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STAT3 participates in transcriptional activation of the C-reactive protein gene by interleukin-6

Interleukin-6 (IL-6) is the major cytokine inducing transcription of human C-reactive protein (CRP) during the acute phase response. STAT (signal transducers and activators of transcription) family members, recently shown to be important mediators of the effects of many cytokines including IL-6, generally induce their effects by binding to palindromic sequences with TT(N)5AA motifs. We report an IL-6 responsive element in the proximal region of the human CRP 5'-flanking region that bears a TT(N)4AA motif, which we have termed CRP acute phase response element (CRP-APRE). In Hep3B cells, IL-6 but not interferon-gamma was capable of activating CAT constructs driven by the CRP promoter containing CRP-APRE. Overexpressed STAT3 was able to transactivate CRP-chloramphenicol acetyltransferase constructs through the CRP-APRE and was able to enhance endogenous CRP mRNA accumulation in response to IL-6. STAT3 (or an antigenically related molecule) bound to the CRP-APRE in response to IL-6. Overexpression of STAT3 in the presence of IL-6 was capable of inducing expression of a construct consisting of the CRP-APRE and a minimal thymidine kinase promoter lacking a C/EBP site. Taken together, these findings indicate that STAT3 participates in the transcriptional activation of CRP in response to IL-6.

Interleukin-6 (IL-6) is the major cytokine inducing transcription of human C-reactive protein (CRP) during the acute phase response. STAT (signal transducers and activators of transcription) family members, recently shown to be important mediators of the effects of many cytokines including IL-6, generally induce their effects by binding to palindromic sequences with TT(N) 5 AA motifs. We report an IL-6 responsive element in the proximal region of the human CRP 5flanking region that bears a TT(N) 4 AA motif, which we have termed CRP acute phase response element (CRP-APRE). In Hep3B cells, IL-6 but not interferon-␥ was capable of activating CAT constructs driven by the CRP promoter containing CRP-APRE. Overexpressed STAT3 was able to transactivate CRP-chloramphenicol acetyltransferase constructs through the CRP-APRE and was able to enhance endogenous CRP mRNA accumulation in response to IL-6. STAT3 (or an antigenically related molecule) bound to the CRP-APRE in response to IL-6. Overexpression of STAT3 in the presence of IL-6 was capable of inducing expression of a construct consisting of the CRP-APRE and a minimal thymidine kinase promoter lacking a C/EBP site. Taken together, these findings indicate that STAT3 participates in the transcriptional activation of CRP in response to IL-6.
A large number of systemic and metabolic changes, collectively referred to as the acute phase response (APR), 1 begin to occur within hours after an inflammatory stimulus (1)(2)(3). Among these changes is a reprogramming of the pattern of plasma protein gene expression in hepatocytes, with consequent changes in blood concentrations of these proteins. C-reactive protein (CRP) is a major acute phase protein in humans, its concentration increasing more than 1000-fold in severe inflammatory states.
Interleukin-6 (IL-6) appears to be the principal regulator of most acute phase proteins (3,4), although other inflammationassociated cytokines also contribute to this process. IL-1␤ and tumor necrosis factor-␣ have been found to participate in induction of a broad subset of acute phase proteins, and both transforming growth factor-␤ and ␥-interferon can induce limited subsets of acute phase proteins (2). IL-6 has been shown to activate members of the C/EBP family of transcription factors in hepatoma cell lines (5). It has recently been found that STAT (signal transducers and activators of transcription) family members may also play a major role in mediating IL-6 effects (6 -9). The binding of IL-6 to its receptor complex leads to phosphorylation of Janus kinase kinases, with subsequent rapid (15-60 min) phosphorylation, dimerization, and nuclear translocation of a transcription factor originally named acute phase response factor (8) and since designated STAT3 (6). STAT3 then binds to specific response elements in the promoter regions of cytokine responsive genes. The promoter regions of a number of human and rat acute phase genes contain TT(N) 5 AA sequences (10 -13) capable of binding STAT proteins. STAT1 (14) also appears to be activated by IL-6 as well as by IFN-␥ with similar rapid kinetics and binds to a similar consensus motif called the ␥-interferon activation site (GAS) (15)(16)(17). Both STAT3 and STAT1 have been found to be activated by a broad spectrum of cytokines and growth factors, including IFN-␥, epidermal growth factor, and IL-6-related cytokines (17,18). We have previously shown in the human hepatoma cell line Hep3B that IL-6 activates transcription of CRP, that IL-1␤, which has no effect alone, synergistically enhances CRP transcription in the presence of IL-6, and that the proximal 157 bp of the 5Ј-flanking region of CRP was sufficient to confer IL-6 induction and IL-1␤ synergistic activation on CRP-CAT (19). Several cis-elements and trans-activators that were required for CRP transcription in response to monocyte conditioned medium and IL-6 have been characterized. Two C/EBP binding sites in the proximal region of the CRP promoter have been shown to bind IL-6-inducible C/EBP family members (20), and two HNF-1 sites have been shown to be adjacent to the C/EBP sites and to be necessary for CRP transcription (21,22). Other regions containing positive and negative cis-elements have also been found in the 5Ј-and 3Ј-flanking regions of the CRP gene (23,24).
