K. Hultman (1, 2), A. Tjarnlund-Wolf (1, 2), J. Odeberg (3), P. Eriksson (3), C. Jern (1, 2)
(1) Institute of Neuroscience and Physiology, Department of Clinical Neuroscience and Rehabilitation, the Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; (2) Department of Clinical Genetics, Sahlgrenska University Hospital, Gothenburg, Sweden; (3) Atherosclerosis Research Unit, Department of Medicine, Karolinska Institute, Stockholm, Sweden
Plasminogen activator inhibitor type 1, Human astrocytes, haploChIP, transcriptional activity
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.
Ryuta Nakamura1,2,3 , Kazuo Umemura 1 , Hisakuni Hashimmoto 2 ,Tetsumei Urano 3
Thromb Haemost 2006 95 4: 637-642
Egbert K. O. Kruithof
Thromb Haemost 2008 100 6: 969-975