Activation of NF-κB by IL-1β blocks IL-6-induced sustained STAT3 activation and STAT3-dependent gene expression of the human γ-fibrinogen gene

Cellular Signalling 19 (2007) 1866 – 1878 www.elsevier.com/locate/cellsig

Activation of NF-κB by IL-1β blocks IL-6-induced sustained STAT3 activation and STAT3-dependent gene expression of the human γ-fibrinogen gene Ute Albrecht a , Xiangping Yang c , Rosanna Asselta d , Verena Keitel a , Maria Luisa Tenchini d , Stephan Ludwig b , Peter C. Heinrich c , Dieter Häussinger c , Fred Schaper c,1 , Johannes G. Bode a,⁎,1 a

Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, 40255 Düsseldorf, Germany b Department of Molecular Virology, University of Münster, 48149 Münster, Germany c Department of Biochemistry, Medical School RWTH University Aachen, 52074 Aachen, Germany d Department of Biology and Genetics for Medical Sciences, University of Milan, 20133 Milan, Italy Received 7 April 2007; accepted 23 April 2007 Available online 1 May 2007

Abstract Despite the essential role of the fibrinogen γ-chain as a blood clotting factor, the fibrinogen γ-chain contains a number of interaction sites to recruit other factors such as leukocytes important for prevention of pathogen entry and propagation of the repair process. Interleukin-6 (IL-6) is known as the major inducer of γ-fibrinogen synthesis in hepatocytes, whereas IL-1β has been shown to act as a potent inhibitor of γ-fibrinogen expression. Studies on the rat fibrinogen γ-chain promoter suggest that nuclear factor (NF)-κB replaces the signal transducer and activator of transcription (STAT) 3 from binding to overlapping NF-κB/STAT3 binding sites within the 5′ regulatory region of the rat γ-chain gene promoter. However, despite its physiological relevance, the underlying mechanism responsible for the inhibitory effect of IL-1β in humans is still not understood and apparently more complex. In contrast to the mechanism described for the rat gene our results indicate that IL-1β suppresses the IL6-induced activation of the human γ-fibrinogen gene particularly by blocking the late phase STAT3-tyrosine phosphorylation NF-κB-dependently but independent from de novo protein synthesis. Consequently, blocking NF-κB activation restores specifically late phase STAT3 activation as well as the induction of the human γ-fibrinogen gene. In contrast, specifically early STAT3 activation could be restored by a block of the p38 mitogen-activated protein kinase (p38MAPK) pathway. In summary, our results indicate that expression of the γ-fibrinogen gene is mainly controlled by the strength of late phase STAT3 activation, which in turn is negatively regulated by the extent of IL-1β-mediated NF-κB activity. © 2007 Elsevier Inc. All rights reserved. Keywords: Acute phase response; Signal transduction; Transcription factors; Gene regulation; Inflammation; Cytokines

1. Introduction Abbreviations: APP, acute phase protein; IL, interleukin; Jak, Janus kinase; NF-κB, nuclear factor-κB; PIAS, protein inhibitor of activated STATs; SHP, SH2-containing protein tyrosine phosphatase; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor; CRP, C-reactive protein; SAA, serum amyloid A. ⁎ Corresponding author. Department of Gastroenterology, Hepatology und Infectiology; Heinrich-Heine-University, Moorenstraβe 5, D-40225 Düsseldorf, Germany. Tel.: +49 211 81 18952; fax: +49 211 8117517. E-mail address: [email protected] (J.G. Bode). 1 Both senior authors contributed equally. 0898-6568/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2007.04.007

One of the most important and immediate processes to combat tissue injury is the formation of a provisional seal of the wound to prevent hemorrhage. Additionally, clot formation constitutes the prerequisite for the following tissue repair process to prevent pathogen entry and finally scar formation. The creation of this provisional seal is achieved by the initiation of a cascade which at the end leads to the activation of platelets, thrombin-mediated activation of fibrinogen and clot formation at the site of injury [1].

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Fibrinogen is the precursor of the main clot structural protein, fibrin. It is a hexameric protein composed of two of each protein subunits Aα, Bβ, and γ [2]. All three subunits are crucial for blood clotting. To complete the primary barrier against pathogen entry the γ-chain has additional functions in the recruitment of leukocytes [3], other clotting factors, growth factors and inflammatory cytokines, such as interleukin (IL)-1β [4]. Given these particular important properties of the fibrinogen γ-chain in humans it is important to understand the mechanisms underlying the regulated expression of γ-fibrinogen. In addition to the basal expression of the human γ-fibrinogen, its production is greatly increased during the acute phase response and is therefore regarded as an acute phase protein (APP). Whereas IL-6 is the major mediator of hepatic APP synthesis in response to sterile inflammatory stimuli, the regulation of APP expression by systemic inflammation elicited e.g. through bacterial components such as lipopolysaccharides (LPS) is more complex and involves additional mediators, particularly the proinflammatory cytokines TNFα and IL-1β [5,6]. Induction of APP synthesis by these cytokines occurs in a synergistic or antagonistic manner, dependent on the specific APP gene. For instance, IL-1β and IL-6 act synergistically on the expression of the acute phase proteins C-reactive protein (CRP), serum amyloid A (SAA) [7,8] and α1-acid glycoprotein, whereas the IL-6-induced expression of the fibrinogen γ-chain, anti-chymotrypsin (ACT) or the rat-specific α2-macroglobulin (α2M) is suppressed by IL-1β [9–12]. In summary, the expression pattern of the different APPs depends on the cytokine pattern released in response to the inflammatory stimulus. Unfortunately, the molecular mechanisms underlying this gene-

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specific regulation by IL-1β and IL-6 are not well understood so far. The coordinated activation of the transcription factors nuclear factor (NF)-κB, NF-IL6 and signal transducer and activator of transcription (STAT) 3 to the promoter, appears to be important for the synergistic effect of IL-1β and IL-6 on CRP expression, whereas the cooperative activation of the SAA promoter depends on NF-κB-mediated recruitment of the transcription factor STAT3 [13,14]. In contrast, attenuation of rat APP gene expression by IL-1β relies on an antagonistic action of NF-κB and STAT3 as shown for the rat α2-macroglobulin [15,16] and the rat fibrinogen γ-chain [10] gene activation. In both cases, NF-κB is thought to counteract STAT3 DNA binding to overlapping STAT3/NF-κB binding sites located within the 5′ regulatory region of these genes. The potential of this regulatory mechanism is intriguing since it would represent a very rapid and gene-specific option by which IL-1β could modulate IL-6induced gene expression. The molecular mechanism identified for the regulation of the rat fibrinogen γ-chain gene is not applicable to the human gene since the 5′ regulatory region of the human γ-fibrinogen gene does not comprise any comparable overlapping STAT3/NF-κB binding sites [17] (Fig. 1). Thus, the underlying mechanism for IL-1β-mediated suppression of IL-6-induced γ-fibrinogen expression in humans remains to be elucidated. Particularly, with respect to the pathophysiological importance of the γ-chain in wound healing and repair processes it is of special interest to resolve this issue. The purpose of the present study was to further elucidate the molecular mechanisms underlying the regulation of the hepatic γ-

Fig. 1. Comparison of the 5′ regulatory regions of the rat and human γ-fibrinogen gene. The respective 5′ regulatory regions of the human (upper sequence) and rat (lower sequence) fibrinogen γ-chain are depicted. The respective STAT3 responsive elements described for the human [17] and the rat [37] genes are marked by underlying grey boxes. The NF-κB site is framed in black. TATA boxes and sites of transcription start are marked by black boxes. The transcription start site is labeled with +1.

