Scrape-loading Dye Transfer Research Articles

Upregulation of Connexin 43 Expression ViaC-Jun N-Terminal Kinase Signaling inPrionDisease.

Prion infection leads to neuronal cell death, glial cell activation, and the accumulation of misfolded prion proteins. However, the altered cellular environments in animals with prion diseases are poorly understood. In the central nervous system, cells connect the cytoplasm of adjacent cells via connexin (Cx)-assembled gap junction channels to allow the direct exchange of small molecules, including ions, neurotransmitters, and signaling molecules, which regulate the activities of the connected cells. Here, we investigate the role of Cx43 in the pathogenesis of prion diseases. Upregulated Cx43 expression, which was dependent on c-Jun N-Terminal Kinase (JNK)/c-Jun signaling cascades, was found in prion-affected brain tissues and hippocampal neuronal cells. Scrapie infection-induced Cx43 formed aggregated plaques within the cytoplasmic compartments at the cell-cell interfaces. The ethidium bromide (EtBr) uptake assay and scrape-loading dye transfer assay demonstrated that increased Cx43 has functional consequences for the activity of Cx43 hemichannels. Interestingly, blockade of PrPSc accumulation reduced Cx43 expression through the inhibition of JNK signaling, indicating that PrPSc accumulation may be directly involved in JNK activation-mediated Cx43 upregulation. Overall, our findings describe a scrapie infection-mediated novel regulatory signaling pathway of Cx43 expression and may suggest a role for Cx43 in the pathogenesis of prion diseases.

Mutant connexin 50 (S276F) inhibits channel and hemichannel functions inducing cataract.

This study was designed to detect the expression, detergent resistance, subcellular localization, and channel and hemichannel functions of mutant Cx50 to understand the forming mechanism for inducing congenital cataract by a novel mutation p.S276F in connexin 50 (Cx50) reported previously by us. HeLa and human lens epithelial (HLE) cells were transfected with wild-type Cx50 and mutant Cx50 (S276F). We examined the functional characteristics of mutant Cx50 (S276F) in comparison with those of wild-type Cx50 using immunoblot, confocal fluorescence microscopy, dye transfer analysis and dye uptake assay. The mutant and wild-type Cx50 were expressed in equal levels and could efficiently localize to the plasma membrane without transportation and assembly problems. Scrape loading dye transfer was significantly evident in cells transfected with wild-type Cx50 compared to those in cells transfected with mutant Cx50 and cotransfected with wild-type and mutant Cx50. The dye uptake was found to be significantly lower in cells transfected with mutant Cx50 than in cells transfected with wild- type Cx50 and cells cotransfected with wild-type and mutant Cx50. The transfected HeLa and HLE cell lines showed similar performance in all the experiments. These results indicated that the mutant Cx50 (S276F) might inhibit the function of gap junction channel in a dominant negative manner, but inhibit the hemichannel function in a recessive negative manner.

Benzalkonium chloride suppresses rabbit corneal endothelium intercellular gap junction communication.

PurposeGap junction intercellular communication (GJIC) plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK) alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects.MethodsThirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM). Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43) and tight junction zonula occludens-1 (ZO-1) gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP). Primary rabbit corneal endothelial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT) was used to assess GJIC activity.ResultsTopical administration of BAK (0.05%, 0.1%) dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity.ConclusionsThe dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations should be used with caution to avoid declines in corneal transparency resulting from losses in GJIC activity and endothelial function.

Effects of High Glucose-Induced Cx43 Downregulation on Occludin and ZO-1 Expression and Tight Junction Barrier Function in Retinal Endothelial Cells

