Gap Junction Intracellular Communication Research Articles

Abstract 660: Injury Induced Connexin 43 Expression Plays A Role In Vascular Endothelial Wound Healing

Vascular surgeries, such as stent placement and coronary artery bypass, damage the endothelial lining of blood vessels. Impaired healing of endothelial damage is a primary contributor to vascular stent and bypass failure, putting patients at risk for myocardial infarction and death. As such, it is necessary to identify regulators of normal endothelial healing and understand how these factors are dysregulated when healing is impaired. Retrospective analysis of RNAseq data from a previous study investigating non-injured vs. regenerating endothelial cells (EC) in mouse aortas revealed a significant increase in the expression of connexin 43 (Cx43) throughout the EC healing process. Although Cx43 functions are important for skin wound healing, the role of connexin 43 in large artery endothelial wound healing has never been investigated. Here, we test the hypothesis that injury-induced increases in Cx43 expression and gap junction functions promote vascular endothelial wound healing. To study Cx43 in the regenerating endothelium in vivo, we developed a novel model of endothelial cell (EC) injury in mouse carotid arteries. In this model, carotids are ligated for a period of one minute, resulting in an EC denudation injury that heals over 24-hours. Western blotting of EC isolated from damaged carotids demonstrated an increase in Cx43 protein in injured mouse EC while immunofluorescent imaging revealed increases in Cx43 staining at EC borders indicative of increased gap junction signaling. Human coronary artery EC scratch assays were also used to study the process of endothelial wound repair in vitro. As Cx43 phosphorylation is the primary regulator of gap junction channel signaling, we used pharmacological strategies (novel peptide treatments) to increase protein kinase C (PKC) phosphorylation at Cx43 serine 368 (Cx43-pS368), associated with gap junction closure and reduced intracellular communication. In cultured EC, increasing Cx43-pS368 resulted in slower wound healing times, suggesting that limiting Cx43 gap junction communication impairs EC wound healing. In conclusion, our data indicate that injury-induced EC upregulation of Cx43 and increases in gap junction intracellular communication may play a key role in EC wound healing responses.

Gap junction is essential for the antidepressant effects of fluoxetine.

Fluoxetine has been used as the first line for the therapy of depression. However, lack of therapeutic efficacy and time lag still limit the application of fluoxetine. Gap junction dysfunction is a potentially novel pathogenic mechanism for depression. To clarify the mechanism underlying these limitations, we investigated whether gap junction was related to the antidepressant effects of fluoxetine. After chronic unpredictable stress (CUS), animals showed decreases in gap junction intracellular communication (GJIC). Treatment with fluoxetine 10 mg/kg significantly improved GJIC and anhedonia of rats until six days. These results indicated that fluoxetine improved gap junction indirectly. Furthermore, to test the role of gap junction on antidepressant effects of fluoxetine, we blocked gap junction using carbenoxolone (CBX) infusion in the prefrontal cortex. CBX dampened fluoxetine-induced decrease in immobility time of mice in tail suspension test (TST). Our study suggested that gap junction dysfunction blocks antidepressant effects of fluoxetine, contributing to understanding the mechanism underlying the time lag of fluoxetine.

