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1 Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
2 Department of Pathology, and
3 Department of Developmental Molecular Anatomy, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
4 CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
5 Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
6 Department of Biomedical Chemistry, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734-8551, Japan
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Abstract |
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Introduction |
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The founder member of the Nox family is gp91phox (also termed Nox2), which is predominantly expressed in professional phagocytes such as neutrophils and also in B lymphocytes, but to a much lesser extent (Nauseef 2004). The phagocyte oxidase is dormant in resting cells, and becomes activated during phagocytosis of invading microbes to produce superoxide, which is converted to microbicidal ROS. The importance of the phagocyte oxidase in host defense is evident from the fact that recurrent and life-threatening infections occur in patients with chronic granulomatous disease (CGD), whose phagocytes lack the superoxide-producing activity (Nauseef 2004). gp91phox/Nox2 is complexed with p22phox, another membrane-integrated protein, to form flavocytochrome b558; via the complex formation, gp91phox/Nox2 stabilizes p22phox, and vice versa. Successful electron transfer in gp91phox/Nox2 for superoxide production requires its association with the three cytoplasmic proteins p47phox, p67phox, and the small GTPase Rac, which translocate to the membrane during phagocytosis or in response to cell stimulants such as phorbol 12-myristate 13-acetate (PMA). Although p22phox does not seem to be directly involved in the electron transfer from NADPH to O2, it plays a crucial role in oxidase regulation by providing an anchorage site for p47phox (Nauseef 2004; Sumimoto et al. 2005).
Nox1, the first identified mammalian homolog of gp91phox, is expressed in colon epithelial cells and at lower levels in various types of cells including vascular smooth muscle cells (Suh et al. 1999; Bánfi et al. 2000). Nox1 seems to function by forming a heterodimer with p22phox (Takeya et al. 2003; Ambasta et al. 2004; Hanna et al. 2004), and is considered to be involved in host defense at the colon and signal transduction leading to hypertrophy and angiogenesis (Lambeth 2004). Nox3, expressed in the inner ear as well as the fetal kidney (Paffenholz et al. 2004), also forms a functional complex with p22phox (Ueno et al. 2005). When these Nox oxidases are ectopically expressed in cells, they release large amounts of superoxide under appropriate conditions (Bánfi et al. 2003; Geiszt et al. 2003; Takeya et al. 2003; Cheng et al. 2004): Nox3- or Nox1-dependent superoxide production does not require cell stimulants, but can be increased by cell treatment with PMA (Takeya et al. 2003; Ueno et al. 2005). In contrast, Nox4 excretes only a trace amount of superoxide outside the cells transfected with its cDNA. Nox4 was initially identified as a kidney NAD(P)H oxidase that might be involved in oxygen sensing and cellular senescence (Geiszt et al. 2000; Shiose et al. 2001). Recent studies have shown that Nox4 is also abundant in vascular cells, especially endothelial cells (Ago et al. 2004; Griendling 2004), and implicated in vascular pathologies (Szöcs et al. 2002; Etoh et al. 2003; Gorin et al. 2003; Lassègue & Clempus 2003). However, little is known how Nox4 functions in cells.
Here we show that a major part of Nox4 localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), where the oxidase likely forms a functional complex with p22phox. The nuclear fraction prepared from HUVECs exhibits an NAD(P)H-dependent superoxide-producing activity, which is up-regulated upon cell stimulation with PMA but impaired by knockdown of Nox4 by RNA interference (RNAi). In addition, Nox4 seems to regulate gene expression in a manner dependent on the regulatory DNA sequence Maf-recognition element (MARE), constituting a part of oxidative stress response (Igarashi et al. 1994; Itoh et al. 1999; Hoshino et al. 2000). The transcriptional activity is increased by PMA, and the increased activity as well as the basal one is prevented by Nox4 silencing.
