Sp3 transcription factor is crucial for transcriptional activation of the human NOX4 gene Masato Katsuyama 1,2 , Hideyo Hirai 3 , Kazumi Iwata 2 , Masakazu Ibi 2 , Kuniharu Matsuno 2 , Misaki Matsumoto 2 and Chihiro Yabe-Nishimura 2 1 Radioisotope Center, Kyoto Prefectural University of Medicine, Japan 2 Department of Pharmacology, Kyoto Prefectural University of Medicine, Japan 3 Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Japan Introduction Reactive oxygen species, including superoxide (O À 2 ) and hydrogen peroxide, have been recognized as intrin- sic signaling molecules that modulate multiple cellular responses. NADPH oxidases are the major source of O À 2 in various tissues [1–3]. They catalyze the reduction of molecular oxygen to O À 2 using NADPH as an elec- tron donor. A wealth of data has been collected on the phagocyte NADPH oxidase, an essential component of the host antimicrobial defense system [4]. The phago- cyte oxidase consists of the catalytic subunit gp91phox (NOX2), the regulatory subunits p22phox, p47phox, p67phox and p40phox, and the small GTPase, Rac. Recent studies in nonphagocytic cells identified several homologs of the catalytic subunit NOX2. Among them, NOX4 was first identified as ‘‘renal NOX’’ and was later found to be expressed in various tissues [5–8]. In contrast to NOX1, of which expression is induced by various bioactive factors in specific cell types, NOX4 is constitutively expressed in numerous cell lineages. It was reported that NOX4 requires only p22phox to exert its full enzymatic activity. Recently, polymerase (DNA-directed) delta-interacting protein 2 (Poldip2) was reported to interact with p22phox and enhance the activity of NOX4 ⁄ NADPH oxidase [9]. Keywords GC-box; NADPH oxidase; NOX4; Sp3; transcriptional regulation Correspondence M. Katsuyama, Radioisotope Center, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan Fax: 81 75 251 5381 Tel: 81 75 251 5381 E-mail: mkatsuya@koto.kpu-m.ac.jp (Received 6 December 2010, revised 7 January 2011, accepted 11 January 2011) doi:10.1111/j.1742-4658.2011.08018.x NOX is the catalytic subunit of NADPH oxidase, the superoxide-generat- ing enzyme. Among several isoforms of NOX, NOX4 is abundantly expressed in various tissues. To clarify the mechanisms of constitutive and ubiquitous expression of NOX4, the promoter activities of the human NOX4 gene were analyzed by reporter assays. The 5’-flanking and non-cod- ing regions of the human NOX4 gene are known to contain multiple GC bases. Among them, three GC-boxes containing putative Sp ⁄ Klf-binding sites, which were not found in rodent genes, were suggested to be essential for the basal expression of the NOX4 gene in SH-SY5Y and HEK293 cells. Electrophoresis mobility shift assays demonstrated that Sp1 and Sp3 could bind to GC-boxes at positions )239 ⁄ )227 and +69 ⁄ +81 in these cells. Chromatin immunoprecipitation assays showed that Sp1 and Sp3 could also bind to GC-boxes at positions )239 ⁄ )227 and +69 ⁄ +81 in vivo. The promoter activity of the NOX4 gene was reduced in SH-SY5Y and HEK293 cells by transfection of an anti-Sp3 short hairpin RNA-expression plasmid. Taken together, these results suggest that Sp3 plays a key role in the expression of NOX4 in various cell lineages in humans. Abbreviations ChIP, chromatin immunoprecipitation; EMSA, electrophoresis mobility shift assay; GADPH, glyceraldehyde-3-phosphate dehydrogenase; shRNA, short hairpin RNA; Sp, specificity protein. 