MOLECULAR AND CELLULAR BIOLOGY,0270-7306/01/$04.0010 DOI: 10.1128/MCB.21.5.1621–1632.2001

Mar. 2001, p. 1621–1632 Vol. 21, No. 5

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Essential Role of STAT3 in the Control of the Acute-PhaseResponse as Revealed by Inducible Gene Activation in

the LiverTONINO ALONZI,1† DIEGO MARITANO,1 BARBARA GORGONI,1 GABRIELLA RIZZUTO,2

CLAUDE LIBERT,2‡ AND VALERIA POLI1*

School of Life Sciences, Wellcome Trust Biocenter, University of Dundee, Dundee DD1 5EH, Scotland,1 andIstituto Ricerche di Biologia Molecolare Angeletti, Pomezia, Italy2

Received 17 July 2000/Returned for modification 3 October 2000/Accepted 8 December 2000

We generated mice carrying a STAT3 allele amenable to Cre-mediated deletion and intercrossed them withMx-Cre transgenic mice, in which the expression of Cre recombinase can be induced by type I interferon.Interferon-induced deletion of STAT3 occurred very efficiently (more than 90%) in the liver and slightly lessefficiently (about 70%) in the bone marrow. Analysis of the induction of liver acute-phase genes in response tobacterial lipopolysaccharide unequivocally identifies STAT3 as a fundamental mediator of their induction. Thedifferent degrees of defectiveness displayed by the various genes allowed us to differentiate them into threeseparate groups according to their degree of dependence on STAT3. Induction was totally defective for groupI genes, defective at 24 h but almost normal at earlier time points for group II genes, and only slightly defectivefor group III genes. This division was in good agreement with the known structures of the respective promoters.We also found that the overall induction of the transcription factors C/EBPb and -d was only minimallydefective in the absence of STAT3. Finally, even though corticosterone levels and action were found to benormal in the conditional-mutant mice, production of both proinflammatory and antiinflammatory cytokineswas increased and prolonged, probably as a result of STAT3 deletion in macrophages.

The acute-phase (AP) proteins are liver plasma proteinswhose levels of expression are either positively or negativelyregulated by cytokines during inflammation, chiefly throughthe regulation of the activities of their cognate genes (13).Interleukin 1 (IL-1) and IL-6 are the main inflammatory me-diators involved in this transcriptional induction (27), actingsynergistically to activate a subset of AP genes known as classI. In contrast, class II genes are solely responsive to IL-6-typecytokines. In addition, corticosteroid hormones are induced byinflammatory cytokines and participate in the induction ofmost AP genes, being required for their optimal induction andin turn exerting an inhibitory effect on cytokine production,thus activating a negative-feedback loop (3, 4, 35). Two mainkinds of cytokine-responsive elements have been characterizedon the promoters of AP genes (recently reviewed in reference36). Type I IL-6-responsive elements (IL-6REs) are bindingsites for CAAT/enhancer binding protein (C/EBP) transcrip-tion factors, which can mediate transcriptional induction byboth IL-6 and IL-1, and have been identified on the promotersof most class I genes, such as those for haptoglobin (HP) (25),a1-acid glycoprotein (AGP) (38, 47), hemopexin (Hpx) (37),complement component 3 (C3) (24), C-reactive protein (CRP)(34), and serum amyloids A (SAA) (21) and P (SAP) (34). Onthe other hand, class II genes, such as a-2 macroglobulin

(a2M) (45) and fibrinogens (FBs) (10, 32, 50), appear to bemainly regulated by type II IL-6REs, which are binding sitesfor members of the signal transducers and activators of tran-scription (STAT) family of transcription factors and particu-larly for STAT3-APRF (45). Type II IL-6RE–STAT3 siteshave also been identified on class I gene promoters.

IL-6-type cytokines, which share the receptor signaling sub-unit gp130, are known to elicit the activation of two majorsignaling pathways through the activation of kinases belongingto the JAK family: tyrosine phosphorylation and activation ofSTAT factors, mainly STAT3 (19, 41), and activation of themitogen-activated protein kinase (MAPK) pathway throughrecruitment of the SH2-containing protein tyrosine phospha-tase 2 (SHP-2) as a molecular adapter (14, 26). Both pathwayslead to the activation of transcription factors involved in theregulation of AP genes: STAT3-APRF is activated directly byJAK family kinases, while C/EBPb and -d, the two C/EBPfamily members that are induced during inflammation, areactivated through the MAPK pathway (see reference 36 andreferences therein). However, on the basis of a number of invitro data, STAT3 has been proposed to be the main mediatorof AP gene induction downstream of IL-6 and other gp130cytokines (29, 30), and indeed, in IL-6-deficient turpentine-treated mice, failure to activate the AP genes correlated withdefective STAT3 activation (2, 12). On the other hand, theanalysis of C/EBPb-deficient mice has failed to reveal anydramatic defect in the activation of AP genes, although a finalconclusion on the overall role of C/EBPs in the regulation ofAP promoters cannot be drawn from these data, since C/EBPdmight well be able to compensate for the absence of C/EBPb(8).

STAT3 can be activated by many cytokines and growth fac-

* Corresponding author. Mailing address: School of Life Sciences,Wellcome Trust Biocenter, University of Dundee, Dow St., DundeeDD1 5EH, Scotland. Phone: 44-1382-345787. Fax: 44-1382-345893.E-mail: v.poli@dundee.ac.uk.

† Present address: Laboratory of Gene Expression, I. R. C. C. S.“L. Spallanzani,” Rome, Italy.

‡ Present address: Department of Molecular Biology, University ofGhent, 9000 Ghent, Belgium.

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tors in addition to gp130 cytokines (see reference 1 for a recentreview), and indeed, STAT3 inactivation by gene targetingleads to early embryonic lethality (44). Cre-mediated inactiva-tion of STAT3 in different cell types has been recently de-scribed, demonstrating important roles for this factor in medi-ating functions of IL-6, IL-2, epidermal growth factor (EGF),and prolactin (9, 40, 42, 43). We describe here the independentgeneration of conditional STAT3 mutant mice in which inac-tivation of the gene can be induced in several tissues by treat-ment with synthetic double-stranded RNA [poly(I z C)], whichtriggers the expression of an interferon (IFN)-inducible Crerecombinase (28). Although STAT3 deletion obtained throughrepeated poly(I z C) treatments eventually triggers the devel-opment of a fulminant form of ulcerative colitis (T. Alonzi etal., unpublished data), short-term treatment causes preferen-tial deletion in the liver and macrophages. We made use of thismodel to directly assess in vivo the role of STAT3 in regulatingtranscription of AP genes in response to treatment with re-combinant IL-6 or bacterial lipopolysaccharide (LPS). Wedemonstrate that STAT3 is indeed essential for the inductionof all tested genes downstream of IL-6. On the other hand,STAT3 also plays an important role in the activation of mostgenes in response to LPS, which triggers the production of amuch more complex repertoire of inflammatory cytokines, in-cluding IL-6, IL-1, and tumor necrosis factor alpha (TNF-a).In this case, however, its relative functional relevance varied inagreement with the structures of the different promoters.Moreover, we show that STAT3 is only marginally involved inthe transcriptional induction of C/EBPb and -d and that cor-ticosterone (CS) production is normal in STAT3 conditional-mutant mice.

MATERIALS AND METHODS

Generation of the targeting vector and of the targeted ES cells and mice. Agenomic library from the 129/SV mouse strain (Stratagene Cloning Systems, LaJolla, Calif.) was screened with a cDNA clone for the mouse STAT3, and severaloverlapping positive clones were identified. A SalI-StuI fragment of approxi-mately 10 kb, containing exons 6 to 14, was subcloned into a Bluescript plasmid.An 88-bp fragment containing a single loxP site, engineered to contain an EcoRVrestriction site, was obtained by PCR from the plasmid pGEM-30 (18) andcloned into the EcoRI site in intron 14. A SalI-XbaI fragment containing apMC1/Neo expression cassette flanked by loxP sites from the plasmid pL2-Neo(17) was inserted into the EcoRI site of intron 11. Finally, a pMC1 herpessimplex virus thymidine kinase cassette (33) was inserted downstream of the 39homology region to generate the targeting vector. This was linearized with SalIand electroporated into E14 embryonic stem (ES) cells (20) according to stan-dard protocols. Clones resistant to both G418 and ganciclovir were screened forhomologous recombination by Southern blotting of EcoRV-digested genomicDNA and probed with a cDNA fragment containing exons 15 to 24, not includedin the region of homology. The predicted fragment sizes were as follows: wild-type, allele, 15 kb; replaced allele, 11 kb. By using a 59 probe (not shown), correctrecombination was confirmed to have also occurred at the 59 side. Three targetedclones were transiently transfected with pMC1-Cre (18) to delete the Neo cas-sette. Clones which had became sensitive to G418 were further analyzed bySouthern blotting upon digestion with EcoRV and hybridization with a 2.1-kbEcoRV/EcoRI fragment from intron 11 (upstream of the Neo insertion) as aprobe. The predicted sizes for the different alleles were as follows: wild type, 11kb; replaced, 5.2 kb; floxed, 4.1 kb; deleted, 2.2 kb. Four STAT3 clones carryingthe floxed allele, derived from two distinct replaced clones, were microinjectedinto CB6F1 (C57BL/6 3 BALB/c) blastocysts to generate chimeric mice. Germline transmission was obtained from all of them. STAT3fl/1 mice derived fromtwo distinct clones were then intercrossed to obtain mice homozygous for thefloxed mutation (STAT3fl/fl). After verifying that both STAT3fl/fl lines were viableand fertile, the one derived from clone 138 was chosen for further studies.

Animals and treatments. STAT3fl/fl mice generated as described above werecrossed with MX-Cre mice (28). MX1 (i.e., deletable) and MX2 (i.e., nondelet-able littermate controls) STAT3fl/fl mice for the experiments were generated bymating MX1 STAT3fl/fl males with MX2 STAT3fl/fl females. Genetic screeningfor the Cre transgene was performed by PCR using the following oligonucleo-tides: CRE1, 59-AGGCGTTTTCTGAGCATACC-39; CRE10, 59-TAGCTGGCTGGTGGCAGATG-39.

The mice were bred and maintained under specific-pathogen-free conditionsin our facility under a 12-h light-dark cycle and provided irradiated food andautoclaved water ad libitum. Procedures involving animals and their care wereconducted in conformity with national (Home Office) and international laws andpolicies and were approved by the Faculty Ethical Committee. Eight- to 10-week-old mice were used for the experiments. MX1 and MX2 STAT3fl/fl mice wereinjected intraperitoneally (i.p.) once with 250 mg of poly(I z C) 4 days before LPStreatment. LPS (Escherichia coli serotype O26:B6; Sigma Chemical Co., St.Louis, Mo.) was resuspended in sterile pyrogen-free saline solution and injectedi.p. at a dose of 1 mg/kg of body weight. Human recombinant IL-6 was injectedat a dose of 10 mg/mouse as described previously (2), and dexamethasone (Dec-adron Shock Pack; MSD, Brussels, Belgium) (750 mg/mouse) was injected i.p. 30min prior to LPS treatment. The mice were sacrificed by CO2 asphyxiation, bloodwas collected by cardiac puncture, and the livers were immediately removed.

Total liver protein extraction and Western blot analysis. The frozen liverswere lysed by homogenization in a buffer containing 50 mM Tris (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% sodium dodecylsulfate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM NaF, 1 mM sodiumvanadate, and a 40-mg/ml protease inhibitor cocktail (Sigma) and cleared bycentrifugation. The protein concentration was determined by Bradford assay(Bio-Rad Laboratories, Hercules, Calif.). Proteins were fractionated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellu-lose. The antibodies used were as follows: anti-STAT3 monoclonal antibodydirected against the amino-terminal part of the protein and anti-STAT1 poly-clonal serum (Signal Transduction Laboratories, San Diego, Calif.) and anti-phospho-STAT3 (Tyr 705), anti-phospho-p44/42 MAPK, and anti-p44/42 MAPK(New England Biolabs, Beverly, Mass.).