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Results |
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Previous studies using reverse transcription (RT)-PCR have indicated that Nox4 is the predominant member among the Nox family oxidases in vascular endothelial cells (Ago et al. 2004; Griendling 2004). To confirm this, we performed Northern blot analysis using a variety of human vascular endothelial cells such as HUVECs, aortic endothelial cells, dermal microvascular endothelial cells, and coronary artery endothelial cells. As shown in Fig. 1, Nox4 was highly expressed in these endothelial cells: the major form of the Nox4 mRNA was approximately 2.4 kb in length with a minor form of about 4.4 kb (Fig. 1), as observed in the human kidney (Shiose et al. 2001). On the other hand, the mRNA for gp91phox/Nox2 was not detected in any of these vascular endothelial cells under the conditions where a high amount of the message was observed in human neutrophils and a much smaller but significant amount of the mRNA was detectable in B lymphocytes (Fig. 1). The conclusion that Nox4 is the predominant oxidase is also supported by the present RT-PCR analysis: transcripts for the Nox exzymes other than Nox4 were barely detected in these kinds of endothelial cells, whereas p22phox as well as Nox4 was abundantly expressed (data not shown).
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To clarify the subcellular localization of endogenous Nox4 in vascular endothelial cells, we prepared three distinct antisera raised against different C-terminal regions of Nox4 and purified them by affinity chromatography using their corresponding antigens as follows: the extreme C-terminal peptide of amino acids 559-578 for the anti-Nox4-C antibody; the peptide of a putative loop region (amino acids 499-511) for the anti-Nox4-loop antibody; and the C-terminal domain (amino acids 406-578) for the anti-Nox4-CTD (for detail, see Experimental procedures). These antibodies all interacted with the C-terminal cytoplasmic domain of Nox4 expressed as a glutathione S-transferase (GST)-fusion protein but not with those of the other Nox family proteins (Fig. 2A), indicative of their specificity to Nox4. Using these antibodies, we performed immunochemical analysis of cultured HUVECs. As shown in Fig. 2B, all the anti-Nox4 antibodies gave strong signals at the nucleus of these cells. However, these signals were not obtained when pre-immune sera or the antibody pre-absorbed with the corresponding antigens were used (Fig. 2B). Although the cytoplasm was also stained in a vesicular pattern, the cytoplasmic signals were weaker than those of the nucleus. These findings suggest that Nox4 localizes preferentially to the nucleus in HUVECs. As indicated on confocal microscopy, Nox4 appears to exist inside the nucleus of HUVECs (Fig. 2C). This idea is supported by immunoelectron microscopic analysis: immunogold labeling with the anti-Nox4-C antibody was localized inside the nucleus of HUVECs (Fig. 2D). The labeling with the anti-Nox4-loop antibody provided an almost identical result (data not shown).
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p22phox has been initially identified as a protein forming a complex with gp91phox/Nox2 in phagocytes, known as flavocytochrome b558 (Nauseef 2004). By the heterodimer formation, p22phox and gp91phox/Nox2 stabilize each other at the protein level: the gp91phox/Nox2 protein is absent in phagocytes lacking p22phox, while the p22phox protein is not detected in the absence of gp91phox/Nox2 (Nauseef 2004). A recent study has shown that ectopically expressed Nox4 is capable of directly interacting with p22phox and stabilizing this protein (Ambasta et al. 2004).
It therefore seemed possible that p22phox forms a complex with Nox4 in the nucleus of HUVECs. To test this possibility, we performed immunocytochemical analysis using an anti-p22phox antibody (Imajoh-Ohmi et al. 1992). As shown in Fig. 2B, p22phox localized in the nucleus of HUVECs as Nox4 did. Moreover, we knocked down Nox4 by RNAi and estimated its effect on p22phox. The RNAi-mediated silencing of Nox4 resulted in a significant decrease in the protein level of p22phox as determined by Western blot (Fig. 4B) and immunocytochemical analysis (Fig. 4C), although this treatment did not alter the amount of the p22phox mRNA (Fig. 4A). These observations suggest that Nox4 forms a heterodimer with p22phox in the nucleus of HUVECs, which formation stabilizes p22phox at the protein level.
Superoxide production by the nuclear fraction of HUVECs
To investigate the superoxide-producing activity of Nox4 present in the nucleus of HUVECs, we prepared the nuclear fraction from these cells. To verify the authenticity of this fraction, we performed Western blot analyses using antibodies to nucleoporin p62 (a marker of the nucleus), iron sulfur protein of mitochondrial respiratory complex II (a marker of the mitochondrion), and VE-cadherin (a marker of the plasma membrane). As expected, nucleoporin p62 was found exclusively in the nuclear fraction but not in the postnuclear supernatant (PNS) fraction, while solely the PNS fraction contained the mitochondrial and plasma membrane markers (Fig. 5A).
