Role of Bach-1 in Regulation of Heme Oxygenase-1 in Human Liver Cells

Abstract

Heme oxygenase-1 is an antioxidant defense enzyme that converts heme to biliverdin, iron, and carbon monoxide. Bach-1 is a bZip protein that forms heterodimers with small Maf proteins and was reported recently to down-regulate the HO-1 gene in mice. Using small interfering RNAs targeted to human Bach-1 mRNA, we investigated whether modulation of human hepatic Bach-1 expression by small interfering (si)RNA technology influences heme oxygenase-1 gene expression. We found that Bach-1 siRNAs transfected into Huh-7 cells significantly reduced Bach-1 mRNA and protein levels ∼80%, compared with non siRNA-treated cells. In contrast, transfection with the same amounts of nonspecific control duplexes or LaminB2-duplex did not reduce Bach-1 mRNA or protein levels, confirming the specificity of Bach-1 siRNA. Expression of the heme oxygenase-1 gene in Bach-1 siRNA-transfected cells was up-regulated 7-fold, compared with cells without Bach-1 siRNA. The effect of increasing concentrations of heme to up-regulate levels of heme oxygenase-1 was more pronounced when Bach-1 siRNA was present. Taken together, these results indicated that Bach-1 has a specific and selective ability to repress expression of human hepatic heme oxygenase-1. Silencing of Bach-1 by siRNAs is a useful method for up-regulating HO-1 gene expression. Exogenous heme produces additional up-regulation, beyond that produced by Bach-1 siRNAs, suggesting that heme does not act solely through its effects on Bach-1. Heme oxygenase-1 is an antioxidant defense enzyme that converts heme to biliverdin, iron, and carbon monoxide. Bach-1 is a bZip protein that forms heterodimers with small Maf proteins and was reported recently to down-regulate the HO-1 gene in mice. Using small interfering RNAs targeted to human Bach-1 mRNA, we investigated whether modulation of human hepatic Bach-1 expression by small interfering (si)RNA technology influences heme oxygenase-1 gene expression. We found that Bach-1 siRNAs transfected into Huh-7 cells significantly reduced Bach-1 mRNA and protein levels ∼80%, compared with non siRNA-treated cells. In contrast, transfection with the same amounts of nonspecific control duplexes or LaminB2-duplex did not reduce Bach-1 mRNA or protein levels, confirming the specificity of Bach-1 siRNA. Expression of the heme oxygenase-1 gene in Bach-1 siRNA-transfected cells was up-regulated 7-fold, compared with cells without Bach-1 siRNA. The effect of increasing concentrations of heme to up-regulate levels of heme oxygenase-1 was more pronounced when Bach-1 siRNA was present. Taken together, these results indicated that Bach-1 has a specific and selective ability to repress expression of human hepatic heme oxygenase-1. Silencing of Bach-1 by siRNAs is a useful method for up-regulating HO-1 gene expression. Exogenous heme produces additional up-regulation, beyond that produced by Bach-1 siRNAs, suggesting that heme does not act solely through its effects on Bach-1. Heme oxygenase (HO, 1The abbreviations used are: HO, heme oxygenase; HeRE, hemeresponsive elements; siRNA, small inhibitory RNA; RT-PCR, reverse transcription PCR.1The abbreviations used are: HO, heme oxygenase; HeRE, hemeresponsive elements; siRNA, small inhibitory RNA; RT-PCR, reverse transcription PCR. E.C. 1.14.99.3) is the rate-controlling enzyme of heme catabolism (1Tenhunen R. Marver H.S. Schmid R. Proc. Natl. Acad. Sci. U. S. A. 1968; 61: 748-755Crossref PubMed Scopus (1501) Google Scholar, 2Tenhunen R. Marver H.S. Schmid R. J. Biol. Chem. 1969; 244: 6388-6394Abstract Full Text PDF PubMed Google Scholar, 3Elbirt K.K. Bonkovsky H.L. Proc. Assoc. Am. 