An internal enhancer regulates heme- and cadmium-mediated induction of human heme oxygenase-1

Nathalie Hill-Kapturczak,1 Eric Sikorski,1 Christy Voakes,1 Jairo Garcia,1 Harry S. Nick,2 and Anupam Agarwal1

1Department of Medicine, Division of Nephrology, Hypertension, and Transplantation, and 2Department of Neuroscience, University of Florida, Gainesville, Florida 32610

Submitted 4 April 2003 ; accepted in final form 21 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, releasing iron, carbon monoxide, and biliverdin. Induction of HO-1 is an adaptive and beneficial response in renal and nonrenal settings of tissue injury. The purpose of this study was to characterize the regulation of the human HO-1 gene in renal proximal tubule and aortic endothelial cells in response to heme and cadmium. Evaluation of multiple human HO-1 promoter-reporter constructs up to -9.1 kb demonstrated only a partial response to heme and cadmium. In an effort to mimic endogenous stimulus-dependent levels of HO-1 induction, we evaluated the entire 12.5 kb of the human HO-1 gene, including introns and exons, in conjunction with a -4.5-kb human HO-1 promoter and observed significant heme- and cadmium-mediated induction of the reporter gene, suggesting the presence of an internal enhancer. Enhancer function was orientation independent and required a region between -3.5 and -4.5 kb of the human HO-1 promoter. Our studies identified a novel enhancer internal to the human HO-1 gene that, in conjunction with the HO-1 promoter, recapitulates heme- and cadmium-mediated induction of the endogenous HO-1 gene. Elucidation of the molecular regulation of the human HO-1 gene will allow for the development of therapeutic strategies to manipulate HO-1 gene expression in pathological states.

gene transcription; renal proximal tubule cells; endothelial cells; heme proteins; molecular regulation


THE CELLULAR CONTENT of heme (ferriprotoporphyrin IX), derived from heme-containing proteins such as hemoglobin, myoglobin, cytochromes, and enzymes such as nitric oxide synthase, catalase, peroxidases, respiratory burst oxidase, and pyrrolases, requires a balance between heme synthesis and degradation (31). In pathological states, destabilization of heme proteins leads to liberation of free heme, which has potential prooxidant effects (22). Increases in renal heme content are observed in rhabdomyolysis, ischemiareperfusion injury, and nephrotoxin-induced acute renal failure (2, 29, 33, 40). Heme damages multiple cellular targets including lipid bilayers, mitochondria, cytoskeleton, nuclei, and several intracellular enzymes (33). Although multiple enzymes are involved in heme synthesis, the major biochemical pathway for heme detoxification is via the heme oxygenase (HO) enzyme system (28).

HO opens the heme ring, producing equimolar quantities of biliverdin, iron, and carbon monoxide (CO). Biliverdin is subsequently converted to bilirubin by biliverdin reductase. Recent studies have highlighted the important biological effects of the HO reaction product(s) that possesses antioxidant, anti-inflammatory, and antiapoptotic functions (reviewed in Ref. 21). Two isoforms of HO have been characterized: an inducible enzyme, HO-1, and a constitutive isoform, HO-2 (28). A putative isozyme, HO-3, isolated from rat brain and sharing ~90% homology with HO-2, has also been described (30). HO-1 and HO-2 are products of different genes and share ~40% amino acid homology (28). We and others demonstrated that induction of HO-1 by chemical inducers (2, 32) or selective overexpression (1, 41) is cytoprotective both in vitro and in vivo, findings that have been further substantiated by studies in HO-1 knockout mice and a patient with HO-1 deficiency (34, 41, 48).

The mechanisms underlying HO-1 induction are complex and tightly regulated at the transcriptional level. Adding to the complexity, regulation of the HO-1 gene is species and cell specific. The promoter of the rat HO-1 gene, for instance, has a heat shock responsive element that is not functional in the human HO-1 gene (50). A GT repeat region identified at position -258/-198 in the human HO-1 promoter is absent in the mouse HO-1 gene (49). Interestingly, length polymorphisms of this GT repeat correlate with disease development in atherosclerosis (16), emphysema (49), and vascular restenosis (18) in humans.

