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PubMed2002

The Armadillo family protein p0071 is a VE-cadherin- and desmoplakin-binding protein.

p0071, a member of the armadillo protein family, localizes to both adherens junctions and desmosomes in epithelial cells and exhibits homology to the adherens junction protein p120 and the desmosomal protein plakophilin-1. p0071 is also present at dermal microvascular endothelial intercellular junctions and colocalizes with VE-cadherin, an endothelium-specific cadherin that associates with both actin and intermediate filament networks. To define the role of p0071 in junction assembly, p0071 was tested for interactions with other components of the endothelial junctional complex. In transient expression assays, p0071 colocalized with and formed complexes with both VE-cadherin and desmoplakin. Deletion analysis using the yeast two-hybrid system revealed that the armadillo repeat domain of p0071 bound directly to VE-cadherin. Site-directed mutagenesis experiments demonstrated that p0071 and p120 bound to the same region on the cytoplasmic tail of VE-cadherin and that overexpression of p0071 could displace p120 from intercellular junctions. In contrast to VE-cadherin, desmoplakin was found to associate with the non-armadillo head domain of p0071. Cotransfections and triple-label immunofluorescence analysis revealed that VE-cadherin colocalization with desmoplakin in transfected COS cells required p0071, suggesting that p0071 may couple VE-cadherin to desmoplakin. Based on previous findings that both VE-cadherin and desmoplakin play central roles in vasculogenesis, these new results suggest that p0071 may play an important role in endothelial junction assembly and in the morphogenic events associated with vascular remodeling.

Adherens Junctions↗
PubMed1981

The development of cell junction during nephrogenesis.

The differentiation and distribution of intercellular junctions especially during the early development stages of the rabbit nephron was studied by freeze-fracture electron microscopy. Metanephrogenic cells were found to be connected by sporadic focal tight junctions. During the formation of the renal vesicle similar tight junctions occurred on the periphery as well as near the developing lumen. These focal tight junction increased in size and coalesced to broad zonulae occludentes lining the vesicular lumen at a later stage. Broad occluding junctions were also observed in the different nephron segments of the S-shaped stage. Ultrastructurally, these early maculae and zonulae occludentes consisted of beaded rows of particles. As development progressed, continuous tight junctions formed, whereas the number of strands decreased with the exception of the distal tubule. In contrast to the parietal glomerular epithelium, the initial occluding zonules of the visceral glomerular cells were gradually reduced to maculae occludentes, and finally disappeared. These results suggest that zonulae occludentes appear synchronously with the establishing lumen; the ultrastructural differentiation of tight junction strands seems to be completed with the onset of glomerular filtration.

Animals↗
PubMed2004

Comparative analysis of armadillo family proteins in the regulation of a431 epithelial cell junction assembly, adhesion and migration.

p0071 is an armadillo family protein related to both the adherens junction protein p120ctn and to the desmosomal proteins plakophilins 1-3. p0071 assembles into both adherens junctions and desmosomes, suggesting that this protein may regulate the balance between adherens junction and desmosome formation. Furthermore, this subfamily of proteins may also regulate cell functions directly influenced by intercellular junctions, including the strength of cell adhesion and the ability of cells to migrate. These possibilities were tested by expressing exogenous p0071 in A431 epithelial cells and monitoring the effects on adhesive junction assembly in comparison to other closely related armadillo family proteins. In this model system, p0071 specifically enhanced adherens junction assembly but dramatically compromised desmosome assembly, resulting in keratin filament retraction from regions of cell-cell contact. Protein interaction studies revealed that p0071 bound to the first 160 amino-terminal residues of desmoplakin and also interacted directly with plakoglobin, suggesting that p0071 may regulate desmosome assembly by controlling plakoglobin availability. Using an in vitro assay to measure the strength of cell-cell contacts, both plakophilin-1 and p120ctn were found to increase the strength of adhesion. Interestingly, p0071 expression caused no overall changes in adhesive strength, but dramatically inhibited the ability of A431 cells to close an in vitro wound. These results suggest that p120ctn/plakophilin family proteins interact with intercellular junction binding partners to differentially modulate the adhesive and migratory behavior of epithelial cells.

