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A SAF Binding Site in the Promoter Region of Human -Fibrinogen Gene Functions as an IL-6 Response Element ¹

Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of fibrinogen is highly induced during inflammation, and such abnormal expression of this protein is considered as a major cardiovascular risk factor. IL-6 is one of the main mediators of abnormal expression of fibrinogen leading to the pathogenic conditions. Transient transfection and EMSA were performed to investigate the molecular mechanism of IL-6-induced -fibrinogen gene expression in hepatic cells. Using progressively deleted 5' fragments of the -fibrinogen promoter coupled to chloramphenicol acetyltransferase gene, an IL-6 responsive element located between positions -273 and -259 was identified. Mutation of this element abrogates IL-6 responsiveness of the -fibrinogen promoter. Interaction of this promoter with a zinc finger transcription factor, serum amyloid A activating factor (SAF)-1, was demonstrated by EMSA. Furthermore, overexpression of wild-type SAF-1 in transfected liver cells can increase transcription of the -fibrinogen promoter. These data show that transcription factor SAF-1 is involved in the regulation of IL-6-mediated induction of the human -fibrinogen gene in liver cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fibrinogen, a plasma glycoprotein, is the precursor of fibrin that is involved in blood clot formation. Statistical analyses of clinical findings show that high circulating levels of fibrinogen pose an increased risk of myocardial infarction and stroke (1, 2). In addition, fibrinogen levels are higher in patients with diabetes, hypertension, hyperlipoproteinaemia, or among smokers. This physiologically significant protein is constitutively synthesized in the liver but its expression is increased several fold in response to a variety of physiologic and pathologic stimuli, including growth hormone, inflammation, infection, glucocorticoids, and trauma (3, 4, 5). Increased expression of this protein under pathophysiological conditions leads to the diseased state. Understanding the mechanism of fibrinogen induction thus becomes important for efficient control of cardiovascular disease and other physiological abnormalities associated with overexpression of fibrinogen.

The fibrinogen molecule is composed of three polypeptide chains, , ß and , and is secreted into the blood as a dimer (A-Bß-)₂ following its synthesis in liver cells. Each polypeptide is encoded by a single gene. Plasma level of three fibrinogen polypeptides rises coordinately in response to many inflammatory conditions and exposure to fibrinogen degradation products (6, 7). Increase of fibrinogen synthesis is due primarily to the increased transcription of the genes of three polypeptide chains. In cultured liver cells, addition of IL-6 can mimic this response, implicating involvement of IL-6 in inducing fibrinogen expression (8). During inflammation, transcription of the -, ß-, and -chain of fibrinogen is seen to be coordinately induced (9). The highly coordinated induction of the three fibrinogen genes and presence of some highly conserved sequences suggested that fibrinogen genes are transcriptionally regulated in a similar fashion during inflammation (10). As yet, however, no such common regulatory transcription factor interacting with the structurally similar promoter elements present in each fibrinogen gene has been identified. Functional analysis of the promoter of the -chain of human fibrinogen (11) showed the presence of a upstream stimulatory factor/adenovirus major late promoter transcription factor DNA binding element, a CAAT binding element, an IL-6 response element, and a negative element between sequences -348 and -390. The constitutive basal expression of the -fibrinogen gene is regulated by the upstream stimulatory factor/adenovirus major late promoter transcription factor (12, 13). To date, there is no known report of any transcription factor that regulates the human -fibrinogen gene under cytokine-inducible conditions.

The present study was designed to characterize the mechanism through which induction of the human -fibrinogen gene is regulated in the liver cells. Usually, the tissue-specific and temporally regulated inducible expression of eukaryotic genes results from the interaction of induced or activated transcription factors and the cis-acting DNA elements present in the promoter. During inflammation, infection, or trauma, a wide variety of cytokines released by different cell types stimulate the synthesis of inflammation-responsive genes via activation of a number of transcription factors, including C/EBP (14), NF-B (15), and STAT3 (16). Recently another family of transcription factors, called serum amyloid A activating factor (SAF)³ (17, 18) has been reported, and it is activated in many cell types in response to various inflammatory agents. These proteins contain multiple Cys₂-His₂-type zinc finger domains at their C-terminal half. In general, SAF family members are inflammation responsive and activated by LPS (19), cytokines, like IL-1 or IL-6 (20), or minimally modified low density lipoprotein treatment (21). In this study, by deletion mapping and site-directed mutagenesis the existence of an IL-6-responsive cis-acting element in the human -fibrinogen promoter and interaction of SAF-1 transcription factor with this element are shown. Overexpression of SAF-1 in liver cells markedly induced expression of the -fibrinogen promoter. Taken together, these results identify a mediator of the signal transduction pathway for regulating IL-6-mediated induction of the -fibrinogen gene in liver.

