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PubMed1984

Effects of extracellular calcium and magnesium on junctional intercellular communication in human fibroblasts.

The role of extracellular calcium and magnesium in intercellular communication via permeable junctions was studied by measuring metabolic co-operation between ASS- and ASL- human fibroblasts. Communication through pre-existing junctions was not affected by the removal of extracellular calcium and magnesium from the medium. On the other hand, the establishment of communication through new permeable junctions, when ASS- and ASL- cells were mixed, was dependent on the presence of extracellular calcium and magnesium.

Calcium↗
PubMed1990

Ultrastructural differences in junctional intercellular communication between highly and weakly metastatic clones derived from rat mammary carcinoma.

We examined by electron microscopy the differences in junctional intercellular communications among highly metastatic clones, weakly metastatic clones, and the parent clone obtained from a spontaneously developed rat mammary carcinoma. We also investigated intercellular communications of the highly and weakly metastatic clone cells with normal fibroblasts. The results showed that ultrastructural changes of the highly metastatic clone cells, such as microvilli, microfilaments, and small organelles including endoplasmic reticulum, Golgi apparatus, and mucous particles, were more distinct than those of the weakly metastatic clone cells, and that the numbers of desmosome and gap junctions of weakly metastatic clone cells were significantly greater than those of highly metastatic clone cells. The formation of gap junctions and desmosomes was found only between weakly metastatic clone cells and normal fibroblasts. When both highly and weakly metastatic clone cells were cultured with normal fibroblasts, a tight junction was observed only in the culture of weakly metastatic clone cells and normal fibroblasts. These results suggest that ultrastructural differences are related to the proliferation and detachment of tumor cells from the primary site in the initial stage of tumor metastasis.

Animals↗
PubMed2002

Decreased gap junctional intercellular communication in hexachlorobenzene-induced gender-specific hepatic tumor formation in the rat.

Hexachlorobenzene (HCB), an epigenetic carcinogen, HCB induces the formation of liver tumors in female rats, whereas only a small percentage of males are responsive. Intercellular communication via gap junctions is decreased in carcinogenesis. Gap junctions are composed of proteins termed connexins (Cxs). The objectives of this study were (i) to determine if HCB-induced tumor development is associated with a loss of gap junctional communication; (ii) to assess if HCB causes a gender-specific decrease in the expression of Cx32 and Cx26; and (iii) to establish if these effects result from gender differences in the constitutive expression of these Cxs. Rats were given HCB by gavage for five consecutive days. In the first experiment, control and HCB-treated female rats were sampled on day 100. Intercellular communication was significantly decreased in HCB-treated females compared to controls. To investigate if changes in Cx levels occur prior to day 100, experiments done using male and female rats sampled on day 50. Hepatic mRNA levels for Cx26 and Cx32 were significantly lower only in HCB-treated females as compared to controls. Cx26 mRNA levels were 3-fold higher and Cx32 mRNA levels were 8-fold lower in females compared with males. In a third experiment, ovariectomy abolished any differences between male and female controls for both Cxs, while estradiol had a partial role in the regulation of Cx32. This suggests that the sexual dimorphism in hepatic Cx levels is determined by the ovarian hormones. However, the HCB-induced decrease in Cx32 and Cx26 mRNA levels was maintained in ovariectomized rats, suggesting that the HCB effects are not mediated via an ovary-dependent pathway. Overall results show that HCB exposure induces gender-specific long-term alterations in intercellular gap junctional communication in female rat liver. This effect appears to be a critical mechanism of HCB-induced liver carcinogenesis and tumor promotion.

Animals↗
PubMed2001

Effective reduction of neuronal death by inhibiting gap junctional intercellular communication in a rodent model of global transient cerebral ischemia.

