Role of a 5′-enhancer in the transcriptional regulation of the human endothelial cell protein C receptor gene

Abstract

The endothelial cell protein C receptor (EPCR) is expressed by endothelial cells of large blood vessels and by hematopoietic stem cells. DNaseI hypersensitive (DH) site mapping across 38 kb of the human EPCR gene (hEPCR) locus identified 3 potential regulatory elements. By itself, the DH region spanning the proximal promoter (PP) was unable to direct cell-specific transcription in transgenic mice. A second DH element, located upstream of PP and termed ؊5.5HS was hypersensitive only in endothelial cells (ECs) and immature hematopoietic cell lines. Transgenes expressing LacZ under the control of ؊5.5HS coupled to either PP or the SV40 promoter were able to direct ␤-galactosidase activity to the endothelium of large vessels during embryogenesis and adulthood. The ؊5.5HS exhibited enhancer activity that was conferred by the interplay of transcription factors interacting with conserved Ets and composite GATA/Tal1 motifs. The third DH element, located in intron 2, was primarily hypersensitive in EPCR-negative cells, and capable of initiating antisense transcription, suggesting a role in hEPCR silencing. This study identifies critical elements required for the tissue specificity of hEPCR and suggests a mechanism for endothelial and hematopoietic stem cell-specific transcriptional regulation that reflects the common origin of these cell types. IntroductionThe protein C (PC) anticoagulant pathway plays a crucial role in the regulation of blood coagulation and inflammation. 1 Activated PC (APC) generated in this pathway serves to confine the hemostatic plug to the site of vascular injury by inhibiting the cofactor function of clotting factors Va and VIIIa on intact endothelium. In addition, APC may also exert antiapoptotic and neuroprotective functions that influence inflammatory responses. 2,3 The important regulatory roles of APC in coagulation and inflammation are illustrated by the increased risk of thrombosis incurred by individuals with deficiencies in components of the PC pathway 4 and by the improved outcome of patients with severe sepsis treated with APC. 5 PC is activated by thrombin, but only when thrombin is bound to its receptor, thrombomodulin, present on endothelial cells (ECs). Activation is further enhanced by another EC receptor, the endothelial cell protein C receptor (EPCR). 6 By binding PC, EPCR helps present PC to the thrombin:thrombomodulin complex and so reduces the K m for PC activation. 7 In this way, EPCR enhances APC generation by at least 5-fold in vitro. 7,8 In vivo, blocking protein C-EPCR interactions results in an 88% decrease in circulating APC levels generated in response to thrombin infusion. 9 EPCR function is critical for embryo development because EPCR knockout mice die in midgestation. 10 The normal distribution of EPCR is highly tissue specific. During embryogenesis, EPCR is expressed by the embryonic giant trophoblast cells from approximately E7.5, and by certain embryonic EC from approximately E11.5. 11 In adults, EPCR is expressed almost exclusively by ECs, particularly those o...

The cytoprotective protein C pathway

“…10,[20][21][22][23] No studies have defined the tissue distribution or cellular localization of EPCR relative to thrombomodulin or PAR-1. It will be interesting to establish the extent to which thrombomodulin and EPCR colocalize in lipid membrane rafts in different tissues where protein C activation can be localized versus the colocalization of PAR-1 and EPCR.…”

Omental implantation of BOECs in hemophilia dogs results in circulating FVIII antigen and a complex immune response

“…These cells were transduced with lentiviral vectors (MOI 20) containing either the CMV, TM, or EF1a promoters, and functional cFVIII expression was measured in culture media 2 weeks after transduction. Relative to pooled canine plasma, levels of cFVIII expression were 0.1, 0.25, and 0.4 U per 10 6 cells over 24 hours for constructs containing the TM, CMV, and To further improve cFVIII expression, we included an endothelial enhancer element (-5.5HSCR) 21 in 3 separate locations within the EF1a promoter. Increases of 104-and 10.6-fold were observed when 1 and 2 copies, respectively, of -5.5HSCR were incorporated into the EF1a promoter; no apparent increase in cFVIII was observed with 3 copies of the enhancer element ( Figure 1G).…”

“…20 The CMV promoter was excised and replaced with the human elongation factor 1 alpha (EF1a) promoter. Three additional constructs were engineered that contain either 1, 2, or 3 copies of the 164-bp endothelial-specific protein C receptor enhancer element (-5.5HSCR) 21 ( Figure 1A). Lentiviral vectors were produced, and titers were determined as previously described.…”

Endothelial Cell Protein C Receptor

“…23 It would be interesting to find out whether the same site in hEPCR is responsible for driving hEPCR expression in human SMCs. 22 The present findings by Bretschneider et al 22 raise not only a possibility that EPCR could play an important role in regulating the biology of vascular SMCs, but also suggest that its expression in SMCs might have implications for the pathogenesis of cardiovascular diseases such as atherosclerosis, and by extension, cerebral amyloid angiopathy in Alzheimer's disease.…”