Eur J Biochem 270, 2274–2286 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03595.x Gene transcription of fgl2 in endothelial cells is controlled by Ets-1 and Oct-1 and requires the presence of both Sp1 and Sp3 Mingfeng Liu1, Julian L Leibowitz2, David A Clark1,4, Michael Mendicino1, Qin Ning1, Jin Wen Ding1, Cheryl D’Abreo3, Laisum Fung1, Philip A Marsden3 and Gary A Levy1 Multi Organ Transplant Program, Toronto General Hospital and The University of Toronto, Canada; 2Department of Pathology and Laboratory Medicine, Texas A & M University System College of Medicine, USA; 3Renal Division and Department of Medicine, St Michael’s Hospital and University of Toronto, Canada; 4McMaster University, Ontario, Canada The immune coagulant fgl2/fibroleukin has been previously shown to play a pivotal role in the pathogenesis of murine and human fulminant hepatitis and fetal loss syndrome Constitutive expression of fgl2 transcripts at low levels are seen in cytotoxic T cells, endothelial, intestinal and trophoblast cells, while specific factors (such as virus and cytokines) are required to induce high levels of fgl2 expression in other cell types including monocytes/macrophages To address the transcriptional mechanisms that regulate constitutive expression of fgl2, murine genomic clones were characterized and the transcription start site was defined by 5¢-RACE and primer extension A comprehensive assessment of basal fgl2 promoter activity in murine vascular endothelial cells defined a minimal 119 bp region responsible for constitutive fgl2 transcription A complex positive regulatory domain (PRD) spanning a 39-bp sequence from )87 to )49 (relative to the transcription start site) was identified Electrophoretic mobility shift assay studies in vascular endothelial cells revealed that the nucleoprotein complexes that form on this positive regulatory domain (PRD) contain Sp1/Sp3 family members, Oct-1, and Ets-1 Heterologous expression studies in Drosophila Schneider cells confirmed that the constitutive expression of this gene is controlled by Ets-1 and requires the presence both of the Sp1 and Sp3 transcription factors The presence of this complex multicomponent PRD in the fgl2 proximal promoter is consistent with the observation that, in vivo, fgl2 expression is tightly regulated Moreover, viral induced fgl2 expression also requires the presence of this PRD These results clearly demonstrate that multiple cis DNA elements in a clustered region work cooperatively to regulate constitutive fgl2 expression and interact with inducible elements to regulate viral-induced fgl2 expression in endothelial cells Activation of the coagulation system represents an important facet of immune and inflammatory reactions, accounting for the fibrin deposition that is commonly observed in these reactions [1] Cellular procoagulants and the soluble factors of the coagulation cascade are important participants in a number of human diseases including allograft rejection [2,3], glomerulonephritis [4,5], septic shock [6,7], and bacterial and viral infections [8] For example, tissue factor (TF) [9], the transmembrane receptor of factor VII, is the major procoagulant of the classical extrinsic pathway of coagulation resulting in thrombin generation, and subsequent fibrin deposition Furthermore, TF has important roles in the regulation of angiogenesis in cancer, embryonic blood vessel development, and intracellular signaling [10–12] Thrombin is involved in many biological processes In addition to its function in blood coagulation and wound healing, thrombin has diverse functions in many different cell types For instance, thrombin induces proliferation of fibroblasts and smooth muscle cells, and neurite retraction and synapse reduction in neurons [12] Moreover, thrombin is a chemotactic agent for inflammatory cells such as macrophages and neutrophils [12] fgl2/fibroleukin was originally described as a fibrinogenlike protein, constitutively expressed in cytotoxic T cells [13] This gene was subsequently determined to encode an immune coagulant and was localized to the proximal region of mouse chromosome [14,15] Fgl2 is a member of the fibrinogen family, which includes tenascin, cytotoxin, and fibrinogen [16–18] Fibrinogen-like protein family members all contain fibrinogen-related domains (FRED), known to represent key regions for protein– protein interaction As a procoagulant, fgl2 is involved in cleaving prothrombin to thrombin endogenously and when functionally expressed in a heterologous system [14,19] Thus, an important biological function of fgl2 might be to regulate the production of thrombin via a pathway independent of TF, especially in settings where TF-independent procoagulant activity has been documented For instance, fgl2 plays a critical role in the pathogenesis of both human and mouse fulminant viral Correspondence to G A Levy, Toronto General Hospital, 621 University Ave., NU-10–116, Toronto, Ontario, Canada M5G 2C4 Fax: 416 340 3378, Tel.: 416 340 5166, E-mail: glfgl2@attglobal.net Abbreviations: EMSA, electrophoretic mobility shift assays; fgl2, fibrinogen-like protein; LUC, luciferase; MHV-3, murine hepatitis virus strain 3; NE, nuclear extracts; PRD, positive regulatory domain; Stat3, signal transducer and transactivator (Received December 2002, revised March 2003, accepted 27 March 2003) Keywords: fibroleukin; fgl2; hepatitis; immune coagulant; transcription regulation Ó FEBS 2003 fgl2 expression regulation in endothelial cells (Eur J Biochem 270) 2275 hepatitis [20] We have previously demonstrated the pivotal role of fgl2 in a murine model for murine hepatitis virus strain (MHV-3) induced fulminant hepatic failure [21,22] Administration of neutralizing antibodies against fgl2 conferred protection against MHV-3 induced fulminant hepatic failure in susceptible BALB/cJ mice [23] Human fgl2 was cloned and shown to be expressed in endothelial cells and macrophages of livers from patients with hepatitis [20,24] Expression of fgl2 during both mouse and human fulminant hepatitis correlates with fibrin deposition, a typical feature of fulminant hepatic failure [20,21,25,26] As a multifunctional protein, fgl2 has a transcription regulation mechanism that seems to reflect its function in different cells and developmental stages Fgl2 mRNA is constitutively expressed in some cell types [13,20,26], but transcription can also be robustly induced by viruses and cytokines, such as interferon-c (IFN-c) [26–28] Constitutive activity of the fgl2 promoter suggests that fgl2 functions as a matrix/adhesion protein, possibly necessary for development of a normal fetus and in regulating immune responses within the intestine Induction of fgl2 in macrophages in response to viruses results in the production of a potent coagulant activity with the characteristics of a prothrombinase This combination of constitutive expression and induction may permit a rapid response to inflammation However, the mechanisms regulating fgl2 expression, either constitutive or induced, remain unclear A comprehensive understanding of the general transcription machinery on the native fgl2 promoter is necessary for developing further insight into the TF-independent coagulation pathway in diseased states We report here the structural and functional characterization of the 5¢-flanking region of the mouse fgl2 gene in vascular endothelial cells Through a series of studies focusing on constitutive expression