964 FEBS Journal 278 (2011) 964–972 ª 2011 The Authors Journal compilation ª 2011 FEBS Another study demonstrated that the activity of NOX4 is regulated by the level of mRNA [10]. Thus, the expression level of NOX4 seems to be directly linked to its enzymatic activity. The mechanisms of transcriptional regulation of NOX4, however, are poorly understood. A study has reported the involve- ment of the transcription factor E2F1 in rodent vascu- lar smooth muscle cells [11], but there seem to be species differences in the transcriptional regulation of NOX enzymes, as found for NOX1 [12,13]. This led us to undertake an investigation of the molecular basis of the constitutive and ubiquitous expression of NOX4 in human tissues. We report here the predominant role of the transcription factor specificity protein (Sp) 3 in the expressional regulation of the human NOX4 gene. Results GC-boxes are essential for transcriptional activation of the human NOX4 promoter Figure 1 shows the alignment of the 5¢-flanking and 5¢-noncoding sequences of the human, rat and mouse NOX4 genes. Compared with the high similarity between the rat and mouse sequences, the similarity of the human sequence was relatively low. Therefore, we isolated the promoter region of the human NOX4 gene and examined its transcriptional activity in human neuroblastoma SH-SY5Y cells, which highly express NOX4. Approximately 2-kb region encompassing the 5¢-flanking and noncoding regions were subcloned into a luciferase vector and deletion mutants were con- structed. As demonstrated in Fig. 2, the transcriptional activity of the NOX4 promoter in SH-SY5Y cells was dramatically decreased by deletion up to nucleotide )226, but not to nucleotide )309. The activity of the 5¢-noncoding sequence alone was, however, still 10-fold higher than that of the basic vector. As shown in Fig. 1, GC-boxes were found in the human NOX4 gene at positions )239 ⁄ )227 (GC-box1), +69 ⁄ +81 (GC-box2) and +221 ⁄ +233 (GC-box3). To examine the contribution of these GC-boxes to the transcrip- tional activity of the NOX4 promoter, additional dele- tion mutants were constructed. As shown in Fig. 3A, the transcriptional activity was dramatically decreased by the deletion of GC-box1. The activity was further decreased by the deletion of GC-box2 (Fig. 3B). As shown in Fig. 3C, the transcriptional activity of the 1 kb of the 5¢-flanking region was dramatically decreased by the deletion of GC-box3, the GC-rich Fig. 1. Alignment of the 5¢-nucleotide sequences of the human, rat and mouse NOX4 genes. The numbers on the left are nucleotide posi- tions relative to the putative transcriptional start sites [7,11]. Nucleotides conserved in all genes are marked with asterisks below the mouse sequence. Three putative GC-boxes found in the human gene are indicated above the sequence. Transcription start sites are indicated by an open arrowhead (human) or by closed arrowheads (rat and mouse). Translation start codons are underlined. M. Katsuyama et al. Sp3 regulates transcription of NOX4 ⁄ NADPH oxidase FEBS Journal 278 (2011) 964–972 ª 2011 The Authors Journal compilation ª 2011 FEBS 965 region adjacent to the start codon. All three GC-boxes were also shown to be essential in the transcriptional activity of the NOX4 promoter in HEK293 cells (Fig. 4). Binding of Sp1 and Sp3 to GC-boxes 1 and 2 in the NOX4 promoter The GC-box is known to bind Sp ⁄ Klf transcription factors, especially Sp1, Sp3 and Sp4 [14,15]. To exam- ine which Sp transcription factors bind to the GC- boxes in the NOX4 promoter, electrophoresis mobility shift assays (EMSAs) were carried out