Báo cáo khoa học: Sp3 transcription factor is crucial for transcriptional activation of the human NOX4 gene doc

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

activation of the human NOX4 gene

Masato Katsuyama1,2, Hideyo Hirai3, Kazumi Iwata2, Masakazu Ibi2, Kuniharu Matsuno2,

Misaki Matsumoto2and Chihiro Yabe-Nishimura2

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 (O2)

and hydrogen peroxide, have been recognized as

intrin-sic signaling molecules that modulate multiple cellular

responses NADPH oxidases are the major source of

O2 in various tissues [1–3] They catalyze the reduction

of molecular oxygen to O2 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 NOX4gene 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.

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, additranscrip-tional 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.

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 plasplas-mids 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 NOX4in 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.

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

0 10 20 30 40

*

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

Relative luciferase activity

0 10 20 30

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.

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

ATG +1

GC box hNOX4

hNOX4 + 76/ATG

hNOX4 + 64/ATG

luc

pGL3-basic

//

//

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.

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,

Biomedi-cals (Solon, OH, USA)

Cell Culture SH-SY5Y cells, purchased from the European Collection of

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 H2O 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

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.

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

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

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

25C 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

CAA-3¢ Sense and antisense oligonucleotides containing the hairpin sequence, the terminator sequence and 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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