Connexin 43 ; genetics ; Connexins ; genetics ; Heart Defects, Congenital ; genetics ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; genetics ; Humans ; Infant ; Infant, Newborn ; Mutation ; genetics ; Myocardium ; metabolism ; Transcription Factors ; genetics

OBJECTIVETo observe the influence of Cx26/Cx32 gap junction channel on the antineoplastic effect of etoposide in Hela cervical cancer cells.

METHODSFluorescence trace was used to assay the gap junction intercellular communication mediated by Cx26/Cx32 in Hela cells and its functional modulation by the pharmacological agents (oleamide, retinoid acid). A standard colony-forming assay was applied to determine the cell growth-inhibiting effect of etoposide in Hela cells with functional modulation of the gap junction. Hoechst 33258 staining was used to assess the changes in etoposide-induced apoptosis of Hela cells with altered gap junction functions.

RESULTSOleamide markedly decreased while retinoid acid obviously increased the gap junction function in Hela cells. Standard colony-forming assay showed that etoposide produced a lowered antiproliferative effect in Hela cells with reduced gap junction and an increased antiproliferative effect in cells with enhanced gap junction function. In cells with a reduced gap junction function, etoposide induced a lowered apoptosis rate, which increased obviously in cells with an enhanced gap junction function.

CONCLUSIONThe antineoplastic effect of etoposide is reduced in Hela cells with a decreased gap junction intercellular communication mediated by Cx26/Cx32 and is enhanced in cells with an increased gap junction intercellular communication.

Antineoplastic Agents, Phytogenic ; pharmacology ; Connexin 26 ; Connexins ; genetics ; metabolism ; physiology ; Etoposide ; pharmacology ; Gap Junctions ; physiology ; HeLa Cells ; Humans ; Transfection

Animals ; Cell Communication ; Cell Transformation, Neoplastic ; Connexins ; genetics ; metabolism ; Cytoplasm ; metabolism ; Gap Junctions ; chemistry ; classification ; metabolism ; physiology ; Gene Expression Regulation, Neoplastic ; Humans ; Mutation ; Neoplasms ; etiology ; metabolism ; pathology

Hearing impairment (HI) affects 1/1000 children and over 2% of the aged population. We have previously reported that mutations in the gene encoding gap junction protein connexin-31 (C×31) are associated with HI. The pathological mechanism of the disease mutations remains unknown. Here, we show that expression of C×31 in the mouse inner ear is developmentally regulated with a high level in adult inner hair cells and spiral ganglion neurons that are critical for the hearing process. In transfected cells, wild type C×31 protein (C×31wt) forms functional gap junction at cell-cell-contacts. In contrast, two HI-associated C×31 mutants, C×31R180X and C×31E183K resided primarily in the ER and Golgi-like intracellular punctate structures, respectively, and failed to mediate lucifer yellow transfer. Expression of C×31 mutants but not C×31wt leads to upregulation of and increased association with the ER chaperone BiP indicating ER stress induction. Together, the HI-associated C×31 mutants are impaired in trafficking, promote ER stress, and hence lose the ability to assemble functional gap junctions. The study reveals a potential pathological mechanism of HI-associated C×31 mutations.
Animals ; Connexins ; genetics ; Ear, Inner ; metabolism ; Endoplasmic Reticulum ; physiology ; Gap Junctions ; genetics ; metabolism ; physiology ; Golgi Apparatus ; genetics ; metabolism ; Hearing Loss ; genetics ; metabolism ; pathology ; Mice ; Mutation ; Neurons ; metabolism ; Protein Transport ; genetics ; Stress, Physiological