We report here the finding of a STAT3 response element in the human CRP promoter with the sequence TTCCCGAA, which is necessary for optimal IL-6-induced transcription of CRP. Oligonucleotides with the TT(N) 4 AA motif have recently been reported to specifically bind STAT3 (25). This finding indicates that STAT3 or a closely related molecule participates in mediation of the transcriptional effect of IL-6 on human CRP.
Cell Culture-Human hepatoma Hep3B cells were kindly provided by Dr. G. J. Darlington (Baylor College of Medicine, Houston, TX). HepG2 cells were gifts from Dr. R. Hanson (Case Western Reserve University). Hep3B cells and HepG2 cells were maintained in RPMI 1640 medium and Dulbecco's modified Eagle's medium/F12, respectively, supplemented with 10% fetal bovine serum. Cells were subcultured weekly after trypsinization.
Transfection and Cytokine Induction-Electroporation was performed according to published procedures (28). Briefly, 10 7 cells subcultured 3 days earlier (80 -90% confluent) were trypsinized and mixed with 20 g of DNA (15 g of CAT reporter DNA, 5 g of RSV␤-Gal, and 2 g of expression DNA or control vector) plus 200 g of carrier DNA (Herring Testis DNA, Sigma) in total volume of 0.5 ml of Hepes-buffered saline. Electroporation was performed at V ϭ 260 V, C ϭ 960 microfarads using Gene Pulser Apparatus (Bio-Rad, Richmond, CA). Following electroporation, cells were seeded onto eight 35-mm-diameter dishes for 6 h in RPMI 1640 medium supplied with 10% fetal bovine serum and for 12 h in RPMI 1640 medium without serum.
For cytokine induction, the transfected cells were incubated with serum-free medium and exposed to 10 ng (100 units)/ml of IL-6, 10 ng (100 units)/ml IL-1␤, or 10 ng (100 units)/ml IFN-␥ for up to 24 h. STAT3 stable cell lines were generated by transfecting Hep3B cells with Rc/CMV-STAT3 by electroporation as described above, followed by G418 (400 g/ml) selection. Single colonies were picked and expanded.
Assay for CAT-CAT assay was performed using the phase extraction method (29). Cell extracts were prepared by three cycles of freezing and thawing. 15 l from 100 l of the cell extract (for 10 6 cells) was used to determine ␤-galactosidase activity as described (30). The remainder of the cell extract was assayed for CAT activity in a reaction containing 0.25 mg/ml of butyryl CoA and 0.02 Ci of [ 3 H] chloramphenicol. The ␤-Gal activity was used to normalize CAT activity to control for transfection efficiency.
Northern Analysis-Northern blotting was performed as described previously (19).
Preparation of Nuclear Extracts-Nuclear extracts were prepared according to Shapiro et al. (33) with the following modifications. Hep3B or HepG2 cells (3 ϫ 10 7 ) were washed with cold phosphate-buffered saline, collected, and pelleted by centrifugation at 4,000 ϫ g for 5 min at 4°C. The cell pellet was resuspended in 3 ϫ packed cell volume of buffer A (20 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM NaVO 4 , 1 mM EDTA, 0.2% Nonidet P-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). The suspension was placed on ice for 5 min. Nuclei were pelleted by centrifugation at 12,000 ϫ g for 2 min. The nuclear pellet was resuspended in 3 packed nuclear volume of buffer B (420 mM NaCl, 20% glycerol, 20 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM NaVO 4 , 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). The suspension was rocked gently for 30 min at 4°C followed by centrifugation at 12,000 ϫ g for 5 min, after which the supernatant was collected and stored at Ϫ70°C.