	Materials and Methods

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Cell culture and transfection

Human liver-derived HepG2 cells were obtained from American Type Culture Collection (Manassas, VA). These cells were cultured in DMEM containing high glucose (4.5 g/L) supplemented with 7% FCS. For induction, cells were stimulated with 50 ng/ml IL-6 (Promega, Madison, WI). Transient transfections were conducted by the calcium phosphate method (22) using a mixture of DNAs containing 1 µg of chloramphenicol acetyltransferase (CAT) reporter plasmid, 1 µg of pSV-ß-gal plasmid (Promega) as a control for measuring transfection efficiency, and carrier DNA so that the total amount of DNA in each transfection remained constant. In some transfection assays, in addition to the CAT reporter and pSV-ß-gal plasmids, varying concentrations of pCMV-SAF, a plasmid that expresses a functionally active transcription factor SAF-1 (18), or pCMV-SAF(mut) that contains a SAF-1 sequence in reverse orientation, were included. Cells were harvested at 24 h posttransfection, and CAT activity was determined from cell extracts as described previously (22). For CAT assays, extracts were heated at 60°C for 10 min to inactivate endogenous acetylase and assayed for ß-galactosidase expression. IL-6 had no effect on the ß-galactosidase expression. All values reported have been corrected for background activity.

Plasmid construction

The progressively deleted CAT reporter plasmids were constructed by cloning various segments of the -fibrinogen promoter into the HindIII and BamHI site of promoterless pBLCAT3 plasmid vector (23). The progressively deleted segments were prepared by PCR using different appropriate 5'-end primers and a constant 3'-end primer. The large template DNA containing human -fibrinogen DNA (11) was kindly provided by Dr. D. W. Chung (Department of Biochemistry, University of Washington, Seattle, WA). The sequence of the primers are described below in Oligonucleotides. The (-300/-200)CAT, (-285/-200)CAT, (-270/-200)CAT, (-255/-200)CAT, (-240/-200)CAT, and (-225/-200)CAT reporter constructs were prepared by ligating the respective sequences into pBLCAT2 vector (23). Specific clustered mutations were generated by PCR using a method as described (24) and -fib(-285/-255)CAT or -fib(-900/+30)CAT as template. Briefly, PCR amplification of DNA was conducted using two primers located "back-to-back" on the duplex, with appropriate 5' ends and 3' ends oriented for extension in opposite orientations around the plasmid circle. One set of primer represented the wild-type sequence and the other primer represented the mutated sequence containing desired mismatches. The primers with mismatched sequences are shown in Fig. 4A, which describes their use. Following amplification, ends of the amplified DNAs were flushed with Klenow fragment of DNA polymerase I, 5'-end phosphorylated with T₄ polynucleotide kinase, circularized with T₄ DNA ligase, and used to transform competent Escherichia coli cells. All constructs were verified by DNA sequencing.

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Mutational analysis of the IL-6 responsive region. A, Sequence of -285/-255, the region responsive to IL-6, is shown along with several mutant derivatives. Site-directed mutagenesis was performed as described in Materials and Methods. B, HepG2 cells were transiently transfected in duplicate with wild-type and various mutant reporter plasmids and tested for their IL-6 responsiveness as described in Fig. 1. These results represent an average of three independent experiments. C, The wild-type -fibrinogen promoter containing sequences from -900 to +30 and a mutant derivative that contains a mutation at the IL-6-responsive element, located between positions -273 and -259 and indicated by a circle, were used in the transient transfection of HepG2 cells. The transfected cells were then tested for their IL-6 responsiveness as described in Fig. 1. Results of triplicate transfection assays are presented.

