Diurnal Regulation of the Early Growth Response 1 (Egr-1)Protein Expression by Hepatocyte Nuclear Factor 4� (HNF4�)and Small Heterodimer Partner (SHP) Cross-talk in LiverFibrosis*

Received for publication, April 20, 2011, and in revised form, June 30, 2011 Published, JBC Papers in Press, July 3, 2011, DOI 10.1074/jbc.M111.253039

Yuxia Zhang‡1, Jessica A. Bonzo§, Frank J. Gonzalez§, and Li Wang‡2

From the ‡Departments of Medicine and Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine,Salt Lake City, Utah 84132 and the §Laboratory of Metabolism, Center for Cancer Research, NCI, National Institutes of Health,Bethesda, Maryland 20892

Early growth response 1 (Egr-1) protein is a critical regulatorof genes contributing to liver fibrosis; however, little is knownabout the upstream transcriptional factors that control itsexpression. Here we show that Egr-1 expression is tightly regu-lated by nuclear receptor signaling. Hepatocyte nuclear factor4� (HNF4�) activated the Egr-1 promoter through three DR1response elements as identified by trans-activation assays. Dele-tion of these response elements or knockdown of HNF4� usingsiRNA largely abrogated Egr-1 promoter activation. HNF4�activity, as well as its enrichment on the Egr-1 promoter, weremarkedly repressed by small heterodimer partner (SHP) co-ex-pression. Egr-1 mRNA and protein were transiently induced byHNF4�. On the contrary, HNF4� siRNA reduced Egr-1 expres-sion at both themRNAandprotein levels, and overexpression ofSHP reversed these effects. Conversely, knockdown of SHP bysiRNA elevated Egr-1 protein. Interestingly, Egr-1 mRNAexhibited diurnal fluctuation, which was synchronized to thecyclic expression of SHP and HNF4� after cells were releasedfrom serum shock. Unexpectedly, the levels of Egr-1mRNA andprotein were highly up-regulated in Hnf4��/� mice. BothHNF4� and Egr-1 expression were dramatically increased inSHP�/� mice with bile duct ligation and in human cirrhotic liv-ers, which was inversely correlated with diminished SHPexpression. In conclusion, our study revealed control networkfor Egr-1 expression through a feedback loop between SHP andHNF4�.

Hepatic fibrosis or cirrhosis is a scarring process character-ized by both increased deposition of extracellular matrix pro-teins and reduced breakdown of extracellular matrix after liverinjury (1). The early growth response 1 (Egr-1)3 gene, a zinc

finger transcription factor, was shown to play roles in multiplepathways and processes, including proliferation, differentia-tion, and inflammation during cholestatic liver injury (2–5).In addition to its critical role in mediating inflammatoryresponses, Egr-1 is also involved in regulating the expression ofgenes contributing to liver fibrosis, such as transforminggrowth factor �1 (TGF-�), platelet-derived growth factor(PDGF), and fibroblast growth factor (FGF) (6). Recent studieshave shown that TGF-� induces a rapid but transient expres-sion of Egr-1 that results in stimulation of collagen gene expres-sion (7). Insulin-like growth factor (IGF)-binding protein 5induces a fibrotic phenotype via the activation ofMAPK signal-ing and the induction of Egr-1, resulting in activation of genesinvolved in fibrogenesis (8). These studies suggest that Egr-1may play an important role in the pathogenesis of fibrosis.However, the function of Egr-1 in hepatic fibrosis is not wellunderstood. Identification of the signaling pathways that con-trol Egr-1 expression could help for a better understanding ofthe molecular basis of liver fibrosis.Nuclear receptor small heterodimer partner (SHP, NROB2)

is a unique member of the nuclear receptor superfamily thatcontains the dimerization and ligand binding domain, but lacksthe conserved DNA binding domain. Numerous studies haveshown that SHP is a transcriptional repressor of a number ofgenes critical to hepatic bile acid, cholesterol, triglyceride, glu-cose, and drug metabolism (9–15). Recent studies demon-strated that SHP has a preventative role in liver fibrosis (10).Activation of hepatic stellate cells, themajor source of extracel-lular matrix in the liver, plays an important role in the develop-ment of cirrhosis through a progressive trans-differentiationfrom a resting phenotype toward a myofibroblast-like pheno-type. Exposure of hepatic stellate cells to farnesoid X receptor(FXR) ligands causes a 3-fold increase of SHP and reduces �(I)collagen and TGF-�, indicating a protective effect of SHP inliver fibrosis (16). In addition, SHP�/� mice showed increasedsensitivity to liver damage induced by bile duct ligation (BDL)(17). These studies provide evidence that SHPmay play a role inprotecting against liver fibrosis. However, the target genes ofSHP involved in regulating fibrosis remain to be determined.In this study, we show that Egr-1 is a direct target of SHP.

SHP represses Egr-1 gene transcription via cross-talk with hep-atocyte nuclear factor 4� (HNF4�). HNF4� binds to and acti-

* This work was supported, in whole or in part, by National Institutes of HealthGrant DK080440 (to L. W.).

1 Supported by Multidisciplinary Cancer Research Training Program GrantT32CA092347.

2 To whom correspondence should be addressed: 30 North 1900 East, SOM3C310, Salt Lake City, UT 84132. Tel.: 801-585-4615 or 4616; Fax: 801-585-0187; E-mail: l.wang@hsc.utah.edu.

3 The abbreviations used are: Egr-1, early growth response 1; BDL, bile ductligation; Co-IP, co-immunoprecipitation; FXR, farnesoid X receptor; h,human; HNF4�, hepatocyte nuclear factor 4�; Luc, luciferase; m, mouse;qPCR, quantitative PCR; RAR, retinoic acid receptor; RXR, retinoid X recep-tor; SHP, small heterodimer partner; WB, Western blot.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 24, pp. 29635–29643, August 26, 2011Printed in the U.S.A.