The human HO-1 gene, located on chromosome 22q12 (26), has five exons and spans ~14 kb. A potential cadmium response element (TGCTAGATTT) has been identified between -4.5 and -4.0 kb of the 5'-flanking region (44) of the human HO-1 gene; however, the cadmium response element in the mouse HO-1 gene is located downstream of this sequence (13). A threefold increase in luciferase activity was reported with a -4.5-kb human HO-1 construct in response to cadmium, with only a minimal response to hemin, sodium arsenite, or cobalt protoporphyrin in HeLa cells (44). We previously showed that the identical 4.5-kb human HO-1 gene promoter fragment responds, in part, to both heme and cadmium with a two- to threefold increase in reporter gene activity compared with ~20- to 30-fold increase in endogenous HO-1 mRNA in human aortic endothelial and renal proximal tubular cells (4). Furthermore, this 4.5-kb promoter fragment does not respond to other stimuli such as 13-hydroperoxyoctadecadienoic acid (13-HPODE), hydrogen peroxide (H2O2), and hyperoxia that directly increase de novo HO-1 gene transcription (4, 19). We report here the identification of an enhancer region internal to the human HO-1 gene that, together with the 4.5-kb promoter, recapitulates levels of heme and cadmium induction observed using steady-state Northern analysis of the endogenous gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. Hemin (ferriprotoporphyrin IX chloride), cadmium chloride (CdCl2), linoleic acid, lipoxidase, H2O2, and DEAE-dextran were purchased from Sigma (St. Louis, MO). Transforming growth factor-{beta}1 (TGF-{beta}1) was obtained from R&D Systems (Minneapolis, MN). RNeasy mini and Maxi-prep kits were obtained from Qiagen (Valencia, CA).

Cell culture. Human renal proximal tubule cells (HPTC) (Clonetics, Walkersville, MD) were grown in renal epithelial basal medium supplemented with 5% FBS, gentamicin (50 µg/ml), amphotericin B (50 µg/ml), insulin (5 µg/ml), transferrin (10 µg/ml), triiodothyronine (6.5 ng/ml), hydrocortisone (0.5 µg/ml), epinephrine (0.5 µg/ml), and human epidermal growth factor (10 ng/ml). These cells are positive for {gamma}-glutamyltranspeptidase, an enzyme marker for proximal tubule cells. The presence of microvilli, abundant mitochondria, lysosomes, and endocytotic vacuoles, morphological features of proximal tubule cells, were confirmed by electron microscopy in these cells. Human aortic endothelial cells (HAEC) [derived from segments of human aorta obtained from heart transplant donors and previously characterized by positive staining for factor VIII-related antigen and acetylated LDL (46)] were grown in endothelial basal medium (Clonetics), supplemented with 10% FBS, gentamicin (50 µg/ml), amphotericin B (50 µg/ml), hydrocortisone (1 µg/ml), human epidermal growth factor (10 ng/ml), and bovine brain extract (6 µg/ml) at 37°C in 95% air-5% CO2. Studies were performed over a range of no more than six to eight passages. Sixteen hours before treatment with HO-1 inducers, growth media was changed to a 0.5 and 1% FBS-containing media for HPTC and HAEC, respectively.

Preparation of HO-1 inducers. Hemin, CdCl2, and 13-HPODE were prepared fresh daily. A stock solution of hemin (1 mM) was prepared using 10 mM NaOH. CdCl2 (5 mM stock solution) and H2O2 (10 mM stock) were prepared in water and sterile filtered. 13-HPODE was prepared as previously described (4). Lyophilized TGF-{beta}1 was dissolved in 4 mM HCl containing 0.1% bovine serum albumin to obtain a 2 ng/µl stock solution, aliquoted, and stored at -80°C.

Cloning and characterization of a human HO-1 genomic clone. A 4.5-kb fragment of the 5'-flanking region of the human HO-1 gene, including the transcription initiation site, was generated by long-range PCR (PerkinElmer, Foster City, CA) using human genomic DNA as a template, as described previously (4). To obtain additional genomic sequences for the human HO-1 gene, two single-copy probes were designed, one from the -4.5/-4.0-kb region and one from an internal exon, and a commercially available human P1 bacteriophage library (Genome Systems, St. Louis, MO) was screened. Two P1 clones (P1 11715 and 11716) were identified by both probes. These clones were characterized by restriction map and sequence analysis, and the presence of the entire human HO-1 gene, as well as >15 kb of the 5'-flanking region, was verified in P1 11716. More recently, the efforts of the human genome project resulted in the sequencing of a bacterial artificial chromosome (BAC) clone, containing portions of chromosome 22 (accession no. Z82244 [GenBank] ). With the use of this BAC clone, sequences of the human HO-1 gene promoter fragments were confirmed, and larger fragments, further upstream, were generated by long-range PCR and restriction digestion (see Fig. 2).



