Regulation Of Cx43 Expression Research Articles

Alterations in connexin 43 gene and protein expression in the chicken oviduct following tamoxifen treatment

Connexins (Cxs) are a group of gap junction proteins involved in the direct exchange of small molecules between neighboring cells. Information concerning the expression and regulation of Cxs in the chicken oviduct is lacking, but likely has potential implications for functioning of the oviduct and the quality of the egg laid by commercially used hens. The present study was designed to examine whether selected Cxs are present in the chicken oviduct and, if so, whether expression of the most abundant Cx changes following tamoxifen (TMX; estrogen receptor modulator) treatment. Hy-Line Brown laying hens were injected (s.c.) daily with a vehicle (n = 6) or with TMX (n = 6), at a dose of 6 mg/kg of body weight for 7 days until complete cessation of egg laying by TMX-treated hens. All oviductal segments (infundibulum, magnum, isthmus, shell gland, and vagina) were collected from hens on day 8 of the experiment. First, the gene expression of GJA1 (i.e. Cx43 protein), GJA4 (Cx39), GJB1 (Cx32), and GJD2 (Cx36) was investigated by real-time PCR in tissues of control birds. The results demonstrated gene- and oviductal segment-dependent expression of GJB1, GJD2, GJA4, and GJA1 mRNA. Since the GJA1 transcript was the most abundant in all oviductal parts, subsequently, the Cx43 expression and localization were examined in the oviduct of all hens. The relative expression of GJA1 mRNA in control hens was highest in the infundibulum and vagina and lowest in the magnum. The pattern of Cx43 protein abundance evaluated by Western blot was similar to that of mRNA. Treatment of hens with TMX decreased the GJA1 mRNA levels in the magnum and isthmus, and Cx43 protein abundances were reduced in the isthmus and vagina. Immunofluorescence demonstrated cell- and segment-dependent localization of Cx43 protein in the oviductal wall; the most intense immunoreactivity was observed in the muscle cells of the shell gland and vagina. In TMX-treated hens, the immunoreactivity for Cx43 in all oviductal segments was slightly reduced and had a different signal pattern compared with control chickens. These results suggest that Cx43 likely takes part in the regulation of oviduct functioning, especially in the coordination of muscle contraction required for egg transport and oviposition. In addition, the results suggest a contribution of estrogen in the regulation of Cx43 expression and/or fates in the chicken oviduct. New insights into the expression and regulation of Cxs in the hen oviduct, indicating their potential involvement in the mechanisms of egg formation and transport that may affect poultry production, were obtained in this study.

Plasmalogens Regulate Retinal Connexin 43 Expression and Müller Glial Cells Gap Junction Intercellular Communication and Migration.

Plasmalogens are a specific glycerophospholipid subtype characterized by a vinyl-ether bound at their sn-1 moiety. Their biosynthesis is initiated in the peroxisome by dihydroxyacetone phosphate-acyltransferase (DHAPAT), which is encoded by the DAPAT gene. Previous studies have shown that plasmalogen-deficient mice exhibit major physiological dysfunctions including several eye defects, among which abnormal vascular development of the retina and a reactive activation of macroglial Müller cells. Interestingly, plasmalogen deficiency in mice is also associated with a reduced expression of brain connexin 43 (Cx43). Cx43 is the main connexin subtype of retinal glial cells and is involved in several cellular mechanisms such as calcium-based gap junction intercellular communication (GJIC) or cell migration. Thus, the aim of our work was 1) to confirm the alteration of Cx43 expression in the retina of plasmalogen-deficient DAPAT−/- mice and 2) to investigate whether plasmalogens are involved in crucial functions of Müller cells such as GJIC and cell migration. First, we found that plasmalogen deficiency was associated with a significant reduction of Cx43 expression in the retina of DAPAT−/- mice in vivo. Secondly, using a siRNA targeting DHAPAT in vitro, we found that a 50%-reduction of Müller cells content in plasmalogens was sufficient to significantly downregulate Cx43 expression, while increasing its phosphorylation. Furthermore, plasmalogen-depleted Müller cells exhibited several alterations in ATP-induced GJIC, such as calcium waves of higher amplitude that propagated slower to neighboring cells, including astrocytes. Finally, in vitro plasmalogen depletion was also associated with a significant downregulation of Müller cells migration. Taken together, these data confirm that plasmalogens are critical for the regulation of Cx43 expression and for characteristics of retinal Müller glial cells such as GJIC and cell migration.

