The Role Of RK1/2 Signaling Pathways In The GATA4 Gene Regulatory The Endocardial Cushions Development

Objective:(1) To investigate the effects of GATA4 gene in the endocardial cushions development;(2) To explore the mechanism of EGF MAPK/ERK1/2 signaling pathways in the GATA4 gene regulation of the endocardial cushions developed into atrioventricular valve/septal;(3) To explore the mechanism of BMP4 signaling pathways in the GATA4 gene regulation of the gene which is important in endocardial cushions developed into atrioventricular valve/septal.Methods:(1)Target gene eukaryote expression vectors were constructed by pc DNA3.1(-) vector plasmid, and were identified by DNA sequence analysis. Recombinant plasmids were transfected into Hela cells with lipofectamine 2000, meanwhile Hela cells transfected with empty vector or those without transfection were served as transfection control group and blank control group respectively. Real-time quantitative PCR was used for the measurement of m RNA, Western blotting was used for the measurement of protein of transcription factors GATA4, Sox9, Scleraxis and extracellular matrix(ECM) protein Aggrecan or Tenascin in each group.(2) Hela cells were divided into three groups when they reached 50% confluence: control(untreated), EGF group and U0126+EGF group. After treating with these cytokines for 12 hours, collected cells and extracted the RNA and protein respectively. The expression of phosphorylated ERK1/2, phosphorylated Smad1/5/8 as well as the expressions of ERK1/2, Smad, GATA4, Sox9,Scleraxis, Aggrecan and Tenascin were detected using Western blotting and real-time quantitative PCR analysis.(3) Hela cells were divided into three groups when they reached 50% confluence: control(untreated), BMP4 group, U0126+BMP4 group. After treating 6 hours, collected these cells and extracted the RNA and protein respectively. The expression of phosphorylated ERK1/2, phosphorylated Smad and the expressions of ERK1/2, Smad, GATA4, Sox9, Scleraxis, Aggrecan, Tenascin were detected using Western blotting and real-time quantitative PCR analysis.Results:(1)GATA4 m RNA has a pronounced increasing in GATA4 over-expression group, while GATA4 H436 Y mutation decreased this high expression. The relative m RNA and protein expression of Sox9, Scleraxis, Aggrecan and Tenascin in both experimental group was significantly higher than that in transfection control group and blank control group(P<0.05), and GATA4 H436 Y mutation group showed a pronounced decrease of these gene expression, while there was no significant difference in transfection control group and blank control group(P>0.05).(2)GATA4 gene expression was reduced after EGF treatment, after pre-treatment with U0126 inhibit the promotion of EGF, the expression of GATA4 was up-regulated but was still inferior to control group; with the action of EGF+U0126, the expression of Sox9 、Scx、Agc and Tnc were up regulated significantly compared to EGF group and control group.(3) After BMP4 treatment, the GATA4 and p ERK1/2 expression were up-regulated, p Smad1/5/8 was down-regulated, but U0126 inhibit the effect of BMP4; with the action of BMP4, the expression of Sox9 、Scx、Agc and Tnc were up regulated significantly, similarly, but the promoting effects will be inhibited by U0126.Conclusion: GATA4 H436 Y mutation will lessen its transcriptional activation, which serves as a theoretical framework to demonstrate that GATA4 gene play a pivotal role in the development process of the endocardial cushions. EGF can regulate the expression of GATA4 through MAPK/ERK1/2 signaling pathway, and the EGF act as negative regulators of endocardial cushions formation. BMP4 can regulate GATA4 gene expression through ERK1/2 MAPK signaling pathway and to further adjust the expression of Sox9, Scx, Agc and Tnc, and may play an important role in the process of the endocardial cushions developed into atrioventricular valve/intervals.