using nuclear extracts obtained from SH-SY5Y and HEK293 cells. As shown in Fig. 5A, the binding of nuclear proteins to GC-boxes 1 and 2 was detected by doublet bands in both cell lines. The extent of binding of nuclear pro- teins to GC-box 3 was much lower than that for other GC-boxes. When specific binding of nuclear proteins to GC-boxes 1 and 2 was verified, the bands almost completely diminished in the presence of excess unla- beled probes, but not in the presence of excess mutated probes (Fig. 5B). Pre-incubation of the nuclear extract with an anti-Sp1 IgG decreased the intensity of the upper band for both GC-boxes 1 and 2 in both cell lines, suggesting that the binding of Sp1, detected by the upper band, was inhibited by the antibody. On the other hand, pre-incubation with an anti-Sp3 IgG gen- erated supershifted bands (as indicated by arrowheads) and decreased the intensity of the lower band (Fig. 5B). These results suggest that Sp1 and Sp3 are the major transcription factors that bind to GC-boxes 1 and 2 in the NOX4 promoter. In vivo binding of Sp1 and Sp3 to GC-boxes 1 and 2 in the NOX4 promoter To examine whether Sp1 and Sp3 bind to GC-boxes 1 and 2 in the NOX4 promoter, chromatin immunopre- cipitation (ChIP) assays were performed. As shown in Fig. 6, specific binding of Sp1 and Sp3 to GC-boxes 1 and 2 in the NOX4 promoter, but not to a region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was demonstrated. All regions were shown to bind RNA polymerase II, as expected. Specificity of the binding of Sp1 or Sp3 was verified by control IgG. Gene silencing of Sp3 attenuated the promoter activity of the NOX4 gene To examine whether Sp1 and Sp3 are actually involved in transcription of the NOX4 gene, an anti-Sp1 or anti-Sp3 short hairpin RNA (shRNA)-expression plas- mid was cotransfected with luciferase plasmids. As shown in Fig. 7A, the anti-Sp1 shRNA-expression plasmid suppressed the expression of Sp1 and the anti- Sp3 shRNA-expression plasmid suppressed the expres- sion of Sp3. The promoter activity of the NOX4 gene was significantly reduced by cotransfection with the anti-Sp3 shRNA-expression plasmid, but not by co- transfection with the anti-Sp1 shRNA-expression plas- mid (Fig. 7B). These results highlight the pivotal role of Sp3 in the transcription of the NOX4 gene. Discussion The major lines of evidence provided by this study are that (a) three GC-boxes in the 5¢-flanking and non- coding regions of the human NOX4 gene are essential for the basal expression of the NOX4 gene, (b) EMSAs demonstrated that Sp1 and Sp3 could bind to GC- boxes at positions )239 ⁄ )227 and +69 ⁄ +81 of the NOX4 gene, (c) Sp1 and Sp3 were also shown to bind to GC-boxes at positions )239 ⁄ )227 and +69 ⁄ +81 in vivo by ChIP assays and (d) RNA interference against Sp3 suppressed the transcriptional activity of the NOX4 gene in SH-SY5Y and HEK293 cells. Based on these findings, it is reasonable to conclude that Sp3 plays a key role in the constitutive expression of NOX4 in various cell types. Among Sp ⁄ Klf family transcription factors, Sp1, Sp3 and Sp4 have been reported to bind to the GC- box [14,15]. Among them, Sp1 and Sp3 are widely dis- tributed in the body. In contrast to the role of Sp1 as a transcriptional activator, the functional role of Sp3 has been controversial [16]. Sp3 has been considered to Relative luciferase activity 0 10 20 30 40 pGL3-basic hNOX4+1/+ATG * * Fig. 2. Analyses of the NOX4 promoter activity in SH-SY5Y cells. The 2 kb of the 5¢-flanking region of the human NOX4 gene