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DNA Cell Biol. 2002; 21: 307-321Crossref PubMed Scopus (111) Google Scholar) have highlighted important biological effects of these HO reaction products, which display antioxidant, anti-inflammatory, and anti-apoptotic functions. Three isoforms of HO, termed HO-1, -2, and -3, have been described (7Shibahara S. Yoshizawa M. Suzuki H. Takeda K. Meguro K. Endo K. J. Biochem. (Tokyo). 1993; 113: 214-218Crossref PubMed Scopus (179) Google Scholar, 8McCoubrey Jr., W.K. Maines M.D. Gene (Amst.). 1994; 139: 155-161Crossref PubMed Scopus (108) Google Scholar, 9McCoubrey Jr., W.K. Huang T.J. Maines M.D. Eur. J. Biochem. 1997; 247: 725-732Crossref PubMed Scopus (735) Google Scholar). Among the three isoforms of HO, only HO-1 is highly inducible. Earlier work from our and other laboratories established that HO-1 could be up-regulated markedly by a variety of stressful stimuli, as well as by heme or certain other metalloporphyrins, particularly, cobalt protoporphyrin (10Lincoln B.C. Healey J.F. Bonkovsky H.L. Biochem. J. 1988; 250: 189-196Crossref PubMed Scopus (51) Google Scholar, 11Lu T.H. Shan Y. Pepe J. Lambrecht R.W. Bonkovsky H.L. Mol. Cell Biochem. 2000; 209: 17-27Crossref PubMed Google Scholar, 12Shan Y. Pepe J. Lambrecht R.W. Bonkovsky H.L. Arch. Biochem. Biophys. 2002; 399: 159-166Crossref PubMed Scopus (25) Google Scholar, 13Shan Y. Pepe J. Lu T.H. Elbirt K.K. Lambrecht R.W. Bonkovsky H.L. Arch. Biochem. Biophys. 2000; 380: 219-227Crossref PubMed Scopus (80) Google Scholar, 14Shan Y. Lambrecht R.W. Pepe J.A. Bonkovsky H.L. Hepatology. 1999; 30: 512AGoogle Scholar). The primary mechanism for up-regulation of the HO-1 gene is by increased transcription of the gene (15Srivastava K.K. Cable E.E. Donohue S.E. Bonkovsky H.L. Eur. J. Biochem. 1993; 213: 909-917Crossref PubMed Scopus (41) Google Scholar), and the induction by such stressors as sodium arsenite or other arsenicals (which produce a chemical oxidative stress), by transition metals, such as cadmium or cobalt, hydrogen peroxide, other reactive oxygen species, or heat shock are clearly different in mechanism from the up-regulation produced by metalloporphyrins (13Shan Y. Pepe J. Lu T.H. Elbirt K.K. Lambrecht R.W. Bonkovsky H.L. Arch. Biochem. Biophys. 2000; 380: 219-227Crossref PubMed Scopus (80) Google Scholar, 14Shan Y. Lambrecht R.W. Pepe J.A. Bonkovsky H.L. Hepatology. 1999; 30: 512AGoogle Scholar, 16Otterbein L.E. Bach F.H. Alam J. Soares M. Tao L.H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Nat. Med. 2000; 6: 422-428Crossref PubMed Scopus (1817) Google Scholar, 17Lu T.H. Lambrecht R.W. Pepe J. Shan Y. Kim T. Bonkovsky H.L. Gene (Amst.). 1998; 207: 177-186Crossref PubMed Scopus (31) Google Scholar, 18Elbirt K.K. Whitmarsh A.J. Davis R.J. Bonkovsky H.L. J. Biol. Chem. 1998; 273: 8922-8931Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 19Shan Y. Lambrecht R.W. Hong L.T. Bonkovsky H.L. Arch. Biochem. Biophys. 1999; 372: 224-229Crossref PubMed Scopus (14) Google Scholar). For example, earlier work from our laboratory showed that cMyc/Max and upstream stimulatory factor elements in the 5′-untranslated region of the HO-1 gene played the key role in inductions by cadmium or cobalt (11Lu T.H. Shan Y. Pepe J. Lambrecht R.W. Bonkovsky H.L. Mol. Cell Biochem. 2000; 209: 17-27Crossref PubMed Google Scholar). In contrast, inductions by sodium arsenite or phenylarsene oxide depend primarily upon activation of the mitogen-activated protein kinases leading to increased levels of AP-1 proteins, which bind to several AP1-consensus elements found in the HO-1 promoter (18Elbirt K.K. Whitmarsh A.J. Davis R.J. Bonkovsky H.L. J. Biol. Chem. 1998; 273: 8922-8931Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 20Lu T.H. Pepe J.A. Gildemeister O.S. Tyrrell R.M. Bonkovsky H.L. Biochim. Biophys. Acta. 1997; 1352: 293-302Crossref PubMed Scopus (22) Google Scholar, 21Gildemeister O.S. Pepe J.A. Lambrecht R.W. Bonkovsky H.L. Mol. Cell Biochem. 