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fibrinogen expression in humans and to contribute to a better understanding of the cross-talk between NF-κB and STAT3 signaling in the acute phase response. Although this study supports the notion that NF-κB activation is also crucial for the inhibitory effect of IL-1β on IL-6-induced synthesis of the human fibrinogen γ-chain, the proposed mechanism differs substantially from the model suggested for the regulation of the corresponding rat gene promoter. Moreover, evidence is provided that activated NF-κB affects the kinetics of STAT3-tyrosine phosphorylation independent of de novo protein synthesis. 2. Materials and methods 2.1. Materials Restriction enzymes were purchased from New England Biolabs (Frankfurt, Germany); Taq Polymerase was from Roche (Mannheim, Germany); oligonucleotides were obtained from MWG-Biotech (Ebersberg, Germany). Dulbecco's modified Eagle medium (DMEM) and DMEM/nutrient mix F-12 were from Invitrogen (Karlsruhe, Germany); fetal calf serum was from Perbio (Bonn, Germany); the internal control plasmid DNA pCH110 was from Amersham Biosciences (Uppsala, Sweden). The specific antibodies against STAT3 phosphorylated at tyrosine 705 and against p65 phosphorylated at serine 536 were from Cell Signaling Technology (Beverly, MA, USA). The antibodies against STAT3α, inhibitor of κB (IκB) α, p65 and gp130 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to GAPDH (glyceraldehyd-3-phosphat dehydrogenase) was obtained from Biodesign (Saco, ME, USA) and antibody to γ-fibrinogen was from Upstate (Charlottesville, VA, USA). Recombinant human IL-1β, recombinant human IL-6 and recombinant mouse erythropoietin were from Roche. Cycloheximide and the p38MAPK inhibitor SB 202190 were from Calbiochem (Schwalbach, Germany).

2.2. Cell culture, stimulation and preparation of cells The human hepatoma cells HepG2 were grown in DMEM/nutrient mix F-12 supplemented with 10% (v/v) heat-inactivated fetal calf serum. Medium was changed and adjusted to 3 ml serum free medium 16 h before experiments were carried out. Cells grown in a 60 mm dish were stimulated with IL-1β or IL-6 at the concentrations indicated. Nuclear extracts were prepared as described by Andrews and Faller [18]. Protein concentration was determined with a BioRad Protein Assay (BioRad; Munich, Germany).

2.3. Isolation and culture of human hepatocytes Liver tissue was obtained from patients undergoing hemihepatectomy or lobectomy for removal of liver metastasis after informed consent and in accordance with the guidelines of the Ethics Committee of the University of Düsseldorf, Germany, and the Declaration of Helsinki. Liver samples (15 cm3) were taken from tumor free tissue and visible vessels were cannulated with 16 gauge-intravenous catheters and perfused for 10 min with buffer A (0.2 mM EGTA, 10 mM HEPES, 7 mM KCl, 143 mM NaCl, pH 7.33), then for 5 min with buffer B (5 mM CaCl2, 50 mM HEPES, 7 mM KCl, 100 mM NaCl) and for another 15 min with buffer B containing 50 mg/150 ml collagenase IV (125 CDU [collagen digestion unit]/mg; Sigma; Taufkirchen, Germany). After removal of the hepatic capsule the hepatocytes were mechanically removed by shaking in the collagenase solution. Afterwards the cell suspension was filtered through a 70 μm cell strainer (BD Falcon; Bedford, MA, USA), pelleted by centrifugation (500 g, 4 °C, 3 min) and washed several times. Cell viability was N90% as determined by trypan blue exclusion. Hepatocytes were resuspended in William's Eagle Medium containing 5% (v/v) FCS, 1% (v/v) penicillin/streptomycin, glutamine (2 mM), insulin (100 nM), dexamethason (100 nM) and were seeded at a density of 2.5 × 106 cells/ dish on collagen type-VII-coated 6 cm dishes. After 3 h cells were washed with PBS and medium was replaced by dexamethason free William's Eagle Medium containing 5% (v/v) FCS, 1% (v/v) penicillin/streptomycin, glutamine (2 mM), insulin (100 nM). Experiments were performed after 10 h.

2.4. Plasmids Standard cloning procedures were performed as outlined by Sambrook and Russel [19]. The sequences of all constructs were controlled by sequencing (MWG; Ebersberg, Germany). pGL2-373 Luc contains a 381 bp-long fragment of the 5′ regulatory region of the γ-fibrinogen gene comprising the sequence from position − 373 to +8 of the human fibrinogen γ-chain gene fused to the luciferase-encoding sequence and was described previously [20]. The pCAGSHA-STAT3 dn was kindly provided by Dr. Hirano (Osaka, Japan) and encodes a dominant-negative (dn) mutant of STAT3 where tyrosine 705 was exchanged to phenylalanine. The cDNA for dominant-negative p38MAPK mutant tagged with the flag epitope was cloned into the KRSPA expression vector as described in Ludwig et al. [21]. The expression vector pRc/CMV-EG encoding the chimeric EpoR/gp130 receptor (pRc/CMV-EG (YYYYYY)) has been described previously [22]. The IκBα (S/A) expression vector encodes for an IκBα mutant, where serine 32 and 36 have been mutated to alanine to avoid phosphorylation and subsequent degradation of IκBα (Upstate Laboratories Inc.; Lake Placid, NY, USA). The suppressor of cytokine signaling (SOCS) 3specific siRNA was cloned into the p-Super vector as described recently [23].