was cloned into pGL3-basic and the reporter construct was transfected into SH-SY5Y cells. The b-galactosidase expression vector was cotransfected as an internal control. The bars represent the means ± standard error of three determinations. * P < 0.05. Sp3 regulates transcription of NOX4 ⁄ NADPH oxidase M. Katsuyama et al. 966 FEBS Journal 278 (2011) 964–972 ª 2011 The Authors Journal compilation ª 2011 FEBS act as a transcriptional repressor of Sp1-dependent transcription [17]. This inhibitory action is, however, considered to be mediated by two short isoforms of Sp3, which lack the activation A domain at the N-ter- minus [16]. In fact, analyses using gene-disrupted mice revealed that Sp3 is essential for postnatal survival and tooth development [18], hematopoiesis [19] and myo- cardial development [20]. The involvement of Sp1 and ⁄ or Sp3 in transcriptional activation is gene-spe- cific. While both Sp1 and Sp3 were able to activate the transcription of some genes [21,22], an Sp3-dependent transcription was documented for other genes [23,24]. Our results suggest that NOX4 is expressed in an Sp3- dependent manner. It was reported that Sp3 acts as a transcriptional activator when the lysine residue at the KEE motif is acetylated [25]. We observed that tri- chostatin A (TSA), a histone deacetylase inhibitor that is known to acetylate Sp3 [26], significantly induced the expression of NOX4 mRNA in HEK293 cells (data not shown). Thus, Sp3 seems to act as a transcrip- tional activator for the expression of NOX4. It has been reported that the DNA-binding activity of Sp1 and Sp3 is enhanced under oxidative stress induced by glutathione depletion or hydrogen peroxide [27,28]. This raises the possibility of a positive feed- back loop in which Sp3 activation by oxidative stress leads to further superoxide production via NOX4 ⁄ NADPH oxidase. There appears to be species specificity in the regula- tion of NOX4 gene expression. GC-boxes 1 and 2 were not found in rodents, and the binding site for the E2F1 transcription factor was not identified in the human NOX4 gene [11] We observed, by reporter assays, that GC-box3, the GC-rich region adjacent to ATG luc pGL3-basic +1 GC box hNOX4 5'-UTR Relative luciferase activity 010203040 * A GC box1 5'-UTR Relative luciferase activity 0 5 10 15 20 hNOX4+76/ATG hNOX4+64/ATG hNOX4+1/ATG ATG luc pGL3-basic +1 GC box hNOX4 * B * GC box2 C GC box hNOX4 Relative luciferase activity 0102030 ATG luc pGL3-basic +1 * GC box3 5'-UTR // // // // // Fig. 3. Three GC-boxes are involved in the promoter activity of the NOX4 gene in SH-SY5Y cells. (A) The promoter activity of human (h)NOX4–243 ⁄ ATG (containing GC-box1) was significantly higher than that of hNOX4–226 ⁄ ATG (lacking GC-box1). (B) The promoter activity of hNOX4+64 ⁄ ATG (containing GC-box2) was significantly higher than that of hNOX4+76 ⁄ ATG (lacking GC-box2). (C) The promoter activity of hNOX4–1k ⁄ ATG (containing GC-box3) was significantly higher than that of hNOX4–1k ⁄ +212 (lacking GC-box3). Bars represent means ± standard error of three determinations. The experiment was repeated three times and a representative result is shown. *P < 0.05. M. Katsuyama et al. Sp3 regulates transcription of NOX4 ⁄ NADPH oxidase FEBS Journal 278 (2011) 964–972 ª 2011 The Authors Journal compilation ª 2011 FEBS 967 the start codon, was essential for NOX4 expression. EMSAs, however, showed that the extent of binding of nuclear proteins to this region was much lower than that for other GC-boxes. Thus, GC-box3 might act as a sequence