The Functional Analysis Of GEFT In Heart

The heart is the first organ to form during embryogenesis, and play an important role in the whole process of life. Cardiac organogenesis is characterized by the precise temporal and spatial-specific regulation of cell proliferation, migration, death, and differentiation. The process of cardiac organogenesis involves a lot of regulatory factors and related signaling pathways.The genetic mutation or abnormal expression of those factors will lead to abnormal heart development. The Rho family of small GTPases, which includes RhoA, Rac1 and Cdc42, controls a wide range of cellular processes in eukaryotic cells, such as normal cell growth, proliferation, differentiation, gene regulation and cell fate determination. The activation of Rho-GTPases requires the exchange of GDP to GTP, a process catalyzed by guanine nucleotide exchange factors. GEFT is a newly identified guanine nucleotide exchange factor, which can specifically activate Rho family of small GTPase. It is highly expressed in the excitable tissue as heart and skeletal muscle. It has been reported that GEFT plays important roles in many cellular processes, such as cell proliferation, migration, and cell fate decision. However, the roles in vivo of GEFT remains unknown. Here, we generated a GEFT conditional knockout mouse through Cre-mediated deletion of the GEFT gene in heart. The hearts of GEFT knockout mice exhibit normal phenotype, which indicated GEFT is dispensable for the development of heart in mouse. But we found that the hearts in the GEFT conditional knockout mice show serious myocardial hypertrophy after isoproterenol stimuli suggesting that GEFT knockout mice might has losed some fuctions of inhibition to hypertrophic stimulis. By western-blot, we find the expression of Cdc42 is mildly repressed in the heart of GEFT conditional knockout mice. However, the inhibition become obvious after isoproterenol treatment. In addition, using RT-RCR to test myocardial hypertrophy related factors, we find the expression of p21, SRF and ANF are up-regulated in GEFT specifically knockout mice. We also detected the ability of GEFT to regulate expression of a luciferase reporter controllded by the p21、ANF、3TP or BRE promoter in 293 A cells overexpressded GEFT. All of the assays all show that GEFT significantly suppresses the transcriptional activity of p21、ANF、3TP and BRE.These results together indicated that serious myocardial hypertrophy after isoproterenol stimuli in GEFT knockout mouse was associated with up-regulation of p21 and other hypertrophy related factors. This research is the firstly time shows the functions of GEFT in vivo, and explored the downstream mechanism of GEFT.