2.5. Transfection procedure and reporter gene assay For transient transfection of HepG2 cells, cells were grown on 6-well plates to 30% confluence and transfected in DMEM supplemented with 10% fetal calf serum. Calcium phosphate precipitation was performed with 1.0 μg of the reporter construct, 0.75 μg of the β-galactosidase expression vector (pCR3lacZ, Amersham Biosciences) and 2.25 μg of the respective expression vector as indicated in the figure legends. Each transfection mixture was adjusted with control vector to normalize amounts of transfected DNA. Cells were incubated with the precipitate for 16 h, washed twice with phosphate buffered saline (PBS), and incubation was continued for additional 10 to 24 h in fresh medium. For reporter gene assays, cells were stimulated for 8 or 16 h as indicated in the figure legends. Cell lysis and luciferase assays were carried out using the luciferase kit (Promega; Madison, WI, USA) as described in the manufacturer's instructions. All expression experiments were done at least in triplicates. Luciferase activity values were normalized to transfection efficiency monitored by the co-transfected β-galactosidase expression vector. Results are expressed as percentage (means ± S.D.) of IL-6-induced luciferase activity.

2.6. EMSA (electrophoretic mobility shift assay) Preparation of nuclear extracts and EMSAs were performed as described previously [24]. The protein concentration of the nuclear extracts was determined with a BioRad reagent. 10.0 μg of protein was incubated with double-stranded 32 P-labeled κB site (5′-GATCCATGGGGAATTCCCCATG-3′) for NF-κB binding. The protein/DNA complexes were separated on a 4.5% polyacrylamide gel containing 7.5% glycerol in 0.25-fold TBE (20 mM Tris base, 20 mM boric acid, 0.5 mM EDTA, pH 8.0) at 20 V/cm for 3.5 h. Gels were fixed in 10% methanol, 10% acetic acid, and 80% water for 30 min, dried, and autoradiographed.

2.7. Retroviral gene transfer/generation of HepG2 cells The pCFG5-IEGZ retroviral vector allowing expression of GFP from the same mRNA as the gene of interest has been described earlier [25]. ϕNX producer cells plated at a density of 1 × 106/10 cm plate were transfected using the calcium phosphate precipitation method with 10.0 μg of pCFG5-IEGZ vector, pCFG5-IEGZ IκBα(S/A) or pCFG5-IEGZ IKKβ(KD) plasmid DNA [26]. 24 h later transfection efficiencies were determined by monitoring GFP expression. Subsequently, 1 mg/ml zeocin (Invitrogen) was added to the cells, which were then grown in the presence of the antibiotic for additional 2 weeks until all the cells were positive for GFP expression. Retroviral infection of HepG2 cells was performed with supernatants from ϕNX producer cells essentially as described [26]. Briefly, supernatants were filtered through a 0.45 μm filter, and 5 μg/ml polybrene (Sigma) were added to the filtrate. Thereafter, medium of HepG2 cells plated in 6-well plates was replaced by ϕNX cell supernatant containing the IκBα(S/A)-cDNA or IKKβ(KD)-cDNA containing retrovirus (giving HepG2-IκBα(S/A), HepG2-IKKβ(KD) or control

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Fig. 2. IL-1β inhibits IL-6-induced activation of STAT3 and STAT3-dependent expression of the human γ-fibrinogen gene. A) and D) HepG2 cells were stimulated with IL-6 (200 U/ml) and/or IL-1β (100 U/ml) for the times indicated and whole cellular protein extracts were prepared. 50 μg of protein/lane were subjected to immunoblot analysis using antibodies specific for the fibrinogen γ-chain, Tyr705-phosphorylated STAT3, total STAT3, gp130 and GAPDH in the indicated panels. B) Primary human hepatocytes were isolated as described in the Materials and methods section and treated with IL-6 and/or IL-1β as indicated. Thereafter protein extracts were prepared and analyzed by immunoblot as outlined for A and D. C) HepG2 cells were transfected with 1.0 μg of a luciferase reporter gene driven by a 381 bp fragment of the 5′ regulatory region of the γ-fibrinogen gene together with 2.25 μg of an expression vector encoding a dominant-negative mutant of STAT3 (STAT3 dn) or the respective control vector and an expression vector for β-galactosidase (0.75 μg) for monitoring transfection efficiency. One day after transfection cells were stimulated with IL-6 (200 U/ml) and/or IL-1β (100 U/ml) for 16 h. Cellular extracts were prepared and luciferase activity was determined and normalized to β-galactosidase activity as described in the Materials and methods section. Results are expressed as percentage (means ± S.D.) of IL-6-induced luciferase activity. virus (giving HepG2-mock). Culture plates were centrifuged at 1000 ×g for 3 h. Medium was then replaced and HepG2 cells were subsequently cultured in the presence of the selection marker zeocin until all cells were positive for GFP.

3. Results

2.8. Western blot analysis

3.1. IL-1β inhibits IL-6-induced STAT3 activation and the expression of the human γ-fibrinogen chain

At the end of experimental treatment, cells were washed twice with PBS supplemented with 0.1 mM Na3Vo4 and solubilized in 500 μl of lysis buffer [1% Triton X-100, 20 mM Tris/HCl (pH 7.4), 136 mM NaCl, 2 mM EDTA, 50 mM βglycerolphosphate, 20 mM sodium pyrophosphate, 1 mM Na3VO4, 4 mM benzamidine, 0.2 mM pefabloc, 5 μg/ml aprotinin, 5 μg/ml leupeptin and 10% glycerol] at 4 °C. Protein concentration was estimated by using the BioRad protein Assay. For immunoblot analysis equal amounts of protein (50 μg) were subjected to SDS/PAGE (8% gel). The electrophoretically separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes by the semidry Western blotting method. Non-specific binding was blocked with 5% (w/v) non-fat dry milk powder in TBS-T (20 mM Tris/HCl, pH 7.4, 137 mM NaCl, and 0.1% Tween) or 5% bovine serum albumin (BSA) overnight at 4 °C. The blots were incubated overnight at 4 °C or for 2 h at room temperature in TBS-T supplemented with primary antibodies at the dilution indicated. After extensive rinsing with TBS-T, blots were incubated with secondary antibodies, goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase for 1 h (Dako; Glostrup, Denmark). After further rinsing in TBS-T, the immunoblots were developed with the enhanced chemiluminescence system (ECL; Amersham Biosciences) following the manufacturer's instructions.