STEM-04. CONNEXIN 43 DRIVES GLIOBLASTOMA CANCER STEM CELL PHENOTYPES THROUGH A WNK1 SIGNALING AXIS

Abstract Tumors represent a highly dynamic system that relies on coordinated signaling to drive growth and adapt to selective pressures, including those induced by anti-cancer therapies. Given the need for tumors to engage in rapid and coordinated cell-cell communication, mechanisms including gap junction intracellular communication would be essential. Surprisingly, the connexin proteins that make up gap junctions have traditionally been considered tumor suppressors. This hypothesis was based on the loss of GJIC in cancer cells and, frequently, lower connexin expression in tumor cells compared to non-neoplastic tissue, including in glioblastoma (GBM), where connexin 43 (Cx43) was shown to be decreased. However, recent data suggest that this tumor-suppressive effect is context dependent and that connexins can function in a tumor-promoting role. We previously found that GBM cancer stem cells (CSCs) rely on gap junctional intercellular communication through connexin 46 (Cx46) for survival. We now identify a subset of GBM patient-derived xenograft (PDX) CSC models that also rely on Cx43 in addition to Cx46. This dependence on Cx43 is independent of Cx43 expression level. Knockdown of Cx43 using shRNA in these PDX models compromises their survival and self-renewal while inducing apoptosis. We leveraged a protein kinase phospho array to identify signaling changes associated with Cx43 knockdown and observed reductions in phosphorylation of WNK lysine-deficient protein kinase 1 (WNK1) in addition to ERK1/2, AKT, and p38MAPK. Immunoblotting validated that WNK1 phosphorylation is lost in the absence of Cx43. Treatment with a WNK1 inhibitor was toxic to CSCs. Current work is focused on interrogating the link among Cx43, WNK1 phosphorylation, and downstream signaling events and the therapeutic window of WNK1 inhibition. This work identifies a potential new signaling axis downstream of Cx43 in glioblastoma CSCs and further strengthens the hypothesis that connexins may act as both tumor suppressors and tumor drivers depending on context.

Trastuzumab and Doxorubicin Sequential Administration Increases Oxidative Stress and Phosphorylation of Connexin 43 on Ser368.

Human epidermal growth factor receptor-2 (HER2) is overexpressed in up to 30% of breast cancer cases, causing a more aggressive tumour growth and poor prognosis. Trastuzumab, the humanized antibody targeted to HER2, increased the life expectancy of patients, but severe cardiotoxicity emerged as a long-term adverse effect. Clinical evidence highlights that Trastuzumab-induced cardiotoxicity drastically increases in association with Doxorubicin; however, the exact mechanisms involved remain incompletely understood. In order to analyse the molecular mechanisms involved and the possible adaptative responses to Trastuzumab and Doxorubicin treatment, in this study, H9c2 cardiomyoblasts were used. Results showed that Trastuzumab and Doxorubicin sequential administration in cardiomyoblast increased cytosolic and mitochondrial ROS production, intracellular calcium dysregulation, mitochondrial membrane depolarization, and the consequent apoptosis, induced by both Trastuzumab and Doxorubicin alone. Furthermore, in these conditions, we observed increased levels of Connexin43 phosphorylated on Ser368 (pCx43). Since phosphorylation on Ser368 decreases gap junction intracellular communication, thus reducing the spread of death signals to adjacent cells, we hypothesized that the increase in pCx43 could be an adaptative response implemented by cells to defend neighbouring cells by Trastuzumab and Doxorubicin sequential administration. However, the other side of the coin is the resulting conduction abnormalities.

IL-10 and TGF- β differentially regulate gap junction formation and membrane transfer in macrophages and macrophage-like cells

Abstract In macrophages, the expression and regulation of the gap junction protein connexin43 (Cx43) is poorly understood. Here we demonstrate that inflammatory and anti-inflammatory mediators differentially regulate gap junction plaque formation in primary bone marrow-derived macrophages (BMDMs) and the macrophage-like cell line RAW264.7. RAW264.7 macrophage-like cells were transfected with GFP-tagged Cx43 to visualize Cx43 trafficking. LPS, an inflammatory mediator, is a potent inducer of Cx43 in BMDMs and RAW264-Cx43-GFP but demonstrates limited trafficking into gap junction plaques. Interestingly, the further addition of IL-10, an anti-inflammatory cytokine, resulted in no difference in protein expression but significantly increased gap junction plaque formation. In contrast to IL-10, the addition of TGF-β (beta) decreased Cx43 protein levels and prevented gap junction plaque formation. We utilized a dye transfer assay to assess gap junction intracellular communication. We could not demonstrate any channel-associated transfer of an intracellular dye, calcein. Uniquely, macrophages were observed transferring endosome-like particles containing membrane between cells as visualized with the membrane-bound dye DiI. This appeared to be through long tube-like structures, possibly tunneling nanotubes. This effect was enhanced with IL-10 treatment and decreased with TGF-β. These results suggest that IL-10 and TGF-β differentially regulate Cx43 plaque formation and that this corresponds to changes in the transfer of cellular components in a gap junction channel-independent way. Overall these results suggest a complex and novel regulation of Cx43 gap junction formation in macrophages and macrophage-like cells.