in endothelial cells, we identified that Ets-1 involves in the fgl2 constitutive expression within a positive regulatory domain formed by Sp1, Sp3, Ets-1, and Oct-1 In addition, we demonstrated that this positive regulatory domain is also required for MHV-3 nucleocapsid proteininduced fgl2 expression Materials and methods Mice Female BALB/cJ mice, 6–8 weeks of age were purchased from Jackson Laboratories (Bar Harbor, ME), and were housed in the animal facility at the Toronto Hospital Research Institute, University of Toronto Cells Peritoneal macrophages used for primer extension and 5¢-RACE were harvested from BALB/cJ mice days after intraperitoneal administration of 1.5 mL of 5% thioglycollate (Difco Laboratories, Detroit, Michigan) as previously described [29] Macrophages were greater than 95% pure as determined by morphology and viability exceeded 98% by trypan blue exclusion MHV-3 was grown and titrated as described previously [8] The murine SVE-10 endothelial cell line and Schneider’s Drosophila line (SL2) were obtained from American Type Culture Collection (ATCC Rockport, MD) Cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium) (SVE-10, ATCC) or Schneider’s Drosophila medium (Invitrogen) supplemented with 10% fetal bovine serum and maintained as described [30] Sequencing Plasmid DNA fragments were sequenced by cycle sequencing on an automated DNA sequencer (Model 377, Applied Biosystems, Foster City, CA) using dideoxy dye terminator chemistry and primer-directed strategies Sequences were determined on both DNA strands Primer extension analysis A 30 base oligonucleotide (5¢-CCTCCACCGCTCGGCA GGCAGCGAGGACGG-3¢) complementary to nucleotides 1363–1392 (GenBank Accession AF025817) at the 5¢ end of the mouse fgl2 coding sequence was purified by polyacrylamide gel electrophoresis and used for the primer extension reaction End labelling and primer extension reactions were performed as described previously [31] Labelled oligonucleotide (100 000 c.p.m.) was hybridized with 50 lg of total RNA from MHV-3 infected (6 h) BALB/cJ mouse peritoneal macrophages Nucleic acids were recovered by ethanol precipitation and reverse transcribed with 100 U of MMLV reverse transcriptase (MMLV-RT) The primer extension products were extracted with phenol/chloroform, precipitated with ethanol and analyzed in 8% sequencing gel The same primer was used to create a dideoxynucleotide chain termination-sequencing ladder from a double stranded genomic DNA fragment loaded adjacent to the primer extension sample 5¢-RACE analysis 5¢-RACE analysis was performed as previously described with minor modifications [31,32] Briefly, 0.5 lg of total RNA extracted from MHV-3 treated (at 10 pfu for 24 h) Balb/cJ liver and peritoneal macrophages infected MHV-3 for h were heated to 95 °C for min, cooled on ice and followed by mixing with one picomole of an antisense primer (5¢-ATCTCGATGGTCGTCAGCC-3¢), which corresponds to nucleotides 1627–1646 (GenBank Accession AF025817) of the BALB/cJ fgl2 gene Reverse transcription was carried out for h at 42 °C with unit of MMLV-RT The sample was heated to 95 °C for to inactivate the MMLV-RT and digested with unit of RNase at 37 °C for 20 First round PCR was performed in a total volume of 100 lL containing the following: 50 lL of H2O, 2.5 pmole PCR (sense primer: 5¢-GACTCGAGTCGACGAATTCAAT-3¢), 25 pmole PCR (5¢-GACTCGAGTCGACGAATT CAA-3¢), 25-pmole of gene specific antisense primer PCA2 (5¢-TGCCACTGCTTCCTTGAGG-3¢), 10 lL of 10 · PCR buffer, 10 lL 25 mM MgCl2, lL of 10 mM dNTP, 20 lL tailed first strand cDNA and 0.5 units of Taq polymerase The second PCR amplification was performed using the same conditions except that a different antisense primer was used (PCA3, 5¢-AGCAC CTCCTCCATGCTGC-3¢) with an annealing temperature Ĩ FEBS 2003 2276 M Liu et al (Eur J Biochem 270) of 57 °C The PCR product was recovered and digested with restriction endonucleases SacII and EcoRI The fragment was subcloned into the pBluescript plasmid (Strategen) The transformants were subjected to DNA sequence analysis RT-PCR detection of fgl2 Expression of fgl2 mRNA was assessed using RT-PCR Total RNA from BALB/cJ macrophages, macrophages infected with MHV-3, SVE-10 murine endothelial cells, and murine small intestine RNA (Clontech, Palo Alto, CA) were used to synthesize the first strain cDNA PCR was then performed in 50-lL reactions using 1-lL portions of cDNA and the primers fgl2318 (5¢-TGCCCACGCTGACCATC CA-3¢) corresponding to nucleotides 318–336 of BALB/cJ fgl2 cDNA (M15761) and fgl21224 (5¢-GAGACAAC GATCGGTACCCCT-3¢) corresponding to nucleotides 1224–1244 of BALB/cJ fgl2 cDNA (M16238), which yield a 906-base pair band in 1% agarose DNA gel after 30 cycles of PCR reaction Amplification products were not obtained when reverse transcriptase was omitted (data not shown) RT-PCR for b-actin was set up as an internal control to ensure equal loading and first strand synthesis with forward primer, 5¢-ATGTTTGAGACCTTCAACAC-3¢, and reverse primer, 5¢-CACGTCACACTTCATGAT GGA-3¢ then excised from PCR2.1 by digestion with HindIII and SalI and transferred into the pGL2-Basic utilizing the HindIII and XhoI sites A 3¢-deleted promoter fragment was amplified from a pM166 template using a common sense primer 5¢-GAATAAGGAGGGCAGGGTGAA-3¢ (positions )1320 to )1302 in GenBank accession no AF025817), and the antisense primers 5¢-TAGTGGGGAAAGA GTTGGAACG-3¢ for fragment )1320/)274 The PCR products representing this promoter fragment was subcloned into PCR2.1 and transferred into pGL2-Basic using the KpnI and XhoI sites in the vector and KpnI and SalI sites in the PCR2.1 clones of the promoter fragments All promoter constructs were sequenced to confirm their orientation and to verify their sequences pCR3.1A59 MHV nucleocapsid protein (N protein) expression vector was reported previously [33] The N gene fragments were subcloned into the expression vector pCR3.1 (Invitrogen), under the control of the cytomegalovirus promoter and bovine growth hormone 3¢-processing signals Drosophila Eukaryotic Expression Constructs Expression cassettes for Sp1, Sp3 variants, and Ets-1 were based upon pPacUO, a transient episomal vector which contains the 2.6-kb Drosophila actin 5C promoter, a 0.7-kb 5¢-UTR Ultrabithorax (Ubx) internal ribosome entry site, the first eight codons of the Ubx open reading frame and 1.1 kb of 3¢-UTR from the actin 5c gene These constructs have been reported previously [30] DNA constructs A single SalI site was introduced into pM166 after the initiating ATG by site directed mutagenesis The restriction fragment extending 3.5 kb upstream from the introduced SalI site was excised by digestion with SalI and SmaI (utilizing the SmaI contained in the pBluescript vector) and subcloned into the luciferase reporter vector pGL2-Basic using the SmaI and XhoI sites in this vector to construct the plasmid pfgl2()3500/+9)Luc A 1.3-kb fragment was also released from pM166 by digestion with SalI and EcoRV and subcloned into pGL2-Basic using the SmaI and XhoI sites in this vector to construct a shorter reporter pfgl2()1320/+9)Luc 5¢-Truncation of the fgl2 promoter in pGL2-Basic was created by PCR 5¢-deleted promoter fragments were amplified from a pM166 template using a common antisense primer (5¢-GCCACAACCAACCAGGAAG-3¢, positions 1335–1353 in GenBank Accession AF025817) and a series of sense primers at varying distances upstream Upstream sense primers were 5¢-TCTTGGGAAATCTGG TTAGAG-3¢ for fragment