Distal AP-1 Binding Sites Mediate Basal Level Enhancement and

THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 267, No. 30, Issue of October 25, pp. 21894-21900,1992

Printed in U. S.A.

Distal AP-1 Binding Sites Mediate Basal Level Enhancement and TPA Induction of the Mouse Heme Oxygenase-1 Gene*

(Received for publication, May 29, 1992)

Jawed Alam and Den Zhining From the Department of Molecular Genetics, Alton Ochsner Medical Foundation, New Orleans, Louisiana 70121

Basal expression of a chimeric gene (pMH04CAT) consisting of approximately 7 kilobase pairs (kbp) of the 6”flanking region of the mouse heme oxygenase-1 (HO-1) gene fused to the bacterial chloramphenicol acetyltransferase gene is 2- to 10-fold greater than that of an analogous construct containing only 1287 bp of the 6”flanking region (pMHO1CAT) in transiently transfected cultured cells. The enhancer activ- i ty has been localized to a 268-base pair (bp) fragment positioned approximately 4 kilobase pairs upstream of the transcription initiation site. This fragment contains two high affinity protein binding sites, regions A and B, as determined by DNase I protection assays using nuclear protein extracts from rat C6 glioma cells. Both sites include core sequence elements, TGAGTCA (region A) and TGTGTCA (region B), that resemble the consensus binding site, TGA(G/C)TCA, of the Jun/ Fos (AP-1) family of transcription factors. Purified, bacterially expressed AP-1 (c- Jun homodimer) specifically binds to both elements, exhibiting greater affinity for the region A motif. The expression of pMH04CAT, but not of pMHOlCAT, is stimulated by the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), and the 268-bp enhancer fragment confers TPA inducibility and c- Jun/c-Fos transactivation to the heterologous SV40 promoter. These functions are mediated by the AP-1 binding sites as multiple copies of the region A motif also confer TPA induction and c- Jun/c-Fos transactivation upon a heterologous promoter.

Tumor-promoting phorbol esters, such as TPA,’ induce a variety of changes in cell morphology, metabolism, and gene expression (reviewed in Refs. 1 and 2). These alterations are largely a consequence of the activation of protein kinase C, the principal cellular target of TPA. As various extracellular stimuli, including antigens, hormones, and growth factors, utilize the protein kinase C signal transduction pathway, the changes in gene expression mediated by these agents overlap with those induced by TPA. Some of the proteins intrinsic to growth factor-dependent proliferative responses that are also

* This work was supported in part by Public Health Service Grant DK-43135 and by funds from the Cancer Association of Greater New Orleans. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTM/EMBL Data Bank with accession number(s) The nucleotide sequence(s) reported in thispaper has been submitted

X66847. The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-

acetate; heme, ferriprotoporphyrin IX; TRE, TPA-responsive element; CAT, chloramphenicol acetyl transferase; bp, base pair(s); kbp, kilobase pair(s).

stimulated by TPA include those encoded by the proto-on- cogene c-fos (3), the human metallothionein IIA gene (4), and the collagenase gene (5 ) . Transcriptional activation of these genes by TPA is mediated by a discrete DNA element, termed the TPA-responsive element (TRE), that was initially recog- nized as the binding site for the transcription factor AP-1 (6, 7).

Although purified preparations of AP-1 from HeLa cells are highly enriched for a single polypeptide (7), subsequent studies (reviewed in Refs. 8 and 9) have shown that AP-1 represents a family of transcription factors comprised of homo- and heterodimers of individual members of the Jun- and Fos-related families of proteins. Of these proteins, c-Jun and C-FOS, products of the proto-oncogenes c-jun and c-fos, respectively, have been most extensively characterized. c-Jun (and other Jun-related proteins) form thermostable homodimers, whereas c-Fos does not homodimerize and only forms stable heterodimers with the members of the Jun family. The stability of the c-Jun:c-Fos heterodimer is significantly greater than that of the c-Jun homodimer (10) although both complexes appear to have equal affinity for the TRE (11). The activity of certain members of this family of transcription factors is increased either indirectly, through protein kinase C activation (12), or directly by mitogen activated kinases (13). These observations are consistent with the notion that the AP-1 family of proteins modulate the transcription of select cellular and viral genes in response to environmental cues (8).