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Fig. 2. Genomic structure of the human HO-1 gene and reporter gene constructs. Top: schematic representation of the human HO-1 genomic clone isolated from the P1 and bacterial artificial chromosome (BAC) clones with a partial restriction map extending from -11.614 to +16.9 kb. The 5 exons are indicated (E). The restriction sites for NheI, N; BamHI, B; PstI, P; XbaI, Xb; EcoRI, R are also indicated. Bottom: plasmid constructs used to assess human HO-1 promoter and enhancer activity. Details for the generation of these constructs are provided in MATERIALS AND METHODS.

 

Plasmid construction. Five'-flanking promoter fragments of the human HO-1 gene, extending from -4.0, -4.5, and -9.1 kb to +80-bp position, were generated using oligonucleotide primers (Table 1) specific to the HO-1 gene by long-range PCR (PerkinElmer). The 4.0- and 4.5-kb PCR products were ligated into TA cloning vectors (Invitrogen, Carlsbad, CA), and the 9.1-kb PCR product was cloned into pCR-XLTOPO (Invitrogen). BamHI sites were incorporated in the primers for the 4.0- and 4.5-kb fragments and SalI sites were incorporated in the primers for the 9.1-kb fragment to enable subcloning into a promoterless human growth hormone (hGH) reporter gene, generating pHOGH/4.0, pHOGH/4.5, and pHOGH/9.1. A 155-bp HO-1 promoter hGH plasmid was generated by deletion of a 4.345-kb HO-1 promoter fragment using NdeI followed by religation, resulting in the formation of pHOGH/155. The 4.5-kb promoter fragment was also cloned into the BglII site in a luciferase reporter (pGL3) generating pHOGL3/4.5. The 9.1-kb promoter fragment (with SalI sites on each end) was cloned into the XhoI site of pGL3, generating pHOGL3/9.1. A 3.5-kb HO-1 promoter reporter vector was generated by cutting the pHOGL3/9.1 with NheI, which cuts HO-1 at ~3.5 kb 5' from the start site and cuts pGL3 once 5' from the XhoI site, and religating to generate pHOGL3/3.5.


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Table 1. Primers used for long-range PCR for human HO-1 constructs

 

The entire +1- to +12.5-kb region of the human HO-1 gene (including all exons and introns) was generated using long-range PCR with oligonucleotide primers specific to the HO-1 gene (Table 1). The 12.5-kb PCR product was cloned into pCR-XL-TOPO and then subcloned into pHOGH/4.5, pHOGL3/4.5, or pHOGL3/3.5, generating pHOGH/4.5/+12.5, pHOGL3/4.5/+12.5, and pHOGL3/3.5/+12.5. SalI sites were included at each end of the 12.5-kb fragment to enable cloning into the SalI site of the hGH and luciferase vectors. We obtained both 5'-3' and 3'-5' orientations of the +12.5-kb fragment in the hGH vector containing the -4.5-kb HO-1 promoter region [pHOGH/4.5/+12.5 and pHOGH/4.5/+12.5 (3'-5'), respectively]. The +12.5-kb fragment was also cloned into the HindIII site of an hGH vector containing a heterologous herpes virus thymidine kinase (TK) minimal promoter to generate pHOTKGH/+12.5. All constructs were verified for orientation by sequencing and restriction analysis.

Transfection of the reporter gene. HPTC and HAEC were transiently transfected with the reporter gene vectors by the DEAE-dextran method using a batch transfection protocol (4). The DEAE-dextran method was optimized to achieve reproducible transfection efficiency (~20-25%) in HPTC and HAEC as assessed by cotransfection with pcDNA3.1/Lac-z (Invitrogen). The transfection protocol for HPTC was as follows: 60-70% confluent cells in 150-mm tissue culture plates were washed once with HBSS and once with Tris-buffered saline solution (TBS). A 1.0-ml solution containing equimolar amounts of the plasmid(s) (normalized to 8.1 µg of pHOGH/4.5) and DEAE-dextran (10 mg/ml) was added per plate and rocked for 1 h at room temperature. The cells were shocked for 1 min at room temperature with 10% DMSO in TBS and then washed once with HBSS. The cells were incubated in complete media (18 ml/plate) containing chloroquine diphosphate (100 µM) to inhibit lysosomal degradation of the DNA. After 4-h incubation at 37°C in 5% CO2-room air, the cells were washed twice with HBSS and incubated in normal growth media overnight.