It is well known that IL-1β impairs IL-6-induced γ-fibrinogen gene expression in rat and human hepatocytes and hepatoma cell lines [9,12,27,28]. For rat hepatocytes this inhibition has been attributed to IL-1β-induced activation of NF-κB, which by competition prevents activated STAT3 from binding to overlapping NF-κB/STAT3 binding sites located within the 5′ regulatory region upstream of the rat γ-fibrinogen gene [10]. However, in the human promoter of the fibrinogen γ-chain the IL6 responsive elements [17] strongly differ from those of the rat promoter and do not consist of similar overlapping binding motifs (Fig. 1). It is therefore unlikely that the mechanism described for the regulation of the rat γ-fibrinogen gene promoter is transferable to the regulation of the respective human gene. As shown in Fig. 2A, the reduced expression of γ-fibrinogen expression in the presence of IL-1β correlates with reduced

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tyrosine phosphorylation of STAT3 (Fig. 2A, compare lanes 1–7 with 10–15; Fig. 2B, compare lanes 1–4 with 5–8) when analyzed in the human hepatoma cell line HepG2 (Fig. 2A) as well as in primary human hepatocytes (Fig. 2B). STAT3 activation by IL-6 reached its maximum after 40 min of stimulation (Fig. 2A: lanes 2 + 3) and declines thereafter to a weaker but sustained state of activation (Fig. 2A: lanes 4 to 7; Fig. 2B lanes 2 to 4). Notably,

IL-1β suppresses both, the initial activation of STAT3 (Fig. 2A: compare lanes 2 + 3 with lanes 10 + 11) as well as the sustained phase (Fig. 2A: compare lanes 4 to 7 with lanes 12 to 15; Fig. 2B compare lanes 2 to 4 with lanes 6 to 8) observed after continued stimulation with IL-6. Hence, we hypothesized that suppression of IL-6-induced STAT3 activation by IL-1β could be a mechanism for the inhibition of human γ-fibrinogen expression.

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To judge the contribution of STAT3 to the activation of the human γ-fibrinogen promoter, reporter gene assays were performed with a γ-fibrinogen promoter fused to a luciferasecoding cDNA in the absence or presence of a dominant-negative STAT3 mutant in HepG2 cells. The crucial role of STAT3 was corroborated by the fact that co-transfection of the dominantnegative mutant of STAT3 almost completely abrogated IL-6induced activation of the human γ-fibrinogen gene promoter (Fig. 2C). Thus, one could expect that inhibition of STAT3 activation by IL-1β contributes to the IL-1β-dependent reduction of IL-6-induced γ-fibrinogen gene expression. While expression of the γ-fibrinogen gene and activation of STAT3 is strongly affected by IL-1β, IL-1β neither influences the total STAT3 protein levels (Fig. 2A) nor the expression levels of the IL-6signal-transducing receptor subunit gp130 (Fig. 2D). These data suggest that inhibition of IL-6-induced STAT3 activation by IL1β and the IL-1β-mediated block of γ-fibrinogen expression might be functionally linked in human hepatic cells. We went on to clarify whether the cells have indeed to be primed with IL-1β to achieve the inhibitory effect on IL-6 signaling. To answer this question IL-1β was added up to 450 min after IL-6 treatment and STAT3 activation, γ-fibrinogen expression and γ-fibrinogen promoter activation was analyzed 8 h later. Fig. 3A indicates that the inhibitory effect of IL-1β on the expression of the fibrinogen γ-chain gene does not only occur if IL-1β is added prior to IL-6. IL-1β still significantly inhibits IL-6mediated activation of STAT3, γ-fibrinogen promoter activity and expression of endogenous γ-fibrinogen even if added as late as 2 to 4 h after IL-6. Moreover, the inhibitory effect of IL-1β on the expression of endogenous γ-fibrinogen (Fig. 3A, second panel) and on STAT3 activation (Fig. 3A, third panel) when added 2 h after IL-6 was as pronounced as if it was given 10 min prior to IL-6 stimulation. These data therefore suggest that although expression of the fibrinogen γ-chain strictly depends on STAT3 activation, the inhibition of the sustained STAT3 activity in the later phase by IL-1β is sufficient to suppress IL-6-induced expression of the γ-chain of human fibrinogen. To elucidate whether the sustained phase of STAT3 activation really contributes to γ-fibrinogen expression, cells were stimulated with an IL-6 pulse for 30 up to 360 min. After withdrawal of IL-6 culture was continued and γ-fibrinogen reporter gene activation (Fig. 3B) and protein expression (Fig. 3C) was analyzed 480 min after initiating the experiment by adding IL-6. In all cases, the lack of IL-6 in the last hours of the assay led to reduced γ-fibrinogen promoter activity. Interestingly, elimination of IL-6 in the very last two hours of the time course is sufficient to significantly reduce promoter activation when compared to

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ongoing IL-6 stimulation (compare bar 2 and 10, Fig. 3B). In summary, these observations are indicative for a crucial role of late STAT3 activity for efficient γ-fibrinogen expression. To follow this idea, we analyzed endogenous γ-fibrinogen expression as well as STAT3 activation in a similar assay (Fig. 3C). In line with the promoter activation in Fig. 3B endogenous γ-fibrinogen expression was reduced when IL-6 was withdrawn at different time points before the very end of the assay (480 min). Again, this supports the idea that IL-6 has to be constantly present to achieve maximal expression levels of γ-fibrinogen and further argues for the importance of late signaling events. According to these observations, withdrawal of the cytokine led to an immediate loss of STAT3 activation (Fig. 3C, first panel) and further argues for a crucial role of late STAT3 activation to achieve maximal gene expression. 3.2. Inhibition of γ-fibrinogen expression is independent from activation of p38MAPK and SOCS3 expression The data outlined above strongly suggest that inhibition of IL6-induced γ-fibrinogen gene expression by IL-1β is mediated through attenuation of STAT3 activity. The mechanism by which IL-1β affects activation of STAT3 is not fully understood. Several reports indicate that activation of the p38MAPK might play a crucial role for the inhibitory effect of IL-1β on IL-6induced STAT3 activation — at least with respect to the initial STAT3 activation occurring within the first 20 to 40 min [29–31]. Hence, p38MAPK activation could also be relevant for the inhibitory effect of IL-1β on γ-fibrinogen gene expression. To address this issue, we analyzed whether γ-fibrinogen promoter activity inhibited by IL-1β could be restored by blocking p38 MAPK activity either with pharmacological inhibitors (Fig. 4A) or by the expression of a dominant-negative mutant of p38MAPK (Fig. 4B). However, interruption of p38MAPK activation did not affect the inhibitory potential of IL-β on IL-6mediated γ-fibrinogen promoter activity. Essentially similar results were obtained when we compared the expression of endogenous γ-fibrinogen in the absence or presence of SB 202190. Again, this p38MAPK inhibitor did not affect the potential of IL-1β to inhibit IL-6-induced γ-fibrinogen expression (Fig. 4C, second panel). The fact that, in accordance to previous reports [29,31], inhibition of p38MAPK activity at least partially rescues STAT3 activation during the early activation phase (Fig. 4D), whereas late STAT3 activity remains unaffected (Fig. 4C, first panel), may further suggest that early STAT3 activation is not primarily relevant for the transcriptional control of the fibrinogen γ-chain gene.

