The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes.

Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes.

Mural granulosa cells support to maintain the viability of growing porcine oocytes and its developmental competence after insemination.

PURPOSE: To enhance the in vitro growth of porcine oocytes, we studied the effect of mural granulosa cells (MGCs) on the viability of oocytes attached to granulosa cells (oocyte-granulosa cell complexes, OGCs) that were obtained from early antral follicles. METHODS AND RESULTS: When OGCs were cultured with MGCs for 12 days, there were significant improvement (P < 0.05) in the robustness of gap junctional communication between the oocyte and the granulosa cells (82% vs. 59%), the survival rate of oocytes (57% vs. 39%), and the diameter of survived oocytes (118 µm vs. 112 µm). The rate of oocyte release of OGCs cultured with MGCs on the 12th day (1.9%) was significantly lower than that of OGCs cultured without MGCs (26%). Complete meiotic arrest was maintained in the group with MGCs (100%), while partial resumption of spontaneous meiosis was noticed in the absence of MGCs (10-19%). Furthermore, the presence of MGCs increased the oocyte maturation rate after maturation culture in both 12- and 14-day culture groups (P < 0.05, 85-88%) compared to OGCs cultured without MGCs (48-60%). MGCs also significantly improved the blastocyst formation rate (day 7) after ICSI (P < 0.05). CONCLUSIONS: The data of this study thus shows that the presence of MGCs during in vitro oocyte growth plays a crucial role in supporting the developmental competence of growing porcine oocytes attached to the granulosa cells via enhancement of their viability.

Connexin 43 enhances oxaliplatin cytotoxicity in colorectal cancer cell lines.

Oxaliplatin, a platinum-based chemotherapeutic agent, is an important first-line drug in the treatment of colorectal cancers, but drug resistance causes treatment failure. It has been reported that gap junctional communication can enhance the cytotoxicity of platinum drugs.  The gap junction formed of connexin proteins provides a direct pathway for electrical and metabolic cell-cell interaction. The voltage-dependent gating of gap junction allows small hydrophilic molecules and ions to permeate to adjacent cells. Connexin 43 is a diagnostic marker for cancer therapy and the predominant connexin isoform in many cell types. The purpose of this study was to investigate the role of connexin 43 in oxaliplatin activity by using colorectal cancer cell lines. LoVo and HCT116 cell lines were used for analysis. Connexin 43 expression was confirmed by western blot and immunocytochemistry. MTT, western blot, "Parachute" dye-coupling assays and reactive oxygen species measurement were used to detect cytotoxicity and the inhibition of connexin 43 expression induced by oxaliplatin. Results showed that connexin 43 enhanced oxaliplatin cytotoxicity through gap junctional communication function and high concentration of oxaliplatin inhibited connexin 43 expression to counteract its cytotoxicity. This study suggested that connexin 43 could be considered a molecular target of oxaliplatin activity in colorectal cancer.

Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions.

STUDY QUESTION: Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? SUMMARY ANSWER: Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. WHAT IS KNOWN ALREADY: Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. STUDY DESIGN, SIZE, DURATION: This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators; 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. MAIN RESULTS AND THE ROLE OF CHANCE: Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P < 0.05), regardless of pre-IVM duration. The final blastocyst yields (proportion of blastocysts/immature oocyte) were 26.3% for the control, compared with 39.2, 35.2 and 34.2%, for the 2, 4 and 6 h pre-IVM FSK + IBMX treatments, respectively. In contrast to standard IVM (control), pre-IVM with cAMP modulators maintained open gap junctions between cumulus cells and oocytes for the duration (6 h) of pre-IVM examined, and persisted for a further 8 h in the IVM phase. Cyclic AMP-modulated pre-IVM increased intra-oocyte GSH levels at the completion of both pre-IVM and IVM, in a pre-IVM duration-dependent manner (P < 0.05), which was ablated when GJC was blocked using CBX (P < 0.05). By 4 h of pre-IVM treatment with cAMP modulators, oocyte H2O2 levels were reduced compared the control (P < 0.05), although this beneficial effect was lost when oocytes were co-treated with BSO. Inhibiting glutathione synthesis with BSO during pre-IVM ablated any positive benefits of cAMP-mediated pre-IVM on oocyte developmental competence (P < 0.01). LIMITATIONS, REASONS FOR CAUTION: It is unclear if the improvement in oocyte antioxidant defence and developmental competence reported here is due to direct transfer of total and/or reduced glutathione from cumulus cells to the oocyte via gap junctions, or whether a GSH synthesis signal and/or amino acid substrates are supplied to the oocyte via gap junctions. Embryo transfer experiments are required to determine if the cAMP-mediated improvement in blastocyst rates leads to improved live birth rates. WIDER IMPLICATIONS OF THE FINDINGS: IVM offers significant benefits to infertile and cancer patients and has the potential to significantly alter ART practice, if IVM efficiency in embryo production could be improved closer to that of conventional IVF (using ovarian hyperstimulation). Pre-IVM with cAMP modulators is a simple and reliable means to improve IVM outcomes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants and fellowships from the National Health and Medical Research Council of Australia (1007551, 627007, 1008137, 1023210) and by scholarships from the Chinese Scholarship Council (CSC) awarded to H.J.L. and the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad awarded to S.S. The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. We acknowledge partial support from the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Saffron and natural carotenoids: Biochemical activities and anti-tumor effects.

