Transforming Growth Factor-β Induces Transcription Factors MafK and Bach1 to Suppress Expression of the Heme Oxygenase-1 Gene*

Background: TGF-β suppresses early carcinogenesis but accelerates malignant progression by activating invasion and metastasis. HO-1 is induced in response to oxidative stress and protects cells from oxidative injury. Results: TGF-β suppresses tBHQ-inducible expression of HO-1 through induction of MafK and Bach1. Conclusion: TGF-β suppresses a protective response to electrophiles. Significance: This work provides the first evidence that TGF-β affects electrophilic responses.

Transforming growth factor-␤ (TGF-␤) regulates multiple biological functions such as cell proliferation, differentiation, apoptosis, and morphogenesis (1,2). Upon ligand binding, type II serine/threonine kinase receptors activate type I receptors, and activated type I receptors phosphorylate Smad proteins. Phosphorylated receptor-regulated Smads (R-Smads; Smad2 and Smad3) form heteromeric complexes with common partner Smad (Co-Smad; Smad4) and accumulate in the nucleus. Activated Smad complexes regulate expression of target genes by binding to specific DNA sequences together with various cobinding transcription factors and recruiting coactivators or corepressors (3). Differential expression of cobinding transcription factors contributes to the cell type-and context-dependent cellular responses to TGF-␤.
TGF-␤ is a potent inhibitor of epithelial cell proliferation; therefore, it acts as a tumor suppressor in the early stages of carcinogenesis. On the other hand, cancer cells develop resistance to TGF-␤-inducible growth inhibition in the advanced stages of carcinogenesis. At these later stages, TGF-␤ promotes epithelial-mesenchymal transition, invasion, and metastasis in certain types of cancer cells without TGF-␤ receptor abnormalities (4). Therefore, TGF-␤ is thought to act as a double-edged sword in cancer development (5). However, the multifunctional effects of TGF-␤ on cancer initiation and progression have not been fully elucidated.
Detoxification and export of xenobiotics are crucial for the maintenance of cellular homeostasis and protection against carcinogenic agents (6). Nrf2, a member of the cap 'n' collar family of basic region leucine zipper transcription factors (7), is a key transcriptional regulator of detoxification enzymes, transporters, and antioxidative molecules. Nrf2 forms heterodimers with small Maf proteins (MafF, MafG, and MafK), binds to antioxidant response elements (AREs), 2 and activates transcription of target genes. Mice lacking Nrf2 fail to induce phase II detoxifying enzymes and antioxidative molecules in response to oxidative stress, indicating that Nrf2 has a critical role in cellular defense against xenobiotics and oxidative stress (8).
ARE-mediated transcriptional activities are regulated by the combinations and relative levels of cap 'n' collar molecules and small Maf proteins. The Nrf2-small Maf heterodimer is essential for the activation of ARE-mediated transcription. On the other hand, small Maf homodimers have been reported to suppress ARE-mediated transcription (7). Other cap 'n' collar molecules, including Bach1, form heterodimers with small Maf proteins and suppress ARE-mediated transcription (9). For example, the MafK-Bach1 heterodimer interacts with AREs in the enhancer region of the heme oxygenase-1 (HO-1) gene and suppresses its transcription. Heme, an inducer of HO-1, displaces Bach1 from the AREs, which is followed by binding of Nrf2 to the AREs, and increases in HO-1 expression (10 -12). HO-1 catalyzes the rate-limiting step in heme catabolism and generates carbon monoxide, ferric iron, and biliverdin. Carbon monoxide and ferric iron can activate Nrf2, suggesting that HO-1 acts as a cytoprotective factor in both suppression of oxidative stress and activation of Nrf2 (13)(14)(15).
In this study, we examined the effect of TGF-␤ on the expression of HO-1. We found that TGF-␤ induces expression of MafK and Bach1 and that these genes are essential for suppression of HO-1 by TGF-␤ signaling.

EXPERIMENTAL PROCEDURES
Cells and Culture-293T and NMuMG cells were obtained from the American Type Culture Collection. These cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Invitrogen). Mouse mammary carcinoma JygMC(A) cells were cultured as described previously (16).
