Background: The bone morphogenetic protein (BMP) pathway plays crucial roles in cardiac development. Recent studies have reported that mutations in Smad9, one of the regulatory Smad specific for the BMP pathway, might result in cardiovascular diseases. However, both regulation and function of Smad9 in the cardiovascular system have not been elucidated.
Methods and Results: We conducted DNA microarray using P19CL6 cells with forced expression of Smad9. Microarray analysis using Ingenuity Pathway Analysis elucidated that 19 genes including Tbx2 were related to BMP pathway and showed significantly altered expression levels by transient expression of Smad9. We confirmed by qRT-PCR that only Tbx2, but not other Tbx families, were induced by Smad9. Importantly, the expression of Tbx2 was more up-regulated by Smad9 than by Smad1. Moreover, we identified Asb2 as a specific E3 ligase that targets Smad9, but not Smad1/5, for proteasomal degradation. The in situ hybridization using murine embryo revealed that Asb2 is expressed predominantly in the heart during embryonic development, suggesting that Asb2 quantitatively regulates Smad9 in the developing heart. Biochemical analysis demonstrated that Tbx2 expression induced by Smad9 was attenuated by Asb2, which was restored by the treatment with proteasome inhibitor, lactacystin. Developmental studies using both P19CL6 cells and zebrafish showed that the ablation of Asb2 leads accumulation of Smad9 resulting in the up-regulation of Tbx2, which attenuates myocardial development while induces non-myocardial tissue including cardiac cushion. Indeed, alcian blue staining of morpholino-mediated knockdown of zebrafish Asb2 showed significantly dilated ventricle and thinned ventricular wall, accompanied with decreased myocardium and increased cardiac jelly.
Conclusions: Smad9 induces the expression of Tbx2 during cardiac development and is temporally and quantitatively regulated by its specific E3 ligase Asb2. This is the first study to show both the target gene and specific E3 ligase of Smad9.