2001; 226: 17-26Crossref PubMed Scopus (4) Google Scholar). In contrast, the up-regulation of the HO-1 gene produced by heme or cobalt protoporphyrin is not mediated by these classic stress pathways or kinase cascades but rather depends upon several heme-responsive elements (and a metalloporphyrin-responsive element), which are distinct from other consensus promoter elements, found in the 5′-untranslated region of rodent, human, and avian HO-1 (12Shan Y. Pepe J. Lambrecht R.W. Bonkovsky H.L. Arch. Biochem. Biophys. 2002; 399: 159-166Crossref PubMed Scopus (25) Google Scholar). Further progress in understanding the molecular mechanisms that underlie the up-regulation of the HO-1 gene by heme has come from the characterization of Bach-1, one of the family of leucine b-Zip proteins. Bach-1 was isolated as a brown protein and was shown to contain heme bound to multiple cysteine-proline motifs (22Oyake T. Itoh K. Motohashi H. Hayashi N. Hoshino H. Nishizawa M. Yamamoto M. Igarashi K. Mol. Cell. Biol. 1996; 16: 6083-6095Crossref PubMed Scopus (520) Google Scholar, 23Igarashi K. Hoshino H. Muto A. Suwabe N. Nishikawa S. Nakauchi H. Yamamoto M. J. Biol. Chem. 1998; 273: 11783-11790Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 24Sun J. Hoshino H. Takaku K. Nakajima O. Muto A. Suzuki H. Tashiro S. Takahashi S. Shibahara S. Alam J. Taketo M.M. Yamamoto M. Igarashi K. EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (511) Google Scholar). It is highly conserved, and Bach-1 proteins have now been described in avian through mammalian species. Recent work of Igarashi and others (22Oyake T. Itoh K. Motohashi H. Hayashi N. Hoshino H. Nishizawa M. Yamamoto M. Igarashi K. Mol. Cell. Biol. 1996; 16: 6083-6095Crossref PubMed Scopus (520) Google Scholar, 23Igarashi K. Hoshino H. Muto A. Suwabe N. Nishikawa S. Nakauchi H. Yamamoto M. J. Biol. Chem. 1998; 273: 11783-11790Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 24Sun J. Hoshino H. Takaku K. Nakajima O. Muto A. Suzuki H. Tashiro S. Takahashi S. Shibahara S. Alam J. Taketo M.M. Yamamoto M. Igarashi K. EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (511) Google Scholar, 25Ogawa K. Sun J. Taketani S. Nakajima O. Nishitani C. Sassa S. Hayashi N. Yamamoto M. Shibahara S. Fujita H. Igarashi K. EMBO J. 2001; 20: 2835-2843Crossref PubMed Scopus (414) Google Scholar, 26Sun J. Brand M. Zenke Y. Tashiro S. Groudine M. Igarashi K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1461-1466Crossref PubMed Scopus (287) Google Scholar) has been interpreted to indicate that Bach-1, under baseline conditions, forms heterodimers with small proteins of the Maf family, and these heterodimers repress transcription of the HO-1 gene by binding to the heme-responsive elements (HeRE) in the 5′-untranslated region of the HO-1 promoter. Under conditions of excess heme, increased binding of heme to Bach-1 leads to a conformational change and a decrease in DNA-binding activity. This permits other Maf-Maf, Nrf2-Maf, and other activating heterodimers to occupy the HeRE sites in the HO-1 promoter and leads to increased transcription and up-regulation of expression of the gene (24Sun J. Hoshino H. Takaku K. Nakajima O. Muto A. Suzuki H. Tashiro S. Takahashi S. Shibahara S. Alam J. Taketo M.M. Yamamoto M. Igarashi K. EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (511) Google Scholar, 25Ogawa K. Sun J. Taketani S. Nakajima O. Nishitani C. Sassa S. Hayashi N. Yamamoto M. Shibahara S. Fujita H. Igarashi K. EMBO J. 2001; 20: 2835-2843Crossref PubMed Scopus (414) Google Scholar, 26Sun J. Brand M. Zenke Y. Tashiro S. Groudine M. Igarashi K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1461-1466Crossref PubMed Scopus (287) Google Scholar). An additional or alternative mechanism for the heme effect, namely, a stabilization of Nrf2 proteins, which are positive transcriptional regulators of HO-1, has recently been proposed by Alam et al. (27Alam J. Killeen E. Gong P. Naquin R. Hu B. Stewart D. Ingelfinger J.R. Nath K.A. Am. J. Physiol. 