Proc. Natd. Acad. Sci. USAVol. 91, pp. 5987-5991, June 1994Cell Biology

Identification of binding sites for transcription factors NF-cB andAP-2 in the promoter region of the human heme oxygenase 1 gene

(eyhroeukemlc c /stre proein/derenatn)

YAN LAVROVSKY*, MICHAL L. SCHWARTZMANt, RICHARD D. LEVERE*, A1TALLAH KAPPAS*,AND NADER G. ABRAHAM*t*The Rockefeller University Hospital, New York, NY 10021; and tDepartment of Pharmacology, New York Medical College, Valhalla, NY 10595

Communicated by Eugene P. Cronkite, January 6, 1994

ABSTRACT Heme oxygenase (HO) is the rate-limitingenzyme in heme catabois and its activity is induced by manyagents, including its substrate heme, heavy metals, UV radi-ation, and other injurious oxidant conditions. We examined thepresence of several regulatory elements in the promoter regionof the human HO-1 gene which could possibly account for itsinduction in response to diverse agents or influences. Hemetreatment increased both HO activity and HO-1 mRNA in thehuman erythroleukemic cell line K562. Electrophoretic mobil-ity-shift assays of nuclear protein extracts from heme-treatedand control cells with specific oligonuceotide probesconingbinding sites for known tanscription factors, incuding AP-1,AP-2, Spl, NF-acB, CTF/NF1, TFY , OKT1, and CREB, andoligonucleotides containing serum-, metal-, and glcocorticoid-responsive elements demonstrated a specific and marked in-crease in the NF-ucB and AP-2 tanscription factors and, to alesser extent, an increase in AP-1. No significant increase inother nscription factors over the control, untreated cells wasobserved. DNase I footprint assays using purified transriptionfactors revealed the presence of NF-ucB and AP-2 binding sitesin the proximal part ofthe promoter region ofthe human HO-1gene. Moreover, nuceotide sequence analysis of the HO-1promoter region showed that the protected regions encom-passed NF-#cB and AP-2 consensus binding sites. The presenceof regulatory sequences for the binding of trnscription factorssuch as NF-HB and AP-2, whose activation is aocited withthe immeiate response of the cell to an injury, may be anindication of the important role which HO-1 may play indefense mechanisms against tissue injury.

Heme oxygenase (HO, EC 1.14.99.3) is the initial and rate-limiting enzyme in heme catabolism. The enzyme oxidativelyruptures the heme to form biliverdin, which is subsequentlyconverted to bilirubin by biliverdin reductase. The hememolecule has a central role in biological processes, serving asthe prosthetic moiety of hemeproteins involved in cell res-piration, energy generation, oxidative biotransformation,and growth differentiation processes. The regulation ofHO isthus fundamental for the homeostasis of the cell. In experi-mental animals, HO is increased in conditions such asspontaneous or chemically induced liver tumors and Gram-positive bacterial infections (1-5). HO activity is also in-creased in whole animal tissues following treatment with itsnatural substrate, heme, as well as metals, xenobiotics,endocrine factors, and synthetic metalloporphyrins (1-3).Many cells in culture including hemopoietic, hepatic, epithe-lial, and endothelial cells respond to these agents in a similarway-i.e., by a marked increase in HO activity (1-8). Fur-thermore, HO is a heat shock protein (9, 10), and also a stress

protein induced following oxidative damage with free radicalsand with UV radiation (11, 12).Two HO isozymes, the products of two distinct genes,

have been described (13, 14). HO-1 is the inducible formwhich is ubiquitously distributed in mammalian tissues,whereas HO-2 is believed to be constitutively expressed, isnot inducible by HO-1 inducers, and is present in tissues suchas the brain and testis (14).One of the mechanisms by which hormones, growth fac-

tors, and other stimuli induce the expression of genes is byactivating various transcription factors. This is a rapid pro-cess which frequently involves transcriptional or structuralactivation of the factor and allows its presence or transfer tothe nucleus. These processes may be part of the mechanismby which various agents, including heme, increase HO ex-pression and activity. Previous studies have shown thepresence of AP-1-binding sequences and interleukin 6-, met-al-, and heat-responsive elements in the HO-1 promoterregion and have suggested the involvement of these nuclearfactors in the regulation of several genes, including thatencoding HO-1 (8-10, 13-18). Erythropoietic cells are en-dowed withHO activity which is inducible by heme (6,7). Wetherefore used a human-derived erythroleukemic cell line,K562, to examine the presence of transcription factors whichmight be involved in heme-induced HO-1 expression and todetermine whether binding sequences for these factors werepresent in the promoter region of the human HO-1 gene.

