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PubMed1990

The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium.

The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.

Animals↗
PubMed2001

FAT is a component of glomerular slit diaphragms.

BACKGROUND: Slit diaphragms are intercellular junctions of podocytes of the renal glomerulus. The molecular composition of slit diaphragms is still elusive. Slit diaphragms are characterized by the presence of a wide intercellular space. The morphological feature is shared by desmosomes and adherens junctions, which contain members of the cadherin superfamily. Thus, we have hypothesized that some components of slit diaphragms belong to the cadherin superfamily. Consequently, we have isolated cDNA encoding FAT from reverse-transcribed (RT) glomerular cDNA by homology polymerase chain reaction (PCR) using primers based on conserved sequences in cadherin molecules. FAT is a novel member of the cadherin superfamily with 34 tandem cadherin-like extracellular repeats, and it closely resembles the Drosophila tumor suppressor fat. METHODS: Expression of FAT was examined in glomeruli of the adult rat kidney by the ribonuclease protection assay and in situ hybridization. To localize the FAT protein in podocytes minutely, we prepared affinity-purified antibody against FAT by immunizing rabbits against an oligopeptide corresponding to the C-terminal 20 amino acids. RESULTS: Expression of FAT mRNA was detected in total RNA from glomeruli. In situ hybridization revealed significant signals in podocytes. Western blot analysis using solubilized glomeruli showed a single band, in which the molecular weight was more than 500 kD. Immunostaining of cultured epithelial cells from rat kidney (NRK52E) revealed FAT accumulation in cell-cell contact sites. In the glomerulus, FAT staining was observed distinctly along glomerular capillary walls. Double-label immunostaining using monoclonal antibody against slit diaphragms (mAb 5-1-6) showed identical localization of anti-FAT antibody and mAb 5-1-6. Furthermore, the double-label immunogold technique with ultrathin cryosections demonstrated that gold particles for FAT cytoplasmic domain were located at the base of slit diaphragms labeled by mAb 5-1-6 and that the cytoplasmic domain of FAT colocalized with ZO-1, a cytoplasmic component associated with slit diaphragms. CONCLUSION: The molecular structure of FAT and its colocalization with 5-1-6 antigen and ZO-1 indicate that FAT is a component of slit diaphragms.

Animals↗
PubMed2003

Expression of connexin 43 in breast cancer in comparison with mammary dysplasia and the normal mammary gland.

Gap junctional intercellular communication (GJIC) plays a critical role in tissue development and differentiation and probably in carcinogenesis. The purpose of the study was to evaluate the expression and localisation of Cx43 in 40 cases of mammary dysplasia and 29 cases of breast cancer (without primary chemotherapy). The tissue sections were investigated for Cx43 expression by immunohistochemistry. In the normal mammary gland there was an intercellular, punctate staining pattern, mainly between myoepithelial cells, characteristic of functional gap junctions. In dysplasias there was mainly mixed (cytoplasmic and intercellular) staining and in most cases of breast cancer we observed diffuse or granular, but cytoplasmic, staining of Cx43. Our results demonstrated that expression of Cx43 in dysplasias and breast cancer is changed and GJIC is probably impaired because of disruption of functional gap junction formation especially between breast cancer cells.

Adenocarcinoma↗
PubMed1982

Freeze-fracture analysis of the interendothelial junctions in the blood vessels of the iris in Macaca mulatta.

The interendothelial cell junctions in the blood vessels of the rhesus monkey iris were examined by both conventional electron microscopy and the freeze-fracturing technique. Endothelial cells of iris blood vessels were joined by two types of intercellular junctions: zonular tight junctions and gap junctions. The zonulae occludentes were represented by a complex network of branching and anastomosing strands that remained preferentially associated with the E fracture face. They varied in complexity from a single strand to eight or more but usually consisted of from two to four strands. Small gap junctions were inserted within the tight junctional network. Junctions similar to those typical of muscular and visceral venules were absent. Thus the intercellular clefts of all iris vessels are closed by zonulae occludentes that exhibit a degree of complexity intermediate between the endothelial junctions of the cerebral cortex and those of striated muscles and viscera. This finding strongly suggests that the blood vessels of the iris participate only minimally in aqueous humor dynamics.