pfgl2()985/+9), 5¢-GGTCAGT ATGCACAAGTGAG-3¢ for fragment pfgl2()723/+9), 5¢-GAGCTGAGTGATGGGGAAGGA-3¢forpfgl2()681/ +9), 5¢-CCACTGACGATTACATAGCC-3¢ for fragment pfgl2()612/+9), 5¢-GGACCTTTGTTCTGATTAGGG GC-3¢ for fragment pfgl2()498/+9), 5¢-CGCAGACATT TAGACGTTCC-3¢ for pfgl2()360/+9), 5¢-GGGCACTG GTATTACAACTGT-3¢ for pfgl2()294/+9), 5¢-CTCCT CCTGTGTGGCGTCTGA-3¢ for fragment pfgl2()119/ +9), and 5¢-GAACGCCTGAGTCAGGCGGCGG-3¢ for fragment pfgl2()58/+9) The PCR products representing these different promoter fragments were subcloned into the plasmid PCR2.1 (Invitrogen) These fragments were Linker scanning analysis Seven site-directed mutants were created within a 70-bp region ()119 to )41) of the fgl2 promoter in the plasmid pfgl2()1320/+9)Luc using primers listed in Table Each mutant contains the sequence GGTACC, a KpnI restriction site These mutations were introduced using paired primers according to the manufacturer’s instructions (Invitrogen) All mutants were verified by sequencing Transient transfection and luciferase assay Transient transfections were carried out using Lipofectamine according to the manufacturer’s instruction (Canadian Life Technology, Burlington, Canada) Cells were plated at · 105 per well in six-well plates 18 h before transfection Transfection conditions were optimized using the SV40 promoter/enhancer luciferase control plasmid, pGL2-Control, to confirm that increasing amounts of templates resulted in proportional increases in reporter activity Endothelial cells were cotransfected with lg of reporter construct and 0.5 lg of pRSV-b-gal DNA to control for transfection efficiency as previously described [30] For Drosophila Schneider studies, cells were cotransfected with lg of experimental luciferase construct, the indicated amount of expression plasmids, and 0.5 lg pRSVb-gal The total amount of DNA transfected was kept constant (2 lg) with the addition of pPacUO Each transfection experiment was performed in triplicate and repeated a minimum of three times Luciferase and b-galactosidase assays were carried out 48 h post transfection as described previously [30] Ó FEBS 2003 fgl2 expression regulation in endothelial cells (Eur J Biochem 270) 2277 Table Primer pairs used to construct linker-scanning fgl2 promoter mutants Underlined are actually mutated sequences Name Primer sequences Mu-S)108/)99 Mu-AS)108/)99 Mu-S)97/)91 Mu-AS)97/)91 Mu-S)87/)80 Mu-AS)87/)80 Mu-S)77/)71 Mu-AS)77/)71 Mu-S)68/)59 Mu-AS)68/)59 Mu-S)57/)50 Mu-AS)57/)50 Mu-S)50/)44 Mu-AS)50/)44 CTC CTC CTG TAA GGT ACC ACA GAC TGT GAT GC GCA TCA CAG TCT GTG GTA CCT TAC AGG AGG AG GTG GCG TCT GAG GTA CCA ATG CAA ATG CGC GCG CAT TTG CAT TGG TAC CTC AGA CGC CAC GAG ACT GTG ATG CGG TAC CTC CCG CCC TTT C GAA AAG GGC GGG AGG TAC CGC ATC ACA GTC TC GCA AAT GCG CCA GGT ACC TTC TGG GAA CTC GAG TTC CCA GAA GGT ACC TGG CGC ATT TGC CCG CCC TTT TGA GGT ACC AGA GAA CGC CTG CAG GCG TTC TCT GGT ACC TCA AAA GGG CGG CTG GGA ACT CAT GGT ACC ACA GTC AGG CGG CCG CCT GAC TGT GGT ACC ATG AGT TCC CAG CTC AGA ACG CCA GGT ACC CGC GGC GGT GGC GCC ACC GCC GCG GGT ACC TGG CGT TCT GAG Table Oligonucleotides used in EMSA analysis Numbers after fgl2 in the name column are the 5¢-ending and 3¢-ending nucleotide location of each oligo used Underlined are the potential cis-DNA elements identified m after fgl2 stands for mutant Mutated sequences are the same as the mutated sequences in Table used in the luciferase assay Ets core GGAA is italicized to indicate its overlapping with consensus Stat3 binding site underlined Name fgl2 fgl2 fgl2 fgl2 fgl2 fgl2 fgl2 fgl2 fgl2 )82/)55 )98/)69 )57/)32 )85/)66 m)85/)66 )97/)77 m)97/)77 )76/)56 m)76/)56 Electrophoretic mobility shift assays (EMSA) Nuclear extracts from endothelial SVE-10 cells were collected as described [34] For EMSA, double stranded oligonucleotide probes (Table 2) were 5¢-end-labelled with [c-P32]ATP (Amersham) using T4 polynucleotide kinase For each EMSA reaction 2–5 lg of nuclear extracts were incubated for 15 on ice in 20 lL of binding buffer · 104 d.p.m of probe was added to each reaction and the mixtures were incubated at room temperature for 30 For supershifts, lL of antibodies were incubated with nuclear extracts for 45 before adding DNA probe Anti-Sp1, Sp3, Stat3, Oct-1, Oct-2, Ets1/2, and PU.1 Ig are from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA) For competition, 100 · cold oligo were added to the reaction Consensus double stranded oligos Oct-1, 5¢-TGT CGAATGCAAATCACTAGAA-3¢; Sp1, 5¢-ATTCGATC GGGGCGGGGCGAGC-3¢; Ets/Pea3, 5¢-GATCTCGAG CAGGAAGTTCGA-3¢; Ets (PU.1), 5¢-GGGCTGCTTG AGGAAGTATAAGAAT-3¢; Stat3, 5¢-GATCCTTCTG GGAATTCCTAGATC-3¢; and C/EBP, 5¢-TGCAGATT GGGCAATCTGCA-3¢ are from Santa Cruz Biotechnology The binding reactions were size-fractionated on a nondenaturing, 5% acrylamide gel, run at 150 V at room temperature for h in · Tris/borate/EDTA buffer Actual mouse fgl2 sequences (5¢ to 3¢-sense strand) GCG CCC GCC CTT TTC TGG GAA CTC AGA A AGA CTG TGA TGC AAA TGC GCC CGC CCT TTT AGA ACG CCT GAG TCA G GCG GCG GTG GC AAT GCG CCC GCC CTT TTC TG AAT GCG CCA GGT ACC TTC TG GAC TGT GAT GCA AAT GCG CCC GAC TGT GAT GCG GTA CCT CCC GCC CTT TTC TGG GAA CTC AGA GCC CTT TTG AGG TAC CTC AGA Results Mapping the transcription start site of fgl2 gene To map the transcription start site of the fgl2 gene and exclude any 5¢-mRNA sequence diversity we employed 5¢-RACE Clones representing the 5¢-end of the fgl2 mRNAs were isolated by 5¢-RACE as described in Materials and methods The results are summarized in Fig 1A Twenty-six of the 33 cDNA clones initiated with an A at position 1320, 34 nucleotides downstream of a putative TATTAAA box, which appears to represent the major transcription initiation site For the remainder of this report we will refer to the transcriptional start site at nucleotide 1320 in AF025817 as position +1 Although there is a second potential upstream TATA box at position )314 with respect to transcription initiation, the usage of this TATA box was not observed in our studies Determination of the 5¢-end of mRNAs isolated from liver tissue and cloned by 5¢-RACE showed similar results (data not shown) Thus, we observed no differences in the fgl2 transcription start site using both in vivo and in vitro RNA sources To complement these findings, we performed primer extension analysis As shown in Fig 1B, compared to uninfected macrophages, major fgl2 transcripts only appeared in mRNA from Ó FEBS 2003 2278 M Liu et al (Eur J Biochem 270) Fig Mapping of the transcription start site of fgl2 mRNA (A) 5¢-RACE of fgl2 mRNA from macrophages Stars indicate the 5¢-terminus of 33 clones isolated after 5¢-RACE (B) Primer extension analysis of fgl2 mRNA The left side of the figure is the sequence ladder using the same primer and the pM166 clone as template Arrow indicates the nucleotide that is matched to the band present in the primer extension reaction on the right The sequence of the primer is 5¢-CCTCCACCGCTCGG CAGGCAGCGAGGACGG-3¢ The lanes on the right correspond to primer extension reactions performed with RNA extracted from uninfected macrophages (M); MHV-3 infected macrophages (M + MHV-3) MHV-3 infected macrophages and transcription was shown to initiate at a site consistent with our 5¢-RACE results (lane T, corresponding to a nucleotide A at that position) Mapping the region essential for fgl2 transcription in endothelial cells Recently, BALB/cJ mouse endothelial cells have been shown to express fgl2 by in situ hybridization [26] Using RT-PCR, we confirmed that the mouse endothelial cell line, SVE-10, constitutively expresses fgl2 (Fig 2A) To identify functionally important DNA regions necessary for the basal level of fgl2 transcription in endothelial cells, we constructed a series of plasmids containing a luciferase reporter under the control of successively deleted fgl2 promoters (Fig 2B) These constructs contain fgl2 promoter all the way to the beginning of ATG start codon to avoid the discrepancy of minor transcription start variance between macrophages and endothelial cells Endothelial cells transiently transfected with the longest fragment of the 5¢-fgl2 flanking region ()3500/+9) resulted in luciferase activity that averaged 10% of that observed in cells transfected with constructs under the control of the strong-heterologous viral SV40 promoter/enhancer (not shown) As shown in Fig 2, serial 5¢-deletion with regions spanning )3.5 bp to )119 bp resulted in only minor changes in fgl2 promoter activity, implying that elements in this region are not important for constitutive activity of the fgl2 promoter However, further deleting regions from )119 bp to )58 bp significantly reduced fgl2 promoter activity to approximately 20% of that observed with the longer constructs To further confirm the 5¢-truncation results, we assessed the activity of pfgl2()1320/)274)LUC relative to pfgl2()1320/+9)LUC in SVE-10 cells Results indicated that deletion of regions spanning )274/+9 abrogated fgl2 promoter activity (Fig 2D), suggesting that this region is necessary for fgl2 transcription A detailed sequence inspection of the region spanning )120 nucleotide to )40 nucleotide indicated many potential cis-acting elements, including Sp1, Ets, Stat3, Octamer [35] binding sites and AP1 sites (Fig 3) and these motifs might be critical for fgl2 expression Together, the 5¢-deletion and 3¢-deletion analyses indicate that sequences from )119 through +9 contain sequences that are sufficient for constitutive fgl2 transcription Linker-scanning and EMSA analyses To gain further insight into the regulation of the fgl2 promoter, the region between )119 to )41 bp, just upstream of the putative TATA box (Fig 4A), was subjected to systematic site-directed mutagenesis [30] Seven constructs were created in pfgl2()1320/+9)LUC so that each construct contained an 8–10 bp mutation (Table 1) These constructs were transfected into SVE-10 cells and luciferase activity was assessed Mutation of position )108 to )99 and )97 to )91 had no significant impact on fgl2 promoter activity Mutation of positions )87 bp to )80 (containing an Octamer motif 5¢-ATGCAAAT-3¢) Ĩ FEBS 2003 fgl2 expression regulation in endothelial cells (Eur J Biochem 270) 2279 Fig Functional analysis of fgl2 promoter (A) Analysis of fgl2 RNA by semiquantitative RT-PCR RT-PCR was performed with lg of each RNA sample M, macrophages; M + MHV-3, macrophages infected with MHV-3; endothelial cells, RNA from murine endothelial SVE-10 cells; intestine, total mouse intestinal RNA (B) Fgl2 promoter-luciferase constructs The numbers give the 5¢-ending and 3¢-ending nucleotide of each construct (C) Relative luciferase activity of 5¢-truncations of fgl2 promoter constructs in endothelial cells Activity of pGl2()1320/+9)Luc is set at 100% (D) Luciferase activity of 3¢-truncation of fgl2 promoter construct in endothelial cells The data is the average of at least three separate experiments each performed in triplicate The data was corrected for variations in transfection efficiency by normalization to the activity of a cotransfected plasmid expressing b-galactosidase Fig Schematic drawing representing the proximal promoter of the mouse fgl2 gene Positive regulatory region (PRD) and functional cisregulatory DNA elements are shown Identified cis elements are underlined Boxes indicate mutated regions Probes used in EMSA are also shown as underline with specific numbers significantly reduced promoter activity The mutation at positions )77 to )71 (containing a Sp1 binding site: 5¢-GCCCGCCC-3¢) reduced the promoter activity to 20% of the parental wild-type promoter The mutations at positions )68 to )59 (containing potential Ets family binding site, and a Stat3-like motif: 5¢-TTCTGGGAACT-3¢) and from )57 to )49 (which is overlapping the Ets/Stat3 binding sites) similarly decreased promoter activity to 40% and 50% of that observed with the wild-type promoter However, mutating the AP1 site (from )50 to )44: 5¢-TGAGTCAG-3¢) did not have any impact on fgl2 promoter activity in vascular endothelial cells (Fig 4B) Thus, these results suggest that a 39-bp positive regulatory region spanning nucleotides )87 to )49 contains cis-acting elements necessary for fgl2 constitutive promoter activity in SVE-10 endothelial cells EMSAs were used to examine the nature of the functionally important nucleoprotein complexes that form at cis-regulating elements important for constitutive fgl2 promoter activity in SVE-10 endothelial cells Using EMSA probes spanning fgl2–82/)55 and )98/)69 we detected protein–DNA complexes with endothelial cell nuclear extracts (not shown) These DNA-protein complexes were specific as they were eliminated by competing unlabelled oligonucleotide containing these sequences but not with mutant oligonucleotides (fgl2 m-97/)77, m-85/ )66, and m-76/)56, Table 2) As both probes contain the Sp1 motif, we predict that DNA-protein complexes could be formed with Sp1 and Sp3 transcription factors as seen 2280 M Liu et al (Eur J Biochem 270) Fig Linker-scanning analysis of important cis elements in fgl2 promoter (A) Linker scanning mutations of fgl2 promoter All mutant constructs were made using pfgl2()1320/+9)Luc as template The numbered boxes indicate the specific nucleotides mutated which were also shown in Table and Fig (B) Luciferase activity of wild-type and mutated fgl2 promoter in SVE-10 endothelial cells Luciferase activity was analyzed as described in Fig in the case of eNOS in vascular endothelial cells [30] To test this hypothesis, we performed an EMSA experiment with a probe containing the fgl2 Sp1 binding site (probe )85/)66, Table and Fig 3) As shown in Fig 5, at least four nucleoprotein complexes were seen using endothelial cell nuclear extracts (lane 2) These complexes were competed away by the same unlabelled probe (lane 3) but not by an unrelated nonspecific probe (lane 4) The addition of anti-Sp1 IgG1 shifted the slowest migrating complex to a higher position in the gel (lane 5) while antiSp3 antibody detected two forms of Sp3 complexes, the second nucleoprotein complex (lane 6) and an additional Sp3 complex, which is in close proximity to the slowest moving Sp1 and did not clearly separate in the gel utilized The same Sp1/Sp3 pattern has been demonstrated for the eNOS promoter in endothelial cells [30] Consistent with our linker-scanning analysis, which suggested that AP1 is not functioning in the constitutive expression of fgl2, no DNA–protein complex was observed with a Ó FEBS 2003 Fig EMSA analysis of cis elements overlapping the 38 bp identified in Fig of the fgl2 promoter Nuclear extracts (NE) from SVE-10 endothelial cells were incubated with probes shown in Table and Fig Arrows indicate the bands that are interacting with these probes All the lanes have labelled probes The adding of extracts and cold probe are indicated in the top of the panel Supershift, ss-Sp1 and ss-Sp3, were shown in presence of anti-Sp1 and anti-Sp3 