EMSA-Complementary oligonucleotides were annealed and labeled by filling in 5Ј protruding ends with Klenow enzyme, using [␣-32 P]dCTP (3000 Ci/mmol). EMSA was carried out according published procedures (34). Nuclear extract (5 g of protein) was incubated with about 200 fmol (50,000 cpm) of probe in gel shift incubation buffer (40 mM KCl, 20 mM Hepes, pH 7.9, 1 mM MgCl 2 , 0.1 mM EGTA, 0.5 mM dithiothreitol, 4% Ficoll, 0.1 g of poly(dI-dC)⅐poly(dI-dC); Pharmacia Biotech Inc.) for 15 min at room temperature. In supershift experiments, antiserum was added at the same time as the probe. In competition experiments, an excess of cold oligonucleotides was added to the binding reactions. The DNA-protein complexes were resolved by electrophoresis on a 4% polyacrylamide gel in 0.25 ϫ TBE (1 ϫ TBE ϭ 89 mM Tris-HCl, 89 mM boric acid, and 2 mM EDTA) at 10 V/cm. The gels were dried and autoradiographed.

RESULTS
The Ϫ123 to Ϫ85 Region Is Required for Optimal IL-6 Response-A series of 5Ј deletion constructs of CRP-CAT were transiently transfected into Hep3B cells, followed by the addition of IL-1␤, IL-6, or their combination. IL-1␤ alone had no effect on CAT expression, whereas IL-6 caused a 4-fold increase in CAT activity (Fig. 1). Deletion of the region between Ϫ157 and Ϫ123 led to a decreased IL-6 response with little effect on basal activity. IL-1␤ enhanced the effect of IL-6 on both constructs. In contrast, when the region between Ϫ123 and Ϫ85 was deleted, IL-6 induction of CAT expression was markedly diminished. Basal CAT activity was also decreased to about one-third of that seen with Ϫ123/ϩ3 CRP-CAT, and IL-1␤ synergy was not observed. Deletion of the region Ϫ85 to Ϫ50 abolished any measurable cytokine effect and decreased basal activity to that of the vector alone (not shown). These results were consistent with the existence of known IL-6 responsive cis-elements upstream of Ϫ123 and downstream of Ϫ85 (see top of Fig. 1) but also suggested that a cis-element(s) in the region of Ϫ123 to Ϫ85 was necessary for constitutive expression of CRP, for IL-6 induction, and for IL-1␤ synergy.
A GAS-like Sequence (CRP-APRE) Is Localized in the Region of Ϫ123 to Ϫ85-Four "block mutations" were constructed such that 5-7 nucleotides were replaced by irrelevant nucleotides at four positions in the interval between Ϫ123 and Ϫ85 (Table I). These mutant constructs, designated A, B, C, and D, were transfected into Hep3B cells, which were then studied for their responses to IL-6 (Table I). Although some decreased IL-6 response was seen with every mutant, the B mutant showed substantially diminished IL-6 inducibility as well as decreased basal activity.
The sequence disrupted in the B mutant, TTCCCGAA, was found to be homologous to members of the family of acute phase response elements (APREs) first identified in the rat ␣2M gene as well as to the GAS (Table II). It is noteworthy that the CRP element has only four nucleotides between the conserved TT . . . AA, although the surrounding sequences closely resemble ␣2M APRE. Therefore, we named the element disrupted in mutant B CRP-APRE.