The transfection protocol for HAEC was as follows: 60-70% confluent cells in 150-mm tissue culture plates were washed once with PBS. Complete media containing 10% NuSerum (BD Biosciences, San Jose, CA) was added to the plates. The transfection solution, 1.5 ml DEAE-dextran solution (10 mg/ml) containing equimolar amounts of the plasmid(s) (normalized to 8.1 µg of pHOGH/4.5), was added to each dish dropwise and then gently swirled to ensure that the DNA/DEAE-dextran was uniformly applied to the cells. Chloroquine diphosphate (100 µM) was then added to the medium. Cells were incubated for 4 h at 37°C in 5% CO2-room air and then shocked for 1 min at room temperature with 10% DMSO in PBS, washed twice with PBS, and incubated in growth media containing 10% FBS overnight.

At twenty-four hours posttransfection, the cells (both cell types) were passaged from 150-mm dishes into several 100-mm dishes (for hGH assays) or 12-well trays (for luciferase assays) to ensure equal transfection efficiency between experimental treatments. Cotransfection with pcDNA3.1/Lac-z demonstrated equal amounts of DNA in each of the wells. After the cells were allowed to recover for 24 h, they were treated with stimulus or vehicle (control). Sixteen to seventy-two hours following stimulus exposure, cells were evaluated for hGH mRNA content by Northern analysis, levels of secreted hGH protein in the culture media, as described previously (4), and luciferase activity was monitored using the luciferase reporter assay system (Promega) according to the manufacturer's instructions. Luminescence was measured with a Sirius Luminometer (Berthold Detection Systems, Pforzheim, Germany).

RNA isolation, molecular probes, and Northern analysis. Total cellular RNA was isolated by the method of Chomczynski and Sacchi (17). Samples were electrophoresed on a 1% agarose formaldehyde gel and electrotransferred to a charged nylon hybridization membrane. The cDNA probes for hGH, human HO-1, and human GAPDH were radiolabeled with [{alpha}-32P]dATP using a random primer labeling kit, according to the instructions of the manufacturer (Invitrogen), and purified over a G-50 column (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were hybridized overnight at 60°C, washed in a high-stringency buffer (0.04 M sodium phosphate, 1 mM EDTA, 1% SDS) at 65°C, and subjected to autoradiography. To quantitate expression levels, autoradiographs were scanned on a Hewlett-Packard ScanJet 4C using DeskScan II software and densitometry was performed using National Institutes of Health Image 1.63 software. Experiments were internally controlled by hybridization with GAPDH, normalized, and expressed as a percentage of maximal expression. All experiments were repeated with at least two to three independent RNA preparations to show reproducibility.

Statistical analysis. Data are expressed as means ± SE. Statistical analyses were performed using ANOVA and the Student-Newman-Keuls test. All results are considered significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of HO-1 mRNA by heme in HPTC and HAEC. Heme serves not only as a substrate for the HO enzyme, but it is also a potent inducer of the HO-1 gene both in vivo and in vitro (11, 32, 50). To understand the molecular mechanism involved in heme-mediated induction of HO-1, HPTC and HAEC were treated with heme (in the form of hemin, 5 µM) for 4 h. Northern analysis demonstrated an ~20- to 30-fold induction of HO-1 mRNA in both cell types (Fig. 1A). Figure 1, B and C, demonstrates that the induction of HO-1 mRNA by hemin was both time and dose dependent in HPTC. Similar results were obtained in HAEC (data not shown). Sodium hydroxide (40 µM) alone, the vehicle used in the preparation of hemin, did not induce HO-1 mRNA (Fig. 1B).



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Fig. 1. Induction of heme oxygenase (HO)-1 mRNA by hemin. A: confluent human aortic endothelial cells (HAEC; left) or human renal proximal tubule cells (HPTC; right) were incubated in 1 and 0.5% FBS-containing media, respectively, with hemin (5 µM) for 4 h as described in MATERIALS AND METHODS. B: confluent HPTC incubated in 0.5% FBS medium containing hemin (0, 1, 2.5, 5, 7.5, or 10 µM) and vehicle, NaOH (40 µM), for 4 h. C: time course of HO-1 mRNA induction in HPTC exposed to hemin (5 µM) at the indicated times. D: graphic representation showing the half-life of HO-1 mRNA following hemin stimulation. HPTC were preincubated with hemin (5 µM) for 4 h, washed, and exposed to actinomycin D (4 µM) in the absence ({bullet}, solid line) or presence ({circ}, dashed line) of additional hemin (5 µM). The percentage of maximal expression of HO-1 mRNA corrected for the internal control (GAPDH) vs. time is plotted. RNA was isolated and subjected to Northern blot analysis with a 32P-labeled cDNA specific for HO-1 or GAPDH as described in MATERIALS AND METHODS. Results are representative of at least 2-4 independent experiments.

