title: Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.
abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.
authors.affiliation: Heart Centre, Children's Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, PR China; Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, PR China.
authors.forename: Lina
authors.initials: L
authors.lastname: Si
authors.affiliation: Heart Centre, Children's Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, PR China; Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, PR China.
authors.forename: Jin
authors.initials: J
authors.lastname: Shi
authors.affiliation: Heart Centre, Children's Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, PR China; Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, PR China.
authors.forename: Wenqun
authors.initials: W
authors.lastname: Gao
authors.affiliation: Heart Centre, Children's Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Zheng
authors.affiliation: Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, PR China.
authors.forename: Lingjuan
authors.initials: L
authors.lastname: Liu
authors.affiliation: Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Heart Centre, Children's Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014, PR China. Electronic address: jietian@cqmu.edu.cn.
authors.forename: Jie
authors.initials: J
authors.lastname: Tian
mesh_terms: D000107:Acetylation; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism*; D002460:Cell Line; D050991:GATA4 Transcription Factor / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D006657:Histones / Q000378:metabolism*; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D032383:Myocytes, Cardiac / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D051901:Smad4 Protein / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D050991:GATA4 Transcription Factor; C084365:Gata4 protein, rat; D006657:Histones; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C524161:Nkx2-5 protein, rat; D051901:Smad4 Protein; C490811:Smad4 protein, rat; D014157:Transcription Factors
keywords: BMP2; GATA4; Histone H3 acetylation; Nkx2.5; Smad4