Animals↗
PubMed1983

The role of cAMP and Ca on the modulation of junctional conductance: an integrated hypothesis.

The role of cAMP and Ca in the modulation of junctional permeability is discussed. An integrated hypothesis is presented which proposes that cAMP modulates the junctional conductance through the activation of specific kinases and phosphorylation of gap junction proteins. A close-loop feed-back between cAMP and Ca is assumed to be relevant in the regulation of junctional conductance under physiological conditions. According to this hypothesis hormones modulate the junctional permeability through variations in the intracellular concentration of cAMP. It is known that in several tissues the cells are connected through low resistance intercellular junctions (Loewenstein, 1966; Bennett, 1973; De Mello, 1975, 1932a). Ions and small molecules can flow freely from cell-to-cell across narrow hydrophilic channels (De Mello, 1982a). This type of intercellular coupling is essential for the fast propagation of the impulse and the synchronization of electrical activity in excitable tissues (Bennett, 1973; De Mello, 1982a). It has been proposed that the exchange of chemical signals between cells is important for metabolic cooperation (Gilula et al. 1972) and growth control (Loewenstein, 1979). Therefore, the modulation of junctional conductance is a significant feature of cell biology. Evidence has been provided that the increase in free [Ca2+]i can produce cell decoupling in Chironomus salivary gland (Loewenstein et al., 1967) and in mammalian cardiac fibers (De Mello, 1972, 1975). The free [Ca2+]i required to suppress cell-to-cell coupling is difficult to determine because Ca ions are continuously taken up by mitochondria, sarcoplasmic reticulum or are extruded from the cell. In salivary gland a concentration of free [Ca2+]i of about 5-8 X 10(-5) M was found to be associated with cell decoupling (Loewenstein et al., 1967). The major difficulty here is that the concentration of the ion determined in the bulk of the cytosol is not necessarily the same near the gap junctions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes.

The signal transduction pathways and activation of the MAP kinase or PI3 kinase signaling cascade regulate a variety of cellular processes, including proliferation and differentiation in hepatocytes. To elucidate the mechanisms of signal transmission required for the regulation of gap and tight junctions during DNA synthesis in rat hepatocytes, we determined changes of expression and function of gap and tight junctions of cells grown in primary culture, using inhibitors of signaling pathways for MAP kinase (PD98059) and PI3 kinase (LY294002). During the stimulation of DNA synthesis induced by epidermal growth factor (EGF), immunoreactivity and mRNAs of gap junction protein Cx32 and of tight junction protein claudin-1 markedly decreased with reduction of gap junctional intercellular communication (GJIC) and the fence function of tight junctions. In Western blots, whole-cell lysate of claudin-1 protein decreased and phosphorylated Cx32 protein in the insoluble fraction of Triton X-100 increased during the stimulation of DNA synthesis. During reinhibition of DNA synthesis, the changes of Cx32 and claudin-1 returned to control levels, as did both functions. In treatment with the inhibitors before DNA synthesis, PD98059 inhibited the changes of expression and function of Cx32, but not claudin-1, without inhibition of cell growth, whereas LY294002 completely inhibited cell growth. These findings indicate that the PI3 kinase pathway rather than the MAP kinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of Cx32 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP kinase. Furthermore, Cx32, but not claudin-1, protein may be a target of activated MAP kinase in hepatocytes.

Animals↗
PubMed1991

Outgrowth of cells from human conjunctival explants onto cornea in vitro.

The epithelium was removed from human corneas, and samples of conjunctival tissue were cultured as explants on the denuded corneal surface for 1 and 2 weeks. Cells migrating from the conjunctival explants onto the corneal surface produced a multilayer where cells on the surface generally showed a flattened appearance. The apical membrane of these cells demonstrated villi as well as microplicae. Surface projections were also detected on cells in the deeper layers of the epithelium. Neighbouring cells were connected by junctional complexes. After 2 weeks, however, a lack of intercellular junctions in some areas resulted in the formation of intraepithelial cystoid spaces. Basal cells were connected to the underlying basement membrane by hemidesmosomes. Although transdifferentiation of the cells into a corneal epithelium was not observed within the 2 weeks, the present system provides a tool for studies on factors affecting reepithelialization of corneal epithelial defects by conjunctival cells.