Lipopolysaccharide-induced redistribution of myocardial connexin43 is associated with increased macrophage infiltration in both normotensive and spontaneously hypertensive rats.

We investigated whether changes in gap junction alpha-1 protein (Cx43) expression may be associated with macrophage-induced inflammation in the heart of spontaneously hypertensive rats (SHR). To examine mutual interactions of macrophage infiltration with Cx43 expression and redistribution, we applied a bolus of bacterial lipopolysaccharide (LPS) to SHR and age-matched normotensive Wistar rats. The results demonstrated association of Cx43 downregulation with increased infiltration of cardiac CD-68 macrophages and upregulation of nuclear factor-κB (NFκB) and tumor necrosis factor-α (TNF-α) expression in the heart of SHR. LPS application to SHR caused further degradation and redistribution of Cx43 accompanied with extensively increased macrophage infiltration and NFκB and TNF-α expression. LPS administration to Wistar rats resulted in elevation of cardiac CD-68 macrophages but it did not significantly affect total Cx43 expression. Our results are suggestive of regulation of Cx43 expression with macrophages-related inflammation in the heart of SHR. The data also indicate that SHR can be more sensitive to LPS than are normotensive rats.

Systemic inflammation disrupts oligodendrocyte gap junctions and induces ER stress in a model of CNS manifestations of X-linked Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMT1X) is a common form of inherited neuropathy resulting from different mutations affecting the gap junction (GJ) protein connexin32 (Cx32). A subset of CMT1X patients may additionally present with acute fulminant CNS dysfunction, typically triggered by conditions of systemic inflammation and metabolic stress. To clarify the underlying mechanisms of CNS phenotypes in CMT1X we studied a mouse model of systemic inflammation induced by lipopolysaccharide (LPS) injection to compare wild type (WT), connexin32 (Cx32) knockout (KO), and KO T55I mice expressing the T55I Cx32 mutation associated with CNS phenotypes. Following a single intraperitoneal LPS or saline (controls) injection at the age of 40–60 days systemic inflammatory response was documented by elevated TNF-α and IL-6 levels in peripheral blood and mice were evaluated 1 week after injection. Behavioral analysis showed graded impairment of motor performance in LPS treated mice, worse in KO T55I than in Cx32 KO and in Cx32 KO worse than WT. Iba1 immunostaining revealed widespread inflammation in LPS treated mice with diffusely activated microglia throughout the CNS. Immunostaining for the remaining major oligodendrocyte connexin Cx47 and for its astrocytic partner Cx43 revealed widely reduced expression of Cx43 and loss of Cx47 GJs in oligodendrocytes. Real-time PCR and immunoblot analysis indicated primarily a down regulation of Cx43 expression with secondary loss of Cx47 membrane localization. Inflammatory changes and connexin alterations were most severe in the KO T55I group. To examine why the presence of the T55I mutant exacerbates pathology even more than in Cx32 KO mice, we analyzed the expression of ER-stress markers BiP, Fas and CHOP by immunostaining, immunoblot and Real-time PCR. All markers were increased in LPS treated KO T55I mice more than in other genotypes. In conclusion, LPS induced neuroinflammation causes disruption of the main astrocyte-oligodendrocyte GJs, which may contribute to the increased sensitivity of Cx32 KO mice to LPS and of patients with CMT1X to various stressors. Moreover the presence of an intracellularly retained, misfolded CMT1X mutant such as T55I induces ER stress under inflammatory conditions, further exacerbating oligodendrocyte dysfunction and pathological changes in the CNS.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0369-5) contains supplementary material, which is available to authorized users.

