Gap Junctional Intercellular Communication Research Articles

Inhibition of Gap Junction–Mediated Intercellular Communication by Poly(I:C) in Cultured Human Corneal Fibroblasts

ABSTRACT Purpose/Aim Corneal stromal fibroblasts are connected to each other via gap junctions, which contribute to maintenance of corneal homeostasis. Viral infection of the corneal stroma can result in inflammation and scarring. The effects of polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, on gap junctional intercellular communication (GJIC) in cultured human corneal fibroblasts (HCFs) were examined. Materials and Methods Cultured HCFs were exposed to poly(I:C) in the absence or presence of inhibitors of mitogen-activated protein kinase (MAPK) signaling or the antioxidant N-acetyl-L-cysteine (NAC). Expression of the gap junction protein connexin 43 (Cx43) was examined by immunoblot and immunofluorescence analyses. The level of Cx43 mRNA or microRNA-21 or −130a was determined by quantitative reverse transcription–polymerase chain reaction analysis. GJIC was measured with a dye coupling assay. The amount of malondialdehyde and the activity of superoxide dismutase (SOD) were measured with assay kits. Results Exposure of HCFs to poly(I:C) resulted in down-regulation of Cx43 expression and GJIC activity as well as in up-regulation of microRNA-21 expression. Poly(I:C) increased the amount of malondialdehyde and reduced the activity of SOD in the cells, and these effects were prevented by NAC. The inhibitory effects of poly(I:C) on both Cx43 expression and GJIC activity were attenuated by NAC and by c-Jun NH2-terminal kinase (JNK) inhibitor II. Conclusions Poly(I:C) inhibited Cx43 expression and GJIC in cultured HCFs, possibly as a result of the associated up-regulation of microRNA-21. Poly(I:C) also increased oxidative stress in these cells, and such stress together with signaling by the MAPK JNK was implicated in the effects of poly(I:C) on Cx43 expression and GJIC activity. Down-regulation of GJIC activity among corneal fibroblasts by double-stranded RNA may thus contribute to the disruption of stromal homeostasis during viral infection of the cornea.

A plant pan-genome immunity landscape

2543 BACKGROUND: The vascular endothelial growth factor (VEGF) is a mediator of angiogenesis and is associated with cancer progression and poor prognosis in patients with primary breast cancer. VEGF is a heparin-binding glycoprotein that is secreted as a homodimer of 45Kda. Most types of cells, but usually not endothelial cells themselves, secrete VEGF. VEGF, particularly VEGF-A, increases vascular permeability, vasodilatation in endothelial cells and can also stimulate cell migration and inhibit apoptosis. We previously reported that elderly women with advanced breast cancer have high levels of VEGF and that correction of anaemia in these patients results in a relevant decrease of VEGF’s plasmatic levels. AIMS: According to these outcomes we would now investigate if variation of VEGF may also affects time to progression’s disease and fatigue that is a frequent symptom in elderly people with cancer. METHODS: VEGF concentrations were analyzed by an enzyme-linked immunosorbent assay (ELISA) in 79 volunteers (mean age: 70 yrs) and 84 elderly breast cancer patients stage III in EDTA plasma specimens. A dosage of 400-500 IU/Kg/t.i.w. of rHuEPO s.c. was administered in order to reach at less 13-14g/dl Hb. Level of serum iron was also adjusted to 24±2 μmol/L if necessary. All patients were treated with liposomal doxorubicin (75 mg/sqm) and cyclophosphamide (600 mg/sqm) every 3 weeks for 6 months. Time-to-progression (TTP) of disease and Fatigue was evaluated with Kaplan-Meyer formula and FACT and FACIT questionary respectively. VEGF was quoted at baseline, after Hb adjustment (a), at the end of therapy (aa) and after 18 months follow-up (aaa). RESULTS: 52 pts (A group) showed: VEGF(baseline): 607±21 pg/ml - VEGF(a): 512±19 pg/ml - VEGF(aa): 319±18 pg/ml - VEGF(aaa): 185±10 pg/ml - Hb (baseline) Mean Values (MV) were: 8.7±2 g/dl. 32 pts (B group) showed: VEGF(baseline): 216±31 pg/ml - VEGF(a): 204±20 pg/ml - VEGF(aa): 185±18 pg/ml - VEGF(aaa): 107±9 pg/ml - Hb (baseline) Mean Values (MV) were: 10.4±1.5 g/dl. Volunteers: VEGF 94±17 pg/ml - Hb 12.6±1.7 g/dl. TTP (A group): 11±1.5 mths - TTP (B group): 14±1.5 mths. Fatigue’s evaluation was as following: 50±10 (A group baseline) 70±10 (A group after HB adjustment) - 80±10 (B group) - 90±10 (Volunteers). CONCLUSION: These findings support a role for hypoxia and anaemia induced VEGF in human breast cancer and also suggest that adjustment of low Hb levels may lead to better results in cancer management through the abrogation of VEGF induced functions. Scrutiny of GJIC (gap junction intercellular communication) activity in human breast cancer cell lines by the Scrape-Loading/Dye Transfer (SL/DT) method in basic condition and after treatment with a specific VEGF-Ab is ongoing in order to confirm these preliminary considerations.

