Influence of inflammatory stimuli and statins on HIVEP1 expression B. Schmitz1,2, A. Salomon3, A. Hillmann4, P.-E. Morange5, F. Cambien6, L. Tiret6, D.-A. Trégouët6, T. Pap4, E. Brand2, S.-M. Brand1 1Institute of Sports Medicine, Department of Molecular Genetics of Cardiovascular Disease, University of Muenster; 2University Hospital Muenster, Internal Medicine D, Department of Nephrology, Hypertension and Rheumatology; 3Medical Faculty of the Westfalian Wilhelms-University of Muenster; 4Institute of Experimental Musculoskeletal Background Medicine, University of Muenster; 5INSERM, UMR_S 626, Marseille; 6INSERM, UMR_S 525, Paris We recently identified a tagging SNP (rs169713), positioned 90 kb upstream of the under different proinflammatory conditions in vascular endothelial cells (EA.hy926) HIVEP1 , to be replicatively associated with venous thrombosis (Morange et and in mice harbouring high TNFαlevels. Since certain statins are suggested to al. , AJHG 2010). In a further genetic study using a larger population size and more harbour antiinflammatory and venous thrombosis preventing capacities, we genetic markers (dense fine map) in formerly proposed phenotype-associated loci, analyzed the effects of clinically prescribed statins on HIVEP1 mRNA and the HIVEP1 gene was confirmed to be highly associated (HIVEP1 exon 4) with expression. Reporter gene assays were conducted to characterize the HIVEP1 venous thrombosis (Germain et al. , PLoS One 2011). As a protein, promoter structure. To identify transcription factors, we performed overexpression, HIVEP1 binds specific DNA sequences including the NF-ĸB consensus sequence bandshift and ChIP assays in EA.hy926 cells. Given the convicting data, our and is suggested to modulate transcriptional regulation of involved in primary goal is to evaluate the causative role of HIVEP1 in venous thrombosis inflammatory processes. HIVEP1 is expressed in human carotid atheroma plaques (inflammatory condition). suggesting a link to cardiovascular disease. We analyzed the expression of HIVEP1 Results

A Figure 1: HIVEP1 mRNA expression is induced 1 A 2 by proinflammatory factors in endothelial cells and mice A Stimulation with TNFα and interleukin IL1-β led to upregulation of endogenous expression of HIVEP1 in EA.hy926 cells. RNA was isolated after treatment with TNFα or IL1-β (10 ng/mL) for 24h. PCR was performed using HIVEP1 specific primers B ***: p<0.001 based on cDNA. hRP27PCR served as loading control. Ø: basic conditions. B Real time PCR revealed a significant increased HIVEP1 mRNA expression in heart tissue of tristetraprolin (TTP)deficient (p<0.001; ~4.0fold) and human TNFα-transgenic (hTNFtg) (p<0.001; ~2.0-fold) mice, compared to wild type (WT). RNA was extracted from heart tissue of 12-weak-old hTNFtg and WT as well as from 20weak-old TTPdeficient mice. HIVEP1 mRNA expression was normalized to reference gene βactin, expressed as fold induction (FI) values relative to WT (FI=1).

3 B *** ** ***: p<0.001 5 *** *** ***: p<0.01 4 ***: p<0.05 ns: not significant ns * 3 ***: p<0.01 *** ns: not significant 2 (fold induction) HIVEP1 298 kDa 1

0

 T S A T P S+T A+ P+T

Figure 2: TNFα-induced HIVEP1 mRNA and protein expression is decreased by statins A Simvastatin and to a lower extent rosuvastatin dose-dependently decreased basal and TNFα-induced HIVEP1 mRNA expression in EA.hy926 cells, while pravastatin and aspirin had no effect on HIVEP1 mRNA expression. In Figure 3: Interaction of nuclear with NF-κB binding sites in the HIVEP1 TNFα-treated cells, low-dose atorvastatin (9 µM) decreased and high-dose atorvastatin (18 µM) increased HIVEP1 promoter is altered by simvastatin treatment mRNA expression. RNA was extracted after treatment of EA.hy926 cells with statins (24h). TNFα was given for 6h. PCR was performed using HIVEP1 specific primers based on cDNA. hRP27PCR served as loading control. EMSA probe AB was designed harbouring both predicted NF-κB binding sites in the HIVEP1 5'-flanking region. Three specific band shifts were detected under basic conditions in EA.hy926 cells. The NF-κB B TNFα-induced HIVEP1 full length protein expression (298 kDa) was significantly decreased predominantly by consensus site (c) competed two of the three band shifts detected (black arrows). Upon stimulation of cells simvastatin and to a lesser extent by atorvastatin and pravastatin in western blot analysis. EA.hy926 cells were with simvastatin (2.4 µM, 24h), in the presence or absence of TNFα (10 ng/mL, 6h), the intensity of the incubated with TNFα (T, 10 ng/mL) for 24h or statins (S: simvastatin, 2.4 μM; A: atorvastatin, 18 μM; P: pravastatin, three band shifts was significantly reduced to 50% compared to basic conditions (p<0.01), while TNFα (10 15 μM) for 30h in combination with TNFα (10 ng/mL), added after 24h for 6h. β-actin served as loading control. ng/mL, 24h) had no influence on the interaction of nuclear proteins with the EMSA probe. In densitometric Densitometric measurement was performed for full length HIVEP1 (298 kDa) (Ø=1). c: cytoplasmic, n: nuclear, measurement basic condition (Ø ) was used as reference and set 1. Ø: basic conditions.