Effect of platelet-derived growth factor-BB on gap junction and connexin43 in rat penile corpus cavernosum smooth muscle cells.

We explored whether platelet-derived growth factor (PDGF)-BB regulates corpus cavernosum smooth muscle cell gap junctions and can ameliorate erectile dysfunction and how it modulates connexin43 (CX43) after bilateral cavernous neurectomy. Primary cultured rat corpus cavernosum smooth muscle cells were treated with PDGF-BB with or without a PDGFR inhibitor, Akt siRNA or the depletion or promotion of β-catenin. PDGF-BB improved CCSMCs gap junction coupling and increased CX43 and PDGFRβ expression; inhibition of PDGFR activity down-regulated CX43 and decreased Akt and nuclear β-catenin. Knockdown or promotion of β-catenin down-regulated and up-regulated CX43 expression respectively. Moreover, β-catenin activation induced CX43 nuclear accumulation, which impeded CX43 down-regulation induced by PDGFR inhibition, suggesting that CX43 expression is positively correlated with nuclear β-catenin expression. Furthermore, CX43 promoter luciferase and chromatin immunoprecipitation assays indicated that β-catenin regulates CX43 transcription by directly interacting with its promoter. Male rats underwent bilateral cavernous neurectomy. After 12weeks, they were injected with PDGF-BB, CX43 and PDGFRβ expression was significantly lower than in the control group, which was reversed by PDGF-BB injection. These results suggested that PDGF-BB contributed to the improvement of gap junction intracellular communication among corpus cavernosum smooth muscle cells, increased CX43 through PDGFRβ/Akt/nuclear β-catenin signalling, and ameliorated cavernous nerve injury-induced erectile dysfunction.

Connexin 43 gap junctions contribute to brain endothelial barrier hyperpermeability in familial cerebral cavernous malformations type III by modulating tight junction structure.

Familial cerebral cavernous malformations type III (fCCM3) is a disease of the cerebrovascular system caused by loss-of-function mutations in ccm3 that result in dilated capillary beds that are susceptible to hemorrhage. Before hemorrhage, fCCM3 lesions are characterized by a hyperpermeable blood-brain barrier (BBB), the key pathologic feature of fCCM3. We demonstrate that connexin 43 (Cx43), a gap junction (GJ) protein that is incorporated into the BBB junction complex, is up-regulated in lesions of a murine model of fCCM3. Small interfering RNA-mediated ccm3 knockdown (CCM3KD) in brain endothelial cells in vitro increased Cx43 protein expression, GJ plaque size, GJ intracellular communication (GJIC), and barrier permeability. CCM3KD hyperpermeability was rescued by GAP27, a peptide gap junction and hemichannel inhibitor of Cx43 GJIC. Tight junction (TJ) protein, zonula occludens 1 (ZO-1), accumulated at Cx43 GJs in CCM3KD cells and displayed fragmented staining at TJs. The GAP27-mediated inhibition of Cx43 GJs in CCM3KD cells restored ZO-1 to TJ structures and reduced plaque accumulation at Cx43 GJs. The TJ protein, Claudin-5, was also fragmented at TJs in CCM3KD cells, and GAP27 treatment lengthened TJ-associated fragments and increased Claudin 5-Claudin 5 transinteraction. Overall, we demonstrate that Cx43 GJs are aberrantly increased in fCCM3 and regulate barrier permeability by a TJ-dependent mechanism.-Johnson, A. M., Roach, J. P., Hu, A., Stamatovic, S. M., Zochowski, M. R., Keep, R. F., Andjelkovic, A. V. Connexin 43 gap junctions contribute to brain endothelial barrier hyperpermeability in familial cerebral cavernous malformations type III by modulating tight junction structure.