Ig Cold and mutated competitors are indicated at the top of the figure probe )57/)32 (Fig 3) which contains the putative AP1 binding site (data not shown) To test whether the Octamer motif, which corresponds to the mutant (Fig 4), is functional in the fgl2 promoter, a cross competition EMSA was performed As seen in Fig 6A, protein–DNA complexes formed by consensus Oct-1 motif were specifically blocked by both excess unlabelled consensus Oct-1 oligo or fgl2 oligo)97/)77 as shown in Table and Fig 3, which contains an identical Oct-1 motif ATGCAAAT (lanes and 4) Competition was not observed with an excess of unlabelled mutant fgl2–97/ )77 oligonucleotide (Table 2) Consistent with this result, unlabelled consensus Oct-1 oligonucleotide, or fgl2 probe ()97/)77), were able to inhibit formation of the nucleoprotein A formed by labelled fgl2–97/)77 probe, but not by the mutated fgl2–97/)77 oligo (lane 8–10) These results suggest that complex A is formed by DNA binding proteins functionally related to members of the Octamer family In addition, similar to that of the consensus Oct-1 probe, the addition of an antiOct-1 antibody significantly reduced the intensity of complex A of probe )97/)77 (Fig 6B, lane and 7) Thus, we infer that complex A represents the interaction of Oct-1 with the fgl2 Octamer DNA motif The Ó FEBS 2003 fgl2 expression regulation in endothelial cells (Eur J Biochem 270) 2281 Fig EMSA analysis of the function of Octamer motif in the fgl2 promoter Lanes 2–5 of panel A and lanes 2–5 of panel B, NE were incubated with labelled consensus Oct-1 Lanes 6–12 of panel A and lanes 6–8 of panel B, NE were incubated with 32P-labelled fgl2 fragment )97/)77 All lanes contained hot probes Addition of NE, cold probe competitors, and antibodies are indicated at the top of the panel (A) Cross competition with consensus Oct-1 and fgl2–97/)77 (B) Antibody analysis of Oct-1 binding in the presence of antiOct-1 and antiOct-2 antibody Arrows indicate the specific Oct-1 DNA-protein complexes All probes are shown in Table and Fig nucleocomplex B and C in panel A could represent binding of the Sp1 protein family, given that the probe contains a half site of a Sp1 motif (Fig 3) Consistent with this view, a consensus Sp1 oligonucleotide blocked the formation of these complexes (Fig 6A, lane 12) To assess this possibility, we performed a supershift assay using antibodies against Sp1 and Sp3 We did not see a supershift or blocking of the complexes B and C (data not shown) Thus, it can be concluded that these complexes are not formed by the binding of Sp1 or Sp3 However, we cannot rule out a Sp1 related transcription factor or an unknown factor at this time Future studies are required to address this issue We next examined whether the motifs located between )68/)57 (Table and Fig 3), corresponding to mutant and 6, are functional As shown in Fig 7A, two DNAprotein complexes were detected Both complexes were competed away by a cold fgl2 probe (lane 3) but only partially affected by a mutant fgl2 probe (Table 2) (lane 4) The slower moving complex was blocked by the addition of two Ets consensus oligonucleotides (Ets1/Pea3 and PU.1, lanes and 6) We observed that formation of the faster moving complex was blocked by a Stat3 consensus oligonucleotide (lane 7) Unlabelled fgl2 probe )76/)57, but not a mutant fgl2–68/)57 probe, blocked the binding to a labelled Stat3 consensus oligonucleotide and provided further evidence for the involvement of Stat3 cis DNA elements in fgl2 expression (Fig 7B) Importantly, we were unable to detect either a supershift or shift abrogation using antibodies directed against Ets (Ets1/Pea3, PU.1, data not shown) This was not unexpected as it has been reported previously that Ets antibodies rarely work in EMSA [30] We were also unable to detect Stat3 supershift with antibody against Stat3 (data not shown) An EMSA probe spanning )57/)32 (Table and Fig 3) did not demonstrate nucleoprotein complex formation in EMSA (data not shown) This may suggest that the effect of mutant in Fig 4B could represent the edge effect of mutation in this area that affected the binding of Ets/Stat3 to its motif Ets-1 is critical in regulating fgl2 expression and requires the presence of both Sp1 and Sp3 Collectively the linker-scanning and EMSA analysis suggest that Oct 1, Sp1/Sp3, Ets family members, and a Stat3-like protein may form a multicomponent nucleoprotein complex upon the 39 bp PRD region and functionally contribute to the constitutive expression of fgl2, especially in vascular endothelial cells To further evaluate this hypothesis, we employed the Drosophila Schneider cell line (SL2), which is deficient in constitutive Sp1, Sp3, and Ets-1 transcription factors [36,37] As shown in Fig 8A, cotransfection of the pfgl2LUC()1320/+9) promoter, along with increasing amounts of Sp1 expression cassette resulted in a concentration-dependent increase in functional fgl2 promoter activity (22-fold maximum effect) Increasing amounts of a Sp3 expression cassette also increased fgl2 promoter activity in a concentration-dependent fashion, though to a lesser extent (Figs 8B, fivefold increase) However, cotransfection of increasing amounts of an Ets-1 expression cassette alone had no important effect on fgl2 promoter activity (Fig 8C) To examine whether Sp1, Sp3, and Ets-1 exert an interactive effect on fgl2 promoter activity, we cotransfected combinations of the varied transcription factors As shown in Fig 9A, cotransfection 2282 M Liu et al (Eur J Biochem 270) Ó FEBS 2003 Fig EMSA analysis of the function of Ets/Stat3 cis elements (A) Cross competition of consensus Ets/PEA3, Ets/PU1, and Stat3 cis elements with corresponding transcription factors NEs from endothelial cells were incubated with labelled fgl2 probe )76/)57 (Table and Fig 5) The adding of NE and cold probes are indicated at the top of the panel Arrows indicate specific Ets, Stat3 DNA-protein complexes (B) Cross competition of Stat3 DNA cis element with its binding protein Consensus Stat3 was labelled and competes with cold fgl2 oligonucleotide )76/)57 (Fig 5) Fig Sp1, Sp3, and Ets-1 transactivate mouse fgl2 promoter/reporter luciferase constructs in Drosophila Schneider cells Assay of promoter activity of pfgl2LUC()1320/+9) promoter/reporter luciferase construct cotransfection with increasing amounts of pPacUSp1 (10–250 ng, panel A), pPacUSp3 (10–250 ng, panel B), and pPacUEts-1 (25–250 ng, panel C) Luciferase activity was analyzed as described in Fig of increasing amounts of Sp3 along with 50 ng of Sp1 (halfmaximum amount) demonstrated increased activity of fgl2 promoter compared to Sp1 or Sp3 alone (Fig 9A) In contrast, increasing amounts of Ets-1 along with 50 ng of Sp1 or a threshold amount of Sp1 (10 ng) did not increase fgl2 promoter activity (data not shown) To examine the possibility that Ets-1 functions with Sp3, we performed a cotransfection assay using Ets-1 and Sp3 expression vectors in combination with an fgl2 promoter construct in SL2 cells Similar to that of Sp1, Ets-1 had no incremental effect on fgl2 promoter activity when used with either 50 ng or a threshold amount of Sp3 (10 ng) (Fig 9A) However, when both threshold amounts (10 ng) of Sp1 and Sp3 were added together, with increasing quantities of Ets-1, a cooperative positive functional interaction was evident (Fig 9B) To define the fgl2 cis-element implicated in the responses of the fgl2 promoter to these transcription