Liver nuclear extracts and electrophoretic mobility shift assays (EMSAs).Nuclear extracts were prepared after LPS or saline treatment from freshly re-moved livers as described previously (16) with modifications. Briefly, 1 g of liverwas homogenized in 2.5 ml of homogenization buffer (10 mM HEPES [pH 7.6],15 mM KCl, 2 mM EDTA, 2 M sucrose, 10% glycerol, 0.5 mM spermidine, 0.15mM spermine, 0.5 mM dithiothreitol, 0.5 mM PMSF, and 1% aprotinin) using aglass-Teflon Dounce homogenizer. The nuclei were pelleted by ultracentrifuga-tion over a cushion of the same buffer and directly lysed in 10 mM HEPES (pH7.9), 400 mM NaCl, 0.1 mM EGTA, 5% glycerol, 0.5 mM dithiothreitol, 0.5 mMPMSF, and 1% aprotinin at 4°C for 30 min. The lysates were cleared by centrif-ugation and frozen in liquid nitrogen. The protein concentration was determinedby Bradford assay.

EMSAs were carried out by incubating 6 mg of each extract in a 20-ml finalvolume of a solution of 20 mM HEPES (pH 7.9), 50 mM NaCl containing 3 mgof poly(dI-dC), and 2 mg of salmon sperm DNA for 10 min on ice. 32P-labeleddouble-stranded oligonucleotides (2 3 104 cpm) were added, and the mixturewas incubated for 15 min at room temperature. DNA-protein complexes wereseparated by electrophoresis on a 6% polyacrylamide gel in 0.253 TBE buffer(25 mM Tris, 25 mM boric acid, 0.6 mM EDTA) and visualized by autoradiog-raphy. Antibodies for supershift experiments were purchased from Santa CruzBiotechnology (Santa Cruz, Calif.) and added to the preincubation mixture for30 min on ice prior to addition of the probe.

The double-stranded oligonucleotides were labeled by filling in with Klenowpolymerase. The sequences of the upper strands were as follows: NF-kB bindingsite, 59-GATCCGCTGGGGACTTTCCAGGCG-39; STAT site, 59-GATCGATTTCCCCGAAAT-39; C/EBP site, 59-GGGCATAGTGGCGCAAACTCCCTTACTG-39.

Slot blot analysis. Total RNA was prepared from frozen livers using a Qiagenkit according to the manufacturer’s instructions. Five or 20 mg, respectively, oftotal RNA was analyzed by slot blotting or by Northern blotting as previouslydescribed (12). The different cDNA probes used have been previously described(8, 12) and were labeled by random priming. The relative abundances of thedifferent mRNAs were measured by phosphorimager analysis and normalized toGAPDH (glyceraldehyde-3-phosphate dehydrogenase).

Cytokine and CS measurements. Cytokines were measured by enzyme-linkedimmunosorbent assay (ELISA) using kits purchased from PharMingen (SanDiego, Calif.) according to the manufacturer’s instructions. IL-1b levels wereassayed by a two-sided sandwich ELISA using antibody pairs purchased fromR&D Systems (Minneapolis, Minn.). CS was measured by radioimmunoassay

1622 ALONZI ET AL. MOL. CELL. BIOL.

using a kit from ICN (Costa Mesa, Calif.) according to the manufacturer’sinstructions.

Statistical analysis. Results were analyzed by the analysis of variables testusing the Statview computer program (Abacus Concept). A P value of ,0.05 wasconsidered statistically significant.

RESULTS

Generation of a mouse line amenable to Cre-mediated con-ditional inactivation of STAT3. A targeting vector was con-structed in which the region corresponding to exons 12 to 14(encoding the putative DNA binding domain) was flanked withtwo loxP sites, while a loxP-Neo cassette was inserted intointron 11 (Fig. 1A). Homologous recombinant E14 embryonicstem cell clones were identified by Southern blot analysis (Fig.

1B and C) and subjected to transient transfection with a Cre-encoding plasmid. Colonies which had lost resistance to G418were amplified and analyzed by Southern blotting, thus iden-tifying those clones which had the Neor cassette but not theexonic region deleted (Fig. 1D). The resulting STAT3 floxedallele (STAT3fl) was expected to be functional but amenable toinactivation by Cre-mediated recombination through removalof the region corresponding to exons 12 to 14 and generationof a STAT3 “deleted” (D) allele. Transcription from thisSTAT3D allele generates a shorter, frameshifted mRNA thatshould be unable to encode a functional protein. Embryonicstem cells carrying a floxed allele were injected into recipientblastocysts, and the resulting chimeras were crossed toBALBC/A mice to generate mice carrying the STAT3fl allele in

FIG. 1. Generation of STAT3fl/1 ES cells and of MX1 or MX2 STAT3fl/fl mice. (A) The structure of the replacement targeting vector alongwith the structures of the wild-type, replaced, floxed, and deleted alleles, the position of the probe used, and the predicted sizes of the restrictionfragments are depicted. The replaced allele was detected by Southern blotting on EcoRV-digested genomic DNA using as a probe a cDNAfragment spanning exons 15 to 24. Cre-mediated deletion was expected to generate STAT3 floxed and deleted alleles and was diagnosed bySouthern blotting using the intronic probe 2.1. (B, C, and D) Southern blot analysis of genomic DNA from targeted ES cells either before (B andC) or after (D) Cre-mediated deletion. The probes used and the relevant alleles detected are indicated (wt, wild type; repl, replaced; fl, floxed;del, deleted). (E and F) Western blot of liver extracts from poly(I z C)-treated mice. MX1 or MX2 STAT3fl/fl mice were injected once i.p. withpoly(I z C) followed 4 days later by either LPS or apyrogenic saline solution. The livers from the indicated mice were collected after 4.5 and 9 h(F) or after 24 h (E), and total extracts were subjected to Western blot analysis with anti-STAT3 (E) or anti-phospho-(Tyr 705) STAT3 (P-STAT3)(F) antibodies. The blots were stripped and reprobed with anti-STAT1 (E) or anti-STAT3 (F) antibodies. C, STAT3-enriched control extract.