Fig. 3. Expression of the fibrinogen γ-chain gene is impaired upon discontinued STAT3 activation either due to its inhibition by IL-1β or to discontinued IL-6 stimulation. A) HepG2 cells were transfected as described for Fig. 2C. HepG2 cells were stimulated for 8 h with IL-6 (200 U/ml) 1 day after transfection. IL-1β (100 U/ml) was added after the indicated time points. Subsequently, total whole cellular lysates were prepared and analyzed for expression of endogenous γ-fibrinogen and STAT3 activation by Western blot as well as for activation of the γ-fibrinogen promoter reporter. Results are expressed as percentage (means ± S.D.) of IL-6-induced luciferase activity. B) HepG2 cells were transfected as described for A) lanes 1 to 4: cells were stimulated with IL-6 (200 U/ml) and/or IL-1β (100 U/ml) added 10 min prior to IL-6 for 8 h. Lanes 5 to 10: cells were cultivated for 8 h in the presence of 200 U/ml IL-6 for the times indicated. After removing of IL-6 the cells were cultured in a pre-conditioned medium for the remaining time. Whole cellular extracts were prepared and γ-fibrinogen promoter activity was determined as described for A. C) Lanes 1 to 8: HepG2 cells were treated with IL-6 (200 U/ml) for the times indicated. Lanes 9 to 14: cells were cultured for 8 h and treated with 200 /ml IL-6 for the times indicated. After removing of IL-6 the cells were cultured in a pre-conditioned medium for the remaining time. STAT3 activation and expression of endogenous γ-fibrinogen expression was monitored by Western blot as described above.

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Fig. 4. IL-1β mediates its inhibitory effects on IL-6-induced expression of the fibrinogen γ-chain expression independent of p38MAPK. A) HepG2 cells were transfected with 1.0 μg of the γ-fibrinogen promoter reporter and 0.75 μg of an expression vector for β-galactosidase. One day after transfection HepG2 cells were pretreated for 40 min with the p38MAPK inhibitor SB 202190 (10 μM) or DMSO (1 μl/ml) for control. Thereafter, cells were stimulated with IL-6 (200 U/ml) and/or IL-1β (100 U/ml) as indicated for 16 h and promoter activity was determined by analyzing luciferase activity, which was normalized to β-galactosidase activity. Results are expressed as percentage (means ± S.D.) of IL-6-induced luciferase activity. B) HepG2 cells were co-transfected as described for A but additionally with expression vectors encoding a dominant-negative mutant of p38MAPK or the empty expression vector for control (2.25 μg). Stimulation and analysis was performed as described for A. C) and D) HepG2 cells were cultivated for 40 min in a medium containing the p38MAPK inhibitor SB 202190 (10 μM). Afterwards cells were stimulated with IL1β (100 U/ml) and 200 U/ml IL-6 for the times indicated. Cellular extracts were prepared, and 50 μg of the cellular protein were subjected to SDS/PAGE. Proteins were analyzed for the presence of Tyr705-phosphorylated STAT3, γ-fibrinogen and GAPDH by probing Western blots with antibodies recognizing Tyr705-phosphorylated STAT3, γ-fibrinogen or GAPDH. E) HepG2 cells were transfected with 1.0 μg vector of γ-fibrinogen promoter reporter together with a vector ending SOCS3 siRNA or a control vector. An expression vector for β-galactosidase (0.75 μg) was transfected in order to monitor transfection efficiency. After one day, cells were stimulated with IL-1β (100 U/ml) and IL-6 (200 U/ml) or both for 16 h as indicated. Promoter activity was determined as described above. (E1) HepG2 cells were transfected with an Epo/gp130 receptor chimera and an expression vector for SOCS3 siRNA or a vector control respectively. After reaching 70% confluence, cells were stimulated with 1 U/ml erythropoietin (Epo) and IL-1β (100 U/ml) for the times indicated. Cellular extracts were prepared and 50 μg of protein were subjected to SDS/PAGE. Expression of endogenous γ-fibrinogen was monitored by Western blotting with an antibody specific for the human γ-fibrinogen chain. For loading control the membrane was reprobed with a GAPDH-specific monoclonal antibody.

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Another mechanism negatively controlling STAT3 activation is mediated by the negative feedback inhibitor of STAT-dependent signaling SOCS3. SOCS3 expression has recently been demonstrated to be enhanced upon stimulation with IL-1β [23]. To confirm the potential relevance of SOCS3 for IL-1β-mediated inhibition of γ-fibrinogen, induction of SOCS3 expression was antagonized by co-transfection of a vector encoding a SOCS3specific small interfering RNA [23]. As expected for STAT3dependent gene expression, SOCS3 knockdown strongly enhanced activation of the γ-fibrinogen promoter by IL-6 (Fig. 4E, compare second and fifth bar) confirming the efficiency of the siRNA used [23]. Nevertheless, IL-1β treatment leads to reduced promoter activation in the absence as well as in the presence of SOCS3 siRNA. To analyze STAT3 activation in the presence or absence of SOCS3 siRNA, we transfected the siRNA encoding vector together with expression vectors coding for an EpoR/ gp130 chimeric receptor. This approach allows to specifically stimulate the population of transfected cells when monitoring activation of endogenous STAT3. Since no influence of SOCS3 on the inhibitory effect of IL-1β could be observed we conclude that SOCS3 counter-regulates γ-fibrinogen expression in response to IL-6 but does not play a significant role for the influence of IL-1β on γ-fibrinogen expression. Despite of SOCS3, the induction of other inhibitory proteins could mediate the regulatory effect of IL-1β on STAT3-dependent gene activation. In order to evaluate whether de novo protein synthesis is required for the inhibitory effect of IL-1β, protein synthesis was blocked by cycloheximide. As shown in Fig. 5 neither the inhibitory effect of IL-1β on the initial phase of IL-6induced STAT3 activation nor the IL-1β-mediated inhibition of late phase STAT3 activity could be blocked by cycloheximide. 3.3. NF-κB is essential for the inhibitory effect of IL-1β on IL-6-induced expression of the human γ-fibrinogen gene To analyze whether, similar to the rat γ-fibrinogen gene, activation of NF-κB may contribute to the inhibitory effect of IL-1β, a non-degradable mutant of the inhibitor of NF-κB was co-transfected with the human γ-fibrinogen gene promoter construct. Interruption of NF-κB completely blocked the inhib-

