Differential expression of connexin 43 in mouse mammary cells

In this study we have employed suppressive subtractive hybridization (SSH) analysis to investigate differential gene expression in primary mouse mammary epithelial cells (PMMEC) cultured under mildly apoptotic/quiescent and differentiating conditions. Among a small group of genes whose expression was differentially regulated was connexin 43. In vitro, connexin 43 mRNA and protein were detectable in PMMEC cultured under proliferative or mildly apoptotic conditions. The level of connexin 43 mRNA expression in vivo was also investigated. High levels of expression were found to be associated with the periods of greatest glandular plasticity (pubertal expansion of the mammary tree, early pregnancy and during early involution). Thus, terminally differentiated cells in vivo and in vitro did not express connexin 43 mRNA suggesting that connexin 43 expression, and perhaps facilitated gap junction communication, is associated with undifferentiated progenitor cell populations.     

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.     

Effect of progesterone and oestradiol on expression of connexin43 in cultured human myometrium cells

In human myometrium, the formation of gap junctions at various stages of labour and in correlation with the concentration of progesterone and oestradiol in maternal blood was described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated myometrial tissue to progesterone and oestradiol on the number of gap junction plaques in human myometrium cells in vitro. The presence of gap junctions was evaluated by immunocytochemistry with antibodies against gap junction protein, connexin43 (Cx43). Human myometrial cells were isolated from biopsies obtained from term pregnant women who had an elective caesarean operation in the 37th or 40th week of pregnancy. The dispersed myometrial cells that were obtained by limited enzymatic digestion of the myometrial samples were maintained in monolayer culture for 1, 3 and 6 days in the presence of medium that contained foetal bovine serum and the steroids at different concentration. In primary culture, as well as after several passages, the characteristics of these cells were morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue. The obtained results showed that the cells in culture responded synchronously to the increased concentrations of oestradiol/progesterone mixtures. The number of gap junctions increased significantly on days 1, 3 and 6 in culture and showed positive correlation (p < 0.05) with the cell number when the concentration of oestradiol was raised to 1 microgram/mL in the progesterone ratio (1.0 microgram/0.5 microgram/mL). No significant correlation, however, in connexin43 gap junction number versus cell number was observed between the six experimental groups treated with progesterone only.     

Short-term pacing in the mouse alters cardiac expression of connexin43

Background

Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time.

Results

The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10–15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias.

Conclusion

Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease.     

Relationship of connexin43 expression to phenotypic modulation in cultured human aortic smooth muscle cells

Transition of arterial smooth muscle cells from the contractile to the synthetic phenotype in vivo is associated with up-regulation of the gap-junctional protein, connexin43 (Cx43). However, the role of increased Cx43 expression in relation to the characteristic features of the synthetic phenotype – altered growth, differentiation or synthetic activity – has not previously been defined. In the present study, growth was induced in cultured human aortic smooth muscle cells by treatment with thrombin and with PDGF-bb; growth arrest was induced by serum deprivation and contact inhibition. Alterations in Cx43 expression and gap-junctional communication were analyzed in relation to expression of markers for contractile differentiation and extracellular matrix synthesis. Treatment with thrombin, but not PDGF-bb, led to up-regulation of Cx43 gap junctions, increased synthetic activity yet also enhanced contractile differentiation. Inhibition of growth by deprivation of serum growth factors in sub-confluent cultures had no effect on Cx43 expression or contractile differentiation. Growth arrest by contact inhibition led to progressive reduction in Cx43 expression, in parallel with progressive increase in expression of differentiation markers but no alteration in synthetic activity. Of a range of stimuli examined, only thrombin had the combined effect of increasing Cx43 gap-junction communication, growth and synthesis, yet it also enhanced contractile differentiation. Down-regulation of Cx43 and improved contractile differentiation occurred only when growth arrest was induced through the contact–inhibition pathway, though, in this instance, synthesis remained undiminished. We conclude that Cx43 levels, though having common correlates, are not exclusively linked to the cell phenotype or the state of growth.     

Inhibition of connexin 43 expression and function in cultured rat dental pulp cells by antisense oligonucleotide

Connexins are gap-junction proteins forming hexameric structures in the plasma membranes of adjacent cells, thereby creating intercellular channels. Connexin 43 (CX43) is expressed in pulp tissue. However, its function in dental pulp tissue has yet to be fully investigated. We have employed antisense oligonucleotides (AS) against rat CX43 to study the role of CX43 in dental pulp cells. Cultured dental pulp cells were treated with AS or sense (S) oligonucleotides. The number of cells in the AS-treated groups was approximately 1.3-fold that in the S-treated controls. Growth rates were significantly different between the AS- and S-treated groups at 48 h (P < 0.01). An alkaline phosphatase assay revealed that AS-treated pulp cells dramatically decreased at 48 h after AS incorporation, whereas S-treated pulp cells showed no marked changes. Western blot analysis revealed that heat-shock protein 25 was highly expressed in S-treated cells but was only weakly expressed in AS-treated cells at 48 h. Furthermore, AS-treated cells highly expressed CX45, whereas S-treated cells exhibited high expression of CX32. These results suggest that CX43 is involved in cell growth, mineralization, and differentiation to odontoblasts in rat pulp cells, and that CX43 plays the opposite role to that of CX45.     