To investigate whether high glucose (HG)-induced downregulation of connexin 43 (Cx43), a gap junction protein, alters ZO-1 and occludin expression and cell monolayer permeability. Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM) medium, high glucose (HG; 30 mM) medium, N medium transfected with Cx43 siRNA, or N medium transfected with scrambled siRNA. To determine Cx43, occludin, and ZO-1 protein expression, Western blot (WB) analysis and immunostaining were performed. Gap junction intercellular communication (GJIC) was determined using scrape load dye transfer (SLDT) assay. In parallel, cell monolayer permeability was assessed in the four groups of cells, and in cells transfected with Cx43 plasmid or dominant negative Cx43 plasmid. Connexin 43 protein expression was significantly reduced in cells grown in HG (67 ± 15% of control), and a significant reduction in Cx43 was achieved when cells grown in N medium were transfected with Cx43 siRNA (76 ± 12% of control), with concomitant decrease in GJIC activity. Cells grown in HG showed significant reduction in occludin (77 ± 9% of control) and ZO-1 (80 ± 11% of control) protein level compared with cells grown in N media. Importantly, cells transfected with Cx43 siRNA and grown in N medium showed significant downregulation in occludin (78 ± 8% of control) and ZO-1 (81 ± 6% of control) expression, and exhibited increased cell monolayer permeability. Furthermore, Cx43 upregulation protected cells against HG-induced excess cell monolayer permeability. Our findings indicate that HG-induced downregulation of Cx43 expression and GJIC may contribute to the breakdown of endothelial barrier tight junctions associated with diabetic retinopathy.

Formation of functional gap junctions in amniotic fluid‐derived stem cells induced by transmembrane co‐culture with neonatal rat cardiomyocytes

Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2-week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co-culture group. Protein expression of cardiac calsequestrin 2 was up-regulated in direct transmembrane co-cultures and media control cultures compared to the other experimental groups, but all groups were up-regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape-loading dye transfer assay, was significantly increased in direct transmembrane co-cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co-culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co-culture does enhance connexin-43-mediated gap junction communication between AFSC.

Abstract 4801: Bronchoalveolar lavage fluid (BALF) from subchronic exposure to butylated-hydroxytoluene (BHT) inhibits gap junctional communication in a toll-like receptor 4-dependent manner.

Abstract Subchronic dosing of BHT in mice is a well-known model used to discretely study the tumor promotion stage of lung adenocarcinoma. Using this model, we have previously shown that functional toll-like receptor 4 (TLR4) is protective against inflammatory sequelae and primary tumor formation in the mouse lung. Gap junctional intercellular communication (GJIC) has been shown to regulate the signal transduction pathways of cells, and inhibition of this communication by toxicant exposure has been linked to dysregulation and eventual carcinogenic endpoints. As part of our research on the protective nature of TLR4 during tumor promotion, we utilized the BHT subchronic model (4 weekly i.p. doses of BHT) to examine the early effects on BALF in both BALB/c (BALB; Tlr4 sufficient) and C.C3H-Tlr4Lps-d mice (BALBLps-d-d; Tlr4 mutant). Analysis of the BALF 24 hours after the final dose for protein content and cytology was indicative of an inflammatory profile in all mice treated with BHT. Magnitude of response to BHT was highest in the BALBLps-d mice; the BALB appeared resistant. In an effort to further characterize the response of the pulmonary epithelium, the BALF samples were used to treat cultured C10 cells, a murine immortalized line derived from BALB/c alveolar type II cells, which are a precursor cell type for adenocarcinoma. Using the scrape-load dye transfer assay, a well-defined method to measure GJIC, we demonstrated marked inhibition of cellular communication after a 4 hour exposure to cell-free BALF that is both treatment and strain dependent. The direction and magnitude of effect is in parallel with that of the direct analysis of the BALF, i.e. we observed more inhibition in BALF from BALBLps-d compared to BALB mice. In addition, quantitative PCR (QPCR) analysis of C10 cells exposed to cell-free BALF for 24 hours indicates alterations in the transcription patterns in genes of innate immunity that are also treatment and strain dependent. These novel findings suggest endocrine signaling from the inflammatory milieu are complicit in the inhibition of GJIC during the promotion phase of carcinogenesis. Furthermore, these data establish a link between active gap junctions, tumor promotion, and the protective nature of TLR4 in an ex vivo model which will facilitate a mechanistic examination of these protective effects at the molecular level. Funded by ACS RSG-10-162-01-LIB (AKB). Citation Format: Thomas Hill, Carla-Maria Alexander, Alison K. Bauer. Bronchoalveolar lavage fluid (BALF) from subchronic exposure to butylated-hydroxytoluene (BHT) inhibits gap junctional communication in a toll-like receptor 4-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4801. doi:10.1158/1538-7445.AM2013-4801