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vates the Egr-1 promoter, inducing Egr-1 mRNA and protein.This transcriptional induction is inhibited by SHP. We furthershow that Egr-1 is strongly up-regulated in human cirrhoticlivers, which is associatedwith the up-regulation ofHNF4� anddown-regulation of SHP. This study reveals a novel pathway bywhich SHP andHNF4� regulate liver fibrosis through targetingEgr-1.

EXPERIMENTAL PROCEDURES

Cell Lines, Animals, and Human Liver Specimens—Humancervix adenocarcinoma cells (HeLa, ATCC CCL-2), humanhepatoma cells (Huh7, Health Science Research ResourcesBank JCRB0403; HepG2, ATCC HB-8065), and mouse hepa-toma cell line Hepa1 (ATCC CRL-1830) were maintained inDulbecco’s modified Eagle’s medium with 100 units of penicil-lin G-streptomycin sulfate/ml and 10% heat-inactivated FBS.Human stellate LX2 cells (a gift from Dr. Scott Friedman) weremaintained in 2% FBS. The SHP�/� (WT) and SHP�/� micehave been described previously (15, 18). Livers were obtainedfrom 6-week-oldmale liver-specificAlb-Hnf4aF/F (Alb-WT) orAlb-Hnf4a�/� (knock-out) mice. An acute, conditional liver-specific Hnf4� knock-out was obtained by treating ErT2-Alb-Hnf4a F/F,cre� (ErT2-WT) or ErT2-Alb-Hnf4aF/F,cre� (induc-ible-knock-out) mice with a purified diet containing 0.1%tamoxifen citrate for 5 days, removing the diet, and killing themice 2 weeks later. Protocols for animal use were approved bythe Institutional Animal Care and use Committee at the Uni-versity of Utah. Livers from five normal livers and eight cir-rhotic specimens were obtained through the Liver Tissue CellDistribution System (Minneapolis, MN).Plasmids, siRNAs, and Antibodies—The mouse Egr-1 (Gene

ID 13653) promoter luciferase reporter (Egr-1Luc) and its dele-tion mutation constructs were engineered in our laboratory.Each DNA fragment of the mouse Egr-1 promoter was insertedinto the KpnI and XhoI sites of pGL3-basic (Promega). Theintegrity of the recombinant plasmids was verified by sequenceanalysis. Expression plasmids for FXR, RXR, RAR, HA-HNF4�,HNF4� S78D, HNF4� S304D, and FLAG-SHP were describedpreviously (19–21). The following antibodies were used for co-immunoprecipitation (Co-IP), chromatin IP (ChIP), andWest-ern blotting (WB): M-280 sheep anti-rabbit or mouse IgGDynabeads (Invitrogen Dynal As), rabbit normal IgG (Sigma,R-2004), and antibodies against FLAG (Sigma, F-7425), HA(Sigma,H-6908), Egr-1 (Cell Signaling, 4153), HNF4� (Cell Sig-naling, 3113), HNF4� (Santa Cruz Biotechnology, sc-6556),�-actin (Sigma, A-1978), tubulin (Sigma, T-6199), and SHP(PPMX, L:-76571). HNF4� (SASI_Hs01_00124507) and non-specific (SIC001) siRNAs were purchased from Sigma. SHPsiRNA (ON-TARGETplus SMARTpool NR0B2, L-003410)was purchased from Thermo Scientific Dharmacon RNAiTechnologies.Transient Transfection and Promoter Activity Assays—For

transient transfection assays, HeLa, Huh7, or LX2 cells wereco-transfected with the mouse Egr-1 Luc reporter, pcDNA3-HA-HNF4�, or pcDNA3-FLAG SHP as indicated in the figurelegends. Empty vector DNA was added as needed so that thesame amounts of expression vector DNA were present in eachtransfection. Transfection was carried out using Lipofectamine

2000 (Invitrogen) in 24-well plates. Thirty-six hours after trans-fection, cells were collected, and luciferase activities were mea-sured and normalized against Renilla activities (Promega).Consistent results were observed in three independent tripli-cate transfection assays in each experiment.ChIP Assays—ChIP assays were carried out essentially as

described previously (20). Briefly, HeLa cells orHuh7 cells wereincubated with 1% formaldehyde at 25 °C for 10 min. Nucleiwere isolated and sonicated to shear the DNA into 0.3–1.0 kb.Chromatin was precleared in the presence of 20 �l of normalserum and 30 �l of M-280 sheep anti-rabbit or mouse IgGDynabeads. Precleared chromatin samples were subjected toimmunoprecipitation at 4 °C overnight in the presence of 2 �gof antibodies against FLAG, HA, HNF4�, or rabbit normal IgG.After the complex was collected by incubation in 30�l of sheepanti-rabbit or mouse IgG Dynabeads and centrifugation, thebeadswerewashed five times, and the chromatin immune com-plex was eluted. Then the cross-links were reversed, and theDNA was purified and used as a template in PCR. Real-timePCR and PCR were performed using ChIP primer sets specificfor the HNF4� binding sites in mouse Egr1 (mEgr-1) andhuman EGR1 (hEGR-1) promoters: mouse p1 forward, 5�-TG-CCCACCACTCTTGGATG-3� and reverse, 5�-CAAGGGTC-TGGAACAGCACG-3�; mouse p2 forward, 5�-AAGGTGGG-ATCCTCAACCG-3� and reverse, 5�-GCAGGGTCACTTTC-CAGGTG-3�; mouse p3 forward, 5�-TAAACGGGTCCTCCG-CACT-3� and reverse, 5�-AGGCTATTCCCTCCGTCCTG-3�;human p1 forward, 5�-CGGTCCTGCCATATTAGGGC-3�and reverse, 5�-CCCGGATCCGCCTCTATTT-3�; human p2forward, 5�-AGTGGCCGTGACTTCCTATCC-3� and reverse,5�-TATCGCTGTCATCCAGGGC-3�; human p3 forward, 5�-CCACCTGGACTGGATAAAGGG-3� and reverse, 5�-CGAA-AGGCTGTTCCCTAGTCC-3�. Control regions from �1665to�1462 in themEgr-1 promoter (forward, 5�-TCAATCTCC-TTCCACACAGGC-3� and reverse, 5�-TGATGGCAGAGAG-ATCCCG-3�) and �1893 to �1717 in the hEGR-1 promoter(forward, 5�-GACGAGGGAGTCCTGGTTCAT-3� and re-verse, 5�-AGAGGGAAGGGAGGAGGTGA-3�) serve as nega-tive controls.Real-time Quantitative PCR (qPCR) Analysis—Quantitative