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itory effect of IL-1β on IL-6-induced transcriptional activation of the human γ-fibrinogen gene (Fig. 6A). Hence, in line with the findings regarding regulation of the rat γ-fibrinogen gene [10], activation of NF-κB seems to play a pivotal role for the inhibition of the IL-6-dependent expression of human γfibrinogen by IL-1β. However, as outlined above, it is unlikely that the underlying mechanism responsible for NF-κB-mediated inhibition of the human fibrinogen γ-chain resembles that proposed for the rat gene, since regulation of the latter requires overlapping STAT3/NF-κB DNA binding sites within the 5′ regulatory region. To understand the contribution of NF-κB for the regulation of the human γ-fibrinogen gene in more detail, human hepatoma cell lines were prepared to be defective for NF-κB activation. Expression cassettes for a non-degradable mutant of IκBα or a dominant-negative mutant of the IκBkinase (IKK)β were retrovirally transferred to HepG2 cells. As visualized in Fig. 6B and C (right panel) stimulation of these cells with IL-1β does not result in the degradation of endogenous IκBα, whereas in mock-infected HepG2 cells IL1β-induced phosphorylation and subsequent degradation of IκBα is detectable within the first two minutes of stimulation (left panels of Fig. 6B and C). In line with impaired IκBα degradation, both cell lines are incapable to activate NF-κB in response to IL-1β from IκBα as monitored by EMSA using a NF-κB-specific DNA probe derived from the Ig kappa chain (Fig. 6B and C lower panels). Moreover, in accordance to previous findings demonstrating that IKKβ mediates phosphorylation of p65 at serine 536 [32] IL-1β-induced phosphorylation of this serine residue is strongly impaired in hepatoma cells expressing the dominant-negative mutant of IKKβ, whereas it remains largely unaffected in mock-transfected cells or cells stably expressing the non-degradable mutant of IκBα (Fig. 6D). These data demonstrate that in both cell lines generated NF-κB activation in response to IL-1β is sufficiently blocked at different levels of the NF-κB activating cascade. Corroborating the assumption that NF-κB activation is crucial for the inhibitory effect of IL-1β on IL-6-induced expression of γ-fibrinogen, abrogation of NF-κB activation – either by a non-degradable mutant of IκBα or a dominant-negative mutant of IKKβ – completely blocks the inhibitory effect of IL-

Fig. 5. Inhibition of STAT3 activation is independent from de novo protein synthesis. HepG2 cells were cultivated for 30 min in a medium containing cycloheximide (20 μM) in order to inhibit de novo protein synthesis. Afterwards, cells were stimulated with IL-1β (100 U/ml) and IL-6 (200 U/ml) for the times indicated. Cellular extracts were performed, and 50 μg of protein were subjected to SDS/PAGE and Western blotting was performed. Proteins were analyzed for the presence of Tyr705phosphorylated STAT3, STAT3 and GAPDH by probing Western blots with antibodies recognizing specifically Tyr705-phosphorylated STAT3, STAT3 and GAPDH, respectively.

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Fig. 6. A) Inactivation of NF-κB blocks the inhibitory effect of IL-1β on the activation of the human γ-fibrinogen promoter. HepG2 cells were co-transfected with 1.0 μg of the γ-fibrinogen promoter reporter, 0.75 μg of an expression vector for β-galactosidase and with an expression vector encoding a dominant-negative mutant of p38MAPK or empty expression vector for control (2.25 μg). Cells were stimulated with IL-6 (200 U/ml) and/or IL-1β (100 U/ml) as indicated for 16 h and promoter activity was determined by analyzing luciferase activity which was normalized to β-galactosidase activity. Results are expressed as percentage (means ± S.D.) of IL-6induced luciferase activity. B) to D) Generation and characterization of HepG2 cells with impaired NF-κB signaling at the level of IKKβ activation or the release of NF-κB from IκBα. HepG2 cells were infected with a retroviral control vector (mock), with a vector coding for a non-degradable mutant of IκBα (IκBα nd) or for dominant-negative, kinase deficient IKKβ (IKKβ dn) as outlined in the Materials and methods section. Cells were stimulated with IL-1β (100 U/ml) for the times indicated. Whole cellular or nuclear extracts were prepared as outlined in the Materials and methods section. 50 μg of protein were separated by SDS/PAGE and analyzed by Western blotting using antibodies specific for the indicated proteins (B and C upper panels, D). 10.0 μg of nuclear extracts were analyzed for DNA binding of NF-κB by EMSA with the NF-κB binding element of the Ig heavy chain (IgH) promoter (lower panel in B and C). NF-κB/DNA complexes are indicated by arrows. D) The indicated cells were stimulated with IL-1β for the times indicated. Whole cellular extracts were prepared and analyzed for Ser536-phosphorylated p65 by Western blotting.

1β on IL-6-induced promoter activation (Fig. 7A) and endogenous γ-fibrinogen protein expression (Fig. 7B andC). 3.4. NF-κB is crucial for the inhibition of IL-6-induced late phase STAT3 activation As already demonstrated in Fig. 2, inhibition of IL-6-induced γ-fibrinogen expression by IL-1β is paralleled by inhibition of

STAT3-tyrosine phosphorylation. Thus, it was easily conceivable that inhibition of STAT3 activation and expression of the fibrinogen γ-chain are functionally linked — a hypothesis which is further substantiated by the fact that IL-6-induced expression of the fibrinogen γ-chain gene absolutely requires functional STAT3 (Fig. 2C). Nevertheless, we could not exclude that rescuing IL-1β-dependent inhibition by blocking NF-κB activation, changes the cells to STAT3-independent γ-

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Fig. 7. IKKβ activation and IκBα degradation are crucial for the inhibitory effect of IL-1β on γ-fibrinogen promoter activation and expression. A) The HepG2 cells characterized in Fig. 5 were transfected and analyzed for γ-fibrinogen promoter activity as described for Figs. 2–4B and C. Mock-transfected HepG2 cells and HepG2 cells expressing the non-degradable mutant of IκBα (A) or the dominant-negative mutant of IKKβ (C) were stimulated with IL-6 and/or IL-1β as indicated. Cellular extracts were prepared and 50 μg of protein were analyzed by Western blotting using a monoclonal antibody against the fibrinogen γ-chain (upper panels). For loading control the membrane was reprobed with a GAPDH-specific monoclonal antibody (lower panel).