OBJECTIVE: To investigate the expressions of tumor suppressor gene CX26 mRNA and coding protein in laryngeal squamous cell carcinoma, and to explore the relationship between CX26 gene and the biological behaviors of laryngeal squamous cell carcinoma for understanding the tumorigenicity and development of laryngeal squamous cell carcinoma. METHOD: Laryngeal carcinoma tissues (studying group), which takeda from the center of tumors and laryngeal normal tissues (control group) takeda at the place of 1.0 cm out of the edge of the tumors, were took from 38 patients with laryngeal squamous cell carcinoma while they were in operation. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression level of CX26 mRNA, and immunohistochemical staining (frozen section) was used to detect the expression of CX26 protein in laryngeal carcinoma tissues and laryngeal normal tissues of 38 cases, respectively. RESULT: mRNA of CX26 gene was all positively expressed in laryngeal carcinoma tissues and laryngeal normal tissues of 38 cases by RT-PCR. However, CX26 mRNA was obviously down-regulated in laryngeal carcinoma tissues than that in laryngeal normal tissues (P < 0.05). Immunohistochemical staining showed CX26 protein was strong-positively expressed in laryngeal normal tissues in 34 cases (89.5%), while it was positively expressed in laryngeal carcinoma tissues in 18 cases (47.4%), and with the location alteration of CX26 protein in laryngeal carcinoma cells. There was significant difference between the expression rate of CX26 protein in laryngeal carcinoma tissues and in laryngeal normal tissues (P < 0.05). Meanwhile, the expression level of CX26 mRNA and the positive-expressed rate of CX26 protein of the laryngeal carcinoma tissues in the advanced stage patients group (III stage and IV stage) were significantly lower than these in the early stage patients group (I and II) (P < 0.05), and it was significantly lower in those who have a cervical lymph node metastasis than those without metastasis. (P < 0.05). Moreover, the expression level of CX26 mRNA and the positive-expressed rate of CX26 protein reduced along with the reduction of pathological differentiation, and there was significant difference among the well-differentiated group, moderately-differentiated group and poorly-differentiated group (P < 0.05). CONCLUSION CX26 gene may play an important role in the pathogenesis and development of laryngeal carcinoma and may be related to its prognosis.
Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Connexin 26 ; Connexins ; metabolism ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; RNA, Messenger ; genetics

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 quantitatively detect the expression of connexins (Cx) mRNA in the posterior nodal extension (PNE) of adult rat heart and understand the relationship between Cx expression and atrial ventricular nodal reentrant tachycardia (AVNRT).

METHODSPNE was separated from adult rat heart by means of laser microdissection (LCM), and the cells were also isolated from the atrioventricular node (AVN), sinoatrial node (SAN), Purkinje fiber (PF), right atrium (RA) and right ventricle (RV), to serve as the controls. The Cx mRNA level was detected in these cells with quantitative real-time PCR (QRT-PCR).

RESULTSThe cells were successfully isolated from the PNE and other regions of adult rat heart, where heterogeneous expression of the 3 Cx isoforms (Cx43, Cx45, and Cx40) were observed. Cx45 mRNA showed higher expression in the PNE than in the working myocardium, whereas Cx43 mRNA level was about 25 times higher (P<0.05) in the working myocardium and 18 times higher (P<0.05) in the PF than in the PNE. In the PF, Cx40 mRNA level was proximately 6.8 times (P<0.01) as much as that in the PNE. Cx expression in the PNE was, however, similar to that in the SAN and AVN.

CONCLUSIONCx mRNAs exhibit heterogeneous expression in the PNE to allow the formation of the slow pathway. In addition, Cx expression in the PNE is very different from that in the adjacent myocardium, resulting in conduction discontinuity at the cellular junction, where, on certain occasion, unidirectional block may occur to cause AVNRT.

Animals ; Atrioventricular Node ; cytology ; metabolism ; Connexin 43 ; genetics ; Connexins ; genetics ; Female ; Male ; Myocardium ; cytology ; metabolism ; Purkinje Fibers ; cytology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sinoatrial Node ; cytology ; metabolism

OBJECTIVETo screen and identify the proteins that interact with connexin 26 (CX26) and to analyze the expressions of these proteins in cochlea so as to explore the proteins that relate to the trafficking, assembly, localizing and gap junction functions of CX26.

METHODSThe whole coding region of GJB2 (CX26) gene was amplified from normal human genomic DNA by polymerase chain reaction (PCR) and then directionally subcloned into the vector pGBKT7 plasmid of the Match Maker Ga14 Two-Hybrid System 3 as a target to screen the interactive proteins of CX26 from the human fetal brain cDNA library by the yeast two hybrid technique. The false positive clones were discarded from the preys by repeated yeast two hybrid method between CX26 and everyone of the preys respectively. The DNAs of the insert of the identified positive clone were sequenced and BLAST analyzed against the GenBank. Lastly, the mRNA of the gene encoding the identified protein was analyzed in the murine inner ear by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe insert of one positive clone contained 867 bp with the former 525 bp being coding region. The DNA sequence and the open reading frame of the insert were identical to the 525 bp before the stop codes (including the stop codes) and the 238 bp after the stop codes of RTN4 gene which encoded Nogo protein. And the 174 amino acid residues encoded by the insert were those of the C-terminal of Nogo protein: Nogo-A, Nogo-B and Nogo-C. RTN4 mRNA expressed in the murine inner ear was confirmed by RT-PCR method.