PCR was performed as described previously (18, 20, 21). Inbrief, total RNA was isolated using TRIzol, cDNA was synthe-sized, and real-time PCRwas carried out using the SYBRGreenPCR master mix (Applied Biosystems). The melting curve datawere collected to checkPCR specificity. Each cDNAsamplewasrun in triplicate, and the corresponding no-reverse transcrip-tase (RT) mRNA sample was included as a negative control.The amount of PCR products was measured by threshold cycle(Ct) values, and the relative ratio of specific genes toHPRT1 foreach samplewas then calculated. The sequences for the primersare as follows: forward primer, 5�-CGTCGTGATTAGCGAT-GATGA-3� and reverse primer, 5�-CACACAGAGGGCCAC-AATGT-3� for mouse Hprt1; forward primer, 5�-TGACACT-GGCAAAACAATGCA-3� and reverse primer, 5�-GGTCCTT-TTCACCAGCAAGCT-3� for humanHRPT1; forward primer,5�-GACGAGTTATCCCAGCCAAA-3� and reverse primer,5�-GGCAGAGGAAGACGATGAAG-3� for mouse Egr-1; for-ward primer, 5�-AGCACCTGACCGCAGAGTCT-3� and

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reverse primer, 5�-AGATGGTGCTGAGGACGAGG-3� forhuman EGR-1; forward primer, 5�-CACCTGCATCTCACAG-CCACT-3� and reverse primer, 5�-GCCAACCCAAGCAGGA-AGA-3� for mouse SHP; forward primer, 5�-GGTGCCCAGC-ATACTCAAGAA-3� and reverse primer, 5�-GGACTTCACA-CAGCACCCAGT-3� for human SHP; forward primer 5�-TGGCCAAGATTGACAACCTG-3� and reverse primer, 5�-AGGTGAGAGGGCATCGTGTT-3� for mouse HNF4�;forward primer, 5�-CAGGAGCTGCAGATCGATGA-3� andreverse primer, 5�-CAGCAGCAGCTCTCCAAAGC-3� forhumanHNF4�; and forward primer, 5�-GCTGCAACTGCTT-TCGGAG-3� and reverse primer, 5�-GATGTCTCCTGGCAT-GAGGTC-3� for mouse E2F1.Co-IP Assays—Co-IP was performed as described previously

with minor modifications (18). Extracts from Huh7 or HepG2cells were incubated in lysis buffer (20 mM KOH-HEPES, pH8.0, 0.2 mM EDTA, 5% glycerol, 250 mM NaCl, 0.5% NonidetP-40, 0.25% sodium deoxycholate, 1 mM dithiothreitol, andprotease inhibitors) with antibody against HNF4� or rabbitimmunoglobulin G (IgG) at 4 °C for 4 h to overnight, and theimmune complex was collected by incubation with 30 �l ofM-280 sheep anti-rabbit IgG Dynabeads for 4 h. Immunopre-cipitates were washed four times with lysis buffer supple-mented with NaCl to 400 mM and subjected to Westernblotting.Western Blotting in Cells and Liver Tissues—Hepa1 or Huh7

cells were pelleted by centrifugation and resuspended in lysisbuffer (50 mM Tris, pH 8.0, 1% Nonidet P-40, 150 mM NaCl,0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitors(Thermo Fisher Scientific, 78410). After incubation on ice for10 min followed by sonication and centrifugation, the proteinconcentration of cell lysates was determined using the DC pro-tein assay (Bio-Rad, 500-0112). Cell lysates (30 �g) wereresolved by SDS-PAGE and transferred to nitrocellulose mem-branes according to standard procedures. Membranes werewashed in Tris-buffered saline containing 0.05% Tween 20(TBST), blocked for 1 h with TBST containing 5% nonfat milk,and then incubated with primary antibodies at a 1:1,000 dilu-tion in TBST containing 5% nonfat milk overnight at 4 °C.Membranes were then washed with TBST before incubationwith horseradish peroxidase-conjugated secondary antibodyfor 1 h at room temperature. Membranes were washed fourtimes with TBST before antibody binding was visualized withSuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific 34080) according to themanufacturer’s proto-col. For Western blotting using mouse livers, 100 mg of liverfragments from three to four individual 6-week-old Alb-HNF4��/�, ErT2-HNF4��/� mice or their respective controlswere homogenized in lysis buffer and subjected to Westernblotting as described above. ForWestern blotting using humanliver specimens, five normal donor livers and eight cirrhoticlivers were homogenized and subjected to Western blotting.Equal loading of protein was verified with �-actin or tubulin,respectively.BDL—The experiments were performed at the University of

