Glycogen synthase kinase 3b and b-catenin pathway is involved in toll-like receptor 4-mediated NADPH oxidase 1 expression in macrophages Jin-Sik Kim 1 *, Seungeun Yeo 1 *, Dong-Gu Shin 2 , Yoe-Sik Bae 3 , Jae-Jin Lee 4 , Byung-Rho Chin 4 , Chu-Hee Lee 1 and Suk-Hwan Baek 1 1 Department of Biochemistry & Molecular Biology, Yeungnam University, Daegu, Korea 2 Department of Internal Medicine, Yeungnam University, Daegu, Korea 3 Department of Biochemistry, Dong-A University, Busan, Korea 4 Department of Dentistry, Yeungnam University, Daegu, Korea Introduction NADPH oxidase (Nox) is an essential enzyme in phagocytes and generates reactive oxygen species (ROS) for host defense [1]. Extensive evidence has now shown that Nox is also one of the major sources of ROS production in various nonphagocytic cells, including smooth muscle cells and hepatocytes [2,3]. Members of the Nox family are transmembrane pro- teins that catalyze the NADPH-dependent one-electron reduction of oxygen to form superoxide [4]. To date, seven members of this family have been described: Nox1–5 and dual oxidase (Duox)-1 and -2. Among these, the most studied is Nox2. Nox1, the first-recognized homologue of Nox2 [5], conserves the structural domain common to the cata- lytic core of Nox2. Nox1-derived ROS were initially reported to have tumorigenic and angiogenic functions [6,7]. However, further studies suggest that Nox1 regu- lates inflammation [8], atherosclerosis [9] and vascular Keywords glycogen synthase kinase 3b; NADPH oxidase 1; reactive oxygen species; toll-like receptor 4; b-catenin Correspondence Suk-Hwan Baek, Department of Biochemistry & Molecular Biology, College of Medicine, Yeungnam University, 317-1 Daemyung-5 Dong, Daegu 705-717, Korea Fax: +82 53 623 8032 Tel: +82 53 620 4523 E-mail: sbaek@med.yu.ac.kr *These authors contributed equally to this work (Received 25 February 2010, revised 23 April 2010, accepted 27 April 2010) doi:10.1111/j.1742-4658.2010.07700.x Macrophage activation contributes to the pathogenesis of atherosclerosis. In the vascular system, the major source of reactive oxygen species is the NADPH oxidase (Nox) family. Nox1 is induced by lipopolysaccharide (LPS) in macrophages, but the expression mechanism is not fully under- stood. We found that LPS causes b-catenin accumulation by glycogen syn- thase kinase 3b (GSK3b) inactivation, and that b-catenin accumulation increases Nox1 expression. LPS induced Nox1 mRNA expression and reac- tive oxygen species generation in Raw264.7 cells. Using bone marrow- derived macrophages from toll-like receptor 4 mutant mice, we also tested whether LPS-induced Nox1 expression is toll-like receptor 4 dependent. LPS caused GSK3b phosphorylation, induced b-catenin accumulation and increased nuclear translocation. The GSK3b inhibitor LiCl potentiated LPS-induced Nox1 expression in accordance with b-catenin accumulation and nuclear translocation. Conversely, ectopic expression of a constitutively active GSK3b mutant severely attenuated Nox1 expression. These findings identify a novel regulatory pathway controlling Nox1 expression by LPS- stimulated macrophages. Abbreviations BMDMs, bone marrow-derived monocytes; DAPI, 4¢,6-diamidino-2-phenylindole; GSK3b, glycogen synthase kinase 3b; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; Nox, NADPH oxidase; ROS, reactive oxygen species; TLR, toll-like receptor. 2830 FEBS Journal 277 (2010) 2830–2837 ª 2010 The Authors Journal compilation ª 2010 FEBS tone [10]. It has been shown to regulate smooth muscle cell growth, both hypertrophy and hyperplasia, and migration [11]. In addition, Nox1 may be important in regulating blood pressure [12]. Furthermore, Nox1 shows a relationship with toll-like receptor (TLR) in controlling innate immunity. Kawahara et al. [13] have shown that lipopolysaccharide (LPS) from pathogenic Helicobacter pylori strains may potently stimulate ROS production, mediated by Nox1, through a TLR4- dependent pathway. Lee et al. [14] also reported that the TLR9 agonist CpG ODN causes