Transforming growth factor-β1 up-regulates connexin43 expression in human granulosa cells.

Does transforming growth factor-β1 (TGF-β1) up-regulate connexin43 (Cx43) to promote cell-cell communication in human granulosa cells? TGF-β1 up-regulates Cx43 and increases gap junction intercellular communication activities (GJIC) in human granulosa cells, and this effect occurs via the activin receptor-like kinase (ALK)5-mediated Sma- and Mad-related protein (SMAD)2/3-SMAD4-dependent pathway. TGF-β1 and its receptors are expressed in human granulosa cells, and follicular fluid contains TGF-β1 protein. In human granulosa cells, Cx43 gap junctions play an important role in the development of follicles and oocytes. This is an experimental study which was performed over a 1-year period. Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing IVF in an academic research center were used as the study models. Cx43 mRNA and protein expression levels were examined after exposure of SVOG cells to recombinant human TGF-β1. An activin/TGF-β type I receptor inhibitor, SB431542, and small interfering RNAs targeting ALK4, ALK5, SMAD2, SMAD3 and SMAD4 were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance. TGF-β1 treatment increased phosphorylation of SMAD2/3 (P < 0.0001) and up-regulated Cx43 mRNA and protein levels (P < 0.001) in SVOG cells and these stimulatory effects were abolished by the TGF-β type I receptor inhibitor SB431542. In addition, the up-regulatory effect of TGF-β1 on Cx43 expression (mRNA and protein) was confirmed in primary cultures of human granulosa-lutein cells (P < 0.05). The small interfering RNA-mediated knockdown of ALK5, but not ALK4, abolished the TGF-β1-induced phosphorylation of SMAD2/3 and the up-regulation of Cx43. Furthermore, knockdown of SMAD2/3 or the common SMAD, SMAD4, abolished the stimulatory effects of TGF-β1 on Cx43 expression in SVOG cells. The TGF-β1-induced up-regulation of Cx43 contributed to the increase of GJIC between SVOG cells (P < 0.001). The results of this study were generated from in vitro system and may not reflect the intra-ovarian microenvironment in vivo. Our studies represent the first comprehensive research of molecular mechanisms of TGF-β1 in the regulation of Cx43 expression and GJIC in human granulosa cells and demonstrate that TGF-β1 may play a crucial role in the local modulation of cell-cell communication. Deepening our understanding of the molecular determinants will offer important insights into ovarian physiology and lead to the development of potential therapeutic methods for fertility regulation. This research was supported by an operating grant from the Canadian Institutes of Health Research to P.C.K.L. There are no conflicts of interest to declare. NA.

AR Pathway Is Involved in the Regulation of CX43 in Prostate Cancer.

CX43 plays a critical role in tumor progression. Previous studies imply that AR pathway may be involved in regulation of CX43. This study was focused on determining the relationship between AR pathway and CX43. The result showed that the expression of CX43 in malignant cells was higher than that in normal cells, and in nonmalignant and malignant cells, not only is the expression level of CX43 different, but the localization of CX43 can also be changed. After androgen stimulation and inhibition of AR pathway, expression of CX43 can also be changed. Thus, AR pathway plays an important role in regulation of CX43 expression in prostate cancer cells. AR may be the upstream signal of CX43.

Influence of the antiandrogen flutamide on connexin 43 (Cx43) gene and protein expression in the porcine placenta and uterus during pregnancy.