CONCLUSIONThe C-terminal of Nogo protein interacts with CX26. Nogo protein expresses in the inner ear and may take part in the trafficking of CX26 or CX26 gap junction function.

Animals ; Base Sequence ; Connexin 26 ; Connexins ; genetics ; metabolism ; Ear, Inner ; metabolism ; Gene Expression ; Humans ; Mice ; Molecular Sequence Data ; Myelin Proteins ; genetics ; metabolism ; Nogo Proteins ; Protein Binding ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Two-Hybrid System Techniques

OBJECTIVETo investigate the relationship between intracellular Ca2+ concentration ([Ca2+]i) mediated by connexin 40/43( Cx40/43) of VSMC and endothelium-dependent vascular contractive response of superior mesenteric arteries (SMA) in hemorrhagic shock rats.

METHODSThird to fifth passage culture of vascular endothelial cells (VEC) and VSMC from SD rats were used as study subject, the changes in contractive response of SMA and VSMC against hypoxia were observed. The expression of Cx40/43 in SMA,VEC,VSMC were blocked by Cx40/43 ASODN, then the effect of Cx40/43 on contractive response of hypoxic SMA and [Ca2+]i of VSMC were observed.

RESULTSThe contractive responses of SMA and VSMC after hypoxia were first increased, then decreased. Hypoxia induced calcium overload in VSMC [(82 +/- 4)% in normal control group, (115 +/- 8)% in hypoxia group at 30 min, (133 +/- 13)% in hypoxia group at 2 h]. Cx40 ASODN increased [Ca2+]i in VSMC and contractive response of SMA towards myricetin, while that of Cx43 ASODN showed opposite tendency.

CONCLUSIONSCx40/43 can regulate the SMA endothelium-dependent vascular contractive response through [Ca2+]i of VSMC after hemorrhagic shock.

Animals ; Calcium ; metabolism ; Connexin 43 ; genetics ; pharmacology ; Connexins ; genetics ; pharmacology ; Endothelium, Vascular ; Female ; Hypoxia ; metabolism ; Male ; Mesenteric Artery, Superior ; drug effects ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; Oligoribonucleotides, Antisense ; Rats ; Rats, Sprague-Dawley ; Shock, Hemorrhagic ; metabolism ; Signal Transduction ; Vasoconstriction

Gap junction channels formed with connexins directly link to the cytoplasm of adjacent cells and have been implicated in intercellular signaling. Connexin 37 (Cx37) is expressed in the gas-exchange region of the lung. Recently, Cx37 has been reported to be involved in the pathogenesis of inflammatory disease. However, no data are available on the role of Cx37 in allergic airway inflammatory disease. In the present study, we used a murine model of ovalbumin (OVA)-induced allergic airway disease and primary murine epithelial cells to examine the change of Cx37 in allergic airway disease. These mice develop the following typical pathophysiological features of asthma: airway hyperresponsiveness, airway inflammation, and increased IL-4, IL-5, IL-13, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, eotaxin, and RANTES levels in lungs. Cx37 protein and mRNA expression were decreased in OVA-induced allergic airway disease. Immunoreactive Cx37 localized in epithelial layers around the bronchioles in control mice, which dramatically disappeared in allergen-induced asthmatic lungs. Moreover, the levels of Cx37 protein in lung tissues showed significantly negative correlations with airway inflammation, airway responsiveness, and levels of Th2 cytokines in lungs. These findings indicate that change of Cx37 may be associated with the asthma phenotype.
Airway Resistance ; Allergens/toxicity ; Animals ; Asthma/etiology/genetics/immunology/*metabolism ; Base Sequence ; Bronchoalveolar Lavage Fluid/cytology ; Cell Adhesion Molecules/metabolism ; Cells, Cultured ; Chemokines/metabolism ; Connexins/genetics/*metabolism ; Cytokines/metabolism ; DNA Primers/genetics ; Disease Models, Animal ; Epithelial Cells/metabolism ; Female ; Lung/immunology/metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Ovalbumin/immunology/toxicity ; RNA, Messenger/genetics/metabolism ; Trachea/metabolism