Cx32 inhibits TNFα-induced extrinsic apoptosis with and without EGFR suppression.

Tumor necrosis factor α (TNFα) and TNF-related apoptosis-inducing ligand (TRAIL) can trigger the extrinsic apoptosis pathway. Our previous study indicated that connexin32 (Cx32) inhibited streptonigrin-induced intrinsic apoptosis via the epidermal growth factor receptor (EGFR) pathway. However, whether Cx32 can exert effects on the extrinsic apoptosis pathway through EGFR signaling remains unclear. In the present study, we investigated the role of Cx32 in extrinsic apoptosis induced by treatment with TNFα + cycloheximide (CHX) or afatinib in human cervical cancer (CaCx) cells. In stable inducible Cx32-transfected HeLa cells (HeLa-Cx32), Cx32 expression was induced by treatment with doxycycline (Dox). Furthermore, C-33A cells, which natively express high levels of Cx32, were used as a cell model for knockdown of Cx32 with siRNA. To determine the non-junctional function of Cx32 in apoptosis, 18α-glycyrrhetinic acid (18α-GA), a gap junction intracellular communication (GJIC) inhibitor, was used. Our results showed that Cx32 could inhibit apoptosis induced by TNFα + afatinib with or without the GJIC inhibitor. In clinical cervical tissue samples, we found that the expression of survivin was markedly higher in CaCx than in normal cervix tissue, which was in accordance with the expression of Cx32 in our previous study. In HeLa-Cx32cells, we also found that Cx32 upregulated the expression of Cox-2. In addition, Cx32 upregulated EGFR expression in low-density culture (lacking GJ formation). Cx32 could also promote the expression of EGFR, phospho-STAT3 and phospho-ERK in HeLa-Cx32 cells following TNFα treatment. After knocking down Cx32 in C-33A cells, the expression levels of survivin and TNFα were downregulated. The present study verifies that Cx32 exerts an inhibitory effect on extrinsic apoptosis in CaCx cells, and suggests that Cx32 may regulate the progression and micro-environment of CaCx cells.

Cx32 suppresses extrinsic apoptosis in human cervical cancer cells via the NF‑κB signalling pathway.

Tumour necrosis factor α (TNFα) and TNF‑related apoptosis inducing ligand (TRAIL) usually trigger either survival or apoptosis signals in various cell types, and nuclear factorκB (NF‑κB) is a key factor that regulates their biological effects. Connexin32 (Cx32) is a gap junction (GJ) protein that plays vital roles in tumourigenesis and tumour progression. Our previous study explored abnormal Cx32 expression in para‑nuclear areas, exacerbated prognostic parameters and suppressed streptonigrin/cisplatin-induced apoptosis in human cervical cancer (CaCx) cells. In this study, we investigated the role of Cx32 in the extrinsic apoptosis pathway of CaCx cells. In transgenic HeLa cells and C-33A cells, Cx32 expression was manipulated using doxycycline or Cx32 siRNA. GJinhibitors or low density culturing was used to change the status of gap junction intracellular communication(GJIC). We found that apoptosis induced by TNFα and TRAIL was suppressed by Cx32 expression despite the presence or absense of GJIC. We also found that Cx32 upregulated the expression of nuclear NF‑κB and its downstream targets c-IAP1, MMP‑2, and MMP‑9 in HeLa‑Cx32 and C-33A cells. Following our previous study design, our clinical data showed that NF‑κB and MMP‑2 levels increased in human CaCx specimens with high Cx32 expression compared to levels in para‑carcinoma of cervical specimens. SC75741 and JSH-23, NF‑кB signalling pathway inhibitors, inhibited the anti-apoptotic effects of Cx32. In conclusion, Cx32 suppressed TNFα /TRAIL-induced extrinsic apoptosis by upregulating the NF‑κB signalling pathway. This study demonstrates a novel mechanism for Cx32's anti-apoptotic effect and provides a reasonable explanation for the pro-tumour effect of Cx32 in human CaCxcells.