Gap junctions assemble astrocytes into syncytia, allowing exchange of metabolites, catabolites, and second-messenger molecules. Connexin43 is the predominant connexin of astrocytic gap junctions. The distribution of gap junction protein connexin43 was analyzed in different subfields of the hippocampal formation as a function of time after transient forebrain ischemia. One decisive key step in understanding why an ischemic insult gradually expands may be to establish how gap junction channels permit dying cells in the ischemic focus to communicate, in particular, with viable cells. The role of gap junctional intercellular communication in the hippocampus under ischemic conditions could be decisive for cell death propagation. We found that the vulnerable CA1/CA2 subfields have a higher density of gap junctions than the resistant CA3/CA4 areas, that changes in the distribution of connexin43 immunoreactivity may correlate with the phenomenon of selective vulnerability, and that inhibition of astrocytic gap junction permeability by octanol restricts the flow of undesirable neurotoxins that could potentially exacerbate neuronal damage. This provides a novel perspective for analysis of the pathophysiology of cerebral ischemia.

Animals↗
PubMed2003

Reduced gap junctional intercellular communication and altered biological effects in mouse osteoblast and rat liver oval cell lines transfected with dominant-negative connexin 43.

Gap junctional intercellular communication (GJIC) maintains normal growth and differentiation of cells in a tissue. The intercellular molecules traversing gap junctions are largely unknown, but the molecular weight (MW) cutoff is normally 1200 Da. No differences in dye transfer were observed in normal or vector controls of WB-F344 rat liver epithelial or mouse osteoblastic MC3T3-E1 cells with either Lucifer Yellow (LY) with a MW of 457 Da (LY-457) or LY with a MW of 649 Da (LY-649). Transfection of a dominant negative-connexin 43 (Cx43) gene decreased GJIC (>50%) when LY-649 was used, however, normal GJIC was observed in both cell lines when LY-457 was used. Therefore, the MW cut off in these clones was considerably less than the wild type. The dominant negative clones of the MC3T3-E1 cells exhibited over 90% less alkaline phosphatase (ALPase) activity and calcium deposition after the induction of differentiation. Similarly, dominant negative Cx43 inhibited gene expression of ALPase and bone sialoprotein but not osteocalcin in MC3T3-E1. WB-F344 cells normally exhibit a biphasic response to 12-O-tetradecanoylphorbol-13-acetate (TPA) where inhibition of GJIC recovers after 2 h, but the dominant negative clones showed no recovery from inhibition of GJIC by TPA. Dominant negative Cx43 also inhibited the formation of network-like structures by WB-F344 cells on Matrigel. These results demonstrate that the dominant negative gene transfected into cell types containing the wild-type connexins result in diminished channel sizes, thus allowing the determination of whether distinct biological endpoints, i.e., differentiation, are dependent upon either small or high MW intercellular signals.

Animals↗
PubMed2003

Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro.

We investigated gene expression associated with trophectoderm epithelial intercellular junction formation in single human embryos at different stages of cleavage using RT-PCR methods based upon magnetic bead separation of polyA+ RNA. Trophectoderm tight junction (TJ) and desmosome biogenesis contribute to intercellular sealing and tissue integrity, critical for vectorial transport and blastocoel cavity formation. Expression of the various genes throughout human preimplantation development showed differing levels of sensitivity of detection; these genes included claudin-1, occludin (TM4+ and TM4 isoforms), ZO-1 (ZO-1alpha+ and ZO-1alpha- isoforms), ZO-2 and JAM (junction adhesion molecule), and the desmosome junction gene, DSC2 (desmocollin 2). Some transcripts appeared to be expressed throughout preimplantation development (claudin-1, JAM, occludin TM4+ and TM4, ZO-1alpha- isoform) while others tended to be expressed preferentially in later cleavage and associated with blastocyst formation (ZO-2, ZO-1alpha+ isoform, DSC-2), illustrating an expression pattern broadly similar to mouse cleavage stages. Human embryo transcript detection was significantly decreased when reverse transcription was performed in solid phase to generate a bead/cDNA transient library rather than after mRNA elution from beads. Transcript detection tended to be positively correlated with embryo morphological grade using the solid phase method. In blastocysts, occludin TM4-, ZO-1alpha+ and DSC2 transcripts were the most susceptible to failure of detection, indicative of low levels of expression which may impact on trophectoderm differentiation competence. Immunoconfocal microscopy analysis of selected adhesion and TJ proteins in human embryos indicated poor membrane assembly compared with mouse blastocysts, which may further affect embryo viability.

Blastocyst↗
PubMed1991

Sequential changes of gap-junctional intercellular communications during multistage rat liver carcinogenesis: direct measurement of communication in vivo.

