Gap Junction Expression Research Articles

The expression of gingival epithelial junctions in response to subgingival biofilms

Periodontitis is an infectious inflammatory disease that destroys the tooth-supporting tissues. It is caused by the formation of subgingival biofilms on the surface of the tooth. Characteristic bacteria associated with subgingival biofilms are the Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, collectively known as the “red complex” species. Inter-epithelial junctions ensure the barrier integrity of the gingival epithelium. This may however be disrupted by the biofilm challenge. The aim of this in vitro study was to investigate the effect of subgingival biofilms on the expression of inter-epithelial junctions by gingival epithelia, and evaluate the relative role of the red complex. Multi-layered human gingival epithelial cultures were challenged with a 10-species in vitro subgingival biofilm model, or its variant without the red complex, for 3 h and 24 h. A low-density array microfluidic card platform was then used for analyzing the expression of 62 genes encoding for tight junctions, gap junctions, adherens junctions, and desmosomes. Although there was a limited effect of the biofilms on the expression of tight, adherens and gap junctions, the expression of a number of desmosomal components was affected. In particular, Desmoglein-1 displayed a limited and transient up-regulation in response to the biofilm. In contrast, Desmocollin-2, Desmoplakin and Plakoglobin were down-regulated equally by both biofilm variants, after 24 h. In conclusion, this subgingival biofilm model may down-regulate selected desmosomal junctions in the gingival epithelium, irrespective of the presence of the “red complex.” In turn, this could compromise the structural integrity of the gingival tissue, favoring bacterial invasion and chronic infection.

Proinflammatory cytokines downregulate connexin 43-gap junctions via the ubiquitin-proteasome system in rat spinal astrocytes

Astrocytic gap junctions formed by connexin 43 (Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders are associated with dysfunctional Cx43-gap junctions. However, the mechanism modulating Cx43-gap junctions in spinal astrocytes under pathological conditions is not entirely clear. A previous study showed that treatment of spinal astrocytes in culture with pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased both Cx43 expression and gap junction intercellular communication (GJIC) via a c-jun N-terminal kinase (JNK)-dependent pathway. The current study further elaborates the intracellular mechanism that decreases Cx43 under an inflammatory condition. Cycloheximide chase analysis revealed that TNF-α (10 ng/ml) alone or in combination with IFN-γ (5 ng/ml) accelerated the degradation of Cx43 protein in cultured spinal astrocytes. The reduction of both Cx43 expression and GJIC induced by a mixture of TNF-α and IFN-γ were blocked by pretreatment with proteasome inhibitors MG132 (0.5 μM) and epoxomicin (25 nM), a mixture of TNF-α and IFN-γ significantly increased proteasome activity and Cx43 ubiquitination. In addition, TNF-α and IFN-γ-induced activation of ubiquitin-proteasome systems was prevented by SP600125, a JNK inhibitor. Together, these results indicate that a JNK-dependent ubiquitin-proteasome system is induced under an inflammatory condition that disrupts astrocytic gap junction expression and function, leading to astrocytic dysfunction and the maintenance of the neuroinflammatory state.

Dominant De Novo Mutations in GJA1 Cause Erythrokeratodermia Variabilis et Progressiva, without Features of Oculodentodigital Dysplasia

Genetic investigation of inherited skin disorders has informed understanding of skin self-renewal, differentiation, and barrier function. Erythrokeratodermia variabilis et progressiva (EKVP) is a rare, inherited skin disease characterized by transient figurate patches of erythema, localized or generalized scaling, and frequent palmoplantar keratoderma. By employing exome sequencing, we show that de novo missense mutations in GJA1 (gap junction protein alpha 1) cause EKVP. The severe, progressive skin disease in EKVP subjects with GJA1 mutations is distinct from limited cutaneous findings rarely found in the systemic disorder oculodentodigital dysplasia, also caused by dominant GJA1 mutations. GJA1 encodes connexin 43 (Cx43), the most widely expressed gap junction protein. We show that the GJA1 mutations in EKVP subjects lead to disruption of Cx43 membrane localization, and aggregation within the Golgi. These findings reveal a critical role for Cx43 in epidermal homeostasis, and provide evidence of organ-specific pathobiology resulting from different mutations within GJA1.