Gingko biloba extracts protect auditory hair cells from cisplatin-induced ototoxicity by inhibiting perturbation of gap junctional intercellular communication

Gap junctional intercellular communication (GJIC) may play an important role in the hearing process. Cisplatin is an anticancer drug that causes hearing loss and Gingko biloba extracts (EGb 761) have been used as an antioxidant and enhancer for GJIC. The purpose of this study was to examine the efficiency of EGb 761 in protecting against cisplatin-induced apoptosis and disturbance of GJIC. House Ear Institute-Organ of Corti 1 auditory cells were cultured and treated with cisplatin (50μM) and EGb (300μg/ml) for 24h, and then analyzed by immunocytochemistry (Annexin V/propidium iodide) and Western blots. GJIC was evaluated by scrape-loading dye transfer (SLDT). Basal turn organ of Corti (oC) explants from neonatal (p3) rats were exposed to cisplatin (1–10μM) and EGb (50–400μg/ml). The number of intact hair cells was counted by co-labeling with phalloidin and MyoVIIa. EGb prevented cisplatin-induced apoptosis in immunostaining and decreased caspase 3 and poly-ADP-ribose polymerase bands, which were increased in cisplatin-treated cells in Western blots. EGb prevented abnormal intracellular locations of connexin (Cx) 26, 30, 31, and 43 in cells treated with cisplatin and increased quantities of Cx bands. EGb also prevented cisplatin-induced disturbance of GJIC in SLDT. In oC explants, EGb significantly prevented hair cell damage induced by cisplatin. In animal studies, EGb significantly prevented cisplatin-induced hearing loss across 16 and 32kHz. These results show that cisplatin induces ototoxicity including hearing loss as well as down-regulation of GJIC and inhibition of Cxs in auditory cells. EGb prevents hearing loss in cisplatin-treated rats by inhibiting down-regulation of Cx expression and GJIC. The disturbance of GJIC or Cx expression may be one of the important mechanisms of cisplatin-induced ototoxicity.

Biphasic increase of gap junction coupling induced by dipyridamole in the rat aortic A-10 vascular smooth muscle cell line

The rat aortic smooth muscle cell line A-10 was used to investigate the effect of dipyridamole on the gap junction coupling of smooth muscle cells. The scrape loading/dye transfer (SL/DT) technique revealed that dipyridamole concentrations between 5μM and 100μM significantly increased gap junction coupling. The adenosine receptor antagonist MRS 1754, as well as the PKA inhibitors Rp-cAMPS and H-89 were able to inhibit the dipyridamole-related increase in coupling, while forskolin and Br-cAMP also induced an enhancement of the gap junction coupling. Regarding the time-dependent behaviour of dipyridamole, a short-term effect characterised by an oscillatory reaction was observed for application times of less than 5h, while applications times of at least 6h resulted in a long-term effect, characterised by a constant increase of gap junction coupling to its maximum levels. This increase was not altered by prolonged presence of dipyridamole. In parallel, a short application of dipyridamole for at least 15min was found to be sufficient to evoke the long-term effect measured 6h after drug washout. We propose that in both the short-term and long-term effect, cAMP-related pathways are activated. The short-term phase could be related to an oscillatory cAMP effect, which might directly affect connexin trafficking, assembly and/or gap junction gating. The long-term effect is most likely related to the new expression and synthesis of connexins. With previous data from a bovine aortic endothelial cell line, the present results show that gap junction coupling of vascular cells is a target for dipyridamole.

