Abstract 21260: SIRT1 Regulates Notch4 Signaling In Endothelial Cells
Signalling by Notch receptors is a key cell-cell communication mechanism essential for the development and homeostasis of the cardiovascular system. In a previous study we identified the NAD+-dependent deacetylase SIRT1 as a novel negative regulator of endothelial Notch1 signaling. We showed that the Notch1 intracellular domain (N1ICD) is reversibly acetylated and that SIRT1 binds to N1ICD to promote its deacetylation and degradation during blood vessel growth. However, endothelial cells (ECs) also express Notch4 and genetic mouse models demonstrate that when deleted along with Notch1, Notch4 aggravates the vascular defects observed in Notch1 deficient mice suggesting that the two receptors have non-redundant functions and that Notch4 controls important processes during vessel formation. To study whether regulation of Notch signaling by SIRT1 is a general mechanism of Notch regulation, we investigated if endothelial Notch4 signaling is regulated by reversible acetylation and targeted by SIRT1. To this end, we first analyzed whether the Notch4 intracellular domain (N4ICD), like the N1ICD, is acetylated by the lysine acetyltransferases PCAF and p300. Co-expression of N4ICD and PCAF or p300 resulted in a robust acetylation of the N4ICD. This acetylation coincided with increased N4ICD protein levels suggesting that acetylation controls N4ICD protein stability. As the N1ICD, N4ICD interacts with with SIRT1, indeed overexpressed and endogenous SIRT1 associated with the N4ICD and SIRT1 dose-dependently decreased N4ICD protein levels, while leaving the Notch4 mRNA expression unaffected. Conversely, knock down of endogenous SIRT1 in ECs cells led to a robust increase in N4ICD protein levels confirming the SIRT1-mediated destabilization of the N4ICD. More importantly, the increase in N4ICD stability in SIRT1-deficient ECs was paralleled by a profound increase in Notch activity and direct Notch target gene expression. Collectively, these data demonstrate that N4ICD, like N1ICD, is negatively regulated by SIRT1 and imply that the acetylation-dependent regulation of Notch responses by the opposing actions of lysine acetyltransferases and SIRT1 is a general regulatory mechanism to control the signaling dynamics of different Notch receptors.
- © 2010 by American Heart Association, Inc.