Death of Neurons following Injury Requires Conductive Neuronal Gap Junction Channels but Not a Specific Connexin.

Pharmacological blockade or genetic knockout of neuronal connexin 36 (Cx36)-containing gap junctions reduces neuronal death caused by ischemia, traumatic brain injury and NMDA receptor (NMDAR)-mediated excitotoxicity. However, whether Cx36 gap junctions contribute to neuronal death via channel-dependent or channel-independent mechanism remains an open question. To address this, we manipulated connexin protein expression via lentiviral transduction of mouse neuronal cortical cultures and analyzed neuronal death twenty-four hours following administration of NMDA (a model of NMDAR excitotoxicity) or oxygen-glucose deprivation (a model of ischemic injury). In cultures prepared from wild-type mice, over-expression and knockdown of Cx36-containing gap junctions augmented and prevented, respectively, neuronal death from NMDAR-mediated excitotoxicity and ischemia. In cultures obtained form from Cx36 knockout mice, re-expression of functional gap junction channels, containing either neuronal Cx36 or non-neuronal Cx43 or Cx31, resulted in increased neuronal death following insult. In contrast, the expression of communication-deficient gap junctions (containing mutated connexins) did not have this effect. Finally, the absence of ethidium bromide uptake in non-transduced wild-type neurons two hours following NMDAR excitotoxicity or ischemia suggested the absence of active endogenous hemichannels in those neurons. Taken together, these results suggest a role for neuronal gap junctions in cell death via a connexin type-independent mechanism that likely relies on channel activities of gap junctional complexes among neurons. A possible contribution of gap junction channel-permeable death signals in neuronal death is discussed.

Platelet‐derived growth factor receptor‐α‐positive cells: new players in nerve‐mediated purinergic responses in the colon

Platelet‐derived growth factor receptor‐α‐positive cells: new players in nerve‐mediated purinergic responses in the colon

Role of gap junction communication in hepatocyte/fibroblast co-cultures: Implications for hepatic tissue engineering

The success of cell-based therapies for treatment of liver disease hinges on the phenotypic stability of isolated hepatocytes; however, primary hepatocytes rapidly lose many liver-specific functions following enzymatic isolation from the liver. Previous studies have demonstrated that liver-specific functions can be stabilized by cocultivation with non-parenchymal cells. Although the precise mechanisms underlying these effects have not yet been elucidated, it is believed that gap junctional intercellular communication plays an important role in cell-cell communication. In this study, indirect immunofluorescence was used to identify connexin 32 and 26 as homotypic gap junction proteins that were present between hepatocytes during co-culture with 3T3 fibroblasts, but not in pure hepatocyte cultures. Additionally, dye transfer of Lucifer yellow revealed that these gap junctions were functionally coupled. Although previous studies reported abundant connexin 43 expression in 3T3 fibroblasts and functional coupling by dye transfer, we did not observe heterotypic gap junction (connexin 43) expression or its functional coupling by either immunofluorescence or dye transfer. Incubation with a gap junction inhibitor, 18β-glycyrrhetinic acid, in hepatocyte co-culture diminished the level of liverspecific markers such as albumin production and P450 activity relative to control co-cultures. Overall, these findings suggest that homotypic gap junctions are present in hepatocytes co-cultured with fibroblasts and might play a functional role in homotypic hepatocyte communication. In conclusion, homotypic gap junctional communication may be important to phenotypic stability of isolated hepatocytes in co-culture and crucial to the design of tissue-engineered therapies for treatment of liver failure.

The role of cellular coupling in the spontaneous generation of electrical activity in uterine tissue.