Adherens Junctions↗
PubMed1980

Junctional structures in digestive epithelia of a cephalopod.

Intercellular junctions have been studied in the epithelia of digestive organs of Sepia officinalis (digestive gland, digestive duct appendages and caecum) by conventional staining, lanthanum tracer and freeze-fracturing techniques. In the three organs studied the same junctional complex occurs, consisting of a belt desmosome, a septate junction and gap junctions. The septate junction isd of pleated-sheet type and the gap junction has its particles on the P face of the fracture. Circular structures have been found in the digestive gland septate junctions. Neither continuous nor tight junctions have been found. These results show that Cephalopods have junctional structures very close to those of other Molluscs and of Annelids. Some small differences between the septate junctions of the three organs could be related to their different physiology.

Animals↗
PubMed1997

The small GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts.

Cadherins are calcium-dependent cell-cell adhesion molecules that require the interaction of the cytoplasmic tail with the actin cytoskeleton for adhesive activity. Because of the functional relationship between cadherin receptors and actin filament organization, we investigated whether members of the Rho family of small GTPases are necessary for cadherin adhesion. In fibroblasts, the Rho family members Rho and Rac regulate actin polymerization to produce stress fibers and lamellipodia, respectively. In epithelial cells, we demonstrate that Rho and Rac are required for the establishment of cadherin-mediated cell-cell adhesion and the actin reorganization necessary to stabilize the receptors at sites of intercellular junctions. Blocking endogenous Rho or Rac selectively removed cadherin complexes from junctions induced for up to 3 h, while desmosomes were not perturbed. In addition, withdrawal of cadherins from intercellular junctions temporally precedes the removal of CD44 and integrins, other microfilament-associated receptors. Our data showed that the concerted action of Rho and Rac modulate the establishment of cadherin adhesion: a constitutively active form of Rac was not sufficient to stabilize cadherindependent cell-cell contacts when endogenous Rho was inhibited. Upon induction of calcium-dependent intercellular adhesion, there was a rapid accumulation of actin at sites of cell-cell contacts, which was prevented by blocking cadherin function, Rho or Rac activity. However, if cadherin complexes are clustered by specific antibodies attached to beads, actin recruitment to the receptors was perturbed by inhibiting Rac but not Rho. Our results provide new insights into the role of the small GTPases in the cadherin-dependent cell- cell contact formation and the remodelling of actin filaments in epithelial cells.

3T3 Cells↗
PubMed2000

Analysis of tight junctions during neutrophil transendothelial migration.

Intercellular junctions have long been considered the main sites through which adherent neutrophils (PMNs) penetrate the endothelium. Tight junctions (TJs; zonula occludens) are the most apical component of the intercellular cleft and they form circumferential belt-like regions of intimate contact between adjacent endothelial cells. Whether PMN transmigration involves disruption of the TJ complex is unknown. We report here that endothelial TJs appear to remain intact during PMN adhesion and transmigration. Human umbilical vein endothelial cell (HUVEC) monolayers, a commonly used model for studying leukocyte trafficking, were cultured in astrocyte-conditioned medium to enhance TJ expression. Immunofluorescence microscopy and immunoblot analysis showed that activated PMN adhesion to resting monolayers or PMN migration across interleukin-1-treated monolayers does not result in widespread proteolytic loss of TJ proteins (ZO-1, ZO-2, and occludin) from endothelial borders. Ultrastructurally, TJs appear intact during and immediately following PMN transendothelial migration. Similarly, transendothelial electrical resistance is unaffected by PMN adhesion and migration. Previously, we showed that TJs are inherently discontinuous at tricellular corners where the borders of three endothelial cells meet and PMNs migrate preferentially at tricellular corners. Collectively, these results suggest that PMN migration at tricellular corners preserves the barrier properties of the endothelium and does not involve widespread disruption of endothelial TJs.

Cell Adhesion↗
PubMed1994

Endothelial gaps and permeability of venules in rat tracheas exposed to inflammatory stimuli.

