BACKGROUND: Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. METHODS: Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca 2+ -related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. RESULTS: Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca 2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. CONCLUSIONS Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.
Animals ; Humans ; Mice ; Apoptosis ; Bone Marrow Cells ; Cell Communication ; Connexin 43/genetics* ; Gap Junctions/metabolism* ; Imatinib Mesylate/therapeutic use* ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology* ; Mesenchymal Stem Cells/metabolism* ; Tumor Microenvironment ; Calcium/metabolism*

BACKGROUND: Dysfunction of the gap junction channel protein connexin 43 (Cx43) contributes to myocardial ischemia/reperfusion (I/R)-induced ventricular arrhythmias. Cx43 can be regulated by small ubiquitin-like modifier (SUMO) modification. Protein inhibitor of activated STAT Y (PIASy) is an E3 SUMO ligase for its target proteins. However, whether Cx43 is a target protein of PIASy and whether Cx43 SUMOylation plays a role in I/R-induced arrhythmias are largely unknown. METHODS: Male Sprague-Dawley rats were infected with PIASy short hairpin ribonucleic acid (shRNA) using recombinant adeno-associated virus subtype 9 (rAAV9). Two weeks later, the rats were subjected to 45 min of left coronary artery occlusion followed by 2 h reperfusion. Electrocardiogram was recorded to assess arrhythmias. Rat ventricular tissues were collected for molecular biological measurements. RESULTS: Following 45 min of ischemia, QRS duration and QTc intervals statistically significantly increased, but these values decreased after transfecting PIASy shRNA. PIASy downregulation ameliorated ventricular arrhythmias induced by myocardial I/R, as evidenced by the decreased incidence of ventricular tachycardia and ventricular fibrillation, and reduced arrythmia score. In addition, myocardial I/R statistically significantly induced PIASy expression and Cx43 SUMOylation, accompanied by reduced Cx43 phosphorylation and plakophilin 2 (PKP2) expression. Moreover, PIASy downregulation remarkably reduced Cx43 SUMOylation, accompanied by increased Cx43 phosphorylation and PKP2 expression after I/R. CONCLUSION PIASy downregulation inhibited Cx43 SUMOylation and increased PKP2 expression, thereby improving ventricular arrhythmias in ischemic/reperfused rats heart.
Rats ; Male ; Animals ; Myocardial Reperfusion Injury/metabolism* ; Connexin 43/genetics* ; Sumoylation ; Down-Regulation ; Rats, Sprague-Dawley ; Arrhythmias, Cardiac/drug therapy* ; Myocardial Ischemia/metabolism* ; RNA, Small Interfering/metabolism*

Adult ; Connexin 43 ; genetics ; Erythrokeratodermia Variabilis ; genetics ; Female ; Heterozygote ; Humans ; Male ; Mutation ; genetics ; Mutation, Missense ; genetics ; Pedigree

OBJECTIVE: Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43 (Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay (FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively. RESULTS: Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with siRNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1 (Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with siRNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown. CONCLUSION Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1.
Caspase 1 ; genetics ; metabolism ; Connexin 43 ; genetics ; metabolism ; Connexins ; genetics ; metabolism ; Gene Expression Regulation ; radiation effects ; Human Umbilical Vein Endothelial Cells ; physiology ; radiation effects ; Humans ; Nerve Tissue Proteins ; genetics ; metabolism ; Pyroptosis ; X-Rays ; adverse effects

OBJECTIVETo explore the genetic basis for a patient with oculodentodigital dysplasia.

METHODSGenomic DNA was extracted from peripheral blood samples from the patient and his parents. Whole-exome sequencing was carried out for the trio family. Suspected mutation was verified by Sanger sequencing.

RESULTSA de novo c.412G>A mutation of the GJA1 gene was identified in the patient, which was validated by Sanger sequencing.

CONCLUSIONThe c.412G>A mutation of the GJA1 gene probably underlies the disease in the patient.