All-trans retinoic acid restores gap junctional intercellular communication between oral cancer cells with upregulation of Cx32 and Cx43 expressions in vitro

Objective: All-trans retinoic acid (ATRA) has been demonstrated to inhibit tumor growth by restoration of gap junctional intercellular communication (GJIC) via upregulation of connexin (Cx) expression in some solid tumors. However, the relationship between ATRA and GJIC remains unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the effect of ATRA on the GJIC function of OSCC. Study design: We measured the effects of ATRA on the viability and cell cycle distribution of SCC9 and Tca8113 OSCC cells. The GJIC function was observed using the scrape-loading dye transfer technique, and the mRNA and protein levels of Cx32 and Cx43 were detected by qRT-PCR, Western blot, and immunofluorescence assays. Results: ATRA inhibited the growth of OSCC cells in a dose- and time-dependent manner (P <0.05) and caused cell cycle arrest. ATRA-treated cells showed a 2.69-fold and 2.06-fold enhancement of GJIC in SCC9 and Tca8113 cells, respectively (P <0.05). Moreover, ATRA induced upregulation of Cx32 and Cx43 at both the mRNA and protein levels in OSCC cells. Conclusion: Our results indicated that restoration of GJIC via enhanced Cx32 and Cx43 expression might serve as a novel mechanism for the anti-tumor effect of ATRA in OSCC. Key words:All-trans retinoic acid, oral squamous cell carcinoma, connexin, gap junctional intercellular communication.

Bronchoalveolar Lavage Fluid Utilized Ex Vivo to Validate In Vivo Findings: Inhibition of Gap Junction Activity in Lung Tumor Promotion is Toll-Like Receptor 4-Dependent.

TLR4 protects against lung tumor promotion and pulmonary inflammation in mice. Connexin 43 (Cx43), a gap junction gene, was increased in Tlr4 wildtype compared to Tlr4-mutant mice in response to promotion, which suggests gap junctional intercellular communication (GJIC) may be compromised. We hypothesized that the early tumor microenvironment, represented by Bronchoalveolar Lavage Fluid (BALF) from Butylated hydroxytoluene (BHT; promoter)-treated mice, would produce TLR4-dependent changes in pulmonary epithelium, including dysregulation of GJIC in the Tlr4-mutant (BALBLps-d) compared to the Tlr4-sufficient (BALB; wildtype) mice. BHT (4 weekly doses) was injected ip followed by BALF collection at 24 h. BALF total protein and total macrophages were significantly elevated in BHT-treated BALBLps-d over BALB mice, similar to previous findings. BALF was then utilized in an ex vivo manner to treat C10 cells, a murine alveolar type II cell line, followed by the scrape-load dye transfer assay (GJIC), Cx43 immunostaining, and quantitative RT-PCR (Mcp-1, monocyte chemotactic protein 1). GJIC was markedly reduced in C10 cells treated with BHT-treated BALBLps-d BALF for 4 and 24 h compared to BALB and control BALF from the respective mice (p < 0.05). Mcp-1, a chemokine, was also significantly increased in the BHT-treated BALBLps-d BALF compared to the BALB mice, and Cx43 protein expression in the cell membrane altered. These novel findings suggest signaling from the BALF milieu is involved in GJIC dysregulation associated with promotion and links gap junctions to pulmonary TLR4 protection in a novel ex vivo model that could assist in future potential tumor promoter screening.

Se-methylselenocysteine induces the Connexin 43 expression and inhibits cell growth in prostate cancer DU145 cells

Objective To study the effect of the Se-methylselenocysteine (MSC) treatment on Connexin 43 (Cx43) and gap junctional intercellular communication (GJIC) in the prostate cancer DU145 cells.Methods Cx43 mRNA and protein expression levels were examined by using reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blotting,and GJIC function by the scrape-loading/dye transfer (SL/DT) after 0,5,10,25 μmol/L MSC treatment,respectively.The growth of prostate cancer DU145 cells was tested by using methyl thiazol tetrazolium (MTT) and clone formation assay,and the B lymphocytes/leukemia-2 (bcl-2) protein level by Western blotting after MSC treatment and Cx43 transfection.Results 5,10,and 25 μmol/L MSC or exogenous transfection of Cx43 in DU145 cells could induce the Cx43 mRNA and protein expression,but not the GJIC function.MSC treatment and up-regulation of Cx43 could reduce the bcl-2 level and inhibit the DU145 cell growth and proliferation (P ＜ 0.05).Conclusion MSC can inhibit the DU145 cell growth and reduce the BCL-2 by up-regulating of Cx 43,which shows a new pathway of MSC in the anti-cancer effect. Key words: Prostate cancer; Selenium; Connexin