Therapeutic strategies for locally recurrent and metastatic de-differentiated liposarcoma with herpes simplex virus–thymidine kinase–expressing mesenchymal stromal cells

Background aimsMajor challenges in de-differentiated liposarcoma (DDLPS) therapy are the high rate of sequential recurrence (>80%) and metastasis (20–30%) following surgical removal. However, well-defined therapeutic strategies for this rare malignancy are lacking and are critically needed. MethodsWe investigated a new approach to DDLPS therapy with mesenchymal stromal cells expressing herpes simplex virus–thymidine kinase (MSC-TK). In an effort to evaluate this efficacy, in vitro cytotoxicity of MSC-TK against DDLPS cells was analyzed using an apoptosis assay. For pre-clinical study, the MSC-TK–induced reduction in recurrence and metastasis was validated in a recurrent DDLPS model after the macroscopic complete resection and lung metastasis DDLPS model. ResultsMSC-TK induced apoptosis in DDLPS cells by bystander effects via gap junction intracellular communication (GJIC) of toxic ganciclovir (GCV). Recurrent DDLPS models following no residual tumor/microscopic tumor resection and lung metastasis DDLPS models were established, which suggested clinical relevance. MSC-TK markedly reduced locoregional recurrence rates and prolonged recurrence-free survival, thus increasing overall survival in the recurrent DDLPS model. MSC-TK followed by GCV treatment yielded a statistically significant reduction in early- and advanced-stage lung metastasis. DiscussionThis therapeutic strategy may serve as an alternative or additional strategy by applying MSC-TK to target residual tumors following surgical resection, thus reducing local relapse and metastasis in these patients.

Mild hypothermia preserves myocardial conduction during ischemia by maintaining gap junction intracellular communication and Na+ channel function.

Acute cardiac ischemia induces conduction velocity (CV) slowing and conduction block, promoting reentrant arrhythmias leading to sudden cardiac arrest. Previously, we found that mild hypothermia (MH; 32°C) attenuates ischemia-induced conduction block and CV slowing in a canine model of early global ischemia. Acute ischemia impairs cellular excitability and the gap junction (GJ) protein connexin (Cx)43. We hypothesized that MH prevented ischemia-induced conduction block and CV slowing by preserving GJ expression and localization. Canine left ventricular preparations at control (36°C) or MH (32°C) were subjected to no-flow prolonged (30 min) ischemia. Optical action potentials were recorded from the transmural left ventricular wall, and CV was measured throughout ischemia. Cx43 and Na+ channel (NaCh) remodeling was assessed using both confocal immunofluorescence (IF) and/or Western blot analysis. Cellular excitability was determined by microelectrode recordings of action potential upstroke velocity (dV/dtmax) and resting membrane potential (RMP). NaCh current was measured in isolated canine myocytes at 36 and 32°C. As expected, MH prevented conduction block and mitigated ischemia-induced CV slowing during 30 min of ischemia. MH maintained Cx43 at the intercalated disk (ID) and attenuated ischemia-induced Cx43 degradation by both IF and Western blot analysis. MH also preserved dV/dtmax and NaCh function without affecting RMP. No difference in NaCh expression was seen at the ID by IF or Western blot analysis. In conclusion, MH preserves myocardial conduction during prolonged ischemia by maintaining Cx43 expression at the ID and maintaining NaCh function. Hypothermic preservation of GJ coupling and NaCh may be novel antiarrhythmic strategies during resuscitation.NEW & NOTEWORTHY Therapeutic hypothermia is now a class I recommendation for resuscitation from cardiac arrest. This study determined that hypothermia preserves gap junction coupling as well as Na+ channel function during acute cardiac ischemia, attenuating conduction slowing and preventing conduction block, suggesting that induced hypothermia may be a novel antiarrhythmic strategy in resuscitation.

