Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites

Rance, James B.; Follows, George; Cockerill, Peter N.; Bonifer, Constanze; Lane, David A.; Simmonds, Rachel E.

doi:10.1182/blood-2002-05-1570

Cited by 13 publications

(19 citation statements)

References 37 publications

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“…15 For reporter gene assays, cells were grown in 24-well plates and transfected with the SEAP reporter constructs using Lipofectamine 2000 transfection reagent (Invitrogen, Karlsruhe, Germany). For normalization, cells were cotransfected with pGL3-control vector (Promega, Mannheim, Germany) encoding firefly luciferase under the control of the SV40 promoter and enhancer.…”

Section: Cell Culture Transfection and Reporter Assaysmentioning

confidence: 99%

“…EPCR is, however, also expressed by primitive hematopoietic stem cells (HSCs). 13 Initial reports on the transcriptional regulation of the murine EPCR (Procr, here termed mEPCR) 14 and human EPCR (PROCR, here termed hEPCR) 15 genes have failed to define the molecular mechanisms responsible for the rather unique in vivo cellular distribution of EPCR.…”

Section: Introductionmentioning

confidence: 99%

“…17 Transcription of hEPCR is initiated from 2 major sites located at Ϫ79 and Ϫ82 bp (base pair) relative to the translation initiation point, and from an additional minor site at Ϫ162 bp. 16,18 Previous investigations of the hEPCR 5Ј-flanking region characterized the 2.3-kb region upstream of the translation initiation site, 15 referred to as the proximal promoter (PP). In transfection studies, PP (and also truncations of this region down to Ϫ572 bp) was transcriptionally active in ECs (human umbilical vein endothelial cell [HUVEC] and EA.hy926) but not in non-ECs (HepG2).…”

Section: Introductionmentioning

confidence: 99%

“…15 Briefly, naked genomic DNA or cultured cells (HUVEC and HepG2) were treated with DMS, following which methylated guanines were digested with piperidine. 22 DMS reactivity was visualized by ligation-mediated polymerase chain reaction (PCR), using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA).…”

Section: In Vivo Dimethylsulphate (Dms) Footprintingmentioning

confidence: 99%

“…The previously generated PP-pSB construct, containing Ϫ2336 to Ϫ9 bp of the hEPCR PP cloned upstream of SEAP, was also used. 15 DNA sequences under investigation were PCR-amplified from either genomic DNA or the PAC clone 212-C5 (which contains hEPCR and its 5Ј-flanking region) using primers that introduced XhoI or SalI restriction sites for cloning. Mutation of transcription factor binding sites (Ets no.…”

Section: Plasmid Constructsmentioning

confidence: 99%

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

Mollica

Crawley

Liu

et al. 2006

Blood

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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...

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Section: Cell Culture Transfection and Reporter Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: In Vivo Dimethylsulphate (Dms) Footprintingmentioning

confidence: 99%

Section: Plasmid Constructsmentioning

confidence: 99%

See 3 more Smart Citations

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

Mollica

Crawley

Liu

et al. 2006

Blood

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Vascular smooth muscle cell‐specific regulation of cyclin‐dependent kinase inhibitor p21^WAF1/Cip1 transcription by Sp1 is mediated via distinct cis‐acting positive and negative regulatory elements in the proximal p21^WAF1/Cip1 promoter