Kansas Medical Center, and the studies were approved by theInstitutional Animal Care and Use Committee of KUMC.Micewere maintained on standard laboratory chow on a 12-h light/

dark cycle. Hepatic fibrosis was induced by BDL in 8–12-week-old male C57BL/6 and SHP�/� mice. Briefly, mice were anes-thetized with isoflurane, and an upper abdominal midlineincision was used to expose the common bile duct, which wasligated twice with a 3-0 surgical silk suture and then cutbetween the ligatures. Sham-operated mice received the samelaparoscopic procedure, except that the bile duct was manipu-lated, but not ligated, and sectioned. Mice were killed 14 daysafter BDL, livers were collected, and mRNA and protein wereisolated by common standard methods.Serum Shock—Huh7 cells were grown to confluence in high

glucose DMEM supplemented with 10% FBS. Cells werestarved in the same medium with no FBS for 18 h, 50% horseserum was added for 2 h, and the medium was then changedback to the starvationmedium (22). Cells were harvested at 4-hintervals, and total RNAs were isolated for qPCR analysis ofmRNA levels.Histological Analysis of Liver Sections—Fresh mouse livers

from the indicated genotypes were either formalin-fixed over-night for paraffin sectioning or immediately embedded in OCTCompound (Tissue-Tek, Sakura Finetek) and frozen on dry icefor frozen sectioning. For H&E staining and Sirius Red staining(fibrosis), paraffin sections were cut at a thickness of 4 �m andsubjected to xylene and ethanol rehydration prior to staining.Frozen sections were cut at 10 �m for Oil red O staining oflipids with hematoxylin counterstain.

FIGURE 1. SHP repression of Egr-1 promoter activity by HNF4�. A, tran-sient transfection assays of Egr-1 promoter luciferase (Luc) reporter activity bynuclear receptors. HeLa cells were transfected with expression plasmids FXR/RXR (200, 400 ng), RAR/RXR (200, 400 ng), and HNF4� (200, 400 ng), alone orin combination with the expression plasmid of SHP (200, 400 ng). B, transienttransfection assays of Egr-1 promoter luciferase reporter activity by HNF4�(200 ng), S78D (200 ng), S304D (200 ng), and SHP (200 ng) in Huh7 cells. S78Dand S304D are DNA binding domain mutant HNF4�. C, transient transfectionassays of Egr-1 promoter luciferase reporter activity by HNF4� (100 ng) in theabsence or presence of HNF4�-siRNA (20 pmol) in Huh7 cells. D, transienttransfection assays of Egr-1 promoter luciferase reporter by HNF4� (400 ng)and SHP (400 ng) in human stellate LX2 cells. A–D, Luciferase (Luc) activities(act) were determined and normalized by Renilla activities. Data are repre-sented as means � S.E. (error bars). *, p � 0.01 versus control pcDNA group; ¥,p � 0.01 versus HNF4� alone. The experiments were repeated three times(triplicate wells/time) with similar results. One representative result is shown.E, immunoprecipitation (IP) and Western blotting to determine the directassociation of SHP with HNF4� protein in Huh7 and HepG2 cells. Anti-HNF4�antibodies were used to immunoprecipitate the endogenous HNF4�, and theprotein levels of SHP and HNF4� were detected by Western blotting usinganti-SHP or anti-HNF4� antibodies, respectively.

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Statistical Analysis—Data are expressed as mean � S.E. Sta-tistical analyseswere carried out using Student’s unpaired t test;p � 0.01 was considered statistically significant.

RESULTS

SHP Inhibits HNF4� Trans-activation of the Egr-1 Promoter—We searched nuclear receptor binding motifs and identifiedpotential binding sites for FXR, RAR, and HNF4� within themouse Egr-1 gene 5�-flanking region. To determine the role ofthese nuclear receptors in regulating Egr-1 promoter activity,HeLa cells were co-transfected with the Egr-1 promoter lucif-erase reporter (Egr-1Luc) and the expressionplasmids for FXR/RXR, RAR/RXR, and HNF4� at the indicated concentrations(Fig. 1A). Compared with pcDNA controls (�), overexpressionof FXR/RXR andRAR/RXRdid not affectEgr-1 promoter activ-

ity. In contrast, HNF4� significantly increased luciferase activ-ity of the Egr-1 Luc reporter in a dose-dependent manner. SHPwas previously shown to interact with and inhibit HNF4�trans-activation (21). As expected, co-expression of SHP abro-gated the activity of HNF4�. Similar results were observed inHuh7 cells (Fig. 1B). On the other hand, S78D and S304D, thetwo HNF4� DNA binding domain mutants, failed to activatethe Egr-1 promoter, confirming that the HNF4� DNA bindingdomain is required for HNF4� activity. To validate this findingfurther, HNF4� siRNA was used to knockdown the endoge-nous HNF4� in Huh7 cells expressing high levels of HNF4�(18). The Egr-1 promoter activity was markedly decreased byHNF4� siRNA but not by a negative control siRNA (Fig. 1C).The efficiency ofHNF4� knockdown is presented in Fig. 4. SHPalso repressed the transactivation of HNF4� in human stellateLX2 cells (Fig. 1D), suggesting that a similar regulatory mecha-nism may exist in stellate cells. A physical association of SHPwith the HNF4� protein was confirmed in Huh7 and HepG2cells using Co-IP and Western blotting (Fig. 1E), consistentwith an early result using mammalian two-hybrid assays (23).

FIGURE 2. Attenuation of the Egr-1 promoter activity by deletion ofHNF4� response elements. A, diagram showing the location of three puta-tive HNF4� binding sites in the Egr-1 promoter and its deletion mutationconstructs. B and C, mutagenesis analysis. Transient transfection assays ofmEgr-1 promoter luciferase reporter activity by HNF4� (200 ng) in HeLa (B)and Huh7 (C) cells are shown. The experiments were done as in Fig. 1. *, p �0.01 versus control pcDNA group; ¥, p � 0.01 versus HNF4� activation of Egr-1Luc-1731. D, mutagenesis analysis of mEgr-1 Luc-787 mutation constructs.Top, transient transfection assays of mEgr-1 Luc-787 activity by HNF4� (200ng) and SHP (200 ng) in HeLa cells. *, p � 0.01 versus control pcDNA group; ¥,‡,p � 0.01 versus HNF4� activation of Egr-1 Luc-787. Bottom, HNF4� site 1 wasdeleted (del-12), partially deleted (del-5), mutated (mut-1), or mutated to aconserved DR1 site (DR1), the latter was used as a positive control. Underlin-ing, mutated nucleotides.

