Abstract
Expression of the tissue-type plasminogen activator gene (t-PA; gene name PLAT) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between PLAT methylation and PLAT expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal PLAT gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the PLAT transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the PLAT promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the PLAT promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient PLAT gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter.
Highlights
Tissue-type plasminogen activator (t-PA) produced by vascular endothelial cells plays an important role in the removal of intravascular fibrin deposits [1,2]
human umbilical vein EC (HUVEC) were isolated de novo as previously described [24,25] and cultured in EGM2 medium (Cambrex); peripheral blood monocytes were isolated from blood buffy coat by cold aggregation [26] and cultured in RPMI1640 and 10% fetal bovine serum (FBS); human foreskin fibroblasts were generated from skin biopsies and cultured in Dulbecco’s modified Eagle’s medium (DMEM) + 10% FBS, as previously described [27]; human astrocytes and hepatocytes were purchased from Lonza and cultured in AGM and HCM medium (Lonza), respectively
Before studying the influence of PLAT gene promoter/enhancer methylation on PLAT gene expression, we investigated the PLAT transcriptional start site (TSS) by 5’RACE analysis using total RNA from five t-PA expressing cell types: astrocytes, Bowes melanoma cells, HeLa cells, HUVEC and HT1080 cells (Fig 1)
Summary
Tissue-type plasminogen activator (t-PA) produced by vascular endothelial cells plays an important role in the removal of intravascular fibrin deposits [1,2]. Some information is available on promoter/enhancer elements regulating agonist-mediated changes in PLAT expression in different cell types. The effect of HDAC inhibition appears to be direct because it is correlated with changes in histone acetylation at the PLAT promoter [18,19,20]. This implies that epigenetic mechanisms suppress t-PA production in EC. A recent study by Magnusson et al [23] observed that culturing human endothelial cells led to a demethylation of the MHRE, which was associated with an increase in t-PA expression
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