A novel role of SIRT2 in regulating gap junction communications via connexin-43 in bovine cumulus-oocyte complexes.

SIRT2, the predominantly cytosolic sirtuin, plays important role in multiple biological processes, including metabolism, stress response, and aging. However, the function of SIRT2 in gap junction intercellular communications (GJICs) of cumulus-oocyte complexes (COCs) is not yet known. The purpose of the present study was to evaluate the effect and underlining mechanism of SIRT2 on GJICs in COCs. Here, we found that treatment with SIRT2 inhibitors (SirReal2 or TM) inhibited bovine oocyte nuclear maturation. Further analysis revealed that SIRT2 inactivation disturbed the GJICs of COCs during in vitro maturation. Correspondingly, both the Cx43 phosphorylation levels and MEK/MER signaling pathways were induced by SIRT2 inhibition. Importantly, SIRT2-mediated Cx43 phosphorylation was completely abolished by treatment with MEK1/2 inhibitor (Trametinib). Furthermore, treatment with SIRT2 inhibitors resulted in the high levels of MEK1/2 acetylation. Functionally, downregulating the MER/ERK pathways with inhibitors (Trametinib or SCH772984) could attenuate the closure of GJICs caused by SIRT2 inactivation in partly. In addition, inhibition of SIRT2 activity significantly decreased the membrane and zona pellucida localization of Cx43 by upregulating the levels of Cx43 acetylation. Taken together, these results demonstrated a novel role that SIRT2 regulates GJICs via modulating the phosphorylation and deacetylation of Cx43 in COCs.

Uremic toxin indoxyl sulfate suppresses myocardial Cx43 assembly and expression via JNK activation

Heart rhythm disturbances have been widely recognized as major triggers of cardiovascular (CV) mortality in chronic kidney disease (CKD) patients. Connexin 43 (Cx43)-composed gap junctions are essential in cardiomyocyte synchronization and may be involved in the pathological response to uremic toxins. Indoxyl sulfate (IS) is one of the most dominant uremic toxins that contribute to CKD-related cardiovascular diseases. In primary cultures of rat neonatal cardiomyocytes, we demonstrated that IS treatment decreased spontaneous contraction without impairing viability. In addition, there was disruption of gap junction intercellular communication (GJIC) between cardiomyocytes after 30 min of IS stimulation. IS caused time- and dose-dependent Cx43 redistribution, and the patterns of Cx43 immunostaining returned to baseline while IS stimulation was removed. Furthermore, IS exposure downregulated Cx43 protein and mRNA levels. Elevated JNK1 and JNK2 phosphorylation was further identified after IS exposure in both rat cardiomyocytes and H9c2 cells. The above changes as well as GJIC and Cx43 suppression were reversed by pretreatment with a JNK inhibitor (SP600125). Inhibition of p-JNK attenuated IS-mediated downward trends in Cx43 transcription and translation. In cardiac muscle from nephrectomy-induced CKD mice, an alteration in Cx43 level was identified at intercalated discs. Our findings disclosed that JNK activation might participate in the remodeling of gap junction and Cx43 expression by uremic toxin-IS both in vitro and in vivo.