The spontaneous emergence of contraction-inducing electrical activity in the uterus at the beginning of labor remains poorly understood, partly due to the seemingly contradictory observation that isolated uterine cells are not spontaneously active. It is known, however, that the expression of gap junctions increases dramatically in the approach to parturition, by more than one order of magnitude, which results in a significant increase in inter-cellular electrical coupling. In this paper, we build upon previous studies of the activity of electrically excitable smooth muscle cells (myocytes) and investigate the mechanism through which the coupling of these cells to electrically passive cells results in the generation of spontaneous activity in the uterus. Using a recently developed, realistic model of uterine muscle cell dynamics, we investigate a system consisting of a myocyte coupled to passive cells. We then extend our analysis to a simple two-dimensional lattice model of the tissue, with each myocyte being coupled to its neighbors, as well as to a random number of passive cells. We observe that different dynamical regimes can be observed over a range of gap junction conductances: at low coupling strength, corresponding to values measured long before delivery, the activity is confined to cell clusters, while the activity for high coupling, compatible with values measured shortly before delivery, may spread across the entire tissue. Additionally, we find that the system supports the spontaneous generation of spiral wave activity. Our results are both qualitatively and quantitatively consistent with observations from in vitro experiments. In particular, we demonstrate that the increase in inter-cellular electrical coupling observed experimentally strongly facilitates the appearance of spontaneous action potentials that may eventually lead to parturition.

Temporal sequence of activation of cells involved in purinergic neurotransmission in the colon.

Platelet derived growth factor receptor α (PDGFRα(+) ) cells in colonic muscles are innervated by enteric inhibitory motor neurons. PDGFRα(+) cells generate Ca(2+) transients in response to exogenous purines and these responses were blocked by MRS-2500. Stimulation of enteric neurons, with cholinergic and nitrergic components blocked, evoked Ca(2+) transients in PDGFRα(+) and smooth muscle cells (SMCs). Responses to nerve stimulation were abolished by MRS-2500 and not observed in muscles with genetic deactivation of P2Y1 receptors. Ca(2+) transients evoked by nerve stimulation in PDGFRα(+) cells showed the same temporal characteristics as electrophysiological responses. PDGFRα(+) cells express gap junction genes, and drugs that inhibit gap junctions blocked neural responses in SMCs, but not in nerve processes or PDGFRα(+) cells. PDGFRα(+) cells are directly innervated by inhibitory motor neurons and purinergic responses are conducted to SMCs via gap junctions. Interstitial cells, known as platelet derived growth factor receptor α (PDGFRα(+) ) cells, are closely associated with varicosities of enteric motor neurons and suggested to mediate purinergic hyperpolarization responses in smooth muscles of the gastrointestinal tract (GI), but this concept has not been demonstrated directly in intact muscles. We used confocal microscopy to monitor Ca(2+) transients in neurons and post-junctional cells of the murine colon evoked by exogenous purines or electrical field stimulation (EFS) of enteric neurons. EFS (1-20Hz) caused Ca(2+) transients in enteric motor nerve processes and then in PDGFRα(+) cells shortly after the onset of stimulation (latency from EFS was 280ms at 10Hz). Responses in smooth muscle cells (SMCs) were typically a small decrease in Ca(2+) fluorescence just after the initiation of Ca(2+) transients in PDGFRα(+) cells. Upon cessation of EFS, several fast Ca(2+) transients were noted in SMCs (rebound excitation). Strong correlation was noted in the temporal characteristics of Ca(2+) transients evoked in PDGFRα(+) cells by EFS and inhibitory junction potentials (IJPs) recorded with intracellular microelectrodes. Ca(2+) transients and IJPs elicited by EFS were blocked by MRS-2500, a P2Y1 antagonist, and absent in P2ry1((-/-)) mice. PDGFRα(+) cells expressed gap junction genes, and gap junction uncouplers, 18β-glycyrrhetinic acid (18β-GA) and octanol blocked Ca(2+) transients in SMCs but not in neurons or PDGFRα(+) cells. IJPs recorded from SMCs were also blocked. These findings demonstrate direct innervation of PDGFRα(+) cells by motor neurons. PDGFRα(+) cells are primary targets for purinergic neurotransmitter(s) in enteric inhibitory neurotransmission. Hyperpolarization responses are conducted to SMCs via gap junctions.