The most prominent and earliest change in gene expression, as determined by one-dimensional sodium dodecyl sulfate- polyacrylamide gel electrophoresis, observed after treatment of BALB/c 3T3 mouse fibroblasts with TPA (14) and indole alkaloid and polyacetate tumor promoters (15), is the increased synthesis of a 32-kDa protein, subsequently identified as heme oxygenase-1 (HO-1) (16). TPA also induces the steady-state level of HO-1 mRNA in human skin fibroblast cells (17). In this report, we have identified two DNA elements within the 5”flanking region of the mouse HO-1 gene, located approximately 4 kbp upstream of the transcription initiation site, that resemble the consensus TRE, TGA(G/C)TCA (6). Results from several lines of investigation strongly indicate that these elements mediate TPA activation of HO-1 gene transcription: 1) purified, bacterially expressed AP-1 (c-Jun homodimer) binds specifically to these elements, 2) a 268-bp fragment containing both AP-1 binding sites, or multiple copies of a single motif, confers TPA inducibility upon homologous and heterologous promoters, and 3) these same constructs are transactivated by overexpression of c-Jun and c-Fos proteins in cotransfection studies. These and other observations are described below in more detail.

EXPERIMENTAL PROCEDURES

Materials-Restriction endonucleases and other DNA-modifying enzymes were purchased from New England Biolabs. RNases A and

21894

JunlFos Regulation of HO-1 Gene 21895

T1 were from GIBCO/BRL and RNase-free DNase I was from Worthington. All radiochemicals were obtained from Du Pont-New England Nuclear. DNA sequence analysis was carried out by the chain-termination method (18) using the Sequenase version 2.0 kit from United States Biochemicals. All enzymes and reagents for CAT and luciferase assays were purchased from Sigma. Purified, bacterially expressed AP-1 (c-Jun homodimer) was obtained from Promega Biotech. All other chemicals were reagent grade.

Plasmid Constructs-The plasmid constructs described below are illustrated (see Fig. 2). pMHOlCAT was constructed by cloning a BamHIIXhoI fragment (-1287 to +73) of the mouse HO-1 gene between the BamHIISalI sites of the promoterless CAT vector, pSKCATASph (19, 20; hereafter referred to as pSKCAT). A 2.2-kbp BamHIIBamHI fragment (-3.5 kb to -1283) was cloned into the BamHI site of pMHOlCAT to yield pMH03CAT. A 7-kbp XhoI/ XhoI fragment was blunt-ended by the fill-in reaction using the Klenow Fragment of Escherichia coli DNA polymerase 1 and partially digested with BamHI. A 5800-bp XhoI (blunt-ended)/BamHI fragment (-7 kb to -1283) was cloned between the Not1 (blunt-ended)/ BamHI sites of pMHOlCAT to generate pMH04CAT. pMH04CAT was digested with BamHI and religated to produce pMH04CATAB. Similarly, pMH04CAT was digested with BamHI and XbaI, blunt- ended, and religated to generate pMH04CATABX. The remainder of the constructs diagrammed (see Fig. 2) were prepared in the following manner: the 5”upstream fragments were isolated after digestion of the 7-kbp XhoI/XhoI fragment with the appropriate restriction endonucleases (see legend to Fig. 2) and cloned into the BamHI (blunt-ended) site (blunt-ended fragments AX1 and BSl), or into the XbaI site (fragment X1) or between the XbaI and SacI sites (fragments SX2, SX3, and SX4) of pMHO1CAT.

SmaIlSalI sites of pSKCAT to give pMHOlCATA-33. pSV2CAT An RsaIIXhoI fragment (-33 to +73) was cloned between the

contains the enhancer and promoter elements of the SV40 early gene regulatory sequences and has been described previously (21). The corresponding enhancerless construct, pSKSVCAT, was derived by cloning a 206-bp fragment (containing the TATA box and the 21-bp repeats) of pSV2CAT into pSKCAT. Derivatives of pMHO1CATA- 33 and pSKSVCAT were generated by cloning appropriate restriction endonuclease fragments in a manner analogous to that described in the preceding paragraph. Plasmid p4xACAT contains four tandem copies of an AP-1 binding site (derived from region A of the HO-1 enhancer fragment) cloned in the BamHI site upstream of the SV40 promoter in plasmid pSKSVCAT. The complementary oligonucleo- tides used to prepare this construct are 5”GATCTTGCTGAGT- CACCCTCT-3’ and 5’-GATCAGAGGGTGACTCAGCAA-3’. Plas- mid pRSVluc was constructed by cloning the Rous sarcoma virus long terminal repeat into the promoterless luciferase reporter vector pXPl (22). The expression of the CAT and luciferase reporter genes in pMHPX4CATA-192 and pMHPX4lucA-192, respectively, is under the control of the mouse hemopexin gene’ regulatory sequences (-192 to +25). The human c-Fos and c-Jun expression plasmids, CMV-Fos and CMV-Jun (23), were kindly provided by Dr. Tom Curran (Roche Institute of Molecular Biology).