STAT3 but Not STAT1␣ Transactivates CRP-CAT in Response to IL-6 -Because both STAT3 and STAT1␣ have been shown to bind to GAS-like sequences in response to both IL-6 and IFN-␥, we evaluated the possible roles of these transcription factors in activating CRP transcription (Fig. 2). Overexpression of STAT3 in the absence of cytokines was sufficient to increase CAT expression 2-3-fold in Ϫ157/ϩ3CRP-CAT. The effect of overexpressed STAT3 was moderately increased by the addition of IL-1␤ and was greatly enhanced by the addition of IL-6. In contrast, overexpression of STAT1␣ had no effect on CAT expression in the presence or the absence of cytokines. As a control experiment, overexpression of STAT1␣ was able to enhance the IFN-␥ response of a CAT reporter construct containing the interferon regulatory factor 1 promoter about 3-fold (data not shown). Studies employing Ϫ123/ϩ3CRP-CAT showed similar results, whereas Ϫ85/ϩ3CRP-CAT was not influenced by co-transfection of either STAT3 or STAT1␣ (data not shown).
Similar observations were made for the endogenous CRP gene in Hep3B cells. In cells stably transfected with a STAT3 expression construct, CRP responses to IL-6 and to the combination of IL-6 ϩ IL-1␤ were markedly increased (Fig. 3).
In contrast, overexpressed STAT1␣ was unable to increase CAT expression of Ϫ123/ϩ3CRP-CAT even in the presence of IFN-␥ (Fig. 4). It was of interest that IFN-␥ as well as IL-6 was able to enhance transactivation of Ϫ123/ϩ3CRP-CAT by STAT3 and that the combination of IL-6 and IFN-␥ had some additive effect in the presence of overexpressed STAT3.
To verify the hypothesis that STAT3 transactivation was exerted through CRP-APRE, the four block mutants of Ϫ123/ ϩ3CRP-CAT were employed in co-transfection experiments (Fig. 5). As expected, the response to cytokines in cells overexpressing STAT3 was greatly abolished in mutant B, while other mutants were only moderately affected. In another approach, we employed an antisense STAT3 expression construct in which the direction of the cDNA was reversed in the expression vector in the co-transfection experiments and found that antisense STAT3 was able to suppress IL-6 induction of CRP-CAT expression to one-third of vector-transfection control levels (data not shown).
STAT3 Binds to CRP-APRE in an IL-6-inducible Manner-EMSAs were employed to determine whether STAT3 binds to CRP-APRE. Because HepG2 cells have been shown to display abundant STAT3 activity in response to IL-6, we initially used nuclear extracts from this cell line to explore this possibility. As shown in Fig. 6, several complexes were observed when nuclear extracts from IL-6-treated (15 min) or untreated cells were incubated with a CRP-APRE probe. Formation of Complex I was induced by IL-6 and was competed by excess amounts (10 -50ϫ) of either self-oligonucleotides or rat ␣2M APRE but not by "CRP ␣" oligonucleotides, which have been shown to bind both C/EBP ␤ and C/EBP ␦ (20). This finding indicated that the binding activity was specific for CRP-APRE and that it shared the binding properties of STAT3, which has been shown to bind rat ␣2M APRE. Antibody against STAT3 abolished the formation of Complex I, whereas a complex with a slower migration rate formed, presumably a "supershifted" antibodycontaining complex. Neither rabbit IgG against ␤-galactosidase nor antibody against STAT5 had any effect. This result demonstrated that STAT3 (or an antigenically related protein) was present in Complex I. Two other complexes (II and in Hep3B cells, III, see below) were observed which were not IL-6-inducible. The significance of these complexes is not clear. A similar pattern of complexes in EMSA was observed for Hep3B cells, although STAT3 binding activity was lower than was seen with

FIG. 1. Activation of CRP-CAT 5 deletion constructs by IL-6 and IL-1␤ in Hep3B cells.
A diagrammatic representation of the CRP promoter (Ϫ157 to ϩ3) is shown at the top. The indicated C/EBP binding sites and HNF-1 site have been described by others (20,21). CRP-CAT constructs were transiently co-transfected with pRSV␤-Gal into Hep3B cells. Cells were treated with cytokines (IL-1␤, 100 units/ ml; IL-6, 100 units/ml) for 24 h. CAT activity (percentage of butyrylated chloramphenicol) was measured as described under "Experimental Procedures." CAT activities were normalized by ␤-galactosidase activity to control for differences in transfection efficiencies. The values are the averages of four independent experiments performed in duplicate. The error bars indicate the standard errors.