FIGURE 3. SHP inhibition of HNF4� recruitment to the Egr-1 promoter.A, schematic showing the putative HNF4�-binding elements in the Egr1 pro-moter. B, ChIP assays to determine the enrichment of HNF4� and SHP to theEgr-1 promoter. HNF4� and SHP were exogenously expressed in HeLa cellswith HA-HNF4� plasmid (5 �g, 10-cm plate) and/or FLAG-SHP plasmid (5 �g,10-cm plate), along with the Egr-1 promoter. The cross-linked chromatin wasco-immunoprecipitated using anti-HA or anti-FLAG antibodies. The enrichedDNA was amplified by PCR using primers mP1, mP2, and mP3. mP4 served asa negative control. C, ChIP assays to determine the recruitment of endoge-nous HNF4� to the human EGR1 promoter. Huh7 cells were transfected withnegative (neg) control or HNF4� siRNA. The enriched DNA was quantified byreal-time qPCR using EGR1 promoter primers hP1, hP2, and hP3. The amountof immunoprecipitated DNA in each sample is represented as signal relativeto the total amount of input chromatin, expressed as Relative (Rel) enrich-ment. Error bars represent S.E. from three independent measurements (*, p �0.01 versus neg siRNA).

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Because Egr-1 is predominantly localized in hepatocytes, sub-sequent studies focused on using hepatocyte derived Hepa1and Huh7 cells.Deletion of HNF4� Response Elements Attenuated Egr-1 Pro-

moter Activity—Three putative HNF4� DR1 binding sites werelocated in the Egr-1 promoter (Fig. 2A). We generated threemutant Egr-1 Luc constructs (Luc-934, Luc-787, and Luc-721).Deletion of HNF4� site 3 did not decrease the Luc-934 activityin HeLa cells, but markedly decreased it in Huh7 cells (Fig. 2, BversusC). Double deletion of site 3 and site 2 or triple deletion ofall three sites attenuated Luc-787 or Luc-721 promoter activa-tion by HNF4� in both HeLa and Huh7 cells. Although themutant promoters showed somewhat different activities inHeLa and Huh7 cells, the data strongly suggest that all threesites bind HNF4�.To confirm further that site1 is important for HNF4� bind-

ing, we generated several site1 mutation constructs based onEgr-1 Luc-787. The Egr-1 Luc-787 promoter activity was abol-ished by deletion of site1 (del-12) (Fig. 2D). Deletion half of site1 (del-5) or mutation of several nucleotides within the site 1(mut-1) significantly decreased Egr-1 Luc-787 activity. We alsomutated site 1 to a conserved DR1, which resulted in an

increased Egr-1 Luc-787 activity by HNF4� compared with thenative Egr-1 Luc-787.SHP Decreases HNF4� Recruitment to the Egr-1 Promoter—

The proximal Egr-1 promoter contains three putative HNF4�-binding elements (Fig. 3A). ChIP assays were used to determinebinding ofHNF4� and SHP to the Egr-1 promoter using primersets mP1, mP2, and mP3 covering each HNF4� binding site,respectively. The mP4 primers were located upstream of themouse Egr-1 promoter region which contains no HNF4� bind-ing site and served as a negative control. Recruitment ofHNF4�to all three sites was observed. Co-expression of SHPmarkedlyattenuatedHNF4� recruitment to all sites (Fig. 3B). In contrast,mP4 yielded no PCR products confirming specificity of theassay. SHP was shown to recruit mSin3A-Swi/Snf co-repressorcomplex to its target promoter (24). It is postulated that a sim-ilar co-repressor complex may be recruited to the Egr-1 pro-moter by SHP to compete for HNF4� binding.Three potential HNF4� response elements were also pre-

dicted in the human EGR-1 promoter (Fig. 3C, left). HNF4�siRNA was used to knockdown endogenous HNF4� in Huh7cells, and anti-HNF4� antibodieswere used to immunoprecipi-tate HNF4� for ChIP analysis. Consistent with the results

FIGURE 4. Induction of Egr-1 expression by HNF4� and inhibition by SHP. A, qPCR of Egr-1 mRNA (left) and Western blots (WB) of Egr-1 protein (right) inmouse hepatoma Hepa1 cells transfected with a HA-HNF4� plasmid (1 �g). Antibodies against Egr-1, HNF4�, or �-actin were used, respectively. B, WB of EGR-1protein in human hepatoma Huh7 cells transfected with a HA-HNF4� plasmid (1 �g). Antibodies against EGR-1, HNF4�, or �-actin were used, respectively.C, qPCR of HNF4� and EGR-1 mRNA in Huh7 cells with HNF4� knockdown using siRNAs (�, nonspecific siRNA control; �, 100 pmol; ��, 200 pmol, 60-mmplate). D, WB of EGR-1 protein in Huh7 cells with HNF4� (left) or SHP (right) knockdown using siRNAs (�, nonspecific siRNA control; �, 200 pmol, 60-mm plate).Anti-EGR-1, anti-HNF4�, or anti-SHP antibodies were used, respectively. E, left, WB of EGR-1 protein in Huh7 cells that were overexpressed with HA-HNF4� (0.5�g) and/or FLAG-SHP (0.5 �g) expression vectors. Antibodies against EGR-1, HA, FLAG, or �-actin were used, respectively. Right, band intensities weremeasured by densitometry, and the intensities relative to that of the control vector were plotted. F and G, qPCR of EGR-1 mRNA in Huh7 cells that wereoverexpressed with HA-HNF4� (0.5 �g) and/or FLAG-SHP (0.5 �g) expression vectors at 12 h (F) and 24 h (G). H, qPCR of cyclic expression of SHP, HNF4�, andEGR-1 mRNAs in Huh7 cells. Serum shock was described under “Experimental Procedures. ” At the indicated times, three wells were harvested to measure SHP,HNF4�, and EGR-1 mRNAs by qPCR in triplicate. Arrows indicate peak expression. qPCR data are represented as mean � S.E. (error bars) of three independentassays (n � triplicate/assay). A, C, and H, normalized by HPRT1; F and G, normalized by GAPDH. *, p � 0.01 versus control (�); ¥, p � 0.01 versus HNF4� alone.

