title: Functional analyses of three Csx/Nkx-2.5 mutations that cause human congenital heart disease.
abstract: A homeodomain-containing transcription factor Csx/Nkx-2.5 is an important regulator of cardiogenesis in mammals. Three different mutants, Gln170ter (designated A) and Thr178Met (designated B) in the helix 2 of the homeodomain and Gln198ter mutation (designated C) just after homeodomain, have been reported to cause atrial septal defect with atrial ventricular block. We here examined the functions of these three mutants of Csx/Nkx-2.5. The atrial natriuretic peptide (ANP) promoter was activated by wild type Csx/Nkx-2.5 (WT, approximately 8-fold), B ( approximately 2-fold), and C ( approximately 6-fold) but not by A. When A, B, or C was cotransfected into COS-7 cells with the same amount of WT, WT-induced activation of the ANP promoter was attenuated by A and B (A > B), whereas C further enhanced the activation. Immunocytochemical analysis using anti-Myc tag antibody indicated that transfected Myc-tagged WT, B, and C were localized in the nucleus of both COS-7 cells and cardiomyocytes of neonatal rats, whereas A was distributed diffusely in the cytoplasm and nucleus in COS-7 cells. Electrophoretic mobility shift assay showed that Csx/Nkx-2.5-binding sequences were bound strongly by WT and C, weakly by B, but not by A. Immunoprecipitation and GST pull-down assay revealed that WT and all mutants interacted with GATA-4. The synergistic activation of the ANP promoter by WT and GATA-4 was further enhanced by C but was inhibited by A and B. In the cultured cardiomyocytes, overexpression of C but not WT, A, or B, induced apoptosis. These results suggest that although the three mutants induce the same cardiac phenotype, transactivation ability and DNA binding ability are different among the three mutants and that apoptosis may be a cause for C-induced cardiac defect.
authors.affiliation: Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo 113-8655, Japan.
authors.forename: W
authors.initials: W
authors.lastname: Zhu
authors.forename: I
authors.initials: I
authors.lastname: Shiojima
authors.forename: Y
authors.initials: Y
authors.lastname: Hiroi
authors.forename: Y
authors.initials: Y
authors.lastname: Zou
authors.forename: H
authors.initials: H
authors.lastname: Akazawa
authors.forename: M
authors.initials: M
authors.lastname: Mizukami
authors.forename: H
authors.initials: H
authors.lastname: Toko
authors.forename: Y
authors.initials: Y
authors.lastname: Yazaki
authors.forename: R
authors.initials: R
authors.lastname: Nagai
authors.forename: I
authors.initials: I
authors.lastname: Komuro
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D009320:Atrial Natriuretic Factor / Q000235:genetics*; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D050991:GATA4 Transcription Factor; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006331:Heart Diseases / Q000151:congenital* / Q000209:etiology*; D006344:Heart Septal Defects, Atrial / Q000235:genetics; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008856:Microscopy, Fluorescence; D009154:Mutation; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D026362:Serum Response Factor; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D029867:Xenopus Proteins
chemical_list: D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; D009687:Nuclear Proteins; D018047:Receptors, Purinergic P1; D026362:Serum Response Factor; D014157:Transcription Factors; D029867:Xenopus Proteins; D009320:Atrial Natriuretic Factor; D005982:Glutathione Transferase