fibrinogen expression. To check whether γ-fibrinogen promoter activation is also STAT3-dependent in cells deficient for NF-κB activation, we co-expressed the non-degradable IκB-inhibitor together with dominant-negative STAT3 and screened for γfibrinogen promoter activation in response to IL-6 alone or in combination with IL-1β. As shown in Fig. 8, activation of the γ-fibrinogen reporter gene construct by IL-6 is strongly dependent on STAT3 in any case. Fig. 9 shows detailed comparative analyses of the time course of IL-6-induced STAT3 activation in the presence or absence of IL-1β. Whereas IL-1β affects both, the immediate (Fig. 9A: compare lanes 2 + 3 with lanes 8 + 9) and the sustained STAT3 activation (Fig. 9A: compare lanes 4 to 6 with lanes 10 to 12) in mock-infected cells, STAT3 activation in the late phase was rescued by blocking NF-κB activation in cells expressing non-degradable IκB (Fig. 9A: compare lanes 10 to 12 with lanes 22 to 24 and Fig. 9B: compare lanes 5 + 6 and lanes 11 + 12). Similar observations were made in cells expressing a dominantnegative IKKβ (Fig. 9C: compare lanes 5 + 6 and lanes 11 + 12). In summary, these data suggest that in human hepatocytes activation of NF-κB by IL-1β suppresses IL-6-induced STAT3dependent expression of γ-fibrinogen particularly by inhibition of late phase STAT3 activation. Therefore, with respect to the γfibrinogen gene expression, inhibition of the initial activation

Fig. 8. The rescue of IL-6-induced γ-fibrinogen promoter activation from the inhibitory effect of IL-1β by blocking NF-κB activation depends on STAT3. HepG2 cells expressing the non-degradable mutant of IκBα were transiently transfected with 1.0 μg vector of the γ-fibrinogen promoter reporter construct, 2.25 μg of either an control vector control (left panel) or an expression vector encoding a dominant-negative mutant of STAT3 (right panel). An expression vector for β-galactosidase (0.75 μg) was co-transfected for monitoring transfection efficiency. Promoter activity was determined as in the experiments presented in the previous figures.

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Fig. 9. NF-κB is responsible for the inhibition of IL-6-induced STAT3 activation by IL-1β in the late phase of STAT3 activation. A) Mock-transfected HepG2 cells (left part) or HepG2 cells expressing the non-degradable mutant of IκBα (right part) were stimulated with 100 U/ml IL-1β and IL-6 (200 U/ml) for the times indicated. Whole cellular extracts were prepared, and analyzed for STAT3 activation as already described for Fig. 2B and C. Mock-transfected HepG2 cells and HepG2 cells either stably expressing the non-degradable mutant of IκBα (B) or the dominant-negative mutant of IKKβ (C) were stimulated with 100 U/ml IL-1β and IL-6 (200 U/ ml) for the times indicated and analyzed for STAT3-tyrosine phosphorylation as described above.

phase of STAT3 by IL-1β seems to be less important. This observation is further supported by the fact that IL-1β inhibits IL-6-induced expression of the fibrinogen γ-chain efficiently, even if added 120 min after IL-6 (Fig. 3A, second lane). 4. Discussion The control of human fibrinogen γ-chain gene expression by IL-6 and IL-1β is incompletely understood, despite the importance of these cytokines for the regulation of acute phase protein synthesis. In contrast to gene expression of C-reactive protein and serum amyloid A, which are synergistically induced by IL-6 and IL-1β [13,14,33], IL-6-initiated expression of the human fibrinogen γ-chain is strongly inhibited by IL-1β in primary human hepatocytes and hepatoma cells [9,12,27,28,34] (Fig. 2). Thus, IL-1β acts as a gatekeeper to enhance the expression of a certain set of acute phase proteins (i.e. CRP and SAA) coevally down-regulating the expression of another set of acute phase proteins (i.e. γ-fibrinogen, α2-macroglobulin). Recent studies on the inhibitory mechanisms of IL-1β suggested a mechanism by which NF-κB displaces activated STAT3 from DNA binding by competing for overlapping STAT3/ NF-κB binding sites located within the 5′ regulatory region of the rat γ-fibrinogen gene promoter [10]. A similar mechanism has been proposed for the regulation of the rat α2-macroglobulin gene promoter [15,16]. This mechanism is meaningful since it allows

the inhibition of the expression of specific genes without affecting the activation of STAT3. However, although rat and human γ-fibrinogen proteins are highly homologous (92%) their 5′ regulatory regions differ conspicuously. In particular the three identified STAT3-responsive elements within the 5′ regulatory region of the human fibrinogen γ-chain gene [17] do not comprise any overlapping NF-κB binding sites as found in the rat promoter (Fig. 1). Thus, it is unlikely that the regulatory mechanism identified for the rat gene is applicable to the human gene. Previous studies provided sufficient evidence that the inhibitory effect of IL-1β on the initial STAT3 activation during the first 30 to 40 min largely depends on the activation of the p38MAPK cascade [29,31]. In this context p38MAPK seems to execute its inhibitory effects on STAT3 activation particularly by down regulation of the surface expression of gp130, the signaltransducing subunit of the IL-6 receptor complex (Radtke, Yang, et al. unpublished data). Therefore, it was reasonable to test whether the reduced expression of γ-fibrinogen in the presence of IL-1β could be restored by inhibition of the p38MAPK activity. Interestingly, blocking p38MAPK activity restored neither IL-6induced expression of the fibrinogen γ-chain (Fig. 4) nor late STAT3 activation but at least partially restored immediate early STAT3 activation (Fig. 4). Thus, IL-6-induced STAT3 activation exhibits two distinct phases: I) the initial STAT3 activation phase which can be inhibited by p38MAPK activation [29,31] and II) the late STAT3 activation phase which is insensitive towards