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observed with the mouse Egr-1 promoter, HNF4� wasrecruited to all sites in the EGR-1 promoter, and its associationwith site 3, particularly site 2, but not site 1, was dramaticallyreduced by HNF4� siRNA (Fig. 3C, right). Taken together, theresults suggest that both the mouse Egr-1 and human EGR-1genes may be regulated by HNF4� in a similar fashion, i.e. bydirect binding of HNF4� to its response elements in thepromoters.The Induction of Egr-1 Expression by HNF4� Is Repressed by

SHP—To determine further the regulation of Egr-1 by HNF4�,we overexpressed HNF4� in mouse hepatoma Hepa1 cellswhich had no detectable levels of HNF4� (data not shown) andperformed qPCR analysis of Egr-1 mRNA. Interestingly, theendogenous Egr-1mRNAwas transiently induced byHNF4� at8 h after transfection, which reachedmaximal induction by 24 hand declined to basal levels by 32 h (Fig. 4A, left). The Egr-1protein showed parallel induction by HNF4� with time,although its increase prolonged to 32 h (Fig. 4A, right). Simi-larly, a time-dependent reduction of HNF4� protein wasobserved by 32 h. The stimulation of endogenous humanEGR-1 protein by HNF4� was observed in Huh7 cells as well(Fig. 4B). At 24 h after knockdown ofHNF4� by siRNA (Fig. 4C,left), EGR-1 mRNA (Fig. 4C, right) and protein (Fig. 4D, left)decreased in Huh7 and HepG2 cells (data not shown). SHPprotein was also subsequently decreased by HNF4� siRNA,supporting SHP as a target of HNF4� (25, 26). In addition,knockdown of SHP using siRNA elevated EGR-1 protein butdid not alter HNF4� protein levels (Fig. 4D, right), suggestingthat SHP inhibition of EGR-1 is most likely through repressingHNF4� transactivation of the Egr-1 promoter (Figs. 1–3), butnot by regulating HNF4� expression.

In agreement with the above observations, the induction ofEGR-1 protein byHNF4�was antagonized by SHP overexpres-

sion (Fig. 4E, fourth versus second lane). The relative moderateincrease in EGR-1 protein by HNF4� may be attributed tolower amounts of exogenously expressed HNF4� in the cells(Fig. 4,E versus B). BecauseHNF4�was able to up-regulate SHPprotein (data not shown), the increased SHPmay also counter-act the effect of HNF4� by masking the activation of EGR-1 byHNF4�.

During this study, we noticed that there appeared to be adiscoordination with regard to the time HNF4� and SHPexhibited their maximal efficacy. For instance, at 12 h aftertransfection, HNF4� induced EGR-1 mRNA, whereas SHP didnot repress basal EGR-1 expression (Fig. 4F). By 24 h, the abilityof HNF4� to elevate EGR-1 declined, whereas SHP exhibited astronger ability to reduce basal EGR-1 levels (Fig. 4G). Thus, thehighest activation of EGR-1 by HNF4� seemed to occur earlier,whereas the strongest ability of SHP inhibition of EGR-1occurred later. In addition, the basal endogenous EGR-1mRNAdropped about 4-fold at 24 h comparedwith 12 h (Fig. 4,F versus G, first lane). Because the expression of SHP shows adiurnal cycle in the liver (12), we examinedEGR-1 expression inHuh7 cells that were briefly exposed to 50% horse serum, acondition that was used to induce cyclic SHP expression (12).SHP mRNA showed a small but nonsignificant increase 4 hafter the cells were released from serum shock (Fig. 4H).Intriguingly, SHP, HNF4�, and EGR-1 mRNAs showed similardiurnal cycles and reached their highest levels at the same timepoint (32 h). A noticeable difference was that the EGR-1 leveldropped sharply at 36 h after its peak expression and reachedminimal by 44 h, whereas SHP and HNF4� levels remainedhigh after 32 h but decreased gradually with time. Based on thisresult, we propose that the up-regulation of EGR-1 at 32 h ismainly contributed by HNF4� activation, whereas its down-regulation after 32 h is more controlled by SHP inhibition.

FIGURE 5. Paradoxical activation of Egr-1 expression in HNF4� mice. A, qPCR of hepatic Egr-1 mRNA (left) and WB of Egr-1 protein (right) in liver-specificHNF4� mice (n � 4/genotype). B, qPCR of hepatic Egr-1 mRNA (left) and WB of Egr-1 protein (right) in acute liver conditional Ert2-HNF4� mice (n � 4/genotype).A and B, antibodies against Egr-1, HNF4�, or �-actin were used, respectively. C and D, qPCR of hepatic SHP (C) and E2F-1 (D) mRNA in HNF4� (left) andErt2-HNF4� (right) mice. E, qPCR of Cyclin E and Cdk1 mRNAs in ErT2-HNF4� mice. Data are represented as mean � S.E. (error bars) of three independent assays(n � triplicate/assay). *, p � 0.01 versus ErT2-WT.