Cell Culture, Transfection, and Enzyme Assays-Mouse fibroblast L929 cells, rat C6 glioma cells, and mouse hepatoma Hepa cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 0.35% glucose. Mouse F9 teratocarcinoma cells were cultured in the same medium on gelatin-coated flasks/plates. Human hepatoma Hep G2 cells were cultured in Earle’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Transient transfections were carried out by the calcium phosphate- precipitation technique (24). CAT activity in cell extracts was determined by the procedure of Nordeen et al. (25), and luciferase assays were carried out as previously described (26) using the Monolight 2010 luminometer (Analytical Luminescence Laboratory). Additional details are provided in the figure legends.

Preparation and Analysis of RNA-Total RNA from transiently transfected cells was isolated by a modification (27) of the procedure of Chomczynski and Sacchi (28). The level of correctly initiated mRNA species was measured by RNase protection assays using standard techniques (29). The in vitro generated RNA probe used for these assays is 151 bases in length and contains sequences complementary to residues -63 to +73 of the mouse HO-1 gene and 15 bases of polylinker sequence from pMHO1CAT. This single probe detects both correctly initiated endogenous HO-1 mRNA (73 bases) and HO- 1/CAT fusion mRNA (88 bases).

J. Alam, unpublished data.

DNase I Protection Analysis-Nuclear protein extracts from rat C6 glioma cells were prepared according to Dignam et al. (30). The extracts were fractionated with 50% ammonium sulfate, and the precipitated proteins were resuspended and dialyzed (2 X) for 5 h against 20 mM Hepes-KOH (pH 7.9) containing 0.1 M KC1, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine, and 20% glycerol. The dialysate was clarified by centrifugation and stored a t -70 “C. Fragment SX2 probes were generated as follows: plasmid pMHOlCAT+SX2 (see Fig. 2) was linearized by digestion with XbaI or BssHII (which cleaves at a vector encoded sequence upstream of the SacI site). A 1-pg sample of each linear plasmid was labeled by the “end fill” reaction using the Klenow fragment of E. coli DNA polymerase I and a dNTP mixture containing [ c Y - ~ * P ] ~ C T P and [cY-~*P]~ATP. The probe fragments, individually labeled on the noncoding strand (XbaI-digested plasmid) or the coding strand (BssHII-digestedplasmid) were liberated by subsequent digestion with SacI or XbaI, respectively, and purified by polyacrylamide gel electrophoresis. The HO-1 proximal promoter fragment, which contains genomic sequences from -149 to +73 was labeled and purified in an analogous manner. Protein binding reactions and DNase I digestion were carried out as described by Lee et al. (7) with slight modifications. Briefly, the labeled fragment (20,000 cpm) was mixed with varying amounts of crude protein extract or purified protein in a total volume of 50 pl with a final buffer concentration of 10 mM Tris-HC1/8 mM Hepes-KOH (pH 7.9), 0.5 mM EDTA, 40 mM KCl, 0.7 mM dithiothreitol, 1 pg of poly[d(I-C)], 10% glycerol, and 2% polyvinyl alcohol. The mixture was prepared on ice and incubated for 20 min at ambient temperature. The DNA/protein mixture was digested for 30 s with addition of 100 pl of freshly prepared solution (70-550 ng/ml) of DNase I in 10 mM MgCl, and 4 p~ CaC1,. The reaction was terminated by addition of 150 p1 of a solution containing 8 M urea, 0.5% sodium dodecyl sulfate, 5 mM EDTA, and 10 pg/ml denatured salmon sperm DNA and subsequently extracted with phenol/chloroform (1:l mixture). Digestion products (4000 cpm) were co-electrophoresed with the purine sequence ladders (31) of the DNA probes on a 6% polyacrylamide/8 M urea gel.

RESULTS

5”Distal Sequences Enhance Basal Activity of the HO-1 Gene Promoter-Induction of the mouse HO-1 gene by heme and heavy metals is controlled primarily at the level of transcription initiation (32). However, an isolated genomic clone, containing the entire coding portion of the mouse HO-1 gene and approximately 3 and 1.5 kbp of the 5‘- and 3”flanking regions, respectively, is not inducible by either agent after transient transfection into cultured cells3 No induction by Cd2+ or Zn2+ is observed despite the fact that this clone contains two sequence motifs that resemble the metal regulatory elements of the metallothionein genes (33).