essential for stabilization of the NOX4 mRNA rather than as a cis-acting element of tran- scription. The pathophysiological roles of NOX4 seem to be dependent on cell types or tissues. In some cell types, NOX4 acts as a regulator of proliferation, hypertrophy or cell survival [29–34]. In other cell types, NOX4 acts as a regulator of apoptosis or differentiation [35–39]. Such discrepancies were also reported in NOX4-trans- genic or knockout mice. Up-regulation of NOX4 by hypertrophic stimuli or pressure overload has been shown to exacerbate cardiac dysfunction by causing mitochondrial dysfunction and apoptosis in cardiac myocytes [40,41]. On the other hand, NOX4 has been found to protect against chronic load-induced stress in mouse hearts by enhancing angiogenesis [42]. The rea- son for these discrepancies remains to be elucidated. AT G +1 GC box hNOX4 hNOX4 + 76/ATG hNOX4 + 64/ATG luc pGL3-basic // // 0 5 10 15 20 Relative luciferase activity GC box1 GC box2 GC box3 5'-UTR Fig. 4. Three GC-boxes are involved in the promoter activity of the NOX4 gene in HEK293 cells. Promoter activities were compared between human (h)NOX4 – 243 ⁄ ATG and hNOX4 – 226 ⁄ ATG, hNOX4+64 ⁄ ATG and hNOX4+76 ⁄ ATG, and hNOX4 – 1k ⁄ ATG and hNOX4 – 1k ⁄ +212 for the involvement of GC-box1, GC-box2 and GC-box3, respectively. A Nuclear ext. Sp Origin Free probe GC-box1 GC-box2 GC-box3 Antibody x100 cold Sp1 Sp3 Sp3 supershift Origin SH-SY5Y HEK293 B Origin GC-box1 GC-box2 Sp3 supershift Sp1 Sp3 Fig. 5. Sp1 and Sp3 transcription factors bind to GC-boxes 1 and 2 of the NOX4 pro- moter in vitro. (A) Nuclear proteins that bind strongly to GC-boxes 1 and 2, but not to GC-box3, were detected by EMSA. (B) Effects of anti-Sp1 and anti-Sp3 IgGs. The upper band disappeared in the presence of an anti-Sp1 IgG, while the supershift of the lower band in the presence of an anti-Sp3 IgG was depicted. The binding specificity was evaluated with a 100-fold excess of unlabeled oligonucleotide. Nuclear extracts were pre-incubated in the presence or absence of an anti-Sp1, anti-Sp3 or anti-Sp4 IgG (0.5 lg). The experiment was repeated three times and a representative result is shown. Nuclear ext., nuclear extact; wt, wild type; mut, mutant. Sp3 regulates transcription of NOX4 ⁄ NADPH oxidase M. Katsuyama et al. 968 FEBS Journal 278 (2011) 964–972 ª 2011 The Authors Journal compilation ª 2011 FEBS Yet, the ubiquitous expression of NOX4 and its tran- scriptional regulation by Sp3, which is involved in the expression of various genes, raise the importance of NOX4 in cellular functions. In addition, the presence of species specificity in the regulation of NOX4 gene expression raises the possibility that the results obtained from animal studies cannot be applied to humans. Thus, our findings may provide useful infor- mation for the development and clinical application of NOX4-selective inhibitors expected for the treatment of fibrosis, cancers, and cardiovascular and metabolic diseases. Materials and methods Materials An antibody against Sp1 was purchased from Active Motif (Carlsbad, CA, USA). Antibodies against Sp3 and Sp4 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). [ 32 P]ATP[cP] was purchased from MP Biomedi- cals (Solon, OH, USA). Cell Culture SH-SY5Y cells, purchased from the European Collection of Cell Cultures, were cultured in Ham’s F12 ⁄ Eagle’s medium with Earle’s salts (1 : 1, v ⁄ v) supplemented with nonessential amino acids and 15% fetal bovine serum. HEK293 cells were SH-SY5Y HEK293 NOX4 GC-box1 NOX4 GC-box2 GAPDH