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J. Biol. Chem., Vol. 281, Issue 26, 17635-17643, June 30, 2006

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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^*

From the Hemostasis and Thrombosis Research Center, Department of Hematology, Leiden University Medical Center, Albinusdreef 2, 2300 RC Leiden, The Netherlands

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.

Received for publication, March 31, 2006 , and in revised form, April 24, 2006.

^* This work was supported by Dutch Thrombosis Foundation Grant TSN 98.002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Hemostasis and Thrombosis Research Center, Dept. of Hematology, Leiden University Medical Center, C2-R139, P. O. Box 9600, 2300 RC, Leiden, The Netherlands. Tel.: 31-71-526-1895; Fax: 31-71-526-6755; E-mail: h.l.vos{at}lumc.nl.

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