ABSTRACT
RATIONALE: The gene encoding the helix-loop-helix transcription factor Id3 (inhibitor of differentiation-3) is located within atherosclerosis susceptibility loci of both mice and humans, yet its influence on atherosclerosis is not known. OBJECTIVE: The present study sought to determine whether polymorphisms in the ID3 gene were associated with indices of atherosclerosis in humans and if loss of Id3 function modulated atherogenesis in mice. METHODS AND RESULTS: Six tagging single-nucleotide polymorphisms (SNPs) (tagSNPs) in the human ID3 gene were assessed in participants of the Diabetes Heart Study. One tagSNP, rs11574, was independently associated with carotid intima-media thickness (IMT). The human ID3 variant at rs11574 results in an alanine to threonine substitution in the C terminus. To determine the effect of this polymorphism on the basic function of Id3, site-directed mutagenesis of the human ID3 gene at rs11574 was performed. Results demonstrated a significant reduction in coimmunoprecipitation of the known E-protein partner, E12, with Id3 when it contains the sequence encoded by the risk allele (Id3105T). Further, Id3105T had an attenuated ability to modulate E12-mediated transcriptional activation compared to Id3 containing the ancestral allele (Id3105A). Microarray analysis of vascular smooth muscle cells from WT and Id3(-/-) mice revealed significant modulation of multiple gene pathways implicated in atherogenesis. Moreover, Id3(-/-)ApoE(-/-) mice developed significantly more atherosclerosis in response to 32 weeks of Chow or Western diet feeding than Id3(+/+)ApoE(-/-) mice. CONCLUSIONS: Taken together, results provide novel evidence that Id3 is an atheroprotective factor and link a common SNP in the human ID3 gene to loss of Id3 function and increased IMT.
Subject(s)
Carotid Arteries/pathology , Carotid Artery Diseases/genetics , Carotid Artery Diseases/pathology , Diabetes Mellitus, Type 2/complications , Inhibitor of Differentiation Proteins/genetics , Neoplasm Proteins/genetics , Polymorphism, Single Nucleotide , Tunica Intima/pathology , Tunica Media/pathology , Aged , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Carotid Artery Diseases/prevention & control , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/pathology , Disease Models, Animal , Female , Gene Expression Profiling , Gene Expression Regulation , Gene Frequency , Genetic Predisposition to Disease , Humans , Immunoprecipitation , Inhibitor of Differentiation Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Muscle, Smooth, Vascular/metabolism , Mutagenesis, Site-Directed , NIH 3T3 Cells , Neoplasm Proteins/metabolism , Phenotype , Protein Binding , Risk Assessment , Risk Factors , TransfectionABSTRACT
Adiponectin is an adipocyte-derived cytokine with beneficial effects on insulin sensitivity and the development of atherosclerosis. Id3 is a helix-loop-helix factor that binds to E-proteins such as E47 and inhibits their binding to DNA. Although the helix-loop-helix factor sterol regulatory element binding protein (SREBP)-1c is a known activator of adiponectin transcription, this study provides the first evidence of a role for Id3 and E47 in adiponectin expression. Decreased Id3 in differentiating adipocytes correlates with increased adiponectin expression and forced expression of Id3 inhibits adiponectin expression. Moreover, Id3-null mice have increased adiponectin expression in visceral fat tissue and in serum. We demonstrate that E47 potentiates SREBP-1c-mediated adiponectin promoter activation and that Id3 can dose-dependently inhibit this action via interaction with E47. Mutation of a consensus E47 binding site results in nearly complete loss of promoter activation. Furthermore, we demonstrate E47 binding to the endogenous adiponectin promoter both in vitro and in vivo by chromatin immunoprecipitation analysis. Binding is not detected in undifferentiated cells which express Id3 but peaks during differentiation in parallel with Id3 decline. This promoter binding can be completely abolished by the overexpression of Id3 and is enhanced in adipose tissue null for Id3. These data establish Id3 and E47 as novel regulators of SREBP-1c-mediated adiponectin expression in differentiating adipocytes and provide evidence that Id3 regulates adiponectin expression in vivo.
