Allele-specific transcription of the PAI-1 gene in human astrocytes

Summary

The 4G allele of the PAI-1 –675(4G/5G) insertion/deletion promoter polymorphism has been associated with elevated plasma levels of PAI-1 and an increased risk of myocardial infarction. However, this allele has also been associated with a reduced risk of ischaemic stroke. In the brain, PAI-1 is mainly produced by astrocytes, and can reduce the neurotoxic effects exerted by tissue-type plasminogen activator during pathophysiologic conditions. The aim of the present study was to investigate whether the PAI-1 –675(4G/5G) polymorphism and the linked –844A/G polymorphism affect transcriptional activity of the PAI-1 gene in human astrocytes. Haplotype chromatin immunoprecipitation (haploChIP) was used in order to quantify allele-specific promoter activity in heterozygous cells. Protein-DNA interactions were investigated by electrophoretic mobility shift assay (EMSA). A clear allele-specific difference in PAI-1 gene expression was observed in astrocytes, where the haplotype containing the 4G and the –844A alleles was associated with higher transcriptional activity compared to the 5G and –844G-containing haplotype. EMSA revealed an allele-specific binding of nuclear proteins to the 4G/5G site as well as to the –844A/G site. Supershift experiments identified specific binding of the transcription factors Elf-1 and Elk-1 to the –844G allele. The relative impact of the different sites on allele-specific PAI-1 promoter activity remains to be determined. We demonstrate that common polymorphisms within the PAI-1 promoter affect transcriptional activity of the PAI-1 gene in human astrocytes, thus providing a possible molecular genetic mechanism behind the association between PAI-1 promoter variants and ischaemic stroke.

Keywords

Human astrocytes - haploChIP - plasminogen activator inhibitor type 1 - transcriptional activity

• References

• 1 Ye Z, Liu EH, Higgins JP. et al. Seven haemostatic gene polymorphisms in coronary disease: meta-analysis of 66,155 cases and 91,307 controls. Lancet 2006; 367: 651-658.
• 2 Boekholdt SM, Bijsterveld NR, Moons AH. et al. Genetic variation in coagulation and fibrinolytic proteins and their relation with acute myocardial infarction: a systematic review. Circulation 2001; 104: 3063-3068.
• 3 Henry M, Chomiki N, Scarabin PY. et al. Five frequent polymorphisms of the PAI-1 gene: lack of association between genotypes, PAI activity, and triglyceride levels in a healthy population. Arterioscler Thromb Vasc Biol 1997; 17: 851-858.
• 4 Anderson JL, Muhlestein JB, Habashi J. et al. Lack of association of a common polymorphism of the plasminogen activator inhibitor-1 gene with coronary artery disease and myocardial infarction. J Am Coll Cardiol 1999; 34: 1778-1783.
• 5 Grubic N, Stegnar M, Peternel P. et al. A novel G/A and the 4G/5G polymorphism within the promoter of the plasminogen activator inhibitor-1 gene in patients with deep vein thrombosis. Thromb Res 1996; 84: 431-443.
• 6 Abboud N, Ghazouani L, Saidi S. et al. Association of PAI-1 4G/5G and –844G/A gene polymorphisms and changes in PAI-1/tissue plasminogen activator levels in myocardial infarction: a case-control study. Genet Test Mol Biomarkers 2010; 14: 23-27.
• 7 Verschuur M, Jellema A, Bladbjerg EM. et al. The plasminogen activator inhibitor-1 (PAI-1) promoter haplotype is related to PAI-1 plasma concentrations in lean individuals. Atherosclerosis 2005; 181: 275-284.
• 8 Haselbauer A, Haberbosch W, Tillmanns H. et al. The impact of the PAI-1 A((-844))G promoter polymorphism on the risk and extent of coronary heart disease. Thromb Haemost 2002; 88: 697-698.
• 9 Eriksson P, Kallin B, van ’t Hooft FM. et al. Allele-specific increase in basal transcription of the plasminogen-activator inhibitor 1 gene is associated with myocardial infarction. Proc Natl Acad Sci USA 1995; 92: 1851-1855.
• 10 Dawson SJ, Wiman B, Hamsten A. et al. The two allele sequences of a common polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene respond differently to interleukin-1 in HepG2 cells. J Biol Chem 1993; 268: 10739-10745.
• 11 McCormack LJ, Semple JI, Stickland MH. et al. The effect of number of days in culture and plasminogen activator inhibitor-1 (PAI-1) 4G/5G genotype on PAI-1 antigen release by cultured human umbilical vein endothelial cells. Thromb Res 1998; 92: 199-206.
• 12 Bentley P, Peck G, Smeeth L. et al. Causal relationship of susceptibility genes to ischemic stroke: comparison to ischemic heart disease and biochemical determinants. PLoS One 2010; 05: e9136.
• 13 Roest M, van der Schouw YT, Banga JD. et al. Plasminogen activator inhibitor 4G polymorphism is associated with decreased risk of cerebrovascular mortality in older women. Circulation 2000; 101: 67-70.
• 14 Gravanis I, Tsirka SE. Tissue plasminogen activator and glial function. Glia 2005; 49: 177-183.
• 15 Chmielewska J, Ranby M, Wiman B. Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for ‘second-site’ interactions. Biochem J 1988; 251: 327-332.
• 16 Calabresi P, Napolitano M, Centonze D. et al. Tissue plasminogen activator controls multiple forms of synaptic plasticity and memory. Eur J Neurosci 2000; 12: 1002-1012.
• 17 Tsirka SE, Rogove AD, Bugge TH. et al. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J Neurosci 1997; 17: 543-552.
• 18 Yepes M, Sandkvist M, Moore EG. et al. Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein. J Clin Invest 2003; 112: 1533-1540.
• 19 Yang D, Nemkul N, Shereen A. et al. Therapeutic administration of plasminogen activator inhibitor-1 prevents hypoxic-ischemic brain injury in newborns. J Neurosci 2009; 29: 8669-8674.
• 20 Nagai N, De Mol M, Lijnen HR. et al. Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice. Circulation 1999; 99: 2440-2444.
• 21 Docagne F, Nicole O, Gabriel C. et al. Smad3-dependent induction of plasminogen activator inhibitor-1 in astrocytes mediates neuroprotective activity of transforming growth factor-beta 1 against NMDA-induced necrosis. Mol Cell Neurosci 2002; 21: 634-644.
• 22 Knight JC, Keating BJ, Rockett KA. et al. In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading. Nat Genet 2003; 33: 469-475.
• 23 Weeks JR, Hardin SE, Shen J. et al. Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing. Genes Dev 1993; 07: 2329-2344.
• 24 Jaffe EA, Nachman RL, Becker CG. et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745-2756.
• 25 Wolf AT, Medcalf RL, Jern C. The t-PA –7351C>T enhancer polymorphism decreases Sp1 and Sp3 protein binding affinity and transcriptional responsiveness to retinoic acid. Blood 2005; 105: 1060-1067.
• 26 Hultman KBF, Nilsson M, Wilhelmsson U. et al. Expression of PAI-1 and PN-1 in human astrocytes; response to injury-related factors. J Neurosci Res 2010; 88: 2441-2449.
• 27 Morange PE, Saut N, Alessi MC. et al. Association of plasminogen activator inhibitor (PAI)-1 (SERPINE1) SNPs with myocardial infarction, plasma PAI-1, and metabolic parameters: the HIFMECH study. Arterioscler Thromb Vasc Biol 2007; 27: 2250-2257.
• 28 Buchwalter G, Gross C, Wasylyk B. Ets ternary complex transcription factors. Gene 2004; 324: 1-14.