Alpha-lipoic acid protects against cadmium-induced hepatotoxicity via calcium signalling and gap junctional intercellular communication in rat hepatocytes.

This study investigated the protective effect of alpha-lipoic acid (LA) on cadmium (Cd)-induced hepatotoxicity in BRL 3A rat liver cells. We demonstrated that LA ameliorated Cd-induced cellular injury in cell viability and nuclear fragmentation in BRL 3A cells. Furthermore, LA markedly ameliorated Cd-induced gap junctional intercellular communication (GJIC) inhibition and Cx43 mRNA expression decrease, as well as disassembly of gap junctions. The gap junction blocker carbenoxolone disodium (CBX) as well as LA protected healthy cells from Cd-exposed cells in Transwell co-culture system. LA also protected BRL 3A cells against Cd-induced elevation of the intracellular concentration of free calcium ([Ca(2+)]i). Pretreatment with a chelater of intracellular Ca(2+) BAPTA-AM or chelater of extracellular Ca(2+) EGTA attenuated Cd-induced cytotoxicity and GJIC inhibition. CBX exacerbated the decrease in cell viability and further elevated the increase in [Ca(2+)]i induced by Cd, whereas BAPTA-AM partly attenuated these phenomena, while EGTA had little effects. These results suggested that Cd-induced hepatotoxicity via GJIC inhibition and [Ca(2+)]i elevation, which originates mainly from intracellular stores. GJIC inhibition has dual effects: (i) it restricts release of Ca(2+) from the cell, which exacerbates the [Ca(2+)]i elevation and cytotoxicity induced by Cd; and (ii) it protects healthy cells from their dangerous neighbors by blocking intercellular communication. Above all, our results indicated that LA partly prevented Cd-induced cytotoxicity via GJIC and calcium signaling in BRL 3A rat liver cells.

Spinal astrocyte gap junction and glutamate transporter expression contributes to a rat model of bortezomib-induced peripheral neuropathy

There is increasing evidence implicating astrocytes in multiple forms of chronic pain, as well as in the specific context of chemotherapy-induced peripheral neuropathy (CIPN). However, it is still unclear what the exact role of astrocytes may be in the context of CIPN. Findings in oxaliplatin and paclitaxel models have displayed altered expression of astrocytic gap junctions and glutamate transporters as means by which astrocytes may contribute to observed behavioral changes. The current study investigated whether these changes were also generalizable to the bortezomib CIPN. Changes in mechanical sensitivity were verified in bortezomib-treated animals, and these changes were prevented by co-treatment with a glial activation inhibitor (minocycline), a gap junction decoupler (carbenoxolone), and by a glutamate transporter upregulator (ceftriaxone). Immunohistochemistry data at day 30 in bortezomib-treated animals showed increases in expression of glial fibrillary acidic protein (GFAP) and connexin 43 but a decrease in GLAST expression. These changes were prevented by co-treatment with minocycline. Follow-up Western blotting data showed a shift in connexin 43 from a non-phosphorylated state to a phosphorylated state, indicating increased trafficking of expressed connexin 43 to the cell membrane. These data suggest that increases in behavioral sensitivity to cutaneous stimuli may be tied to persistent synaptic glutamate resulting from increased calcium flow between spinal astrocytes.

Correlations of differentially expressed gap junction connexins Cx26, Cx30, Cx32, Cx43 and Cx46 with breast cancer progression and prognosis.

Background and AimsConnexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers.Materials and MethodsMeta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models.ResultsThe expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other’s prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively.ConclusionDifferential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers.