 

We previously demonstrated the importance of de novo transcription by nuclear run-on analysis in HAEC and HPTC treated with hemin (5 µM) and observed increased HO-1 gene transcription (4). To evaluate the role of message stability in the induction of HO-1 mRNA following stimulation with hemin, the half-life of HO-1 mRNA was measured. Confluent HPTC were exposed to hemin (5 µM) for 4 h and washed with HBSS followed by the addition of fresh media containing actinomycin D (4 µM) with or without additional hemin. As shown in Fig. 1D, the half-life of HO-1 mRNA was similar (~4 h) with and without additional hemin treatment, suggesting that message stability is not involved. These findings are consistent with previous studies in other cell types (10) and enable us to use these human primary cultured cells for further studies to evaluate the transcriptional activation of the human HO-1 gene by hemin.

Identification of the enhancer region internal to the human HO-1 gene. To identify regulatory elements in the human HO-1 gene that mediate its transcription following stimulation with hemin, multiple promoter fragments (155 bp, 4.0 kb, 4.5 kb, and 9.1 kb) were generated and incorporated into promoterless reporter vectors as shown in Fig. 2 and described in MATERIALS AND METHODS. Growth hormone mRNA levels, measured by Northern analysis, were used as a direct measure of reporter gene transcription rates. HAEC transfected with hGH vectors containing -155-bp or -4.0-kb promoter constructs demonstrated no hGH mRNA induction in response to hemin (data not shown) and the -4.5- and -9.1-kb constructs resulted in a modest hemin-mediated induction in hGH mRNA (~6.7- and ~9.9-fold, respectively), as shown in Fig. 3A.



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Fig. 3. Analysis of human growth hormone (hGH) mRNA in cells transfected with hGH-HO-1 reporter constructs. HAEC were transiently transfected with equimolar amounts of pHOGH/4.5 (-4.5-kb HO-1 promoter in hGH vector) or pHOGH/9.1 (-9.1-kb HO-1 promoter in hGH vector; A) or pHOGH/4.5/+12.5 (enhancer with -4.5-kb HO-1 promoter in hGH vector; B) using DEAE/dextran and a batch transfection protocol as described in MATERIALS AND METHODS. Transfected cells were exposed to hemin (5 µM), cadmium chloride (CdCl2; 10 µM), transforming growth factor-{beta}1 (TGF-{beta}1; 2 ng/ml), or 13-hydroperoxyoctadecadienoic acid (13-HPODE; 25 µM) for 16 h and analyzed for reporter gene expression by Northern analysis. C: HPTC were transiently transfected with equimolar amounts of pHOGH/4.5 or pHOGH/4.5/+12.5 using DEAE/dextran and a batch transfection protocol as described in MATERIALS AND METHODS. Cells were exposed to hemin (5 µM) or CdCl2 (10 µM) for 16 h before collection of RNA for reporter gene analysis. Total RNA was isolated and Northern analysis was performed using 32P-labeled hGH and GAPDH cDNA probes. Results are representative of at least 3 independent experiments.

 

Because levels of reporter gene activity did not correlate with steady-state Northern levels of HO-1 induction with hemin, we hypothesized that additional regulatory sequences might exist within the HO-1 gene. Therefore, a construct containing the 12.5-kb region internal to the HO-1 gene with the 4.5-kb HO-1 promoter (pHOGH/4.5/+12.5) was generated. Unfortunately, we were unable to subclone the 12.5-kb fragment with the 9.1-kb HO-1 promoter. The construct, pHOGH/4.5/+12.5, demonstrated significant hemin- as well as CdCl2-inducible hGH mRNA in HAEC (16.1- and 32.4-fold, respectively; Fig. 3B). Similar results were observed in HPTC: pHOGH/4.5 yielded a 6.6- and 4.3-fold increase in response to hemin and CdCl2, respectively, and pHOGH/4.5/+12.5 resulted in a 27- and 18-fold increase in response to hemin and CdCl2, respectively, over control (untreated cells) transfected with pHOGH/4.5 (Fig. 3C). These results indicate the presence of sequences internal to the HO-1 gene that potentially function as an enhancer. Interestingly, the putative enhancer region does not function for all known inducers of HO-1 as pHOGH/4.5/+12.5 failed to respond to TGF-{beta}1 (2 ng/ml), H2O2 (200 µM), or 13-HPODE (25 µM) (Fig. 3B).

