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

【摘要】  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.

Conclusion- IL-6 induces PS expression via STAT3. A possible function for IL-6-induced PS expression in cell survival is discussed.

The important anticoagulant Protein S forms inactive complexes with complement component 4b-binding protein (C4BP), which is a known acute phase reactant. This raises the question how Protein S gene ( PROS1 ) transcription is regulated during acute phase. Here we show that PROS1 transcription is upregulated by IL-6 via the STAT3 pathway.

【关键词】  protein S PROS IL STAT C/EBPß

Introduction

The protein C anticoagulant pathway is indispensable in maintaining the hemostatic balance. 1,2 In this pathway, protein S (PS) functions as a nonenzymatic cofactor for activated protein C in the inactivation of coagulation factors VIIIa and Va. 3-6 The clinical importance of PS became apparent by the association of venous thrombo-embolism with (partial) PS deficiency. 7-9 The major source of circulating plasma PS is the hepatocyte. 10 In human plasma, PS circulates in an active free form (40%) and a C4b-binding protein (C4BP)-bound inactive form (60%). 11-13

The acute phase response (APR) is characterized by a rapid increase of hepatic proteins, which serves to restore the physiological balance and limit the deleterious effects of tissue injury and infection. In the liver, synthesis of several plasma proteins, eg, 1-antitrypsin, 2-macroglubulin, plasminogen, and fibrinogen, undergoes dramatic changes during the APR. 14 Several of these proteins are also involved in coagulation or fibrinolysis. The changes during the APR are mainly mediated by the action of cytokines, such as interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-.

In vitro studies in cultured human hepatoma cell line HepG2, primary human umbilical vein endothelial cells, and in the human microvascular endothelial cell line HMEC-1, have shown increased PS production on IL-6 stimulation. 15-17 C4BP is an established acute phase reactant with 2- to 3-fold elevated plasma levels during inflammation. 18-21 The C4BP protein contains 6 or 7 identical -chains and a single ß-chain, although 17% of the C4BP molecules lack the ß-chain. 13 PS is bound to C4BP via the ß-chain. Several studies have demonstrated similar or slightly increased total PS levels, but reduced free PS levels in plasma of patients during inflammation. 22-24 The observed reduction in free PS levels has been explained as a direct consequence of an increase in the ß-chain containing C4BP-form. Other studies, however, provide evidence for stable free PS levels during inflammation, showing only a rise in C4BP -chain levels, but not in ß-chain levels. 21,25 The observed discrepancy between the various studies may be explained by a differential regulation of C4BP - and ß-chains in different subpopulations of patients during the APR. 26

The IL-6 signaling pathway is well-defined. 27-29 In short, IL-6 is known to exert its stimulatory effect through IL-6-responsive elements (IL-6-RE) in the promoters of IL-6-responsive genes. Two types of IL-6-REs have been identified in genes encoding acute phase proteins. Type I is a binding site for CCAAT/enhancer binding protein (C/EBP) ß (NF-IL6, LAP), whereas the type II IL-6-RE is bound by Signal Transducer and Activator of Transcription 3 (STAT3). 30 Although IL-6 upregulates nuclear C/EBPß levels through both elevated C/EBPß gene transcription and C/EBPß phosphorylation, 29 increased nuclear STAT3 levels are mainly attributable to rapid STAT3 phosphorylation. 28 C/EBPß and STAT3 have been shown to affect IL-6-induced transcription by binding to, among others, the promoters of the genes encoding C-reactive protein, 31,32 plasminogen, 33 haptoglobin, 34 and fibrinogen. 35 In this report we investigate the mechanism by which IL-6 upregulates PS protein levels.

Two copies of the PS gene are located on chromosome 3. The active PS gene ( PROS1 ) shares 96% homology with the inactive pseudogene ( PROS2 ). 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 tissue-specific 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.

Methods

Plasmids

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 Site-Directed Mutagenesis kit from Stratagene, with forward primer 5'-CCA GCT CCG AAA AGC CG C CTG G TG C TG TCC TTG TTA TCA C-3'and reverse primer 5'-GTG ATA ACA AGG ACA GCA CCA GG C G GC TTT TCG GAG CTG G-3'. Successful incorporation of the mutations was confirmed by automated sequencing (Beckman).

Cell Culture

The human hepatoblastoma cell line HepG2 was purchased from the American Type Culture Collection (ATCC). Cells were grown in minimal essential medium (MEM), 10% fetal bovine serum (FBS), 100 µg/mL penicillin, 100 µg/mL streptomycin, and 1 x MEM nonessential amino acids (all purchased from Gibco, Invitrogen; Carlsbad, Calif, USA). For induction experiments, cells were washed twice with phosphate buffered saline, after which fresh medium was added that lacked FBS, but contained 0.1% human serum albumin and 5 or 100 ng/mL recombinant human IL6 (Strathmann; Hamburg, Germany).