We have developed a simple method to measure gap-junctional intercellular communication (GJIC), by means of microinjection/dye transfer assay, in liver slices freshly removed from the rat. Using this method and immunostaining of connexin 32 (cx32), the major liver gap junction protein, we studied sequential changes of GJIC during chemical hepatocarcinogenesis in male Fischer-344 rats under a modified Solt-Farber protocol (3 weeks 4 day exposure regimen). Four weeks after commencement of the protocol, there was a substantial decrease in GJIC in the liver parenchyma, which was free from focal lesions. The decrease in GJIC persisted up to at least the 15th week of treatment, while a decrease in the number of immunoreactive cx32 spots was evident only at 4 weeks of post-protocol commencement. Most enzyme-altered (GST-P-positive) focal lesions showed markedly lower GJIC and a significantly lower number of cx32-positive spots than surrounding hepatocytes. Most GST-P-positive foci showed a selective lack of GJIC with surrounding heptocytes. Hepatocellular carcinomas arising 1 year after the carcinogenic regimen had significantly reduced communicational capacity accompanied by a large decrease in cx32 expression. These results suggest that a progressive decrease in homologous as well as heterologous GJIC in preneoplastic lesions occurs during rat hepatocarcinogenesis, and that preneoplastic lesions with the most prominent disorders in GJIC may be more likely to develop into carcinomas.

Animals↗
PubMed1996

Modulation of gap junctional intercellular communication by EGF in human kidney epithelial cells.

Modulation of gap junctional intercellular communication (GJIC) was studied in a multistep model of human renal epithelial carcinogenesis. We report that the majority of primary human kidney epithelial cells (NHKE) grown from fetal kidney explants did not communicate through gap junctions. Communication could, however, be observed within a subpopulation of the cells. Ni(II)-immortalized cells (IHKE) showed GJIC at a level of 10-20 communicating cells, but with heterogeneous regions on the dish, with regard to both communication and distribution of connexin43. The heterogeneity was less pronounced in a ras-transfected tumourigenic cell line (THKE), which also showed communication of approximately 10-20 dye-coupled cells. Communication within the IHKE sub-clone K7 decreased from 55 dye-coupled cells communicating on day 1 after seeding to approximately 13 in cells grown for 4 days. Daily change of growth medium reduced the decrease in GJIC. EGF enhanced communication following a lag period which depended on days in culture. The largest increase in GJIC was observed in 2-day-old cultures, where the number of communicating cells in some experiments increased from approximately 45 to 130 dye-coupled cells 4 h following change to medium with EGF. The induction was concentration dependent and communication was enhanced gradually between 2 and 7 h after exposure to EGF. A 15 min pulse of EGF was sufficient to induce the GJIC increase if the total incubation period was unchanged. Cycloheximide completely blocked the EGF-induced enhancement of communication, while actinomycin D had no effect. EGF exposure resulted in an increase in the cellular level of connexin43 protein in parallel with the enhancement in communication. Together, these results indicate that the EGF-induced enhancement of GJIC in human kidney epithelial cells was mediated through translational control of connexin43 expression.

Cell Communication↗
PubMed1989

A human endothelial cell-restricted, externally disposed plasmalemmal protein enriched in intercellular junctions.

We have raised an mAb to a previously undescribed 135-kD externally disposed integral membrane protein that is enriched in the intercellular junctional domain of cultured human umbilical vein endothelial cells. This protein localizes at the appositional surfaces of cells as they become confluent and is stably expressed in the junctional zones of confluent monolayers. This protein is expressed in situ on continuous endothelia of all blood vessels in all human tissues examined. Moreover, this protein, as determined by mAb immunocytochemistry, is not expressed by any other cell type. This protein may mediate endothelial-specific functions restricted to the intercellular domain. It may also serve as a unique cell surface marker for the identification and purification of human endothelial cells.

Antibodies, Monoclonal↗
PubMedDate not supplied

Enhancement of gap junctional intercellular communication by dibutyryl cyclic AMP in lung epithelial cells.