MATERIALS AND METHODSCell Culture. K562 cells were obtained from the American

Type Culture Collection and grown in RPMI 1640 mediumwith 10% fetal bovine serum (GIBCO) supplemented with 20mM Hepes. Cells were seeded at 105 cells per ml in 75-cm2culture flasks and incubated in a humidified atmosphere at5% C02/95% air for 24 hr. Heme (10 pM; Sigma) was addedto the experimental flasks for 0, 5, 10, 20, 30, 40, and 80 min.RNA Ex io and Northern Blot Analysis. Total RNA

was isolated by lysis of the cells in 4 M guanidinium isothio-cyanate and quantitated by spectrophotometry (19). Tenmicrograms of total RNA was denatured and size-separatedby electrophoresis in 1.2% agarose gels containing 2.2 Mformaldehyde. To verify the integrity of the samples, gelswere stained for 15 min with ethidium bromide (0.5 pg/ml) indiethyl pyrocarbonate-treated water, destained, and photo-graphed. RNA was transferred to nylon membranes (Gene-ScreenPlus membranes, NEN) in 20x SSC [lx SSC (stan-dard saline citrate) is 0.15 M NaCl/0.15 M trisodium citrate,

Abbreviations: HO, heme oxygenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility-shiftassay; GRE, glucocorticoid-responsive element; MRE, metal-responsive element; SRE, serum-responsive element; HSE, heatshock-responsive element; fpu, footprint unit(s).tTo whom reprint requests should be addressed.

5987

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5988 Cell Biology: Lavrovsky et al.

pH 7.0]. The membranes were then baked for 2 hr at 80(C.Blots were hybridized with the EcoRI-EcoRI cDNA frag-ment of the human HO-1 gene (7) (specific activity, 5 x 108cpm/pug) at 600C overnight and washed according to Churchand Gilbert (20). The hybridization mixture consisted of 1%bovine serum albumin, 7% SDS, and 1 mM EDTA in 3 x SSC.Filters were exposed to Kodak XAR film at -70TC. As acontrol, all filters were reprobed with cDNA encoding glyc-eraldehyde-3-phosphate dehydrogenase (GAPDH) (Clon-tech) to ensure that equal amounts of RNA had been loadedonto each lane.

Eectrophoretic Mobility-Shift Assay (EMSA). The syn-thetic oligonucleotides corresponding to the phorbol 12-myristate 13-acetate ("12-O-tetradecanoyl 13-acetate,"TPA)-responsive element (TRE) of the metallothionein pro-moter region (5'-GATCCATGAGTCAGAG-3'), the serum-responsive element (SRE) of the c-fos promoter region (5'-CGGATGTCCATATTAGGACATCTGCGTCAGCAG-3')(21), and the metal-responsive element (MRE) (5'-CGATCTCTGCACTCCGCCCGA-3') (22) were synthesizedby a standard automated method. Double-stranded oligonu-cleotides corresponding to the AP-2, Spl, NF-icB, TFIID,OKT1, CREB, and CTF/NF1 binding sites and to the glu-cocorticoid-responsive element (GRE) were purchased fromPromega. All oligonucleotides were end-labeled with[y-32P]ATP and polynucleotide kinase and purified by poly-acrylamide gel electrophoresis followed by reverse-phaseHPLC (Silasorb C8, 0-50%6 gradient of acetonitrile with 20mM LiCl04). Nuclear protein extracts were prepared fromcontrol and heme-treated cells by the method ofDigam et al.(23). End-labeled double-stranded oligonucleotides (10,000cpm; specific activity, %3000 Ci/mmol; 1 Ci = 37 GBq)corresponding to the various binding sites (described above)were incubated with 2 /g of sonicated calf thymus DNA and5 pg ofnuclear protein extract in 20mM Hepes, pH 8.0/5mMdithiothreitol/i mM EDTA/0.5 mM phenylmethanesulfonylfluoride/50 mM KCI/5 mM MgC12/5% (vol/vol) glycerol(EMSA buffer) for 15 min at room temperature. Controlcompetition experiments included 10- and 100-fold molarexcess of specific and nonspecific oligonucleotides. Reactionproducts were fractionated by electrophoresis in 3% Meta-phor agarose (FMC) in 25 mM Tris/25 mM boric acid/i mMEDTA, pH 8.3. Gels were dried and exposed to KodakX-Omat film at -70TC with an intensifying screen.DNase I Footprint Analysis. A 590-bp probe was prepared