This study determined the number of endothelial gaps in venules of the rat trachea and related these values to the amount of plasma extravasation after an inflammatory stimulus (neurogenic inflammation). From 1 to 30 min after the stimulus, vessels were fixed by vascular perfusion, and endothelial cell borders were stained with silver nitrate, which made it possible to quantify the number and distribution of endothelial gaps. It also was possible to quantify the leukocyte attachment sites, to measure the size, shape, and number of endothelial cells, and to delineate the architecture of the tracheal vasculature. Sites of increased vascular permeability were localized with Monastral blue B, india ink, or fluorescent microspheres. After the stimulus, the silver lines around endothelial cells of postcapillary venules and collecting venules were interrupted by stereotyped silver dots (diam, 1.4 +/- 0.03 microns; +/- SE), which were found by electron microscopy to be silver deposits at endothelial gaps. The dots were most abundant in the smallest postcapillary venules (diam, 7-20 microns) where Monastral blue extravasation was greatest. The number of silver dots (14.4 +/- 0.7 dots/endothelial cell) and the amount of extravasation were maximal 1 min after the stimulus. However, the dots disappeared more slowly (half-life, 3.2 min) than did the extravasation (half-life, 1.3 min). In addition to the silver dots, 64% of the sites at which leukocytes were attached to the endothelium were stained with silver. These sites were marked by silver rings (diam, 3.4 +/- 0.2 microns) and were most numerous in the largest postcapillary venules (diam, 20-40 microns). Most (95%) of the silver rings were located at intercellular junctions but usually were not sites of Monastral blue extravasation. The results indicate that endothelial gaps at intercellular junctions are focal openings, which occupy < 3% of the luminal surface and are distinct from sites of leukocyte attachment. The reduction in Monastral blue extravasation that precedes the closure of the gaps could result from a decrease in the driving force for the convective movement of the tracer or from a decrease in the conductance of the gaps, perhaps due to the accumulation of sievelike substances within the gaps.

Animals↗
PubMed2004

Induction of aryl hydrocarbon receptor-mediated and estrogen receptor-mediated activities, and modulation of cell proliferation by dinaphthofurans.

A group of heterocyclic aromatic compounds, dinaphthofurans (DNFs), recently have been identified as potentially significant contaminants in freshwater sediments. In the present study, a battery of in vitro assays was used for detection of toxic effects of DNFs that are potentially associated with endocrine disruption and tumor promotion. Dinaphthofurans were found to act as relatively potent inducers of aryl hydrocarbon receptor (AhR)-mediated activity in the chemical-activated luciferase reporter gene expression DR-CALUX assay. The relative AhR-inducing potencies of DNFs were similar or even higher than relative potencies of unsubstituted polycyclic aromatic hydrocarbons (PAHs), with dinaphtho[1,2-b;2'3'-d]furan being the most potent AhR agonist. Two compounds, dinaphtho[2,1-b;2'3'-d]furan and dinaphtho[1,2-b;1'2'-d]furan, induced estrogen receptor (ER)-mediated activity in the estrogen receptor-mediated CALUX (the ER-CALUX) assay. Two types of potential tumor-promoting effects of DNFs were investigated, using in vitro bioassays for detection of inhibition of gap-junctional intercellular communication and detection of a release from contact inhibition. Although the acute inhibition of gap-junctional intercellular communication was not observed, all six tested DNFs were able to release rat liver epithelial WB-F344 cells from contact inhibition at concentrations as low as 100 nM. In summary, the present study indicated that DNFs can exert multiple biological effects in vitro, including induction of the AhR-mediated activity, release of cells from contact inhibition, and induction of ER-mediated activity.

Animals↗
PubMed2005

[Establishment of three-dimensional culture models related to different stages of nasopharyngeal carcinogenesis].