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To clarify the role of Nox4 in superoxide production by the nuclear fraction of HUVECs, we tested the effect of inhibitors for several potential sources of superoxide. The nuclear superoxide-producing activity was drastically blocked by diphenylene iodonium (DPI), an inhibitor of Nox enzymes (Lambeth 2004) (Fig. 5E). It is formally possible that the blockade is not due to the inhibition of Nox oxidases, since DPI is known also to prevent other superoxide-producing flavoproteins such as nitric oxide synthases, xanthine oxidase and mitochondrial respiratory chain complex I NADH reductase (Lassègue & Clempus 2003). However, as shown in Fig. 5E, the nuclear superoxide-producing activity was not inhibited by rotenone (a blocking agent of mitochondrial respiratory chain complex I) (Degli Esposti 1998), L-NAME (an inhibitor of nitric oxide synthases) (Griffith & Kilbourn 1996), or oxypurinol (an inhibitor of xanthine oxidase) (Berry & Hare 2004), indicating that these flavoproteins are not involved in the superoxide production. These findings suggest that Nox enzymes are the major source of the superoxide production by the nuclear fraction of HUVECs. Since Nox4 is the predominant member among the Nox family oxidases in HUVECs, Nox4 is a probable candidate as the enzyme responsible for the nuclear superoxide production.
This proposal is strongly supported by the following finding: the nuclear fraction prepared from HUVECs transfected with siRNA targeting Nox4 exhibited about a several-fold smaller superoxide-producing activity than that of the cells transfected with control RNA (Fig. 6A,B). On the other hand, transfection with siRNAs against gp91phox/Nox2 did not affect the superoxide production (data not shown). Thus Nox4 appears to be involved in the superoxide production in the nucleus of HUVECs.
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The protein kinase C activator PMA is known to be capable of eliciting activation of endogenous gp91phox/Nox2 in phagocytes and ectopically expressed gp91phox/Nox2 in non-phagocytic cells (Takeya et al. 2003; Ueno et al. 2005). Although cell stimulants are dispensable to the superoxide-producing activity of Nox1 and Nox3, the addition of PMA to cells leads to enhancement of both Nox1- and Nox3-dependent superoxide production (Takeya et al. 2003; Ueno et al. 2005). To test the effect of PMA treatment on the Nox4 activity, we prepared the nuclear fraction from HUVECs treated with or without PMA, and compared their superoxide-producing activities. As shown in Fig. 7, the treatment with PMA resulted in an increased superoxide production by the nuclear fraction, suggesting that the Nox4 activity can be enhanced by PMA.
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We finally investigated the role of the superoxide-producing activity of Nox4 in HUVECs. Superoxide produced likely causes oxidative stress in cells by itself and/or by its derivatives, namely other ROS. Oxidative stress is known to elicit various responses, a part of which is executed by gene expression via the regulatory DNA sequence MARE (Igarashi et al. 1994; Itoh et al. 1999; Hoshino et al. 2000). To test the effect of the ROS-producing enzyme Nox4 on the MARE-dependent transcription, we performed a transfection reporter assay using the plasmid pRBGP2, carrying a TATA box and three copies of MARE (Igarashi et al. 1994; Itoh et al. 1999; Hoshino et al. 2000). As shown in Fig. 7C, the reporter luciferase activity was blocked by treatment of HUVECs with siRNA for Nox4, suggesting that Nox4 participates in the gene expression in HUVECs. However, the Nox4-dependent transcription was not observed, when the reporter pRBGP4, containing mutations within the MAREs (Igarashi et al. 1994; Itoh et al. 1999; Hoshino et al. 2000), was used instead of pRBGP2 (data not shown); thus Nox4 likely acts via MARE. Furthermore, PMA enhanced the gene expression via MARE by about twofold (Fig. 7C), which extent is similar to that of the PMA-induced enhancement of superoxide production by Nox4 (Fig. 7B). Again, the siRNA-mediated silencing of Nox4 blocked the MARE-dependent transcription in the presence of PMA (Fig. 7C). These findings suggest that Nox4 promotes the MARE-dependent gene expression by producing superoxide, which event can be regulated by cell stimulation.