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Therefore, theEGR-1 gene displays a diurnal response to serumshock involving an early induction by HNF4� followed by asubsequent repression of SHP.The Expression of Egr-1 Is Paradoxically Increased in

HNF4��/� Mice—Thus far, our in vitro studies identifiedHNF4� and SHP cross-talk in regulating Egr-1 expression. Totest whether this regulatory mechanism exists in vivo, we per-formed qPCR and immunoblotting of Egr-1mRNAand proteinin liver-specific Alb-Hnf4a�/� mice. Unexpectedly, both Egr-1mRNA and protein were markedly increased in livers of Alb-Hnf4a�/� mice (Fig. 5A). Similar results were obtained usingthe acute conditionally deleted ErT2-Alb-Hnf4a�/� mice (Fig.5B). Another interesting observation was that SHP mRNA wasmarkedly diminished inAlb-Hnf4a�/� (Fig. 5C, left), but not inErT2-Alb-Hnf4a�/� mice (Fig. 5C, right). The decreased SHPmay derepress Egr-1 and mask the lost activation of Egr-1 byHNF4� in Alb-Hnf4a�/� mice. The transcription factor E2F-1is able to activate Egr-1 expression.4 E2F-1mRNAwas dramat-ically increased in ErT2-Alb-Hnf4a�/� mice (Fig. 5D, right),which may in part contribute to the high level of Egr-1. TheE2F-1 target genes, including Cyclin E and Cdk1 (27, 28), wereup-regulated in ErT2-Alb-Hnf4a�/� mice compared with theErT2-WT mice, which was in agreement with the increasedE2F-1 expression (Fig. 5E).Hnf4a�/� Mice Exhibit Hepatocyte Hypertrophy—Histology

of liver sections was performed in Alb-Hnf4a�/� and ErT2-Alb-Hnf4a�/� mice. H&E staining revealed hepatocyte hyper-trophy in both knockouts (Fig. 6A), which was more severe inthe Alb-Hnf4a�/� mice. Neutral lipid accumulation appearedto be lower in Alb-Hnf4a�/� mice relative to ErT2-Alb-Hnf4a�/� mice, as examined by Oil red O staining (Fig. 6B).SHP deficiency resulted in diminished fatty liver (9, 14). Thedecreased SHP mRNA in Alb-Hnf4a�/� liver (Fig. 5C) may inpart protect the mice from developing steatosis. Sirius Redstaining did not identify liver fibrosis in eitherHnf4� knock-outmouse model (Fig. 6C). A stress condition is likely required toassess the development of liver fibrosis in both mouse models.Increased hepatocyte proliferation was observed in ErT2-Alb-Hnf4a�/� mice,5 which may be associated with the up-regula-tion of E2F-1 (Fig. 5D, left).The Expression of HNF4� and EGR-1 Is Inversely Correlated

with SHP Expression in Human Cirrhotic Livers—We nextdetermined whether HNF4� was elevated under a cholestaticcondition that ultimately leads to fibrosis and cirrhosis. BDLwas performed in SHP�/� mice to induce cholestasis. HNF4�mRNA was induced in WT mice with BDL, but to a muchgreater extent in SHP�/� mice without or with BDL (Fig. 7A).Consistent with the mouse models, up-regulation of HNF4�was also observed in human cirrhotic livers (Fig. 7B). Egr-1protein expression was markedly high in SHP�/� livers (Fig.7C) and in human cirrhotic livers (Fig. 7D), with a concomitantreduction of SHP expression (Fig. 7D). Taken together, theseresults establish an inverse correlation betweenHNF4�/EGR-1and SHP expression under cholestatic/cirrhotic conditions.

4 Y. Zhang and L. Wang, unpublished data.5 J. A. Bonzo and F. J. Gonzalez, unpublished data.

FIGURE 6. Histology of liver sections from wild-type and Hnf4� mice.A, H&E staining to examine liver morphology. Large vacuoles are a result ofglycogen deposition. Large areas of cell death were observed in HNF4� mice.B, Oil red O staining to examine neutral lipid accumulation. Mild steatosis wasobserved in ErT2-HNF4� mice. C, Sirius Red staining to examine liver fibrosis.There was no evidence of fibrotic collagen fibers in either knockout.

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DISCUSSION

Accumulating evidence suggests a critical role for Egr-1 infibrosis. Elevated Egr-1 protein and/or mRNA has been widelydetected in fibrotic tissues, including ureteral obstructionfibrotic kidneys in rats, lung tissues from patients with emphy-sema, and skin fibroblasts from patients with diffuse sclero-derma (29–31). In addition to directly stimulating �-smoothmuscle actin expression and myofibroblast trans-differentia-tion, Egr-1 also controls the expression of extracellular matrixgenes contributing to fibrogenesis (29). Targeting the Egr-1gene with specific DNA enzyme in a ureteral obstruction ratmodel blocked Egr-1 expression with a concomitant reductionin TGF-�, �-smooth muscle actin, and type I collagen mRNAexpression, consequently inhibited interstitial fibrosis (29).Further studies showed that fibrosis of the lungs induced byTGF-� or interleukin-13 was markedly attenuated in Egr-1�/�

mice (32, 33). Despite the importance of Egr-1 in fibrosis, themechanisms responsible for elevated Egr-1 expression infibrotic tissues remain largely unknown.Here we elucidate a transcriptional mechanism that controls

Egr-1 expression through nuclear receptor-mediated signaling.Using a series of in vitro and in vivo approaches, we demon-strate that SHP directly represses Egr-1 promoter activity andgene transcription through HNF4�. Although liver fibrosis-as-sociated Egr-1 induction results from elevated bile acids (34),the underlying mechanism is unclear. SHP plays a critical rolein the negative feedback regulation of bile acid synthesis (11),and bile acid levels are elevated in SHP�/� mice due to loss ofSHP inhibition of Cyp7a1 and Cyp8b expression (15, 35). SHPexpression is decreased, whereas HNF4� is increased in chole-static and cirrhotic livers. Therefore, the mechanism contrib-uting to bile acid-induced Egr-1 in fibrotic and cirrhotic livers isat least in part attributable to the diminished SHP inhibition,with a concomitant activation by HNF4�.