Reporter activity, as measured by hGH protein or luciferase activity, yielded results similar to hGH mRNA measurements. No promoter activity was detected in cells transfected with hGH constructs containing -155-bp or -4.0-kb promoter fragments and treated with hemin (5 µM) (Fig. 4A). Cells transfected with vectors containing the -4.5- or -9.1-kb promoter constructs and exposed to hemin (5 µM) demonstrated a 3.6- and 4.5-fold, respectively, increase in hGH protein over untreated cells (Fig. 4A). The construct containing the 12.5-kb region internal to the HO-1 gene, in conjunction with the -4.5-kb HO-1 promoter region, demonstrated elevated basal levels as well as significant hemin-inducible hGH protein levels, ~5.1-fold over untreated cells (Fig. 4A). Luciferase reporter vectors containing HO-1 gene fragments were generated to confirm the results with another reporter gene as well as ease of cloning and simplicity of the assay. Luciferase/HO-1 vectors yielded results similar to hGH reporter vectors in both HAEC and HPTC (Fig. 4, B and C, respectively).



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Fig. 4. Analysis of reporter activity in cells transfected with HO-1 reporter constructs in response to hemin. A: HAEC were transiently transfected with equimolar amounts of pHOGH/155 (-155-bp HO-1 promoter), pHOGH/4 (-4.0-kb HO-1 promoter), pHOGH/4.5 (-4.5-kb HO-1 promoter), pHOGH/9.1 (-9.1-kb HO-1 promoter), and pHOGH/4.5/+12.5 (enhancer with -4.5-kb HO-1 promoter) as described in MATERIALS AND METHODS. Cells were exposed to hemin (5 µM) for 72 h, and secreted hGH protein in the media was measured using a radioimmunoassay kit as described previously (4). Results are derived from 2 independent experiments. HAEC (B) and HPTC (C) were transiently transfected with equimolar amounts of pHOGL3/4.5 (-4.5-kb HO-1 promoter in luciferase vector) or pHOGL3/4.5/+12.5 (enhancer with -4.5-kb HO-1 promoter in luciferase vector), exposed to hemin (5 µM) for 16 h, and luciferase activity was measured as described in MATERIALS AND METHODS. Results are derived from 2 independent experiments with 3-12 replicates per group. Open bars represent untreated (control) cells, and filled bars represent hemin-stimulated cells. #P < 0.05 control vs. hemin; *P < 0.001 vs. other groups (ANOVA).

 

Orientation-independent effects of the HO-1 enhancer region. To determine whether the +12.5-kb fragment functions as a true enhancer, both orientations (5'-3' and 3'-5') were cloned into pHOGH/4.5 to give pHOGH/4.5/+12.5 and pHOGH/4.5/+12.5 (3'-5'). Similar levels of heme-mediated induction, as measured by Northern analysis of hGH mRNA, were observed, regardless of orientation (Fig. 5). In addition, to determine whether the enhancer could function with a heterologous promoter, the +12.5-kb fragment was cloned into a hGH vector containing a TK promoter (pHOTKGH/+12.5), which is a 200-bp minimal, TATA-containing promoter. Transient transfection of the 12.5-kb fragment in conjunction with a TK promoter failed to respond to hemin, as assessed by Northern analysis of hGH mRNA (data not shown), suggesting that the enhancer only functions with the HO-1 promoter.



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Fig. 5. Orientation-independent effects of the HO-1 enhancer. HAEC were transiently transfected with equimolar amounts of pHOGH/4.5 (-4.5-kb HO-1 promoter in hGH vector), pHOGH/4.5/+12.5 (5'-3' orientation of the enhancer with -4.5-kb promoter in hGH vector), and pHOGH/4.5/+12.5 (3'-5') (3'-5' orientation of the enhancer with -4.5-kb promoter in hGH vector) as described in MATERIALS AND METHODS. Cells were exposed to hemin (5 µM) for 16 h, and total RNA was isolated for Northern analysis using 32P-labeled hGH and GAPDH cDNA probes.

 

To further define the relevant sequences in the HO-1 promoter region that are required for enhancer function, the +12.5-kb enhancer fragment was evaluated with a 3.5-kb promoter of the human HO-1 gene pHOGL3/3.5/+12.5. As shown in Fig. 6, the +12.5-kb fragment requires the 4.5-kb HO-1 promoter fragment to function, because the 3.5-kb promoter with the +12.5-kb enhancer did not demonstrate any basal or hemin-inducible activity. The results demonstrate that enhancer sequences internal to the human HO-1 gene (within 12.5 kb), in conjunction with regions between -3.5 and -4.5 kb of the HO-1 promoter, mediate transcriptional activation of the human HO-1 gene by hemin and the enhancer is likely composed of a complex set of interacting elements.