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Objectives Gap junctions (GJ) are intercellular channels that contribute to the robust vasodilation found in pregnancy in several vascular beds. GJs allow for the coordination of endothelial cell responses by directing cell-cell communication via facilitated diffusion of signaling molecules (e.g. cAMP, cGMP). The phosphorylation of GJ protein Cx43 at serine (Ser) residues regulates both open (Ser365) and closed gating (Ser368), thus allowing the passage of signaling molecules between cells. Although the phosphorylation events of cAMP are well studied, little is known about cGMPs role on gap junction intracellular communication (GJIC) and function. Hypothesis: The inhibition of cAMP and cGMP-mediated pathways will modulate the phosphorylation and expression of Cx43 at their respective phosphorylation sites and thus increase or reduce GJIC in pregnant ovine uterine artery endothelial cells (P-UAECs) and human umbilical vein endothelial cells (HUVECs). Methods P-UAECs were pretreated with the respective cAMP and cGMP nucleotide cyclase inhibitors SQ22536 and ODQ (0.1–100 μ M) followed by treatment with 100 μ M of ATP or Forskolin, or 1 μ M–1 mM 8-Bromo-cAMP or 8-Bromo-cGMP and analyzed using ELISA, western analysis, or scrape-loading/dye transfer technique. Results ELISA revealed that the inhibitors SQ22536 and ODQ specifically and completely abrogated (P 0.01) cAMP and cGMP productions, respectively. Western analysis showed that SQ22536, decreased (P 0.05) the ATP, Forskolin, and cAMP-induced phosphorylation at Ser365 in P-UAECs and HUVECs, while GJIC function was reduced. ODQ decreased (P 0.05) ATP and cGMP-induced elevation at Ser365 in P-UAECs and HUVECs. In contrast, ODQ alone had no effect on GJIC. Conclusions cAMP via adenylyl clycase and cGMP via guanylyl cyclase differentially regulate Cx43 phosphorylation and expression, which is important in regulating GJIC. Although both cyclic nucleotides may serve as important regulation mechanisms in controlling vascular tone, cAMP may play the most important functional role in GJICs. These findings also suggest an expanded role of cGMP signaling on GJ function. Disclosures B. Ampey: None. I. Bird: None. R. Magness: None.

A Connexin43 Mimetic Peptide Promotes Regenerative Healing and Improves Mechanical Properties in Skin and Heart

Evidence is building that the gap junction protein connexin43 (Cx43) is an important molecule in regenerative healing of skin and heart. Excess scarring from skin wound healing is a continuing clinical problem. Humans generally lack the ability to regenerate tissue following injury, and some degree of fibrotic repair occurs. In the skin, this results in unsightly scars with inferior mechanical properties. In the heart, scarring causes disruption in the contractility of cardiac muscle and increases the risk of deadly arrhythmia. Therapies that tip the balance of wound healing away from scar tissue and toward regeneration would thus represent a significant medical advance. A cell-permeant peptide, αCT1 (alpha connexin carboxyl-terminal peptide), based on the carboxyl-terminus of connexin43, has been shown to elicit changes in gap junction organization and intracellular communication. In the skin, αCT1 applied at acute time points results in decreased inflammatory response, reduced area of scar progenitor tissue, and restoration of more normal dermal structure and mechanical strength. αCT1 application to infarcted hearts improved cardiac contractility, reduced the propensity for arrhythmia, and increased conduction velocity through the injured heart. Application of therapies like αCT1 could reduce cutaneous scarring and improve mechanical properties of healed skin and the contractile function and electrical stability of the heart following injury or surgery. αCT1 is a potential therapy for cutaneous wounds that could lead to reduced scarring and improvements in the mechanical properties of healed skin. For injured myocardial tissues, this Cx43 mimetic peptide may also provide a therapeutic approach for targeting pathological fibrosis and reducing the likelihood of sudden death from cardiac arrhythmias.