TABLE I Activation of block mutation constructs (A-D) by IL-6
Ϫ123/ϩ3CRP-CAT and mutants A-D (see "Experimental Procedures") were transfected into Hep3B. Cells were treated with IL-6 (100 units/ml) for 24 hours and harvested, and CAT activity was determined and normalized to co-transfected pRSV-␤Gal. The values are the averages of three independent experiments performed in duplicate with standard errors of less than 10% of each mean value.

STAT3 Activates CRP Transcription
HepG2 (data not shown). To examine the time course of the binding between STAT3 and CRP-APRE, Hep3B cells were treated with IL-6 for various lengths of time. EMSA studies (Fig. 7) revealed that IL-6induced binding of STAT3 to CRP-APRE was present within 15 min of IL-6 induction and was still detectable 18 h later.
CRP-APRE Is Sufficient to Mediate STAT3 Transactivation in Response to IL-6 -To examine whether CRP-APRE was able to confer STAT3 transactivation or IL-6 induction, we inserted two copies of CRP-APRE upstream of the TK basic promoter of 105 bp in length. This element alone did not confer IL-6 responsiveness in Hep3B cells, but CAT expression was increased 2-3-fold by IL-6 in cells overexpressing STAT3 (data not shown). Because a potential C/EBP binding site was present in the Ϫ105 to Ϫ83 region of the TK sequence, we subsequently deleted this region (Ϫ105 to Ϫ77) and generated a new truncated pTK-CAT vector and pAPRE-TK-CAT (see "Experimental Procedures"). The results of transactivation assays with these reconstructed plasmids (pTK(Ϫ77)-CAT and pAPRE-TK(Ϫ77)-CAT) are shown in Fig. 8. The results are similar to those obtained with Ϫ105 constructs; IL-6 alone did not stimulate CAT expression from either construct but did substantially induce the CAT activity of pAPRE-TK(Ϫ77)-CAT in the presence of STAT3 overexpression. These data exclude the possibility that the STAT3 transactivation was exerted through the C/EBP binding site in TK promoter and support the conclusion that a portion of the IL-6 response is mediated by STAT3 through the CRP-APRE. DISCUSSION Our major findings were that overexpressed STAT3 but not STAT1␣ was able to transactivate CRP-CAT constructs in response to IL-6 stimulation through a GAS-like sequence that we have termed CRP-APRE, that overexpressed STAT3 activated the endogenous CRP gene in response to IL-6, that CRP-APRE was able to bind STAT3 in an IL-6 inducible fashion, and that CRP-APRE, in the presence of overexpressed STAT3, conferred IL-6 inducibility on a heterologous promoter lacking C/EBP binding sites in Hep3B cells. Taken together, these findings indicate that STAT3 participates in the transcriptional activation of C-reactive protein in response to IL-6.
Although both STAT3 and STAT1␣ have been shown to bind the same GAS-like elements and activate transcription of a number of genes (9,35), this was not the case for CRP-APRE. Only STAT3 and not STAT1␣ transactivated CRP-CAT containing wild-type CRP-APRE; IL-6 markedly enhanced this transactivation. IFN-␥ in the absence of STAT3 overexpression had no effect on either CRP-CAT constructs or on the endogenous CRP gene (data not shown). Accordingly, we conclude that the ability of IFN-␥ to enhance CAT expression that we observed in STAT3-transfected cells was due to activation of overexpressed STAT3 in Hep3B cells. Similarly, EMSA demonstrated that CRP-APRE bound to STAT3 in a IL-6-dependent manner. This selective response to IL-6 but not IFN-␥ has not been reported for promoters containing other APREs, including ␣2M APRE (36). STAT3 has been shown to be the major STAT member mediating IL-6 signaling, whereas STAT1␣ is the major STAT activated by IFN-␥ (17). It is likely that the selective binding of STAT3 determines the specific response of the CRP gene to IL-6 and not to IFN-␥.
