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Comparative Study
. 2003 Nov 25;100(24):14235-40.
doi: 10.1073/pnas.1835672100. Epub 2003 Nov 12.

A20, a regulator of NFkappaB, maps to an atherosclerosis locus and differs between parental sensitive C57BL/6J and resistant FVB/N strains

Susanne Idel  1 , Hayes M Dansky, Jan L Breslow
Affiliations
Comparative Study

A20, a regulator of NFkappaB, maps to an atherosclerosis locus and differs between parental sensitive C57BL/6J and resistant FVB/N strains

Susanne Idel et al. Proc Natl Acad Sci U S A. .

Abstract

An intercross between atherosclerosis susceptible (C57BL/6J ApoE0) and resistant (FVB/N ApoE0) mice revealed a susceptibility locus on chromosome 10 (11 cM, logarithm of odds 7.8). Surprisingly, the genotypic means for this locus revealed that heterozygosity or homozygosity for the C57BL/6J allele was associated with decreased atherosclerosis. A candidate gene in this region is A20, which is involved in the feedback suppression of NFkappaB activation induced by tumor necrosis factor alpha (TNFalpha). We sequenced the A20 gene coding region from the parental strains and found a single-nucleotide polymorphism resulting in a single amino acid exchange, Glu627Ala (C57BL/6J vs. FVB/N). This mutation introduces a putative casein kinase 2 phosphorylation site in C57BL/6J-A20 not present in FVB/N-A20. NFkappaB reporter gene assays showed that this amino acid change results in less effective termination of TNFalpha-stimulated NFkappaB activation by C57BL/6J-A20. In accordance, the TNFalpha-induced expression of NFkappaB target genes (A20, IkappaBalpha) in vascular smooth muscle cells was prolonged in cells isolated from C57BL/6J compared with FVB/N mice. In light of the genotypic means for atherosclerosis at the chromosome 10 locus in F2 mice from this intercross, the observations now reported suggest that prolonged expression of genes induced by NFkappaB might be antirather than proatherogenic.

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Figures

Fig. 1.
Fig. 1.
Expression of A20 mRNA in different tissues from parental mice. Ratio (C57BL/6J to FVB/N) of A20 mRNA expression levels in various tissues determined by real-time PCR and normalized to β-actin. Averages of three independent experiments ± SD are shown.
Fig. 2.
Fig. 2.
Sequence comparison of C57BL/6J-, FVB/N-, and human A20 cDNA. The amino acid residue 627 that differs between C57BL/6J-A20 and FVB/N-A20 is labeled in red. The putative CKII phosphorylation site introduced in the C57BL/6J sequence is marked by a blue square, whereas the conserved PKC site is framed in green. The asterisks indicate the amino acids that could be phosphorylated by the respective kinases.
Fig. 3.
Fig. 3.
Activity of A20 with mutated phosphorylation sites. HEK293 cells were transfected with the indicated amounts of C57BL/6J-A20, FVB/N-A20, CKII-A20, or noPhos-A20 in addition to an NFκB reporter and a β-gal normalization plasmid. After 4 h of stimulation with TNFα (30 ng/ml), β-gal activity was determined, and NFκB activity was quantified by using a commercial luciferase assay. Shown are averages of nine independent experiments ± SD. (Inset) A20 expression normalized to total protein content (0.01 μg of A20 DNA), visualized by Western blotting, to demonstrate equal expression of different A20 mutants. Lane 1, C57BL/6J-A20; lane 2, CKII-A20; lane 3, FVB/N-A20; lane 4, noPhos-A20. *, P < 0.05; **, P < 0.001.
Fig. 4.
Fig. 4.
Expression of A20 and IκBα after TNFα stimulation in VSMCs. VSMCs isolated from C57BL/6J and FVB/N mice were stimulated with TNFα (30 ng/ml) for the indicated times before preparation of RNA and determination of A20 (A) or IκBα (B) expression levels via real-time PCR. Data shown represent mean ± SD of three independent experiments, i.e., three different cell preparations followed by TNFα incubation, RNA isolation, and expression analysis. *, P < 0.05; **, P < 0.001.
Fig. 5.
Fig. 5.
IκBα protein level after TNFα stimulation in VSMCs. VSMCs isolated from C57BL/6J or FVB/N mice were incubated with TNFα (30 ng/ml) for the indicated times. Afterward, cells were harvested and lysed, and the resulting cell extracts were subjected to Western blot analysis by using an antibody specific for IκBα. Protein expression was quantified by laser densitometry. The data represent means ± SD of three independent experiments. *, P < 0.05; **, P < 0.001.
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