RNA Interference-NMuMG and JygMC(A) cells were transfected with 40 nM small interfering RNA (siRNA) directed against MafK or Bach1 using Lipofectamine 2000 (Invitrogen). In the case of double knockdown of MafK and Bach1, cells were transfected with a 40 nM concentration of each siRNA. Sequences of siRNA are listed in Table 1. Control siRNA was purchased from Invitrogen (Stealth TM RNAi Negative Universal Control Medium). A pSUPER-puro vector (Oligoengine) expressing a short hairpin RNA against human and mouse Smad4 (pSUPER-sh-Smad4) was described previously (22). NMuMG cells transfected with pSUPER-sh-Smad4 were cloned and maintained in the presence of puromycin (1 g/ml).
Reverse Transcription and Polymerase Chain Reaction (RT-PCR)-Total RNA was prepared using ISOGEN (Nippon Gene). RT was performed using High Capacity RNA-to-cDNA Master Mix (Applied Biosystems), and PCR was performed using Ex Taq polymerase (Takara Bio). PCR primers are listed in Table 2.

TGF-␤ Suppresses Electrophile-inducible Expression of HO-1-
To examine the effect of TGF-␤ on expression of HO-1, NMuMG cells were treated with tBHQ in the absence or presence of TGF-␤ signaling. mRNA levels of HO-1 were highly increased 4 h after tBHQ stimulation (Fig. 1A, lanes 2 and 3). However, pretreatment of the cells with TGF-␤ significantly reduced the induction (Fig. 1A, lanes 4 and 5). TGF-␤ alone had no detectable effect on the basal expression levels of HO-1 (Fig.  1A, lanes 6 and 7). A representative result of the densitometric quantification of normalized mRNA levels is shown in Fig. 1A, right panel. TGF-␤-mediated suppression of HO-1 was also examined at the protein level (Fig. 1B). These results indicate that TGF-␤ suppresses tBHQ-inducible expression of HO-1 in NMuMG cells.
TGF-␤ Does Not Affect the Stabilization or Nuclear Accumulation of Nrf2-To examine how TGF-␤ affects HO-1 expression, we examined the expression and subcellular localization of Nrf2 proteins in NMuMG cells. Because Nrf2 activates the transcription of HO-1, we assumed that TGF-␤ would affect the accumulation or nuclear translocation of Nrf2. However, as shown in Fig. 2, neither the stabilization ( Fig. 2A) nor the nuclear localization of Nrf2 (Fig. 2, B and C) was affected by TGF-␤.
TGF-␤ Induces MafK and Bach1-Nrf2 activates transcription of target genes by binding to AREs together with small Maf proteins. However, if small Maf levels rise exceedingly, small Maf homodimers can compete with Nrf2-small Maf heterodimers for the binding to AREs (7). Otherwise, heterodimers of small Mafs and other cap 'n' collar-type transcriptional regulators such as Bach1 can compete for the binding (9). Because TGF-␤ did not affect the nuclear accumulation of Nrf2, we next examined the effect of TGF-␤ on the expression of small Mafs and Bach1 and found that TGF-␤ increased MafK and Bach1 at both mRNA and protein levels (Fig. 3, A and B). Induction of MafK and Bach1 was suppressed by a TGF-␤ type I receptor kinase inhibitor, SD-208 (Fig. 3C).
