1. A20 is an atherosclerosis susceptibility candidate gene at theA20 is an atherosclerosis susceptibility candidate gene at the
chromosome 10 locus revealed in an C57 E0 X FVB E0 intercrosschromosome 10 locus revealed in an C57 E0 X FVB E0 intercross
BreslowBreslow Jan l.Jan l. ,, Dansky HDansky H ,, Idel SusanneIdel Susanne ,, Teupser DanielTeupser Daniel ,,
Aim: assess whether A20 differs between C57 and FVB
Parameter and results:
1. Expression levels:
Both with the same A20 mRNA levels in liver, aorta, thymus and spleen.
Similarly, TNFa stimulation lead to the same fold induction of A20 in liver and cultured vascular smooth
muscle cells (VSMC) from both strain.
2. Sequencing A20 cDNA:
different coding region in E627A, located between zinc fingers 4 and 5, generates a putative Casein-Kinase
2
phosphorylation site in C57 two residues from a conserved PKC phosphorylation site. ,
3. Cotransfecting 293 cells + NFkB reporter gene + an expression vector:
C57-A20 is less effective in shutting down TNFa induced NFkB activity than FVB-A20
4. Caspase 3 activity:
C57 cells are less susceptible to induction of apoptosis by TNFa + cyclohexaminde
Conclusion:
In C57 mice less active A20 leads to increased inflammation and reduced apoptosis due to prolonged expression
of survival and pro-inflammatory genes.
In contrast in FVB mice more active A20 results in decreased inflammation and increased apoptosis.
In this manner A20 variation could contribute to differences in atherosclerosis susceptibility between C57 and
FVB strains.
2. • NF-kB activation is classically defined as a
transient response initiated by the
degradation of IkB inhibitor proteins
leading to nuclear import of NF-kB, and
culminating with the resynthesis of IkBa
and subsequent inactivation of the
transcription factor
3. • The A20 gene product is a novel zinc finger protein originally described as a tumor
necrosis factor alpha (TNF)-inducible early response gene in human umbilical vein
endothelial cells (HUVEC). Its described function is to block TNF-induced apoptosis
in fibroblasts and B lymphocytes, but more recently it has also been shown to play a
role in lymphoid cell maturation. The mechanism of action of A20 is unknown. The
aim of our study was to assess the effect of A20 upon endothelial cell activation. By
transfecting bovine aortic endothelial cells (BAEC) with A20 as well as reporter
constructs consisting of the promoters of genes known to be up-regulated during
endothelial cell activation, i.e. E-selectin, interleukin (IL)-8, tissue factor (TF), and
inhibitor of nuclear factor kappaBalpha (IkappaBalpha), we demonstrate that A20
expression inhibits gene up-regulation associated with TNF, lipopolysaccharide
(LPS), phorbol 12-myristate 13-acetate (PMA), and hydrogen peroxide (H2O2)-
induced endothelial cell (EC) activation. The mechanism of action of A20 is in part, or
totally, due to the blockade of nuclear factor kappaB (NF-kappaB), as shown by its
ability to suppress the activity of a NF-kappaB reporter. This effect is specific, as A20
does not block a noninducible, constitutively expressed reporter, Rous sarcoma virus-
luciferase (RSV-LUC); nor does it block the c-Tat-inducible, NF-kappaB-independent
reporter, human immunodeficiency virus-chloramphenicol acetyltransferase (HIV-
CAT). How A20 blocks NF-kappaB is unclear, although we demonstrate that it does
not affect p65 (RelA)-mediated gene transactivation. The inhibition of endothelial cell
activation by A20 is a novel function for A20.
4. BackgroundBackground
-- NF-kappaB induces the
expression of genes involved
in immune response,
apoptosis, inflammation, and
the cell cycle.
-- Certain NF-kappaB-
responsive genes are
activated rapidly after the cell
is stimulated by cytokines and
other extracellular signals.
However, the mechanism by
which these genes are
activated is not entirely
understood.
--A20, the immediate-early TNF-
alpha-responsive gene.
--A20 is a TNF-induced primary
response gene which encodes a
novel zinc finger protein.
--A20 is a cytoplasmic zinc finger
protein that inhibits nuclear factor
kappaB (NF-kappaB) activity and
tumor necrosis factor (TNF)-
mediated programmed cell death
(PCD).
5. BackgroundBackground
• FVB/NJ ApoE-deficient (FVB E0) mice: had higher total cholesterol, HDL
cholesterol, ApoA1, and ApoA2 levels than C57BL/6J ApoE-deficient (C57
E0) mice
• At 16 weeks of age, mean aortic root atherosclerotic lesion area was 7- to 9-
fold higher in chow diet-fed C57 E0 mice and 3.5-fold higher in Western
diet-fed C57 E0 mice compared with FVB E0 mice fed similar diets
• Lesion area in chow diet-fed first-generation mice from a strain intercross
was intermediate in size compared with parental values.
• The distribution of the lesion area in 150 chow diet-fed second-generation
progeny spanned the range of the lesion area in both parental strains
• There were no correlations between total cholesterol, non-HDL
cholesterol, HDL cholesterol, ApoA1, ApoA2, ApoJ, or anti-cardiolipin
antibodies and lesion area in the second-generation progeny. Thus, a
genomic approach may succeed in identifying the genes responsible for the
variation in atherosclerosis susceptibility in these 2 strains of ApoE-deficient
mice, which could not be explained by measured plasma parameters.
Beyaert R. Heyninck K. Van Huffel S.
Biochemical Pharmacology. 60(8):1143-51, 2000 Oct 15.
6. BackgroundBackground
• Control (or regulation) of gene expression can occur at multiple
stages (i.e. activation of gene structure > initiation of transcription >
processing of transcripts > transport to the cytoplasm > translation
of mRNA). Initiation of transcription is one of the most important of
these regulatory events.
• Transcription factors bind DNA and activate transcription through
DIFFERENT INDEPENDENT DOMAINS.
• Transcription factors must recognise specific target sequences
(such as REs) (DNA binding DOMAIN). Having bound the DNA, the
protein activates transcription by binding to other components of the
transcription apparatus (activating DOMAIN).
• the zinc finger motif: one of the DNA binding domain.
• Zinc finger motifs are comprised of a small group of conserved
amino acids which bind a zinc ion, and form an independent domain
in the protein. The ‘finger’ description comes from the loop of amino
acids which protrude from the zinc binding site
7. BackgroundBackground
• Control (or regulation) of gene expression can occur at multiple
stages (i.e. activation of gene structure > initiation of transcription >
processing of transcripts > transport to the cytoplasm > translation
of mRNA). Initiation of transcription is one of the most important of
these regulatory events.
• Transcription factors bind DNA and activate transcription through
DIFFERENT INDEPENDENT DOMAINS.
• Transcription factors must recognise specific target sequences
(such as REs) (DNA binding DOMAIN). Having bound the DNA, the
protein activates transcription by binding to other components of the
transcription apparatus (activating DOMAIN).
• the zinc finger motif: one of the DNA binding domain.
• Zinc finger motifs are comprised of a small group of conserved
amino acids which bind a zinc ion, and form an independent domain
in the protein. The ‘finger’ description comes from the loop of amino
acids which protrude from the zinc binding site