Visualization of Protein-Lipid Interactions in Connexin-46/50 Intercellular Channels by Cryo-EM and MD-Simulation

Cell-to-cell communication by gap junctions is facilitated by a unique macromolecular architecture, where intercellular channels directly couple the plasma membranes of two neighboring cells. In each membrane, a “hemi-channel” is formed by the oligomerization of six individual subunits (called connexins). To gain insight toward how gap junctions interact with their local membrane environment, we used lipid nanodisc technology to incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid membrane system – closely mimicking a native cell-to-cell junction. Structural characterization of Cx46/50 lipid-embedded channels by single particle CryoEM revealed a drastic lipid-induced stabilization to the channel architecture, resulting in a 3D reconstruction at 2.1 Å resolution – providing an unprecedented level of detail for the class of protein. The subunit packing within each hemi-channel is buttressed by a bouquet of highly-stabilized lipid acyl-chains. Remarkably, the stabilizing effects to the lipid-bilayer extends well beyond the annular layer of lipids and effectively immobilizes the extra-cellular leaflets of the two connected membranes. In addition, over 150 water molecules are resolved within the channel architecture. Time-averaged densities of the lipids and water obtained by MD simulation display the same pattern of stabilization seen in the CryoEM reconstruction. MD analysis suggest lipid headgroups remain highly dynamic relative to the lipid acyl-chains, rationalizing the absence of headgroup densities in the 2.1 Å CryoEM density map. 3D heterogeneity analysis of the CryoEM data identified 4 distinct classes of lipid headgroup configurations, which were also detected by network analysis of extracellular lipids in MD-simulations. Lipid and water binding sites identified in Cx46/50 are remarkably conserved across the connexin family, suggesting these stabilizing features are key contributors to the structure and function of gap junction intercellular communication.

Gap Junctions in the Bone Marrow Lympho-Hematopoietic Stem Cell Niche, Leukemia Progression, and Chemoresistance.

The crosstalk between hematopoietic stem cells (HSC) and bone marrow (BM) microenvironment is critical for homeostasis and hematopoietic regeneration in response to blood formation emergencies after injury, and has been associated with leukemia transformation and progression. Intercellular signals by the BM stromal cells in the form of cell-bound or secreted factors, or by physical interaction, regulate HSC localization, maintenance, and differentiation within increasingly defined BM HSC niches. Gap junctions (GJ) are comprised of arrays of membrane embedded channels formed by connexin proteins, and control crucial signaling functions, including the transfer of ions, small metabolites, and organelles to adjacent cells which affect intracellular mechanisms of signaling and autophagy. This review will discuss the role of GJ in both normal and leukemic hematopoiesis, and highlight some of the most novel approaches that may improve the efficacy of cytotoxic drugs. Connexin GJ channels exert both cell-intrinsic and cell-extrinsic effects on HSC and BM stromal cells, involved in regenerative hematopoiesis after myelosuppression, and represent an alternative system of cell communication through a combination of electrical and metabolic coupling as well as organelle transfer in the HSC niche. GJ intercellular communication (GJIC) in the HSC niche improves cellular bioenergetics, and rejuvenates damaged recipient cells. Unfortunately, they can also support leukemia proliferation and survival by creating leukemic niches that provide GJIC dependent energy sources and facilitate chemoresistance and relapse. The emergence of new strategies to disrupt self-reinforcing malignant niches and intercellular organelle exchange in leukemic niches, while at the same time conserving normal hematopoietic GJIC function, could synergize the effect of chemotherapy drugs in eradicating minimal residual disease. An improved understanding of the molecular basis of connexin regulation in normal and leukemic hematopoiesis is warranted for the re-establishment of normal hematopoiesis after chemotherapy.

