The Constitutive Expression of Anticoagulant Protein S Is Regulated through Multiple Binding Sites for Sp1 and Sp3 Transcription Factors in the Protein S Gene Promoter

Abstract

Protein S (PS) is a vitamin K-dependent plasma protein that inhibits blood coagulation by serving as a nonenzymatic cofactor for activated protein C in the protein C anticoagulant pathway. Low PS levels are a risk factor for the development of deep venous thrombosis. The regulation of PS levels through transcriptional regulation of the PS gene was investigated in this report. A minimal PS gene promoter 370 bp upstream from the translational initiation codon was sufficient for maximal promoter activity in transient transfections regardless of the cell type. A pivotal role for Sp1 in the constitutive expression of the PS gene was demonstrated through electrophoretic mobility shift assay experiments, transient expression of mutant PS promoter-reporter gene constructs, and chromatin immunoprecipitations in HepG2 cells. At least four Sp-binding sites were identified. The two sites most proximal to the translational start codon were found to be indispensable for PS promoter activity, whereas mutation of the two most distal Sp-binding sites had a negligible influence on basal promoter activity. In addition, all other major promoter-binding proteins that were found by electrophoretic mobility shift assay could be positively identified in supershift assays. We identified binding sites for the hepatocyte-specific forkhead transcription factor FOXA2, nuclear factor Y, and the cAMP-response element-binding protein/activating transcription factor family of transcription factors. Their relevance was investigated using site-directed mutagenesis.The coagulation cascade is a complex system in which the consecutive activation of multiple coagulation factors leads to the production of thrombin and ultimately to the formation of fibrin polymers, the primary component of blood clots (for a recent review see Ref. 1). Protein S (PS)2 is a vitamin K-dependent plasma protein that functions as a nonenzymatic cofactor for activated protein C in the down-regulation of the coagulation cascade via proteolytic inactivation of coagulant factors Va and VIIIa (2-5). PS has also been shown to display activated protein C-independent anticoagulant activity in purified systems as well as in plasma (6 -8). Recent studies indicate that PS may have a second function unrelated to coagulation in the clearance of apoptotic cells (9, 10).Over the past two decades low PS plasma levels have become a well established risk factor for the development of deep venous thrombosis (11-13). However, not all mechanisms underlying low plasma PS levels have been fully characterized. Hereditary PS deficiency has been shown to be an autosomal dominant trait, and many causative genetic mutations have been described in the PS gene (14,15). On the other hand, PS deficiency can also be acquired throughout life by conditions such as oral contraceptive use and liver disease (16). To better understand the different functions of PS and the possible causes of PS deficiency, more information is needed on the regulation of the PS gene, mRNA, and protein.The major source of circulati...

Down-regulation of PROS1 Gene Expression by 17β-Estradiol via Estrogen Receptor α (ERα)-Sp1 Interaction Recruiting Receptor-interacting Protein 140 and the Corepressor-HDAC3 Complex

“…The luciferase assays suggested that the GC-rich motif at Ϫ172 of the PROS1 promoter plays an important role in E 2 -dependent gene repression. In subsequent EMSAs and DNA pulldown analyses, we observed that Sp1 and Sp3 bound to the GC-rich motif of PROS1, which was consistent with the findings of de Wolf et al (38).…”

Interleukin-6 Induction of Protein S Is Regulated Through Signal Transducer and Activator of Transcription 3

“…36 -38 Recently, constitutive expression of the PROS1 promoter was shown to be regulated mainly by Sp1 in vitro, and to contain binding sites for various other transcription factors such as the hepatocyte-specific forkhead transcription factor FOXA2, which are possibly involved in tissuespecific or induced expression of the PROS1 promoter. 39,40 We confirmed earlier findings that PS is upregulated by IL-6 in cultured HepG2 cells and complemented these with quantitative PS mRNA data thereby identifying the regulatory mechanism as pretranslational. Subsequently, we identified a region in the PS promoter spanning nucleotides 229 to 207 upstream from the translational start that binds STAT3, one of the nuclear messengers of IL-6.…”

“…The generation of PROS1 promoter reporter constructs is described in de Wolf et al 40 PROS1 promoter constructs were mutated at a putative STAT3 binding site by use of the QuikChange XL SiteDirected Mutagenesis kit from Stratagene, with forward primer 5Ј-CCA GCT CCG AAA AGC CGC CTG GTG C TG TCC TTG TTA TCA C-3Јand reverse primer 5Ј-GTG ATA ACA AGG ACA GCA CCA GGC GGC TTT TCG GAG CTG G-3Ј. Successful incorporation of the mutations was confirmed by automated sequencing (Beckman).…”

Glucocorticoids Induce Protein S-Dependent Phagocytosis of Apoptotic Neutrophils by Human Macrophages

“…Protein S is produced in the liver and by endothelial cells (41). Production of both protein S and C4BP in the liver appears to be controlled by inflammatory mediators, including IL-6 (42,43). Levels of protein S are reduced in patients with ischemic stroke (44) and in patients with sepsis (45), possibly via the effects of TNF on endothelial cells (46).…”