factors, mutations corresponding to mutant ()77/)71) and ()68/)59) were employed As shown in Fig 9B, mutation of the Sp1/Sp3 binding site (mut4) deceased luciferase activity of the Sp1/ Sp3 interaction or the Sp1/Sp3/Ets-1 interaction by approximately 90% and 80%, respectively Mutation of the Ets-1 binding site (mut5) also blocked the effect of Ets-1 in the presence of Sp1/Sp3 Effect of the fgl2 positive regulatory region on induced expression of fgl2 Previously, our laboratory has provided evidence that fgl2 expression is responsible for the pathogenesis of MHV-3 induced fulminant hepatitis through its procoagulant activity [14,26] We have also recently demonstrated that Ó FEBS 2003 fgl2 expression regulation in endothelial cells (Eur J Biochem 270) 2283 Fig Cooperative activity of Sp1, Sp3, and Ets-1 on fgl2 promoter/reporter luciferase constructs in Drosophila Schneider cells (A) Assay of fgl2 promoter activity upon cotransfection increasing amount of Sp3 with half-maximal amounts of pPacUSp1 (50 ng, left panel) or increasing amount of Ets-1 with 50 ng of either Sp1 or Sp3 (B) Assay of fgl2 promoter activity upon cotransfection of thresholds amount of both Sp1 and Sp3 (10 ng, right panel) and assay of promoter activity of wild-type (pfgl2 ()1320/+9) and linker-scanning mutant and following cotransfection with 10 ng of pPacUSp1, pPacUSp3, and pPacUEts-1 Luciferase activity was analyzed as described in Fig Fig 10 The effect of PRD on nucleocapsid protein induced fgl2 expression Wild-type pfgl2()1320/+9)Luc (which was set as 1) and mutated fgl2 promoter-luciferase construct pfgl2(mut4)Luc were separately cotransfected with pCR3.1A59 into SVE-10 endothelial cells The results were expressed as fold increase compared to cotransfection with pCR3.1 empty vector the nucleocapsid (N) protein of MHV-3 can induce transcription of fgl2 and implicated regions of the fgl2 promoter located )372 to )306 bp upstream of the transcription initiation site [33] Therefore, we undertook studies to determine the contribution of the PRD at nucleotide )87 to nucleotide )49 that is implicated in the constitutive expression of fgl2 to the induced transcription of fgl2 in response to MHV-3 We examined whether a mutation of the PRD would abolish the induction of fgl2 by MHV-3 nucleocapsid protein As shown in Fig 10, we cotransfected plasmid pfgl2()1320/+9) with a constitutively active nucleocapsid protein expression plasmid (pCR3.1A59) into endothelial cells Co-transfection of the N gene expressed by pCR3.1A59 induced a 5.5-fold activation of the fgl2 promoter relative to that observed with cotransfection with empty vector (pCR3.1) Mutation of the Sp1/Sp3 (mut4) site completely abolished this activation Discussion Evidence indicates that inducible expression of fgl2 correlates with the vascular thrombosis of the liver seen in fulminant 2284 M Liu et al (Eur J Biochem 270) viral hepatitis [14,21,26] The function of constitutively expressed fgl2 at low levels in cells or tissues including cytotoxic T cells, trophoblasts, intestinal epithelial cells and endothelial cells remains to be defined We hypothesize that a variety of tissues may express basal levels of a TF/factor VIIindependent procoagulant Indeed, constitutive expression of TF in some cells and tissues is known to play this role, such as in the CNS [9] The biological importance of constitutive fgl2 expression is exemplified by the observation that the fgl2–/– mouse has a developmental defect with measured fetal loss of fgl2–/– embryos due to hemorrhage at the fetomaternal interface (manuscript in preparation) Constitutive fgl2 promoter activity was also seen in a small percentage of the primary endothelial cells isolated from these fgl2–/– mice, in which the fgl2 coding region is replaced with b-galactosidase (unpublished data) The constitutively expressed fgl2 might reflect functional contributions of fgl2 as a matrix/adhesion protein, possibly necessary for development of a normal fetus In this study we have functionally characterized the 5¢-flanking region of the mouse fgl2 gene We mapped the 5¢-terminal region of the fgl2 mRNA using primer extension analysis and 5¢-RACE Sequence analysis revealed a TATA box (TATTAAA) located 33 bp upstream of the major transcription start site The results of transient transfection with a 3¢-deletion construct that removed this major transcription start site and TATA box ()1320/)274) provided functional evidence for this conclusion When we performed functional analysis of the 5¢-flanking region of the fgl2 gene, we demonstrated in murine endothelial cells that a region 119 bp upstream from the transcriptional start site and bp downstream are sufficient for the basal expression of the fgl2 gene This finding is consistent with the observation that fgl2 is constitutively expressed in cultured endothelial cells, as well as in the primary endothelial cells in low level Although the maximal activity we observed with the fgl2 promoter is approximately 10% of the strong SV-40 promoter/enhancer, this is not an unusual finding For example, the promoter of the constitutively expressed human endothelial NOS promoter evidences approximately 10% of the activity of the SV-40 promoter/enhancer [30] A more comprehensive mutational analysis of this fgl2 promoter segment revealed tightly clustered cis-regulatory DNA elements spanning 39 bp in the proximal region of the fgl2 promoter As shown in Fig 4, mutating nucleotides from )87 to )80 (containing Oct-1), )77 to )71 (containing Sp1), )68 to )57 (containing Ets/Stat3) all significantly reduced fgl2 promoter activity The linker-scanning mutant )77 to )71, which contained the Sp1 binding site, had the greatest effect on fgl2 promoter activity, reducing it to the level observed with the deletion construct pfgl2()51/ +9)LUC As mutation of any of the above fragments significantly affected basal expression of fgl2, it is possible that the nucleoprotein complexes that form upon this PRD cis region interact with the core transcription machinery that forms upon the TATA box to direct basal expression of fgl2 [38] The presence of a multicomponent PRD in the fgl2 proximal promoter suggests the complexity of fgl2 as a proinflammatory gene As well as the functional Sp1/Sp3 binding site, we identified that the octamer site (ATGCAAAT) in the fgl2 Ó FEBS 2003 promoter is functional and interacts with the Oct-1 transcription factor (Fig 6) It has been shown that POU homeodomain family proteins bind to this consensus motif and regulate gene transcription in diverse cells through interaction with a variety of partner DNA binding proteins, including Sp1 family members and Ets family members, as observed in the current studies [35,39] In the endothelial cell, Oct-1 has been demonstrated to positively or negatively regulate a variety of genes For example, the expression of endothelial cell-specific TIE2 gene is dependent on Oct-1 and an endothelial cell-restricted cofactor [40] We also identified that the Ets/Stat3 motifs are functional and contribute to fgl2 constitutive expression in vascular endothelial cells (Fig 7) Ets proteins have been shown to be promiscuous in their binding to the same GGAA core sequence [41] Consistent with reports from others, we did not detect supershifts with antiEts1/2 and antiPU.1 antibody However, we did demonstrate that both Ets1/Pea3 consensus and PU.1 consensus oligonucleotides were able to inhibit the formation of nucleoprotein