Improved multiparametric scrape loading-dye transfer assay for a simultaneous high-throughput analysis of gap junctional intercellular communication, cell density and viability

Gap junctional intercellular communication (GJIC) is a vital cellular process required for maintenance of tissue homeostasis. In vitro assessment of GJIC represents valuable phenotypic endpoint that could be effectively utilized as an integral component in modern toxicity testing, drug screening or biomedical in vitro research. However, currently available methods for quantifying GJIC with higher-throughputs typically require specialized equipment, proprietary software and/or genetically engineered cell models. To overcome these limitations, we present here an innovative adaptation of traditional, fluorescence microscopy-based scrape loading-dye transfer (SL-DT) assay, which has been optimized to simultaneously evaluate GJIC, cell density and viability. This multiparametric method was demonstrated to be suitable for various multiwell microplate formats, which facilitates an automatized image acquisition. The assay workflow is further assisted by an open source-based software tools for batch image processing, analysis and evaluation of GJIC, cell density and viability. Our results suggest that this approach provides a simple, fast, versatile and cost effective way for in vitro high-throughput assessment of GJIC and other related phenotypic cellular events, which could be included into in vitro screening and assessment of pharmacologically and toxicologically relevant compounds.

The Analysis of Gap Junctional Intercellular Communication Among Osteocytes in Chick Calvariae by Fluorescence Recovery After Photobleaching.

This chapter describes the use of fluorescence recovery after photobleaching (FRAP) for analyzing gap junctional intercellular communication (GJIC) among osteocytes in chick calvariae by confocal laser scanning microscope.

The roles of gap junctional intercellular communication in non-alcoholic steatohepatitis (NASH) and liver fibrosis

Non-alcoholic steatohepatitis (NASH) is a common risk factor for fibrosis, cirrhosis, and a predisposing factor for the development of hepatocellular carcinoma. Recently, incidence of NASH has increased due to an increase in metabolic syndrome. Connexin (Cx)32, a hepatocyte gap-junction protein, plays an important role in liver tissue homeostasis; Cx32 dominant-negative transgenic rat (Cx32ΔTg) has much decreased gap-junctional intercellular communication, and high susceptibility to carcinogens. We found for the first time that Cx32 has play suppressive roles in inflammation and fibrosis of NASH using Cx32ΔTg received methionine-choline deficient diet (MCDD). Elevation of reactive oxygen species (ROS) play important roles in progression of NASH and elimination of ROS by antioxidant luteolin inhibited NASH in the Cx32ΔTg-MCDD model. This model had histological changes similar to those of human NASH, but was not accompanied by the metabolic syndrome such as obesity and insulin resistance. Therefore, we further established an improved NASH model. Cx32ΔTg rats and wild-type rats were fed a high-fat diet (HFD) and dimethylnitrosamine to induce NASH with metabolic syndrome. The HFD and DMN increased body, liver, and visceral fat weights in both genotypes. Serum insulin level and HOMA-IR score in Cx32ΔTg rats were higher than those in wild-type rats. Elevation of serum hepatic enzymes (AST, ALT), inflammatory cytokine expressions, α-smooth muscle actin expression, progression of steatohepatitis and fibrosis were induced by HFD and dimethylnitrosamine especially in Cx32ΔTg rats. These results indicate Cx32 dysfunction promoted the development of NASH and fibrosis accompanied by metabolic syndrome through accumulation of oxidative stress.

The Role of Proteostasis in the Regulation of Cardiac Intercellular Communication.

Given the low mitotic activity of cardiomyocytes, the contractile unit of the heart, these cells strongly rely on efficient and highly regulated mechanisms of protein degradation to eliminate unwanted potentially toxic proteins. This is particularly important in the context of disease, where an impairment of protein quality control mechanisms underlies the onset and development of diverse cardiovascular maladies. One of the biological processes which is tightly regulated by proteolysis mechanisms is intercellular communication. The different types of cells that form the heart, including cardiomyocytes, endothelial cells, fibroblasts, and macrophages, can communicate directly, through gap junctions (GJ) or tunneling nanotubes (TNT), or at long distances, via extracellular vesicles (EV) or soluble factors.The direct communication between cardiomyocytes is vital to ensure the anisotropic propagation of the electrical impulse, which allows the heart to beat in a coordinated and synchronized manner, as a functional syncytium. The rapid and efficient propagation of the depolarization wave is mainly conducted by low resistance channels called GJ, formed by six subunits of a family of proteins named Cxs. Dysfunctional GJ intercellular communication, due to increased degradation and/or redistribution of connexin43 (Cx43), the main Cx present in the heart, has been associated with several cardiac disorders, such as myocardial ischemia, hypertrophy, arrhythmia, and heart failure. Besides electrical coupling, a fine-tuned exchange of information, namely proteins and microRNAs, conveyed by EV is important to ensure organ function and homeostasis. Disease-induced deregulation of EV-mediated communication between cardiac cells has been implicated in diverse processes such as inflammation, angiogenesis, and fibrosis. Therefore, a better understanding of the mechanisms whereby proteolysis modulates the cross talk between cardiac cells is of utmost importance to develop new strategies to tackle diseases caused by defects in intercellular communication.

