Id3 is a novel atheroprotective factor containing a functionally significant single-nucleotide polymorphism associated with intima-media thickness in humans

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.

Figure 1. Tagging SNPs within the human ID3 gene

A, Schematic depicting the six tagging SNPs in the human ID3 gene. Approximate sites of RNA transcripts and protein products are noted beneath the gene sequence. B, LD plot depicting the two haplotype blocks encompassing the six ID3 tagging SNPS in the DHS analysis. Numbers reflect D' values.

Figure 2. The variant ID3 allele at rs11574 demonstrates decreased binding to E12

A, NIH3T3 cells were transfected with either the pEF4-Id3105A or pEF4-Id3105T plasmids and analyzed by Western blotting 48 hours after transfection. To control for potential differences in transfection efficiency between Id3105A and Id3105T, expression of Id3 was normalized to expression levels of ShBle in the same samples. β-actin was used as a loading control. Results are representative of three independent experiments in triplicate. B, NIH3T3 fibroblasts were transfected with either Id3105A or Id3105T constructs and FLAG-E12. Forty-eight hours after transfection, total lysates were precipitated using G sepharose beads that had been pre-conjugated to an Id3 antibody. Immunoprecipitates and total lysates were separated by SDS-PAGE and immunoblotted with FLAG or Id3 antibodies. Results are representative of three independent experiments. C, Quantitation of co-IP experiments in B. p=0.0002.

Figure 3. Id3105T has a decreased ability to inhibit transcription compared with Id3105A

NIH3T3 cells were transfected in triplicate with a smooth muscle alpha actin promoter-reporter (pCAT-SMaA) as well as with Id3 and FLAG-E12 expression vectors as indicated (values in μg). Thirty-six hours after transfection, cells were harvested and assayed for CAT activity. CAT activity was normalized to protein concentration and is presented as fold activation relative to the first group (promoter plus vector only). Samples were immunoblotted with anti-FLAG and anti-Id3 antibodies in parallel to confirm the expected expression patterns. Representative Western blots are shown above their corresponding samples. *:p=0.034. **:p=0.002.

Figure 4. Id3^-/- ApoE^-/- mice have significantly more atherosclerosis than Id3^+/+ ApoE^-/- mice in the descending aorta

Beginning at eight weeks of age, Id3^+/+ ApoE^-/- and Id3^-/- ApoE^-/- mice were fed a chow or Western diet for 16 or 32 weeks. Aortas were perfused with paraformaldehyde, harvested, opened longitudinally and stained with Sudan IV. En face lesion area was quantitated using Image-Pro 5.0 software. A, Representative vessels from Id3^+/+ ApoE^-/- and Id3^-/- ApoE^-/- mice. Regions shown are of the descending aorta from the bifurcation of the iliac arteries to the bifurcation of the left subclavian (left to right). B, Quantitation of lesion area by en face analysis in Id3^+/+ ApoE^-/- and Id3^-/- ApoE^-/- mice after 16 weeks of chow diet, 32 weeks of chow diet or 32 weeks of Western diet. Each point represents one animal. *: p = 0.001. **: p = 0.005.

Figure 5. Id3^-/- ApoE^-/- mice have significantly more atherosclerosis than Id3^+/+ ApoE^-/- mice in the aortic arch

Id3^+/+ ApoE^-/- and Id3^-/- ApoE^-/- mice were fed a chow or Western diet for 16 weeks. Aortas were perfused with paraformaldehyde and the ascending portions from the aortic cusp to the bifurcation of the brachiocephalic artery were removed and paraffin embedded. Embedded tissue was sectioned into five μm thick intervals. Ten sections, 150 μm apart were stained by the Movat method and analyzed using Image-Pro 5.0 software. A, Representative cross sections from matched regions of the aortas of Id3^+/+ ApoE^-/- and Id3^-/- ApoE^-/- mice after 16 weeks of Western feeding. Quantitation of: B atherosclerosis in Id3^+/+ ApoE^-/- and Id3^-/- ApoE^-/- mice after 16 weeks of Western diet for cross sectional lesion area (*:p = 0.0002), C, cellular vs. acellular content, and D, percent plaque that is cellular vs. acellular.