Homocysteine downregulates cardiac homeobox transcription factor NKX2.5 via IGFBP5.

Homocysteine (Hcy) is an independent risk factor of congenital heart disease (CHD), but its exact underlying mechanism is unclear. In this study, we collected amniotic fluid (AF) supernatant samples from pregnant women carrying CHD-affected (n = 16) or normal (n = 16) fetuses. We found that Hcy concentrations were higher in the AF of the CHD group when compared with normal pregnancies. Also, Western blot showed that NK2 homeobox 5 (NKX2.5) was decreased and insulin-like growth factor binding protein 5 (IGFBP5) was increased in the AF of the CHD group. In the H9C2 cell culture experiment, 500 μmol/L Hcy downregulated NKX2.5 and upregulated IGFBP5. Real-time PCR and Western blot showed that NKX2.5 expression was reduced in H9C2 cells treated with IGFBP5. Luciferase reporter gene demonstrated that IGFBP5 decreased the transcription of the NKX2.5 promoter. Chromatin immunoprecipitation and electrophoretic mobility shift assay suggested that IGFBP5 binds to the NKX2.5 promoter region. Thus, the data indicated that one of the possible mechanisms by which Hcy is involved in CHD may be that Hcy inhibits NKX2.5 expression partly through IGFBP5.NEW & NOTEWORTHY We found that Hcy and IGFBP5 were increased, whereas NKX2.5 was decreased, in AF of CHD. Meanwhile, Hcy could upregulate IGFBP5 but downregulate NKX2.5, and IGFBP5 inhibited NKX2.5 expression in vitro. Moreover, IGFBP5 can bind to the NKX2.5 promoter region and reduce NKX2.5 transcriptional activity.