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Discussion |
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The idea that Nox4 localizes to the nucleus of vascular endothelial cells is based on the following findings. First, nuclei of cultured HUVECs give strong signals with three distinct affinity-purified anti-Nox4 antibodies in immunocytochemical analyses, although the cytoplasm is also stained but to a weaker extent (Fig. 2); the signals are attenuated by knockdown of Nox4 by transfection of the cells with siRNA (Fig. 4). The nuclear localization of Nox4 is supported by immunoelecrton microscopy (Fig. 2D). In addition to cultured HUVECs, the nuclear staining was also observed in endothelial cells in situ (Fig. 3). Second, the present biochemical analysis shows that siRNA-mediated silencing of Nox4 results in a significant decrease in the NAD(P)H-dependent superoxide-producing activity of the nuclear fraction of HUVECs (Figs 5 and 6). The nuclear localization of Nox family oxidases has been also suggested in several types of cells. Immunofluorescence staining with an anti-Nox1 antibody has recently shown that Nox1 is preferentially distributed in the nuclei of transformed human keratinocytes but not in those of more differentiated cells (Chamulitrat et al. 2003). Hilenski et al. (2004) have recently demonstrated that, in rat vascular smooth muscle cells, Nox4 localizes to the nuclei as well as focal adhesions of rat vascular smooth muscle cells. In addition, it has been also reported that majorities of gp91phox/Nox2 and p22phox are recovered in the nucleus-rich fraction prepared from vascular endothelial cells (Li & Shah 2002), whereas, in phagocytes, they are present solely in the plasma and granule membranes (Nauseef 2004).
Since Nox4 contains multiple transmembrane segments, this protein is expected to be integrated into membranous structures such as the nuclear envelope. The present findings, however, suggest that Nox4 is located in the nucleoplasm but not on the nuclear envelope (Fig. 2C,D). Although the nucleoplasm is considered to be almost membrane-free, it is known that there exist intranuclear membranous structures (Isaac et al. 2001). Nox4 may be incorporated into such structures. Given the membrane topology of Nox4 (Bokoch & Knaus 2003; Geiszt & Leto 2004; Lambeth 2004; Sumimoto et al. 2005), superoxide should be released to the lumen side of intranuclear membranes, and subsequently dismuted into hydrogen peroxide. Because hydrogen peroxide can freely move across the membrane, it reaches the nucleoplasm, probably serving as a messenger for signal transduction (Rhee et al. 2000).
In HUVECs, Nox4 is likely complexed with p22phox in the nucleus, since knockdown of Nox4 by RNAi resulted in a loss of the nuclear p22phox (Fig. 4) as well as a decreased superoxide production (Fig. 6). Such Nox4-mediated stabilization of p22phox has been recently shown by Ambasta et al. using co-transfected cells (Ambasta et al. 2004). They have also shown that Nox4 was co-immunoprecipitated with p22phox, and that Nox4-dependent ROS production is facilitated a few folds by ectopic expression of p22phox. This is consistent with a finding that ROS production of rat aortic endothelial cells is decreased by treatment with a p22phox anti-sense oligonucleotide as well as with a Nox4 anti-sense oligonucleotide (Ago et al. 2004). Cooperation between Nox4 and p22phox is also suggested by a finding that Nox4 and p22phox are concomitantly increased at the protein level in the kidney of diabetic rats (Etoh et al. 2003). Thus it is probable that Nox4 forms a functional complex with p22phox to produce superoxide in the nucleus of vascular endothelial cells as well.
As shown in the present study, the Nox4-dependent superoxide production in the nucleus is increased by treatment of HUVECs with PMA, an agent capable of up-regulating other Nox oxidases such as gp91phox/Nox2, Nox1, and Nox3 (Takeya et al. 2003; Ueno et al. 2005). It is known that PMA induces conversion of Rac into the active form (Akasaki et al. 1999) and conformational change of the oxidase regulator p47phox (Nauseef 2004; Sumimoto et al. 2005), both events of which serve as a switch for activation of gp91phox/Nox2. Although the mechanism for Nox4 activation by PMA is presently unknown, Rac may mediate the PMA effect; the involvement of Rac in Nox4-mediated superoxide generation has been suggested in renal mesangial cells stimulated with angiotensin II (Gorin et al. 2003). On the other hand, p47phox does not seem to be involved in the PMA-mediated Nox4 activation at least in HUVECs, because these cells scarcely express p47phox (J. Kuroda & H. Sumimoto, unpublished observation).