Preparation of Nuclear Extracts

Nuclear extracts (NE) were prepared according to the method of Dignam et al. 41 The final nuclear extract buffer contained 20 mmol/L HEPES (pH 7.9), 0.2 mmol/L EDTA, 100 mmol/L KCl, 0.5 mmol/L DTT, 0.2 mmol/L PMSF and a 1 x concentration of an EDTA-free protease inhibitor cocktail (Roche). NE were aliquoted and frozen at -80°C until further use. Protein concentration of the NE was measured with the BCA assay (Pierce Biotech). NE were prepared from HepG2 cells cultured in the absence or presence (30 minutes or 14 hours) of 100 ng/mL IL-6.

Immunologic Detection of STAT3 and C/EBPß

NE (10 µg protein) were fractionated on a denaturing 7.5% polyacrylamide slab gel and transferred onto a nitrocellulose membrane. After blocking for 1 hour in phosphate-buffered saline (PBS) containing 1% nonfat dry milk, 1% bovine serum albumin, and 0.05% Tween 20, the membrane was incubated for 1 hour at room temperature with the first antibody. C/EBPß, p-STAT3, and Sp1 were detected with the polyclonal antibodies sc-150G (1:1000), sc-7993 (1:1000), and sc-59 (1:1000), respectively (Santa Cruz Biotech). As secondary antibody, horseradish peroxidase-conjugated goat anti-rat IgG (Dako, Glostrup, Denmark) was used at a dilution of 1:1000. Enhanced chemoluminescence was used for detection by incubating the membrane for 1 minute with freshly mixed 1.25 mmol/L 3-aminophtalhydrazide, 0.2 mmol/L p-coumaric acid, and 0.01% v/v H 2 O 2 in 0.1 mol/L Tris-HCl pH 8.5.

Reporter Gene Assays

Cells were transfected at 60% to 80% confluence. Each transfection was performed in triplicate in 12-wells plates. All assays were conducted with 2 different DNA preparations of each construct. Transfections were carried out using 3 µL Tfx-20 lipids (Promega) per µg transfected DNA. In each transfection, an equimolar concentration of construct was used, supplemented with pUC13-MCS vector to obtain a fixed amount of transfected DNA. In pUC13-MCS the multiple cloning site had been removed by digestion with Pvu II and religation. Control vector pRL-SV40 (Promega), expressing the Renilla luciferase, was cotransfected for correction of transfection efficiency in a 1:500 ratio to total transfected DNA. The cell extracts were harvested at 24 hours (HepG2) after transfection. During induction experiments, medium was aspirated and replaced by fresh medium with or without IL-6 eight hours after transfection and cell extracts were harvested 48 hours after IL-6 addition. Luciferase activity was measured according to the Dual Luciferase Assay System Protocol (Promega). Cells were lysed in 250 µL Passive Lysis Buffer/well, after which 20 µL was used to measure luciferase activity. Activity was measured using a Lumat LB9507 luminometer (Berthold, Bad Wildbad, Germany).

PS Measurements

Total PS antigen levels in IL-6-stimulated and unstimulated culture media were determined by enzyme-linked immunosorbent assay (ELISA) as described previously 42 with the following modifications. ELISA plates were coated with 10 µg/mL goat anti-human PS IgG (Kordia, Leiden, The Netherlands) overnight at 4°C. A second coating with 2.5% ovalbumin (Sigma-Aldrich) at 37°C for 1 hour was performed to reduce background absorbance. Immobilized PS was detected with horseradish peroxidase-conjugated rabbit anti-human IgG (Dako, Glostrup, Denmark). Absorbance at 450 nm was determined with an Organon Teknika plate reader (Turnhout, Belgium).