Primer sets used for PCR synthesis of the progressively deleted fragments of the human -fibrinogen (11) promoter are: -900/+30, 5'-GAAGCTTCTGGAGGCATTTCTCCA-3' and 5'-CGGATCCAGCCTTGTAGTGTCAGC-3'; -700/+30, 5'-GAAGCTTCACAGGAACAATGAAGT-3' and5'-CGGATCCAGCCTTGTAGTGTCAGC-3'; -600/+30, 5'-GAAGCTTGAGAAGTGAGAGCCTATGA-3' and 5'-CGGATCCAGCCTTGTAGTGTCAGC-3'; -400/+30, 5'-GAAGCTTTTGGTAATTCAGGTGAT-3' and 5'CGGATCCAGCCTTGTAGTGTCAGC-3'; -300/+30, 5'-GAAGCTTGCATCACACAGCCTCCAG-3' and 5'-CGGATCCAGCCTTGTAGTGTCAGC-3'; -200/+30, 5'-GAAGCTTGAGCTGGGCCAAAAAGG3' and 5'-CGGATCCAGCCTTGTAGTGTCAGC-3'; -100/+30, 5'-GAAGCTTCCTGCCCACCCTTCTGGT-3' and 5'-CGGATCCAGCCTTGTAGTGTCAGC-3'.

Nuclear extract preparation and EMSA

Nuclear extract was prepared from uninduced and IL-6-induced cells as described (17). Protein content was measured by the Bradford method (25). ³²P-labeled double-stranded DNA probes for EMSA were prepared by filling in the overhangs at the termini with Klenow and [-³²P]dCTP. In EMSA, equal protein amounts of nuclear extracts were incubated with the radiolabeled DNA probe, and the resulting incubation products were electrophoresed in a nondenaturing 6% polyacrylamide gel as described (17). In some reactions, competitor oligonucleotides were included. For Ab interaction studies, Abs were added to the reaction mixture during a preincubation period of 30 min on ice. Antisera against C/EBP, STAT3, NF-B, Egr-1, Sp1, HNF-1, and HNF-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-SAF-1 Ab was prepared as described (18). Preabsorption of anti-SAF Ab was conducted with purified recombinant SAF-1.

Western blot assay

Cell extracts (30 µg of protein) were fractionated in a SDS 5%/12% polyacrylamide gel and transferred onto a nitrocellulose membrane using an electroblotter (Research Products, Madison, WI). For evaluation of the relative amounts of protein in each lane, proteins were stained with Coomassie blue. Immunoblotting was performed using anti-SAF-1 Ab and HRP-conjugated secondary Ab. Chemiluminescence reaction was performed with the enhanced chemiluminescence detection kit using the manufacturer’s protocol (Amersham, Arlington Heights, IL).

RNA isolation and Northern blot analysis

Total RNA was isolated from uninduced and IL-6-induced HepG2 cells by using guanidinium thiocyanate method (26). Fifty micrograms of each sample of RNA was fractionated in a 1.1% agarose gel containing 2.2 M formaldehyde and transferred onto a nylon membrane. The blot was hybridized using human -fibrinogen cDNA probe (a gift from Dr. D. W. Chung). The same membrane was subsequently hybridized with an actin cDNA probe to ensure the quantity and quality of each RNA sample loaded on the gel.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
-Fibrinogen mRNA is induced by IL-6 in HepG2 liver cells

To study the regulation of IL-6-stimulated transcription of the -fibrinogen gene, it was necessary to determine the induction level of -fibrinogen mRNA. Human liver-derived HepG2 cells were incubated in the absence or presence of 50 ng/ml IL-6 for 24 h, and the level of -fibrinogen expression was monitored by RNA blot assay (Fig. 1). When treated with IL-6, HepG2 cells synthesized a much higher level of -fibrinogen mRNA (Fig. 1A, lane 2) than the control untreated cells (Fig. 1A, lane 1). Although -fibrinogen mRNA was constitutively synthesized by HepG2 cells, IL-6 caused marked induction of this level. These results established that the -fibrinogen gene was indeed induced by IL-6 in the present culturing conditions in HepG2 liver cells.