Influence of Endogenous Cardiac Glycosides, Digoxin, and Marinobufagenin in the Physiology of Epithelial Cells.

Cardiac glycosides are a group of compounds widely known for their action in cardiac tissue, some of which have been found to be endogenously produced (ECG). We have previously studied the effect of ouabain, an endogenous cardiac glycoside, on the physiology of epithelial cells, and we have shown that in concentrations in the nanomolar range, it affects key properties of epithelial cells, such as tight junction, apical basolateral polarization, gap junctional intercellular communication (GJIC), and adherent junctions. In this work, we study the influence of digoxin and marinobufagenin, two other endogenously expressed cardiac glycosides, on GJIC as well as the degree of transepithelial tightness due to tight junction integrity (TJ). We evaluated GJIC by dye transfer assays and tight junction integrity by transepithelial electrical resistance (TER) measurements, as well as immunohistochemistry and western blot assays of expression of claudins 2 and 4. We found that both digoxin and marinobufagenin improve GJIC and significantly enhance the tightness of the tight junctions, as evaluated from TER measurements. Immunofluorescence assays show that both compounds promote enhanced basolateral localization of claudin-4 but not claudin 2, while densitometric analysis of western blot assays indicate a significantly increased expression of claudin 4. These changes, induced by digoxin and marinobufagenin on GJIC and TER, were not observed on MDCK-R, a modified MDCK cell line that has a genetically induced insensitive α1 subunit, indicating that Na-K-ATPase acts as a receptor mediating the actions of both ECG. Plus, the fact that the effect of both cardiac glycosides was suppressed by incubation with PP2, an inhibitor of c-Src kinase, PD98059, an inhibitor of mitogen extracellular kinase-1 and Y-27632, a selective inhibitor of ROCK, and a Rho-associated protein kinase, indicate altogether that the signaling pathways involved include c-Src and ERK1/2, as well as Rho-ROCK. These results widen and strengthen our general hypothesis that a very important physiological role of ECG is the control of the epithelial phenotype and the regulation of cell-cell contacts.

Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48-linked NADPH oxidase 4 polyubiquitination and degradation.

Nox4 is the major isoform of NADPH oxidase found in the kidney and contributes to the pathogenesis of diabetic nephropathy. However, the molecular mechanisms of increased Nox4 expression induced by hyperglycaemia remain to be elucidated. Here, the role of the connexin32-Nox4 signalling axis in diabetic nephropathy and its related mechanisms were investigated. Diabetes was induced in mice by low-dose streptozotocin (STZ) combined with a high-fat diet. Effects of connexin32 on Nox4 expression and on renal function and fibrosis in STZ-induced diabetic mice were investigated using adenovirus-overexpressing connexin32 and connexin32-deficient mice. Interactions between connexin32 and Nox4 were analysed by co-immunoprecipitation and immunofluorescence assays. Connexin32 was down-regulated in the kidneys of STZ-induced diabetic mice. Overexpression of connexin32 reduced expression of Nox4 and improved renal function and fibrosis in diabetic mice, whereas connexin32 deficiency had opposite effects. Down-regulation of fibronectin expression by connexin32 was not dependent on gap junctional intercellular communication involving connexin32. Connexin32 interacted with Nox4 and reduced the generation of hydrogen peroxide, leading to the down-regulation of fibronectin expression. Mechanistically, connexin32 decreased Nox4 expression by promoting its K48-linked polyubiquitination. Interestingly, Smurf1 overexpression inhibited K48-linked polyubiquitination of Nox4. Furthermore, connexin32 interacted with Smurf1 and inhibited its expression. Connexin32 ameliorated renal fibrosis in diabetic mice by promoting K48-linked Nox4 polyubiquitination and degradation via inhibition of Smurf1 expression. Targeting the connexin32-Nox4 signalling axis may contribute to the development of novel treatments for diabetic nephropathy.