VOL. 21, 2001 STAT3 IS REQUIRED TO INDUCE MOST ACUTE-PHASE GENES 1623

their germ line. STAT3fl/fl mice were obtained from heterozy-gous matings at a Mendelian ratio and were phenotypicallyindistinguishable from wild-type or heterozygous littermates,indicating that the STAT3fl allele is functional.

Inducible STAT3 inactivation. In order to generate animalsin which the STAT3 gene could be inactivated in an inducibleway, STAT3fl/fl mice were bred to Mx-Cre transgenic mice,which express the Cre recombinase under the control of theIFN-responsive Mx-1 promoter (28), thus generating MX1

(i.e., where the STAT3 allele is deletable) or MX2 (control)STAT3fl/fl mice. Cre expression was induced in adult mice bya single injection of a synthetic double-stranded RNA[poly(I z C)], which is known to induce a strong and transientproduction of type I IFN. The efficiency of Cre-mediated de-letion in different tissues was evaluated by Southern blot anal-ysis of genomic DNA extracted 2 days after the treatment andwas found to be almost 100% in the liver and 70 to 80% in theadipose tissue and bone marrow (not shown). Western blotanalysis of liver protein extracts 4 (not shown) and 5 (Fig. 1E)days after poly(I z C) treatment confirmed that liver STAT3protein levels were greatly reduced, indicating that the deletionallele is unable to encode a detectable protein. Therefore,MX1 STAT3fl/fl mice that have been treated with poly(I z C)as described above will hereafter be referred to as STAT3conditional-mutant mice. STAT3 levels were slightly in-duced by LPS treatment in both kinds of mice (Fig. 1E and F).In addition, STAT3 activation as detected in liver nuclearextracts by specific anti-phosphotyrosine STAT3 antibodieswas strongly induced by LPS in the MX2 control mice at both4.5 and 9 after LPS treatment (Fig. 1F). In contrast, STAT3phosphorylation was barely detectable at 4.5 h and not at all by9 h in the STAT3 conditional-mutant mice (Fig. 1F), thusconfirming that very low levels of active STAT3 were presenteven under the inflamed conditions.

Analysis of the AP response in the STAT3 conditional-mu-tant mice. In order to compare the induction of AP mRNAs inthe presence and absence of STAT3 in the liver, MX1 andMX2 STAT3fl/fl mice were treated once with poly(I z C) totrigger STAT3 inactivation and injected with LPS after 4 days.Liver and blood samples were then collected at different times,and total RNA from the liver was subjected to slot blot analysiswith cDNA probes directed to different AP mRNAs. The re-sults were quantified and normalized to GAPDH as an internalcontrol, and the normalized arbitrary levels are plotted in Fig.2. It is worth noticing that poly(I z C) treatment did not in-crease the basal levels of the different AP mRNAs tested,which were equivalent in the saline-treated mice injected andnot injected with poly(I z C) (not shown). All mRNAs testedwere increased by severalfold in the control mice, althoughwith slightly different time courses. Induction of all tested APmRNAs was affected in the STAT3 conditional-mutant mice,and the respective genes could be divided into three groups.Group I comprised genes whose activation was always totallydefective in the absence of STAT3, including those for SAP,one of the major AP reactant in the mouse, and FBa and -g.Group II comprised genes whose activation was comparable tothat of the controls at early time points but declined muchfaster and was not detectable at 24 h, including those for HP,AGP, and FBb. Finally, group III comprised genes whoseactivation was only slightly defective and whose mRNAs were

induced at levels comparable to those of the controls 6 or 12 hafter LPS treatment and remained only slightly defective at24 h. The SAA, Hpx, and C3 genes belonged to this category.The SAA probe used in this experiment recognized all threemajor forms of SAA, which in the mouse are encoded by threehomologous genes, SAA1, -2, and -3. To study the specificcontributions of the different SAA forms to the total SAARNA level, probes preferentially recognizing each of the threeisoforms were used. As shown at the bottom of Fig. 2, SAA3induction was the least affected by the absence of STAT3,being normal at 6 and 12 h and only reduced by about 50% at24 h. In contrast, SAA1 induction was almost totally defective,and the SAA2 mRNA showed an intermediate behavior, beinginduced at 6 h but failing to increase any further.

Although the data reported above clearly show a critical rolefor STAT3 in the induction of most AP mRNAs, LPS leads tosystemic production of several inflammatory cytokines, andSTAT3 in our model is not inactivated exclusively in the liver.The possibility therefore exists that some of the effects we areassessing are due to indirect modes of action caused by alter-ations of the systemic response rather than by a direct role ofSTAT3 in gene transcription in the liver. In order to ensurethat this is not the case and to provide a direct test of thetranscriptional role of STAT3 downstream of gp130 cytokines,we have analyzed the induction of AP mRNAs upon treatmentwith recombinant IL-6. Induction of all tested mRNAs wasdramatically impaired in the conditional-mutant mice (Fig. 3),thus providing a striking confirmation of the crucial role ofSTAT3 in mediating IL-6 induction of AP genes. Interestingly,although most mRNAs were totally unresponsive to IL-6 in theabsence of STAT3, some (for HP, AGP, and Hpx) did show aslight activation which followed the same trend as in the LPStreatments, i.e., it occurred early but not late. Moreover, underthese experimental conditions, C3 did not appear to be in-duced by IL-6 in either kind of mouse.