Adolescent↗
PubMed1996

Characterization of ret oncogenic activation in MEN2 inherited cancer syndromes.

Germline mutations of c-ret, encoding a receptor-type tyrosine kinase, were found to be associated with variants of multiple endocrine neoplasia type 2 (MEN2A, MEN2B), and familial medullary thyroid carcinoma. NIH/3T3 stable transfectants expressing RET with a mutation of MEN2A (MEN2A/RET) or MEN2B (MEN2B/RET) gained a transformed morphology, formed colonies in soft agar, and formed tumors in nude mice. These results confirmed that both MEN2A/RET and MEN2B/RET exert dominant transforming activities in NIH/3T3 cells. However, in contrast to their clinical manifestation, transfectants expressing MEN2A/RET exhibited a higher tumorigenicity in nude mice than transfectants expressing MEN2B/RET may depend on the presence of its ligand and/or substrates that are absent in NIH/3T3 cells. No change in the cellular localization of the mutated RET proteins was observed compared to c-RET. Interestingly, ret activation in NIT/3T3 cells appeared to be associated with up-regulation of homologous gap-junctional intercellular communication and increased expression of a gap-junctional protein, connexin43.

3T3 Cells↗
PubMed1995

Inhibition of intercellular communication by condensates of high and low tar cigarettes.

Inhibition of gap junctional intercellular communication (GJIC) is a predictive short term test for tumor promoting activity. A new metabolic cooperation assay has been developed, which takes the cytochrome P-450 metabolism into account. In this assay the inhibitory activity of tobacco smoke condensates (CSC) and CSC fractions from high and low tar cigarettes was tested. CSC of both high and low tar cigarettes and fractions thereof contained tumor promoting activity. The tar yield of the cigarettes did not closely reflect the effects in the GJIC assay and the major constituent nicotine had no effect. The effect was only marginally greater in cells expressing different cytochrome P-450 enzymes, indicating that the active substances are not metabolized by these enzymes. The activities of CSC fractions were considerably lower than the activities in the unfractionated CSC. This may indicate that compounds in the CSC act strongly synergistically. Furthermore, CSC and CSC fractions synergistically inhibit GJIC with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, indicating different mechanisms of action.

Cell Communication↗
PubMed2004

Decreased intercellular communication and connexin expression in mouse aortic endothelium during lipopolysaccharide-induced inflammation.

The role of gap junctional intercellular communication during inflammatory processes is not well understood. In particular, changes in the expression and function of vascular endothelial connexins (gap junction proteins) in response to inflammatory agents has not been fully investigated. In this study, we used intercellular dye transfer methods to assess interendothelial communication in aortic segments isolated from mice treated with or without intraperitoneal lipopolysaccharide (LPS), a potent inflammatory mediator. LPS treatment resulted in a 49% decrease in endothelial dye coupling 18 h after injection. Western blots indicated that LPS treatment also caused a reduction in endothelial connexin40 (Cx40) levels to 33% of control levels. Connexin37 (Cx37) levels decreased only slightly after LPS treatment to 79% of control levels. We also examined endothelial communication in aortic segments isolated from Cx37-/- and Cx40-/- mice. LPS treatment caused a significantly greater decrease in dye transfer in endothelium isolated from Cx37-/- animals compared with endothelium from Cx40-/- animals (71 vs. 26% decrease). LPS injection caused a reduction in Cx40 levels in Cx37-/- endothelium, whereas LPS actually increased Cx37 levels in Cx40-/- endothelium. These results suggest that LPS mediates changes in endothelial gap junction-mediated communication, at least in part, through modulation of Cx40 and Cx37 levels.

Animals↗
PubMed2006

Altered intercellular communication in lung fibroblast cultures from patients with idiopathic pulmonary fibrosis.