Reasoning that distal sequences may be necessary for induction of the mouse HO-1 gene by heavy metals and heme, we have isolated additional recombinant X clones containing up to 13 kbp of the 5“flanking region of this gene (data not shown). A chimeric CAT gene containing approximately 7 kbp of mouse HO-1 promoter sequence (pMH04CAT) is still uninducible by Cd” or heme after transient transfection into mouse L cells (Fig. 1) or human hepatoma (Hep G2) cells (data not shown). The apparent induction of the fusion gene by Cd2+ (compare lanes 2 and 4 ) has not been observed consistently and, in this experiment, may reflect variations in transfection efficiency. The high basal expression of the HO- 1/CAT fusion gene, compared to that of the endogenous HO- 1 gene, suggests that the transfected HO-1 promoter is aber- rantly regulated (e.g. loss of repression). This conclusion, however, is tentative at best as the increased expression of the chimeric gene may be due to other factors such as the relatively high copy number of transfected DNA. In any case, we have observed that the basal activity of pMH04CAT is consistently 2- to 4-fold greater than that of constructs containing 1287 bp (pMHO1CAT) or 3.5 kb (pMHO3CAT) of

J. Alam, submitted for publication.

21896 JunlFos Regulation of HO-1 Gene

” i - e

? -

1 2 3 4 5 6 7 8 010 Y 112

HOCAT 3

Ho- 59

59

FIG. 1. Effect of heme or CdC12 on the expression of heme oxygenase promoter/CAT fusion genes. L929 cells (approximately 1.5 X 106/100-mm plate) were plated 16 h prior to transfection. Cells were transfected with a total of 20 pg of DNA containing 1 pmol of pSKCAT (lane I), pMHOlCAT (lanes 2-4), pMH04CAT (lanes 5-7), or pMH03CAT (lams 8-10) and the appropriate amount of pBSSK- for 4 h. After incubation for 16 h in complete medium, the cells were exposed for 3 h to serum-free medium containing vehicle (lams 1,2,5, and 8), 10 p~ heme (lanes 3 ,6 , and 9) , or 10 p~ CdClz (lams 4, 7, and 10). RNA was isolated and analyzed as described under “Experimental Procedures.” The endogenous HO-1 mRNA (HO) and the transfected fusion gene mRNA (HOCAT) are marked. The size, in bases, of the marker fragments ( M ; Hue111 digestion products of plasmid Bluescript I1 SK- (pBSSK-)) is indicated.

the HO-1 gene 5”flanking region (Fig. 1, compare lune 5 with lunes 2 and 8). This result suggests that 5’-distal sequences enhance the basal expression of the mouse HO-1 gene.

In order to further localize the putative enhancer element(s), subfragments of the distal 5”flanking region were cloned upstream of pMHOlCAT and analyzed for enhancer activity in transient expression assays. As the enhancer activity was found to be significantly greater in rat C6 glioma cells, most of the analysis was carried out using this cell line. A -270-bp fragment (SX2), located approximately 4 kpb upstream of the transcription initiation site, increases the expression of pMHOlCAT by approximately 10-fold (Fig. 2). For comparison, an adjacent fragment of similar size (SX3) is without effect. Fragment SX2 is equally effective in either orientation (data not shown). To test if SX2 can enhance the activity of a heterologous promoter, this fragment was cloned upstream of a chimeric CAT gene under the control of the SV40 early gene regulatory sequences (pSKSVCAT; this fusion gene lacks the SV40 72-bp enhancer and is only weakly active in most cell types). The resulting construct, pSKSVCAT+SX2, produces a high level of CAT activity in C6 and Hepa cells (Fig. 3). Similarly, the SX2 fragment can activate an inactive promoter. pMHO1CATA-33, which contains only 33 bp of the HO-1 5”flanking sequence, including the TATA box, is inactive in several cells examined: but under the influence of the SX2 fragment (pMHO1CATA- 33+SX2) generates a significant level of CAT activity. As before, the control fragment, SX3, exhibits no enhancer activity. These data demonstrate that a 5”distal sequence element(s) regulates the transcription of the mouse HO-1 gene and that this element functions as a classical enhancer. (Al- though we have not examined the activity of the SX2 fragment at a position downstream of the CAT gene, it is clear that this fragment is equally effective at various distances from the transcription initiation site.) The transcription factor(s) that interacts with the enhancer element appears to be ubiquitous as enhancer activity is observed in four different cell lines, derived from three types of tissues.

