title: Analysis of the rat connexin 43 proximal promoter in neonatal cardiomyocytes.
abstract: 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 beta-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.
authors.affiliation: Department of Medical Physiology, University Medical Center Utrecht, P.O. Box 85060, 3508 AB Utrecht, The Netherlands. B.E.J.Teunissen@med.uu.nl
authors.forename: Birgit E J
authors.initials: BE
authors.lastname: Teunissen
authors.forename: Anita T
authors.initials: AT
authors.lastname: Jansen
authors.forename: Shirley C M
authors.initials: SC
authors.lastname: van Amersfoorth
authors.forename: Terrence X
authors.initials: TX
authors.lastname: O'Brien
authors.forename: Habo J
authors.initials: HJ
authors.lastname: Jongsma
authors.forename: Marti F A
authors.initials: MF
authors.lastname: Bierhuizen
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation; D015870:Gene Expression; D005786:Gene Expression Regulation; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D020218:Response Elements / Q000235:genetics; D016415:Sequence Alignment; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D018031:Connexin 43; D004268:DNA-Binding Proteins; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; C484469:Nkx2-5 protein, mouse; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; C497123:Sp3 protein, rat; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D051705:Sp3 Transcription Factor