Abstract
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.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Actins / genetics
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Actins / metabolism*
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Basic Helix-Loop-Helix Transcription Factors / genetics
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Basic Helix-Loop-Helix Transcription Factors / metabolism*
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Differentiation
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Cells, Cultured
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Coculture Techniques
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Endothelial Cells / metabolism
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Enzyme Inhibitors / pharmacology
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Histone Deacetylase Inhibitors
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Histone Deacetylases / metabolism
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Humans
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Hydroxamic Acids / pharmacology
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Immunoglobulin J Recombination Signal Sequence-Binding Protein / genetics
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Immunoglobulin J Recombination Signal Sequence-Binding Protein / metabolism*
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Muscle, Smooth, Vascular / drug effects
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Muscle, Smooth, Vascular / enzymology
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Muscle, Smooth, Vascular / metabolism*
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Myocytes, Smooth Muscle / drug effects
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Myocytes, Smooth Muscle / enzymology
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Myocytes, Smooth Muscle / metabolism*
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Promoter Regions, Genetic
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Protein Binding
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Protein Structure, Tertiary
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Proto-Oncogene Proteins / metabolism
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RNA, Messenger / metabolism
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Receptor, Notch1 / metabolism
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Receptor, Notch2 / metabolism
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Receptor, Notch4
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Receptors, Notch / chemistry
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Receptors, Notch / genetics
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Receptors, Notch / metabolism*
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Signal Transduction* / drug effects
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Time Factors
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Transcription, Genetic
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Transduction, Genetic
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Up-Regulation
Substances
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Actins
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Basic Helix-Loop-Helix Transcription Factors
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Cell Cycle Proteins
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Enzyme Inhibitors
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HEY1 protein, human
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Histone Deacetylase Inhibitors
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Hydroxamic Acids
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Immunoglobulin J Recombination Signal Sequence-Binding Protein
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NOTCH1 protein, human
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NOTCH2 protein, human
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NOTCH4 protein, human
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Proto-Oncogene Proteins
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RBPJ protein, human
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RNA, Messenger
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Receptor, Notch1
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Receptor, Notch2
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Receptor, Notch4
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Receptors, Notch
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trichostatin A
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Histone Deacetylases