RATIONALE: Gap junctions are membrane channels formed by an array of connexins which links adjacent cells realizing an electro- metabolic synapse. Connexin-mediated communication is crucial in the regulation of cell growth, differentiation, and development. The activation and proliferation of phenotypically altered fibroblasts are central events in the pathogenesis of idiopathic pulmonary fibrosis. We sought to evaluate the role of connexin-43, the most abundant gap-junction subunit in the human lung, in the pathogenesis of this condition. METHODS: We investigated the transcription and protein expression of connexin-43 and the gap-junctional intercellular communication (GJIC) in 5 primary lung fibroblast lines derived from normal subjects (NF) and from 3 histologically proven IPF patients (FF). RESULTS: Here we show that connexin-43 mRNA was significantly reduced in FF as demonstrated by standard and quantitative RT-PCR. GJIC was functionally evaluated by means of flow-cytometry. In order to demonstrate that dye spreading was taking place through gap junctions, we used carbenoxolone as a pharmacological gap-junction blocker. Carbenoxolone specifically blocked GJIC in our system in a concentration dependent manner. FF showed a significantly reduced homologous GJIC compared to NF. Similarly, GJIC was significantly impaired in FF when a heterologous NF line was used as dye donor, suggesting a complete defect in GJIC of FF. CONCLUSION: These results suggest a novel alteration in primary lung fibroblasts from IPF patients. The reduced Cx43 expression and the associated alteration in cell-to-cell communication may justify some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical practice.

Adaptation, Physiological↗
PubMed1985

The effects of thermal stimulation on the ultrastructure of the human atrichial sweat gland. II. The duct.

The duct of the human atrichial sweat gland, after thermal stimulation, exhibited increased cytoplasmic vesiculation, particularly of the luminal cells, a widening of the intercellular spaces, and the presence of particulate matter in the lumen. The luminal cells of the coiled zone also displayed varying degrees of apical disruption, most probably due to filtration of cell fluid through the terminal web of microfilaments. This zone seems to have a secretory as well as an absorptive role. Specialized junctions (intercellular bridges, gap junctions and tight junctions) were found amongst the basal and luminal cells of the wall of the ascending duct.

Cytoplasm↗
PubMed2002

Keratinocyte junctions and the epidermal barrier: how to make a skin-tight dress.

Although intercellular junctions are known to be the major regulators of permeability of simple epithelia, they had not been thought to be important in regulating the permeability of stratified mammalian epithelia. Furuse et al. (2002)(this issue) demonstrate that functional tight junctions may indeed be a necessary part of the permeability barrier of the skin.

Animals↗
PubMed2001

Determination of a potential role of the CCN family of growth regulators in connexin43 transfected C6 glioma cells.

Tumour cells often exhibit erratic cell growth, as well as decreased gap junctional intercellular communication (GJIC). C6 glioma cells are characterized by low levels of gap junction mRNA and protein, and decreased amounts of GJIC when compared with astrocytes. Previous work has shown that C6 glioma cells transfected with connexin43 (C6-Cx43) exhibit decreased proliferation in vivo and in vitro, as well as genes that are differentially expressed between these cells. In this study, RNA levels of two CCN (connective tissue growth factor [CTGF], Cyr61/Cef-10, nephroblastoma overexpressed [NOV]) gene family members are shown to be upregulated in C6-Cx43 cells: Cyr61 and Nov. Cyr61 has previously been shown to increase adhesion, migration and growth in many cell types, whereas NOV has growth suppressive capacities. Cyr61 RNA expression is shown here to respond to serum in quiescent cells in an immediate early gene fashion, independent of Cx43 expression. In contrast, Nov RNA levels remain constant, reflective of transfected Cx43 expression. Furthermore, confocal microscopy indicates that NOV colocalizes with Cx43 plaques at the cell membrane. These findings provide insight into the possible role of Nov and Cyr61 in tumour cells.

Animals↗
PubMed2000

Gap junctions in the chicken pineal gland.

The chicken pineal gland, which contains a heterogeneous cell population, sustains a circadian rhythm of activity. Synchronization of cellular activity of heterogeneous cells might be facilitated by gap junctional intercellular channels which are permeable to ions and second messengers. To test this possibility, we looked for morphologically identifiable gap junctions between the different pineal cells, used antibodies and cDNA probes to screen for the presence of connexins, and tested for functional intercellular coupling. By transmission electron microscopy and immunocytochemistry, gap junctions and connexins were observed between pinealocyte cell bodies, stromal cells, astrocytes, and astrocyte and pinealocyte processes. Two gap junctional proteins, connexin43 and connexin45, were detected by immunocytochemistry, immunoblotting and RNA blot analysis. Functional intercellular coupling was observed in the gland by transfer of low molecular weight dyes. Dye transferred between homologous and heterologous cells. These data suggest that homologous and heterologous gap junctions may provide a mechanism for coordination of the cellular responses of the elements of the biological clock which are induced by lighting cues to produce the circadian rhythm of pineal activity.