Saffron, a spice derived from the flower of Crocus sativus, is rich in carotenoids. Two main natural carotenoids of saffron, crocin and crocetin, are responsible for its color. Preclinical studies have shown that dietary intake of some carotenoids have potent anti-tumor effects both in vitro and in vivo, suggesting their potential preventive and/or therapeutic roles in several tissues. The reports represent that the use of carotenoids without the potential for conversion to vitamin A may provide further protection and avoid toxicity. The mechanisms underlying cancer chemo-preventive activities of carotenoids include modulation of carcinogen metabolism, regulation of cell growth and cell cycle progression, inhibition of cell proliferation, anti-oxidant activity, immune modulation, enhancement of cell differentiation, stimulation of cell-to-cell gap junction communication, apoptosis and retinoid-dependent signaling. Taken together, different hypotheses for the antitumor actions of saffron and its components have been proposed such as a) the inhibitory effect on cellular DNA and RNA synthesis, but not on protein synthesis; b) the inhibitory effect on free radical chain reactions; c) the metabolic conversion of naturally occurring carotenoids to retinoids; d) the interaction of carotenoids with topoisomerase II, an enzyme involved in cellular DNA-protein interaction. Furthermore, the immunomodulatory activity of saffron was studied on driving toward Th1 and Th2 limbs of the immune system. In this mini-review, we briefly describe biochemical and immunological activities and chemo-preventive properties of saffron and natural carotenoids as an anticancer drug.

Bioelectrical model of head-tail patterning based on cell ion channels and intercellular gap junctions.

Robust control of anterior-posterior axial patterning during regeneration is mediated by bioelectric signaling. However, a number of systems-level properties of bioelectrochemical circuits, including stochastic outcomes such as seen in permanently de-stabilized "cryptic" flatworms, are not completely understood. We present a bioelectrical model for head-tail patterning that combines single-cell characteristics such as membrane ion channels with multicellular community effects via voltage-gated gap junctions. It complements the biochemically-focused models by describing the effects of intercellular electrochemical coupling, cutting plane, and gap junction blocking of the multicellular ensemble. We provide qualitative insights into recent experiments concerning planarian anterior/posterior polarity by showing that: (i) bioelectrical signals can help separated cell domains to know their relative position after injury and contribute to the transitions between the abnormal double-head state and the normal head-tail state; (ii) the bioelectrical phase-space of the system shows a bi-stability region that can be interpreted as the cryptic system state; and (iii) context-dependent responses are obtained depending on the cutting plane position, the initial bioelectrical state of the multicellular system, and the intercellular connectivity. The model reveals how simple bioelectric circuits can exhibit complex tissue-level patterning and suggests strategies for regenerative control in vivo and in synthetic biology contexts.

Connexin 43 enhances paclitaxel cytotoxicity in colorectal cancer cell lines.

Colorectal cancer has a relatively low sensitivity to paclitaxel. The purpose of this study was to investigate the role of connexin 43 (Cx43), which is a structural component of gap junctional communication (GJC), in paclitaxel cytotoxicity in colorectal cancer cells. Three colorectal cancer cell lines (HCT106, HCT116 and LoVo) were transfected with Cx43 and used to examine paclitaxel cytotoxicity. A western blot assay was used to confirm Cx43 expression in transfected cell lines as well as the expression of several proteins that are associated with paclitaxel cytotoxicity. A parachute dye-coupling assay was used to measure GJC function. An MTT assay was used to analyze the viability of paclitaxel-treated cells. Cx43 expression level and GJC function were significantly upregulated by the transfection (P<0.05). The viability of transfected cells was significantly inhibited compared with that of untransfected cells when treated with paclitaxel (20 or 80 nM) at high culture density but not at low culture density (P<0.05). Cx43 transfection significantly increased the mitotic arrest, tubulin polymerization and apoptosis effects of paclitaxel (P<0.05). It was also found that paclitaxel had an inhibitory effect on GJC function after 12 h of treatment in LoVo cells (P<0.05). These results indicate that Cx43 may serve as a target of paclitaxel chemotherapy for colorectal cancer.

Gap junctional intercellular communication dysfunction mediates the cognitive impairment induced by cerebral ischemia-reperfusion injury: PI3K/Akt pathway involved.