by PCR using forward primer A (5'-TGACATTTTAGG-GAGCTGGA-3') and reverse primer D (5'-TTGCCT-GTCGGGTTGC-3'), corresponding to positions -480 and+ 110 ofthe human HO-1 gene, respectively (ref. 16, see Fig.3). Primers were chosen by using the OLIGOPRIMERS program(Clontech). PCR was performed by incubating 100 ng ofeachprimer and 10 ng of plasmid containing the human HO-1 gene(16) as follows: 5 min at 94°C; 30 sec at 94WC; 30 sec at 50°C;and 1 min at 72°C for 20 cycles; and 10 min at 72°C. The PCRproduct was purified with the Magic PCR Preps DNA puri-fication system (Promega) and was end-labeled with[y.32P]ATP and polynucleotide kinase. The labeled PCRproduct was digested with Pst I restriction endonuclease.Alternatively, the PCR product was first digested with Pst IandXho I and then end-labeled by use of [a-32P~dCTP, dTTP,and DNA polymerase I Klenow fiagment. Approximately 104cpm of end-labeled, polyacrylamide gel-purified probe wasincubated with 1 footprint unit (fpu) ofAP-1, AP-2, or NF-KB(p5O) protein (Promega) in EMSA buffer (described above)for 15 min on ice and then for 2 min at 25°C. DNase I (1unit/ml) was added for 1 mim at 25°C, and the reactions wereterminated by the addition ofan equal volume ofstop solution(0.5 M sodium acetate/0.1 M EDTA/1% SDS containingyeast tRNA at 100 pg/ml). DNA was extracted with phenol/chloroform, precipitated with ethanol, and analyzed by 8 M

urea/6% acrylamide gel electrophoresis in 90 mM Tris/90mM boric acid/i mM EDTA, pH 8.0.

RESULTSEffect of Heme on HO-i mRNA and Transcription Factor

Binding Activity. Treatment of K562 cells with heme resultedin an increased amount of HO-1 mRNA as compared withuntreated cells, confirming previous reports from our andother laboratories using other cell lines, including erythro-leukemic cells (6, 7, 13, 16). Northern hybridization of totalRNA with acDNA probe for human HO-1 revealed a markedincrease in HO-1 mRNA caused by heme within 20 min (Fig.1). Rehybridization of the membranes with a cDNA probe forGAPDH showed no difference in the amount of RNA be-tween control and heme-treated cells (Fig. 1). The maximalincrease in HO-1 mRNA levels occurred after 30-40 min, aspreviously described (6).

It is possible that transmission ofthe heme signal to inducethe HO-1 gene is mediated by activation of specific transcrip-tional factors. We therefore measured the effect of heme onthe activation of transcriptional factors by EMSA. We useddouble-stranded oligonucleotides corresponding to bindingsites of transcription factors which might be involved in theHO-i promoter. These include AP-1, AP-2, NF-KB, Spl,CTF/NF1, TFIID, OKT1, and CREB binding sites, SRE,MRE, and GRE (24). A significant increase in AP-1, CTF/NF1, and GRE binding activities was found at 30 min, as wasa slight decrease in nuclear factor binding to SRE and CREBoligonucleotides in heme-treated K562 cells (Fig. 2a). Themost pronounced effect of heme, however, was on NF-KBand AP-2 binding activities. Moreover, this increase wasstrikingly evident within 5 min and was sustained over a i-hrperiod (data not shown), supporting the contention that hemeis a powerful activator of these transcription factors.The binding specificity to AP-2 and NF-KB was confirmed