2003; 284: F743-F752Crossref PubMed Scopus (151) Google Scholar) based upon studies with immortalized proximal tubular epithelial cells. The purpose of the work described in this paper was to investigate whether down-regulation of Bach-1 expression in human hepatocytes would lead to up-regulation of the HO-1 gene. We chose small interfering RNAs (siRNA) directed at Bach-1 as a convenient means to down-regulate the Bach-1 gene. A portion of this work has been presented in abstract form (28Shan Y. Lambrecht R.W. Ghaziani T. Donohue S. Bonkovsky H.L. Hepatology. 2004; 40: 527APubMed Google Scholar). Materials—The human hepatoma cell line, Huh-7, was purchased from the Japan Health Research Resources Bank (Osaka, Japan). Ferric (Fe+3)-protoporphyrin IX·Cl (heme) was from Porphyrin Products (Logan, UT). Dimethyl sulfoxide (Me2SO) was purchased from Fisher-Biotech (Fair Lawn, NJ). RNAzol was from Biotecx (Houston, TX). Goat anti-Bach-1, anti-β-actin polyclonal antibodies, and rabbit anti-goat IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse anti-HO-1 monoclonal antibody (OSA-111) was purchased from Stressgen (Victoria, Canada). Rabbit anti-mouse IgG and ECL-Plus were purchased from Amersham Biosciences. Nonspecific control duplexes-XIII and LaminB2 duplex were purchased from Dharmacon (Lafayette, CO). Cell Cultures and Preparation of Chemicals—Huh-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% (v/v) fetal bovine serum (Invitrogen) and were routinely passaged twice a week. Heme was dissolved in Me2SO and stored at –20 °C; it was further diluted with 1% human serum albumin in 40 mm Tris-HCl (pH 7.4) just before use. Bach-1 siRNAs Preparation and Transfection—We used Bach-1 siRNAs, targeting the following positions of the human Bach-1 mRNA (Accession Number NM_001186), 331–349, 573–591, 1673–1691, and 2296–2314. The forward sequences of Bach-1-duplex siRNAs were GAACATTACTCTTCCAGAA, AAACAGACCTTAAACTTTC, GAACAAGAATGTGAGGTAA, and GCAGATGACTGATAAATGT. All Bach-1 siRNAs were synthesized and annealed at Dharmacon. Huh-7 cells were plated the day before transfections and grown to 50–70% confluence in 12-well plates. Transfections were carried out with Lipofectamine Plus reagent (Invitrogen) as described previously (12Shan Y. Pepe J. Lambrecht R.W. Bonkovsky H.L. Arch. Biochem. Biophys. 2002; 399: 159-166Crossref PubMed Scopus (25) Google Scholar, 19Shan Y. Lambrecht R.W. Hong L.T. Bonkovsky H.L. Arch. Biochem. Biophys. 1999; 372: 224-229Crossref PubMed Scopus (14) Google Scholar). Transfected cells were grown for 24–72 h in a 37 °C incubator with 5% CO2 and changed to serum-free medium just before heme treatment. Cells were harvested, total RNA and protein was extracted, and these samples were stored at –80 °C for subsequent quantitative RT-PCR and Western blotting. RNA Preparation and Quantitative Real Time PCR Analysis—Total RNA from treated cells was extracted by applying 500 μl of RNAzol directly onto the cells and following the manufacturer's instructions with the addition of DNase digestion steps. RNA concentration and purity were determined spectrophotometrically by measuring absorbance at 260/280 nm. The minimal required value of this ratio was 1.8. Total RNA (2 μg) was reverse transcribed into cDNA in a total volume of 50 μl using M-MLV Reverse Transcriptase (Invitrogen), according to the manufacturer's instructions. Real time quantitative