Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca 2+ -dependent gap junction intercellular communication / 中华医学杂志(英文版)

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.

A spatial model showing differences between juxtacrine and paracrine mutual oocyte-granulosa cells interactions.

The bidirectional communication between oocytes and granulosa cells are mediated by several factors via a local feedback loop(s). The current model was carried out to study the spatial mutual interaction of porcine denuded oocytes and granulosa cells either in direct contact (juxtacrine) or paracrine co-culture using transwell system. Transwell 0.4 µm polyester membrane inserts were used to permit oocytes-granulosa cells paracrine communication with a distance of 2 mm between them in co-culture. Oocytes were cultured with granulosa cells in a defined basic maturation medium for 44 h. In results, oocyte secreted factors (OSFs; GDF9 and BMP15) temporal expression showed progressive decrement by the end of culture in case of direct contact with granulosa cells while it was increased progressively in the paracrine co-culture groups. However, oocytes that were cultured in direct contact showed a significant increase in blastocyst development after parthenogenetic activation than the paracrine co-cultured ones (20% vs. 11.5%, respectively). By the end of culture, granulosa cell count in direct contact showed a significant decrease than the indirect co-culture group (1.2 × 105 cell/mL vs. 2.1 × 105 cell/mL, respectively). Steroids (P4 and E2) and steriodogenesis enzymes mRNA levels showed significant temporal alterations either after 22 h and 44 h of IVM in both juxtacrine and paracrine co-culture systems (P ≤ 0.05). CX43 was much more highly expressed in the granulosa of the direct contact group than the indirect co-culture group. These results indicate the difference in mutual communication between oocytes and granulosa cells that were cocultured either in direct contact (juxtacrine) or with a short distance (paracrine) and propose a new paradigm to study different ovarian follicular cells interaction.

A low-protein diet during pregnancy prevents modifications in intercellular communication proteins in rat islets

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O tabagismo está associado com a remodelação de junções comunicantes no coração de ratos: explicação do paradoxo dos fumantes? / Smoking is associated with remodeling of gap junction in the rat heart: smoker's paradox explanation?

FUNDAMENTO: Em estudo anterior, utilizando o modelo de ratos, a exposição à fumaça do cigarro durante 5 semanas aumentou a sobrevida após IAM, apesar da idade similar e tamanho do infarto entre fumantes e não fumantes, e da ausência de reperfusão. OBJETIVO: Dessa forma, o presente estudo teve como objetivo analisar os efeitos da exposição à fumaça do cigarro sobre a intensidade, distribuição ou fosforilação da conexina 43 no coração de ratos. MÉTODOS: Ratos Wistar, pesando 100 g, foram distribuídos aleatoriamente em 2 grupos: 1) Controle (n = 25); 2) Expostos à fumaça do cigarro (ETS), n = 23. Depois de 5 semanas, foram conduzidas análise morfométrica do ventrículo esquerdo, imuno-histoquímica e Western blot para conexina 43 (Cx43). RESULTADOS: A fração do volume de colágeno, as áreas transversais e o peso ventricular não foram estatisticamente diferentes entre os grupos controle e ETS. O grupo ETS apresentou uma coloração de menor intensidade da Cx43 em discos intercalados (Controle: 2,32 ± 0,19; ETS: 1,73 ± 0,18; p = 0,04). A distribuição da Cx43 em discos intercalados não diferiu entre os grupos (Controle: 3,73 ± 0,12; ETS: 3,20 ± 0,17; p = 0,18). Os ratos do grupo ETS mostraram um nível maior de forma desfosforilada da Cx43 (Controle: 0,45 ± 0,11; ETS: 0,90 ± 0,11; p = 0,03). Por outro lado, o Cx43 total não diferiu entre os grupos de controle e ETS (Controle: 0,75 ± 0,19; ETS: 0,93 ± 0,27; p = 0,58). CONCLUSÃO: A exposição à fumaça do cigarro resultou na remodelação das junções comunicantes cardíacas, caracterizada por alterações na quantidade e fosforilação da Cx43 em corações de ratos. Essa constatação pode explicar o paradoxo dos fumantes observado em alguns estudos.

BACKGROUND: In a previous study utilizing the rat model, exposure to tobacco smoke for 5 weeks increased survival after AMI, despite similar age and infarct size between the smokers and nonsmokers, and absence of reperfusion. OBJECTIVE: Thus, this study aimed to analyze the effects of exposure to tobacco smoke on intensity, distribution or phosphorylation of connexin 43 in the rat heart. METHODS: Wistar rats weighing 100 g were randomly allocated into 2 groups: 1) Control (n = 25); 2) Exposed to tobacco smoke (ETS), n = 23. After 5 weeks, left ventricular morphometric analysis, immunohisthochemistry and western blotting for connexin 43 (Cx43) were performed. RESULTS: Collagen volume fraction, cross-sectional areas, and ventricular weight were not statistically different between control and ETS. ETS showed lower stain intensity of Cx43 at intercalated disks (Control: 2.32 ± 0.19; ETS: 1.73 ± 0.18; p = 0.04). The distribution of CX43 at intercalated disks did not differ between the groups (Control: 3.73 ± 0.12; ETS: 3.20 ± 0.17; p = 0.18). ETS rats showed higher levels of dephosphorylated form of Cx43 (Control: 0.45 ± 0.11; ETS: 0.90 ± 0.11; p = 0.03). On the other hand, total Cx43 did not differ between control and ETS groups (Control: 0.75 ± 0.19; ETS: 0.93 ± 0.27; p = 0.58). CONCLUSION: Exposure to tobacco smoke resulted in cardiac gap junction remodeling, characterized by alterations in the quantity and phosphorylation of the Cx43, in rats hearts. This finding could explain the smoker's paradox observed in some studies.