PURPOSE: Cerebral ischemia/reperfusion (I/R) injury causes hippocampal apoptosis and cognitive impairment, and the dysfunction of gap junction intercellular communication (GJIC) may contribute to the cognitive impairment. We aim to examine the impact of cerebral I/R injury on cognitive impairment, the role of GJIC dysfunction in the rat hippocampus and the involvement of the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) pathway. METHODS: Rats were subjected to a cerebral I/R procedure and underwent cognitive assessment with the novel object recognition and Morris Water Maze tasks. The distance of Lucifer Yellow dye transfer and the Cx43 protein were examined to measure GJIC. Neural apoptosis was assessed with the terminal deoxynucleotide-transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. After rats received inhibitors of the PI3K/Akt pathway, GJIC and cognitive ability were measured again. RESULTS: GJIC promotion by ZP123 significantly reversed cognitive impairment and hippocampal apoptosis induced by cerebral I/R, while the inhibition of GJIC by octanol significantly facilitated cognitive impairment and hippocampal apoptosis. The phosphorylation of Akt was enhanced by cerebral I/R and octanol but inhibited by ZP123. The inhibition of the PI3K/Akt pathway significantly suppressed GJIC and cognitive impairment. CONCLUSION: The PI3K/Akt pathway is involved in cognitive impairment caused by gap junctional communication dysfunction in the rat hippocampus after ischemia-reperfusion injury.

Interactions between thromboxane A2, thromboxane/prostaglandin (TP) receptors, and endothelium-derived hyperpolarization.

Endothelium-dependent smooth muscle hyperpolarization (EDH) increasingly predominates over endothelium-derived nitric oxide (NO) as a participant in vasodilation as vessel size decreases. Its underlying nature is highly variable between vessel types, species, disease states, and exact experimental conditions, and is variably mediated by one or more transferable endothelium-derived hyperpolarizing factors and/or the electrotonic spread of endothelial hyperpolarization into the media via gap junctions. Although generally regarded (and studied) as a mechanism that is independent of NO and prostanoids, evidence has emerged that the endothelium-derived contracting factor and prostanoid thromboxane A2 can modulate several signalling components central to EDH, and therefore potentially curtail vasodilation through mechanisms that are distinct from those putatively involved in direct smooth muscle contraction. Notably, vascular production of thromboxane A2 is elevated in a number of cardiovascular disease states that promote endothelial dysfunction. This review will therefore discuss the mechanisms through which thromboxane A2 interacts with and modulates EDH, and will also consider the implications of such cross-talk in vasodilator control in health and disease.

Cerebral ischemic injury is enhanced in a model of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a rare autosomal dominant disease that results in visible developmental anomalies of the limbs, face, eyes and teeth. Recently analysis of human connexin43 (Cx43) DNA sequences has revealed a number of different missense, duplication and frame shift mutations resulting in this phenotype. A mouse model of this disorder has been created with a missense point mutation of the glycine amino acid at position 60 to serine (G60S). Heterozygote +/G60S mice exhibit a similar ODDD phenotype as observed in humans. In addition to the malformations listed above, ODDD patients often have neurological findings. In the brain, Cx43 is highly expressed in astrocytes and has been shown to play a role in neuroprotection. We were interested in determining the effect of the +/G60S mutation following stroke. Four days after middle cerebral artery occlusion the volume of infarct was larger in mice with the +/G60S mutation. In astrocyte-neuron co-cultures, exposure to glutamate also resulted in greater cellular death in the +/G60S mutants. Protein levels of Cx43 in the mutant mouse were found to be reduced when compared to the normal tissue. Cx43 protein was observed as a continual line of small punctate aggregates in the plasma membrane with increased intracellular localization, which is distinct from the larger plaques seen in the normal mouse astrocytes. Functionally, primary +/G60S astrocytes exhibited reduced gap junctional coupling and increased hemichannel activity, which may underlie the mechanism of increased damage during stroke. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Connexin26-mediated transfer of laterality cues in Xenopus.

A cilia-driven leftward flow of extracellular fluid breaks bilateral symmetry in the dorsal midline of the neurula stage vertebrate embryo. The left-specific Nodal signaling cascade in the lateral plate mesoderm (LPM) is key to asymmetric morphogenesis and placement of organs during subsequent development. The nature of the initial asymmetric cue(s) as well as the transfer of information from the midline to the left side has remained elusive. Gap junctional communication has been previously involved in Xenopus left-right (LR) development, however a function at cleavage stages was inferred from inhibitor experiments. Here we show by heptanol-mediated block of connexin function that flow stages during neurulation represent the critical time window. Flow in Xenopus occurs at the gastrocoel roof plate (GRP), a ciliated sheath of cells of mesodermal fate transiently positioned within the dorsal epithelial lining of the forming archenteron. We reasoned that endodermal cells immediately adjacent to the GRP are important for transfer of asymmetry. A systematic screen identified two connexin genes, Cx26 and Cx32, which were co-expressed in these lateral endodermal cells. Gain- and loss-of-function experiments pinpointed Cx26 as the critical connexin for LR development, while Cx32 had no effect on laterality. Importantly, GRP morphology, ciliation and flow were not affected in Cx26 morphants. Our results demonstrate a decisive role of Cx26 in the transfer of laterality cues from the GRP to the left LPM, providing a novel access to the identification of the initial asymmetric signal generated by flow.