RNA Assays

Total RNA was isolated from cell culture using Trizol reagent (Invitrogen) according to the manufacturer?s recommendations. Samples were treated with RNAse-free DNAse I (Amersham) after which RNA was purified with an RNeasy mini kit (Qiagen). PROS1 mRNA levels were determined by real-time quantitative polymerase chain reaction (qPCR) analysis. First, 1 µg total RNA from each cell line was reverse transcribed using Superscript II reverse transcriptase and random hexamers (Invitrogen). 1/20th of the obtained cDNA was subsequently used in a qPCR reaction with primers and probes specific for PROS1. The forward primer was 5'-TGC TGG CGT GTC TCC TCC TA-3', the reverse primer was 5'-CAG TTC TTC GAT GCA TTC TCT TTC A-3', the probe was TET-5'-CTT CCC GTC TCA GAG GCA AAC TTT TTG TC-3'-TAMRA. The primers and probe sequence locations and lengths were determined by using the ABI Primer Express Program (Applied Biosystems). PROS1 qPCR reactions (Eurogentec) were performed in 0.5 mL thin-walled, optical-grade qPCR tubes (Applied Biosystems) in a 50 µL final volume, by addition of the following components: 0.25 U AmpliTaq Gold DNA polymerase, 160 nM TaqMan probe, 300 nM of each primer, and 3 mmol/L MgCl 2. A qPCR of the internal standard, the porphobilinogen deaminase gene (PBGD), was carried out in a similar fashion for each RNA sample with 4 mmol/L MgCl 2. For this qPCR the following primers and probe were used: forward primer 5'-GGC AAT GCG GCT GCA A-3', reverse primer 5'-GGG TAC CCA CGC GAT CAC-3', and probe TET-5'-CTC ATC TTT GGG CTG TTT TCT TCC GCC-3'-TAMRA. An Applied Biosystems Prism model 7700 instrument monitored the reactions. Thermal cycling conditions consisted of 10 minutes at 95°C followed by 50 cycles of 15 s at 95°C and 1 minute at 60°C. Determinations of cycle threshold (C T ) were performed automatically by the instrument. The results are expressed as fold transcript relative to the internal standard PBGD (=2 Ct ).

Electrophoretic Mobility Shift Assays

Electrophoretic mobility shift assays (EMSAs) were performed in a 13-µL binding reaction containing 10 µg NE and 195 ng denatured herring sperm DNA. EMSA buffers were purchased from Active Motif and used according to the manufacturer?s recommendations. Double-stranded (ds) oligonucleotides were end-labeled using 32 P-ATP and T4 polynucleotide kinase. The following ds oligonucleotides were used (only the sense strand is given): -229/-207wt 5'-AAA AGC TTC CTG GAA ATG TCC TTG-3', -229/-207mt 5'-AAA AGC CG C CTG G TG C TG TCC TTG-3', STAT3cons 5'-GAT CCT TCT GGG AAT TCC TAG ATC-3', C/EBPßcons 5'-TGC AGA TTG CGC AAT CTG CA-3'. Reaction mixtures were incubated on ice for 20 minutes in the presence or absence of an unlabeled competitor. Subsequently, the 32 P-labeled ds probe was added and the incubation was continued for another 20 minutes. In antibody supershift experiments, NE was incubated on ice for 10 minutes with the 32 P-labeled ds probe after which an anti-STAT3 or anti-cEBPß antibody (Santa Cruz Biotechnology) was added and the incubation was allowed to continue for another 10 minutes. Samples were loaded on a 5% nondenaturing polyacrylamide gel, which was electrophoresed for 2 hours at 200V, after which gels were vacuum dried and exposed to X-ray film.

Results

IL-6 Induces PROS1 mRNA and PS Protein Levels in HepG2

PS circulates in normal plasma at a concentration of about 0.33 µmol/L. 13 Hepatocytes are the largest contributor to the systemic levels of PS 10; therefore, hepatoma cell line HepG2 was used as a model system. PS mRNA and protein levels under basal culture conditions were compared with those after induction with 5 or 100 ng IL-6 per ml culture medium ( Figure 1 ). A dose-dependent increase in PS mRNA and protein levels became detectable after 16 hours of IL-6 treatment of HepG2 cells. After this time, mRNA levels remained similar, but PS continued to accumulate in the medium up to at least 48 hours after the addition of IL-6. Total fibrinogen antigen levels were measured as a positive control. Fibrinogen is an established acute phase reactant, 43-45 and its levels increased from 3.5 nM to 8.6 or 16.6 nM after induction with 5 or 100 ng/mL IL-6 for 24 hours, respectively.

Figure 1. Induction of PS mRNA and protein expression by IL-6. In the top panel, the expression of PROS1-derived mRNA in HepG2 cells is shown relative to that of the housekeeping gene PBGD, encoding porphobilinogen deaminase. The incubation time (x-axis) is plotted against the PROS1:PBGD transcript ratio (y-axis). The ratio of PROS1 over PBGD transcripts was determined by real-time qPCR. In the lower panel, the production of PS antigen over time is shown. The PS concentration in the culture medium was determined by ELISA. All values represent the average of at least 3 separate wells. The error bars indicate the standard deviation.