Animals↗
PubMed1982

Partial purification of an intercalated disc-containing cardiac plasma membrane fraction.

The sarcolemma of cardiac muscle cells contains a specialised junctional region, the intercalated disc which includes three types of intercellular junction, the macula and fascia adherens and the nexus or gap junction. To facilitate the isolation of these junctions a procedure for the partial purification from mouse hearts of a subcellular fraction containing the intercalated disc region of the sarcolemma was developed. This involved investigating methods of tissue disruption that preserve the integrity of the intercalated disc and minimise myofibrillar entrapment of organelles. Examination of the distribution of marker enzymes showed that relative to the homogenate the intercalated disc fraction prepared by sucrose density centrifugation was only enriched 1.5- to 3-fold in 5'-nucleotidase and (Na+ + K+)-ATPase activities, whereas mitochondrial and sarcoplasmic reticulum marker enzymes were low. The properties of the intercalated disc-containing fraction were compared with the vesicular sarcolemmal fractions devoid of junctional complexes prepared by other methods.

5'-Nucleotidase↗
PubMed1989

Fetal vasculogenesis and angiogenesis in human placental villi.

Placental villi of 5 exactly defined early human specimens ranging from day 21 post conception (p.c.) until day 42 p.c. and from an additional 43 specimens from about 5 to 40 weeks menstrual age have been analyzed ultrastructurally with regard to fetal vasculogenesis and angiogenesis. The following results were obtained: The first cells differentiating at day 21 p.c., probably originating from mesenchymal precursors, are macrophage-like cells. At almost the same time, mesenchymal cells transform into haemangioblastic cell cords which are the forerunners of the capillary endothelium and haematopoietic stem cells. A third cell population related to the fetal circulatory system and derived from the mesenchymal cells are presumptive pericytes. Capillary formation takes place by the aggregation of haemangioblastic cells which are attached to each other by intercellular junctions. The lumen is formed by the dehiscence of the intercellular clefts. A capillary basal lamina cannot be detected earlier than in the last trimester. In this last period of gestation, fetal villous angiogenesis takes place by the proliferation of the existing endothelium and pericytes rather than via haemangioblastic cells.

Blood Vessels↗
PubMed2002

Migration of human hematopoietic progenitor cells across bone marrow endothelium is regulated by vascular endothelial cadherin.

The success of stem cell transplantation depends on the ability of i.v. infused stem cells to engraft the bone marrow, a process referred to as homing. Efficient homing requires migration of CD34(+) cells across the bone marrow endothelium, most likely through the intercellular junctions. In this study, we show that loss of vascular endothelial (VE)-cadherin-mediated endothelial cell-cell adhesion increases the permeability of monolayers of human bone marrow endothelial cells (HBMECs) and stimulates the transendothelial migration of CD34(+) cells in response to stromal cell-derived factor-1alpha. Stromal cell-derived factor-1alpha-induced migration was dependent on VCAM-1 and ICAM-1, even in the absence of VE-cadherin function. Cross-linking of ICAM-1 to mimic the leukocyte-endothelium interaction induced actin stress fiber formation but did not induce loss of endothelial integrity, whereas cross-linking of VCAM-1 increased the HBMEC permeability and induced gaps in the monolayer. In addition, VCAM-1-mediated gap formation in HBMEC was accompanied by and dependent on the production of reactive oxygen species. These data suggest that modulation of VE-cadherin function directly affects the efficiency of transendothelial migration of CD34(+) cells and that activation of ICAM-1 and, in particular, VCAM-1 plays an important role in this process through reorganization of the endothelial actin cytoskeleton and by modulating the integrity of the bone marrow endothelium through the production of reactive oxygen species.

Antigens, CD↗