BACKGROUND & OBJECTIVE: Since three-dimensional culture simulates in vivo microenvironment better than planar culture, the biological character of cells in three-dimensional culture model are more similar with that of living tissues. This study was to establish three-dimensional culture models that represent different stages of nasopharyngeal caicinogenesis, and provide information to elucidate the related molecular events. METHODS: Non-tumor nasopharyngeal biopsy specimens were used to culture for normal nasopharyngeal epithelial cells. Early and late passages of normal nasopharyngeal epithelial cell line NPNE2 from nasopharyngeal biopsy specimens, immortalized nasopharyngeal epithelial cell line NP69SV40T, nasopharyngeal carcinoma (NPC) cell line SUNE-1 and its high metastatic subclone 5-8F were cultured in Matrigel to establish three-dimensional models. RESULTS: The cells grew out of non-tumor nasopharyngeal biopsy specimens displayed the morphologic characteristics as epithelia, and could proliferate in vitro for 8-10 passages before senescence. Immunocytochemical staining of cytokeratin revealed that the cells were of epithelial origin. All of the cells, except late passages of NPNE2 cells, could proliferate in the three-dimensional culture system. NPNE2 and NP69SV40T cells mainly developed reticular structures in morphology, and formed few clones with clear and smooth edges as well as tight intercellular junctions. SUNE-1 and 5-8F cells formed clones with irregular morphology, unclear edge, and loose intercellular junctions. In addition, the clones formed by 5-8F cells also developed a lot of pseudopodia, but developed no reticular structure. Late passages of NPNE2 cells formed no clone and reticular structure in the three-dimensional culture. CONCLUSIONS: Normal nasopharyngeal epithelial cells can be successfully cultured in vitro from naspharyngeal biopsy specimens. The three-dimensional culture models, established with normal nasopharyngeal epithelial cells, immortalized nasopharyngeal epithelial cells, and NPC cells, may represent the different stages of nasopharyngeal carcinogenesis.

Cell Culture Techniques↗
PubMed2006

The cadherin-11 cytoplasmic juxtamembrane domain promotes alpha-catenin turnover at adherens junctions and intercellular motility.

Cadherins mediate homophilic cell adhesion and contribute to tissue morphogenesis and architecture. Cadherin cell adhesion contacts are actively remodeled and impact cell movement and migration over other cells. We found that expression of a mutant cadherin-11 lacking the cytoplasmic juxtamembrane domain (JMD) diminished the turnover of alpha-catenin at adherens junctions as measured by fluorescence recovery after photobleaching. This resulted in markedly diminished cell intercalation into monolayers reflecting reduced cadherin-11-dependent cell motility on other cells. Furthermore, the actin cytoskeleton in cadherin-11 deltaJMD cells revealed a more extensive cortical F-actin ring that correlated with significantly higher levels of activated Rac1. Together, these data implicate the cadherin-11 cytoplasmic JMD as a regulator of alpha-catenin turnover at adherens junctions and actin-cytoskeletal organization that is critical for intercellular motility and rearrangement in multicellular clusters.

Actins↗
PubMed2001

Induction of radio-adaptive response in colony formation by low dose X-ray irradiation.

In this study, we examined the induction of a radio-adaptive response to cell death using a colony formation test in m5S, G401.2/6TG.1 and HeLa cells. When m5S cells were subjected to priming irradiation of 0.05 to approximately 0.15 Gy 4 hr before being irradiated with 4.5 Gy, the survival ratios increased significantly to 39 to approximately 42%. The priming irradiation effect was also observed when G401.2/6TG.1 cells were subjected to priming irradiation of 0.025 to approximately 0.1 Gy 4 hr before being irradiated with 0.8 Gy. This effect showed a two-phasic characteristic, where the first peak was reached at 0.025 Gy, and the second peak was reached at 0.075 Gy. The first peak showed a survival ratio of 56%, while the second peak was at 55%. However, in HeLa cells, this priming irradiation effect was not observed. These results indicated that induction of the radio-adaptive response did not depend on whether cells are normal or cancerous. One of the differences in these cells is that m5S and G401.2/6TG.1 cells have gap-junctional intercellular communication, but HeLa cells do not. Induction of the radio-adaptive response may be related to gap-junctional intercellular communication.

Adaptation, Physiological↗
PubMed2005

Rotigaptide (ZP123) prevents spontaneous ventricular arrhythmias and reduces infarct size during myocardial ischemia/reperfusion injury in open-chest dogs.