Identification of the Enhancer Element as the AP-1 Binding Site-The DNA sequence of fragment SX2 is shown in Fig. 4. In order to identify the enhancer element(s), the protein-

binding properties of this fragment was examined by DNase I footprinting analysis. Partially fractionated nuclear extracts from C6 cells consistently protect four regions from DNase I digestion (Fig. 5 ) . Transcription factors that bind within or in the vicinity of region D may include proteins that interact with CACCC box, an element initially identified in the promoter of the p-globin gene (34). Both regions C and D are only weakly protected by nuclear protein extracts from C6 cells or L929 cells (data not shown) and have not been examined further. Region A exhibits a relatively higher level of protection than region B when equivalent amounts of protein extracts are used. A closer examination of both protected sites reveals core sequences, TGAGTCA (region A) and TGTGTCA (region B), that are identical to, or closely resemble, the consensus binding site, TGA(G/C)TCA for the AP-1 family of transcription factors.

This, potentially large, class of transcription factor is comprised of homo- or heterodimers of individual members of the Jun- and Fos-related families of proteins. Binding of an AP- 1 type transcription factor to the HO-1 enhancer fragment was examined directly by DNase I protection assays using purified, bacterially expressed c-Jun homodimers. Previous studies (35) have shown that the bacterially expressed protein binds DNA with a specificity indistinguishable from that isolated from HeLa cells. The c-Jun homodimer specifically binds to region A and the nucleotide residues protected from DNase I digestion are identical to those protected by partially fractionated nuclear extracts from C6 cells (Fig. 6). The bacterially expressed c J u n also interacts with sequences in region B, albeit with considerably lower affinity. The weak binding affinity is probably due to the fact that this HO-1 sequence motif, TGTGTCA, deviates from the consensus AP- 1 binding site at the third position. Additionally, since purified c-Jun binds to only a subset of region B as defined by the nuclear extracts, other transcription factors may stabilize the interaction of c-Jun (or other AP-1 proteins) at this HO-1 site in vivo. (Purified c-Jun appears to interact with a larger section of region B than indicated in Fig. 6. This extended protection, however, is not observed on the coding strand.) As a control, the c-Jun homodimer does not bind to an HO-1 proximal promoter region that includes a sequence motif, AGAGTCA (nucleotide residues -140 to -134), that also resembles the consensus AP-1 binding site. This result is consistent with previous observations (6, 36) that mutation of the first residue of the consensus AP-1 sequence has deleterious effect on protein binding. Proteins in crude nuclear extracts that interact with this region of the HO-1 promoter3 are likely to be different from members of the AP- 1 family of transcription factors. Taken together, these data strongly imply that the 5’-distal enhancer activity of the HO- 1 gene is mediated by one or more members of the Jun/Fos family of transcription factors. Furthermore, since AP-1 activity is observed in most cell types, this may explain the ubiquitous nature of the HO-1 enhancer activity.

HO-1 AP-1 Binding Sites Confer Responsiveness to TPA- The AP-1 binding site coincides with the TPA-responsive element present in the promoter/enhancer region of genes induced by this phorbol ester (6). Mutational analysis of the TRE indicates that AP-1 protein binding to the TRE is essential for TPA inducibility (5 , 6). As the HO-1 gene is transiently induced after TPA treatment of cultured cells (16, 17), we reasoned that this response is mediated by the AP-1 binding sites in fragment SX2. To test this hypothesis, several different CAT constructs were transiently transfected into Hep G2 cells, and the cells were either untreated or incubated with medium containing 100 ng/ml TPA for 16-24 h. In

JunlFos Regulation of HO-1 Gene - xh X A S X S B X A B A A xh I I I I I I I I I I I I I

I I I -6000 -4000 -2000 t1

CAT pMHO 1 CAT

pMH03CAT

pMH04CAT

pMH04CAT A B

pMH04CAT ABX

pMHOlCAT+AXl

pMHOlCAT+Xl

pMHOlCAT+SX4

pMHOlCAT+SXB

pMHOlCAT+SXS

pMHOlCAT+BSl

21897

CAT ACTIVITY (% PMHOICAT)