β-Catenin Signaling Contributes to Platelet Derived Growth Factor Elicited Bladder Smooth Muscle Cell Contraction Through Up-Regulation of Cx43 Expression

Increased gap junctions contribute to bladder overactivity but the factors and mechanisms involved in gap junction regulation in the bladder are not well established. We examined whether and how platelet derived growth factor regulates connexin43 in bladder smooth muscle cells. Cultured rat bladder smooth muscle cells were treated with growth factors with or without agents that interfere with phosphatidylinositol 3-kinase, mitogen activated protein kinase and β-catenin signaling pathways. Connexin43 expression was examined by Western and Northern blot, and immunochemistry. Functional gap junctions were evaluated by scrape-loading dye transfer assay. Bladder smooth muscle cell contraction was measured by collagen gel contraction. 1) Platelet derived growth factor induced phosphatidylinositol 3-kinase and mitogen activated protein kinase dependent accumulation of nuclear β-catenin. This was followed by increased connexin43 expression. 2) Down-regulation of β-catenin by specific siRNA abolished the connexin43 increasing effect of platelet derived growth factor while β-catenin stimulation due to glycogen synthase kinase inhibition mimicked that effect. 3) Basic fibroblast growth factor and epidermal growth factor also induced connexin43 expression. Their effects were potentiated by platelet derived growth factor. 4) Gap junction inhibition attenuated the bladder smooth muscle cell contraction induced by platelet derived growth factor. Consistently fibroblasts from connexin43 knockout (Cx43-/-) mice showed a much weaker contractile response to platelet derived growth factor than cells from connexin43-wild (Cx43+/+) litter mates. Platelet derived growth factor induces connexin43 expression and bladder smooth muscle cell contraction by activating β-catenin signaling. As a convergence point for many signal pathways, β-catenin may be targeted to treat bladder overactivity.

Developmental Truncations of Connexin 50 by Caspases Adaptively Regulate Gap Junctions/Hemichannels and Protect Lens Cells against Ultraviolet Radiation

The gap junction-forming connexin (Cx) 50 is truncated gradually during lens development. Premature cleavage of lens connexins is thought to be associated with cataract formation. We have shown previously that Cx50 is likely to be cleaved by caspase-3 like protease during chick lens development. Here, using HPLC-electrospray tandem mass spectrometry, we mapped two cleavage sites at the C terminus of Cx50 after Glu-368 and Asp-379 and identified caspase-3 and caspase-1 as the responsible proteases, respectively. The activity of caspase-1, like caspase-3, was detected in the outer cortex increased during lens development, which coincided with the accumulation of the truncated fragments of Cx50 in the core region of the lens. The truncated Cx50 fragments present in older lenses were reproduced in the younger lens after treatment with UV radiation; this cleavage could be partially blocked by caspase-1/3-specific inhibitors. Interestingly, as compared with full-length Cx50, caspase-truncated Cx50 showed a dramatic decrease in gap junction coupling and a loss of hemichannel function. Furthermore, expression of caspase-truncated Cx50 fragments increased cell viability against UV radiation as compared with full-length Cx50. Together, these results suggest that both caspase-1 and -3 are responsible for the cleavage at the C terminus of Cx50 during lens development. The reduction of gap junction coupling and closure of hemichannels formed by truncated Cx50 are likely to adaptively protect cells against elevated oxidative stress associated with lens aging.

