs) is required for effective activation of DCs

Gap junctions, formed by members of the connexin (Cx) family, are intercellular channels allowing direct exchange of signaling molecules. Gap junction-mediated intercellular communication (GJIC) is a widespread mechanism for homeostasis in organs. GJIC in the immune system is not yet fully understood. Although dendritic cells (DC) reportedly form cell-to-cell contact between DCs in nonlymphoid and lymphoid organs, GJIC between DCs remains unknown. In this study we examined whether DCs form GJIC. XS52 and bone marrow-derived DCs (BMDCs) were tested for GJIC by counting intercellular transfer of Lucifer Yellow microinjected into a cell. Either DC became effectively dye-coupled when activated with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma. LPS- plus IFN-gamma-induced dye-coupling was mediated by DC-derived TNF-alpha. In addition, CpG plus IFN-gamma induced dye-coupling in BMDCs, which was also mediated by DC-derived TNF-alpha. LPS- plus IFN-gamma-induced activation of DCs (assessed by CD40 expression) was observed when there was cell-to-cell contact and was significantly blocked by heptanol, a gap junction blocker. These results indicate that cell-to-cell contact and GJIC are required for effective DC activation. In addition, heptanol significantly inhibited the LPS- plus IFN-gamma-induced up-regulation of the other costimulatory (i.e., CD80 and CD86) and MHC class II molecules expressed by BMDCs, and it significantly reduced their allostimulatory capacity. Among Cx members, Cx43 was up-regulated in dye-coupled BMDCs, and Cx mimetic peptide, a blocker of Cx-mediated GJIC, significantly inhibited the dye-coupling and activation, suggesting the involvement of Cx43. Thus, our study provides the first evidence for GJIC between DCs, which is required for effective DC activation.     

Gap junctional intercellular communication is required to maintain embryonic stem cells in a non-differentiated and proliferative state

Pluripotent embryonic stem (ES) cells are capable of maintaining a self-renewal state and have the potential to differentiate into derivatives of all three embryonic germ layers. Despite their importance in cell therapy and developmental biology, the mechanisms whereby ES cells remain in a proliferative and pluripotent state are still not fully understood. Here we establish a critical role of gap junctional intercellular communication (GJIC) and connexin43 (Cx43) in both processes. Pharmacological blockers of GJIC and Cx43 down-regulation by small interfering RNA (siRNA) caused a profound inhibitory effect on GJIC, as evidenced by experiments of fluorescence recovery after photobleaching. This deficient intercellular communication in ES cells induced a loss of their pluripotent state, which was manifested in morphological changes, a decrease in alkaline phosphatase activity, Oct-3/4 and Nanog expression, as well as an up-regulation of several differentiation markers. A decrease in the proliferation rate was also detected. Under these conditions, the formation of embryoid bodies from mouse ES cells was impaired, although this inhibition was reversible upon restoration of GJIC. Our findings define a major function of GJIC in the regulation of self-renewal and maintenance of pluripotency in ES cells.     

Suppression of the transformed phenotype of breast cancer by tropomyosin-1

Gap junctional intercellular communication (GJIC) is thought to play a crucial role in cell differentiation. Small gap junction plaques are frequently associated with tight junction strands in hepatocytes, suggesting that gap junctions may be closely related to the role of tight junctions in the establishment of cell polarity. To examine the exact role of gap junctions in regulating tight junctions, we transfected connexin 32 (Cx32), Cx26, or Cx43 cDNAs into immortalized mouse hepatocytes derived from Cx32-deficient mice and examined the expression and function of the endogenous tight junction molecules. In transient wild-type Cx32 transfectants, immunocytochemistry revealed that endogenous occludin was in part localized at cell borders, where it was colocalized with Cx32, whereas neither was detected in parental cells. In Cx32 null hepatocytes transfected with Cx32 truncated at position 220 (R220stop), wild-type Cx26, or wild-type Cx43 cDNAs, occludin was not detected at cell borders. In stable wild-type Cx32 transfectants, occludin, claudin-1, and ZO-1 mRNAs and proteins were significantly increased compared to parental cells and all of the proteins were colocalized with Cx32 at cell borders. Treatment with a GJIC blocker, 18 beta-glycyrrhetinic acid, resulted in decreases of occludin and claudin-1 at cell borders in the stable transfectants. The induction of tight junction proteins in the stable transfectants was accompanied by an increase in both fence and barrier functions of tight junctions. Furthermore, in the stable transfectants, circumferencial actin filaments were also increased without a change of actin protein. These results indicate that Cx32 formation and/or Cx32-mediated intercellular communication may participate in the formation of functional tight junctions and actin organization.     

Involvement of connexin43 in the EGF/EGFR signalling during self-renewal and differentiation of neural progenitor cells

Neural progenitor cells (NPCs) are sensitive to epidermal growth factor (EGF), which is essential for their self-renewal. Recently we showed that high level of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) are also required to maintain NPCs in a proliferative state. In this study the connection between EGF/EGFR signalling and Cx43 expression was investigated during proliferation and differentiation of cultured ReNcell VM197 human NPCs. We found that EGF, but not basic fibroblast growth factor (bFGF), strongly stimulated both Cx43 expression and GJIC in proliferating cells. This stimulatory effect was blocked by AG1478, a specific inhibitor for EGFR kinase. Notably, knockdown of Cx43 strongly inhibited the cell proliferation promoted by EGF/EGFR signalling. High sensitivity to EGF was still maintained in differentiated NPCs. Administration of EGF to differentiating cells led to a pronounced increase (9-fold) of Cx43 expression and a re-induction of proliferation. This strong impact of EGF was found to correlate with a surprisingly massive 60-fold up-regulation of EGFR expression in differentiated cells. Our data argue for a mutual regulation between Cx43 expression and EGF/EGFR signalling during self-renewal and differentiation of NPCs.