“ L929 C6

100 100

106 126

2% 1104

1047

126

162

1024

112

899

120

100

FIG. 2. Localization of the 5”distal enhancer of the HO-1 gene. Cultured cells (approximately 5 X 105/60-mm plate) were plated 16 h prior to transfection. Cells were transfected with 16 pg of a DNA mixture containing 1 pmol of CAT expression plasmid, 1 pg of pRSVluc, and an appropriate amount of pBSSK- for 4 h. Cells were harvested after 40 h of incubation in complete medium. CAT activity in each extract was normalized to luciferase activity in the same extract. Each data point represents the average value from five to nine independent transfection assays. The location of the enhancer fragment is indicated by a bar above the restriction endonuclease map. A, AccI; B, BarnHI; S , Sad; X , XbaI; Xh, XhoI.

agreement with this hypothesis, the expression of pMH04CAT, but not of pMHOlCAT, is induced 2.5-fold by TPA treatment (Fig. 7). Similarly, fragment SX2, but not fragment SX3, confers TPA responsiveness to the minimal SV40 promoter. Fragment SX2 (in plasmid pMHOlCATA-33+SX2) also confers TPA responsiveness to the homologous, minimal HO-1 promoter (data not shown). Mutational analysis and studies using synthetic oligonucleo- tides have established that the AP-1 binding site represents the minimal TPA-responsive element. Not surprisingly, the AP-1 binding motif of region A is sufficient and necessary for TPA-dependent activation of a heterologous promoter (compare pSKSVCAT with p4xACAT; the latter contains four tandem copies of the sequence spanning DNase I-protected region A). The AP-1 binding motif of region B has not been analyzed in a similar manner. The expression of the positive control construct, pSVBCAT, which contains the entire early gene regulatory region (including the TPA-responsive 72-bp enhancer) of SV40, is increased approximately 3-fold by TPA treatment. The negative control plasmid pMHPX4CATA- 192, in which expression of the CAT gene is under the control of 192 bp of the mouse hemopexin promoter, is highly active in Hep G2 cells but unresponsive to TPA. Surprisingly, nei- ther pMH04CAT nor pSKSVCAT+SX2 is responsive to TPA in C6 cells (data not shown), possibly due to the high level of constitutive AP-1 type activity in these cells.

Transactivation of the HO-1 Promoter by c-Jun and c-Fos- A functional role for the Jun/Fos family of proteins in the regulation of HO-1 gene expression was further established by transactivation studies in F9 teratocarcinoma cells. Undif-

ferentiated F9 cells, which show low to undetectable levels of AP-1 activity (37, 38), were transiently transfected with the appropriate CAT construct either alone or in combination with expression plasmids encoding human c-Jun and/or c- Fos. Consistent with the inability of c-Fos to from active homodimers, overexpression of this protein has little effect on the expression of pSKSVCAT+SX2 (Fig. 8). c-Jun expression produces a modest induction of CAT activity but maxi- mal transactivation is observed with coexpression of c-Jun and c-Fos, presumably due to the greater stability of the c- Jun:c-Fos heterodimer. Multiple copies of the AP-1 binding site of region A (p4xACAT), but not fragment SX3 (pSKSVCAT+SX3), also permit c-Jun/c-Fos transactivation. Surprisingly, transactivation was not observed in every experiment, especially with late passage F9 cells, and as with TPA induction, transactivation of the HO-1 promoter is not observed in C6 cells (or L929 cells). The reason for this variability and cell specificity is unclear, but similar observations have been reported by Curran and colleagues (23) with regard to c-Jun/c-Fos transactivation of the preproren- kephalin gene.

DISCUSSION

In this report we provide compelling evidence for the in- volvement of the Jun/Fos family of transcription factors in the basal and induced expression of the mouse HO-1 gene. Two closely spaced DNA elements, TGAGTCA and TGTGTCA, located approximately 4-kbp upstream of the transcription initiation site of the mouse HO-1 gene, resemble the consensus binding site, TGA(G/C)TCA, of the Jun/Fos

21898 JunlFos Regulation of HO-1 Gene

CAT ACTMTY (cpm x IO -4 )

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C6

0 HEPA

r FIG. 3. Activation of heterologous and homologous minimal promoters by the HO-1 enhancer fragment. Transfections were

carried out as described in the legend to Fig. 2 except that a total of 10 pg of DNA was used per dish. Extracts containing equivalent amounts of luciferase activity were used to measure CAT activity. CAT reactions were incubated for 16 h.