Kavurma

Khachigian

2004

J of Cellular Biochemistry

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Smooth muscle cells (SMC) play a central role in common vascular pathologies such as atherosclerosis and restenosis. Understanding the molecular regulation of SMC proliferation at a transcriptional level may provide important clues for the targeted control of vascular hyperplasia. We recently reported the capacity of the transcription factor Sp1 to down-regulate p21(WAF1/Cip1) production thereby reducing p21(WAF1/Cip1)-cyclin D1-Cdk4 complex formation and inhibiting vascular SMC proliferation (Kavurma and Khachigian [2003] J. Biol. Chem. 278, 32537-32543). We have now localized the Sp1-response elements in the p21(WAF1/Cip1) promoter responsible for p21(WAF1/Cip1) repression in WKY12-22 SMCs. The proximal region of the p21(WAF1/Cip1) promoter contains five distinct Sp1-binding elements that we have termed A, B, C, D, and E. Electrophoretic mobility shift analysis revealed that SMC nuclear Sp1 interacts with all five Sp1-binding sites, and each of these sites is critical for Sp1 repression of the p21(WAF1/Cip1) promoter, since mutation in any one element ablates repression, and in some cases results in activation. In contrast, only elements C, D, and E are bound by Sp1 in endothelial cells. Sp1 overexpression activates the p21(WAF1/Cip1) promoter in this cell type. Furthermore, mutation in any of these five elements is not sufficient to prevent activation of the p21(WAF1/Cip1) promoter by Sp1 in endothelial cells. Surprisingly, double mutations of elements C and E facilitates superactivation by Sp1 in both cell types, whereas triple mutations of C, D, and E inactivate the promoter. These findings demonstrate cell type-specific regulation of p21(WAF1/Cip1) transcription by Sp1 via distinct cis-acting positive and negative regulatory elements in the proximal p21(WAF1/Cip1) promoter.

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The endothelial cell protein C receptor: cell surface conductor of cytoprotective coagulation factor signaling

Gleeson

O’Donnell

Preston

2011

Cell. Mol. Life Sci.

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Increasing evidence links blood coagulation proteins with the regulation of acute and chronic inflammatory disease. Of particular interest are vitamin K-dependent proteases, which are generated as a hemostatic response to vascular injury, but can also initiate signal transduction via interactions with vascular receptors. The endothelial cell protein C receptor (EPCR) is a multi-ligand vitamin K-dependent protein receptor for zymogen and activated forms of plasma protein C and factor VII. Although the physiological role of the EPCR-FVII(a) interaction is not well-understood, protein C binding to EPCR facilitates rapid generation of APC in response to excessive thrombin generation, and is a central requirement for the multiple signal-transduction cascades initiated by APC on both vascular endothelial and innate immune cells. Exciting recent studies have highlighted the emerging role of EPCR in modulating the cytoprotective properties of APC in a number of diverse inflammatory disorders. In this review, we describe the structure-function relationships, signal transduction pathways, and cellular interactions that enable EPCR to modulate the anticoagulant and anti-inflammatory properties of its vitamin K-dependent protein ligands, and examine the relevance of EPCR to both thrombotic and inflammation-associated disease.

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Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites

Abstract: The human endothelial cell protein C receptor (hEPCR) is normally expressed by the endothelium of large blood vessels, but the molecular basis for its in vivo specificity is uncertain. In this study, DNaseI hypersensitive site mapping demon-

Cited by 13 publications

References 37 publications

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

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

Vascular smooth muscle cell‐specific regulation of cyclin‐dependent kinase inhibitor p21^WAF1/Cip1 transcription by Sp1 is mediated via distinct cis‐acting positive and negative regulatory elements in the proximal p21^WAF1/Cip1 promoter

The endothelial cell protein C receptor: cell surface conductor of cytoprotective coagulation factor signaling

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Regulation of the human endothelial cell protein C receptor gene promoter by multiple Sp1 binding sites

Abstract: The human endothelial cell protein C receptor (hEPCR) is normally expressed by the endothelium of large blood vessels, but the molecular basis for its in vivo specificity is uncertain. In this study, DNaseI hypersensitive site mapping demon-

Cited by 13 publications

References 37 publications

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

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

Vascular smooth muscle cell‐specific regulation of cyclin‐dependent kinase inhibitor p21WAF1/Cip1 transcription by Sp1 is mediated via distinct cis‐acting positive and negative regulatory elements in the proximal p21WAF1/Cip1 promoter

The endothelial cell protein C receptor: cell surface conductor of cytoprotective coagulation factor signaling

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Resources

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Vascular smooth muscle cell‐specific regulation of cyclin‐dependent kinase inhibitor p21^WAF1/Cip1 transcription by Sp1 is mediated via distinct cis‐acting positive and negative regulatory elements in the proximal p21^WAF1/Cip1 promoter