4 5 A Figure 5: SP1 is involved in basal HIVEP1 expression regulation A A EMSA probe was designed according to in B silico identification of SP1 and WT1 binding sites in intron 1 (depicted below). Two band shifts were observed indicating ***: p<0.001 sequence-specific interaction of nuclear proteins with SP1/WT1 binding sites. EMSA was performed with EA.hy926 nuclear extracts (5 µg) B ChIP analysis in EA.hy926 cells using 20 fmol of biotinylated probe. EMSA blots demonstrated in vivo binding of TF SP1 were incubated with SP1- or WT1specific to HIVEP1 promoter portions -307 to - B antibody. The detected specific band (A) was 469 and -916 to 1097 under basic identified to involve sequencespecific binding of conditions. SP1 but not WT1. Chromatin treated with magnetic beads Lane without oligonucleotide served as control or with beads and serum served as (Ø). -: 20 fmol probe; +: 4 pmol sequence- negative control (control 1 and 2, ***: p<0.001 specific competitor respectively). ChIP assays were

SP1 performed using 4 µg specfic antibody SP1 SP1 SP1 WT1 SP1 SP1 SP1 SP1 WT1 SP1 WT1 SP1 SP1 (anti-SP1, antiEGR1, anti-WT1). tcggcgcgggctccgcggcgggggcgctgcagctggggagggcggcggggcggaggggggggggggcaggagcacatcccttcggcgggcggggggcgtgcgggcgcg

C Conclusion

***: p<0.001 * : p<0.05 • TNFα increases HIVEP1 mRNA expression in endothelial cells and mice (Fig. 1). • Statins, predominantly simvastatin, decrease the TNFα-induced HIVEP1 mRNA and protein expression in endothelial cells (Fig. 2). • Simvastatin decreases the interaction of NF-κB with its binding sites in the HIVEP1 Figure 4: Overexpression of TF SP1, EGR1 or WT1 promoter region (Fig. 3). A Addition of 342 bp of intron 1 to HIVEP1 promoter deletion constructs led to a significant decrease of transcriptional activity (TA). • Intron 1 is involved in HIVEP1 expression regulation, since inclusion of a defined part of A/B Cotransfection of (TF) SP1 and EGR1 caused a significant mean 3.9-fold and 4.4-fold increase of TA in EA.hy926 cells, respectively. intron 1 leads to a significant decrease of TA (Fig. 4). C Upregulation of TA upon overexpression with WT1 was only observed for • EGR1 increases TA of all constructs, while WT1 upregulates TA of constructs harbouring constructs harbouring the added intron fragment. FI (Fold induction): Values after adjusting each TA to the pGL3-Basic level. intron 1 exclusively (Fig. 4B/C). Transient transfection was performed and TA assessed as relative light units (RLU). • Cotransfection, EMSA and ChIP experiments demonstrate involvement of SP1 in basal White bars indicate pGL3-Control vector; black bars basic activity of pGL3-Basic. HIVEP1 expression regulation (Fig. 4A, Fig. 5A/B).

Morange PE, Bezemer I, Saut N, Bare L, Burgos G, Brocheton J, Durand H, Biron-Adreani C, Schved JF, Pernod G, Galan P, Drouet L, Zelenika D, Germain M, Nicaud V, Heath S, Ninio E, Delluc A, Münzel T, Zeller T, Brand-Herrmann SM, Alessi MC, Tiret L, Lathrop M, Cambien F, Blankenberg S, Emmerich J, Trégouët DA, and Rosendaal FR. A Follow-Up Study of a Genome-wide Association Scan Identifies a Susceptibility Locus for Venous Thrombosis on 6p24.1. American Journal of Human Genetics 2010;86(4):592-5. Germain M, Saut N, Greliche N, Dina C, Lambert JC, Perret C, Cohen W, Oudot-Mellakh T, Antoni G, Alessi MC, Zelenika D, Cambien F, Tiret L, Bertrand M, Dupuy AM, Letenneur L, Lathrop M, Emmerich J, Amouyel P, Trégouët DA, Morange PE. Genetics of Venous Thrombosis: Insights from a New Genome Wide Association Study. PLoS One. 2011;6(9):e25581.