The present study also shows that Nox4-mediated ROS production in the nucleus probably plays a role in gene expression. Oxidative stress is known to elicit various responses, a part of which is executed by gene expression via MARE (Igarashi et al. 1994; Itoh et al. 1999; Hoshino et al. 2000). As shown in the present study, Nox4 likely participates in transcriptional regulation via MARE in HUVECs, since the transcription is impaired by knockdown of Nox4 (Fig. 7C). This proposal is supported by the following findings: PMA enhanced the gene expression via MARE by about twofold (Fig. 7C), which is similar to that of the PMA-induced enhancement of superoxide production by Nox4 (Fig. 7B); and the siRNA-mediated silencing of Nox4 blocked the MARE-dependent transcription in the presence of PMA (Fig. 7C). Thus Nox4 appears to promote the MARE-dependent gene expression by producing superoxide. In addition, it is also possible that ROS produced in the nucleus by Nox4 causes modification of DNA or chromosomes, such as telomere shortening, which is causal for cellular senescence induced by oxidative stress (Dröge 2002). Indeed it has been shown that over-expression of Nox4 in cultured cells develops signs of cellular senescence (Geiszt et al. 2000). Zhang et al. (2002) have proposed that changes in nuclear redox could be manifested primarily through NADH. Since Nox4 utilizes NADH as an electron donor for superoxide production (Fig. 5), it is tempting to postulate that Nox4 might act as a sensor for nuclear redox.
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Experimental procedures |
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Human vascular endothelial cells including HUVECs, human aortic endothelial cells, human dermal microvascular endothelial cells, and human coronary artery endothelial cells were purchased from Kurabo (Osaka). The cells were cultured in HuMedia EG2 (Kurabo) containing human EGF (10 ng/mL), hydrocortisone (1 µg/mL), gentamycin (50 µg/mL), amphotericin B (50 ng/mL), human FGF-B (5 ng/mL), heparin (10 µg/mL), and 2% FBS.
Northern blot analysis
Total RNAs of indicated cells were prepared with TRIZOL reagent (Life Technologies, Inc.). The total RNAs (10 µg) were separated on 1% agarose gel electrophoresis with 0.66 M formaldehyde, and transferred by diffusion blotting onto a nylon transfer membrane (Hybond N+ Amersham Pharmacia Biotech). The blotted membrane was hybridized with a 32P-labeled cDNA fragment encoding Nox4 or gp91phox/Nox2 under high stringency conditions using ExpressHybTM (Clontech). The cDNA probes for Nox4 and gp91phox/Nox2 encode the regions that correspond to amino acids 314-578 and 301-570, respectively.
RT-PCR
Total RNAs were subjected to RT-PCR using a GeneAmp kit (PerkinElmer Life Sciences) with a pair of specific primers: 5'-GCAAGATCTGTTGTTATGCACCCATCCAA-3' (forward primer) and 5'-GCTGGTACCTCAAAAATTTTCTTTGTTGAAGT-3' (reverse primer) for Nox1; 5'-GGAGGATCCGTGGTCACTCACCCTTTCAA-3' (forward primer) and 5'-CCACTCGAGCTCATGGAAGAGACAAGTTAG-3' (reverse primer) for Nox2; 5'-GCAGGATCCGTGGTAAGCCACCCCTCTG-3' (forward primer) and 5'-GCTGAATTCAGAAGCTCTCCTTGTTGTAAT-3' (reverse primer) for Nox3; 5'-GCAGGATCCGTCATAAGTCATCCCTCAGA-3' (forward primer) and 5'-GCTGTTAACGTCGACTCAGCTGAAAGACTCTTTAT-3' (reverse primer) for Nox4; 5'-GCAGGATCCACTATCTGGCTGCACATTCG-3' (forward primer) and 5'-GCTGAATTCCTAGAAATTCTCTTGGAAAAATC-3' (reverse primer) for Nox5; 5'-GCAGGATCCATGGGGCAGATCGAGTGGGC-3' (forward primer) and 5'-GCTGAATTCACACGACCTCGTCGGTCACC-3' (reverse primer) for p22phox.