The study focuses on the expression of connexin 43 (Cx43), a gap junctional protein in the porcine placenta and uterus. The aim was to examine Cx43 mRNA and protein expression after antiandrogen flutamide treatment. Flutamide was injected into pregnant gilts at a daily dose of 50 mg/kg body weight at different stages of pregnancy: between days 43-49 (50 dpc), 83-89 (90 dpc) and 101-107 (108 dpc) of gestation. The animals were sacrificed and tissues were collected one day after the last injection. Cx43 immunostaining was observed in epithelial and stromal cells of the fetal part of the placenta; luminal and glandular epithelial cells of maternal part of the placenta and myometrium of the uterus within placentation sites. Cx43 was also found in glandular epithelium and myometrium of non-placental uterus. Flutamide treatment caused fluctuations in Cx43 expression especially before parturition. Although significant changes in Cx43 mRNA expression were observed only in the fetal part of the placenta, Cx43 protein level was affected within the maternal part of the placenta and non-placental uterus. These results suggest the involvement of androgens in the regulation of Cx43 expression within the feto-maternal compartment in pigs. However, androgen deficiency caused pronounced changes during late pregnancy and before parturition. These results are interesting due to the functional changes in the porcine uterus during the preparturient period that is determined by Cx43 protein.

Hypoxia induces connexin 43 dysregulation by modulating matrix metalloproteinases via MAPK signaling

Connexin 43 (Cx43) is a major structural protein found in the gap junctions of the ventricular myocardium and a major determinant of its electrical properties. The effects of matrix metalloproteinases (MMPs), the mitogen-activated protein kinase (MAPK) signaling pathway, transcription factor NF-kB, and activator protein-1 (AP-1)/c-Jun on the regulation of Cx43 gene expression in H9c2 cardiomyocytes were assessed. The MAPK signaling pathway (MEK/ERK1/2 and PI3K) and transcription factors NF-kB and AP-1/c-Jun were inhibited, then Cx43 expression was assessed using Western blot analysis, and MMP-9 activity was assessed using gelatin zymography. Hypoxia decreased the Cx43 protein level by approximately 30–50 %. Doxycycline (10 μg/mL), an inhibitor of MMP, markedly attenuated the hypoxia-induced downregulation of Cx43 protein expression at 6 h. The hypoxia-induced decrease in Cx43 protein expression was significantly reversed by U0126 (10 μM), a MEK/ERK1/2 inhibitor, at 6 and 12 h; LY294002 (30 μM), a PI3K inhibitor, downregulated Cx43 expression. Hypoxia-induced MMP-9 activation was inhibited by treatment with LY294002, U0126, and, most especially, U0126. JSH-23 (30 μM), an NF-kB inhibitor, and SP600125 (10 μM), an AP-1/c-Jun inhibitor, attenuated the loss of Cx43. These results suggest that MAPK signaling and the activities NF-kB and MMPs play an important roles in the regulation of Cx43 expression.

Post-Transcriptional Regulation of Connexin43 in H-Ras-Transformed Cells

Connexin43 (Cx43) expression is lost in cancer cells and many studies have reported that Cx43 is a tumor suppressor gene. Paradoxically, in a cellular NIH3T3 model, we have previously shown that Ha-Ras-mediated oncogenic transformation results in increased Cx43 expression. Although the examination of transcriptional regulation revealed essential regulatory elements, it could not solve this paradox. Here we studied post-transcriptional regulation of Cx43 expression in cancer using the same model in search of novel gene regulatory elements. Upon Ras transformation, both Cx43 mRNA stability and translation efficiency were increased. We investigated the role of Cx43 mRNA 3′ and 5′Untranslated regions (UTRs) and found an opposing effect; a 5′UTR-driven positive regulation is observed in Ras-transformed cells (NIH-3T3Ras), while the 3′UTR is active only in normal NIH-3T3Neo cells and completely silenced in NIH-3T3Ras cells. Most importantly, we identified a previously unknown regulatory element within the 3′UTR, named S1516, which accounts for this 3′UTR-mediated regulation. We also examined the effect of other oncogenes and found that Ras- and Src-transformed cells show a different Cx43 UTRs post-transcriptional regulation than ErbB2-transformed cells, suggesting distinct regulatory pathways. Next, we detected different patterns of S1516 RNA-protein complexes in NIH-3T3Neo compared to NIH-3T3Ras cells. A proteomic approach identified most of the S1516-binding proteins as factors involved in post-transcriptional regulation. Building on our new findings, we propose a model to explain the discrepancy between the Cx43 expression in Ras-transformed NIH3T3 cells and the data in clinical specimens.