Antimutagenic and anticarcinogenic effect of methanol extracts of Petasites japonicus Maxim leaves

The methanol extract from the leaves of Petasites japonicus Maxim (PJ) was studied for its (anti-)mutagenic effect with the SOS chromotest and reverse mutation assay. The (anti-)carcinogenic effects were evaluated by the cytotoxicity on human cancer line cells and by the function and the expression of gap junctions in rat liver epithelial cell. PJ extracts significantly decreased spontaneous beta-galactosidase activity and beta-galactosidase activity induced by a mutagen, ICR, in Salmonella (S.) typhimurium TA 1535/pSK 1002. All doses of the extract (0.08-100 mg/plate) decreased the reversion frequency induced by benzo (alpha)pyrene (BaP) in S. typhimurium TA 98. It decreased not only the spontaneous reversion frequency but also that induced by BaP in S. typhimurium TA 100. PJ extract showed greater cytotoxic effects on human stomach, colon and uterus cancer cells than on other cancer cell types and normal rat liver epithelial cells. Dye transfers though gap junctions were significantly increased by PJ extracts at concentrations greater than 200 microg/mL and the inhibition of dye transfer by 12-O-tetradecanoylphorobol-13-acetate (TPA) was obstructed in all concentrations of PJ. PJ significantly increased the numbers of gap junction protein connexin 43, and increased the protein expression decreased by TPA in a dose-dependent manner. Based on these findings, PJ is suggested to contain antimutagenic and anticarcionogenic compounds.

Gap junction reduction in cardiomyocytes following transforming growth factor-â treatment and Trypanosoma cruzi infection

Gap junction connexin-43 (Cx43) molecules are responsible for electrical impulse conduction in the heart and are affected by transforming growth factor-â (TGF-â). This cytokine increases during Trypanosoma cruzi infection, modulating fibrosis and the parasite cell cycle. We studied Cx43 expression in cardiomyocytes exposed or not to TGF-â T. cruzi, or SB-431542, an inhibitor of TGF-â receptor type I (ALK-5). Cx43 expression was also examined in hearts with dilated cardiopathy from chronic Chagas disease patients, in which TGF-â signalling had been shown previously to be highly activated. We demonstrated that TGF-â treatment induced disorganised gap junctions in non-infected cardiomyocytes, leading to a punctate, diffuse and non-uniform Cx43 staining. A similar pattern was detected in T. cruzi-infected cardiomyocytes concomitant with high TGF-â secretion. Both results were reversed if the cells were incubated with SB-431542. Similar tests were performed using human chronic chagasic patients and we confirmed a down-regulation of Cx43 expression, an altered distribution of plaques in the heart and a significant reduction in the number and length of Cx43 plaques, which correlated negatively with cardiomegaly. We conclude that elevated TGF-â levels during T. cruzi infection promote heart fibrosis and disorganise gap junctions, possibly contributing to abnormal impulse conduction and arrhythmia that characterise severe cardiopathy in Chagas disease.

Effects of Sinusoidal Electromagnetic Field on Structure and Function of Different Kinds of Cell Lines

This study investigated that whether a 2 mT, 60 Hz, sinusoidal electromagnetic field (EMF) alters the structure and function of cells. This research compared the effects of EMF on four kinds of cell lines: hFOB 1.19 (fetal osteoblast), T/G HA-VSMC (aortic vascular smooth muscle cell), RPMI 7666 (B lymphoblast), and HCN-2 (cortical neuronal cell). Over 14 days, cells were exposed to EMF for 1, 3, or 6 hours per day (hrs/d). The results pointed to a cell type-specific reaction to EMF exposure. In addition, the cellular responses were dependent on duration of EMF exposure. In the present study, cell proliferation was the trait most sensitive to EMF. EMF treatment promoted growth of hFOB 1.19 and HCN-2 compared with control cells at 7 and 14 days of incubation. When the exposure time was 3 hrs/d, EMF enhanced the proliferation of RPMI 7666 but inhibited that of T/G HA- VSMC. On the other hand, the effects of EMF on cell cycle distribution, cell differentiation, and actin distribution were unclear. Furthermore, we hardly found any correlation between EMF exposure and gap junctional intercellular communication in hFOB 1.19. This study revealed that EMF might serve as a potential tool for manipulating cell proliferation.