CBMS-08 INVESTIGATION FOR NICOTINIC EFFECTS ON STEM CELL’S PROPERTY IN HSV-TK/GCV GENE THERAPY

BACKGROUNDHerpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) system is one of feasible therapeutic strategies for defeating malignant gliomas. Stem cells with intrinsic tumor tropism are used for suicide gene vehicles, which make this therapy further realistic. Nicotine is known to affect cellular migration capacity in variety types of cells but whether nicotine impacts on stem cells’ migration capacity to gliomas is not scrutinized. In this research, we investigated nicotinic impact on stem cells’ properties including tumor tropism and gap junctional intercellular communication (GJIC), which is crucial to this therapeutic strategy.METHODSMouse induced pluripotent stem cell (iPSC)-derived neural stem cells (miPS-NSCs) and human dental pulp mesenchymal stem cells (hDPSCs) were used. Nicotine cytotoxicity for 24 hours was evaluated by MTT assay for stem cells and glioma cells; GS-9L and C6 (rat), GL261 (mouse), U251 and U87 (human). Tumor tropism to glioma-conditioned medium (CM) with or without non-toxic nicotine concentrations was assessed using Matrigel Invasion Chamber. Nicotine effect on GJIC was assessed with scrape loading/dye transfer assay (SL/DT assay) for co-culture of stem cells and glioma cells (stem cell/glioma cell) or parachute assay for glioma cells alone using high-content analysis.RESULTSMTT assay revealed 1 μM of nicotine, equivalent to serum nicotine concentration in habitual smoking, is the maximum safe concentration for stem cells and glioma cells. Tumor tropism (miPS-NSCs to GL261-CM, hDPSCs to U251- or U87-CM) and GJIC of co-culture of stem cells and glioma cells (miPS-NSC/GL261, hDPSC/U251) or glioma cells alone (GS-9L, C6, GL261 and U251) were not affected by 1 μM of nicotine.CONCLUSIONSPhysiological nicotine presence did not affect (1) stem cell’s tumor tropism to gliomas and (2) GJIC between stem cells and glioma cells or within glioma cells. HSV-tk/GCV therapy may retain its therapeutic efficacy against gliomas even under physiological nicotine concentrations.

Connexin 26 and Connexin 43 in Canine Mammary Carcinoma.

Incidence of canine mammary carcinoma is two times higher than the rate of human breast cancer. Mammary tumors are the most common type of cancer in intact female dogs and account for about half of all neoplasms in these dogs. Well-established models of breast cancer have shown that neoplastic cells often have a loss of intercellular communication, particularly gap junction proteins. Thus, the objective of this study is to explore the aspect of gap junction intercellular communication in canine mammary carcinoma, non-cancerous (CMEC) and cancerous (CMT12, CMT27, and CF41.Mg) cells, and patient-derived tumors. Both non-cancerous and cancerous mammary cells express connexins 26 and 43 using immunofluorescence; however, the level of expression is significantly different in quantitative analysis using western blot in which connexin 43 in both CMT12 and CMT27 is significantly decreased compared to CMEC. Furthermore, a decrease of gap junction capacity in CMT12 and CMT27 was observed compared to CMEC. Immunostaining of CMT27-xenograft tumors revealed positive Cx26 and negative Cx43 expression. Similarly, immunostaining of spontaneous canine mammary tumors revealed that Cx26 is present in all tumors while Cx43 is present in 25% of tumors. Overall, the study provides for the first time that a differential pattern of connexin expression exists between non-cancerous and cancerous mammary cells in dogs. This study will pave the path for further in vitro work of connexins in comparative canine models and possibly allow for novel therapeutics to be developed.