RT-PCR was performed using an ABI Prism 7900 Sequence Detection System (Applied Biosystems) and QuantiTect™ SYBR Green PCR kit (Qiagen, Valencia, CA). Sequence-specific primers were designed using Applied Biosystems Primer Express software. Each assay was designed such that the product spanned an intron/exon boundary to minimize the possibility of coamplifiying genomic DNA. Primers used were as follows, for Bach-1 sense primer, 5′-GGACACTCCTTGCCAAATGCAG; Bach-1 antisense primer, 5′-TGACCTGGTTCTGGGCTCTCAC; HO-1 sense primer, 5′-CGGGCCAGCAACAAAGTG; HO-1 antisense primer, 5′-AGTGTAAGGACCCATCGGAGAA; glyceraldehyde-3-phosphate dehydrogenase sense primer, 5′-TTGTTGCCATCAATGACCC; glyceraldehyde-3-phosphate dehydrogenase antisense primer, 5′-CTTCCCGTTCTCAGCCTTG; HO-2 sense primer, 5′-CAATGTCAGCGGAAGTGGAAAC, HO-2 antisense primer, 5′-CAGCCATTCTCATTTGGTTCTCC; ALAS-1 sense primer, 5′-GGCAGCACAGATGAATCAGAGAG, and ALAS-1 antisense primer, 5′-TTCAGCAACCTCTTTCCTCACGG; LaminB2 sense primer, 5′-AAGAAGTCCTCGGTGATGCGT-3′; LaminB2 antisense primer, 5′-TCCCCCTGTTGGTGGAAAA-3′. We included no-template controls and no-reverse transcriptase controls, which were expected to produce negligible signals (Cycle threshold values >35). Absolute standard curves of Bach-1, HO-1, and glyceraldehyde-3-phosphate dehydrogenase were constructed with results of parallel PCRs performed on serial dilutions of standard DNA (originally generated by in vitro transcription). Input cDNA concentrations were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA. Protein Preparation and Western Blotting—Cells were washed twice with phosphate-buffered saline and lysed in a lysis buffer (phosphate-buffered saline containing 1% Triton 100, 2 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml each of leupeptin and pepstatin). The lysed cells were sonicated for 10 s, followed by centrifugation for 10 min at 13,000 × g at of were stored at –80 °C Western Total proteins μg) were on onto a were for 1 h in phosphate-buffered saline containing 5% and and for 1 h with primary antibody at The dilutions of the primary were as for for anti-β-actin and for anti-HO-1 with in phosphate-buffered the were for 1 h with a antibody anti-goat or anti-mouse the were washed and the bound were with the ECL-Plus according to the manufacturer's A was used to the of each specific Western blotting. are as the of the control to the value with the cells or which were or of were three for Western included at samples for each results from are were performed with For in values were by of with the for multiple or a was with CA). of were of HO-1 Gene Expression in Liver is a of HO-1 in cell and We have previously inductions of HO-1 gene expression in primary of cells E. Y. Healey J. Bonkovsky H. Biochem. Biophys. PubMed Scopus (25) Google Scholar, E.E. Gildemeister O.S. Pepe J.A. Lambrecht R.W. Bonkovsky H.L. Mol. Cell. Biochem. 1997; PubMed Scopus Google Scholar). we and one the HO-1 promoter region that mediated activation of the HO-1 gene in cells (12Shan Y. Pepe J. Lambrecht R.W. Bonkovsky H.L. Arch. Biochem. Biophys. 2002; 399: 159-166Crossref PubMed Scopus (25) Google Scholar, 13Shan Y. Pepe J. Lu T.H. Elbirt K.K. Lambrecht R.W. Bonkovsky H.L. Arch. Biochem. Biophys. 2000; 380: 219-227Crossref PubMed Scopus (80) Google Scholar). the mechanism heme HO-1 gene expression in human we HO-1 mRNA and protein levels with heme in Huh-7 cells. heme HO-1 mRNA and protein levels in a and not HO-1 mRNA levels to 10 heme for 2 a at and were but significantly increased A concentration of heme as as 1 significantly up-regulated HO-1 mRNA by and the was at heme for The concentration of heme that to up-regulation of HO-1 h of was 2 concentrations of heme produce up-regulation of the HO-1 gene in human Huh-7 as in other cell (13Shan Y. Pepe J. Lu T.H. Elbirt K.K. Lambrecht R.W. Bonkovsky H.L. Arch. Biochem. Biophys. 