Modulation of connexin43 alters expression of osteoblastic differentiation markers

Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43^–). hFOB/Cx43^– cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43^– cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43^– cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor 1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43^–. Osteopontin mRNA levels were increased in hFOB/Cx43^– relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43^– and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells. gap junction communication; alkaline phosphatase activity; osteopontin; osteocalcin; hFOB 1.19     

Inverse relationship between connexin43 and desmin expression in cultured porcine aortic smooth muscle cells.

Our previous work has shown that in vascular tissues the elastic medial regions express high levels of the gap junctional protein, connexin43, but low levels of desmin, while the muscular medial regions express low levels of connexin43 but high levels of desmin. It is uncertain, however, whether this regional difference at the tissue level extends down to the level of the individual cell, or reflects an averaged relationship of groups of cells of different connexin43 and desmin expression. The present study has addressed this question using cultured porcine aortic smooth muscle cells. Immunoconfocal microscopic analysis of single-labeled cells showed that while smooth muscle alpha-actin, calponin and vimentin were positively labeled in the majority of medial smooth muscle cells both in intact porcine aorta and corresponding cultured cells, desmin and connexin43 labeling was highly heterogeneous. In the cultured cells, 0.3-0.5% of cells were found to be desmin-positive, and quantitative analysis after double labeling for desmin and connexin43 revealed that the desmin-positive cells were smaller, and contained significantly lower numbers and smaller sizes of connexin43 gap-junctional spots than did desmin-negative cells. Our findings demonstrate that an inverse expression pattern of connexin43 and desmin holds true at the level of the individual cell. This suggests a close relationship between intrinsic phenotypic control and the regulation of connexin43 expression in the arterial smooth muscle cell.     

Immunohistochemical analysis of connexin26 and 43 expression in the mouse alimentary tract

The spatial pattern of connexins26 (Cx26) and 43 (Cx43) expressions were investigated in the mouse digestive tract by immunocytochemistry. High levels of connexin43 in the epithelium of the oesophagus, non-glandular part of the stomach, and the circular layer of duodenal and ilea muscularis externa were detected. Cx26 was expressed in stratum granulosum of oesophagal folds and in the non-glandular part of the stomach. A low level of immunoreactivity of Cx43 was observed in the circular, and very low in the longitudinal layer of the muscularis externa in the stomach and colon. No immunoreactivity for Cx26 and Cx43 was found in the entire muscularis externa of the oesophagus or in the longitudinal muscle layers of the duodenum and ileum.     

Involvement of connexin43 in the EGF/EGFR signalling during self-renewal and differentiation of neural progenitor cells

Neural progenitor cells (NPCs) are sensitive to epidermal growth factor (EGF), which is essential for their self-renewal. Recently we showed that high level of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) are also required to maintain NPCs in a proliferative state. In this study the connection between EGF/EGFR signalling and Cx43 expression was investigated during proliferation and differentiation of cultured ReNcell VM197 human NPCs. We found that EGF, but not basic fibroblast growth factor (bFGF), strongly stimulated both Cx43 expression and GJIC in proliferating cells. This stimulatory effect was blocked by AG1478, a specific inhibitor for EGFR kinase. Notably, knockdown of Cx43 strongly inhibited the cell proliferation promoted by EGF/EGFR signalling. High sensitivity to EGF was still maintained in differentiated NPCs. Administration of EGF to differentiating cells led to a pronounced increase (9-fold) of Cx43 expression and a re-induction of proliferation. This strong impact of EGF was found to correlate with a surprisingly massive 60-fold up-regulation of EGFR expression in differentiated cells. Our data argue for a mutual regulation between Cx43 expression and EGF/EGFR signalling during self-renewal and differentiation of NPCs.     

Distribution pattern of connexin 43, a gap junctional protein, during the differentiation of mouse heart myocytes

In the cardiac muscle, the electrical coupling of myocytes by means of gap (or communicating) junctions, allows the action potentials to be propagated. Connexin 43 (CX 43) is the major constitutive protein of the gap junctions in the mammalian myocardium. In this organ, the abundance of CX 43 and of its messenger, as well as the spatial expression of this protein, are developmentally regulated. These findings are complemented by the results presented in this article, which deals with the distribution of CX 43 in the ventricular myocytes of mouse heart during differentiation, between the 11 days post coitum embryo stage and adulthood. By immunoelectron microscopy experiments on ultrathin sections of cardiac ventricular tissue of one-week-old mouse, we have provided confirmation that the anti-CX 43 antibodies used here specifically recognized the gap junctions. Double labeling immunofluorescence experiments have been undertaken to localize, within the same cells, either CX 43 and desmin, or CX 43 and Con A or WGA receptor sites. From the earliest stage investigated (11 days post coitum) onwards, expression of CX 43 is always associated with desmin-positive cells, that is, with the myocytes. Up to birth, there is in the ventricular wall a gradient of expression of CX 43 which is superimposable on a gradient of expression of desmin. Immunoreactivity to anti-CX 43 and anti-desmin antibodies is high in the sub-endocardial trabeculae and low (or even undetectable for CX 43, in the early stages) in the sub-epicardial cell layers. In the embryonic stages, the expression sites of CX 43 are visible in the form of small dots, whose abundance increases as development proceeds. During these stages, the immunoreactive sites are distributed in a relatively homogeneous pattern throughout the membrane of the myocytes. One week after birth, the CX 43 expression is restricted to the two ends of the myocytes (where the intercalated discs develop), and the adjacent lateral regions. This polarization of CX 43 is more pronounced at the two and three weeks post natal stages and in the fully differentiated ventricular myocytes (adult stage) CX 43 is only present in the intercalated discs.     