complexes with the fgl2 GGAA core It is known that Ets family members generally serve as coactivators for the transcriptional activator Sp1 and synergistically regulate gene expression [42] The presence of an Ets site immediately proximal to the Sp1 site in the fgl2 promoter may explain the constitutive expression of fgl2 With our EMSA results (Fig 7) suggesting the involvement of Ets family members in the regulation of fgl2 promoter, we found that Ets-1, a major Ets family member in endothelial cells, participated in activation of the fgl2 promoter Our results suggested that Ets-1 can cooperate with Sp1/Sp3 in a positive manner in Drosophila cells that are known to be deficient in constitutively expressed Ets and Sp1 protein family members (Figs and 9) Removing any one of these factors from the system or mutating any of these two activator recognition sites found in the PRD resulted in a marked decrease in functional promoter activity Another motif, TTCTGGGAACT that overlaps the Ets binding site, differed by one nucleotide from the consensus Stat3 binding site (TTCTGGGAATT) [43] and showed specific DNA–protein interactions in endothelial cells Although Stat3 is commonly implicated in activated gene expression, constitutively activated Stat3 has been shown to be present in many cell types, including endothelial cells [43] Importantly, Stat3 is known to regulate constitutive gene expression through cooperative interactions with other transcriptional coactivators [43,44] As we did not detect supershifts with anti-Stat3 antibody, either with fgl2 sequence or a consensus Stat3 oligo, this issue will require further study, such as by utilizing the dominant negative Stat3 expression system to test its function in fgl2 expression Among the identified DNA cis-elements, the Sp1 motif plays an important role in the regulation of many constitutively expressed genes [45] Sp1 might be initially recruited to the fgl2 promoter and then facilitate the further recruitment and binding of other positive trans-regulators, such as Oct-1, Ets-1, and Stat3-like protein (Fig 5) In other systems Sp1 has been shown to physically interact and recruit trans-acting factors, such as AP1, GATA, NF-jB, and Ets-1 [30] For example, in the promoter of parathyroid Ó FEBS 2003 fgl2 expression regulation in endothelial cells (Eur J Biochem 270) 2285 hormone-related protein (PTHrP), Sp1 and Ets-1 proteins cooperate to transactivate the expression of PTHrP [46] Consistent with previous findings, our EMSA also detected two forms of Sp3 protein–DNA complexes with a fgl2 probe (Fig 5) Sp3 is known to act either positively or negatively to regulate gene expression [37,45] and to have at least three human variants with molecular masses of 115, 80 and 78 kDa that are abundantly expressed in a broad number of mammalian cells In EMSA, the two shorter forms comigrate as a fast protein–DNA complex, while the longer form migrates as a slower protein– DNA complex [47] In addition, Sp1 has also been established to be responsible for induced expression of IL-10 [48] Our data from endothelial cells and the Drosophila system, supports the contention that both Sp1 and Sp3 positively affect fgl2 constitutive expression Importantly, the presence of both Sp1 and Sp3 is required for Ets-1 function Taken together, our data suggest that DNA regions spanning 39 bp, from )87 to )49 of the fgl2 promoter, represent functional cis-DNA regulatory elements that interact with Oct-1, Ets-1, and Sp1/Sp3 As fgl2 expression is tightly controlled [49], the interaction of transcription factors Sp1/Sp3 with the fgl2 promoter form a core to recruit both Oct-1 and Ets-1 to form a multicomponent PRD that is sufficient to confer basal level expression of fgl2 in endothelial cells In addition, this PRD is required to interact with inducible elements upon activation to enhance fgl2 expression This mechanism may explain how fgl2 can be both constitutively transcribed and rapidly responsive to proinflammatory stimuli Although the observations presented in this study suggest that this PRD is necessary for the inducible expression of fgl2, we plan to undertake additional studies aimed at more precisely elucidating specific mechanisms regulating induction of fgl2 promoter activity in response to proinflammatory stimuli such as IFN-c or other viral factors Acknowledgements This work was supported in part by grant 13298, MOP 37780 and MOP 37778 from the Canadian Institute of Health Research (CIHR) and grant AI43368 from the National Institutes of Health References Cosgriff, T.M (1989) Viruses and hemostasis Rev Infect Dis Suppl 4, S692 Halloran, P., Aprile, M., Haddad, G & Robinette, M (1982) The significance of elevated procoagulant activity in the monocytes of renal transplant recipients Transplant Proc 14, 669–672 Silverman, R., Cohen, Z., Levy, G., Craig, M., Cullen, J & Langer, B (1987) Immune responses in small intestinal transplantation in the rat: correlation of histopathology and monocyte procoagulant activity Surgery 102, 395–401 Tipping, P.G., Lowe, M.G & Holdsworth, S.R (1988) Glomerular macrophages express augmented procoagulant activity in experimental fibrin-related glomerulonephritis in rabbits J Clin Invest 82, 1253–1259 Cole, E.H., Schulman, J., Urowitz, M., Keystone, E., Williams, C & Levy, G.A (1985) Monocyte procoagulant activity in glomerulonephritis associated with systemic lupus erythematosus J Clin Invest 75, 861–868 Kucey, D.S., Cheung, P.Y & Rotstein, O.D (1992) Plateletactivating factor modulates endotoxin-induced macrophage procoagulant activity by a protein kinase C-dependent mechanism Infect Immun 60, 944–950 Sullivan, B.J., Swallow, C.J., Girotti, M.J & Rotstein, O.D (1991) Bacterial translocation induces procoagulant activity in tissue macrophages A potential mechanism for end-organ dysfunction Arch Surg 126, 586–590 Levy, G.A., Leibowitz, J.L & Edgington, T.S (1981) Induction of monocyte procoagulant activity by murine hepatitis virus type parallels disease susceptibility in mice J Exp Med 154, 1150– 1163 Konigsberg, W., Kirchhofer, D., Riederer, M.A & Nemerson, Y (2001) The TF: VIIa complex: clinical significance, structurefunction relationships and its role in signaling and metastasis Thromb Haemost 86, 757–771 10 Carmeliet, P., Mackman, N., Moons, L., Luther, T., Van Gressens, P.V.I., Demunck, H., Kasper, M., Breier, G., Evrard, P., Muller, M., Risau, W., Edgington, T & Collen, D (1996) Role of tissue factor in embryonic blood vessel development Nature 383, 73–75 11 Huang, X., Molema, G., King, S., Watkins, L., Edgington, T.S & Thorpe, P.E (1997) Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature Science 275, 547–550 12 Ryu, J., Pyo, H., Jou, I & Joe, E (2000) Thrombin induces NO release from cultured rat microglia via protein kinase C, mitogenactivated protein kinase, and NF-kappa B J Biol Chem 275, 29955–29959 13 Koyama, T., Hall, L.R., Haser, W.G., Tonegawa, S & Saito, H (1987) Structure of a cytotoxic T-lymphocyte-specific gene shows a strong homology to fibrinogen beta and gamma chains Proc Natl Acad Sci USA 84, 1609–1613 14 Parr, R.L., Fung, L., Reneker, J., Myers-Mason, N., Leibowitz, J.L & Levy, G (1995) Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity J Virol 69, 5033–5038 15 Qureshi, S.T., Clermont, S., Leibowitz, J., Fung, L.S., Levy, G & Malo, D (1995) Mouse hepatitis virus-3 induced prothrombinase (Fgl2) maps to proximal chromosome Genomics 29, 307–309 16 