Gap junctions mediate STAT5-independent β-casein expression in CID-9 mammary epithelial cells

Crosstalk between gap junction intracellular communication (GJIC), STAT5 and OCT-1 in gap junction (GJ)-dependent β-casein expression was investigated. CID-9 mammary cells plated with prolactin on non-adherent substratum (poly-HEMA) expressed β-casein independent of STAT5 only in the presence of the GJIC inducer, cAMP. Nuclear STAT5 levels were not detectable. By contrast, cells on EHS-drip expressed β-casein in a STAT5-dependent manner and nuclear STAT5 levels were up-regulated. A 75 kDa OCT-1 isoform was detected in conditions that induced β-casein expression regardless of substratum. Interestingly, 40 and 28 kDa OCT-1 isoforms were induced in cells on polyHEMA with cAMP. Electrophoretic mobility shift assays (EMSA) for OCT-1 revealed two band shifts in cells on polyHEMA with cAMP and on EHS-drip, which were repressed by the GJIC inhibitor, 18α-GA. These studies demonstrated that mammary cells on polyHEMA expressed β-casein in response to prolactin in a pathway that involves GJIC and OCT-1 and is independent of STAT5 nuclear translocation.

Naringenin, a flavanone alters the tumorigenic features of C6 glioma cells

The effect of naringenin on the growth of C6 glioma cells was investigated in the current study. Suppression of cell growth was assessed by MTT assay. Induction of cell cycle arrest was assessed by propidium iodide staining using flowcytometric analysis. Induction of DNA damage was assessed by agarose gel electrophoresis. The antiproliferative effect of NGEN was also assessed and confirmed by cyclooxygenase-2 (COX-2) activity assay. Gap junctional analysis was also done using dye transfer technique. The results showed that NGEN inhibited the growth of C6 cells in a dose dependent manner, compared to the control. At 20 and 30 μg concentrations, NGEN induced cell cycle arrest, induced DNA damage in C6 cells, reduced the activity of COX-2 and increased the gap junction intracellular communication (GJIC). Thus NGEN, a component of citrus fruit, suppresses the growth of C6 cells through the inhibition of proliferation.

S-Adenosylmethionine regulates connexins sub-types expressed by hepatocytes

Intercellular communication via GAP Junctions plays an important role in tissue homeostasis, apoptosis, carcinogenesis, cell proliferation and differentiation. Hepatocyte connexins (Cx) 26 and 32 levels are decreased during the de-differentiation process of primary hepatocytes in culture, a situation that is also characterized by a decrease in S-Adenosylmethionine (SAMe) levels. In this current study, we show that SAMe supplementation in cultured hepatocytes every 12 h, leads to an up-regulation of Cx26 and 32 mRNA and protein levels and blocks culture-induced Cx43 expression, although it failed to increase Cx26 and 32 membrane localization and GAP junction intracellular communication. SAMe reduced nuclear β-catenin accumulation, which is known to stimulate the TCF/LEF-dependent gene transcription of Cx43. Moreover SAMe-induced reduction in Cx43 and β-catenin was prevented by the proteasome inhibitor MG132, and was not mediated by GSK3 activity. SAMe, and its metabolite 5′-methylthioadenosine (MTA) increased Cx26 mRNA in a process partially mediated by Adenosine A 2A receptors but independent of PKA. Finally livers from MAT1A knockout mice, characterized by low hepatic SAMe levels, express higher Cx43 and lower Cx26 and 32 protein levels than control mice. These results suggest that SAMe maintains a characteristic expression pattern of the different Cxs in hepatocytes by differentially regulating their levels.

Naringenin, a flavanone alters the tumorigenic features of C6 glioma cells

The effect of naringenin on the growth of C6 glioma cells was investigated in the current study. Suppression of cell growth was assessed by MTT assay. Induction of cell cycle arrest was assessed by propidium iodide staining using flowcytometric analysis. Induction of DNA damage was assessed by agarose gel electrophoresis. The antiproliferative effect of NGEN was also assessed and confirmed by cyclooxygenase-2 (COX-2) activity assay. Gap junctional analysis was also done using dye transfer technique. The results showed that NGEN inhibited the growth of C6 cells in a dose dependent manner, compared to the control. At 20 and 30 μg concentrations, NGEN induced cell cycle arrest, induced DNA damage in C6 cells, reduced the activity of COX-2 and increased the gap junction intracellular communication (GJIC). Thus NGEN, a component of citrus fruit, suppresses the growth of C6 cells through the inhibition of proliferation.