24 ALPHA1-ADRENOCEPTOR STIMULATION IN THE SPINAL CORD ENHANCES THE MICTURITION REFLEX IN SPINAL CORD INJURED RATS

INTRODUCTION AND OBJECTIVES: It has been suggested that an elevated number of gap junctions (GJs) may contribute to bladder overactivity. However, the factors and mechanisms involved in the regulation of GJs in the bladder have not been well established. Here we examined whether and how platelet derived growth factor (PDGF) regulates connexin43 (Cx43) in bladder smooth muscle cells (BSMCs). METHODS: Cultured rat BSMCs were treated with various growth factors in the presence or absence of agents that interfere with phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and -catenin signaling pathways. Cx43 expression was examined by Western blot, Northern blot, and immunochemistry. Functional GJs were evaluated by scrape-loading dye transfer assay. BSMC contraction was measured by collagen gel contraction. RESULTS: 1) PDGF induced a PI3Kand MAPK-dependent accumulation of nuclear -catenin. This was followed by an elevated Cx43 expression (Fig. A). 2) Downregulation of -catenin with specific siRNA abolished (Fig. B D), whereas stimulation of -catenin through inhibition of glycogen synthase kinase (GSK) mimicked the Cx43elevating effect of PDGF. 3) Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) also induced Cx43 expression. Their effects were potentiated by PDGF. 4) Inhibition of GJs attenuated PDGF-induced BSMC contraction. Consistently, fibroblasts from Cx43 knockout (Cx43 / ) mouse displayed a much weaker contractile response to PDGF than cells from Cx43-wild (Cx43 / ) littermates (Fig. E). CONCLUSIONS: PDGF induces Cx43 expression and BSMC contraction through activation of -catenin signaling. As -catenin is a convergence point for many signal pathways, targeting -catenin may hold promise for the treatment of bladder overactivity. Source of Funding: None

23 β-CATENIN SIGNALING CONTRIBUTES TO PDGF-ELICITED BLADDER SMOOTH MUSCLE CELL CONTRACTION THROUGH UPREGULATION OF CX43 EXPRESSION

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Basic Research I1 Apr 201223 β-CATENIN SIGNALING CONTRIBUTES TO PDGF-ELICITED BLADDER SMOOTH MUSCLE CELL CONTRACTION THROUGH UPREGULATION OF CX43 EXPRESSION Kai Li, Jian Yao, Norifumi Sawada, Tatsuya Miyamoto, Masanori Kitamura, and Masayuki Takeda Kai LiKai Li Chuo City, Yamanashi, Japan More articles by this author , Jian YaoJian Yao Chuo, Yamanashi, Japan More articles by this author , Norifumi SawadaNorifumi Sawada Chuo, Yamanashi, Japan More articles by this author , Tatsuya MiyamotoTatsuya Miyamoto Chuo, Yamanashi, Japan More articles by this author , Masanori KitamuraMasanori Kitamura Chuo, Yamanashi, Japan More articles by this author , and Masayuki TakedaMasayuki Takeda Chuo, Yamanashi, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.066AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It has been suggested that an elevated number of gap junctions (GJs) may contribute to bladder overactivity. However, the factors and mechanisms involved in the regulation of GJs in the bladder have not been well established. Here we examined whether and how platelet derived growth factor (PDGF) regulates connexin43 (Cx43) in bladder smooth muscle cells (BSMCs). METHODS Cultured rat BSMCs were treated with various growth factors in the presence or absence of agents that interfere with phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and β-catenin signaling pathways. Cx43 expression was examined by Western blot, Northern blot, and immunochemistry. Functional GJs were evaluated by scrape-loading dye transfer assay. BSMC contraction was measured by collagen gel contraction. RESULTS 1) PDGF induced a PI3K- and MAPK-dependent accumulation of nuclear β-catenin. This was followed by an elevated Cx43 expression (Fig. A). 2) Downregulation of β-catenin with specific siRNA abolished (Fig. B - D), whereas stimulation of β-catenin through inhibition of glycogen synthase kinase (GSK) mimicked the Cx43-elevating effect of PDGF. 3) Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) also induced Cx43 expression. Their effects were potentiated by PDGF. 4) Inhibition of GJs attenuated PDGF-induced BSMC contraction. Consistently, fibroblasts from Cx43 knockout (Cx43−/−) mouse displayed a much weaker contractile response to PDGF than cells from Cx43-wild (Cx43+/+) littermates (Fig. E). CONCLUSIONS PDGF induces Cx43 expression and BSMC contraction through activation of β-catenin signaling. As β-catenin is a convergence point for many signal pathways, targeting β-catenin may hold promise for the treatment of bladder overactivity. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e9 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kai Li Chuo City, Yamanashi, Japan More articles by this author Jian Yao Chuo, Yamanashi, Japan More articles by this author Norifumi Sawada Chuo, Yamanashi, Japan More articles by this author Tatsuya Miyamoto Chuo, Yamanashi, Japan More articles by this author Masanori Kitamura Chuo, Yamanashi, Japan More articles by this author Masayuki Takeda Chuo, Yamanashi, Japan More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