Phosphorylation of STAT3 is Induced and Maintained by IL-6 in HepG2 Cells

The signal transduction pathway of IL-6 signal has 2 main cellular targets: the transcription factors STAT3 and C/EBPß that mediate most of the IL-6-induced alterations in gene expression. Strongly increased nuclear STAT3 levels stimulate transcription of target genes and are attributable to rapid STAT3 phosphorylation by janus kinases (JAKS) that are stimulated by binding of IL-6 to its cognate receptor. 28 As a consequence, induction of gene transcription by binding of phosphorylated STAT3, which leads to dimerization, occurs rapidly after IL-6 signaling. On the other hand, increased C/EBPß activity is achieved by enhanced transcription of the structural gene as well as by phosphorylation of the protein 29; hence, increased trans -activation of gene promoters by C/EBPß generally occurs later in time and lasts longer. 46 Therefore, we tested NE from HepG2 cells, which had been stimulated with IL-6, for the presence of both C/EBPß and phosphorylated STAT3 ( Figure 2 ). Whereas C/EBPß levels remained similar, phosphorylated STAT3 levels were increased at both time points tested, 30 minutes and 14 hours. The presence of strong STAT3 phosphorylation after 14-hours incubation with IL-6 is in concordance with the sustained PROS1 mRNA induction.

Figure 2. Effect of IL-6 treatment on several transcription factors. The effect of IL-6 treatment on the transcription factors Sp1 (as a control), STAT3 and C/EBPß is shown using Western Blot Analysis. Antibodies specific for Sp1, C/EBPß and the phosphorylated form of STAT3 were used. Separate gels and blots were used for each antibody.

An IL-6 Responsive Element is Present in the PROS1 Promoter

Subconfluent HepG2 cells were transfected with promoter-reporter gene plasmids containing a PROS1 5' region of varying length, after which the cells were either induced with IL-6, or grown further under basal conditions ( Figure 3 a). A PROS1 promoter region of 261 bp remained IL-6-responsive. However, transfections with the shortest promoter-reporter gene construct, PS197, did not result in increased luciferase levels on IL-6 induction. These results indicate that an IL-6-responsive element within the PROS1 promoter is located between 261 and 197 bp upstream from the translational startcodon. The stimulatory effect of IL-6 on PS261 was not augmented with increasing promoter size.

Figure 3. Localization of the IL-6-responsive element in the PROS1 promoter. a, Histogram showing PROS1 promoter activity, as assessed using luciferase constructs, in the absence (open bars) or presence (shaded bars) of a physiological concentration of IL-6 (5 ng/mL). A schematic representation of each construct is shown on the left of the histogram. Statistical significance was determined with Student t test. b, Sequence of the PROS1 promoter between -261 and -197 containing the IL-6 responsive element. The double stranded oligonucleotide used in the EMSA assays is indicated.

The IL-6 Response is Mediated by Binding of STAT3 to the IL6-RE in the PROS1 Promoter

Computational analysis using various databases on the internet such as MatInspector professional (hhtp://www.genomatix. com), Transfac (http://www.gene-regulation.com), and tfsitescan (http://www.ifti.org) showed that both a STAT3 and a C/EBPß binding consensus are located in the -261 to -197 bp region in the PROS1 promoter ( Figure 3 b).

Increased binding of a protein complex to a ds oligonucleotide probe, -229/-207wt, overlapping this region was observed in extracts from IL-6-treated HepG2 cells ( Figure 4, left panel). The majority of this protein-DNA complex was shifted with a STAT3 antibody, leaving a weak signal at the original location.

Figure 4. EMSA of the IL-6-resposive element in the PROS1 promoter. EMSA using a double-stranded oligonucleotide representing the IL-6-responsive element and using NE from HepG2 cells treated with or without 100 ng/mL IL-6. The location of the IL-6-specific band is indicated by an arrow. Nonlabeled double-stranded competitor oligonucleotides were used at 100-fold molar excess. wt indicates wild-type, mt, mutant, cs1 is a STAT3 consensus oligonucleotide, cs2 is a C/EBPß consensus oligonucleotide (see the Methods section for the sequences of the oligonucleotides). Antibody 1 was a STAT3-specific antibody and antibody 2 was a C/EBPß specific antibody.

The mutated unlabeled ds oligonucleotide, -229/-207mt, was unable to compete with the PS wild-type probe for protein binding, whereas unlabeled ds oligonucleotides -229/-207wt and STAT3cons containing the PROS1 wild-type STAT3 site and the STAT3 consensus sequence were efficient competitors ( Figure 4 ). The C/EBPß consensus oligonucleotide did not influence protein binding to the -229/-207 probe. However, a slight shift from the original complex in NE from untreated HepG2 cells was observed with a C/EBPß antibody, suggesting that the residual band present at the original location in the STAT3 supershift might contain comigrating C/EBPß-probe complexes ( Figure 4, right panel).