Defective AP mRNA induction is not due to lack of C/EBPbor -d activation. Transcription factors belonging to the C/EBPfamily, and in particular C/EBPb and -d, are believed to playan important role in mediating the cytokine inducibility ofmost AP genes (36). In addition, transcription of both factorsis activated during inflammation and in response to IL-6 bothin vivo and in vitro, and this activation has been proposed, atleast for C/EBPd, to be dependent on STAT3 (7). We there-fore analyzed the induction of both C/EBPb and -d mRNAs inthe livers of the STAT3 conditional-mutant and control miceupon treatment with either LPS or recombinant IL-6. Asshown in Fig. 4A and B, the overall inductions of both geneswere similar in the conditional-mutant and control mice uponboth LPS and IL-6 treatment. This suggests that STAT3 is notstrictly required for transcriptional induction of C/EBPb or -din the liver during inflammation and that the defective expres-sion of AP mRNAs in the conditional-mutant mice is directlydue to lack of STAT3 function and is not mediated by defectiveC/EBP induction. Interestingly, however, induction of C/EBPband C/EBPd by LPS was slightly but significantly blunted at 1.5and 6 h after LPS treatment but then increased and was stillapparent at 24 h, when it had almost returned to basal levels inthe control mice. In contrast, the inductions of both mRNAswere completely comparable in response to recombinant IL-6.These observations suggest that STAT3 may indeed play a role

1624 ALONZI ET AL. MOL. CELL. BIOL.

in mediating full induction of both genes at early time pointsbut apparently not in response to IL-6. It is worth noting thatthe mRNA levels of C/EBPa, a family member thought not tobe involved in the regulation of AP genes and whose transcrip-tion is actually reduced by LPS and IL-6, were equivalent inuntreated STAT3 conditional-mutant and control mice anddecreased to similar extents upon LPS or IL-6 treatment (datanot shown).

In order to verify that the levels of C/EBPb and -d proteinsand their DNA binding capacities reflected the abundance ofthe respective mRNAs, liver nuclear extracts were analyzed byWestern blotting. As shown in Fig. 4C, the levels of bothC/EBPb and -d were strongly increased 4.5 h after LPS treat-ment and slightly decreased by 9 h, and the overall abundances

of the two proteins were comparable in the conditional-mutantand control mice. C/EBPb occurs in three different forms, thefull-length form, a slightly shorter form called LAP, and atruncated form termed LIP, which lacks the activation domainand is thought to act as a dominant negative (11). Interestingly,while the intermediate form, LAP, was already abundant in theliver extracts of untreated mice and was slightly increased onlyat 4.5 h, the other two forms were strongly induced at 4.5 h andstill well above basal levels at 9 h. Finally, we have analyzed byEMSA the C/EBP DNA binding activities present in the livernuclear extracts from mice either untreated or treated withLPS for 4.5 h, when the levels of both the C/EBPb and -dproteins were highest. Using a double-stranded oligonucleo-tide carrying a C/EBP binding site, the DNA-protein com-

FIG. 2. LPS induction of AP mRNAs as measured by slot blot analysis on STAT3 conditional-mutant (open bars) and control (solid bars) mice.The mice were injected with either LPS or saline solution 4 days after poly(I z C) treatment, and their livers were collected after 0, 1.5, 6, 12, and24 h, as indicated. Total RNA was extracted, denatured, transferred to nylon membranes by slot blotting, and hybridized with the indicated cDNAprobes. The results, shown as mean values 1 standard errors of the mean of at least five mice per group, were plotted after normalization withGAPDH as an internal control. All values obtained after saline injection were uniform and were therefore pooled and shown collectively as timezero, representing the steady-state value.

VOL. 21, 2001 STAT3 IS REQUIRED TO INDUCE MOST ACUTE-PHASE GENES 1625

plexes formed with the extracts from untreated mice wereequivalent in the conditional-mutant and control mice (Fig.4D). In both cases, most complexes were supershifted by an-tibodies against either C/EBPa or C/EBPb, suggesting thatunder untreated conditions, C/EBPa and -b homo- and het-erodimers are responsible for the vast majority of the detectedC/EBP DNA binding activities. Upon LPS treatment, the mo-bilities of the complexes were greatly increased, and noC/EBPa binding activity was detected anymore by supershiftanalysis. In contrast, all complexes detected appeared to con-tain C/EBPb, since everything was supershifted by anti-C/EBPb antibodies. Again, no difference was detected betweenextracts from STAT3 conditional-mutant mice and controls.Surprisingly, we were unable to detect any C/EBPd DNA bind-ing activity by supershift analysis despite having attempted itwith two different sources of anti-C/EBPd antibodies expectedto possess supershifting activity, including those that readilydetected the protein by Western blotting. Perhaps the propor-

tion of C/EBPb isoforms present in the extracts is too high forC/EBPd to detectably bind, at least in vitro, under these con-ditions.

Hyperproduction of cytokines and susceptibility to endo-toxic shock in the STAT3 conditional-mutant mice. As alreadymentioned, STAT3 inactivation induced by poly(I z C) was notlimited to the liver but occurred at good efficiency in othercompartments as well, and most notably in the bone marrow(data not shown) and in bone marrow-derived macrophages(Alonzi et al., unpublished data). Since macrophages are themajor cytokine-producing inflammatory cells, it was importantto assess their functionality by measuring the levels of circu-lating inflammatory cytokines produced upon LPS injection inthe STAT3 conditional-mutant mice compared to those in thecontrols. Interestingly, the induction of all proinflammatorycytokines tested was found to be stronger and more sustainedin the STAT3 conditional mutants (Fig. 5A). IL-6 was stillincreasing between 6 and 12 h after injection, at a time when it

FIG. 3. IL-6 induction of AP mRNAs as measured by slot blot analysis on STAT3 conditional-mutant (open bars) and control (solid bars) mice.The mice (three per group) were injected with either recombinant IL-6 or saline solution 4 days after poly(I z C) treatment, and their livers werecollected after 0, 1.5, 6, and 24 h, as indicated. The methods and symbols are described in the legend to Fig. 2.