Chiquet-Ehrismann, R (1995) Tenascins, a growing family of extracellular matrix proteins Experientia 51, 853–862 17 Ruegg, C.E., Gandolfi, A.J., Nagle, R.B., Krumdieck, C.L & Brendel, K (1987) Preparation of positional renal slices for study of cell-specific toxicity J Pharmacol Meth 17, 111–123 18 Xu, X & Doolittle, R.F (1990) Presence of a vertebrate fibrinogen-like sequence in an echinoderm Proc Natl Acad Sci USA 87, 2097–2101 19 Yuwaraj, S., Ding, J., Liu, M., Marsden, P.A & Levy, G.A (2001) Genomic characterization, localization, and functional expression of FGL2, the human gene encoding fibroleukin: a novel human procoagulant Genomics 71, 330–338 20 Levy, G.A., Liu, M., Ding, J., Yuwaraj, S., Leibowitz, J., Marsden, P.A., Ning, Q., Kovalinka, A & Phillips, M.J (2000) Molecular and functional analysis of the human prothrombinase gene (HFGL2) and its role in viral hepatitis Am J Pathol 156, 1217–1225 21 Fingerote, R.J., Abecassis, M., Phillips, M.J., Rao, Y.S., Cole, E.H., Leibowitz, J & Levy, G.A (1996) Loss of resistance to murine hepatitis virus strain infection after treatment with corticosteroids is associated with induction of macrophage procoagulant activity J Virol 70, 4275–4282 22 Pope, M., Rotstein, O., Cole, E., Sinclair, S., Parr, R., Cruz, B., Fingerote, R., Chung, S., Gorczynski, R & Fung, L (1995) Pattern of disease after murine hepatitis virus strain infection 2286 M Liu et al (Eur J Biochem 270) 23 24 25 26 27 28 29 30 31 32 33 34 correlates with macrophage activation and not viral replication J Virol 69, 5252–5260 Fung, L.S., Neil, G., Leibowitz, J., Cole, E.H., Chung, S., Crow, A & Levy, G.A (1991) Monoclonal antibody analysis of a unique macrophage procoagulant activity induced by murine hepatitis virus strain infection J Biol Chem 266, 1789–1795 Ruegg, C & Pytela, R (1995) Sequence of a human transcript expressed in T-lymphocytes and encoding a fibrinogen-like protein Gene 160, 257–262 Schwartz, B.S., Levy, G.A., Fair, D.S & Edgington, T.S (1982) Murine lymphoid procoagulant activity induced by bacterial lipopolysaccharide and immune complexes is a monocyte prothrombinase J Exp Med 155, 1464–1479 Ding, J.W., Ning, Q., Liu, M.F., Lai, A., Leibowitz, J., Peltekian, K.M., Cole, E.H., Fung, L.S., Holloway, C., Marsden, P.A., Yeger, H., Phillips, M.J & Levy, G.A (1997) Fulminant hepatic failure in murine hepatitis virus strain infection: tissue-specific expression of a novel fgl2 prothrombinase J Virol 71, 9223–9230 Clark, D.A., Ding, J.W., Chaouat, G., Coulam, C.B., August, C & Levy, G.A (1999) The emerging role of immunoregulation of fibrinogen-related procoagulant Fgl2 in the success or spontaneous abortion of early pregnancy in mice and humans Am J Reprod Immunol 42, 37–43 Lafuse, W.P., Castle, L., Brown, D & Zwilling, B.S (1995) The cytotoxic T lymphocyte gene FIBLP with homology to fibrinogen beta and gamma subunits is also induced in mouse macrophages by IFN-gamma Cell Immunol 163, 187–190 Li, C., Fung, L.S., Chung, S., Crow, A., Myers-Mason, N., Phillips, M.J., Leibowitz, J.L., Cole, E., Ottaway, C.A & Levy, G (1992) Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection J Exp Med 176, 689–697 Karantzoulis-Fegaras, F., Antoniou, H., Lai, S.L., Kulkarni, G., D’Abreo, C., Wong, G.K., Miller, T.L., Chan, Y., Atkins, J., Wang, Y & Marsden, P.A (1999) Characterization of the human endothelial nitric-oxide synthase promoter J Biol Chem 274, 3076–3093 Marsden, P.A., Heng, H.H., Scherer, S.W., Stewart, R.J., Hall, A.V., Shi, X.M., Tsui, L.C & Schappert, K.T (1993) Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene J Biol Chem 268, 17478–17488 Hall, A.V., Antoniou, H., Wang, Y., Cheung, A.H., Arbus, A.M., Olson, S.L., Lu, W.C., Kau, C.L & Marsden, P.A (1994) Structural organization of the human neuronal nitric oxide synthase gene (NOS1) J Biol Chem 269, 33082–33090 Ning, Q., Liu, M., Kongkham, P., Lai, M.M., Marsden, P.A., Tseng, J., Pereira, B., Belyavskyi, M., Leibowitz, J., Phillips, M.J & Levy, G (1999) The nucleocapsid protein of murine hepatitis virus type induces transcription of the novel fgl2 prothrombinase gene J Biol Chem 274, 9930–9936 Fan, J., Marshall, J.C., Jimenez, M., Shek, P.N., Zagorski, J & Rotstein, O.D (1998) Hemorrhagic shock primes for increased expression of cytokine-induced neutrophil chemoattractant in the lung: role in pulmonary inflammation following lipopolysaccharide J Immunol 161, 440–447 Ó FEBS 2003 35 Clark, M.E & Mellon, P.L (1995) The POU homeodomain transcription factor Oct-1 is essential for activity of the gonadotropin-releasing hormone neuron-specific enhancer Mol Cell Biol 15, 6169–6177 36 Courey, A.J & Tjian, R (1988) Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif Cell 55, 887–898 37 Hagen, G., Muller, S., Beato, M & Suske, G (1994) Sp1-mediated transcriptional activation is repressed by Sp3 EMBO J 13, 3843– 3851 38 Thanos, D & Maniatis, T (1995) Virus induction of human IFN beta gene expression requires the assembly of an enhanceosome Cell 83, 1091–1100 39 Belsham, D.D & Mellon, P.L (2000) Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5¢-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression Mol Endocrinol 14, 212–228 40 Fadel, B.M., Boutet, S.C & Quertermous, T (1999) Octamerdependent in vivo expression of the endothelial cell-specific TIE2 gene J Biol Chem 274, 20376–20383 41 Cieslik, K., Zembowicz, A., Tang, J.L & Wu, K.K (1998) Transcriptional regulation of endothelial nitric-oxide synthase by lysophosphatidylcholine J Biol Chem 273, 14885–14890 42 Gegonne, A., Bosselut, R., Bailly, R.A & Ghysdael, J (1993) Synergistic activation of the HTLV1 LTR Ets-responsive region by transcription factors Ets1 and Sp1 EMBO J 12, 1169– 1178 43 Schaefer, T.S., Sanders, L.K & Nathans, D (1995) Cooperative transcriptional activity of Jun and Stat3 beta, a short form of Stat3 Proc Natl Acad Sci USA 92, 9097–9101 44 Bromberg, J & Darnell, J.E Jr (2000) The role of STATs in transcriptional control and their impact on cellular function Oncogene 19, 2468–2473 45 Tone, M., Powell, M.J., Tone, Y., Thompson, S.A & Waldmann, H (2000) IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3 J Immunol 165, 286–291 46 Schoenherr, C.J & Anderson, D.J (1995) The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuronspecific genes Science 267, 1360–1363 47 Kennett, S.B., Udvadia, A.J & Horowitz, J.M (1997) Sp3 encodes multiple proteins that differ in their capacity to stimulate or repress transcription Nucleic Acids Res 25, 3110– 3117 48 Brightbill, H.D., Plevy, S.E., Modlin, R.L & Smale, S.T (2000) A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages J Immunol 164, 1940–1951 49 Marazzi, S., Blum, S., Hartmann, R., Gundersen, D., Schreyer, M., von Argraves, S.F.V., Pytela, R & Ruegg, C (1998) Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes J Immunol 161, 138–147  ... respectively Mutation of the Ets-1 binding site (mut5) also blocked the effect of Ets-1 in the presence of Sp1/ Sp3 Effect of the fgl2 positive regulatory region on induced expression of fgl2 Previously,... for the basal expression of the fgl2 gene This finding is consistent with the observation that fgl2 is constitutively expressed in cultured endothelial cells, as well as in the primary endothelial. .. contain fgl2 promoter all the way to the beginning of ATG start codon to avoid the discrepancy of minor transcription start variance between macrophages and endothelial cells Endothelial cells