17The brugada syndrome and cardiomyopathy: altered collagen and gap junction expression

Background: Brugada syndrome (BrS) is considered an inherited arrhythmia syndrome, though it may represent a concealed cardiomyopathy confined to the right ventricular outflow tract (RVOT). We aimed to ascertain fibrosis and gap junction expression in ventricular myocardium following sudden cardiac death (SCD) due to BrS. Methods: The BrS cohort consisted of 6 cases of unexplained SCD referred for specialist whole heart autopsy whose family were diagnosed with BrS. Comparisons were made to matched homograft control hearts (n=6) and cases of arrhythmogenic right ventricular cardiomyopathy (ARVC, n=5). Morphometric evaluation of collagen and fat content of left (LV) and right (RV) ventricles and RVOT by picrosirius red-staining was undertaken. Gap junction expression was assessed by Connexin43 (Cx43) immunostaining in the RVOT. Results: Collagen in controls was predominantly perivascular in long fine fibres and diffuse reticular interstitial networks. Interstitial long collagen strands were thickened in BrS, with evidence of epicardial patchy replacement fibrosis. ARVC was characterised by both interstitial and dense patchy replacement fibrosis. Maximal collagen content, indicative of fibrosis, was present in the RVOT (p=0.003) and epicardium (p=0.001) in all three disease groups, with RV more fibrosed than LV (p=0.020). ARVC cases exhibited the most fibrosis (p=0.001), while BrS cases were significantly more fibrosed than controls (OR 1.42; p=0.024). No significant difference in fat content was observed between BrS and controls (p=0.133). BrS cases showed a reduced Cx43 signal in comparison to controls (OR 0.59; p=0.001), which remained significant after adjusting for collagen content (Figure). However, lateralisation and dispersion of Cx43 over the cellular surface was seen in both control and BrS sections. In ARVC, Cx43 signal was severely reduced in comparison to BrS (OR 0.61; p=0.002). Discussion: BrS associates with generalised ventricular myocardial fibrosis and reduced RVOT gap junction expression in comparison with controls, with ARVC demonstrating similar but more severe abnormalities. Thus, BrS may represent a disease of myocardial architecture, with baseline properties of the RVOT predisposing it to more severe fibrosis. We speculate that myocyte electrical uncoupling indicated by gap junction abnormalities and changes in conduction related to fibrosis confer arrhythmic risk. ![Graphic][1] Figure Scatterplot of Cx43:collagen ratio in RVOT by myocardial zone (filled data points=mean) demonstrating significant differences between ARVC, BrS and Control cohorts, but no difference from epi- to endo-cardium. [1]: /embed/inline-graphic-1.gif

Gap junction-mediated death of retinal neurons is connexin and insult specific: a potential target for neuroprotection.

Secondary cell death via gap junctions (GJs) plays a role in the propagation of neuronal loss under a number of degenerative disorders. Here, we examined the role of GJs in neuronal death in the retina, which has arguably the most diverse expression of GJs in the CNS. Initially, we induced apoptotic death by injecting single retinal ganglion cells and glia with cytochrome C and found that this resulted in the loss of neighboring cells to which they were coupled via GJs. We next found that pharmacological blockade of GJs eradicated nearly all amacrine cell loss and reduced retinal ganglion cell loss by ∼70% after induction of either excitotoxic or ischemic insult conditions. These data indicate that the GJ-mediated secondary cell death was responsible for the death of most cells. Whereas genetic deletion of the GJ subunit Cx36 increased cell survivability by ∼50% under excitotoxic condition, cell loss in Cx45 knock-out mouse retinas was similar to that seen in wild-type mice. In contrast, ablation of Cx45 reduced neuronal loss by ∼50% under ischemic insult, but ablation of Cx36 offered no protection. Immunolabeling of the connexins showed differential changes in protein expression consistent with their differing roles in propagating death signals under the two insults. These data indicate that secondary cell death is mediated by different cohorts of GJs dependent on the connexins they express and the type of initial insult. Our results suggest that targeting specific connexins offers a novel therapeutic strategy to reduce progressive cell loss under different neurodegenerative conditions.

