Hairy-related transcription factors inhibit Notch-induced smooth muscle alpha-actin expression by interfering with Notch intracellular domain/CBF-1 complex interaction with the CBF-1-binding site.

Notch signaling regulates smooth muscle cell phenotype and is critical for vascular development. One Notch target is smooth muscle alpha-actin (SMA), a differentiated smooth muscle cell marker. The Notch intracellular domain (NotchICD) forms a complex with CBF-1 (C-promoter-binding factor-1) and directly induces SMA expression. Using primary human smooth muscle cells, we show that expression of the constitutive active ICD of human Notch1, Notch2, or Notch4 receptors increase SMA levels. NotchICD also induce expression of the transcriptional repressors HRT1 (Hairy-related transcription factor 1) and HRT2, in a CBF-1-dependent manner. However, unlike the activating effects of NotchICD, HRT1 or HRT2 represses basal SMA expression, and both are strong antagonists of NotchICD-induced SMA upregulation. This antagonism does not depend on histone deacetylase activity and occurs at the transcriptional level. Competitive coimmunoprecipitation experiments demonstrate that HRT does not disrupt the association of NotchICD and CBF-1, which form a complex in the presence or absence of HRTs. However, HRT suppresses NotchICD/CBF-1 binding to the SMA promoter, as measured by chromatin immunoprecipitation, and transactivation of an SMA promoter reporter spanning sequences -124/+32. SMA expression was regulated similarly following endogenous Notch activation in smooth muscle cells by coculture with endothelial cells, and this effect was also sensitive to HRT inhibition. Temporally defined HRT activity may constitute a negative feedback mechanism of Notch signaling. Our study presents a novel mechanism by which a balance between Notch signaling and HRT activity determines the expression of smooth muscle differentiation markers including SMA.