1626 ALONZI ET AL. MOL. CELL. BIOL.

was reduced to basal levels in the control mice. IL-1b followeda very similar pattern of induction, although its levels werebelow the detection limits of our assay in the control mice.Similarly, TNF-a was still increasing at 6 h, while it had be-come undetectable after 3 h in the control mice. Taken to-gether, these data indicate that the defective activation of APgenes detected in the conditional-mutant mice in response toLPS is most definitely not due to impaired cytokine produc-tion, and if anything they emphasize the fundamental role ofSTAT3, which is absolutely required for the induction of mostAP genes even in the presence of abnormally high levels ofcytokines.

Despite the low dosage used, which was well tolerated by allcontrol mice, most conditional-mutant mice were found to bealmost moribund 24 h after LPS injection, most likely due tothe endotoxic shock caused by TNF-a hyperproduction. Ourresults are in agreement with those reported by S. Akira andcolleagues in mice in which STAT3 had been inactivated spe-cifically in macrophages and granulocytes (42) and can beexplained by their observation that STAT3 is required forresponsiveness to IL-10, a cytokine known for its potent deac-tivating effects on macrophages. IL-10, is also known to exertan inhibitory effect on its own synthesis, and apparently thisaction is also dependent on STAT3, since IL-10 levels in theconditional-mutant mice were abnormally high 12 h after LPStreatment (Fig. 5A).

CS production and action are normal in the STAT3 condi-tional-mutant mice. The activation of the hypothalamic-pitu-itary-adrenal axis by inflammatory cytokines and the conse-quent production of glucocorticoid hormones play animportant dual role in the inflammatory response by partici-pating as coactivators in the transcriptional induction of sev-eral AP genes (3) while at the same time providing negativefeedback for cytokine production (4, 35). STAT3 deletion oc-curring in tissues other than the liver may therefore interfereeither with cytokine-induced CS production or with the actionof CS itself on target cells. We have therefore measured CSlevels in the serum of mice treated with LPS for differentlengths of time and found no differences between the STAT3conditional mutant and the control mice (Fig. 5B), suggestingthat the activation of the hypothalamic-pituitary-adrenal axis isnot compromised. Moreover, administration of dexametha-sone 30 min prior to LPS injection was able to significantlyreduce IL-6 and TNF-a levels in the blood of both the controland the conditional-mutant mice (Fig. 5C), thus demonstratingthat glucocorticoid hormone action is not impaired by STAT3inactivation.

Increased NF-kB and STAT1 activation in the livers of theSTAT3 conditional-mutant mice. As shown in Fig. 4, the over-all induction of both C/EBPb and -d in the conditional mutant

FIG. 4. Induction of C/EBPb and -d as measured by Northern blot,Western blot, and EMSA analyses. (A and B) STAT3 conditional-mutant (open bars) and control (solid bars) mice were treated for theindicated times as described in the legend to Fig. 2 with either LPS (A)or recombinant IL-6 (B) and analyzed by Northern blotting. The dataare shown as mean values 1 standard errors of five (A) orthree (B) mice per group. The values were normalized and plotted asdescribed in the legend to Fig. 2. The symbols indicate statisticallysignificant differences between the two groups of mice at each time

point: #, P , 0.03; p, P , 0.01; ‡, P , 0.0001. (C) Western blot analysisof C/EBPb and C/EBPd. Mice were treated with LPS as described abovefor 4.5 or 9 h, and nuclear extracts were analyzed by Western blotting withspecific antisera. The different polypeptides detected are indicated. 2,negative; 1, positive. (D) EMSA analysis of C/EBP binding activities.Nuclear extracts from mice either untreated or treated with LPS for 4.5 hwere used in an EMSA with a double-stranded oligonucleotide carrying aC/EBP binding site. Where indicated, antisera against different C/EBPproteins were preincubated with the extracts.

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mice was comparable to that in the control mice, and accord-ingly, C/EBP DNA binding activities in liver nuclei before andafter LPS injection were also equivalent. Other transcriptionfactors that are activated by LPS and that may play a role in theinduction of AP genes are NF-kB and STAT1. NF-kB is themain target of TNF-a and IL-1, while STAT1 is activated alongwith STAT3 by IFN-g and gp130 cytokines. LPS injectiontriggered the activation of NF-kB DNA binding activity, whichwas abundant after 4.5 h and slightly decreased after 9 h in thelivers of the control mice (Fig. 6A, top). The same complexes,only much more abundant, were also formed by using nuclearextracts from the STAT3 conditional-mutant mice, suggestinga stronger and more sustained NF-kB activation.

Three different DNA-protein complexes are formed usingliver nuclear extracts from LPS-treated mice with a STATbinding site as a probe and are known to correspond to STAT3homodimers, STAT3-STAT1 heterodimers, and STAT1 ho-modimers. All three complexes were detected when liver nu-clear extracts from the LPS-treated control mice were used(Fig. 6A, bottom). No STAT3 DNA binding activity was de-tected in the extracts from the conditional-mutant mice, thusconfirming that the low levels of tyrosine-phosphorylatedSTAT3 present in the livers of these mice (Fig. 1F) were notsufficent to allow detectable binding. However, the complexcorresponding to STAT1 homodimers was considerably moreintense at both time points analyzed.

The inactivation of STAT docking sites on gp130 has beenproposed to prolong MAPK activation (30), and this might inturn partly explain the slightly prolonged induction observedfor both the C/EBPb and -d mRNAs. Indeed, phosphorylationof ERK1 and ERK2 was more elevated in the absence ofSTAT3, although by 15 h after LPS treatment the activationlevels were equivalent to those found in the control mice, andby 24 h phosphorylation levels were back to their basal valuesin both cases (Fig. 6B).