Prenatal and neonatal exposure to the antiandrogen flutamide alters connexin 43 gene expression in adult porcine ovary

Connexin 43 (Cx43) is the predominant gap junction protein within porcine ovary and is required for proper follicle and corpus luteum (CL) development. Recent research suggests maternally or neonatally mediated effects of antiandrogens on reproductive function during adulthood, notably those dependent on gap junctional communication. The current study was conducted to determine whether late gestational or neonatal exposure to the antiandrogen flutamide influences Cx43 gene expression in the adult porcine ovary. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 posnatally. After animals reached sexual maturity, the ovaries were collected from treated and nontreated (control) pigs. Expression of Cx43 mRNA and protein was determined for preantral and antral follicles and for CLs. In addition, 3β-hydroxysteroid dehydrogenase (3β-HSD) expression and progesterone concentration were determined for luteal tissues. In preantral follicles, Cx43 mRNA was down-regulated ( P < 0.01) following maternal and neonatal flutamide exposure. In large antral follicles, Cx43 mRNA was up-regulated ( P < 0.01) after neonatal flutamide administration. Immunofluorescence showed that Cx43 expression decreased ( P < 0.001) in preantral follicles and increased ( P < 0.001) in large antral follicles following flutamide exposure. In luteal tissues, Cx43 and 3β-HSD expression and progesterone concentration decreased ( P < 0.01) after postnatal flutamide treatment. Overall, these results suggest the involvement of androgens in the regulation of Cx43 expression in pig ovary. Moreover, alteration of Cx43 expression by the administration of flutamide during particular prenatal and neonatal time periods may affect porcine follicle development, as well as CL formation and function.

Abstract 1525: Atrial Rhythm Acutely Modulates Connexin43 Expression and Phosphorylation State in Human Atrial Fibrillation

Background: Adjacent areas of heterogenous conduction in the atria form a substrate for atrial fibrillation (AF). Connexin proteins (Cx) underlie conduction between myocytes; conduction is modulated by connexin expression and phosphorylation. Here we tested the hypothesis that left atrial (LA) expression and/or phosphorylation of Cx43 is distinctly regulated by atrial rhythm. Methods: Cx43 expression and phosphorylation was evaluated in LA tissue homogenates from 3 groups: cardiac surgery patients with history of AF presenting for surgery in AF (AFAF, n=8); history of AF but in sinus rhythm (AFSR, n=8); patients with no history of AF (CTL, n=8). Groups were matched for age, LA size, EF (&gt;50%), CAD, valve disease, and hypertension. Results: Western blot analysis revealed increased total Cx43 expression (t-Cx43) in AFAF (2.03±0.38) compared to AFSR (0.85±0.15, p=0.01) and SR (0.98±0.16, p=0.02) groups. Blots re-probed with an antibody detecting non-phosphorylated Cx43 at S368 (Np-Cx43) revealed a significant decrease in the Np-Cx43/t-Cx43 ratio, indicating increased S368 phosphorylation in the AFAF group (by 23% in AFAF vs. AFSR, p=0.046; by 12% in AFAF vs. CTL, p=0.051). Subcellular compartmentation of t-Cx43 was assessed in 3 samples per group using tissue fractionation and immunoblotting. Cytoskeletal t-Cx43 levels were increased in AFAF vs. AFSR and vs. CTL, ANOVA, p=0.016); t-Cx43 expression was not different between groups in membrane, cytoplasmic or nuclear fractions. Confocal microscopy revealed greater Cx43 lateralization in both AF groups compared to CTL. Conclusion: Our results (increased Cx43 expression and phosphorylation) suggest that AF rhythm at the time of surgery , more than the history of AF, has a distinct impact on LA Cx43 remodeling. Further the dynamic increase restricted to the cytoskeletal compartment suggests that cytoskeletal changes in AF may modulate connexin trafficking to the cell surface. This may affect the anisotropic properties of atrial conduction. Additional electrophysiological studies are needed to determine the functional contribution of lateralized Cx. Our study provides the first evidence linking acute rhythm status with regulation of Cx43 expression and phosphorylation in human atria.