View larger version (68K): [in this window] [in a new window]   FIGURE 1. -Fibrinogen mRNA is induced in HepG2 liver cells in response to IL-6. HepG2 cells were incubated in the presence of 50 ng/ml IL-6 (Promega) for 24 h. Total RNA (50 µg) prepared from untreated (lane 1) and IL-6-treated (lane 2) cells were electrophoresed in a 1.1% fomaldehyde-agarose gel and transferred to a nylon membrane. A, The RNA blot was hybridized using human -fibrinogen cDNA as a probe. B, As control, the same blot was hybridized with an actin cDNA probe after removal of the previous radioactive signal. C, Ethidium bromide stain of the agarose gel.

Analysis of the IL-6-inducible promoter element of the human -fibrinogen gene

A 900-bp DNA fragment containing the upstream 5'-flanking region was cloned in the right orientation into the plasmid vector pBLCAT3. This vector does not contain a functional promoter and is entirely dependent upon the functional promoter activity of the ligated heterologous gene sequence. The recombinant reporter gene was transiently transfected into HepG2 cells to analyze its promoter activity. As a control, the parent plasmid pBLCAT3 was used. Transfected liver cells were grown in the presence or absence of 50 ng/ml IL-6 for an additional 24 h, and CAT activity was measured. Results presented in Fig. 2A show that promoter activity of the -fibrinogen-CAT construct is highly induced when cells were treated with IL-6. These data showed that the DNA sequences present in the 5'-flanking region (-900/+30) of the human -fibrinogen gene contain element(s) necessary for the induction of the reporter gene by IL-6 in liver cells.

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Functional analysis of the promoter region of the human -fibrinogen gene. A, A DNA fragment containing sequences from -900 to +30 of the human -fibrinogen promoter was subcloned in right orientation into plasmid vector pBLCAT3. The resultant plasmid (pFib900-CAT) was used in transfection of HepG2 cells to assess the inducibility of the -fibrinogen gene promoter. HepG2 cells were transfected separately with recombinant pFib900-CAT and pBLCAT3 (as a control) DNA (1 µg of each). Following transfection, cells were incubated in the presence and absence of 50 ng/ml IL-6 (Promega) for an additional 24 h. Fold induction of CAT activity of the transfected cells relative to that of pBLCAT3 alone was determined and plotted as relative CAT activity. Details of the CAT constructs, transfection, and CAT assay procedure are described in Materials and Methods. B, HepG2 liver cells were transiently transfected in duplicate, with CAT reporter plasmids containing progressively deleted fragments of the -fibrinogen promoter. One set of transfected cells was incubated in the presence of 50 ng/ml IL-6 for 24 h and the other set was left untreated. Induction of CAT activity in transfected cells relative to that of uninduced cells was determined and plotted as relative CAT activity. These results represent an average of three independent experiments.

For further definition, five additional deletion constructs were prepared in which progressively deleted sequences of the IL-6-responsive region (-300 to -200) were ligated to the pBLCAT2 vector. pBLCAT2 vector contains a minimal tk promoter and does not respond to IL-6 stimulation (data not shown). As shown in Fig. 3, IL-6 response of these constructs decreased significantly when sequences up to -255 were deleted. Deletion of sequences up to -270 slightly affected IL-6 inducibilty of the reporter gene. Results of these deletion analyses established the presence of an IL-6-inducible cis-acting element between positions -285 to -255 of the -fibrinogen gene.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Mapping of the IL-6-responsive promoter of the human -fibrinogen gene. HepG2 cells were transiently transfected, in duplicate, with six CAT reporter plasmids containing progressive deletions within the -300 to -200 region that confers IL-6 inducibility. Following transfection, one set of transfected cells was incubated in the presence of 50 ng/ml IL-6 and the other left untreated for an additional 24 h. Details of the CAT constructs, transfection, and CAT assay procedure are described in Materials and Methods. These results represent an average of three independent experiments.