Reduced gap junctional intercellular communication (GJIC) occurs in neoplastic cells and contributes to their phenotype. Cyclic AMP agonists inhibit lung cancer cell growth and enhance GIIC in other cell types, but little is known about their effects on lung epithelial cell gap junctions. We have examined whether N6, 2'-O-dibutyryladenosine 3':5'-cyclic mono-phosphate (DBcAMP) affected GJIC, gap junction protein (connexin43) expression, and the growth of non-transformed and neoplastic mouse lung epithelial cells. DBcAMP (0.01.1 mM) stimulated GJIC (assayed by fluorescent dye microinjection) and connexin43 expression (assessed by Northern and Western blotting) and reduced their proliferation. These results suggest an association between cAMP growth inhibition and enhanced GJIC in lung epithelial cells.

Animals↗
PubMed1999

[Endothelial intercellular junctions: relationship between vascular adhesion and morphogenesis].

Endothelial cell-cell junctions are required for integrity of the vascular system. Similar to that of epithelial cells, the interendothelial cleft contains tight, adherens and gap junctions, and the newly-defined complexus adherentes. In the past few years, the molecular components of these organelles were identified as well as their mutual interactions. These data deciphered the complex link between the cell membrane and the cytoskeleton. The contribution of these junctions in vessel formation and permeability is discussed in this review.

Animals↗
PubMed1995

Inhibition of rat liver gap junction intercellular communication by tumor-promoting agents in vivo. Association with aberrant localization of connexin proteins.

BACKGROUND: Gap junctional intercellular communication is believed to play an important role in the maintenance of tissue homeostasis, and disruption of it has been proposed to be involved in carcinogenesis. A number of tumor-promoting agents have been shown to inhibit capacity for intercellular communication in cell culture studies. Recently, we developed a simple dye-transfer technique to evaluate cell-coupling function in fresh liver slices, and we used it to show that inhibition of intercellular communication is associated with rat liver tumor progression. Using this method with analysis of gap junction protein connexin expression, we have examined whether and how different liver-specific tumor-promoting agents inhibit dye-coupling in rat liver in vivo. EXPERIMENTAL DESIGN: Groups of Fischer 344 rats received repeated chronic treatment of phenobarbital (PB), polychlorinated biphenyls (PCB), dichlorodiphenyltrichloroethane (DDT), and clofibrate (CF) for 5 weeks. After 1, 2, and 5 weeks of treatment, intercellular communication via gap junctions was evaluated by the dye-transfer assay in liver slices taken immediately after killing the rats. In parallel, the expression of connexins (cx) 32, 26, and 43 (gap junction proteins expressed in the liver) was studied at the mRNA and protein levels. RESULTS: All four tumor-promoting agents decreased dye-coupling in rat liver. This decrease was associated with a reduced number of gap junctions and aberrant localization of some amount of cx 32 proteins in hepatocytes; cx 32 often was observed in the cytoplasm of hepatocytes instead of at gap junctions in the plasma membrane. Western blot analysis showed only slight changes in the level of cx 32 proteins. Although cx 26 proteins at gap junctions were usually decreased by tumor promoters in rat liver, local induction of cx 26 protein expression in centrolobular groups of hepatocytes after PCB and DDT treatment was observed. The expression of cx 43 was induced in hepatocytes after PCB, DDT, and CF exposure, but this protein was also localized intracytoplasmically, suggesting no functional role. All four tested tumor-promoting agents also increased cell proliferation, as revealed by staining with an anti-Ki 67 antibody. CONCLUSION: The results demonstrate that different types of liver tumor-promoting agents inhibit dye-coupling in rat liver in vivo. This inhibition may be due to aberrant localization of the major liver gap junction protein cx 32, rather than its transcriptional or translational disregulation.

Animals↗
PubMed1996

Prolonged cytokine exposure causes a dynamic redistribution of endothelial cell adhesion molecules to intercellular junctions.