The expression of mutated promoter-reporter gene constructs in HepG2 cell culture was analyzed to investigate the contribution of STAT3 and possibly C/EBPß to transcriptional upregulation of PS levels during IL-6 induction. Although basal promoter activity remained unaltered on mutation of the -229/-207 region, the mutations resulted in abolishment of the IL-6 response in all promoter-reporter gene constructs ( Figure 5 ). This underlines our preliminary conclusion from the data obtained with the wild type PROS1 -reporter gene constructs that the site at -229/-207 is the only IL-6-RE in the proximal PROS1 promoter.

Figure 5. Activity of the PROS1 promoter after mutagenesis of the IL-6-responsive element. Histogram showing the luciferase activity of the wild-type -370 construct and of 3 constructs with a mutated IL-6-responsive element in the presence of a physiological concentration IL-6 (5 ng/mL).

Discussion

Plasma proteins that are upregulated during the APR can be roughly divided into 3 major groups: proteins with an increased level of about 50% (eg, ceruloplasmin and complement factor-3), those with an increase of 2- to 3-fold (haptoglobin, fibrinogen, C4BP), and a group of proteins which responds with a rapid increase of 5-fold to up to 1000-fold (C-reactive protein and serum amyloid A protein). 18,47-50 We found a 2-fold increase in PS levels in the medium after 48 hours incubation of HepG2 cell culture with 5 ng IL-6 per ml medium ( Figure 1 ), which is in agreement with a previous report. 15 Both PROS1 mRNA as well as PS protein levels were elevated up to 48 hours of IL-6 stimulation. Protein S thus seems to belong to the second category of acute phase proteins.

The fact that not only PS protein levels but also PROS1 mRNA levels were upregulated by IL-6 indicated that IL-6 acted at the transcriptional level. Because IL-6 induction is known to be mediated primarily by STAT3 and C/EBPß, we tested the levels of these 2 transcription factors in IL-6-induced NE and found that total C/EBPß levels remained unchanged throughout time, whereas phosphorylated STAT3 levels were increased, also after prolonged IL-6 stimulation ( Figure 2 ). This result is in concordance with the sustained higher PS levels in IL-6-treated versus untreated HepG2 cell culture.

In this study, we identified an IL-6-RE in the PROS1 promoter at a region &220 bp upstream from the PROS1 translational start codon. This region contains both the hallmarks of the STAT3 and the C/EBPß binding sites ( Figure 3 ). We identified STAT3 as the main transcription factor binding to the IL-6-RE ( Figure 4 ). C/EBPß may also bind to this region, but its contribution appears to be relatively minor. Mutation of the STAT3 binding site in the PROS1 promoter completely abolished the IL-6 response in transfection experiments with promoter-reporter gene constructs ( Figure 5 ). This indicates that there are no additional functional STAT3 or C/EBPß binding sites through which the IL-6 effect is mediated in the region investigated. The putative overlap between the STAT3 and the C/EBPß binding sites around position -220 makes it difficult to make definitive statements about the role of the latter protein in the induction of PROS1 transcription in HepG2 cells. Overlapping binding sites might constitute an efficient way to create an IL-6-responsive element that is active both at early and late time points of IL-6 stimulation.

Although PROS1 is clearly IL-6-responsive in HepG2 cell culture, patient studies indicate that total systemic PS levels are only slightly increased or remain at a similar level during inflammation. Moreover, in a subpopulation of patients free PS levels are reduced during inflammation because of increased C4BP ß-chain levels. 22 Also in patients with sepsis, free PS levels (measured as PS activity) were decreased. 51 In both circumstances, inflammation or sepsis, IL-6 is a major player and its induction of PS transcription does not apparently outweigh the above-mentioned in vivo effects. However, it could be argued that anticoagulation would even be worse if PROS1 transcription by IL-6 was not mildly induced. Nevertheless, the possibility that the main function of PROS1 transcriptional regulation by IL-6 may lie in another aspect of the inflammatory response should also be considered.