DISCUSSION

Our previous observation that in IL-6-deficient mice im-paired and normal induction of AP genes in response to dif-ferent stimuli correlated with defective and normal activationof STAT3, respectively (2, 12), pointed towards a fundamentalrole for STAT3 in the induction of AP genes in vivo. Indeed,we found that deletion of STAT3 in the livers of adult micevirtually abolished the ability of IL-6 to induce all AP mRNAsmeasured. More importantly still, AP gene induction was alsodefective upon LPS injection, thus largely confirming the piv-otal role of STAT3 even under conditions that mimic a sys-temically induced inflammatory reaction and lead to the pro-duction of a broad variety of cytokines.

Promoter structure correlates with functional predomi-nance of STAT3 for induction of AP genes. A functional anal-

FIG. 5. (A and B) Serum cytokine and CS levels measured in LPS-treated STAT3 conditional-mutant (open bars) and control (solid bars) mice.The mice were treated as described in the legend to Fig. 2. Blood was collected at the indicated time points, and the cytokine content of the serumwas measured by ELISA (A) or the CS content was measured by radioimmunoassay (B). The data are shown as mean values 1 standard errorsof the mean of at least five (A) or three (B) mice per group. The symbols indicate statistically significant differences between the two groups ofmice at each time point: #, P , 0.01; p, P , 0.001. (C) Cytokine levels upon pretreatment with dexamethasone. Mice were treated with eitherdexamethasone or apyrogen saline solution 30 min prior to LPS injection, and blood was collected 1 h later and subjected to ELISA analysis. 1,present; 2, absent.

1628 ALONZI ET AL. MOL. CELL. BIOL.

ysis of the known structure of the corresponding promoters,whose schematic representation is shown in Fig. 7, indicates asurprisingly good correlation between known functional pro-moter organization and the differential role played by STAT3in their induction. The scheme focuses particularly on C/EBPand STAT sites, which are the two main regulatory elementscommon to most AP genes, but other sites characterized asfunctionally important are indicated as well. Where functionaldata were not available for the mouse promoters, either the rator the human promoter is shown.

According to this analysis, the mouse AP genes can be di-vided into three groups: genes whose promoters have beenshown to only carry STAT3 sites as cytokine-responsive ele-ments appeared to be totally dependent on STAT3 for theirinduction (group I; FBa and -g) (32, 50); genes carrying bothSTAT and C/EBP sites of equivalent functional importance ontheir promoters, whose induction was also totally defective at24 h but was almost normal at earlier time points (group II,including HP [25], AGP [38, 47], and FBb [25]); and finally,genes with no characterized STAT site on their promoters,which were only minimally affected by the absence of STAT3

(group III, including SAA3 [21] and C3 [24, 46]). The only twoexceptions to this rule involved the SAP and Hpx genes, eachof which carries one STAT and one C/EBP site (22, 34) and yetwas totally or minimally defective, respectively. In both caseshowever, one of the sites was found to be dominant over theother (34, 37), which suggests that perhaps in vivo this func-tional predominance may be more stringent. Particularly in thecase of Hpx, while the C/EBP site has been shown to bedominant in the human promoter (37), the reverse is true forthe rat promoter (23). Although no functional data are avail-able for the mouse gene, sequence comparison indicates thatthe C/EBP site on the mouse promoter is more similar to thehuman site than to the rat site, and in contrast to the rat site,it can be predicted to be a strong binding site for C/EBPproteins (Fig. 7B) (F. Altruda, personal communication). Therelative independence from STAT3 shown by the SAA3 andC3 genes is in agreement with previous data obtained withC/EBPb-deficient mice, where in contrast, Hpx induction wascompletely uncompromised (8). This could perhaps reflect adominant role for C/EBPd rather than -b in the induction ofthis gene. Some weak and therefore not-yet-characterized

FIG. 6. EMSAs and anti-MAPK detection with liver extracts from STAT3 conditional-mutant and control mice. The mice were treated asdescribed in the legend to Fig. 2. (A) Livers were collected 4.5 and 9 h after LPS injection, and nuclear extracts were prepared immediately. Totalnuclear extracts were incubated with 32P-labeled double-stranded oligonucletides carrying binding sites for NF-kB (from the human immunode-ficiency virus 39 long terminal repeat) or STAT3-STAT1 (corresponding to the sis-inducible element on the c-fos promoter). (B) To detect MAPKphosphorylation, total extracts from mice treated with LPS for the indicated lengths of time were subjected to Western blot analysis withanti-phospho-(p44/42) MAPK antibodies (P-MAPK). The blots were stripped and reprobed with anti-MAPK antibodies (MAPK). 1, positive; 2,negative.

VOL. 21, 2001 STAT3 IS REQUIRED TO INDUCE MOST ACUTE-PHASE GENES 1629

STAT element might likewise be present on the mouse SAA3and C3 promoters, since their induction is slightly but repro-ducibly impaired in the STAT3 conditional-mutant mice. How-ever, these two promoters appear to be functionally different,since SAA3 was strongly induced by recombinant IL-6 whileC3 was not.

The regulation of the SAA family revealed some unexpectedcharacteristics. C/EBP and NF-kB sites have been shown toplay a major role in the transcriptional induction of all char-acterized SAA genes (mouse SAA3 [21], rabbit SAA [39], ratSAA1 [31], and human SAA2 [5]), which therefore would beexpected not to be affected by the absence of STAT3. Asdiscussed above, this was indeed the case for SAA3, whoseinduction by LPS was only partially reduced in the STAT3conditional-mutant mice. In contrast, induction of both SAA1and SAA2 was profoundly impaired. Although the mouseSAA1 and -2 gene promoters have not been characterized,computer-assisted alignment including the human SAA1 pro-moter revealed two conserved potential STAT elements but noC/EBP or NF-kB sites (6, 48), which is in good agreement withthe substantially defective activation of these two genes in theabsence of STAT3. It is worth noting that, although SAA3induction was only partially impaired by STAT3 inactivation,IL-6-dependent induction was totally defective, suggesting thatthe portion of SAA3 induction due to IL-6 action is dependenton STAT3. On the other hand, the low but reproducible two-

to threefold induction displayed by the Hpx gene in responseto IL-6 seems to suggest that IL-6 is indeed capable of inducingcertain AP genes via a STAT3-independent pathway, mostlikely involving C/EBPb and -d activation, which was normal inthe STAT3 conditional-mutant mice.