The Gap Junctional Protein Connexin(Cx)43 in Testicular Cancer: its Loss Marks Progession from Carcinoma In Situ to Invasive Germ Cell Tumour

Carcinoma‐in‐situ (CIS) of the testis is known to be the pre‐invasive stage of most human germ cell tumours (seminoma and non‐seminoma), but the mechanisms leading to an increased pubertal proliferation of CIS cells after a long latency and to progression of CIS to an invasive malignancy are still not known. Additionally, CIS and seminoma have also been reported in equine testis (Veeramachaneni and Sawyer, 1998). The gap junctional protein and tumour suppressor gene connexin(cx)43 represents the predominant cx in human, canine and rodent testis so far and it is expected to play a key role for the regulation of both proliferation and differentiation of germ cells (spermatogonia and spermatocytes), and its gene‐ and protein‐expression pattern is typical for the pubertal terminal differentiation of somatic Sertoli cells. Using cDNA‐microarray analysis, in‐situ hybridization (ISH), RT‐PCR from tissue homogenate and semi‐quantitative RT‐PCR from well defined microdissected tubules with normal spermatogenesis, CIS, intratubular seminoma (ISe) and from seminoma cells from invasive seminoma we found a downregulation of cx43 starting in intratubular CIS, leading to a complete loss in most invasive seminoma cells. This indicates that regulation of cx43 expression takes place at transcriptional level confirming and expanding earlier studies of protein expression (Brehm et al., 2002). This reduction of cx43‐expression suggests that an early intratubular derangement in cx43‐gene expression and disruption of inter‐cellular communication between Sertoli cells and/or Sertoli cells and pre‐invasive tumour cells via cx43‐gap junctions may play a role in the proliferation of CIS cells and seminoma cells and in the progression phase of testicular seminoma development.References Veeramachaneni, D. N., and H. R.Sawyer, 1998: Carcinoma in situ and seminoma in equine testis. APMIS 106, 183–185.Brehm R., A. Marks, R. Rey, S. Kliesch, M. Bergmann and K. Steger, 2002: Altered expression of connexins 26 and 43 in Sertoli cells in seminiferous tubules infiltrated with carcinoma‐in‐situ or seminoma. J. Pathol. 197, 647–653.

Differential Activation of the Connexin 43 Promoter by Dimers of Activator Protein-1 Transcription Factors in Myometrial Cells

The expression of activator protein-1 (AP-1) transcription factors is increased in the myometrium at term and may therefore regulate the expression of genes, such as connexin 43 (Cx43), required for the onset of labor. The region upstream of the mouse, rat, and human Cx43 genes contains two consensus AP-1 binding sequences, a proximal AP-1, located close to the TATA box, and a distal AP-1, 1 kb upstream. A transient transfection system was developed in which Syrian hamster myometrial cells were transfected with Cx43 promoter-luciferase constructs in combination with expression vectors for the AP-1 family. Transfection with c-Jun or JunB had no effect on transcription from the Cx43 promoter, whereas transfection with JunD or combinations of Jun and Fos family members led to significant increases in transcription. Deletion of the distal AP-1 site did not abrogate transcription driven by Fos/Jun, whereas a 2-bp mutation in the proximal AP-1 site significantly reduced pCx43 transactivation by AP-1 dimers. Dimers comprising Fos/Jun proteins conferred greater transcriptional activity than Jun dimmers, with Fra-2/JunB combination conferring greatest activity. These data suggest that increased expression of Fos family members in the myometrium at term drives the increase in Cx43 transcription and expression during labor. Because expression of Fra-2 increases earlier than other Fos family members and confers the highest transcriptional drive to the Cx43 promoter, our data suggest that Fra-2 is a central component in the regulation of Cx43 expression during labor.

Gap junction-microtubule associations in rat alveolar epithelial cells.