Blockade of connexin 43 expression by stable transfection of antisense cDNA in cultured vascular smooth muscle cells

Gap junctional communication is involved in embryogenesis, cell growth control, and coordinated contraction of cardiac myocytes. It has been hypothesized that gap junctions coordinate responses of vascular cells to constrictor or dilator stimulation. Three connexin (Cx) proteins, 37, 40, and 43, are found in the vasculature. Cx43 gap junctions are widely distributed along the vascular tree, although a precise physiologic role in vascular function is unknown because of a lack of specific functional inhibitors and of suitable animal models. To investigate the role of Cx43 in intercellular communication among vascular smooth muscle (VSM) cells, we selectively modified the expression of the Cx43 gene using antisense cDNA stable transfections in culture. Results show that in cells stably transfected with antisense Cx43 cDNA, gene expression of Cx43 could be reduced to 20% of that observed in vector-transfected cells. In spite of the mRNA and protein reduction, the antisense Cx43 cDNA-transfected cells did not show a significant reduction in dye transfer or a difference in cell growth rate as compared with control. These results suggest either that the residual amount of Cx43 protein is sufficient for dye transfer and growth control or that the dye transfer in these cells can be mediated by Cx40 or other connexin proteins. Therefore, more potent approaches, such as dominant negative and gene knockout, are required to fully block gap junctional communication in VSM cells.     

Decreased connexin43 expression in the mouse heart potentiates pacing-induced remodeling of repolarizing currents

Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose toward arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiological effects of GJR. We paced wild-type (normal Cx43 abundance) and heterozygous Cx43 knockout (Cx43+/-; 66% mean reduction in Cx43) mice for 6 h at 10-15% above their average sinus rate. We investigated the electrophysiological effects of pacing on the whole animal using programmed electrical stimulation and in isolated ventricular myocytes with patch-clamp studies. Cx43+/- myocytes had significantly shorter action potential durations (APD) and increased steady-state (Iss) and inward rectifier (I(K1)) potassium currents compared with those of wild-type littermate cells. In Cx43+/- hearts, pacing resulted in a significant prolongation of ventricular effective refractory period and APD and significant diminution of Iss compared with unpaced Cx43+/- hearts. However, these changes were not seen in paced wild-type mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiological changes, which may contribute to the worsened prognosis often associated with pacing in the failing heart.     

Changes in phospholipid composition during differentiation of cultured mouse myeloid leukemia cells

Mouse myeloid leukemia cells(M1) could be induced by various inducers to form Fc receptors, phagocytize, migrate in agar, produce lysosomal enzyme activities, and change into forms that were morphologically similar to macrophages and granulocytes. When M1 cells were cultured with inducer, the ratio of the percentage of phosphatidylethanolamine to that of phosphatidylcholine was increased about 2-fold. This ratio of the differentiated M1 cells was similar to that of peritoneal macrophages of normal mice or Mm-1 cells, which were established from spontaneously differentiated macrophage-like cells from M1 cells. These changes in phospholipid may be involved in the mechanisms of expression of the differentiation-associated phenotypic properties.     

Epidermal growth factor enhances expression of connexin 43 protein in cultured porcine preantral follicles

Connexin 43 (Cx43) and gap junctional coupling appear to play a critical role in early follicular development because absence of Cx43 disrupts progression of follicles beyond primary stages in transgenic mouse ovaries. Two experimental culture systems were used to determine whether epidermal growth factor (EGF) stimulates expression of Cx43 in early porcine follicular development. Ovarian explants were collected from 32- to 40-day-old gilts and cultured for 6 days on membrane inserts in Waymouth MB 752/1 medium supplemented with 0, 50, or 500 ng/ml mouse EGF. Western blot analysis demonstrated significant increases (P < 0.05) in relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) with 50 and 500 ng/ml of EGF as compared with control cultures. Preantral follicles were enzymatically isolated from 70- to 86-day-old gilts and cultured for 8 days in collagen matrices. Medium and EGF treatments were the same as previously described. Western blot analysis demonstrated a significant increase (P < 0.05) in relative amounts of Cx43 protein with 50 and 500 ng/ml of EGF as compared with control cultures. EGF increased expression of Cx43 protein in secondary preantral follicles in a dose-dependent manner, which suggests that EGF or similar growth factor molecules may modulate early folliculogenesis by stimulating expression of Cx43 gap junctions.