Gap junction mediated transport of shRNA between human embryonic stem cells

Gap junction intracellular communication (GJIC) allows the direct transport of small molecules between adjacent cells. We hypothesized that siRNAs in one hESC could inhibit target RNA expression in another hESC via GJIC. We co-cultured green fluorescent protein (GFP)-expressing ENVY hESC with non-GFP-expressing hESC, which had been transduced to stably express shRNA directed against GFP. We discovered that the GFP shRNA expressing hESC inhibited GFP expression in the adjacent GFP-expressing hESC in a dose-dependent manner. This downregulation of GFP expression in ENVY cells was not observed when the co-cultured cells had been transduced with a non-functional GFP shRNA that was mutated at two nucleotides or when the cells were incubated with the GJIC inhibitor, α-glycyrrhetinic acid (α-GA). We conclude that 21–23 bp double-stranded shRNA/siRNA oligonucleotides are able to move through gap junctions between hESCs and thus can affect gene expression in neighbouring hESC. This novel intercellular gene expression regulatory mechanism may offer new approaches to manipulation of hESC.

Epigenomics and disease, 10th anniversary winter meeting of the UK Molecular Epidemiology Group (MEG), The Royal Statistical Society, London, UK, 8th December 2006

An organism has a unique genome but may have different tissue-specific epigenomes. Distinct from the genotype, epigenomics encompasses the modulation of gene activity through particular global chromatin methylation patterns or histone modifications; these may be known as epigenetic marks. The chromatin pattern of epigenetic marks is modifiable over a lifespan and may influence disease progression at a particular site. The meeting aim was to discuss the role of epigenomics in the aetiology of disease, particularly cancer. Epigenetic marks might be modifiable through dietary intake of methyl donors and aberrant patterns may underlie phenotypical changes resulting in chronic diseases such as cancer. DNA methylation patterns or histone modifications are potentially reversible, but, in certain circumstances, such marks become imprinted and give rise to trans-generational effects. Other reversible effects influencing disease occurrence might be inhibition of gap junction intracellular communication. Environmental and/or dietary factors play a pivotal role in the aetiology of cancer. Most cancers require a mutagenic initiation step. However, it is now recognized that an aberrant pattern of epigenetic marks may link the initiating mutation to the gene expression profile of a disease phenotype. This workshop stressed the need for a human epigenomic project out of which specific aberrant patterns of epigenetic marks might be developed as novel predictors to facilitate the implementation of future disease prevention strategies, to lend new insights into aetiology of disease, to allow more exact diagnosis and to develop better-targeted therapeutic regimens.

Final Report on the Safety Assessment of Glycyrrhetinic Acid, Potassium Glycyrrhetinate, Disodium Succinoyl Glycyrrhetinate, Glyceryl Glycyrrhetinate, Glycyrrhetinyl Stearate, Stearyl Glycyrrhetinate, Glycyrrhizic Acid, Ammonium Glycyrrhizate, Dipotassium Glycyrrhizate, Disodium Glycyrrhizate, Trisodium Glycyrrhizate, Methyl Glycyrrhizate, and Potassium Glycyrrhizinate1

Final Report on the Safety Assessment of Glycyrrhetinic Acid, Potassium Glycyrrhetinate, Disodium Succinoyl Glycyrrhetinate, Glyceryl Glycyrrhetinate, Glycyrrhetinyl Stearate, Stearyl Glycyrrhetinate, Glycyrrhizic Acid, Ammonium Glycyrrhizate, Dipotassium Glycyrrhizate, Disodium Glycyrrhizate, Trisodium Glycyrrhizate, Methyl Glycyrrhizate, and Potassium Glycyrrhizinate1