Mutational analysis of the human -fibrinogen IL-6-responsive element

For precise definition of the IL-6-responsive promoter, clustered mutations were introduced within -285 and -255 region of the -fibrinogen promoter. Sequences of the wild-type and five mutant promoters are shown (Fig. 4A). These promoter-reporter constructs were used in a transient transfection assay to test their IL-6 responsiveness (Fig. 4B). Three of the five mutants, M1, M2, and M5, appeared to retain full IL-6 response, while mutants M3 and M4 showed almost complete loss of reponsiveness to IL-6. Based upon the location of mutations, the above results indicate that the sequences between -273 and -259 contain an IL-6-responsive element. To evaluate the specificity of such clustered mutations, a reporter gene was constructed where the sequences between -273 and -259 of the -fibrinogen promoter spanning sequences from -900 to +30 were altered by in vitro site-directed mutagenesis, and IL-6 response of the mutated construct was evaluated. Results presented in Fig. 4C showed that specific mutation at the -273/-259 region can substantially diminish IL-6-mediated induction of the -fibrinogen promoter, indicating IL-6 responsiveness of this promoter is achieved primarily through this region.

Identification of an inducible NF that binds to the IL-6-responsive element of the -fibrinogen promoter

To identify the transacting factors that can interact with the IL-6-responsive element, EMSA was performed using untreated or IL-6-treated HepG2 cell nuclear extracts and a ³²P-labeled probe containing sequences between -273 and -259. As shown in Fig. 5A, one DNA-protein complex was formed, which was clearly induced by the IL-6 treatment (compare lanes 1 and 2). In contrast, no DNA-protein complex was formed with the mutant probe (lanes 3 and 4). Further, the IL-6-inducible complex was completely inhibited by a molar excess of homologous wild-type oligonucleotide (lane 6) but not by the mutated oligonucleotide (lane 7). These results indicated that sequences from -273 to -259 can indeed serve as a recognition site for a NF whose DNA binding activity is induced by IL-6. To determine the identity of this protein, Abs against a variety of IL-6-inducible and liver-specific transcription factors were used (Fig. 5B). The IL-6-inducible DNA-protein complex was not inhibited by C/EBP-, NF-B-, STAT3-, Egr-1-, Sp1-, HNF-1-, or HNF-3-specific Abs (Fig. 5B, lanes 2–6 and 8 and 9). In lane 2, some supershifting is seen with C/EBP Ab, which is nonreproducible and therefore should be regarded as an electrophoresis artifact. Only an anti-SAF-1 (18) polyclonal Ab (lane 7) could inhibit the formation of this complex. For further verification, preadsorbed anti-SAF-1 Ab was used in the DNA binding assay, and such a preparation of Ab failed to neutralize this complex (lane 12). Also, an oligonucleotide containing the consensus binding element for SAF-1 (18) inhibited this DNA-protein complex (Fig. 5A, lane 8). These results characterized and verified interaction of SAF-1 transcription factor with the IL-6-responsive element (-273/-259) of the human -fibrinogen gene.

View larger version (63K): [in this window] [in a new window]   FIGURE 5. Detection of DNA-protein complexes using EMSA. A, Nuclear extract (10 µg protein) prepared from HepG2 cells, both uninduced (lanes 1 and 3) and IL-6 induced for 24 h (lanes 2 and 4–8), were incubated with a double-stranded wild-type 32P-labeled -fibrinogen (-273/-259) element (lanes 1, 2, and 5–8) or with a double-stranded 32P-labeled mutant probe (lanes 3 and 4). The upper strand sequences of the wild-type and mutant -fibrinogen oligonucleotides are 5'-TGCCCTCCTCCTCAC-3'and 5'-TGCTATACGTATCAC-3', respectively. Underline represents mutation. Lane 5 contains no competitor, lane 6 contains 100x unlabeled wild-type -fibrinogen oligonucleotide, lane 7 contains 100x unlabeled mutant oligonucleotide, and lane 8 contains 100x unlabeled 5'-AGGGGAGGAG-3' oligonucleotide that contains consensus binding element of SAF. The resulting complexes were resolved in a 6% nondenaturing polyacrylamide gel. B, Characterization of the IL-6-induced complexes. Ten micrograms of IL-6-induced HepG2 cell nuclear protein was incubated with a wild-type 32P-labeled -fibrinogen element (-273/-259). Nuclear extracts, in some experiments, were preincubated with specific antisera for 30 min on ice before the addition of the 32P-labeled -fibrinogen probe. Different Abs used in the assay are indicated in the figure. Lanes 1 and 10 contain no Ab.