2000; 380: 219-227Crossref PubMed Scopus (80) Google Scholar, J. Killeen E. Gong P. Naquin R. Hu B. Stewart D. Ingelfinger J.R. Nath K.A. Am. J. Physiol. 2003; 284: F743-F752Crossref PubMed Scopus (151) Google Scholar, K.K. of in the of Heme by in of Scholar, J. Shibahara S. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, K.A. H. A.J. Alam J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). previously in cells (15Srivastava K.K. Cable E.E. Donohue S.E. Bonkovsky H.L. Eur. J. Biochem. 1993; 213: 909-917Crossref PubMed Scopus (41) Google Scholar, E.E. Gildemeister O.S. Pepe J.A. Lambrecht R.W. Bonkovsky H.L. Mol. Cell. Biochem. 1997; PubMed Scopus Google Scholar, Cable E.E. Bonkovsky H.L. Mol. Cell. Biochem. 1993; PubMed Scopus Google Scholar), the up-regulation is a up-regulation for at Silencing Bach-1 Gene with Bach-1 siRNA in Huh-7 siRNAs have to gene expression in mammalian for the of and proteins. RNA is a highly mechanism in gene siRNAs have been shown to act as the in RNA cleavage of mRNA in a that is to be an defense mechanism J. A. T. EMBO J. 2001; 20: PubMed Scopus Google Scholar, J. A. K. T. 2001; PubMed Scopus Google Scholar, A. A. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). RNA cleavage is by the which as a Using siRNA we reduced human Bach-1 gene expression by transfection of siRNA duplexes for Bach-1 into Huh-7 cells. were and compared with of siRNA Bach-1 mRNA concentrations in Bach-1 siRNA-transfected cells using quantitative RT-PCR, we found Bach-1 siRNA at h could reduce Bach-1 mRNA levels by when compared with siRNA treated cells We Bach-1 protein concentrations in Bach-1 siRNA-transfected cells using Western We found that of Bach-1 siRNA reduced Bach-1 protein to at h when compared with siRNA-transfected cells the specificity of the Bach-1 gene by Bach-1 siRNAs, we LaminB2 duplex LaminB2 mRNA by quantitative RT-PCR in Huh-7 cells. The results showed that transfected LaminB2 siRNA for h reduced by LaminB2 mRNA We further Bach-1 gene expression from the cells transfected with siRNA, nonspecific control duplexes and LaminB2 We found that were of Bach-1 mRNA or protein levels in the cells transfected with and 100 nonspecific control duplexes and LaminB2 duplex shown 100 when compared with cells that were not transfected we that of the human Bach-1 gene using Bach-1 mRNA was and of effect of nonspecific siRNAs on Bach-1 mRNA and protein levels in Huh-7 cells. Huh-7 cells were transfected with 100 nonspecific control Bach-1 siRNA, or LaminB2 siRNA for which cells were harvested, and mRNA and protein were as described under quantitative RT-PCR of Bach-1 mRNA are presented as means S.E. from three from other Western of Bach-1 protein isolated from the same cells as in A. were by to a and with and The of Bach-1 normalized to for the Western is shown in the from cells transfected with siRNA was as of HO-1 mRNA with Expression of Bach-1 Gene by Bach-1 transcription Bach-1 forms heterodimers with one of the small Maf proteins or that bind to the Maf and expression of that to heterodimers (24Sun J. Hoshino H. Takaku K. Nakajima O. Muto A. Suzuki H. Tashiro S. Takahashi S. Shibahara S. Alam J. Taketo M.M. Yamamoto M. Igarashi K. EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (511) Google Scholar, 25Ogawa K. Sun J. Taketani S. Nakajima O. Nishitani C. Sassa S. Hayashi N. Yamamoto M. Shibahara S. Fujita H. Igarashi K. EMBO J. 2001; 20: 2835-2843Crossref PubMed Scopus (414) Google Scholar). Sun et al. (24Sun J. Hoshino H. Takaku K. Nakajima O. Muto A. Suzuki H. Tashiro S. Takahashi S. Shibahara S. Alam J. Taketo M.M. Yamamoto M. Igarashi K. EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (511) Google