WNT3A induces a contractile and secretory phenotype in cultured vascular smooth muscle cells that is associated with increased gap junction communication

Evidence suggests a role for Wnt signaling in vascular wound repair and remodeling events. Despite this, very little is known about the effect of Wnt ligands on the structure and function of vascular cells. In this study, we treated vascular smooth muscle cells with 250 ng/ml of recombinant Wnt3a for 72 h and observed changes in the cell phenotype. Our data suggest Wnt3a completely alters the phenotype of vascular smooth muscle cells. The Wnt3a-treated cells appeared larger and had increased formation of stress fibers. These cells also had increased expression of the smooth muscle contractile proteins, calponin and smooth muscle α-actin, and contracted a collagen lattice faster than control cells. The Wnt3a-treated smooth muscle cells displayed increased extracellular matrix synthesis, as measured by collagen I and III mRNA expression, along with increased expression of MMP2 and MMP9, but decreased TIMP2 levels. The Wnt3a-induced change in cell phenotype was associated with increased expression of the gap junction protein connexin 43. Consistent with this, Wnt3a-treated smooth muscle cells displayed enhanced intercellular communication, as measured by the scrape-loading dye transfer technique. The canonical Wnt antagonist, dickkopf-related protein 1, completely reversed the contractile protein and connexin 43 expression seen in the Wnt3a-treated cells, suggesting these changes were dependent on canonical Wnt signaling. Collectively, this data suggest Wnt3a promotes a contractile and secretory phenotype in vascular smooth muscle cells that is associated with increased gap junction communication.

Human Connexin30.2/31.3 (GJC3) does not Form Functional Gap Junction Channels but Causes Enhanced ATP Release in HeLa Cells

Gap junctional intercellular communication has numerous functions, each of which meets the particular needs of organs, tissues, or groups of cells. Connexins (CXs) are homologous four-transmembrane-domain proteins that are the major components of gap junctions. CX30.2/CX31.3 (GJC3) is a relatively new member of the CX protein family. Until now, however, the functional characteristics of CX30.2/CX31.3 have been unclear. To elucidate the properties of CX30.2/CX31.3 channels, their subcellular localization in HeLa cells, their effectiveness in dye transfer, and function on channels were investigated. In the immunofluorescent assay, cells that express CX30.2/CX31.3-GFP exhibited continuous fluorescence along the apposed cell membranes, rather than punctated fluorescence in contacting membranes between two cells. Surprisingly, dyes that can be capable of being permeated by CX26 GJ, according to a scrape loading dye transfer assay in previous studies, are impermeated by CX30.2/CX31.3 GJ, suggesting a difference between the characteristics of CX30.2/CX31.3 GJ and CX26 GJ. Furthermore, a significant amount of ATP was released from the HeLa cells that stably expressed CX30.2/CX31.3, in a medium with low calcium ion concentration, suggesting a hemichannel-based function for CX30.2/CX31.3. Based on these findings, we suggest that CX30.2/CX31.3 shares functional properties with pannexin (hemi) channels rather than gap junction channels of other CXs.

Effects of different types of Helicobacter pylori on the gap junction intercellular communication in GES-1 cells.

