Characterization of the human protein S gene promoter: a possible role of transcription factors Sp1 and HNF3 in liver

SummaryProtein S is a vitamin-K-dependent plasma glycoprotein that serves as a cofactor for activated protein C in the protein C anticoagulation pathway, which regulates blood coagulation by inactivating factors Va and VIIIa. Mechanisms regulating the expression of the protein S gene remain unknown to date. The aim of this study was to characterize the cis-acting DNA elements of the human protein S gene in liver.We found that liver cell lines (HepG2 and PLC) transcribed the human protein S gene to mRNA, whereas non-liver cell lines (HEK293 and HeLa cells) either transcribed the gene weakly or not at all. Isolation and analysis of tissue-specific gene expression in HepG2 and HeLa cells of the 5’-flanking region from-6183 to +294 of the protein S gene indicated that the consensus binding motifs to HNF3 and Sp1 or MAZ transcription factors in the flanking region are essential for protein S gene expression. Exogenous expression of the Sp1 gene augmented the protein S-reporter gene expression in HepG2 or PLC cells but had no effect in HeLa cells. Taken together, we would conclude that transcription factors of HNF3, MAZ, and Sp1 are required for high-level expression of the protein S gene in hepatic cells, but in non-hepatic cells such as HeLa cells, an unknown factor(s) binds to the Sp1 region and disturbs the action of Sp1 and MAZ.

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Regulation of the human protein S gene promoter by liver enriched transcription factors

British Journal of Haematology ◽

10.1111/j.1365-2141.2006.06327.x ◽

2006 ◽

Vol 135 (4) ◽

pp. 538-546 ◽

Cited By ~ 10

Author(s):

Adrian J. Hall ◽

Ian R. Peake ◽

Peter R. Winship

Keyword(s):

Transcription Factors ◽

Gene Promoter ◽

Protein S ◽

Human Protein ◽

S Gene

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GENE STRUCTURE OF VITAMIN K-DEPENDENT PROTEIN S; A REGION HOMOLOGOUS TO SEX HORMONE BINDING GLOBULIN (SHBG) REPLACES THE SERINE PROTEASE REGION OF FACTORS IX, X AND PROTEIN C

10.1055/s-0038-1644640 ◽

1987 ◽

Author(s):

C-M Edenbrandt ◽

S Gershagen ◽

P Femlund ◽

R Wydro ◽

J Stenflo ◽

...

Keyword(s):

Amino Acids ◽

Vitamin K ◽

Protein C ◽

Protein S ◽

Sex Hormone ◽

Human Protein ◽

Sex Hormone Binding Globulin ◽

Hormone Binding ◽

Clotting Factors ◽

S Gene

It has recently been shown that the similarity between coagulation factors IX, X and protein C in the protein sequence is also evident in the organization of their genes. To further elucidate the relation of protein S to the other vitamin K-dependent clotting factors, we are now characterizing the human protein S gene. The size of the gene was estimated to be more than 45 kb, by hybridization of a cDNA for human protein S with chromosomal DNA in a Southern blot.We have isolated three overlapping clones from a human genomic DNA library in bacteriophage λ Charon 4A, which cover approximately 40 kb of the gene. The clones have been mapped by single- and double restriction enzyme digestion. Genomic subclones in pUC 18 which hybridize with cDNA probes for protein S have been isolated and sequenced to establish the intron/exon structure of the gene. The 5’- part of the human protein S gene closely resembles the corresponding part of the genes for factors IX, X and protein C. However, the thrombin sensitive region (amino acids 46-75), which is unique for protein S among the vitamin K-dependent clotting factors, is coded for by a separate exon. The 3'- end of the protein S gene, coding for amino acids 247-635, is not homologous to the catalytic region of the vitamin K-dependent serine proteases but shows a significant homology to human sex hormone binding globulin (SHBG).

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A Procedure for Isolation of Human Protein C and Protein S as by-Products of the Purification of Factors VII, IX, X and Prothrombin

Preparative Biochemistry ◽

10.1080/00327488308064248 ◽

1983 ◽

Vol 13 (3) ◽

pp. 191-214 ◽

Cited By ~ 10

Author(s):

S. P. Bajaj ◽

S. I. Rapaport ◽

S. L. Maki ◽

S. F. Brown

Keyword(s):

Protein C ◽

Protein S ◽

Human Protein ◽

By Products

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Recombinant human protein C, protein S and thrombomodulin as antithrombotics

Perspectives in Drug Discovery and Design ◽

10.1007/bf02171862 ◽

1994 ◽

Vol 1 (3) ◽

pp. 503-520 ◽

Cited By ~ 17

Author(s):

S. Betty Yan ◽

Brian W. Grinnell

Keyword(s):

Protein C ◽

Protein S ◽

Human Protein ◽

C Protein ◽

Recombinant Human Protein

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DEVELOPMENT OF A PROTEIN C CONCENTRATE

10.1055/s-0038-1644313 ◽

1987 ◽

Author(s):

Prabir Bhattacharya ◽

Carolyn L Orthner ◽

Dudley K Strickland

Keyword(s):