MafK and Bach1 Regulate Induction of HO-1-We next examined the effect of MafF, MafG, and MafK on pHO1-luc activities. Transiently expressed MafG and MafK, but not MafF, suppressed the reporter activities (Fig. 4A). MafF, MafG, and MafK all formed heterodimers with Nrf2 (Fig. 4B), but when small Maf proteins were independently expressed, MafK and MafG, but not MafF bound to HO-1 ARE DNA fragments (Fig. 4C). We then established NMuMG cell lines with stable expression of MafK, MafG, or Bach1. Binding of MafK and MafG to HO-1 ARE (E2) was confirmed by chromatin immunoprecipitation (Fig. 4D). In functional analyses, both tBHQ and diethyl maleate (DEM) induced HO-1 expression in  ). B, NMuMG cells were treated as in A. Immunoblot analysis was performed using anti-HO-1 antibody. ␤-Actin was examined as a loading control (left panel). Quantification of the protein levels was performed using NIH ImageJ and normalized to ␤-actin (right panel). All experiments were repeated more than three times to confirm their reproducibility. NMuMG cells. However, these effects were completely blocked by overexpression of MafK (Fig. 5, A and B). MafK also decreased pHO1-luc activities in both the absence and the presence of tBHQ or DEM (Fig. 5C). On the other hand, reduction of MafK by siRNA strikingly enhanced tBHQ-inducible expression of HO-1 mRNA (Fig. 5D). However, stable overexpression of MafG did not suppress tBHQ-and DEM-inducible expression of HO-1 (supplemental Fig. 2). We also established NMuMG cells stably expressing FLAG-Bach1 (Fig. 5E). Bach1 had a nearly identical effect as that of MafK on HO-1 expression (Fig. 5, E-H) except for a stronger induction of HO-1 in the absence of both electrophiles and TGF-␤ (Fig. 5H). Furthermore, double knockdown of MafK and Bach1 highly increased both the basal and the tBHQ-inducible expression of HO-1 and abolished the suppressive effect of TGF-␤ on tBHQ-inducible HO-1 expression (Fig. 5I). Endogenous MafK and Bach1 also suppressed HO-1 in breast cancer cells. Knockdown of MafK or Bach1 in JygMC(A) cells clearly increased expression of HO-1 (Fig. 5J). Recruitment of Nrf2, MafK, and Bach1 to ARE Sites in the HO-1 Promoter-ARE sites (E1 and E2) in the promoter region of HO-1 are essential for its transcriptional regulation. To determine whether changes in HO-1 expression are associated with altered binding of Nrf2, MafK, and Bach1 to ARE sites in vivo, we performed chromatin immunoprecipitation assays. When cells were treated with tBHQ, Nrf2 binding to both E1 and E2 (mainly to E1) increased. These interactions were reduced when cells were treated with TGF-␤ before tBHQ stimulation (Fig. 6A). Binding of MafK to E1 and E2 increased after tBHQ stimulation together with Nrf2, but its binding to E2 was not decreased by TGF-␤ treatment (Fig.  6B). In contrast, Bach1 binding to E2 was highest in the absence of tBHQ and was reduced when cells were treated with tBHQ (Fig. 6C).
Binding of Smad3 and MafK on ARE Sites in the HO-1 Promoter-Binding of MafK or Bach1 and Smad3 was examined by coprecipitation assays. Both MafK and Bach1 bound to Smad3. MafK bound to Smad3 in the presence of TGF-␤ signaling. On the other hand, Bach1 bound to Smad3 in the absence of TGF-␤ signaling (Fig. 7, A and B). Binding of Smad2/3 on ARE (E2) in the promoter region of HO-1 was detected by chromatin immunoprecipitation assays in the presence of TGF-␤ signaling (Fig. 7C).

DISCUSSION
In this study, we proved that TGF-␤ suppresses the electrophile-inducible transcriptional activation of HO-1. This is the first report on the regulation of the Nrf2-mediated oxidative stress responses by TGF-␤ signaling. Intriguingly, TGF-␤ did not reduce the nuclear accumulation of Nrf2 provoked by electrophiles, but the recruitment of Nrf2 to AREs (E1 and E2) in the promoter region of HO-1 was clearly suppressed by TGF-␤. Consistent with the previous report that MafK-Bach1 heterodimers repress HO-1 expression (12), knockdown of MafK and Bach1 enhanced HO-1 expression and abolished the suppressive effect by TGF-␤ (Fig. 5I). These results suggest that TGF-␤ signaling generally antagonizes cytoprotective responses mediated by the Keap1-Nrf2 system. Therefore, we further investigated the effect of TGF-␤ on Nrf2-mediated activation of other Nrf2 target genes. NQO1 and the heavy and light chains of ␥-glutamylcysteine synthetase were examined, and all of these target genes were suppressed by TGF-␤, although the extents of the suppression were different in different genes (data not shown). Furthermore, the constitutively active TGF-␤ type I receptor ALK5T204D significantly suppressed Nrf2-mediated activation of pNQO1-ARE-luc together with Smad3 (data not shown). The reporter activity was further suppressed by the addition of Smad4. These results suggested that TGF-␤ suppresses the transcriptional activity of Nrf2 through activation of the Smad signaling pathway. However, suppression of these Nrf2-target genes was not canceled by FIGURE 2. TGF-␤ does not affect the stabilization and nuclear accumulation of Nrf2. A, NMuMG cells were treated with TGF-␤ (5 ng/ml) for 1 h before stimulation with tBHQ (25 M) and incubated for an additional 4 h. Immunoblot analysis was performed using anti-Nrf2, -phospho-Smad2 (P-Smad2), -Smad2 and -␣-tubulin antibodies as indicated. Arrow, specific band for Nrf2; *, nonspecific bands. B, NMuMG cells were treated as in A. After fixation, cells were serially stained with anti-Nrf2 (green) and anti-Smad2 (red) antibodies. Nuclei were counterstained with DAPI. Scale bar, 50 m. C, NMuMG cells were treated as in A. Nuclear and cytosolic fractions were isolated and analyzed by immunoblotting using antibodies for Nrf2 and Smad2. Lamin A/C was used as a nuclear protein marker, and ␣-tubulin was used as a cytosolic protein marker.
