Characterization and regulation of the 5′-flanking region of the murine endothelial protein C receptor gene

The protein C pathway plays a critical role in the negative regulation of blood coagulation The nucleotide sequence of the murine endothelial protein C receptor (mEPCR) gene was determined for 8.8 kilobase pairs of the genomic structure and 3.4 kilobase pairs of the 5'-flanking region. RNase protection assay revealed six major transcription start sites clustered at -100 to - 109 upstream of the translation initiation site. A series of 5'-promoter deletion fragments were fused to a luciferase reporter gene and transiently transfected into bovine aortic endothelium. Deletion of the sequence from -220 to -180 dramatically reduced luciferase expression in bovine aortic endothelial cells. This region of the murine endothelial protein C receptor gene contains one AP4 site and one SP1 site. Mutations in the core sequence of the AP4 and SP1 sites impaired both nuclear protein binding and luciferase expression. These results suggest important roles for AP4 and SP1 in the constitutive expression of mEPCR. A thrombin response element (CCCACCCC) was found to mediate the induction of mEPCR by thrombin in cell culture. Transgenic mice were developed expressing green fluorescent protein driven by the -350 to -1 or - 1080 to -1 promoter. Thrombin up-regulated mEPCR and the transgene in vivo.