Effects of Sinusoidal Electromagnetic Field on Structure and Function of Different Kinds of Cell Lines

This study investigated that whether a 2 mT, 60 Hz, sinusoidal electromagnetic field (EMF) alters the structure and function of cells. This research compared the effects of EMF on four kinds of cell lines: hFOB 1.19 (fetal osteoblast), T/G HA-VSMC (aortic vascular smooth muscle cell), RPMI 7666 (B lymphoblast), and HCN-2 (cortical neuronal cell). Over 14 days, cells were exposed to EMF for 1, 3, or 6 hours per day (hrs/d). The results pointed to a cell type-specific reaction to EMF exposure. In addition, the cellular responses were dependent on duration of EMF exposure. In the present study, cell proliferation was the trait most sensitive to EMF. EMF treatment promoted growth of hFOB 1.19 and HCN-2 compared with control cells at 7 and 14 days of incubation. When the exposure time was 3 hrs/d, EMF enhanced the proliferation of RPMI 7666 but inhibited that of T/G HA- VSMC. On the other hand, the effects of EMF on cell cycle distribution, cell differentiation, and actin distribution were unclear. Furthermore, we hardly found any correlation between EMF exposure and gap junctional intercellular communication in hFOB 1.19. This study revealed that EMF might serve as a potential tool for manipulating cell proliferation.

Gap junctions, connexins and sudden death caused by coronary heart disease / 法医学杂志

Gap junctions construct hydrophilic trans-membrane channels which adjust the intercellular communication of chemistry and electricity. In the heart, individual cardiac myocytes are linked by gap junctions. These junctions form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all the myocardium, ensuring their synchronous contraction. In recent years, some researchers have found that connexins, the protein molecules of gap junction channels, are reduced in number or redistributed from intercalated disks (ID) to lateral cell borders in a variety of cardiac disease, especially in ischemic heart disease. The gap junction remodeling is considered to be arrhythmogenic. These findings will lead us to a new realm in the diagnostic of sudden death caused by coronary heart disease.

Immunohistochemical study of Cx43 dephosphorylation in human left ventricular myocardium suffered by acute ischemia / 法医学杂志

OBJECTIVE@#This study was performed to detect the phosphorylation state of Cx43 in human left ventricular myocardium among sudden deaths caused by acute myocardial ischemia (AMI) especially sudden coronary death (SCD) and control groups. And then evaluate the significance of these findings in diagnosing the early pathological changes of acute myocardial ischemia.@*METHODS@#Immunohistochemistry (IHC) SP techniques were adopted to detect the phosphorylation state of Cx43 in the left ventricular myocardium samples of 45 deceased, which classified as group I--SCD, group II & III (other two groups of AMI) and Group IV & V (two control groups, sudden death caused by lethal acute cranio-cerebral injury or pathologic intracranial hemorrhage). In addition, we selected anti-Pan-Cadherin (construction protein of adherent junctions on the intercalated disc) and PHA-E+L/Bio, to detect the integration of myocardial mechanical coupling and membranes (applying affinityhistochemistry, AHC) respectively.@*RESULTS@#(1) Phosphorylated Cx43 positive staining was almost invisible in Group I, II and III or scattered in sarcoplasm in few samples; but it was assembling at the IDs clearly in group IV and V. (2) Strongly positive staining of Pan-Cadherin could be observed at the IDs and (3) integrated myocardial membranes were found in all samples.@*CONCLUSION@#These findings suggested that compared with the control groups, the integration of myocardial mechanical coupling and membranes did not alter in AMI. But Cx43, the key protein of electrical coupling on myocardial gap junctions, occurred dephosphorylation remarkably in AMI. Thus applying IHC techniques to detect the Cx43 dephosphorylation in human left ventricular myocardium maybe useful to recognize the onset of arrhythmia in AMI, especially in SCD whose myocardium without apparent morphological changes.

Regulation of gap junctions by protein phosphorylation

Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism) and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various phsysiological tissue and cell functions as well as to be altered under pathological conditions. (AU)Gap junctions are constituted by intercellular channels and provide a pathway for transfer of ions and small molecules between adjacent cells of most tissues. The degree of intercellular coupling mediated by gap junctions depends on the number of gap junction channels and their activity may be a function of the state of phosphorylation of connexins, the structural subunit of gap junction channels. Protein phosphorylation has been proposed to control intercellular gap junctional communication at several steps from gene expression to protein degradation, including translational and post-translational modification of connexins (i.e., phosphorylation of the assembled channel acting as a gating mechanism) and assembly into and removal from the plasma membrane. Several connexins contain sites for phosphorylation for more than one protein kinase. These consensus sites vary between connexins and have been preferentially identified in the C-terminus. Changes in intercellular communication mediated by protein phosphorylation are believed to control various phsysiological tissue and cell functions as well as to be altered under pathological conditions.