Abstract 9798: The E3 lLgase Asb2 Regulates Cardiac Development Through Targeting Smad9 for Proteasomal Degradation
A growing body of evidence revealed that temporal regulation of transcriptional factors is crucially required for cardiac development. Although the induction of transcription factors such as Nkx2.5 and Tbx5 has been of particular interest, the role and mechanism of their timely down-regulation during cardiac development is not fully elucidated. We hypothesized that E3 ligase-dependent degradation of transcription factors plays crucial roles in cardiac development. To identify key E3 ligases regulating cardiac development, we induced three different populations of cardiomyocytes from murine embryonic stem cells treated with any of Noggin, Frizzled or vehicle. We analyzed the temporal changes in gene expressions of them and finally identified an E3 ligase, namely ankyrin repeat and socs box protein2 (Asb2). Both whole mount in situ hybridization of mouse embryos and subsequent RT-PCR revealed that the Asb2β transcript, an isoform of Asb2, is present predominantly in heart during the early embryonic stage. We found that Smad9, an intracellular transducer of the bone morphogenetic protein signals, interact with Asb2β. Biochemical analysis revealed that Asb2β specifically ubiquitinate Smad9, but not Smad1/5. Pulse chase analysis demonstrated that Smad9 was targeted for proteasomal degradation by Asb2β. Knockdown of zebrafish asb2 using morpholino caused significant thinning of the ventricular wall (3.6 ± 0.9 vs. 8.7 ± 1.0μm, p <0.01 ) and remarkable dilatation of the ventricular diameter (23.7 ± 1.1 vs. 11.3 ± 0.7μm, p<0.01 ) compared with wild type siblings. Importantly, simultaneous knockdown of smad9 rescued the impairment of cardiac development due to asb2 knockdown in zebrafish, whereas single knockdown of smad9 caused no abnormal phenotype. These results imply that the suppression of Smad9 is crucial for cardiac development. Also we confirmed that forced expressions of Smad9 in P19CL6 cells failed in cardiac differentiation while that of Asb2β did not affect their fate. We conclude that the accumulation of Smad9 critically impairs the cardiac development and the temporal degradation of Smad9 mediated by E3 ligase Asb2 is critically required for cardiognesis.
- © 2013 by American Heart Association, Inc.