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Fig. 6. Requirement of the -4.5-kb HO-1 promoter for enhancer function. HPTC were transiently transfected with equimolar amounts of pHOGL3/3.5/+12.5 (enhancer with -3.5-kb HO-1 promoter in a luciferase vector) or pHOGL3/4.5/+12.5 as described in MATERIALS AND METHODS. Cells were exposed to hemin (5 µM) for 16 h, and luciferase assays were performed. Results are derived from 2 independent experiments with 6 replicates per group. Open bars represent untreated (control) cells, and filled bars represent hemin-stimulated cells. *P < 0.001 vs. other groups (ANOVA).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of HO-1 is an adaptive and protective response in renal ischemia-reperfusion injury (40), nephrotoxin-induced renal injury (1, 41), organ transplantation (23), acute glomerulonephritis (47), rhabdomyolysis (32, 34), as well as nonrenal settings of tissue injury (3). The protective effects of HO-1 were first recognized due to its robust induction in cells/tissues exposed to a wide variety of stimuli that are otherwise injurious (24, 32). The mechanisms underlying HO-1 induction by multiple inducers, including heme, CdCl2, nitric oxide (NO), oxidized LDL, and cytokines, are complex and regulated predominantly at the transcriptional level. Increased mRNA stability has been reported to contribute to induction of HO-1 by NO donors in primary human embryonic fibroblasts with a strong correlation between the rate of NO release and the half-life of HO-1 mRNA (14). Furthermore, HO-1 mRNA stability was independent of RNA or protein synthesis. Our previous studies using nuclear run-on assays (4) combined with the present half-life experiments with actinomycin D suggest that mRNA stability is not involved in heme-mediated HO-1 induction in renal epithelial and endothelial cells, results consistent with previously published observations in other cell types (10).

Thus far, studies on mammalian HO-1 gene regulation have focused mainly on the mouse HO-1 gene (5-13), where multiple inducer-specific stress response elements have been localized within 10 kb of the 5'-flanking region. Two distal promoter regions, E1 and E2, at -4.0 and -10 kb, respectively, are required for induction of the mouse HO-1 gene in response to heme, heavy metals, H2O2, and sodium arsenite (5-7, 13). It was proposed that activation of the mouse HO-1 gene by most stimuli is mediated exclusively via the E1 and/or E2 regions. These regions contain consensus binding sites for the NF-E2-related factor Nrf2, and overexpression studies coupled with mutations in these binding sites have implicated the involvement of Nrf2 in the induction of the mouse HO-1 gene (12, 20).

To test whether promoter elements in the 5'-flanking region of the human HO-1 gene can analogously control heme-mediated induction, we initially analyzed several human HO-1 promoter constructs up to -9.1 kb, which contain sequences potentially similar to the E1 and E2 regions in the mouse gene. The 4.5- and 9.1-kb promoter fragments demonstrated a modest induction with heme; however, unlike the E1 and E2 regions of the mouse gene, these constructs did not completely recapitulate steady-state Northern levels of HO-1 induction. In addition, unlike the mouse promoter, we also found that the human HO-1 promoter constructs showed no response to other known HO-1 stimuli, including 13-HPODE, hyperoxia, curcumin, and TGF-{beta}1, in human cultured cells (4, 19, and unpublished observations).

In our efforts to mimic endogenous, stimulus-dependent transcription levels, we hypothesized that additional regulatory element(s) were necessary for hemeand cadmium-mediated induction of the human HO-1 gene. To identify other relevant regulatory sequences, we tested the entire +12.5-kb HO-1 gene, including introns and exons, in conjunction with the -4.5-kb human HO-1 promoter and observed significant hemeand cadmium-dependent induction of reporter activity. Furthermore, this enhancer region was specific for heme and cadmium and did not function for other known HO-1 stimuli, such as TGF-{beta}1, H2O2, or 13-HPODE. The large +12.5-kb fragment, in part, satisfied the characteristics of a true enhancer region by functioning in an orientation-independent manner. However, when the HO-1 promoter was replaced with a heterologous TK promoter, no induction by heme was observed, thus demonstrating that this region functions as a gene-specific enhancer. This is substantiated by our findings that the enhancer region requires a portion of the HO-1 promoter to mimic endogenous levels of induction. Specifically, we demonstrated that a region between -4.5 and -3.5 kb is required for enhancer function. Our data also indicated the importance of an additional region between -4.5 and -9.1 kb for heme-mediated HO-1 promoter activity. However, we were unable to evaluate the +12.5-kb enhancer in the context of the -9.1-kb promoter construct due to our inability to successfully subclone both of these large fragments into a single plasmid. Studies to further delineate the regulatory sequences within the enhancer region are currently in progress. As these regions become more well defined, constructs containing the relevant sequences within the enhancer will be evaluated with the -9.1-kb HO-1 promoter.