Fig. 6. Sp1 and Sp3 transcription factors bind to GC-boxes 1 and 2 of the NOX4 promoter in vivo. Formalin cross-linked chromatin pre- pared from SH-SY5Y or HEK293 cells was incubated with anti-Sp1 IgG, anti-Sp3 IgG, anti-RNA polymerase II IgG (positive control) or IgG (negative control). Aliquots of chromatin before immunoprecipi- tation (Input DNA) or sterile H 2 O were used as positive or negative controls for PCR, respectively. Amplified DNA bands for GC-boxes 1 (138 bp) and 2 (129 bp) of the NOX4 promoter and the GAPDH promoter (166 bp) are demonstrated. The experiment was repeated three times and a representative result is shown. 0 0.2 0.4 0.6 0.8 1 1.2 SH-SY5Y HEK293 Relative luciferase activity (fold of Scr-shRNA) Scr-shRNA Sp1-shRNA Sp3-shRNA 100 kDa 75 kDa Sp1 A B 150 kDa 100 kDa 75 kDa Sp3 Sp3 Sp1 * * Fig. 7. Gene silencing of Sp3 attenuated the transcriptional activity of the NOX4 gene. (A) Expression of Sp1 and Sp3 in SH-SY5Y cells infected with the anti-Sp1 or anti-Sp3 shRNA-expressing lentiviral vector. Whole-cell lysate (10 lg) was subjected to western blot analyses. The membranes were stripped and rehybridized with another antibody. (B) Cotransfection with the anti- Sp3 shRNA-expression plasmid suppressed the promoter activity of the NOX4 gene. The luciferase activity of human (h)NOX4– 243 ⁄ ATG, relative to pGL3-basic, when cotransfected with scramble shRNA (Scr-shRNA), was 44.5 ± 5.2 and 10.1 ± 0.2 for SH-SY5Y and HEK293 cells, respectively. The activities are expressed as ‘fold of Scr-shRNA’. The bars represent the means ± standard error of four to five deter- minations. *P < 0.05 versus Scr-shRNA. M. Katsuyama et al. Sp3 regulates transcription of NOX4 ⁄ NADPH oxidase FEBS Journal 278 (2011) 964–972 ª 2011 The Authors Journal compilation ª 2011 FEBS 969 cultured in Eagle’s medium with Earle’s salts supplemented with nonessential amino acids and 10% fetal bovine serum. Reporter constructs and luciferase assay Human genomic DNA was isolated from SH-SY5Y cells using a PUREGENE DNA Isolation Kit (Gentra Systems, Minneapolis, MN, USA). The 5¢-flanking and non-coding regions of the human NOX4 gene were amplified by PCR and cloned into the vector pGL3-basic (Promega, Madison, WI, USA). The 2-kb 5¢-flanking and noncoding regions were cloned into the SmaI ⁄ NcoI site of pGL3-basic. A ser- ies of 5¢-deletion constructs was made by cleavage with restriction enzymes or amplification by PCR. All constructs were subjected to sequencing analyses to verify the orienta- tion and fidelity of the insert. Luciferase plasmids (0.75 lg per well) and a pSV-b-galactosidase control vector (0.25 lg per well; Promega) were cotransfected into SH-SY5Y cells or HEK293 cells using TransIT-LT1 Reagent (Mirus, Madison, WI, USA). The cells were cultured for 48 h, after which the luciferase activity in the cell lysates was deter- mined and normalized relative to the b-galactosidase activ- ity, as described previously [43]. EMSAs EMSAs were performed essentially as described previously [44]. A double-stranded probe containing a GC-box was prepared by annealing complementary synthetic oligonucle- otides. The sense sequences were 5¢-TGTACAAGGGGGC GGCGAGGGTCCC-3¢ (GC-box1), 5¢-GTAGCAGACCCC GCCCGGGCTGGCT-3¢ (GC-box2) and 5¢-AGCGCAGC GCGGCGGGGCCGGCGGC-3¢ (GC-box3), and the seq- uences of the mutated probes were 5¢-TGTACAAGGGttCt GCGAGGGTCCC-3¢ (GC-box1), 5¢-GTAGCAGACCCaG aaCGGGCTGGCT-3¢ (GC-box2) and 5¢-AGCGCAGCGC ttCttGGCCGGCGGC-3¢ (GC-box3). The probes were labeled at the 5¢-end with [ 32 P]ATP[cP] and