IL-6 has been implicated in a variety of cellular processes with a diverse array of regulatory roles. Dependent on the target cell, IL-6 may induce various and sometimes contrasting responses. For instance, IL-6 has a proinflammatory function and it can induce cytotoxic effects, but it is also a cell survival factor. Several studies have demonstrated the neuroprotective effects of IL-6, which are mediated by the stimulation of growth factors and STAT3. 52-56 Recently, Liu et al reported a neuroprotective effect of intravenously injected PS on cortical neurons during ischemic injury in mice. 57 The authors speculated that PS may be acting via a similar mechanism as its structural homologue, growth arrest-specific gene 6 ( gas6 ), which induces cell survival through a signaling pathway mediated by the Tyro3/Axl membrane receptor family. Stimulation of local PS synthesis by IL-6 may therefore explain part of the cell survival properties reported for IL-6. In addition, increasing evidence suggests a function of PS in macrophage phagocytosis of apoptotic cells both directly 58,59 and indirectly by directing complement to the surface of apoptotic cells. 60,61 Exactly what this new role for PS entails has not yet been fully clarified. Based on the cited literature, it seems plausible that the upregulation by IL-6 of local and not systemic PS levels may contribute to processes such as the cell survival of cells in the inflamed region. Obviously, this hypothesis needs to be tested in future research.

Acknowledgments

Sources of Funding

This work was financially supported by the Dutch Thrombosis Foundation (grant application number TSN 98.002).

Disclosures

None.

【参考文献】
  Esmon CT. The protein C pathway. Chest. 2003; 124: 26S-32S.

Dahlbäck B. Progress in the understanding of the protein C anticoagulant pathway. Int J Hematol. 2004; 79: 109-116.

Walker FJ. Regulation of activated protein C by a new protein. A possible function for bovine protein S. J Biol Chem. 1980; 255: 5521-5524.

Walker FJ. Protein S and the regulation of activated protein C. Semin Thromb Hemost. 1984; 10: 131-138.

Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood. 1984; 63: 486-489.

Walker FJ, Chavin SI, Fay PJ. Inactivation of factor VIII by activated protein C and protein S. Arch Biochem Biophys. 1987; 252: 322-328.

Comp PC, Esmon CT. Recurrent venous thromboembolism in patients with a partial deficiency of protein S. N Engl J Med. 1984; 311: 1525-1528.

Engesser L, Broekmans AW, Briët E, Brommer EJ, Bertina RM. Hereditary protein S deficiency: clinical manifestations. Ann Intern Med. 1987; 106: 677-682.

Zöller B. Familial thrombophilia: clinical and molecular analysis of Swedish families with inherited resistance to activated protein C or protein S deficiency. Scand J Clin Lab Invest (Suppl). 1996; 226: 19-46.

Fair DS, Marlar RA. Biosynthesis and secretion of factor VII, protein C, protein S, and the Protein C inhibitor from a human hepatoma cell line. Blood. 1986; 67: 64-70.

Dahlbäck B, Stenflo J. High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein. Proc Natl Acad Sci U S A. 1981; 78: 2512-2516.

Dahlbäck B. Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein. J Biol Chem. 1986; 261: 12022-12027.

Griffin JH, Gruber A, Fernandez JA. Reevaluation of total, free, and bound protein S and C4b-binding protein levels in plasma anticoagulated with citrate or hirudin. Blood. 1992; 79: 3203-3211.

Ramadori G, Christ B. Cytokines and the hepatic acute-phase response. Semin Liver Dis. 1999; 19: 141-155.

Hooper WC, Phillips DJ, Ribeiro M, Benson J, Evatt BL. IL-6 upregulates protein S expression in the HepG-2 hepatoma cells. Thromb Haemost. 1995; 73: 819-824.

Hooper WC, Phillips DJ, Evatt BL. TNF-alpha suppresses IL-6 upregulation of protein S in HepG-2 hepatoma cells. Thromb Res. 1996; 81: 315-326.

Hooper WC, Phillips DJ, Evatt BL. Endothelial cell protein S synthesis is upregulated by the complex of IL-6 and soluble IL-6 receptor. Thromb Haemost. 1997; 77: 1014-1019.

Saeki T, Hirose S, Nukatsuka M, Kusunoki Y, Nagasawa S. Evidence that C4b-binding protein is an acute phase protein. Biochem Biophys Res Commun. 1989; 164: 1446-1451.

Barnum SR, Dahlbäck B. C4b-binding protein, a regulatory component of the classical pathway of complement, is an acute-phase protein and is elevated in systemic lupus erythematosus. Complement Inflamm. 1990; 7: 71-77.

Moffat GJ, Tack BF. Regulation of C4b-binding protein gene expression by the acute-phase mediators tumor necrosis factor-alpha, interleukin-6, and interleukin-1. Biochemistry. 1992; 31: 12376-12384.

Garcia de Frutos P, Alim RI, Hardig Y, Zöller B, Dahlbäck B. Differential regulation of alpha and beta chains of C4b-binding protein during acute-phase response resulting in stable plasma levels of free anticoagulant protein S. Blood. 1994; 84: 815-822.