Adult ; Connexin 43 ; genetics ; Craniofacial Abnormalities ; genetics ; Exome ; Eye Abnormalities ; genetics ; Foot Deformities, Congenital ; genetics ; Humans ; Male ; Mutation ; Sequence Analysis, DNA ; Syndactyly ; genetics ; Tooth Abnormalities ; genetics

Gap junctions mediate direct communication between cells; however, toxicological cascade triggered by nonessential metals can abrogate cellular signaling mediated by gap junctions. Although cadmium (Cd) is known to induce apoptosis in organs and tissues, the mechanisms that underlie gap junction activity in Cd-induced apoptosis in BRL 3A rat liver cells has yet to be established. In this study, we showed that Cd treatment decreased the cell index (a measure of cellular electrical impedance) in BRL 3A cells. Mechanistically, we found that Cd exposure decreased expression of connexin 43 (Cx43), increased expression of p-Cx43 and elevated intracellular free Ca2+ concentration, corresponding to a decrease in gap junctional intercellular communication. Gap junction blockage pretreatment with 18β-glycyrrhizic acid (GA) promoted Cd-induced apoptosis, involving changes in expression of Bax, Bcl-2, caspase-3 and the mitochondrial transmembrane electrical potential (Δψm). Additionally, GA was found to enhance ERK and p38 activation during Cd-induced activation of mitogen-activated protein kinases, but had no significant effect on JNK activation. Our results indicated the apoptosis-related proteins and the ERK and p38 signaling pathways may participate in gap junction blockage promoting Cd-induced apoptosis in BRL 3A cells.
Animals ; Apoptosis/*drug effects ; Cadmium/*toxicity ; Calcium/metabolism ; Cell Communication/drug effects ; Connexin 43/genetics ; Enzyme Activation/drug effects ; Gap Junctions/*drug effects ; Gene Expression Regulation/drug effects ; Hepatocytes/cytology/*drug effects ; Rats ; Signal Transduction/drug effects

OBJECTIVETo investigate the effects of the quinoline derivative PQ1 combined with cisplatin on the proliferation and gap junction communication of prostate cancer PC3 cells.

METHODSWe cultured in vitro prostate cancer PC3 cells and divided them into DMSO blank control, cisplatin control, and cisplatin (10 mg/ml) plus PQ1 (1, 2, 5, 10, and 15 μmol/L) groups. We measured the proliferation of the prostate cancer PC3 cells, determined the expressions of the connexin 43 (Cx43) mRNA and protein by RT-PCR and Western blot, and compared the indexes among different groups.

RESULTSCisplatin combined with PQl at 1 - 10 μmol/L significantly inhibited the proliferation of the PC3 cells and the inhibition rate rose in a concentration- and time-dependent manner, from (48.72 ± 0.98)% vs (50.33 ± 0.62)% at 0 μmol/L to (77.38 ± 1.12)% vs (83.50 ± 1.05)% at 15 μmol/L at 24 and 48 hours (P < 0.05). Compared with the cisplatin control, cisplatin combined with PQ1 at 1, 2, 5, 10, and 15 μmol/L increased the expression of Cx43 mRNA from 0.379 ± 0.113 to 0.669 ± 0.031, 0.831 ± 0. 127, 0.769 ± 0.100, 0.532 ± 0.086, and 0.475 ± 0.134, respectively (P < 0.05), and cisplatin combined with PQ1 at 1, 2, 5, and 10 μmol/L elevated that of Cx43 protein from 0.138 ± 0.146 to 0.263 ± 0.111, 0.306 ± 0.152, 0.415 ± 0.280, and 0.643 ± 0.310, respectively (P < 0.05).

CONCLUSIONThe quinoline derivative PQ1 can promote the gap junction communication of prostate cancer PC3 cells and enhance the killing effect of cisplatin on PC3 cells by upregulating the expressions of Cx43 mRNA and protein.

Aminoquinolines ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Connexin 43 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Gap Junctions ; drug effects ; physiology ; Humans ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; physiopathology ; RNA, Messenger ; metabolism ; Time Factors

BACKGROUNDMicroRNA-206 (miR-206) and connexin 43 (Cx43) are related with the distant metastasis of breast cancer. It remains unclear whether the regulatory effect of miR-206 on Cx43 is involved in metastasis of breast cancer.

METHODSUsing quantitative real-time polymerase chain reaction and Western blot, the expressions of miR-206 and Cx43 were determined in breast cancer tissues, hepatic and pulmonary metastasis (PM), and cell lines (MCF-10A, MCF-7, and MDA-MB-231). MCF-7/MDA-M-231 cells were transfected with lentivirus-shRNA vectors to enhance/inhibit miR-206, and then Cx43 expression was observed. Cell counting kit-8 assay and Transwell method were used to detect their changes in proliferation, migration, and invasion activity. The mutant plasmids of Cx43-3' untranslated region (3'UTR) at position 478-484 and position 1609-1615 were constructed. Luciferase reporter assay was performed to observe the effects of miR-206 on luciferase expression of different mutant plasmids and to confirm the potential binding sites of Cx43.