T4 polynucleo- tide kinase. Nuclear extracts of SH-SY5Y cells or HEK293 cells were prepared as described previously [45]. The nuclear extracts and the labeled probe were incubated at 25°C for 30 min, resolved in a 4% polyacrylamide gel and analyzed using a Fujix BAS 5000 Bio-imaging Analyzer (Fuji Film, Tokyo, Japan). ChIP assay ChIP assays were performed using the ChIP-IP Express kit (Active Motif), essentially according to the manufacturer’s instructions. Immunoprecipitations were carried out using an anti-Sp1 IgG, an anti-Sp3 IgG, an anti-RNA polymer- ase II IgG (positive control), or negative control IgG. Prim- ers used for amplification of the NOX4 promoter were as follows: 5¢-AACAATCAGTCTAAAAGAGCTGTGTCTT CT-3¢ (forward for GC-box1), 5¢-CTCCAAAATACTGG CAAACATGTGAACAAT-3¢ (reverse for GC-box1), 5¢- TGAGTGGGCAGAGCTGACCCGGTGCGGGT-3¢ (for- ward for GC-box2) and 5¢-CGAGGGTCAAAGACTGAG TGGAAGCCCGAA-3¢ (reverse for GC-box2). Primers used for amplification of the GAPDH promoter were provided in the ChIP-IT Control Kit-Human. Aliquots of chromatin before immunoprecipitation were used as input controls. Gene silencing of Sp1 or Sp3 The anti-Sp1 or anti-Sp3 shRNA was designed against nu- cleotides 883-907 of the human Sp1 mRNA (GenBank BC062539) or against nucleotides 1244-1268 of the Sp3 mRNA (GenBank NM_003111). The scramble shRNA sequence was 5¢-TTGGGAATTAATATGCACAGGC CAA-3¢. Sense and antisense oligonucleotides containing the hairpin sequence, the terminator sequence and over- hanging sequences were synthesized. By annealing over- hanging sequences of the synthetic oligonucleotides, PCR was performed to amplify the sequence encoding the shRNA, which was inserted into the pGreenPuro lentiviral vector (System Biosciences, Mountain View, CA, USA). An anti-luciferase control shRNA insert, supplied by System Biosciences, was also inserted. Efficiency of the shRNA- expression plasmids was verified by infection of SH-SY5Y cells and subsequent western blot analyses. shRNA-expres- sion plasmids (0.5 lg per well), luciferase plasmids (0.1 lg per well) and a Renilla luciferase vector (pRL-null; 0.01 lg per well) were cotransfected into SH-SY5Y cells or HEK293 cells. The cells were then cultured for 48 h, after which the luciferase activity in cell lysates was determined and normalized according to Renilla luciferase activity. Statistical analysis Values were expressed as the mean ± standard error. Sta- tistical analysis was performed using the Student’s t-test. Acknowledgements This work was supported in part by a Grant-in-Aid for Young Scientists (B) 21790525 from The Ministry of Education, Culture, Sports, Science and Technology of Japan (M. K.). We thank Dr Yoshihiro Sowa, Kyoto Prefectural University of Medicine, for useful discussion. 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Katsuyama et al. 972 FEBS Journal 278 (2011) 964–972 ª 2011 The Authors Journal compilation ª 2011 FEBS . essential for transcriptional activation of the human NOX4 promoter Figure 1 shows the alignment of the 5¢-flanking and 5¢-noncoding sequences of the human, rat and mouse NOX4 genes. Compared with the. similarity between the rat and mouse sequences, the similarity of the human sequence was relatively low. Therefore, we isolated the promoter region of the human NOX4 gene and examined its transcriptional. ubiquitous expression of NOX4 in human tissues. We report here the predominant role of the transcription factor specificity protein (Sp) 3 in the expressional regulation of the human NOX4 gene. Results GC-boxes