Affinity purification of three distinct anti-Nox4 antibodies
For expression as GST-fusion proteins, the cDNA fragments encoding the C-terminal regions of the following Nox proteins were ligated to pGEX-2T or pGEX-4T-1: Nox1 (amino acids 378-564) (Suh et al. 1999), gp91phox/Nox2 (384-570) (Shiose et al. 2001), Nox3 (382-568) (Cheng et al. 2001), Nox4 (406-578) (Shiose et al. 2001), and Nox5 (536-737) (B·nfi et al. 2001). GST-fusion proteins were expressed in Escherichia coli strain BL21 and purified by glutathione-Sepharose-4B, as previously described (Koga et al. 1999).
Anti-Nox4 rabbit polyclonal antisera, namely, the anti-Nox4-C and the anti-Nox4-loop antisera, were raised against peptides corresponding to amino acids 559-578 and 499-511, respectively, and each anti-Nox4-antibody was purified from a crude anti-Nox4 serum using a column (HiTrap NHS-activated HP; Amersham Biosciences) coupled with the respective peptides. The anti-Nox4-CTD antiserum was raised against the C-terminal domain (amino acids 406-578) fused to GST, and the antibody was purified using a column coupled with a protein of the same region fused to the maltose-binding protein MBP.
To verify that these purified antibodies are specific for Nox4, we performed Western blot analysis. Briefly, the C-terminal regions of Nox proteins expressed as GST-fusions were subjected to SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane, and probed with the purified antibodies. The blots were developed using ECL-plus (Amersham Biosciences) for visualization of the antibodies.
Immunocytochemical analysis
Cultured HUVECs were harvested and washed three times with PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4). The cells were fixed for 20 min in Mildform 10 N (Wako Pure Chemical Industries, Osaka), and were washed four times with PBS containing 0.1% Triton X-100. In experiments using the anti-Nox4-C or anti-Nox4-loop antibody as a primary antibody, the cells were boiled for 10 min with a pressure-cooker to allow the antibodies to interact better with the cells. Then the cells were blocked with PBS containing 1.5% skimmed milk for 60 min. The samples were incubated with antibodies as indicated. An anti-p22phox rabbit polyclonal antibody was prepared as previously described (Imajoh-Ohmi et al. 1992). FITC-conjugated anti-rabbit IgG was used as a secondary antibody. Images were visualized with a Nikon Eclipse TE300 and captured on an ORCA digital camera (Hamamatsu Photonics), or with a LSM5 PASCAL confocal microscope (Zeiss).
Immunoelectron microscopy
Cultured HUVECs were harvested and fixed with 1% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer, as previously described (Rogers et al. 1992; Précigout et al. 1993). Specimens were dehydrated through a series of ethanol and embedded at –20 °C in LR Gold resin (Energy Beam Sciences). Thin sections were mounted on nickel grids, blocked for 30 min with PBS containing 5% dry milk and 0.1% Tween 20, and incubated overnight at 4 °C with the anti-Nox4-C antibody diluted 1 : 100. Sections were subsequently incubated for 1 h with anti-rabbit IgG conjugated with 15-nm gold particles (Nanogold®; Nanoprobes). After staining uranyl acetate and lead citrate, sections were observed in a JEOL 2000 EX electron microscope.
Immunohistochemical analysis
Immunohistochemical analysis was performed as previously described (Chen et al. 1999). Briefly, human umbilical specimens were fixed in Bouin's solution and embedded in paraffin. The procedures of the use of the samples in the present study were in accordance with the guidelines of local Human Subject Committee at Kyushu University. The sections were deparaffinized and incubated for 20 min with 10% normal rabbit serum for prevention of the primary antibody from binding nonspecifically. They were subsequently incubated overnight at 4 °C with the anti-Nox4-C or anti-Nox4-loop antibody in a moisture chamber, followed by 3 washes for 5 min with PBS. The sections were subsequently incubated with 0.3% (wt/vol) H2O2 in methanol, for inhibition of any endogenous peroxidase activity. After washing three times for 5 min with PBS, the sections were incubated for 30 min with Rabbit Envision (Dako), followed by counterstaining with hematoxylin.
The specificity of the antibodies was confirmed by preabsorption of the antibodies with the respective antigens. Preimmune rabbit sera were also used instead of the respective primary antibodies as other negative controls.