knockdown of MafK and Bach1 (data not shown). HO-1 was the only gene in which suppression by TGF-␤ was canceled by knockdown of MafK and Bach1. Therefore, TGF-␤ probably suppresses different target genes in different molecular mechanisms. Consistent with this notion, both HO-1 and NQO1 are regulated by Nrf2, and these genes contain ARE sites in their promoter regions, but Bach1 interacts specifically with AREs in HO-1. Subtle differences in AREs or their flanking sequences affect the binding affinities of the Maf-containing dimers, resulting in different contributions of each dimer to the AREdependent gene regulation. Indeed, both Nrf2-MafG heterodimer and MafG homodimer bind to the consensus Maf recognition element with high affinity but bind differentially to the suboptimal binding sequences degenerated from the con-sensus (26,27). Different Maf complexes may be differently regulated by TGF-␤ signaling.
A discrepancy between the nuclear accumulation and transcriptional activity of Nrf2 has been reported. When human aortic endothelial cells were exposed to oscillating flow, Nrf2 accumulated in the nuclei but did not activate stress response genes. In contrast, when the cells were exposed to laminar flow, Nrf2 accumulated in the nuclei and activated its target genes (28). The analogous finding of the current study suggests that levels of small Mafs and Bach1 or other related transcriptional factors might be involved in nuclear regulation of Nrf2 activities.
TGF-␤ markedly elevated expression of MafK and Bach1 in NMuMG cells. Transcriptional regulation of tissue-specific expression of the MafK gene was analyzed previously in transgenic mice harboring the lacZ gene as a reporter. Two alternative promoters were identified in the MafK gene, and the upstream and downstream promoters mediate the mesodermal and neuronal expressions, respectively (29,30). A hematopoietic enhancer was also identified in the 3Ј-region of the MafK gene (31). However, the regulatory regions responsible for the induction by TGF-␤ have not been identified. We found that Smad4 was indispensable for the expression of MafK and suppression of HO-1 by TGF-␤ (supplemental Fig. 1, C and E). Conversely, Bach1 expression was constitutively activated by knockdown of Smad4 (supplemental Fig. 1E).  The regulatory mechanisms of Bach1 function identified so far are mainly at the posttranscriptional level, i.e. changes in DNA binding affinity, subcellular localization, and protein stability (10,32,33). One report described the transcriptional regulation of BACH1 examined in a reporter assay in K562 cells (34). A GC box residing in the promoter region was critical for the promoter activity, and Sp1 was a transactivating factor binding to the GC box, which could be a target of TGF-␤ signaling. Transcriptional regulation of Bach1 by TGF-␤ signaling might constitute a novel layer of the regulation of Bach1 function in vivo.
TGF-␤ signaling is highly enhanced in many pathological conditions including precancerous lesions associated with ulcerative colitis and viral hepatitis (35,36). The present study demonstrated that TGF-␤ markedly suppresses a cytoprotective gene, HO-1. It should be noted that a single nucleotide polymorphism in the enhancer region of HO-1 that affects HO-1 expression has been implicated in an increased incidence of lung cancer (37). These molecular epidemiological data suggest that suppression of HO-1 may accelerate cancer initiation and progression. Therefore, TGF-␤ might promote cancer initiation in precancerous lesions or expedite cancer progression through suppression of Nrf2 target genes including HO-1.