............... GAGCTCCACC CCCACCCAGG ATTCCAGCCC CCACAGGAGC T W C T T T G T .X3

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CTAOGMTAG TTGGTAAAAG GTTCCGGMC GGCTTTMCT TCAGGCAGM .m

OGMGTGAAA GTTCTAGA .x4

FIG. 4. Nucleotide sequence of the HO-1 5”distal enhancer fragment. The sequence of both strands was determined and that of the noncoding strand (5’ + 3’) is presented. Sequences that strongly or weakly interact with partially fractionated nuclear protein extracts from C6 cells (see Fig. 5) are indicated by continuous or dashed lines, respectively. Lines above or below the sequence delimit regions of the noncoding or coding strands, respectively, that are protected from DNase I digestion. The boundaries of regions C and D may be inexact due to weak protein binding. Carets (A) indicate residues sensitive to DNase I in a larger protected segment. DNA elements that match the consensus AP-1 binding site are highlighted in bold and boxed.

(AP-1) family of transcription factors. These elements interact with proteins in nuclear extracts from C6 cells and specifically bind purified, bacterially expressed c J u n homodimer. The HO-1 AP-1 binding sites, within the context of a 268-bp fragment, enhance basal transcription of the HO-1 promoter approximately 10-fold in C6 cells and between 2- to 4-fold in other cell lines. More importantly, this enhancer fragment or multiple copies of a single AP-1 binding element specifically mediate TPA induction in Hep G2 cells and c-Jun/c-Fos transactivation in F9 cells.

c-Jun and c-Fos are primary response genes (genes whose induction can occur without intervening protein synthesis) whose products are proposed to function as nuclear “third

CODINQ NONGODINQ

FIG. 5. Protein binding analysis of the HO-1 enhancer fragment. The coding and noncoding strands of fragment SX2 were end- labeled individually as described under “Experimental Procedures.” DNase I protection assays were carried out using 0 (lane O ) , 15 pg (lane I), 30 (lane 2), or 60 pg (lane 3) of nuclear protein extract from C6 cells. The digestion products were co-electrophoresed with the G + A chemical sequencing ladder of each strand on a denaturing, 6% polyacrylamide gel and autoradiographed for 48 h. Protected regions are indicated by brackets.

JunlFos Regular a + A0521

PROMOTER ENHANCER

FIG. 6. Specific binding of c-dun homodimers to the HO-1 enhancer elements. The noncoding strand of fragment SX2 (En- hancer) and the coding strand of the HO-1 proximal promoter fragment (Promoter) were digested with DNase I in the presence of 20 pg of total protein comprised of the indicated amount (in micrograms) of c-Jun and the appropriate amount of bovine serum albumin. The digestion products and the G + A chemical sequencing ladder were co-electrophoresed on a denaturing, 6% polyacrylamide gel and autoradiographed for 48 h. SX2 fragment sequences protected by c-Jun are delimited by the inner brackets. Regions A and B protected by partially fractionated nuclear extracts from C6 cells are also presented for comparison. The region of the proximal promoter fragment containing an AP-1-like binding site is marked by a dashed bracket (see text for more detail).

T

FIG. 7. TPA induction of HO-1 gene promoter activity. Hep G2 cells (approximately 2 X 106/60-mm plate) were plated 16 h prior t o transfection. Cells were transfected for 4 h with DNA mixtures containing 5 pg of the indicated CAT expression plasmid, 1 pg of pMHPX4lucA-192, and 4 pg of pBSSK-. Cells were cultured in medium containing 0.5% FBS for 40 h, and vehicle or TPA (100 ng/ ml) was added for the final 16 h of incubation. Luciferase-equivalent portions of cell extracts were used to measure CAT activity. Each measurement represents the average k S.D. from three to five individual transfections or the average value of two transfections (pSKSVCAT, pSKSVCAT+SX3, and pSV2CAT).

’ion of HO-1 Gene 21899

-a- psttsvCrT+SXZ p4UCAT pSKSVCATtSX3

FIG. 8. Transactivation of the HO-1 gene enhancer by c- Fos and c-dun. F9 cells (approximately 5 X 105/60-mm plate) were plated 24 h prior to transfection. Cells were transfected for 16 h with DNA mixtures containing 4 pg of the indicated CAT plasmid, 2 pg of pRSVluc, and a total of 4 pg of the CMV-Fos/CMV-Jun expression plasmids. For co-expression of c-Fos and c-Jun, 2 pg of each plasmid was used. The “control” DNA mixture contained 4 pg of the parent CMV expression vector to equalize for the level of CMV promoter activity. After transfection, the cells were cultured in Dulbecco’s modified Eagle’s medium containing 0.5% fetal bovine serum for 32 h. Background CAT activity (in extracts from pSKCAT transfected cells) was subtracted from each CAT assay value. CAT activity per unit of luciferase activity was calculated for each transfection and that from control cells was arbitrarily given a value of 1. Each data point represents the average +- S.D. from four independent experi- ments (pSKSVCAT+SX2) or the average of two transfections (p4xACAT and pSKSVCAT+SX3).