Changes in gap junction expression and function following ischemic injury of spinal cord white matter.

Gap junctions are widely present in spinal cord white matter; however, their role in modulating the dynamics of axonal dysfunction remains largely unexplored. We hypothesized that inhibition of gap junctions reduces the loss of axonal function during oxygen and glucose deprivation (OGD). The functional role of gap junctions was assessed by electrophysiological recordings of compound action potentials (CAPs) in Wistar rat spinal cord slices with the sucrose gap technique. The in vitro slices were subjected to 30-min OGD. Gap junction connexin (Cx) mRNA expression was determined by qPCR and normalized to β-actin. A 30-min OGD resulted in reduction of CAPs to 14.8 ± 4.6% of their pre-OGD amplitude (n = 5). In the presence of gap junction blockers carbenoxolone (Cbx; 100 μM) and 1-octanol (Oct; 300 μM), the CAP reduction in OGD was to only 35.7 ± 5.7% of pre-OGD amplitude in Cbx (n = 9) and to 37.4 ± 8.9% of pre-OGD amplitude in Oct (n = 10). Both drugs also noticeably prolonged the half-decline time of CAP amplitudes in OGD from 6.0 min in no-drug conditions to 9.6 min in the presence of Cbx and to 7.7 min in the presence of Oct, suggesting that blocking gap junctions reduces conduction loss during OGD. With application of Cbx and Oct in the setting of OGD, expression of Cx30 and Cx43 mRNA was downregulated. Our data provide new insights into the role of gap junctions in white matter ischemia and reveal the necessity of a cautious approach in determining detrimental or beneficial effects of gap junction blockade in white matter ischemia.

Less is more: minimal expression of myoendothelial gap junctions optimizes cell–cell communication in virtual arterioles

Dysfunctional electrical signalling within the arteriolar wall is a major cause of cardiovascular disease. The endothelial cell layer constitutes the primary electrical pathway, co-ordinating contraction of the overlying smooth muscle cell (SMC) layer. As myoendothelial gap junctions (MEGJs) provide direct contact between the cell layers, proper vasomotor responses are thought to depend on a high, uniform MEGJ density. However, MEGJs are observed to be expressed heterogeneously within and among vascular beds. This discrepancy is addressed in the present study. As no direct measures of MEGJ conductance exist, we employed a computational modelling approach to vary the number, conductance and distribution of MEGJs. Our simulations demonstrate that a minimal number of randomly distributed MEGJs augment arteriolar cell-cell communication by increasing conduction efficiency and ensuring appropriate membrane potential responses in SMCs. We show that electrical coupling between SMCs must be tailored to the particular MEGJ distribution. Finally, observation of non-decaying mechanical conduction in arterioles without regeneration has been a long-standing controversy in the microvascular field. As heterogeneous MEGJ distributions provide for different conduction profiles along the cell layers, we demonstrate that a non-decaying conduction profile is possible in the SMC layer of a vessel with passive electrical properties. These intriguing findings redefine the concept of efficient electrical communication in the microcirculation, illustrating how heterogeneous properties, ubiquitous in biological systems, may have a profound impact on system behaviour and how acute local and global flow control is explained from the biophysical foundations.

8-Methyltryptanthrin-induced differentiation of P19CL6 embryonal carcinoma cells into spontaneously beating cardiomyocyte-like cells.