C/EBPb and C/EBPd induction are not dependent onSTAT3. STAT3 has been proposed to play an important role inthe induction of C/EBPd by IL-6, and C/EBPb has been shownto carry a functionally active STAT binding site (7). It wastherefore conceivable that the defective AP gene induction inthe absence of STAT3 might be at least partly mediated byimpaired induction of these two C/EBP family members. Ourdata indicate that STAT3 may play a marginal role in theinduction of both C/EBPb and -d mRNAs, mostly at early timepoints, but suggest that its function is not strictly required,since overall induction was only partially blunted. Moreover,both protein levels and DNA binding activities were compara-ble in STAT3 conditional-mutant and control mice, thus con-firming that defective C/EBP activity could not be an indirectcause of defective AP gene induction. The reason why C/EBPfactors alone are not able to at least weakly activate genes suchas those for AGP, HP, and FBb while they are apparently ableto do so for Hpx, C3, and SAA3 is at present not clear. Possiblythis difference originates from the structures of the respectivepromoters. For example, efficient induction of a given AP geneby C/EBPb or -d might require cooperation with at least an-

FIG. 7. (A) Structure of the analyzed AP promoters from the corresponding mouse (m), rat (r), or human (h) genes. Open ovals, C/EBP–typeI IL-6RE sites; solid rectangles, STAT–type II IL-6RE sites; shaded rectangles, proposed STAT sites which have not been functionally charac-terized; dotted oval on the rat FBa promoter, potential C/EBP site that was found not to be functional. The position and sequence of each STATelement is shown, and previous nomenclature is also given when appropriate. The coordinates of each promoter are indicated, and elementsidentified on the promoters but not involved in cytokine responsiveness are named and indicated by arrows. Both the distal (DRE) and theProximal (PRE) regulatory regions of the rat AGP gene are shown. The glucocorticoid responsive element (GRE) contained within the PRE isrequired for optimal induction. References are given in the text. (B) Sequence comparison of the C/EBP sites on the human, mouse, and rat Hpxpromoters. Bases mutated in the mouse and rat promoters compared to the human sequence are in boldface and underlined.

1630 ALONZI ET AL. MOL. CELL. BIOL.

other type of cytokine-inducible factor. Indeed, in addition tothe C/EBP site(s), the C3 promoter carries an NF-kB-likeIL-1RE (24), and the murine SAA3 promoter carries a bindingsite for SEF-1 (SAA enhancer factor), which is also requiredfor induction (21). In contrast, only C/EBP and STAT sites areinvolved in the induction of the HP, AGP, and FBb genepromoters (10, 25, 38, 47).

Cytokine overproduction and prolonged C/EBP and MAPKactivation. The reason why induction of C/EBPb and -d isprolonged in the absence of STAT3 will require further study.One possible cause could be the prolonged and increased pro-duction of inflammatory cytokines that occurs as a likely con-sequence of the lack of responsiveness to IL-10 of STAT3-deficient macrophages (42). This could also explain thestronger and prolonged NF-kB and ERK activation detected inthe conditional-mutant mice. On the other hand, abnormallyprolonged activation of the MAPK pathway, involved in theinduction of C/EBP factors, has already been shown to occur invitro when a mutant form of gp130 unable to activate STATfactors is used (30). This has been proposed to occur throughphysical interference of STAT3, bound to its membrane-prox-imal docking site, with the association of SHP-2 to its owndocking site, which is situated in close proximity. It is temptingto speculate that, in analogy to the dual role of SHP-2 asactivator of ERKs and down-regulator of the STAT activity,STAT3 activation might in turn exert an inhibitory effect onthe MAPK pathway, the absence of which could explain theprolonged induction of the C/EBP factors.

Finally, the observation that the HP, AGP, and FBb geneswere activated in the absence of STAT3 6 h after LPS treat-ment but were totally defective after 24 h raises the question ofwhich other transcription factor(s) might be involved in theearly induction. Even though IL-6REs are exquisitively respon-sive to STAT3, some have been shown to be able to respond toSTAT1 as well, although at a lower efficiency (49). It is there-fore tempting to propose that perhaps the abnormally highSTAT1 levels present in the conditional-mutant mice, partic-ularly at earlier time points (4.5 h), might be able to partiallycompensate for the absence of STAT3 and could account forthe initial burst of AP gene activation, perhaps in conjunctionwith C/EBP factors. It has been shown that two of the fourgp130 YXXQ domains that act as docking sites for STATfactors are specific for STAT3 while two can recruit bothSTAT3 and STAT1 with similar affinities (15). It is thereforelikely that the absence of STAT3 releases competition for thedocking site and favors recruitment and activation of STAT1.

ACKNOWLEDGMENTS

We are grateful to W. Muller, U. Betz, and K. Rajewsky for pro-viding the Mx-Cre transgenic mice; to H. Baumann, S. Maeda, U.Muller-Eberhard, J. Sipe, W. Liao, K. Yamamoto, S. McKnight, andJ. E. Darnell for the gift of plasmids; to H. van der Putten for providingthe E14 ES cells; to F. Altruda for sharing unpublished sequences; toIan Newton for technical help; and to L. Malone and V. Murray-Taitfor expert mouse care. STAT3 targeted mice were generated at theIstituto Ricerche di Biologia Molecolare. Angeletti by V.P. and T.A.

This work was supported by the Wellcome Trust (Senior ResearchFellowship to V.P.). T.A. and B.G. were the recipients of EC MarieCurie fellowships.

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