messengers’’ in coupling short term signals elicited by cell surface stimulation to long-term alteration in cellular phe- notypes by modulating the expression of select target genes (8, 39). The JunFos transcription factors are, therefore, primary nuclear targets of receptor-mediated signal transduction pathways activated by extracellular ligands. The identification of functional AP-1 binding sites within the HO-1 promoter and the demonstration that c-Jun/c-Fos transacti- vate these sequences suggest that certain extracellular factors may regulate the expression of the HO-1 gene.

One class of physiological activators that utilizes the Jun/ Fos transcription factor pathway to modulate the expression of specific cellular genes are peptide growth factors. Platelet- derived growth factor, for example, stimulates transin gene expression, in a c-Fos-dependent manner (40). Several in uiuo studies (41-43) indicate that endocrine factors, including insulin, stimulate hepatic heme oxygenase activity in rats. In contrast, insulin apparently suppresses basal and Co2+-induced heme oxygenase activity in cultured chick embryo hepatocytes (44). As these studies measured only enzyme activity, the effect of insulin on the specific expression of the heme oxygenase genes, HO-1 and HO-2, is not readily dis- cernible. That insulin may regulate the expression of HO-1 gene expression is suggested by the observation that this peptide stimulates the transcription of the c-fos gene in rat hepatoma cells (45) and that insulin induction of metallothionein gene expression in Hep G2 cells is partly mediated by the protein kinase C signal transduction pathway (4). In any case, a systematic analysis of the effects, if any, of insulin and other growth factors on HO-1 gene expression remains to be examined.

A second class of extracellular ligands that could potentially utilize the Jun/Fos pathway to regulate HO-1 gene expression are the pleiotropic cytokines, interleukin-1, interleukin-6, and tumor necrosis factor. For example, interleukin-1 induces interleukin-2 expression in T lymphocytes via an AP-1 binding site in the interleukin-2 gene promoter (46) and the c-Fos

2 1900 JunlFos Regular

and c-Jun gene products are essential for interleukin-1 stimulation of P-endorphin release from AtT-20 cells (47). These cytokines also mediate the acute-phase response elicited by inflammatory agents such as endotoxin, a known inducer of hepatic heme oxygenase activity (48, 49). Recent studies (50, 51) indicate that these cytokines induce heme oxygenase activity and HO-1 expression in animals and cultured cells. The role of the Jun/Fos family of transcription factors in this induction, however, is questionable as transcriptional stimulation of various genes during the acute phase response is mediated in part by distinct members of the C/EBP family of transcription factors (52, 53).

Finally, the Jun/Fos family of transcription factors may also mediate HO-1 gene induction by agents that do not interact with cell surface receptors. Tyrell and colleagues (17) have recently classified known inducers of HO-1 into two groups: 1) agents which generate active oxygen intermediates (e.g. UV irradiation, hydrogen peroxide, TPA) and 2) agents which interact with and/or modify glutathione levels (e.g. sodium arsenite, iodoacetamide, cadmium chloride). These agents may activate c-Fos and c-Jun function by modulating the redox state of these proteins, a mechanism that was recently shown to regulate c-Fos a n d c J u n DNA-binding activity in uitro (54). Of note, UV irradiation of primary human skin fibroblasts induces the expression of the c-fos gene, and UV-induced transcriptional activation of the collagenase gene (55) and the c-jun gene (56) in HeLa cells is mediated by the AP-1 binding motif.

Although the Jun/Fos family of transcription factors may mediate the induction of HO-1 gene transcription by multiple agents, the existence of conserved metal regulatory elements and heat shock elements within the promoters of several HO- 1 genes (57, 58), including the mouse HO-1 gene,3 suggests that other transcription factor pathways are required for HO- 1 induction by certain agents. Clearly, the presence of the AP-1 binding sites is not sufficient for optimal transcriptional activation of the mouse HO-1 promoter by heme or heavy metals. Studies to identify cis-acting DNA elements that mediate transcriptional activation by these agents are cur-

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