Regulatory sequences functioning as enhancers have been identified within introns of several genes, including manganese superoxide dismutase, platelet-derived growth factor A, and alcohol dehydrogenase-1 (15, 37, 45). We performed sequence comparisons with the 12.5-kb enhancer region and did not identify any sequences that are linked to known heme or cadmium response elements. Alam and Den (8) previously reported that the region analogous to our enhancer region containing the entire protein coding region of the mouse HO-1 gene coupled to the mouse HO-1 promoter did not respond to heme or cadmium in rodent cells. Two possibilities could account for the lack of response. On the one hand, the limited size (~3.0 kb) of the mouse HO-1 promoter used in these studies may account for the negative results (8). On the other hand, perhaps it is a reflection of the differential regulation of the mouse vs. human HO-1 genes.

Our observations indicate that the human HO-1 gene requires regulatory sequences that differ from those previously implicated for the mouse gene. Other interspecies differences in HO-1 gene regulation have also been noted. For example, the human HO-1 promoter contains a GT repeat region that is absent in the mouse HO-1 gene (49). Deletion of this repeat region results in a significant elevation of basal HO-1 promoter activity (unpublished observations), similar to previously reported results using HO-1 promoter constructs with shorter GT repeats (49), suggesting that this human-specific region may function as a negative regulator of HO-1 gene expression. Studies have also demonstrated that HO-1 is induced by hypoxia in rat, bovine, mouse, and monkey cells but is rather repressed in human cells (25, 27). Similarly, HO-1 has been popularized as heat shock protein 32 based on its inducibility by heat shock in rodent cells (38, 39). However, heat shock does not induce HO-1 in human cells (35). Although cytokines induce HO-1 in rodent cells, previous studies have reported that interferon-{gamma} decreases human HO-1 gene expression (43). Furthermore, a putative cadmium response element has been identified in the human HO-1 promoter (44); however, the cadmium response element in the mouse HO-1 gene is immediately 3' to this region and is an Nrf2 consensus sequence (13). Collectively, these observations suggest that the underlying molecular mechanisms that regulate the human HO-1 gene are different from those described for mouse HO-1 gene expression.

Recent studies have demonstrated that the expression of the mouse HO-1 gene is regulated through antagonism of transcription activators and the repressor Bach1 (42). Under normal physiological conditions, HO-1 expression is repressed by Bach1, and increased heme levels displace Bach1 from the E1 and E2 regions, allowing activators to bind to the regulatory sequences (42). The authors suggest that the transcriptional regulation of the mouse HO-1 gene involves a direct sensing of heme levels by Bach1, generating a feedback loop by the substrate, as in the lac operon. Studies to explore the involvement of Bach1 in human HO-1 gene regulation will be of interest.

In summary, our data provide evidence that at least two regions in the human HO-1 promoter, one between -4.5 and -4.0 and another between -9.1 and -4.5, are required for activation of the HO-1 gene by heme. Most importantly, we identified a novel transcriptional enhancer internal to the human HO-1 gene that, in conjunction with the HO-1 promoter, recapitulates a steady-state level expression of HO-1 by heme and cadmium. Studies on regulation of the human HO-1 gene are biologically relevant because of the well-documented beneficial effects of HO-1 activity that include degradation of heme (a toxic prooxidant), generation of bilirubin (an antioxidant), coinduction of ferritin (an intracellular repository for iron), and the antiapoptotic and anti-inflammatory effects of CO (36). We are continuing our investigation of the molecular mechanisms involved in the regulation of the human HO-1 gene in an effort to fine tune endogenous HO-1 gene expression to ultimately optimize its potential as a therapeutic modality.


    DISCLOSURES
 
This work was supported by National Institutes of Health Grants K-08-DK-02446 and R-01-DK-59600 to A. Agarwal and Grant K-01-DK-02902 to N. Hill-Kapturczak.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Agarwal, Div. of Nephrology, Hypertension, and Transplantation, Box 100224 JHMHC, 1600 SW Archer Rd., Univ. of Florida, Gainesville, FL 32610 (E-mail: agarwal{at}nersp.nerdc.ufl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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