Preparation of the nuclear fraction of HUVECs
HUVECs were harvested and suspended in a lysis buffer (300 mM sucrose, 3 mM CaCl2, 2 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, 10 mM Tris-HCl, pH 8.0) containing 0.1% Triton X-100, followed by homogenization with a Dounce homogenizer by 20 strokes. The homogenate was centrifuged for 10 min at 1000 g. The resultant supernatant was used as the PNS fraction. On the other hand, the pellet was suspended in the lysis buffer containing 0.1% Triton X-100 with a Dounce homogenizer. The suspension was subsequently layered over a cushion of a solution composed of 700 mM sucrose, 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM EDTA, and 10 mM Tris-HCl (pH 8.0), followed by centrifugation for 5 min at 1000 g. The pellet was suspended by sonication, and the sonicates were used as the nuclear fraction.
The fractions were subjected to SDS-PAGE, and transferred to a PVDF membrane. The membrane was probed with an anti-nucleoporin p62 antibody (Transduction Laboratories), an anti-iron sulfur protein of mitochondrial respiratory complex II antibody (a generous gift from Dr Dongchon Kang, Kyushu University) (Alam et al. 2003), or an anti-VE-cadherin antibody (Santa Cruz) as markers of the nucleus, the mitochondrion, and the plasma membrane, respectively. The blots were developed using ECL-plus as mentioned above.
Assay for superoxide production
Superoxide production was determined as superoxide dismutase (SOD)-inhibitable chemiluminescence detected with an enhancer-containing luminol-based detection system (DIOGENES; National Diagnostics), as previously described (Koga et al. 1999; Kuribayashi et al. 2002). The nuclear fraction of HUVECs was suspended in 200 µL of an assay buffer composed of 100 mM potassium phosphate (pH 7.0), 10 µM FAD, 1 mM NaN3, and 1 mM EGTA. After pre-incubation with the enhanced luminol-based substrate (200 µL), NADH or NADPH was added at the final concentration of 12.5 µM. The chemiluminescence was continuously monitored using a luminometer (Auto Lumat LB953; EG & G Berthold). The reaction was terminated by the addition of SOD (100 µg/mL).
RNAi for knockdown of Nox4
RNAi was performed according to the method by Elbashir et al. (2001). The double-strand siRNAs targeting Nox4 and gp91phox/Nox2 were prepared as 3'-overhanged form using sense and anti-sense oligoribonucleotides. The sequences were as follows: 5'-GUUCUUAACCUCAAGUGCATT-3' (sense) and 5'-UGCAGUUGAGGUUUAAGAACTT-3' (anti-sense) for Nox4; 5'-UGCCUGAAUUUCAACUGCATT-3' (sense) and 5'-UGCAGUUGAAAUUCAGGCATT-3' (anti-sense) for gp91phox/Nox2. For transfection of HUVECs with siRNA, we used the Nucleofector technology (Amaxa Biosystems), an electroporation-based gene transfer technique, according to the manufacturer's protocol. Briefly, HUVECs were suspended in HUVEC Nucleofector Solution (Amaxa Biosystems) at a final concentration of 0.5–1 x 106 cells/100 µL, and 5 µg of siRNA duplex were added to the sample. The sample was transferred into a certified cuvette, and subjected to the Nucleofector, which was programmed for optimized transfection to HUVECs. After transfection, the cells were cultured for 48 h in the medium described above, and used for the experiments.
Reporter assays
The reporter plasmids used in the present study were pRBGP2, carrying TATA box and three copies of MARE (Igarashi et al. 1994; Itoh et al. 1999; Hoshino et al. 2000), and pRBGP4, containing mutations within the MAREs (Igarashi et al. 1994; Itoh et al. 1999; Hoshino et al. 2000). HUVECs were transfected with either reporter by the Nucleofector technology (Amaxa Biosystems), as described above. The transfected cells were cultured for 48 h and lyzed with the Luciferase Assay System (Promega) according to the manufacturer's protocol. Luciferase activities of the cell lysates were measured with a luminometer (Auto Lumat LB953; EG & G Berthold).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: hsumi{at}bioreg.kyushu-u.ac.jp
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References |
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Received: 30 August 2005
Accepted: 15 September 2005
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