This specificity of CRP-APRE for STAT3 may be explained by its structure, TT(N) 4 AA, which differs from the TT(N) 5 AA motif found in other APREs. In a recent study (25) in which the effects of the spacing between the TT and AA core half-sites on the binding of the STAT complexes were examined using synthetic oligonuleotides, it was found that TT(N) 5

STAT3 Activates CRP Transcription
matched the CRP-APRE defined in this study. These results strongly support the conclusions of this study. These authors also induced expression of a luciferase construct containing four copies of TTCCCGAA in HepG2 cells with IL-6. This result contrasts with our finding that expression of pAPRE-TK-CAT was unaffected by IL-6 in Hep3B cells not transfected with STAT3. This discrepancy could be due to the low level of activated STAT3 in the Hep3B cell line or to the comparatively high copy numbers of TTCCCGAA (four instead of two) in their constructs. To our knowledge, CRP-APRE is the first example of a "natural" TT(N) 4 AA cis-element shown to bind STAT3 in an IL-6-inducible manner, although an element bearing the TTCCTGAC motif in the murine JunB gene was reported recently to bind STAT3 (37).
The binding of STAT3 to CRP-APRE in Hep3B cells was found to last at least 18 h, which contrasts to the duration of 1 h reported in HepG2 cells with the ␣2M APRE (8). This difference may arise from differences in the cell lines used or the assay systems employed or may be due to distinctive binding properties of each DNA oligonucleotide. Long term activation of STAT proteins has been reported using GAS-like elements such as pIRE of the interferon regulatory factor 1 gene in the human breast carcinoma cell line T47D (38). It is conceivable that in our system, continuous activation of STAT3 contributes to the "prolonged" binding detected in EMSA. Alternatively, inactivation of STAT3 signaling (e.g. dephosphorylation of STAT3) may be slow in our cells.
Our findings suggest that the CRP-APRE may also participate in maintenance of basal expression of CRP. Mutation of CRP-APRE not only decreased IL-6 induction of CRP-CAT but also lowered the basal level of CAT expression. We observed additional IL-6-independent complexes not abolished by antibody against STAT3 in EMSA using CRP-APRE as a probe ( Fig. 6 and 7). This finding raises the possibility that binding of an unidentified transcription factor(s) other than STAT3 may account for constitutive basal level expression of CRP-CAT.
The observations presented here should not be taken to imply that STAT3 is the major transcription factor participating in mediation of the CRP response to IL-6. The role of C/EBP binding sites in the CRP response to IL-6 is well documented (22). Our finding that two copies of CRP-APRE in the absence of overexpressed STAT3 were not sufficient to confer IL-6 responsiveness on a truncated minimal TK promoter, although subject to other interpretations, raises the possibility that there is need for one or more other response elements. The fact that a GAS-like IL-6 responsive element exists in close proximity to the two C/EBP binding sites in the CRP promoter (and other IL-6 responsive promoters) points to possible cooperative effects between STAT and C/EBP family members. Relevant to this issue are our preliminary findings, in studies of reporter FIG. 4. Transactivation of ؊123/؉3CRP-CAT by STAT3 but not STAT1 ␣ in response to IL-6 and IFN-␥. Ϫ123/ϩ3CRP-CAT (15 g) was transiently co-transfected with STAT3 expression plasmid Rc/ CMV-STAT3 (2 g) or STAT1␣ expression plasmid pMNC-91 (2 g) plus pRSV-␤GAL (5 g). Cells were treated with cytokines (IL-6, 100 units/ml; IFN-␥, 100 units/ml) for 24 h. CAT activity was determined and normalized to ␤-galactosidase activity. Two independent experiments with duplicate samples were performed, and similar patterns of expression were obtained. CAT activity of the means of duplicate samples in one of these experiments is plotted. constructs containing mutated C/EBP or CRP-APRE sites, which suggest that these sites are cooperative rather than functionally independent. The latter observation, if confirmed, would be consistent with the abundant evidence supporting the fundamental role of interactions between transcription factors in gene-specific transcriptional regulation (39). Among acute phase proteins, for example, an element in the human hemopexin promoter has been shown to bind a complex which contains STAT3 in response to IL-6 (40); the human hemopexin promoter, like CRP, also demonstrates IL-6-inducible C/EBP binding (40). Physical interaction between C/EBP␤ and NF-B has been demonstrated in numerous cases (41)(42)(43). C/EBP␤ has also been shown to physically interact and functionally synergize with the glucocorticoid receptor in the induction of the rat ␣1 acid glycoprotein promoter (44). Whether C/EBP members have similar interaction with STAT3 is under investigation. These examples indicate how difficult it may prove to be to assign relative importance to interacting transcription factors participating in the full CRP response.