Enhancement of cardiac differentiation is critical to stem cell transplantation therapy for severe ischemic heart disease. The aim of this study was to investigate whether several derivatives of tryptanthrin (1), extracted from the medicinal plant Polygonum tinctorium, induce the differentiation of P19CL6 mouse embryonal carcinoma cells into beating cardiomyocyte-like cells. P19CL6 cells were cultured in α-MEM supplemented with 10% FBS including a test compound or vehicle. Drug-induced differentiation was assessed by measuring the number of beating and nonbeating aggregates and the area of beating aggregates, and the expression of genes involved in cardiac differentiation was evaluated by real-time PCR. A 1 μM concentration of 8-methyltryptanthrin (2) induced the differentiation of P19CL6 cells into cardiomyocyte-like cells to a significantly greater degree than 1% dimethyl sulfoxide (DMSO), a conventional differentiation inducer of P19CL6 cells. Furthermore, 2 strongly increased both the number and the area of spontaneously beating aggregates in comparison with DMSO. Two distinct genes of the calcium channel family, Cav1.2 and Cav3.1, underlying cardiac automaticity were significantly expressed in the presence of 2. Gap junction genes GJA1 and GJA5 contributing to the synchronized contraction of the myocardium were also induced significantly by 2. These results suggest that 2 successfully differentiated P19CL6 cells into spontaneously beating cardiomyocyte-like cells by activating the gene expression of pacemaker channels and gap junctions.

Connexins and cyclooxygenase-2 crosstalk in the expression of radiation-induced bystander effects.

Background:Signalling events mediated by connexins and cyclooxygenase-2 (COX-2) have important roles in bystander effects induced by ionising radiation. However, whether these proteins mediate bystander effects independently or cooperatively has not been investigated.Methods:Bystander normal human fibroblasts were cocultured with irradiated adenocarcinoma HeLa cells in which specific connexins (Cx) are expressed in the absence of endogenous Cx, before and after COX-2 knockdown, to investigate DNA damage in bystander cells and their progeny.Results:Inducible expression of gap junctions composed of connexin26 (Cx26) in irradiated HeLa cells enhanced the induction of micronuclei in bystander cells (P<0.01) and reduced the coculture time necessary for manifestation of the effect. In contrast, expression of connexin32 (Cx32) conferred protective effects. COX-2 knockdown in irradiated HeLa Cx26 cells attenuated the bystander response due to connexin expression. However, COX-2 knockdown resulted in enhanced micronucleus formation in the progeny of the bystander cells (P<0.001). COX-2 knockdown delayed junctional communication in HeLa Cx26 cells, and reduced, in the plasma membrane, the physical interaction of Cx26 with MAPKKK, a controller of the MAPK pathway that regulates COX-2 and connexin.Conclusions:Junctional communication and COX-2 cooperatively mediate the propagation of radiation-induced non-targeted effects. Characterising the mediating events affected by both mechanisms may lead to new approaches that mitigate secondary debilitating effects of cancer radiotherapy.

Amitriptyline up‐regulates connexin43‐gap junction in rat cultured cortical astrocytes via activation of the p38 and c‐Fos/AP‐1 signalling pathway

Intercellular communication via gap junctions, comprised of connexin (Cx) proteins, allow for communication between astrocytes, which in turn is crucial for maintaining CNS homeostasis. The expression of Cx43 is decreased in post-mortem brains from patients with major depression. A potentially novel mechanism of tricyclic antidepressants is to increase the expression and functioning of gap junctions in astrocytes. The effect of amitriptyline on the expression of Cx43 and gap junction intercellular communication (GJIC) in rat primary cultured cortical astrocytes was investigated. We also investigated the role of p38 MAPK intracellular signalling pathway in the amitriptyline-induced expression of Cx43 and GJIC. Treatment with amitriptyline for 48 h significantly up-regulated Cx43 mRNA, protein and GJIC. The up-regulation of Cx43 was not monoamine-related since noradrenaline, 5-HT and dopamine did not induce Cx43 expression and pretreatment with α- and β-adrenoceptor antagonists had no effect. Intracellular signalling involved p38 MAPK, as amitriptyline significantly increased p38 MAPK phosphorylation and Cx43 expression and GJIC were significantly blocked by the p38 inhibitor SB 202190. Furthermore, amitriptyline-induced Cx43 expression and GJIC were markedly reduced by transcription factor AP-1 inhibitors (curcumin and tanshinone IIA). The translocation of c-Fos from the cytosol and the nucleus of cortical astrocytes was increased by amitriptyline, and this response was dependent on p38 activity. These findings indicate a novel mechanism of action of amitriptyline through cortical astrocytes, and further suggest that targeting this mechanism could lead to the development of a new class of antidepressants.