After 4 hours of treatment with TNF, newly synthesized endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 molecules are diffusely expressed on the apical surface of cultured umbilical vein endothelial cells. Such cells maintain the epithelioid, cobblestone appearance of untreated endothelial cells and display cytoskeletal actin largely arranged in dense peripheral bands. After 24 to 72 hours of treatment with TNF, cells become elongated and rearrange their actin filaments into longitudinal stress fibers. At this time, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 remain elevated but redistribute to the cell junctions. Intercellular adhesion molecule 2, beta 1 integrins, and beta 3 integrins also redistribute to cell junctions in TNF-treated cultures. IFN-gamma produces morphologic changes similar to those induced by TNF but does not cause surface protein redistribution. Cells treated with TNF plus IFN-gamma become even more elongated and display TNF-like redistributions. We conclude that TNF activates a program of membrane protein redistribution, and we speculate that this dynamic redistribution of adhesion molecules to cell junctions may contribute to the recruitment of leukocytes to sites of inflammation.

Animals↗
PubMed2002

Composition and formation of intercellular junctions in epithelial cells.

The polarized nature of epithelial cells is manifested by the nonrandom partitioning of organelles within the cells, the concentration of intercellular junctions at one pole, and the asymmetric distribution of proteins and lipids within the plasma membrane. These features allow epithelia to fulfill their specific tasks, such as targeted uptake and secretion of molecules and the segregation of different tissue compartments. The accessibility of Drosophila melanogaster and Caenorhabditis elegans to genetic and cell biological analyses, combined with the study of mammalian cells in culture, provides an ideal basis for understanding the mechanisms that control the establishment and maintenance of epithelial cell polarity and tissue integrity. Here, we focus on some of the best-studied junctions and membrane-associated protein complexes and their relation to cell polarity. Comparisons between fly, worm, and vertebrate epithelia reveal marked similarities with respect to the molecules used, and pronounced differences in the organization of the junctions themselves.

Animals↗
PubMed2005

Defective gap junctional intercellular communication in the carcinogenic process.

Gap junctions are membrane structures made of intercellular channels which permit the diffusion from cytoplasm to cytoplasm of small hydrophilic molecules. Nearly 40 years ago, the loss of functional gap junctions has been described in cancer cells and led to the hypothesis that such type of intercellular communication is involved in the carcinogenesis process. From this time, a lot of data has been accumulated confirming that gap junctions are frequently decreased or absent in cancer cells whatever their tissue and species origins. Here, we review such data by insisting on the possible links existing between altered gap-junctional intercellular communication capacity (or the altered expression of their constitutive proteins, the connexins) and the stages of cancer progression in various cancer models. Then, we analyse particular aspects of the disturbance of connexin-mediated communication in cancer such as the cytoplasmic localization of connexins, the lack of heterologous communication between cancer cells and normal cells, the role of connexin gene mutations in cancer. In a separate part of the review, we also analyse the disturbance of gap-junctional intercellular communication during the late stages of cancer (invasion and metastasis processes).

Animals↗
PubMed2007

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.

Cell Communication↗
PubMed1999

Up-regulation of gap junctional intercellular communication by hexamethylene bisacetamide in cultured human peritoneal mesothelial cells.

Gap junctional intercellular communication (GJIC) is believed to be an important means of regulating cell growth and the malignant potential of tumors. This study examined the effect of hexamethylene bisacetamide (HMBA), a hybrid polar compound and a potent differentiation inducer, on GJIC in cultured primary human peritoneal mesothelial cells. The redistribution of fluorescence after photobleaching was used to detect GJIC. After the incubation of confluent cell cultures with 3 or 6 mM HMBA for 3 and 6 days, GJIC was significantly increased in a concentration-dependent manner compared with cultures without HMBA. Western blotting showed that connexin 43 (Cx43), the major functional protein of gap junctions in peritoneal mesothelial cells, was present in unphosphorylated and phosphorylated forms in control cell cultures. The addition of HMBA to cultures induced a significant increase of total Cx43 protein because of an increase of the phosphorylated forms. Immunofluorescence studies showed that HMBA increased the intensity of fluorescence for Cx43 at cell membrane borders. Quantitative reverse transcription and PCR analysis revealed that the addition of HMBA to cultures resulted in the concentration-dependent up-regulation of mRNA for Cx43. These results indicate that HMBA induces the enhancement of GJIC in peritoneal mesothelial cells through both the up-regulation of Cx43 messages and an increase of post-translational phosphorylation. HMBA may contribute to the maintenance of cellular homeostasis through the up-regulation of GJIC.

Acetamides↗