by EMSAs using specific and nonspecific competitor oligo-nucleotides. Incubation of nuclear extracts from heme-treated cells with AP-2 and NF-KB oligonucleotides resultedin a marked increase in binding activity (Fig. 2b, lanes A andE, respectively). Addition of a 100-fold molar excess ofnonspecific competitor did not change AP-2 and NF-KBbinding activity in nuclear extract (lanes B and F, respec-tively). In contrast, addition of 10- and 100-fold excesses ofspecific competitors (unlabeled AP-2 and NF-KB oligonucle-otides) decreased the binding of nuclear extract protein(s) toAP-2 (lanes C and D, respectively) and NF-KB (lanes G andH, respectively) oligonucleotides.

_HO-1

GAPDH

FIG. 1. (Upper) Northern hybridization of a 32P-labeled cDNAprobe of the human HO-i gene with total RNA (10 pg per lane)derived from control (lane 1) or heme-treated (lane 2) K562 cells.Confluent cultures were treated with heme (10 pM) for 30 min andtotal RNA was extracted. (Lower) Control hybridization with aGAPDH cDNA probe.

Proc. Nad. Acad. Sci. USA 91 (1994)

Proc. Nadl. Acad. Sci. USA 91 (1994) 5989

b A B C D E F G Has t- 0 d* f*_

a

A B A B A B A Ba,.:,.^,1' r. ift.

A B A B A B A B A B

AN -4-

A B

mo.1I

AP-1 Spi CTF/NF I AP-2 NF-KB CRE B GRE SRE OKT1 TFIID MRE

FIG. 2. EMSA using oligonucleotides corresponding to various regulatory elements and nuclear extract from control and heme-treated K562cells. (a) Five micrograms of nuclear protein extract from untreated (lanes A) or heme-treated (lanes B) K562 cells was incubated with 10,000cpm of radiolabeled oligonucleotides (3000 Ci/mmol). (b) Competition experiments to demonstrate binding specificity. AP-2 oligonucleotides(lanes A-D) and NF-KB oligonucleotides (lanes E-H) were incubated with nuclear extract from heme-treated K562 cells in the presence of nocompetitor (lanes A and E), a 100-fold molar excess of nonspecific competitor oligonucleotides (anes B and F), or a 10-fold (lanes C and G)or 100-fold (lanes D and H) molar excess of specific oligonucleotides (AP-2 and NF-KB, respectively).

Presence ofAP-2 and NF-ucB Transcription Sites in the HO-1Promoter Region. To determine whether the HO-1 promoterregion contained AP-2 and NF-KB binding sites, the HO-1promoter region was amplified by PCR; two fragments, alarge fragment (bp -1500 to +110) and a small fragment (bp-480 to + 110) (Fig. 3) were examined. Both fragments wereused for DNase I footprinting analysis. The results arepresented in Figs. 4 and 5. DNase I footprinting analysisusing PCR-amplifled, end-labeled promoter fragments (XhoI-Pst I), indicated in Fig. 3, demonstrated that addition ofeither AP-1 or AP-2 proteins to the incubation mixture did notresult in any specific protection (Fig. 4a, lanes 2 and 3,respectively). In contrast, incubation ofNF-KB (p50) proteinwith the same PCR products resulted in specific protectionagainst DNase I digestion (lane 4). The specificity of thisprotection was assessed by incubation with specific NF-KcBcompetitor oligonucleotide and nonspecific AP-1 competitoroligonucleotide. The specific competitor blocked the protec-tion provided by the NF-KB protein (Fig. 4a, lane 5). Incontrast, incubation with the nonspecific competitor did notinfluence the protected band (lane 6). The same result wasobtained with another labeled fragment, extending from theD primer to the Pst I site (Fig. 3). Absence of nuclear proteindid not provide any protection for DNase I digestion (Fig. 4b,