Connexin 43 (Cx43) is a predominant gap junction (GJ) protein expressed by alveolar epithelial cells (AEC) in primary cell culture. Cx43 trafficking, assembly, and turnover are regulated by multiple mechanisms, including those mediated by integrins, by extracellular matrix, and by the cytoskeleton. Immunocytochemical double labeling demonstrates association of microtubules with internalization of Cx43-positive GJ plaques. Antibodies against the alpha 5-integrin subunit block cell-matrix interactions without effect on tubulin expression, whereas inhibition of MAP kinase kinase by PD-98059 reduces tubulin expression, based on both Western blot and immunostaining. To examine direct association of microtubules (MT) with GJ plaques, we treated day 3 AEC for 0.5-24 h with colchicine, an inhibitor of tubulin polymerization. After 60 min, MTs were disassembled, whereas Western blot analysis showed no change in tubulin expression. In parallel, colchicine initiated redistribution of immunopositive Cx43 from the membrane to the cytosol. These observations support the premise that direct association of the cytoskeleton with gap junctions plays a significant role in regulation of Cx43 expression and distribution through integrin-mediated signal transduction pathways.

Analysis of the rat connexin 43 proximal promoter in neonatal cardiomyocytes

Altered transcriptional control is likely to contribute to the down-regulation of connexin 43 (Cx43) expression observed in many forms of heart disease. However, little is known about the factors regulating Cx43 transcription in the heart under (patho)physiological conditions. Therefore, a systematic study of rat Cx43 (rCx43) proximal promoter regulation in rat primary neonatal ventricular cardiomyocytes (NCM) and, for comparison, different cell types was initiated. Luciferase assays revealed that, in NCM, the proximal promoter is preserved in a conserved region extending from 148 nucleotides upstream towards 281 nucleotides downstream relative to the transcription initiation site (TIS). Further deletional analysis suggested the involvement of four putative Sp- and two AP1-binding sites. The binding of both Sp1 and Sp3 to the Sp-binding elements and AP1 to the AP1-binding elements was demonstrated by electrophoretic mobility shift assays (EMSA). Promoter–luciferase assays using the natural rCx43 proximal promoter and mutated derivatives in NCM, HL-1 and A7r5 cells revealed that all sites contribute to basal promoter activity. Trans-activation of the Cx43 proximal promoter with Sp1 and Sp3 in Drosophila Schneider line 2 (SL2) cells demonstrated that Sp1 and, to a lesser extent, Sp3 determine rCx43 promoter activation. Thus Sp1, Sp3 and AP1 determine basal Cx43 expression. In addition, we studied the effect of the cardiac transcription factor Nkx2.5 on Cx43 regulation. NCM were infected with adenovirus encoding either β-galactosidase (control) or Nkx2.5. Cx43 protein and mRNA were significantly decreased after Nkx2.5 infection as shown by Western and Northern blot analyses. Promoter–reporter assays demonstrated that the rCx43 promoter was down-regulated approximately twofold upon Nkx2.5 overexpression. Therefore, in NCM, Nkx2.5 appears to play a role in the regulation of Cx43 expression.

C-Jun N-terminal kinase activation mediates downregulation of connexin43 in cardiomyocytes.

Loss of gap junctions and impaired intercellular communication are characteristic features of pathological remodeling in heart failure as a result of stress or injury, yet the underlying regulatory mechanism has not been identified. Here, we report that in cultured myocytes, rapid loss of the gap junction protein connexin43 (Cx43) occurs in conjunction with the activation of c-Jun N-terminal kinase (JNK), a stress-activated protein kinase, on stress stimulation. To investigate the specific role of JNK activation in the regulation of connexin in cardiomyocytes, an activated mutant of mitogen-activated protein kinase kinase 7 (mutant D), a JNK-specific upstream activator, was expressed in myocytes by adenovirus-mediated gene transfer. JNK activation in infected cardiomyocytes resulted in significant reduction of Cx43 expression at both mRNA and protein levels and impaired cell-cell communication. To evaluate the role of JNK in the regulation of Cx43 expression and gap junction structure in vivo, a Cre-LoxP-mediated gene-switch system was used to establish a transgenic animal model with targeted activation of JNK in ventricular myocardium. The transgenic hearts exhibited significant downregulation of Cx43 expression and loss of gap junctions in myocardium that may contribute to the cardiac dysfunction and premature death phenotype. Our report represents the first evidence, both in vitro and in vivo, implicating JNK as an important mediator of stress-induced Cx43 downregulation and impaired intercellular communication in the failing heart.