The antiarrhythmic and cardioprotective effect of increasing gap junction intercellular communication during ischemia/reperfusion injury has not been studied. The antiarrhythmic peptide rotigaptide (previously ZP123), which maintains gap junction intercellular communication, was tested in dogs subjected to a 60-min coronary artery occlusion and 4 h of reperfusion. Rotigaptide was administered i.v. 10 min before reperfusion as a bolus + i.v. infusion at doses of 1 ng/kg bolus + 10 ng/kg/h infusion (n = 6), 10 ng/kg bolus + 100 ng/kg/h infusion (n = 5), 100 ng/kg bolus + 1000 ng/kg/h infusion (n = 8), 1000 ng/kg bolus + 10 mug/kg/h infusion (n = 6), and vehicle control (n = 5). Premature ventricular complexes (PVCs) were quantified during reperfusion. A series of four or more consecutive PVCs was defined as ventricular tachycardia (VT). The total incidence of VT was reduced significantly with the two highest doses of rotigaptide (20.3 +/- 10.9 and 4.3 +/- 4.1 events; p < 0.05) compared with controls (48.7 +/- 6.0). Total PVCs were reduced significantly from 25.1 +/- 4.2% in control animals to 11.0 +/- 4.4 and 1.7 +/- 1.3% after the two highest doses of rotigaptide. Infarct size, expressed as a percentage of the left ventricle, was reduced significantly from 13.2 +/- 1.9 in controls to 7.1 +/- 1.0 (p < 0.05) at the highest dose of rotigaptide. Ultrastructural evaluation revealed no differences in myocardial injury in the infarct area, area at risk, border zone, or normal zone in vehicle and rotigaptide-treated animals. However, rotigaptide did increase the presence of gap junctions in the area at risk (p = 0.022, Fisher's exact test). Rotigaptide had no effect on heart rate, blood pressure, heart rate-corrected QT interval, or left ventricular end-diastolic pressure. In conclusion, these results demonstrate that rotigaptide is a potent antiarrhythmic compound with cardioprotective effects and desirable safety.

Animals↗
PubMed1996

Connexin expression in epidermal cell lines from SENCAR mouse skin tumors.

Alteration of gap-junctional intercellular communication (GJIC) has long been proposed to be involved in carcinogenesis. Previously, we reported that the level of gap junctional intercellular communication in mouse skin carcinoma cell lines is significantly lower than in papilloma cell lines and normal mouse keratinocytes Klann et al., Cancer Res 49:699-705, 1989). Here, we present data on expression of the gap-junctional protein connexins (Cx) 26, Cx31.1, and Cx43 in a comprehensive panel of keratinocyte cell lines representing different stages of mouse skin carcinogenesis and the effect of different conditions of propagation on Cx phenotype. Northern and western blot analyses and immunostaining showed that all cell lines studied in vitro expressed Cx43 but most did not express Cx31.1 or Cx26. The abundance of Cx43 expression on plasma membranes correlated well with the level of GJIC. In vivo expression of Cx43 and Cx26 was strongly increased. Whereas none of tumorigenic cell lines expressed Cx26 gap junctions in culture, those growing as tumors in nude mice began to express Cx26 protein. The comparison of Cx expression on the keratinocyte membranes in three different groups of tumors (papillomas and squamous cell and spindle cell carcinomas) clearly revealed that the abundance of Cx43 and Cx26 expression directly correlated with the level of tumor differentiation. All studied tumors were Cx31.1 negative. These results suggest that both Cx expression and gap-junction permeability are gradually reduced during the tumor progression stage of mouse skin carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

Effect of hydrostatic pressure gradients and Na2EDTA on permeability of human Schlemm's canal cell monolayers.