lane 1). Similarly, the presence of 1 fpu or 10 fpu ofAP-1 didnot provide any protection (Fig. 4b, lanes 2 and 3). Incontrast, inclusion of 1 fpu of NF-#cB (p50) demonstrated amarked protected sequence (Fig. 4b, lane 4), similar to thatseen in Fig. 4a, lane 4. DNase I footprint assay was alsoperformed with a more distal part of the promoter region(from theA primer to the Pst I site; Fig. 3). No other sequencewas found to be protected in reactions when no nuclearextract protein was added (ane 6) or in the presence ofAP-1,AP-2, or NF-icB protein (Fig. 4b, lanes 7-9, respectively),suggesting that these transcriptional sites are not present inthe distal part of the HO-1 promoter region.DNase I footprint assay also revealed the presence of the

AP-2 transcription site in the proximal part of the humanHO-1 promoter region close to the NF-icB site. Purifiedtranscription factor AP-2 protected the sequence marked inFig. 3 (Fig. 5, lane 2); and this protected region disappearedafter incubation with specific competitor DNA (AP-2 con-sensus oligonucleotide) (lane 3). Addition of nonspecificcompetitor DNA (AP-1 consensus oligonucleotide) to aDNase I reaction mixture (lane 4) did not result in competi-tion with the AP-2 protein in protection, which confirmed thespecificity ofthe reaction seen in lane 2. The location ofAP-2and NF-,cB transcriptional sites in the HO-1 promoter gene is

A HSE NFkB AP-2 TATA ATG D

5' 3'~~~~~~~~~~~~~~~~~~~~~~~~~~3-480 E -380 P 170T 150 .130 A -85+1 85

/ ~~~~~HSE\-390 CCCAGCI LIC TGGAACCIIC TGGGACGCCT GGGGTGCATC

-350 AAGTCCCAAG GGGACAGGGA GCAGAAGGGG GGGCTCTGGA

-310 AGGAGCAAAA TCACACCCAG AGCCTGCAGC TTCTCAGATT

-270 TCCTTAAAGG TTTTGTGTGT GTGTGTGTGT GTGTGTGTGT

-230 GTGTATGTGT GTGTGTGTGT GTGTGTGTGT GTGTTTTCTCNFkB

-190 TAAAAGTCCT ATGGCCAGAC TTTGTTTCCC AAGGGTCATAAP-2

-150 TGACTGCTCC TCTCCACCCC ACACTGGCCC GGGGCGGGCT

-110 GGGCGCGGGC CCTGCGGGTG TTGCAACGCC

FIo. 3. Schematic representation of the human HO-1 gene upstream region. The 5' (A) and 3' (D) primers used for the HO-1 gene promoterregion amplification are shown. HSE, heat shock-responsive element; NF-KB, site protected by NF-KB (p50) in DNase I footprint analysis; AP-2,site protected by AP-2 protein in DNase I footprint analysis; TATA, TATA-like element. ATG, initiation codon. Restriction sites for Eco47Il(E), Pst I (p), EcoTl41 (T), Ava I (Av), and Xho I (X) are indicated.

A B*X'5,

Cell Biology: Lavrovsky et al.

5990 Cell Biology: Lavrovsky et al.

b

a 1 2 3 4 5 6 7

NF-i<B-,(-B

NF

5 -G ACT TTGTTT CC CA A GGGTC AT -3

FiG. 4. DNase I footprinting analysis of the human HO-1 pro-moter in the presence of purified transcription factors. (a) The probeused covered the sequence from the Xho I site to the Pst I site-proximal part of the HO-1 promoter region (Fig. 3). Lane 1, withoutaddition of nuclear protein; lane 2, AP-M protein; lane 3, AP-2protein; lane 4, NF-KB (p50) protein; lane 5, the same as lane 4 plusspecific competitor oligonucleotide; lane 6, the same as lane 4 plusnonspecific competitor oligonucleotide; lane 7, G sequencing reac-tion (43). (b) The probe used covered the sequence from the D primerto the Pst I site-proximal part (lanes 1-5) and from the A primer, inthe distal region, to the Pst I site (lanes 6-11) (Fig. 3). Lanes 1 and6, no nuclear extract protein; lanes 2 and 7, 1 fpu ofAP-1 protein; lane3, 10 fpu of AP-M protein; lane 8, 1 fpu of AP-2 protein; lanes 4 and9, 1 fpu of NF-KB (p50); lanes 5 and 11, G sequencing reactions.

revealed within the nucleotide sequence in the HO-1 pro-moter region (Fig. 3). Among the several regulatory elementsshown in the nucleotide sequence are the AP-2 and NF-KBmotifs, which are located between the TATA-like elementand the HSE.