Inhibition of miR30a-3p by sulforaphane enhances gap junction intercellular communication in pancreatic cancer.

The therapy resistance of pancreatic cancer is associated with the loss of gap junction intercellular communication and connexin 43 expression. The broccoli isothiocyanate sulforaphane restored these features and therapy sensitivity. We investigated whether microRNA signaling is involved. Established cell lines and a patient tissue array (n = 96) were evaluated by miRNA and gene array, bioinformatics, expression studies, in situ hybridization and immunohistochemistry. Sulforaphane inhibited the expression of our top candidate miR30a-3p. Upon transfection of miR30a-3p inhibitors, the gemcitabine bystander effect, Cx43 expression and intercellular communication increased, whereas miR30a-3p mimics inhibited the luciferase activity of a Cx43 3'-UTR construct. miR30a-3p-overexpressing tumor xenografts had a decreased tumor volume and increased gemcitabine sensitivity. In patient tissues, a higher expression of miR30a-3p and a lower expression of Cx43 correlated with malignancy. These findings provide new knowledge and suggest sulforaphane as cotreatment for pancreatic cancer.

TGF-\u03b21 facilitates cell\u2013cell communication in osteocytes via connexin43- and pannexin1-dependent gap junctions

Connexins and pannexins are two families of channel forming proteins that are able to pass small molecules to achieve communication between cells. While connexins have been recognized to mediate gap junctional intercellular communication (GJIC), pannexins are far less known. Our previous study reported the potential role of TGF-β1 in mediating of connexins in osteocytes in vitro. Herein, we aimed to elucidate the influence of TGF-β1 on cell–cell communication based on gap junctions assembled by connexins and pannexins in vitro and ex vivo. We first showed that TGF-β1 positively affected the elongation of dendritic processes of osteocytes. Our data indicated that TGF-β1 increased expressions of connexin43 (Cx43) and pannexin1 (panx1), which are indispensable for hemichannel formation in gap junctions, in osteocytes in vitro and ex vivo. TGF-β1 enhanced gap junction formation and impacted cell–cell communication in living osteocytes, as indicated by the scrape loading and Lucifer yellow transfer assays. TGF-β1 enhanced the expressions of Cx43 and panx1 via activation of ERK1/2 and Smad3/4 signalling. The TGF-β1-restored expressions of Cx43 and panx1 in osteocytes in the presence of an ERK inhibitor, U0126, further demonstrated the direct participation of Smad3/4 signalling. TGF-β1 increased the accumulation of Smad3 in the nuclear region (immunofluorescence assay) and promoted the enrichment of Smad3 at the binding sites of the promoters of Gja1 (Cx43) and Panx1 (ChIP assay), thereby initiating the enhanced gene expression. These results provide a deep understanding of the molecular mechanisms involved in the modulation of cell–cell communication in osteocytes induced by TGF-β1.

Dynamic regulation of connexins in stem cell pluripotency.

Characterization of the pluripotent "ground state" has led to a greater understanding of species-specific stem cell differences and has imparted an appreciation of the pluripotency continuum that exists in stem cells in vitro. Pluripotent stem cells are functionally coupled via connexins that serve in gap junctional intercellular communication (GJIC) and here we report that the level of connexin expression in pluripotent stem cells depends upon the state in which stem cells exist in vitro. Human and mouse pluripotent stem cells stabilized in a developmentally primitive or "naïve" state exhibit significantly less connexin expression compared with stem cells which are "primed" for differentiation. This dynamic connexin expression pattern may be governed, in part, by differential regulation by pluripotency transcription factors expressed in each cell state. Species-specific differences do exist, however, with mouse stem cells expressing several additional connexin transcripts not found in human pluripotent stem cells. Moreover, pharmacological inhibition of GJIC shows limited impact on naïve human stem cell survival, self-renewal, and pluripotency but plays a more significant role in primed human pluripotent stem cells. However, CRISPR-Cas9 gene ablation of Cx43 in human and mouse primed and naïve pluripotent stem cells reveals that Cx43 is dispensable in each of these four pluripotent stem cell types.