Overexpression of SAF-1 can stimulate the -fibrinogen gene

As SAF-1 protein was identified as an interacting factor with the IL-6-responsive element of the human -fibrinogen promoter, the effect of SAF-1 overexpression on the human -fibrinogen transcription was investigated. HepG2 cells were cotransfected with a wild-type -fibrinogen CAT (-900/+30) promoter and a SAF-1 expression vector. As seen in Fig. 6, overexpression of SAF-1 increased expression of the reporter gene in a dose-dependent manner. Transfection of cells with an expression plasmid containing cDNA of SAF-1 in reverse orientation did not increase the expression of the reporter gene. Also, the mutated reporter gene containing an altered IL-6-responsive element was not at all activated by both wild-type and altered SAF expression plasmids. These results indicated that SAF-1 is indeed necessary for transcriptional activation of the -fibrinogen promoter that contains a functional SAF binding element located between -273 and -259.

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Transactivation of the -fibrinogen promoter by SAF-1. HepG2 cells were cotransfected with a wild-type -fibrinogen reporter plasmid (-900/+30)CAT and increasing concentrations (1, 2, and 3 µg) of either pCMV-SAF-1 or pCMV-SAF-1(mut) plasmid DNA. In a parallel experiment, HepG2 cells were cotransfected with a mutant -fibrinogen reporter plasmid (-900/+30)mtCAT containing altered sequences between -273 and -259 and increasing concentrations (1, 2, and 3 µg) of either pCMV-SAF-1 or pCMV-SAF-1(mut) plasmid DNA. Induction of CAT activity in the cotransfected cells relative to that of reporter plasmid alone was determined and plotted as relative CAT activity. These results represent an average of three independent experiments.

The increase of SAF-1 DNA binding activity by IL-6 that resulted in the induction of the -fibrinogen gene could result due to an increased protein content and/or a posttranslational modification of this transcription factor. For evaluation of these possibilities, Western blot analysis of untreated and IL-6-treated cell extracts was performed using an anti-SAF-1 Ab as a probe (Fig. 7A). For verification of loading equal protein amounts of cell extracts, duplicate samples were electrophoresed and stained with Coomassie blue dye (Fig. 7B). Two bands of variable intensity were visualized using this Ab, and there was no change in the levels of these two bands. The dark, faster moving band comigrates with the recombinant SAF-1 protein (data not shown). The lighter, slow moving band could arise due to a cross-reaction of this Ab with a different SAF isoform or it could appear due to some cross-reaction with an unrelated protein. Nonetheless, this result suggested that IL-6 treatment of the cells does not increase the de novo synthesis of SAF-1 protein, and induction of SAF DNA binding activity in the nuclear extract of IL-6-treated cells is not due to any increased expression but may be due to a posttranslational modification of this protein.

View larger version (83K): [in this window] [in a new window]   FIGURE 7. Western immunoblot analysis for SAF-1 in IL-6-treated HepG2 cells. A, Cell extracts (30 µg of protein) prepared from untreated (lane 1) and IL-6-treated (lane 2) HepG2 cells were fractionated in a 5%/12% SDS-polyacrylamide gel, electroblotted onto a nitrocellulose membrane, and probed with an anti-SAF-1 Ab. Arrows indicate location for SAF-1. This Ab detects two isoforms of SAF. B, Coomassie blue staining of proteins in the same amounts of cell extracts as used in A. Molecular masses of standard protein markers are indicated in kilodaltons. Similar patterns of protein in both lanes indicate that they are of equal quantity and quality and suitable for comparative studies.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Epidemiological studies indicate that a mere 2-fold increased level of circulating fibrinogen should be considered as a major cardiovascular risk factor. Furthermore, as atherosclerotic lesions progress, in terms of severity there is increased deposition of fibrin, fibrinogen, and collagen in areas of advanced atherosclerosis (27). As an acute-phase protein, expression of fibrinogen is known to be induced in response to various inflammatory conditions. In the present study, IL-6 used as a mediator of inflammatory response induced the expression of -fibrinogen mRNA in HepG2 liver cells (Fig. 1). The systematic analysis of serially truncated or mutated reporter constructs identified an IL-6-responsive element in the promoter region of the -fibrinogen gene. This element, positioned between -273 and -259, is different from a region previously identified as an IL-6-responsive element of the human -fibrinogen gene (11). Furthermore, the previous report could not detect interaction of any transcription factor with that putative IL-6-responsive element and therefore the mechanism of the signaling pathway of human -fibrinogen gene induction remained incompletely understood. The present study demonstrates that a recognition site for SAF-1 in the human -fibrinogen promoter is essential for IL-6-mediated induction of this gene. SAF is a family of zinc finger transcription factors that is activated during inflammation. One member of this family, SAF-1, is homologous to human MAZ (28) or mouse Pur-1 (29). The broad array of other target genes that this family of proteins regulate are SAA (17, 18, 19, 20, 21), c-myc (28), insulin (29), serotonin 1A receptor (30), and the CD4 receptor (31). SAF-1 protein is inflammation responsive and is activated in several cell types, including the liver. LPS, IL-6, and minimally modified low density lipoprotein particles are a few among the known activating agents of SAF-1 (17, 18, 19, 20, 21). However, because SAF-1 is not expressed exclusively in the liver, liver-specific induction of the -fibrinogen gene may not be mediated solely by this factor and may require some assistance. The presence of an accessory factor that dimerizes and participates in the tissue-specific regulation of HNF-1, a transcription factor implicated in the liver-specific regulation of many genes, is recently documented (32). By analogy, liver-specific induction of the human -fibrinogen gene by SAF-1 may involve the presence of some specific cofactor(s). Identity of such accessory factor(s) remains to be determined.

