Loss of Cardioprotective Effects at the ADAMTS7 Locus as a Result of Gene-Smoking Interactions

BACKGROUND: Common diseases such as coronary heart disease (CHD) are complex in etiology. The interaction of genetic susceptibility with lifestyle factors may play a prominent role. However, gene-lifestyle interactions for CHD have been difficult to identify. Here, we investigate interaction of smoking behavior, a potent lifestyle factor, with genotypes that have been shown to associate with CHD risk. METHODS: We analyzed data on 60 919 CHD cases and 80 243 controls from 29 studies for gene-smoking interactions for genetic variants at 45 loci previously reported to be associated with CHD risk. We also studied 5 loci associated with smoking behavior. Study-specific gene-smoking interaction effects were calculated and pooled using fixed-effects meta-analyses. Interaction analyses were declared to be significant at a P value of <1.0x10(-3) (Bonferroni correction for 50 tests). RESULTS: We identified novel gene-smoking interaction for a variant upstream of the ADAMTS7 gene. Every T allele of rs7178051 was associated with lower CHD risk by 12% in never-smokers (P= 1.3x10(-16)) in comparison with 5% in ever-smokers (P= 2.5x10(-4)), translating to a 60% loss of CHD protection conferred by this allelic variation in people who smoked tobacco (interaction P value= 8.7x10(-5)). The protective T allele at rs7178051 was also associated with reduced ADAMTS7 expression in human aortic endothelial cells and lymphoblastoid cell lines. Exposure of human coronary artery smooth muscle cells to cigarette smoke extract led to induction of ADAMTS7. CONCLUSIONS: Allelic variation at rs7178051 that associates with reduced ADAMTS7 expression confers stronger CHD protection in never-smokers than in ever-smokers. Increased vascular ADAMTS7 expression may contribute to the loss of CHD protection in smokers.Peer reviewe

AA31-51 are required for nuclear localization of TRIB1.

<p>Top, schema of the deletions and insertions introduced in FLAG-tagged TRIB1. Bottom, immunofluorescence analysis of HeLa cells transfected for 48 h with the indicated TRIB1 mutants. FLAG tagged TRIB1 expressing cells were fixed, permeabilized and examined by confocal microscopy using an antibody specific for the FLAG epitope and an anti-mouse secondary (Alexa 647 goat anti-mouse, Molecular Probes). Nuclei were counterstained with Hoechst 33342.</p

TRIB1 NLS is located near its N terminus.

<p>Variants of TRIB1 fused C-terminal to eCFP were expressed in HeLa cells for 24 h, fixed, permeabilized and subjected to fluorescence confocal microscopy using a 440 nm laser. Hoechst 33342 was included to visualize the nucleus. Data represent 2 independent experiments. Schema of the deletions is shown on the right.</p

Proteasome inhibitors increase TRIB1 but do not prevent TRIB1 destabilization in stably transduced cells.

<p>Cells were treated for 5 h with vehicle, CHX or ACTD, in combination with MG132 (20 μM) or Bortezomib (BTZ, 10 μM), as indicated. A, Lysates were subjected to western blot using the indicated antibodies. B, mRNA was isolated and quantified by qRT-PCR for <i>TRIB1</i>. Values are normalized to vehicle alone (DMSO) and represent the mean of biological triplicates ± S.D.</p

Endogenous TRIB1 is undetectable by straight western blot.

<p>A, HeLa, 293T and ASMC lysates were lysed in IP buffer and resolved by SDS-PAGE. Purified GST-TRIB1 fusion (GT1) protein was included as positive control. B, HeLa and 293T cells treated for 48 h with a TRIB1 (ASO1) or a control (NT) antisense oligonucletide were lysed in IP buffer supplemented with protease and phosphatase inhibitor cocktails, resolved by SDS-PAGE and analyzed by western blotting using 2 distinct TRIB1 antibodies. Cell lines stably transduced with TRIB1 and lysed under the same conditions were used as positive control. TRIB1 bands (indicated by side bars) had apparent masses ranging from 43–48 kDa. * indicates residual TUBB signal.</p

Endogenous <i>TRIB1</i> is expressed at low levels in several cell models.

<p>Left, RNA quantification by RNAseq calculated from to the Human Protein Atlas portal data, using PFKM counts for the indicated tissue (SMC, Liver) or cell lines (HepG2, HEK293). Right, RNA quantification by qRT-PCR in the indicated cell models. Abundance is normalized to PPIA. Quantification of 3–6 experiments is shown (± 95% CI).</p

Minimal impact of untranslated regions on TRIB1 stability.

<p>Stable HeLa cell lines expressing TRIB1 with native 5’ and 3’ UTRs (HeLaT1UTR). Cells were treated with MG132 (20 μM) or BTZ (10 μM) or vehicle (0.1% DMSO) in the presence or absence of CHX for 5 h. Cells were lyzed and analyzed by western blotting using TRIB1 or TUBB antibodies. Data are representative of 3 biological replicates.</p

<i>TRIB1</i> transcript is unstable and increased by translation inhibitors.

<p>A, RNA from HeLa cells and ASMC treated with the indicated drugs for 5 h were harvested for quantification by qRT-PCR using primers specific for <i>TRIB1</i>. Values are first normalized to the housekeeping gene <i>PPIA</i> and then expressed relative to the Ctl values (Ctl = 1). B, as in A except that <i>TRIB2</i> and <i>TRIB3</i> transcripts were measured by qRT-PCR. * indicates statistical difference from control (p < 0.05, 2-tailed unpaired Student’s t- test) while in A, all changes were significant relative to the control. Error bars represent 95% confidence intervals.</p

Suppression of βTRCP increases <i>TRIB1</i> expression but does not affect its instability.

<p>HeLaT1 cells incubated for 48 h with siRNA targeting CUL1, βTRCP or a non-target control were treated with vehicle (H₂0) or CHX for 5 h. Parallel lysates were then analyzed by western blot and qPCR. A, qRT-PCR of RNA isolated from cells treated with the indicated siRNAs for 48 h. Quantification of corresponding transcript is shown, normalized to the non-target oligonucleotide (NT). B, western blot of extracts from silenced cells. Data shown are representative of 3 biological replicates. C, βTRCP and CUL1 suppression increase <i>TRIB1</i> transcript abundance. qRT-PCR of <i>TRIB1</i> in cells suppressed for the indicated siRNAs. In A and C, data represent the average of 2 biological replicates ± S.D.</p

TRIB1 is unstable in HeLaT1 and 293T1 <i>in vivo</i> but stable <i>in vitro</i>.

<p>A, stably integrated pools of cells were treated with CHX or ACTD for 5 h and analyzed by western blot using TRIB1(TRIB1g (goat) or TRIB1r (rabbit)) and TUBB specific antibodies. Data are representative of 3 experiments. Quantification presented is for the TRIB1g and TUBB only. B, TRIB1 stable cells were lysed in IP buffer with no additives, diluted in assay buffer and incubated at 37°C for the indicated time. Western blot was performed with the indicated antibodies.</p