PURPOSE: Elevated intraocular pressure in those with glaucoma appears to be a function of increased resistance to movement of aqueous humor through the conventional outflow pathway. The majority of resistance in both normal and glaucomatous eyes is generated in the region between the juxtacanalicular trabecular meshwork and the inner wall of Schlemm's canal. To accommodate transient elevations in pressure, we hypothesize that conventional outflow increases rapidly due to changes in complexity of intercellular junctions between cells of the inner wall of Schlemm's canal. METHODS: To test this hypothesis we examined specifically the effects of hydrostatic pressure gradients and the calcium chelator, Na2EDTA, on permeability of cultured human Schlemm's canal cell monolayers in isolation. Human Schlemm's Canal cells were isolated, cultured and then seeded onto permeable supports and maintained in culture to allow intercellular junctions to mature. With a minimum net transendothelial electrical resistance of 10 Ohm cm2, cells were placed into an Ussing-type chamber and hydraulic conductivity was calculated from pressure and flow measurements that were continuously recorded. Simultaneously, transendothelial electrical resistance was measured manually at fixed intervals. In parallel experiments, cell margins were monitored in real time by videomicroscopy. RESULTS: During the baseline measurement period when cells were exposed to pressure but not Na2EDTA, hydraulic conductivity was constant but transendothelial electrical resistance decreased continuously at rate of 0.24 Ohm cm2/minute. After Na2EDTA treatment, no significant change in transendothelial electrical resistance was measured while, hydraulic conductivity of Schlemm's Canal monolayers increased significantly by 125%; corresponding to noticeable intercellular separations. Restoration of cell-cell contact was observed by videomicroscopy 30 minutes following washout of Na2EDTA and functionally after 2 hours. CONCLUSIONS: Responses of Schlemm's Canal cells to pressure and calcium chelators in vitro are consistent with a role for calcium sensitive junctions in outflow resistance in vivo.

Animals↗
PubMed2000

Induction of skin papillomas, carcinomas, and sarcomas in mice in which the connexin 43 gene is heterologously deleted.

It has been suggested that blocked gap junctional intercellular communication plays a crucial part in multistage carcinogenesis. The mouse skin tumor-promoting phorbol esters are potent inhibitors of gap junctional intercellular communication and this inhibition is considered to be a mechanism by which clonal expansion of "initiated" cells is promoted. We examined whether mice in which the gene for a gap junction protein, connexin 43, is heterozygously deleted are more susceptible to chemical carcinogenesis; connexin 43 is expressed in the basal cell layer and the dermis of the skin. When the back skin was painted with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate, the incidence and yields of both papillomas and carcinomas were similar in connexin 43+/- and connexin 43+/+ mice; for this experiment, the original mice with C57BL/6 genetic background was crossed with CD1 strain for three generations. Subcutaneous injection of 7, 12-dimethylbenz[a]anthracene resulted in induction of fibrosarcomas in connexin 43+/- and connexin 43+/+ mice to a similar extent. All papillomas and carcinomas induced with 7, 12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate contained the 7,12-dimethylbenz[a] anthracene-specific mutation in the ras gene (A to T transversion at the 61st codon). About 50% of fibrosarcomas also contained this mutation, but in the Ki-ras gene; there was no difference in the prevalence of this mutation in tumors from connexin 43+/- and connexin 43+/+ mice. None of the tumors examined, however, showed any mutation in the connexin 43 gene. These results suggest that the deletion of one allele of the connexin 43 gene does not significantly contribute to, nor alter, the molecular events involved in skin carcinogenesis. These results are compatible with previous observations that nongenetic disruption of function rather than mutations of connexins, commonly occurs in cancer cells.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2003

The roles of cadherins and nectins in interneuronal synapse formation.

Cadherins are Ca(2+)-dependent intercellular adhesion molecules (CAMs) and they play key roles in the intercellular junctions of a wide variety of cells, including interneuronal synapses. Nectins are Ca(2+)-independent immunoglobulin-like CAMs and they are also involved in the organization of various types of intercellular junctions, including interneuronal synapses, either in cooperation with or independently of cadherins. Intercellular adhesion through nectins induces activation of Cdc42 and Rac small G proteins, leading to a reorganization of the actin cytoskeleton, gene expression, and cell polarization.

Animals↗
PubMed1989

Lymphangiosarcoma and haemangiosarcoma in a cat.

Ultrastructural findings in a feline ventral abdominal vascular tumour showed lack of basal lamina, few micropinocytotic vesicles and intercellular junctions and a discontinuous endothelial cell layer. A splenic cyst had a continuous basal lamina, numerous micropinocytotic vesicles and intercellular junctions and a continuous endothelial cell layer. These findings were compatible with diagnosis of lymphangiosarcoma (ventral abdomen and metastases) and haemangiosarcoma (splenic cyst).

Animals↗