Egr-1 stimulation is normally acute and transient (7), andsustained Egr-1 expression can be induced by tissue hypoxia orTGF-� (36). We observed a prolonged induction of EGR-1 byexogenously expressed HNF4�. Initially, we were surprised bythe results that in the absence of Hnf4�, Egr-1 mRNA and pro-tein are markedly elevated in both Alb-Hnf4��/� (long terminactivation) and ErT2-Alb-Hnf4��/� (short term inactiva-tion)mice. This suggests that the influence of other factorsmayplay a predominant role in governing Egr-1 expression underHnf4�-deficient conditions. Mice lacking hepatic Hnf4� haveincreased levels of serum bile acids due to decreased Cyp8bactivity (37). The SHP gene is activated by Hnf4� (25), and itslevels are diminished in Alb-Hnf4��/� mice. On the otherhand, E2F-1 expression is induced in ErT2-Alb-Hnf4��/�

mice, which activates Egr-1.4 The decreased SHP or theincreased E2F-1, combined with the elevated bile acids andother unknown factors, may contribute to the elevation ofEgr-1 in Alb-Hnf4��/� mice. Thus, alteration of gene expres-sion in vivo often reflects the consequences derived from bothdirect and indirect effects, and the final net outcome is stronglyinfluenced by the physiological and pathological conditions.Of particular interest, EGR-1 mRNA exhibits cyclic rhythm

in Huh7 cells subjected to serum shock, which has not beenreported before. Its diurnal fluctuation is synchronizedwith thecyclic expression of SHP and HNF4�. However, this is some-what expected considering the fact that SHP is a clock-con-trolled gene and its interaction with HNF4�/LRH-1 showsrhythmic binding to the microsomal triglyceride transfer pro-tein (MTP) promoter causing diurnal regulation of plasma trig-lycerides (12). Diurnal variations of bile acid concentration andcomposition are also observed in mice (38). Egr-1 expressiondisplays circadian dependence in rat retina (39). It is highlypossible that circadian regulation of hepatic Egr-1 expressionexists in vivo in mice as well. It would be of great interest to

FIGURE 7. Up-regulation of EGR-1 and HNF4� expression in human cirrhotic liver. A, qPCR of hepatic HNF4� mRNA in wild type (WT) and SHP�/� micesubjected to BDL or control sham operation 14 days after surgery (n � 10 –15/group). *,£,#, p � 0.01 versus WT sham; †, p � 0.01 versus SHP �/� sham; ¶, p �0.01 versus WT BDL. B, qPCR of hepatic HNF4� mRNA in 5 normal and 8 cirrhotic human livers. Total RNA was isolated from individual liver specimen and usedfor qPCR analysis (n � triplicate/sample). Graph on the right shows the average of the corresponding results on the left. Data are expressed as means � S.E. (errorbars). Each bar represents an individual liver. C, WB of Egr-1 protein in WT and SHP�/� livers. Each lane represents an individual mouse. D, WB of EGR-1 and SHPproteins in 5 normal and 8 cirrhotic human livers. Each lane represents an individual liver. Anti-EGR-1 and anti-SHP antibodies were used, respectively.

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explore in future studies. It should be restated that HNF4�exerts a dual role in activating both Egr-1 and SHP, and theactivated SHP in turn inhibits the activity of HNF4� to induceEgr-1. Such regulation represents a fine tuning control of Egr-1expression through a feedback loop between SHP and HNF4�that is likely mediated by the circadian clock.A recent study showed that overexpression of HNF4� allevi-

ated hepatic fibrosis in rat BDL or dimethylnitrosaminemodels(40). The authors propose that suppression of the epithelial-mesenchymal transition occurs through induction of E-cad-herin. Egr-1 levels, as well as other fibrogenic genes, were notanalyzed in the rat study, leaving unknown the association ofthe observed effect of HNF4� with Egr-1. It is presumable thatHNF4� is a pleiotropic factor that regulates multiple cellularprocesses, and the consequences of its activation can be dra-matically different depending on cellular milieu and contexts.In conclusion, our studies present convincing evidence that

the expression of Egr-1 is regulated by nuclear receptor signal-ing through SHP and HNF4� cross-talk. It helps better ourunderstanding of the molecular basis that controls Egr-1expression and function in liver fibrosis, which may pave theway of developing future therapeutic agents for the treatmentof liver fibrosis.

Acknowledgments—We thank Dr. Scott Friedman for the LX2 cellsand Drs. Frances Sladek and Akiyoshi Fukamizu for the HNF4� con-structs. Normal human liver and cirrhotic liver specimens wereobtained through the Liver Tissue Cell Distribution System (Minne-apolis, MN), which is funded by National Institutes of Health Con-tract N01-DK-7-0004/HHSN267200700004C.

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HNF4� and SHP Regulation of Egr-1

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Yuxia Zhang, Jessica A. Bonzo, Frank J. Gonzalez and Li WangCross-talk in Liver Fibrosis

) and Small Heterodimer Partner (SHP)α (HNF4αHepatocyte Nuclear Factor 4Diurnal Regulation of the Early Growth Response 1 (Egr-1) Protein Expression by

doi: 10.1074/jbc.M111.253039 originally published online July 3, 20112011, 286:29635-29643.J. Biol. Chem. 

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