RESULTSCx43 protein expression in hepatic and PM was significantly higher than that in the primary tumor, while no significant difference was showed in messenger RNA (mRNA) expression. MiR-206 mRNA expression in hepatic and PM was significantly lower than that in the primary tumor. Cx43 mRNA and protein levels, as well as cell proliferation, migration, and invasion capabilities, were all significantly improved in MDA-MB-231 cells after reducing miR-206 expression but decreased in MCF-7 cells after elevating miR-206 expression, which demonstrated a significantly negative correlation between miR-206 and Cx43 expression (P = 0.03). MiR-206 can drastically decrease Cx43 expression of MCF-7 cells but exerts no effects on Cx43 expression in 293 cells transfected with the Cx43 coding region but the lack of Cx43-3'UTR, suggesting that Cx43-3'UTR may be the key in Cx43 regulated by miR-206. Luciferase expression showed that the inhibition efficiency was reduced by 46.80% in position 478-484 mutant, 16.72% in position 1609-1615 mutant; the inhibition was totally disappeared in double mutant (P = 0.02).

CONCLUSIONSMiR-206 can regulate the expression of Cx43, the cytobiological activity, and the metastasis of breast cancer through binding to the two binding sites in Cx43-3'UTR: position 478-484 and position 1609-1615.

Breast Neoplasms ; pathology ; Cell Movement ; Cell Proliferation ; Connexin 43 ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; MCF-7 Cells ; MicroRNAs ; genetics ; physiology ; Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Messenger ; analysis

We studied the function of connexin 43 (Cx43) gene in Xiao Meishan swine bone marrow mesenchymal stem cells (BMSCs) resulting from overexpression. Cx43 eukaryotic expression vector (pEGFP-Cx43) was constructed and transfected into BMSCs by nucleofector, after detecting the transfection efficiency; the expression of Cx43 was verified by RT-PCR, immunofluorescence and western blotting. Furthermore, we detected its cell cycle and apoptosis through flow cytometry. Our results show that pEGFP-Cx43 plasmid was successfully constructed, and green fluorescence in pEGFP-Cx43 transfected BMSCs was highly expressed with 60% transfection efficiency. In transgenic Xiao Meishan swines BMSCs, the expression level of Cx43 mRNA and protein were up-regulated. Meanwhile, the ability of cell proliferation was significantly increased, and the apoptosis rate was significantly reduced. Taken together, Cx43 overexpression could promote the proliferation of Xiao Meishan swine's BMSCs and markedly reduce their apoptosis, which provides evidence for in vivo research.
Animals ; Animals, Genetically Modified ; Apoptosis ; Cell Proliferation ; Connexin 43 ; metabolism ; Flow Cytometry ; Genetic Vectors ; Mesenchymal Stromal Cells ; metabolism ; Plasmids ; Swine ; genetics ; Transfection

OBJECTIVETo explore action mechanism of electroacupuncture (EA) on treatment of Parkinson's disease (PD).

METHODSA total of 40 healthy male SD rats were randomly divided into a normal group, a sham operation group, a model group and an EA group, 10 rats in each group. PD rat model was duplicated by micro injection of 6-hydroxyl dopamine into right striatum of rats, and the rats in the sham operation group were treated with micro injection of 0. 9% NaCl. Rats in the normal group, model group and the sham operation group received no treatment; rats in the EA group were treated by EA at "Fengfu" (GV 16) and "Taichong" (LR 3) with continuous wave, 2 Hz in frequency, 1 mA in intensity for 30 min. The treatment was given once a day for total 2 weeks. Behavioral test was used to evaluate rotational behavior changes of PD rats. RT-PCR method was applied to detect the expression of GFAP (glial fiber acidic protein) mRNA and Cx43 (connexin 43) mRNA in the striatum.

RESULTSThe difference of rotational behavior was not significant before and after treatment in the model group (P>0. 05), while that in the EA group was significant (P<0. 01). The expression of GFAP mRNA and Cx43 mRNA in the model group was significantly higher than that in the normal group and sham operation group (all P<0. 01); after EA treatment, the expression of GFAP mRNA and Cx43 mRNA in the EA group was lower significantly than that in the model group (both P<0. 01).

CONCLUSIONThe action mechanism of EA for prevention and treatment of Parkinson' s disease may be associated with inhibiting the activation of astrocytes.

Acupuncture Points ; Animals ; Astrocytes ; Connexin 43 ; genetics ; metabolism ; Corpus Striatum ; metabolism ; Electroacupuncture ; Humans ; Male ; Nerve Tissue Proteins ; genetics ; metabolism ; Parkinson Disease ; genetics ; metabolism ; therapy ; Rats ; Rats, Sprague-Dawley