Clinical Challenge and Call for Research on Keloid Disorder: Meeting Report from The 3rd International Keloid Research Foundation Symposium, Beijing 2019

Clinical Challenge and Call for Research on Keloid Disorder: Meeting Report from The 3rd International Keloid Research Foundation Symposium, Beijing 2019

Characterization of Adult Canine Kidney Epithelial Stem Cells That Give Rise to Dome-Forming Tubular Cells.

Dome formation can occur in cultured tubular epithelial cells originating from various tissues, including the mammary gland and the kidney. The isolation and characterization of normal kidney epithelial stem cells that give rise to dome-forming tubular cells have never been reported. We attempted to isolate and characterize canine kidney epithelial stem cells using a simple cell culture method that we have previously used to isolate other adult human stem cells. Dome-forming kidney epithelial cells were derived from dissociated adult canine kidney tissues that were cultured in a modified keratinocyte serum-free medium supplemented with N-acetyl-l-cysteine, l-ascorbic acid 2-phosphate, nicotinamide, and fetal bovine serum. These cells exhibited high self-renewal capacity in long-term culture (growth for >13 months and 30 cumulative population doublings) and exhibited characteristics of stem cells, including (1) deficiency in gap junctional intercellular communication, (2) anchorage-independent growth, (3) expression of stem cell markers octamer-binding transcription factor 4 and SRY (sex determining region Y)-box 2, (4) expression of cell surface markers CD24 and CD133, and (5) multipotent differentiation into osteoblasts, adipocytes, chondrocytes, and dome-forming tubular cells. Most of these characteristics are shared by the well-known canine renal tubule-derived immortalized Madin-Darby Canine Kidney cell line. Furthermore, the putative canine kidney stem cells developed in this study formed budding tubule-like organoids on Matrigel and required high cell density (>4,000 cells/cm2) for sustained growth and confluency for dome formation. The signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitor, AG490, inhibited colony-forming efficiency and dome formation, whereas lipopolysaccharide, an activator of STAT3, increased colony-forming efficiency in a dose-dependent manner. These results are consistent with the hypothesis that high cell density induces STAT3 expression, which promotes both stem cell self-renewal and differentiation into tubular cells. Our novel cell culture method should be useful for the future development of normal human kidney stem cells for clinical applications and for studying mechanisms of nephrotoxicity.

Asymmetric dimethylarginine aggravates blood-retinal barrier breakdown of diabetic retinopathy via inhibition of intercellular communication in retinal pericytes.

Blood-retinal barrier breakdown is the main pathological characteristics of diabetic retinopathy (DR). Asymmetric dimethylarginine (ADMA) was reported to be elevated in DR patients. In this study, we observed the dynamic profile of ADMA, retinal morphology and permeability of BRB at 2, 4 or 8week of diabetic rats induced by a single intraperitoneal injection of streptozocin (60mg/kg) and in cultured rat retinal pericytes pretreated with D-glucose (30mM) for 1, 3, 5 and 7days or ADMA (3, 10, 30 μM) for 24, 48 and 72h, trying to explore the effects of ADMA on blood-retinal barrier in DR. Gap junction intercellular communication (GJIC) and the expression of blood-retinal barrier-specific component connexin 43 (Cx43) were examined in diabetic rats or cultured retinal pericytes to elucidate whether ADMA impacted blood-retinal barrier function via damaging Cx43-GJIC. The results showed that with increasing duration of diabetes, the ultrastructure of blood-retinal barrier of diabetic rats appeared cell junction damage, apoptosis of retinal pericytes and breakdown of barrier successively. The increases in retinal permeability, ADMA levels and Cx43 expression, and abnormal GJIC were observed in diabetic rats and retinal pericytes exposed to D-glucose (30mM). A glucose-like effect was seen using ADMA or another L-arginine analogue NG-monomethyl-L-arginine or dimethylarginine dimethylaminohydrolases (DDAHs) siRNA, implicating that ADMA aggravated the breakdown of blood-retinal barrier via damaging Cx43-GJIC.