Similarly, our findings should not be taken to indicate that other as yet unidentified transcription factors do not participate in CRP induction by IL-6. Several studies indicate that unidentified activity other than STAT3 may activate IL-6-induced transcription (45)(46)(47). Although STAT3 has been shown to bind to three CTGGGA elements in the ␥-fibrinogen gene (48), a similar CTGGGA element in A␣-fibrinogen was reported to associate with an unidentified protein that was not STAT3 (47). In another recent report, STAT3 was found to contribute to but not to be sufficient to up-regulate specific IL-6 response element-containing reporter constructs (46). It is likely that various combinations of STAT3, C/EBP, and other as yet undefined IL-6 responsive factors and elements in their unique promoter contexts determine the activation mechanisms for each IL-6 responsive acute phase gene. Our finding that CRP-APRE (2X) is not sufficient to confer IL-6 responsiveness on the 105-bp TK promoter (which contains a C/EBP site) raises the possibility that an optimal response may require elements other than STAT and C/EBP, although other explanations for these findings clearly exist.
Finally, it is premature to conclude, as some have, that IL-6-induced expression of all acute phase response genes requires STAT3 (49). Thus far, as indicated above, it is known that both STAT and C/EBP family members can mediate IL-6induced gene transcription and that other unidentified proteins may have such capabilities as well. Theoretically there may be genes whose response to IL-6 is dependent on transcription factors other than STAT3. This possibility is supported by our continuing studies of Hep3B cells stably transfected with STAT3 in which CRP demonstrated an enhanced response to IL-6 ϩ IL-1␤ (Fig. 3). Preliminary studies in these cells suggest that serum amyloid A does not display such a response, implying that STAT3 does not play a substantial role in the serum amyloid A response to these cytokines.
It should also be noted that the minimal elements required for inducible expression of the CRP gene in hepatoma cells are not sufficient to control expression of the human CRP gene in FIG. 7. Binding of STAT3 to CRP-APRE in response to IL-6 exposure for varying times. Hep3B cells were either untreated or treated with IL-6 (100 units/ml) for the indicated length of time. Nuclear extract (5 g of protein) was incubated with 32 P-labeled CRP-APRE oligonucleotide and analyzed by EMSA. In the competition experiments, 50ϫ molar excess of cold oligonucleotides were included in the binding reaction. 1 g of C-20 anti-STAT3 antibody (Santa Cruz) was used for supershifts.

FIG. 8. Transactivation of pAPRE-TK(؊77)-CAT by STAT3.
pAPRE-TK(Ϫ77)-CAT contains two copies of the CRP-APRE inserted upstream of a 77-bp fragment of the herpes simplex virus thymidine kinase promoter driving the CAT gene (see "Experimental Procedures"). The orientation of the two CRP-APREs was the same as in the CRP promoter. pAPRE-TK(Ϫ77)-CAT (15 g) or the vector plasmid pTK(Ϫ77)-CAT (15 g) was transiently co-transfected by electroporation into Hep3B cells with STAT3 expression plasmid Rc/CMV-STAT3 (2 g) plus pRSV-␤GAL (5 g). 2 g of pRc/CMV vector DNA were used when Rc/CMV-STAT3 was absent. Cells were untreated or treated with IL-6 (100 units/ml) for 24 h. CAT activity was determined and normalized to ␤-galactosidase activity. Two independent experiments with duplicate samples were performed, and similar patterns of expression were obtained. CAT activity of the means of duplicate samples in one of these experiments is plotted. transgenic mice following LPS treatment (24). The reasons for this discrepancy are unclear. One possibility is that the cytokine milieu induced in vivo by LPS may be more complex than the defined medium and defined cytokines used in our cell culture experiments. In addition, many genes are dependent for expression on the presence of distant regulatory elements (both positive and negative), which may be thousands of base pairs away. It should therefore not be surprising that findings of gene regulation employing relatively short DNA sequences cannot be replicated in vivo, where many more regulatory elements, both positive and negative, come into play.