Chronic regulation of the expression of the gap junction protein connexin 43 in transfected HeLa cells

Salameh, Aida; Polontchouk, Lioudmilla; Dhein, Stefan; Hagendorff, Andreas; Pfeiffer, Dietrich

doi:10.1007/s00210-003-0760-1

Cited by 8 publications

(4 citation statements)

References 47 publications

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“…Thereafter, RNA was reverse transcribed from 1 µg total RNA with random hexamers to generate first‐strand cDNA using standard protocols. After first‐strand cDNA was prepared, 1 µL cDNA was mixed with PCR reagents using SYBR Green supermix (BioRad) according to the manufacturer's instruction to make a 25 µL solution, and real‐time PCR was carried out using the following primer pairs (Salameh et al. , 2003):…”

Section: Methodsmentioning

confidence: 99%

The signal transduction cascade regulating the expression of the gap junction protein connexin43 by β‐adrenoceptors

Salameh

Krautblatter

Karl

et al. 2009

British J Pharmacology

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Background and purpose:In mammalian heart, connexin43 (Cx43) represents the predominant connexin in the working myocardium. As the b-adrenoceptor is involved in many cardiac diseases, we wanted to clarify the pathway by which b-adrenoceptor stimulation may control Cx43 expression. Experimental approach: Cultured neonatal rat cardiomyocytes were stimulated with isoprenaline. Cx43 expression as well as activation of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, JUN NH2-terminal kinase (JNK) and nuclear translocation of the transcription factors activator protein 1 (AP1) and CRE-binding protein (CREB) were investigated. Additionally, we assessed Cx43 expression and distribution in left ventricular biopsies from patients without any significant heart disease, and from patients with either congestive heart failure [dilated cardiomyopathy (DCM)] or hypertrophic cardiomyopathy (HCM). Key results: Isoprenaline exposure caused about twofold up-regulation of Cx43 protein with a pEC50 of 7.92 Ϯ 0.11, which was inhibited by propranolol, SB203580 (4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-1H-imidazole) (p38 inhibitor), PD98059 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) (MAPK 1 kinase inhibitor) (Alexis Biochemicals, San Diego, CA, USA) or cyclosporin A. Similar findings were obtained for Cx43 mRNA. Furthermore, Cx43 up-regulation was accompanied by phosphorylation of p38, p42/44 and JNK, and by translocation of AP1 and CREB to the nucleus. Analysis of Cx43 protein and mRNA in ventricular biopsies revealed that in patients with DCM, Cx43 content was significantly lower, and in patients with HCM, Cx43 content was significantly higher, relative to patients without any cardiomyopathy. More importantly, Cx43 distribution also changed with more Cx43 being localized at the lateral border of the cardiomyocytes. Conclusion and implication: b-adrenoceptor stimulation up-regulated cardiac Cx43 expression via a protein kinase A and MAPK-regulated pathway, possibly involving AP1 and CREB. Cardiomyopathy altered Cx43 expression and distribution.

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Section: Methodsmentioning

confidence: 99%

The signal transduction cascade regulating the expression of the gap junction protein connexin43 by β‐adrenoceptors

Salameh

Krautblatter

Karl

et al. 2009

British J Pharmacology

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“…Thereafter, RNA was reverse transcribed from 1 g of total RNA with random hexamers to generate first-strand cDNA using standard protocols. After firststrand cDNA was prepared, 1 l of cDNA was mixed with PCR reagents using SYBR Green SuperMix (Bio-Rad) according to the manufacturer's instructions to make a 25-l solution, and real-time PCR was done using the following primer pairs (Salameh et al, 2003): Cx43 antisense, 5Ј-TTG TTT CTG TCA CCA GTA AC-3Ј and sense, 5Ј-GAT GAG GAA GGA AGA GAA GC-3Ј; and GAPDH antisense, 5Ј-CCG CCT GCT TCA CCA CCT TCT-3Ј and sense, 5Ј-GTC ATC ATC TCC GCC CCT TCC-3Ј.…”

Section: Methodsmentioning

confidence: 99%

Signal Transduction and Transcriptional Control of Cardiac Connexin43 Up-Regulation after α₁-Adrenoceptor Stimulation