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activation of the p38MAPK. Hence, the fact that the inhibitory effect of IL-1β on fibrinogen γ-chain expression is also not affected by inhibition of p38MAPK points to a minor role of the initial STAT3 activation phase for the expression of γ-fibrinogen. Nevertheless, fibrinogen γ-chain expression upon stimulation with IL-6 is strictly STAT3-dependent (Fig. 2C) which points to a substantial role of the other, often neglected late phase of STAT3 activation (Figs. 2A, 3C and 9). We observed that inhibition of the IL-6-induced late STAT3 activation by IL-1β could be restored by inactivation of NF-κB (Fig. 9). Concurrently, the block of NF-κB activation restored γ-fibrinogen gene expression in the presence of IL-1β (Figs. 6 and 7). This indicates that in humans IL-1β inhibits IL-6-induced expression of the γ-fibrinogen gene (Figs. 2 and 3) through NF-κB (Figs. 6 and 7) which antagonizes the late STAT3-tyrosine phosphorylation (Fig. 9). This inhibitory activity of IL-1β requires IKKβ (Figs. 7C and 9C) which initiates the degradation of IκBα (Figs. 6A, 7A,B and 9A,B) and the subsequent release of NF-κB but is independent from the phosphorylation state of the NF-κB subunit p65 at serine 536. Furthermore, the inhibitory activity of IL-1β on γ-fibrinogen expression is independent of the IL-6-induced SOCS3 feedback inhibitor (Fig. 4E) or of other feedback inhibitors which require de novo protein synthesis (Fig. 5). How NF-κB mediates its inhibitory effect on STAT3tyrosine phosphorylation is not yet clear. Interestingly, STAT3 was shown to inhibit transcription of the inducible nitric oxide synthase gene in mesangial cells by direct protein/protein interaction with p65 and p50 upon stimulation with IFNγ plus LPS or IL-1β [35]. In a more recent study it has been shown that the STAT3 DNA binding domain is crucial for the interaction of p65 and STAT3 and the inhibitory potential of STAT3 [36]. Hence, IL-1β-induced interaction of STAT3 with p65 might represent a possible explanation for p65-mediated block of STAT3 activation and STAT3-mediated transcription of the γfibrinogen gene. The interaction of NF-κB with STAT3 could also prevent STAT3 from binding to DNA. This would be in line with the fact that p50 prevents STAT3 from binding to DNA [16] — an observation which has initially been explained simply by a competition of NF-κB and STAT3 for binding to the DNA [10,16]. In summary, these data show that NF-κB activation by IL-1β inhibits IL-6-induced late phase STAT3 activation that results in the suppression of γ-fibrinogen gene expression, which mainly depend on continued STAT3 activation and to a lesser extend on the initial strong STAT3 activation. To our knowledge this is the first study that discriminates the relevance of early and late IL-6induced STAT3 activation on gene expression and regulation by IL-1β. 5. Conclusions Inhibition of IL-6-mediated STAT3 activation by IL-1β is achieved by different molecular mechanisms: the immediate early STAT3 activation can be inhibited by p38MAPK but not by NF-κB activity, whereas the later, sustained STAT3 activation is blocked NF-κB-dependently but resistant to p38MAPK activation. Our data indicate that the induction of the human γ-fibrinogen gene

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particularly depends on sustained STAT3-activation which is sensitive to the IL-1β-meditated release of NF-κB from the IκB/ NF-κB complex. Acknowledgments We thank Marianne Ruhl for her excellent technical assistance. This work was supported by Grants from the Deutsche Forschungsgemeinschaft (Bonn), particularly the Sonderforschungsbereich 542 “Molekulare Mechanismen Zytokin-gesteuerter Entzündungsprozesse: Signaltransduktion und pathophysiologische Konsequenzen”. References [1] J.M. Stassen, J. Arnout, H. Deckmyn, Curr. Med. Chem. 11 (17) (2004) 2245. [2] M.W. Mosesson, J. Thromb. Haemost. 3 (8) (2005) 1894. [3] T.P. Ugarova, V.P. Yakubenko, Ann. N. Y. Acad. Sci. 936 (2001) 368. [4] D.H. Farrell, Curr. Opin. Hematol. 11 (3) (2004) 151. [5] D. Haussinger, R. Kubitz, R. Reinehr, J.G. Bode, F. Schliess, Mol. Aspects Med. 25 (3) (2004) 221. [6] J.G. Bode, P.C. Heinrich, in: I.M. Arias, J.L. Boyer, F.V. Chisari, N. Fausto, D. Schachter, D.A. Shafritz (Eds.), The Liver: Biology and Pathobiology, Lippincott Williams Wilkins, Philadelphia, 2001, p. 565. [7] H.J. Moshage, H.M. Roelofs, J.F. van Pelt, B.P. Hazenberg, M.A. van Leeuwen, P.C. Limburg, L.A. Aarden, S.H. Yap, Biochem. Biophys. Res. Commun. 155 (1) (1988) 112. [8] U. Ganter, R. Arcone, C. Toniatti, G. Morrone, G. Ciliberto, Embo. J. 8 (12) (1989) 3773. [9] A. Mackiewicz, T. Speroff, M.K. Ganapathi, I. Kushner, J. Immunol. 146 (9) (1991) 3032. [10] Z. Zhang, G.M. Fuller, Blood 96 (10) (2000) 3466. [11] T. Andus, T. Geiger, T. Hirano, T. Kishimoto, T.A. Tran-Thi, K. Decker, P.C. Heinrich, Eur. J. Biochem. 173 (2) (1988) 287. [12] J.V. Castell, M.J. Gomez-Lechon, M. David, T. Andus, T. Geiger, R. Trullenque, R. Fabra, P.C. Heinrich, FEBS Lett. 242 (2) (1989) 237. [13] K. Hagihara, T. Nishikawa, Y. Sugamata, J. Song, T. Isobe, T. Taga, K. Yoshizaki, Genes Cells 10 (11) (2005) 1051. [14] A. Agrawal, H. Cha-Molstad, D. Samols, I. Kushner, Immunology 108 (4) (2003) 539. [15] Z. Zhang, G.M. Fuller, Biochem. Biophys. Res. Commun. 237 (1) (1997) 90. [16] J.G. Bode, R. Fischer, D. Haussinger, L. Graeve, P.C. Heinrich, F. Schaper, J. Immunol. 167 (3) (2001) 1469. [17] H.O. Duan, P.J. Simpson-Haidaris, J. Biol. Chem. 278 (42) (2003) 41270. [18] N.C. Andrews, D.V. Faller, Nucleic Acids Res. 19 (9) (1991) 2499. [19] J. Sambrook, D. Russel, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2000. [20] R. Asselta, S. Duga, M. Modugno, M. Malcovati, M.L. Tenchini, Thromb. Haemost. 79 (6) (1998) 1144. [21] S. Ludwig, A. Hoffmeyer, M. Goebeler, K. Kilian, H. Hafner, B. Neufeld, J. Han, U.R. Rapp, J. Biol. Chem. 273 (4) (1998) 1917. [22] J. Schmitz, M. Weissenbach, S. Haan, P.C. Heinrich, F. Schaper, J. Biol. Chem. 275 (17) (2000) 12848. [23] X.P. Yang, U. Albrecht, V. Zakowski, R.M. Sobota, D. Haussinger, P.C. Heinrich, S. Ludwig, J.G. Bode, F. Schaper, J. Biol. Chem. 279 (43) (2004) 45279. [24] U.M. Wegenka, J. Buschmann, C. Lütticken, P.C. Heinrich, F. Horn, Mol. Cell. Biol. 13 (1993) 276. [25] A.W. Kuss, M. Knodel, F. Berberich-Siebelt, D. Lindemann, A. Schimpl, I. Berberich, Eur. J. Immunol. 29 (10) (1999) 3077. [26] A. Denk, M. Goebeler, S. Schmid, I. Berberich, O. Ritz, D. Lindemann, S. Ludwig, T. Wirth, J. Biol. Chem. 276 (30) (2001) 28451. [27] H. Baumann, C. Richards, J. Gauldie, J. Immunol. 139 (12) (1987) 4122.

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