Regulation of connexin43 expression by c-fos and c-jun in myometrial cells.

Labor is associated with a dramatic increase in the myometrial expression of connexin43 (Cx43) which is thought to mediate myocyte contractile coupling. The transcription factor c-fos is also dramatically increased prior to the onset of labor and is therefore a potential regulator of Cx43. The promoter region of Cx43 contains a conserved activator protein-1 (AP-1) site, which binds dimers of Fos and Jun proteins. We constructed expression vectors for c-Fos and c-Jun to investigate the role of these transcription factors in the regulation of Cx43 expression. These expression vectors were then co-transfected into SHM (syrian hamster myocyte) cells with a Cx43 promoter-Luciferase vector. The combinations of c-Fos and c-Jun proteins activated the Cx43 promoter while c-Jun alone had no effect on Cx43 promoter activity. Mutation of the AP-1 site was found to reduce this responsiveness. These data indicate that the transcription factors c-Fos and c-Jun are important in the regulation of Cx43 expression.

Retinoic acid enhances connexin43 expression at the post-transcriptional level in rat liver epithelial cells.

The mechanism by which all-trans retinoic acid (RA) stimulates gap junctional intercellular communication (GJIC) in the rat liver epithelial cell line. IAR203, was investigated. When RA, at 0.1 microM for 24-48 h, enhanced the dye transfer in IAR203 cells (x 1.4), it increased the amount of connexin43 (Cx43) in the cell-cell contact regions of the plasma membrane, as evidenced by analysis by Western blot and by immunofluorescence. It had no effect on the level of Cx43 mRNA. Freeze-fracture analysis of the size of gap junctions revealed an increase of the proportion of small gap junctions in RA-treated cells. We conclude that, in IAR203 cells, RA stimulates GJIC by acting at the post-transcriptional level of Cx43 regulation. The possibility that RA acts indirectly on the regulation of Cx43 expression, and increases the half-life of Cx43 by inducing adhesion molecules is discussed.

Upregulation of gap junctional communication and connexin43 gene expression by carotenoids in human dermal fibroblasts but not in human keratinocytes

Consumption of dietary carotenoids has been statistically associated with decreased risk of cancer at several anatomic sites. In a model murine system of carcinogenesis (the 10T1/2 assay), we have previously shown that carotenoids can inhibit chemically and physically induced neoplastic transformation. This action is strongly correlated with the ability of carotenoids to increase gap-junctional communication (GJC) by induction of connexin43 (Cx43) gene expression. Here we extend these studies to human foreskin-derived dermal fibroblasts and keratinocytes. In fibroblasts, beta-carotene and canthaxanthin at concentrations between 10(-5) and 3 x 10(-6) M were found to strongly enhance GJC in a dose- and time-dependent manner. This was accompanied by an increase in the number of immunofluorescent junctional plaques recognized by an anti-Cx43 antibody and by an increase in Cx43 protein level as determined by western blot analysis. No decrease in proliferation rates was detected by [H3]thymidine labeling. Human keratinocytes grown in monolayer culture did not respond to carotenoids in terms of GJC as measured by dye transfer, immunofluorescent analysis of Cx43 distribution, or Cx43 levels as measured by western blotting. Both cell types accumulated high levels of carotenoids. Because canthaxanthin, which has no known provitamin A activity in mammals, is as active in fibroblasts as is beta-carotene, the carotenoid with the highest provitamin A activity, the induction of GJC and Cx43 expression by carotenoids in human dermal fibroblasts seems unrelated to their provitamin A status. The lack of response of keratinocytes suggests differences in regulation of Cx43 expression or in carotenoid processing.