Internal translation of the connexin 43 transcript.

BackgroundConnexin 43 (Cx43), the most widely expressed gap junction protein, is associated with a number of physiological and pathological conditions. Many functions of Cx43 have been shown to be independent of gap junction formation and only require the expression of Cx43 C-terminal fragments. Recent evidence demonstrated that naturally occurring C-terminal isoforms can be generated via internal translation.FindingsHere, we confirm that C-terminal domains of Cx43, particularly the major 20-kDa isoform, can be independently generated and regulated by internal translation of the same single GJA1 gene transcript that encodes full-length Cx43. Through direct RNA transfection experiments, we provide evidence that internal translation is not due to a bona fide cap-independent IRES-mediated mechanism, as upstream ribosomal scanning or translation is required. In addition to the mTOR pathway, we show for the first time, using both inhibitors and cells from knockout mice, that the Mnk1/2 pathway regulates the translation of the main 20-kDa isoform.ConclusionsInternal translation of the Cx43 transcript occurs but is not cap-independent and requires translation upstream of the internal start codon. In addition to the PI3K/AKT/mTOR pathway, the major 20-kDa isoform is regulated by the Mnk1/2 pathway. Our results have major implications for past and future studies describing gap junction-independent functions of Cx43 in cancer and other pathological conditions. This study provides further clues to the signalling pathways that regulate internal mRNA translation, an emerging mechanism that allows for increased protein diversity and functional complexity from a single mRNA transcript.

Reversal of Ischemic Cardiomyopathy with Sca-1+ Stem Cells Modified with Multiple Growth Factors

BackgroundWe hypothesized that bone marrow derived Sca-1+ stem cells (BM Sca-1+) transduced with multiple therapeutic cytokines with diverse effects will induce faster angiomyogenic differentiation in the infarcted myocardium.Methods and ResultsBM Sca-1+ were purified from transgenic male mice expressing GFP. Plasmids encoding for select quartet of growth factors, i.e., human IGF-1, VEGF, SDF-1α and HGF were prepared and used for genetic modification of Sca-1+ cells (GFSca-1+). Scramble transfected cells (ScSca-1+) were used as a control. RT-PCR and western blotting showed significantly higher expression of the growth factors in GFSca-1+. Besides the quartet of the therapeutic growth factors, PCR based growth factor array showed upregulation of multiple angiogenic and prosurvival factors such as Ang-1, Ang-2, MMP9, Cx43, BMP2, BMP5, FGF2, and NGF in GFSca-1+ (p<0.01 vs ScSca-1+). LDH and TUNEL assays showed enhanced survival of GFSca-1+ under lethal anoxia (p<0.01 vs ScSca-1+). MTS assay showed significant increased cell proliferation in GFSca-1+ (p<0.05 vs ScSca-1+). For in vivo study, female mice were grouped to receive the intramyocardial injection of 15 μl DMEM without cells (group-1) or containing 2.5×105 ScSca-1+ (group-2) or GFSca-1+ (group-3) immediately after coronary artery ligation. As indicated by Sry gene, a higher survival of GFSca-1+ in group-3 on day4 (2.3 fold higher vs group-2) was observed with massive mobilization of stem and progenitor cells (cKit+, Mdr1+, Cxcr4+ cells). Heart tissue sections immunostained for actinin and Cx43 at 4 weeks post engraftment showed extensive myofiber formation and expression of gap junctions. Immunostaining for vWF showed increased blood vessel density in both peri-infarct and infarct regions in group-3. Infarct size was attenuated and the global heart function was improved in group-3 as compared to group-2.ConclusionsAdministration of BM Sca-1+ transduced with multiple genes is a novel approach to treat infarcted heart for its regeneration.