Interleukin-6 induction of protein s is regulated through signal transducer and activator of transcription 3.

OBJECTIVE: The protein C anticoagulant pathway is an essential process for attenuating thrombin generation by the membrane-bound procoagulant complexes tenase and prothrombinase. In this pathway, protein S (PS) serves as a cofactor for activated protein C. PS circulates in plasma both in a free form and in complex with complement component 4b-binding protein (C4BP). C4BP is a known acute phase reactant, thereby suggesting a relation between PS and the acute phase response. Interleukin (IL)-6 has been shown to increase both PS and C4BP gene expression. Our objective was to study the regulation of PS gene expression by IL-6 in detail. METHODS AND RESULTS: IL-6 upregulates both PS mRNA and protein levels in liver-derived HepG2 cells. The promoter of the PS gene (PROS1) was cloned upstream from a luciferase reporter gene. After transfection in HepG2 cells, the luciferase activity was shown to be stimulated by the addition of IL-6. IL-6 exerts its effect through Signal Transducer and Activator of Transcription 3 (STAT3) that interacts with the PROS1 promoter at a binding site in between nucleotides 229 to 207 upstream from the translational start. CONCLUSIONS: IL-6 induces PS expression via STAT3. A possible function for IL-6-induced PS expression in cell survival is discussed.

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.

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.