D?Angelo A, Gerosa S, D?Angelo SV, Mailhac A, Colombo A, Agazzi A, Mazzola G, Chierchia S. Protein S and protein C anticoagulant activity in acute and chronic cardiac ischemic syndromes. Relationship to inflammation, complement activation and in vivo thrombin activity. Thromb Res. 1994; 75: 133-142.

Vila N, Reverter JC, Yague J, Chamorro A. Interaction between interleukin-6 and the natural anticoagulant system in acute stroke. J Interferon Cytokine Res. 2000; 20: 325-329.

Kaba NK, Francis CW, Hall WJ, Falsey AR, Smith BH. Protein S declines during winter respiratory infections. J Thromb Haemost. 2003; 1: 729-734.

Criado-Garcia O, Gonzalez-Rubio C, Lopez-Trascasa M, Pascual-Salcedo D, Munuera L, Rodriguez de Cordoba S. Modulation of C4b-binding protein isoforms during the acute phase response caused by orthopedic surgery. Haemostasis. 1997; 27: 25-34.

Criado-Garcia O, Sanchez-Corral P, Rodriguez de Cordoba S. Isoforms of human C4b-binding protein. II. Differential modulation of the C4BPA and C4BPB genes by acute phase cytokines. J Immunol. 1995; 155: 4037-4043.

Akira S. IL-6-regulated transcription factors. Int J Biochem Cell Biol. 1997; 29: 1401-1418.

Heinrich PC, Behrmann I, Haan S, Hermanns HM, Müller-Newen G, Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J. 2003; 374: 1-20.

Schrem H, Klempnauer J, Borlak J. Liver-enriched transcription factors in liver function and development. Part II: the C/EBPs and D site-binding protein in cell cycle control, carcinogenesis, circadian gene regulation, liver regeneration, apoptosis, and liver-specific gene regulation. Pharmacol Rev. 2004; 56: 291-330.

Derfalvi B, Igaz P, Fulop KA, Szalai C, Falus A. Interleukin-6-induced production of type II acute phase proteins and expression of junB gene are downregulated by human recombinant growth hormone in vitro. Cell Biol Int. 2000; 24: 109-114.

Ramji DP, Vitelli A, Tronche F, Cortese R, Ciliberto G. The two C/EBP isoforms, IL-6DBP/NF-IL6 and C/EBP delta/NF-IL6 beta, are induced by IL-6 to promote acute phase gene transcription via different mechanisms. Nucleic Acids Res. 1993; 21: 289-294.

Zhang D, Sun M, Samols D, Kushner I. STAT3 participates in transcriptional activation of the C-reactive protein gene by interleukin-6. J Biol Chem. 1996; 271: 9503-9509.

Bannach FG, Gutierrez-Fernandez A, Parmer RJ, Miles LA. Interleukin-6-induced plasminogen gene expression in murine hepatocytes is mediated by transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta). J Thromb Haemost. 2004; 2: 2205-2212.

Zauberman A, Lapter S, Zipori D. Smad proteins suppress CCAAT/enhancer-binding protein (C/EBP) beta- and STAT3-mediated transcriptional activation of the haptoglobin promoter. J Biol Chem. 2001; 276: 24719-24725.

Gervois P, Vu-Dac N, Kleemann R, Kockx M, Dubois G, Laine B, Kosykh V, Fruchart JC, Kooistra T, Staels B. Negative regulation of human fibrinogen gene expression by peroxisome proliferator-activated receptor alpha agonists via inhibition of CCAAT box/enhancer-binding protein beta. J Biol Chem. 2001; 276: 33471-33477.

Schmidel DK, Tatro AV, Phelps LG, Tomczak JA, Long GL. Organization of the human protein S genes. Biochemistry. 1990; 29: 7845-7852.

Ploos van Amstel HK, Reitsma PH, van der Logt CP, Bertina RM. Intron-exon organization of the active human protein S gene PS alpha and its pseudogene PS beta: duplication and silencing during primate evolution. Biochemistry. 1990; 29: 7853-7861.

Edenbrandt CM, Lundwall A, Wydro R, Stenflo J. Molecular analysis of the gene for vitamin K dependent protein S and its pseudogene. Cloning and partial gene organization. Biochemistry. 1990; 29: 7861-7868.

Tatewaki H, Tsuda H, Kanaji T, Yokoyama K, Hamasaki N. Characterization of the human protein S gene promoter: a possible role of transcription factors Sp1 and HNF3 in liver. Thromb Haemost. 2003; 90: 1029-1039.

De Wolf CJF, Cupers RMJ, Bertina RM, Vos HL. 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. J Biol Chem. 2006; 281: 17635-17643.

Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983; 11: 1475-1489.

Deutz-Terlouw PP, Ballering L, van Wijngaarden A, Bertina RM. Two ELISAs for measurement of protein S, and their use in the laboratory diagnosis of protein S deficiency. Clin Chim Acta. 1990; 186: 321-324.

Amrani DL. Regulation of fibrinogen biosynthesis: glucocorticoid and interleukin-6 control. Blood Coagul Fibrinolysis. 1990; 1: 443-446.

Karlsson JO, Yarmush ML, Toner M. Interaction between heat shock and interleukin 6 stimulation in the acute-phase response of human hepatoma (HepG2) cells. Hepatology. 1998; 28: 994-1004.

Bas S, Gauthier BR, Spenato U, Stingelin S, Gabay C. CD14 is an acute-phase protein. J Immunol. 2004; 172: 4470-4479.

Niehof M, Streetz K, Rakemann T, Bischoff SC, Manns MP, Horn F, Trautwein C. Interleukin-6-induced tethering of STAT3 to the LAP/C/EBPbeta promoter suggests a new mechanism of transcriptional regulation by STAT3. J Biol Chem. 2001; 276: 9016-9027.

Kushner I, Gewurz H, Benson MD. C-reactive protein and the acute-phase response. J Lab Clin Med. 1981; 97: 739-749.

Dowton SB, Colten HR. Acute phase reactants in inflammation and infection. Semin Hematol. 1988; 25: 84-90.

Korkmaz C, Ozdogan H, Kasapcopur O, Yazici H. Acute phase response in familial Mediterranean fever. Ann Rheum Dis. 2002; 61: 79-81.

Lannergard A, Larsson A, Kragsbjerg P, Friman G. Correlations between serum amyloid A protein and C-reactive protein in infectious diseases. Scand J Clin Lab Invest. 2003; 63: 267-272.

Kinasewitz GT, Yan SB, Basson B, Comp P, Russell JA, Cariou A, Um SL, Utterback B, Laterre PF, Dhainaut JF; PROWESS Sepsis Study Group. Universal changes in biomarkers of coagulation and inflammation occur in patients with severe sepsis, regardless of causative micro-organism [ISRCTN74215569]. Crit Care. 2004; 8: R82-R90.

Gadient RA, Otten UH. Interleukin-6 (IL-6)-a molecule with both beneficial and destructive potentials. Prog Neurobiol. 1997; 52: 379-390.

Carlson NG, Wieggel WA, Chen J, Bacchi A, Rogers SW, Gahring LC. Inflammatory cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha impart neuroprotection to an excitotoxin through distinct pathways. J Immunol. 1999; 163: 3963-3968.

Badache A, Hynes NE. Interleukin 6 inhibits proliferation and, in cooperation with an epidermal growth factor receptor autocrine loop, increases migration of T47D breast cancer cells. Cancer Res. 2001; 61: 383-391.

Thorns V, Walter GF, Licastro F. Effects of IL6 and IL1beta on aFGF expression and excitotoxicity in NT2N cells. J Neuroimmunol. 2002; 127: 22-29.

Sriuranpong V, Park JI, Amornphimoltham P, Patel V, Nelkin BD, Gutkind JS. Epidermal growth factor receptor-independent constitutive activation of STAT3 in head and neck squamous cell carcinoma is mediated by the autocrine/paracrine stimulation of the interleukin 6/gp130 cytokine system. Cancer Res. 2003; 63: 2948-2956.

Liu D, Guo H, Griffin JH, Fernandez JA, Zlokovic BV. Protein S confers neuronal protection during ischemic/hypoxic injury in mice. Circulation. 2003; 107: 1791-1796.

Anderson HA, Maylock CA, Williams JA, Paweletz CP, Shu H, Shacter E. Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells. NatImmunol. 2003; 4: 87-91.

Kask L, Trouw LA, Dahlbäck B, Blom AM. The C4b-binding protein-protein S complex inhibits the phagocytosis of apoptotic cells. J Biol Chem. 2004; 279: 23869-23873.

Webb JH, Blom AM, Dahlbäck B. Vitamin K-dependent protein S localizing complement regulator C4b-binding protein to the surface of apoptotic cells. J Immunol. 2002; 169: 2580-2586.

Webb JH, Blom AM, Dahlbäck B. The binding of protein S and the protein S-C4BP complex to neutrophils is apoptosis dependent. Blood Coagul Fibrinolysis. 2003; 14: 355-359.

作者单位：Hemostasis and Thrombosis Research Center, Department of Hematology, Leiden, The Netherlands.