Scholar) showed that HO-1 is at levels in of suggesting that Bach-1 as a of transcription of the HO-1 gene. We the effect of Bach-1 on expression of human HO-1 by the Bach-1 gene in Huh-7 cells. Bach-1 siRNA significantly increased HO-1 mRNA levels in Huh-7 cells in a and A and The levels of HO-1 mRNA were significantly increased to 50 Bach-1 siRNA and a at of HO-1 mRNA expression was at h to 100 Bach-1 siRNA and a by The expression levels of HO-1 mRNA increased for and this was with the of Bach-1 mRNA expression. whether the increased HO-1 gene expression in the of Bach-1 siRNA a we the levels of HO-2 and ALAS-1 mRNA in the same Bach-1 siRNA-treated cells. Bach-1 siRNA did not HO-2 or ALAS-1 mRNA levels in Huh-7 cells. these results that Bach-1 is a of the HO-1 gene in human hepatic with the results of Sun et al. (24Sun J. Hoshino H. Takaku K. Nakajima O. Muto A. Suzuki H. Tashiro S. Takahashi S. Shibahara S. Alam J. Taketo M.M. Yamamoto M. Igarashi K. EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (511) Google Scholar) in mice. of HO-1 Expression by Heme and Bach-1 the effects of of heme and Bach-1 siRNA on HO-1 gene in Huh-7 cells. HO-1, we Bach-1 mRNA was by more by Bach-1 siRNA. HO-1 mRNA expression levels were markedly up-regulated by with a concentration of heme in the of Bach-1 siRNA and were for of the heme concentrations Using the HO-1 mRNA levels were with heme concentrations in the or of Bach-1 siRNA The curves are significantly different from each other an effect of Bach-1 siRNA and heme on the up-regulation of HO-1 gene expression. HO-1 protein levels were with HO-1 mRNA levels, in cells treated with heme in the or of Bach-1 siRNA et al. K. Sun J. Taketani S. Nakajima O. Nishitani C. Sassa S. Hayashi N. Yamamoto M. Shibahara S. Fujita H. Igarashi K. EMBO J. 2001; 20: 2835-2843Crossref PubMed Scopus (414) Google Scholar) and Sun et al. (24Sun J. Hoshino H. Takaku K. Nakajima O. Muto A. Suzuki H. Tashiro S. Takahashi S. Shibahara S. Alam J. Taketo M.M. Yamamoto M. Igarashi K. EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (511) Google Scholar) reported that Bach-1 is a Heme to cysteine-proline motifs of Bach-1 when heme is the DNA-binding and of is markedly We and one in the 5′-untranslated region of the HO-1 promoter region Y. Lambrecht R.W. Bonkovsky H.L. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar). we found are HeRE but not a in the human HO-1 promoter results are with the that the up-regulation of HO-1 is at in to a which elements to the gene. the of heme HO-1, and the of the binding proteins An alternative is that heme the of the Nrf2 protein (27Alam J. Killeen E. Gong P. Naquin R. Hu B. Stewart D. Ingelfinger J.R. Nath K.A. Am. J. Physiol. 2003; 284: F743-F752Crossref PubMed Scopus (151) Google Scholar) leading to of heterodimers of that bind to the activating the HO-1 gene. results showed that heme produces an additional up-regulation of HO-1, beyond that produced by Bach-1 siRNAs, suggesting that heme does not act solely through its effects on Bach-1, which is with this alternative It that the induction of HO-1 is a of Bach-1 heme binding with subsequent of HO-1 gene and of Nrf2 a in of Nrf2 with subsequent induction of HO-1 gene In heme markedly HO-1 mRNA and protein levels in a and in human Huh-7 cells. Using siRNA we of the human Bach-1 gene by using siRNAs, targeted to different of the human Bach-1 mRNA. Bach-1 has a specific and selective effect to repress the expression of human hepatic The of Bach-1 by siRNAs is a useful method for up-regulating HO-1 gene expression in human Exogenous heme produces additional up-regulation beyond that produced by Bach-1 siRNAs, suggesting that heme does not act solely through its effects on Bach-1. We M. for of Health for