Protein C ◽

Antithrombin Iii ◽

Activated Protein C ◽

Protein S ◽

Exchange Chromatography ◽

Factor Ix ◽

Human Protein ◽

Red Cross ◽

American Red Cross ◽

Protein C Concentrate

A Protein C (PC) concentrate may be useful in treating patients with congenital or acquired Protein C deficiencies. A method for preparation of a human Protein C concentrate has been developed using a by-product of American Red Cross Factor IX production as the starting material (Menache et. al. Blood, 64, 1220). Levels of other vitamin K dependent proteins in the Protein C concentrate were measured and found to be <10 units per 100 units of PC, except for Protein S. The level of Protein S as judged by immunological assay was 30 u/100 u PC. Assay of the PC concentrate using chrcmogenic substrates revealed that levels of thrombin, Factor 3�a and Factor IXa were less than 0.006 u/mL. In addition, Antithrombin III and ax -macroglobulin were not detected. The vivo effects of Protein C concentrate and Protein C activated by thrombin have been tested in anesthetized rabbits. Thrombin was removed from the activated Protein C by ion-exchange chromatography; depletion was verified by S-2238 or by a clotting assay (< 0.006 u/mL). Rabbits were injected with Protein C concentrate (400 ug/kg) or activated Protein C 24 - 48 ug/Kg). The activated partial thromboplastin time (APTT), FactorV (FV) and Factor VIII (FVIII) levels were measured in samples collected over the next three hours. Infusion of PC concentrate elevated the level of PC to 150% of the preinfusion level within 30 min. It did not change the levels of FV, FVIII, fibrinogen or platelet count. In contrast, infusion of activated Protein C produced progressive prolongation of the APTT. Levels of FV and FVIII were decreased to 25% and 50% of preinfusion levels, respectivelv, three hours after the infusion. Fibrinogen and platelet levels were unchanged during that period. These data demonstrate that activated human Protein C concentrate induces an anticoagulant effect that can be readily measured in rabbits.

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Regulation of human protein C gene expression by the mouse WAP promoter

Transgenic Research ◽

10.1007/bf01976765 ◽

1994 ◽

Vol 3 (6) ◽

pp. 335-343 ◽

Cited By ~ 17

Author(s):

Rekha K. Paleyanda ◽

Da-Wei Zhang ◽

Lothar Hennighausen ◽

Robert A. McKnight ◽

Henryk Lubon

Keyword(s):

Gene Expression ◽

Protein C ◽

Human Protein

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It Takes Two: Liver Cell-specific Gene Expression Co-regulated by Unique Transcription Factors♦

Journal of Biological Chemistry ◽

10.1074/jbc.p110.217745 ◽

2011 ◽

Vol 286 (34) ◽

pp. e99963-e99963

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Liver Cell ◽

Specific Gene ◽

Specific Gene Expression

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The role of Stat and C/EBP transcription factors in the synergistic activation of rat serine protease inhibitor-3 gene by interleukin-6 and dexamethasone

Biochemical Journal ◽

10.1042/bj3131019 ◽

1996 ◽

Vol 313 (3) ◽

pp. 1019-1027 ◽

Cited By ~ 37

Author(s):

Tomasz KORDULA ◽

james TRAVIS

Keyword(s):

Transcription Factors ◽

Binding Site ◽

Interleukin 6 ◽

Serine Proteinase ◽

Protein S ◽

Term Treatment ◽

Signal Transducers ◽

Hepatic Cells ◽

Synergistic Activation ◽

Long Term Treatment

The rat serine proteinase inhibitor 3 gene is activated by interleukin 6 (IL-6) and glucocorticoids in hepatic cells. We report here that a 147 bp promoter is sufficient for both IL-6 stimulation and glucocorticoid enhancement of IL-6 induced transcription. Within this region we identified two functional elements binding transcription factors from the C/EBP (CCAAT/enhancer binding proteins) and Stat (signal transducers and activators of transcription) families. Mutations introduced into the Stat binding site resulted in a loss of responsiveness, showing that this element is indispensable for activation. In contrast, the promoter containing the mutated C/EBP binding site was still responsive to IL-6 and glucocorticoids; however, the magnitude of the induction was decreased by 50%. The Stat binding element is an enhancer capable of conferring both responsiveness to IL-6 and partial enhancement of glucocorticoids on to a heterologous promoter. In response to IL-6 this element rapidly binds acute-phase response factor (APRF/ Stat3) and, later, the protein(s) that require ongoing protein synthesis and is recognized by anti-Stat3 antibodies. In addition, long-term treatment with IL-6 results in sustained phosphorylation of APRF/Stat3.

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

Blood ◽

10.1182/blood-2002-05-1570 ◽

2003 ◽

Vol 101 (11) ◽

pp. 4393-4401 ◽

Cited By ~ 11

Author(s):

James B. Rance ◽

George A. Follows ◽

Peter N. Cockerill ◽

Constanze Bonifer ◽

David A. Lane ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Endothelial Cell ◽

Binding Sites ◽

Hepg2 Cells ◽

Protein C ◽

Cell Protein ◽

Hypersensitive Site ◽

Flanking Region

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 demonstrated the presence of a hypersensitive site in the 5′ flanking region of the hEPCR gene in endothelial cells and certain transformed cells (HeLa and U937) known to express hEPCR in vitro. Conversely, this site was only weakly hypersensitive in HepG2 cells, cells which do not express hEPCR mRNA. Functional analysis of this 5′ flanking region by in vivo dimethylsulfate footprinting in cultured endothelial cells identified multiple regions, containing high and low homology consensus Sp1 binding sequences, that were protected from methylation in endothelial cells. These sequences were not protected in HepG2 cells. Reporter gene analysis of this region in endothelial cells demonstrated the presence of promoter activity conferred by the proximal 572 bp but failed to identify a functional TATA-box. This promoter was inactive in HepG2 cells. Electrophoresis mobility shift assays using endothelial cell nuclear extracts identified Sp1 family proteins binding to sites that were protected during footprinting. Sp1 sites were identified in regions at –368, –232, –226, –201, –146, and –102 bp relative to the translation start site. With the exception of the site at –102 bp, each identified Sp1 binding site made a positive contribution to reporter gene expression, although no individual site was critically important. We conclude that transcription factor binding to Sp1 binding sites in the 5′ flanking region is critical for normal hEPCR gene expression in endothelial cells.

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