To determine the effect of different types of Helicobacter pylori (H.pylori) on the gap junction intercellular communication (GJIC) in GES-1 cells, and investigate the types of H.pylori related to the dysfunction of GJIC. Different types of H.pylori clinical strains were isolated and cultured, including the East Asian CagA(-)positive H.pylori (East Asian CagA(+)H.pylori), Western CagA(-)positive H.pylori (Western CagA(+)H.pylori), and the CagA(-)negative H.pylori (CagA(-)H.pylori). We co-cultured these H.pylori strains with GES-1 cells for 24 and 48 h, respectively. The control group was cultured without any H.pylori for 24 and 48 h. Change of the GJIC function in GES-1 cells was detected by the scrape-loading dye transfer (SLDT) technique. The cell proliferation of each group was examined by the methyl thiazolyl tetrazolium bromide (MTT) assay. The control group showed better GJIC function in the GES-1 cells, and the fluorescent dye migrated 4-5 rows to the adjacent cells at 24 and 48 h. Compared with the control group, the GJIC function of GES-1 cells in the CagA(-)H.pylori group decreased and the fluorescent dye migrated 3 rows to the adjacent cells. Compared with the control group and the CagA(-) H.pylori group, the GJIC function of GES-1 cells in the Western CagA(+)H.pylori group decreased and the fluorescent dye migrated 1-2 rows to the adjacent cells. The East Asian CagA(+)H.pylori group showed no GJIC function or weak GJIC function, and most of the fluorescent dye was confined to the area of scratched single row cells and only a few migrated 1-2 rows to the adjacent cells. Difference in the cell proliferation between the CagA(-)H.pylori group and the control group was not significant. The cell proliferation of the Western CagA(+)H.pylori group and the East Asian CagA(+)H.pylori group at bacterium-to-cell ratio of 100:1 and 200:1 was higher than that of the control group. The cell prolife-ration of the East Asian CagA(+)H.pylori group at bacterium-to-cell ratio of 400:1 was significantly lower than that of the control group at 48 h. H.pylori can inhibit the GJIC function in GES-1 cells, which may be associated with CagA(+)H.pylori, especially with East Asian CagA(+)H.pylori. The effect of H.pylori on the proliferation of GES-1 cells is related to virulence factor CagA.

Ultraviolet A exposure induces reversible disruption of gap junction intercellular communication in lens epithelial cells

Gap junction intercellular communication (GJIC) is essential for the proper function of many organs including the lens. Disruption of GJIC can cause lens metabolic disorder and can induce cataracts. The purpose of this study was to investigate the signal transduction pathways involved in GJIC disruption following ultraviolet A (UVA) exposure in lens epithelial cells. Following exposure of human lens epithelial cells to UVA, connexin 43 (Cx43), the main component of gap junctions, was down-regulated at both the mRNA and protein levels. Furthermore, we observed that UVA exposure can increase protein kinase C activity and stimulate reactive oxygen species generation and lipid peroxidation. Using scrape load dye transfer technique, we found that the GJIC is compromised by UVA exposure. In addition, we demonstrated that UVA-induced modulation of GJIC was associated with p38 mitogen-activated protein kinase activation. More importantly, at non-lethal doses (10 J/cm²), the UVA-induced GJIC disruption and the consequent alterations were reversible. Collectively, our data revealed a new signaling pathway in GJIC disruption following UVA exposure, suggesting that UVA-compromised gap junction activity may sensitize human lens to photoaging and cataract formation.

Green tea prevents down-regulation of gap junction intercellular communication in human keratinocytes treated with PMA

It has been suggested that connexin (Cx) gap junction proteins act as tumor suppressors and green tea has a potential to prevent tumor development, however, the studies on their association with human keratinocytes were rare. We evaluated the effects of a tumor promoter, phorbol-12-myristate-13-acetate (PMA), on the expression of Cxs and gap junction intercellular communication (GJIC) in human keratinocytes (HaCaT cells) and explored the preventive effects of green tea extracts-epicatechin (EC) and epigallocatechin-3-gallate (EGCG). We performed neutral red dye uptake assay to determine the optimal concentrations of PMA, EC, and EGCG for this study and confirmed the expression of Cx mRNAs using RT-PCR. We evaluated GJIC quantitatively using the 'scrape-loading dye transfer (SLDT)' technique after 24-h culture of HaCaT cells treated with agents. To analyze the expression change of Cxs, we also performed Western blot and immunocytochemistry. HaCaT cells were found to express Cx26, Cx30, Cx31, and Cx43, but not Cx29. In 'scrape-loading dye transfer' for functional study for GJIC, EC and EGCG significantly prevented PMA-induced down-regulation of GJIC. Western blot analyses revealed that EC and EGCG prevented down-regulation of Cx26 and Cx43 proteins in HaCaT cells treated with PMA. Immunocytochemistry showed decreased expression and abnormal location of Cx26 and Cx43 in HaCaT cells when treated with PMA, and EC and EGCG inhibited its effect. These results suggest an important role of GJIC played in carcinogenesis involving human keratinocytes and green tea as a useful anticancer diet.