IL-6 has been known to activate a group of transcription factors in which STAT3 plays a major role in regulating the expression of several acute-phase genes. Previous several studies identified a highly conserved CTGG(G/A)AA motif in the IL-6-responsive regions of all three fibrinogen genes (10, 33, 34). This CTGG(G/A)AA motif was also identified in the IL-6-responsive promoter region of the ₂-macroglobulin gene (35). Since the discovery of STAT3/APRF (36, 37), which interacts with a consensus element of TT(A/C)(C/T)N(G/A)(G/T)AA sequence, there has been intense effort to verify the role of the putative CTGG(G/A)AA motif and STAT3 in regulating the three fibrinogen genes. For the human -, ß-, and -fibrinogen genes, identity of the transcription factor that can interact with the CTGG(G/A)AA element present in these genes remained elusive (11, 33, 38). In the rat, induction of -fibrinogen was shown to be regulated by a novel transcription factor interacting with one of the CTGG(G/A)AA motifs of this gene (39). The regulatory role of STAT3 was documented only in case of the rat -fibrinogen gene (40). However, it has been suggested that STAT3 may not be the sole regulator of this gene and possibly acts in conjunction with other unidentified transcription factors because many conditions that activate STAT3 do not activate the -fibrinogen gene. In the present study, the role of STAT3 in the regulation of the human -fibrinogen gene was investigated by EMSA and transient transfection analysis (data not shown). Because the outcome of these investigations was negative, it was concluded that STAT3 may have no direct role in directing IL-6-mediated inducible expression of the human -fibrinogen gene.

As yet, limited information is available on the activation mechanism of SAF-1 and its family members. The DNA binding activity of SAF-1, increased by IL-6 (Fig. 5), is not due to any increased expression of SAF-1 protein (Fig. 7). These results strongly suggest that IL-6 causes some posttranslational modification of this protein and this event increases its DNA binding ability. Protein phosphorylation is regarded as a primary mechanism for activation of numerous transcription factors. As IL-6 is known to activate many protein kinases, it is speculated that SAF-1 may be activated by phosphorylation. Enhancement of transactivating ability of SAF-1 in the presence of IL-6 further attests to this possibility (data not shown). Further studies on the nature of protein kinases involved in activating SAF will shed light on the role this factor plays in controlling IL-6-induced expression of the human -fibrinogen gene.

	Acknowledgments

 
I am grateful to Dr. Dominic Chung for human -fibrinogen DNA.

	Footnotes

 
¹ This work was supported in part by U.S. Public Health Service Grant DK49205 and funds from the College of Veterinary Medicine, University of Missouri.

² Address for correspondence and reprint requests to Dr. Alpana Ray, Department of Veterinary Pathobiology, University of Missouri, 313 Connaway Hall, Columbia, MO 65211.

³ The abbreviations used is this paper: SAF, serum amyloid A activating factor; CAT, chloramphenicol acetyltransferase.

Received for publication January 7, 2000. Accepted for publication July 3, 2000.

	References

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