DISCUSSIONThe HO-1 promoter region has recently been studied forpossible regulatory elements which could account for itsinduction by various agents. Functional AP-1 binding siteswithin the HO-1 promoter have been identified, which sug-gested the involvement ofthe Jun/Fos family oftranscriptionfactors in mediating the induction ofHO-1 gene transcriptionby multiple agents (8). Other transcription factors may alsobe involved in the induction of HO-1, as indicated by thepresence ofconserved MREs and HSEs within the promotersof several HO-1 genes. Two interleukin 6-responsive ele-ments were also found in the promoter region of the human

AP-2

__ t,010FIG. 5. DNase I footprinting analysis of the human HO-1 pro-

moter in the presence of AP-2 protein. The probe used covered thesequence from the Xho I site to the Pst I site (Fig. 3). Lane 1, Gsequencing reaction; lane 2, 1 fpu of purified AP-2 transcriptionfactor (Promega); lane 3, the same as lane 2 plus specific competitor;lane 4, the same as lane 2 plus nonspecific competitor; lane 5, noprotein.

HO-1 gene (26). The diversity of HO-1 inducers suggestsmultiple regulatory elements for this gene. Indeed, the pres-ent study identified two additional regulatory elements, bind-ing sites for NF-KB and AP-2, which may be implicated asimportant factors mediating the role ofHO in the response tooxidative stress/injury and in growth/differentiation pro-cesses.The finding ofAP-2 and NF-KB binding sites on the human

HO-1 promoter suggests the importance ofHO-1 in processeswhere these transcription factors are activated. The tran-scription factor AP-2 was first purified from HeLa cells (27).Functional AP-2 binding sites have been identified in theenhancer regions of viral and cellular genes, including simianvirus 40, human metallothionein IIA, human T-cell leukemiavirus type I, murine major histocompatibility complex, andhuman keratin K14 genes (27-33). Phorbol esters, cAMP, andretinoic acid have been shown to induce AP-2 activity, whichis believed to play a role in regulating the expression ofgenesinvolved in cellular differentiation (34-37). The importance ofAP-2 in regulating HO-1 induction and the relation to cellgrowth and differentiation is still unclear and further func-tional studies are needed.NF-KB is a transcription factor that is activated in many

different cell types following a challenge with primary (vi-ruses, bacteria, and stress factors) or secondary (inflamma-tory cytokines) pathogenic stimuli (25, 38-41). The activatedfactor then leads to a rapid induction of genes encodingdefense and signaling proteins, implicating NF-KB as animmediate early mediator of immune and inflammatory re-sponses. There is also evidence that NF-KB and relatedproteins are involved in growth control (39, 40, 42). ThatNF-KB activation occurs shortly after addition ofheme to thecells and that the HO-1 promoter region contains an NF-KB

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Proc. Nad. Acad. Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 5991

binding site suggest a role for this transcription factor in theregulation of HO-1 expression. The latter process shares acommon feature with NF-KB activation-i.e., induction byprimary and secondary pathogenic stimuli. Through a com-puter search, Rizzardini et al. (15) found sequences on theHO-1 gene that perfectly share recognition sequences forNF-IL-6 and NF-KB. It is therefore possible that the imme-diate induction in HO-1 expression which results from cellexposure to injurious stimuli, as well as inflammatory cyto-kines, is mediated in part by the activation of NF-KB and/orAP-2 and its binding to the corresponding regulatory se-quences to bring about an increase in HO-1 transcription.

We thank Dr. S. Shibahara for his generous gift of human HO-1DNA. This work was supported by Grants EY06531 and HL34300from the National Institutes of Health.

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