Salameh

Krautblatter

Baessler

et al. 2008

J Pharmacol Exp Ther

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Syncytial behavior of cardiac tissue is mainly controlled by the expression of cardiac gap junction proteins, and of these, connexin43 (Cx43) represents the predominant connexin in the working myocardium. Because the ␣ 1 -adrenoceptor is involved in many cardiac diseases, the following experiments were performed to clarify the pathway whereby ␣ 1 -adrenoceptor stimulation may control Cx43 expression. Cultured neonatal rat cardiomyocytes were stimulated with phenylephrine for 24 h, and Cx43 expression was investigated. Moreover, we investigated activation of p38 mitogenic-activated protein kinase (MAPK), p42/44-MAPK, and c-JUN NH 2 -terminal kinase (JNK) by phosphospecific enzyme-linked immunosorbent assay and nuclear translocation of the transcription factors c-fos and activator protein 1 (AP1). For verification of our results, a Cx43-promoter-enhanced green fluorescent protein (EGFP) construct using the complete promoter [2771 base pairs (bp)] or fragments (0 -2421 bp) with EGFP under control of the Cx43 promoter was transfected into cardiomyocytes, and fluorescence intensity was investigated. Phenylephrine exposure caused approximately 2-fold up-regulation of Cx43 protein with an EC 50 of approximately 5 nM, which was significantly inhibited by bisindolylmaleimide I [protein kinase C (PKC) inhibitor], 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580; p38 inhibitor), or 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059; p42/44 inhibitor). Similar findings were obtained for Cx43 mRNA. Furthermore, Cx43 up-regulation was accompanied by phosphorylation of p38, p42/44, and JNK. Moreover, we found translocation of c-fos and AP1 to the nucleus. Phenylephrine stimulation of Cx43-promoter EGFP-transfected cardiomyocytes significantly increased fluorescence, depending on the length of promoter fragments. A 91-bp fragment containing the first AP1 binding site produced approximately 50% of the fluorescence intensity of the complete promoter. Therefore, we conclude that ␣ 1 -adrenoceptor stimulation up-regulates cardiac Cx43 expression via a PKC p38-and p42/44 MAPK-regulated pathway, possibly involving AP1.Intercellular communication is an important feature of organization within many kinds of tissue. Gap junction channels form the basis of direct intercellular communication. These channels allow electrical and metabolic coupling between neighboring cells. One complete gap junction channel is composed of two hemichannels (connexons), and each hemichannel consists of six protein subunits, the so-called connexins. A connexin has four transmembrane domains, two extracellular loops, and the N and C terminus at the cytoplasmic side of the cell. The C terminus is the variant part of a connexin and differs in length and amino acid sequence between the various connexin isoforms. Moreover, it is known that the C terminus contains consensus sequences that are susceptible to a number of protein kinases such as protein kinase A, protein kinase B, protein kinase C (PKC), protein kinase...

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“…After first-strand cDNA was prepared, 1µl cDNA was mixed with PCR reagents to make a 25-µl solution containing 1U Taq DNA polymerase (Gibco, Germany), 10× PCR buffer, 1.5 mM MgCl 2 , 0.2 mM dNTP, and 0.5 µl of each primer. The amplification was carried out using the following primer according to Salameh et al (15): Cx43 antisense 5′-TTG TTT CTG TCA CCA GTA AC-3′, sense 5′-GAT GAG GAA GGA AGA GAA GC-3′, GAPDH antisense 5′-CCG CCT GCT TCA CCA CCT TCT-3′, sense 5′-GTC ATC ATC TCC GCC CCT TCC-3′. The cDNA amplification products of Cx43 were predicted to be 600 bp and of GAPDH 415 bp, respectively.…”

Section: Reverse Transcription and Pcr Amplificationmentioning

confidence: 99%

Subchronic alpha‐ and beta‐adrenergic regulation of cardiac gap junction protein expression

Salameh¹,

Frenzel²,

Boldt

et al. 2005

The FASEB Journal

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Gap junction channels are essential for intercellular electrical communication in the heart. The most important cardiac gap junction proteins are connexin43 (predominantly) (Cx43), connexin40 (Cx40), and in early developmental stages connexin45. Since catecholamines play an important role in cardiac (patho)physiology, we wanted to elucidate whether catecholamines may affect expression of Cx43 and Cx40. Cultured neonatal rat cardiomyocytes were exposed for 24 h to increasing concentrations of noradrenaline (1-10000 nM) (physiological agonist at alpha and beta-adrenoceptors), resulting in significantly increased Cx43-expression, while Cx40 was unaffected. In further experiments cells were incubated with either phenylephrine (alpha-adrenergic agonist) or isoproterenol (beta-adrenergic agonist) (0.1-1000 nM) for 24 h. Both catecholamines lead to a concentration-dependent increase in Cx43 protein and mRNA expression (EC50: 10-20 nM). Inhibition experiments showed that the phenylephrine effect was transduced via PKC, while the isoproterenol effect was mediated by PKA. Dual whole-cell voltage clamp demonstrated that increased Cx43-expression was accompanied by significant increases in gap junction current. In additional in vivo experiments, adult rats were subjected to 24-h infusion of isoproterenol or phenylephrine showing again significant increase in Cx43 but not Cx40. Adrenergic stimulation of cardiomyocytes can enhance Cx43 expression thereby increasing cellular coupling, indicating a possible role for catecholamines in the regulation of cardiac gap junction expression in cardiac disease.

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Chronic regulation of the expression of the gap junction protein connexin 43 in transfected HeLa cells

Cited by 8 publications

References 47 publications

The signal transduction cascade regulating the expression of the gap junction protein connexin43 by β‐adrenoceptors

The signal transduction cascade regulating the expression of the gap junction protein connexin43 by β‐adrenoceptors

Signal Transduction and Transcriptional Control of Cardiac Connexin43 Up-Regulation after α₁-Adrenoceptor Stimulation

Subchronic alpha‐ and beta‐adrenergic regulation of cardiac gap junction protein expression

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Chronic regulation of the expression of the gap junction protein connexin 43 in transfected HeLa cells

Cited by 8 publications

References 47 publications

The signal transduction cascade regulating the expression of the gap junction protein connexin43 by β‐adrenoceptors

The signal transduction cascade regulating the expression of the gap junction protein connexin43 by β‐adrenoceptors

Signal Transduction and Transcriptional Control of Cardiac Connexin43 Up-Regulation after α1-Adrenoceptor Stimulation

Subchronic alpha‐ and beta‐adrenergic regulation of cardiac gap junction protein expression

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Signal Transduction and Transcriptional Control of Cardiac Connexin43 Up-Regulation after α₁-Adrenoceptor Stimulation