ROS production via Nox1 gene expression in macrophages and contrib- utes to foam cell formation. These studies suggest a crucial role for Nox1 in TLR-mediated signaling path- ways and its importance in innate immunity and inflammatory responses. The regulation of Nox1 seems to differ significantly from that of Nox2, and the Nox1 regulatory mecha- nism is still poorly defined. Full Nox1 activity is known to need regulatory cofactors, including NoxO1, NoxA1 and Rac1 [5]. The most well-studied activation of Nox1 is that of angiotensin II mediating phospholi- pase C and protein kinase C in vascular smooth mus- cle cell. However, regulation of Nox1 activity by mRNA induction is also important. Nox1 mRNA is most highly expressed in colon epithelia [15], but is also expressed at lower levels in macrophages [16]. Known Nox1-inducing factors are TLR agonists such as angiotensin II, interferon-c and platelet-derived growth factor [17–20]. However, research on the essen- tial proteins involved in Nox1 gene inducement in macrophages is not clear. There have been some reports of the Nox1 expression mechanism in macro- phages. Reports show that IRAK-1 increases Nox1 expression to produce ROS [21], and the increase in Nox1-mediated ROS production is involved in macro- phage differentiation into receptor activator of NF-j-B ligand (RANKL)-induced osteoclasts [22]. Also, we have previously reported that activation of c-Jun NH 2 -terminal kinase (JNK) and cytosolic phospho- lipase A 2 (cPLA 2 ) by CpG ODN is essential in Nox1 mRNA expression and ROS generation in macrophag- es [23]. Recently, we reported that calcium-independent phospholipase A2b-mediated signaling regulates Nox1 expression in macrophages, and that the ROS pro- duced are part of an important process controlling foam cell formation [24]. Glycogen synthase kinase 3 (GSK3)b and the b-cate- nin pathway are crucial regulators in the balance between pro- and anti-inflammatory cytokine produc- tion [25]. Recently, this pathway was shown to have an essential role in inflammation and immune cells [25]. In particular, many groups have shown that GSK3b, through TLR signaling, is necessary in inflam- mation. For example, it has been reported that GSK3b regulates TLR-mediated cytokine production and inac- tivation of GSK3b by LPS has a negative effect on production of the pro-inflammatory cytokine inter- feron-b [26]. b-Catenin is one of the most important downstream molecules of the GSK3b pathway [27]. When b-catenin is released into the cytosol and is not degraded by the proteosome, it may be translocated to the nucleus and form a complex with T-cell factor. The b-catenin ⁄ T-cell factor complex acts as a tran- scriptional activator of many genes [28]. In this study, we show that GSK3b plays a funda- mental role in regulating Nox1 gene expression. LPS causes phosphorylation of GSK3b and accumulation of b-catenin. Inhibition of GSK3b activity potently augmented expression of the Nox1 gene in LPS-stimu- lated macrophages, whereas ectopic expression of a constitutively active GSK3b mutant reduced Nox1 gene expression. Taken together, these findings suggest that GSK3b and the b-catenin pathway are critical reg- ulators controlling Nox1 gene expression. Results Stimulation of macrophages with LPS induces the expression of Nox1 via TLR4 We studied Nox1 expression and ROS formation by TLR4 agonist LPS in Raw264.7 cells. As shown previ- ously [24], LPS increased Nox1 mRNA expression in a time-dependent manner (Fig. 1A) and induced ROS formation (Fig. 1B). The TLR4 dependence of LPS- induced Nox1 expression was confirmed using TLR4 mutant mice. Monocytes separated from the bone mar- row of TLR4 wild-type (C3H ⁄ HeN) and TLR4 mutant (C3H ⁄ HeJ) mice were differentiated into macrophages by adding macrophage colony-stimulating factor (M-CSF). We stimulated each bone marrow-derived monocyte (BMDM), isolated from two types of mice, with LPS, and compared the Nox1 mRNA expression using RT-PCR. The effects of LPS were strong in the BMDM of C3H ⁄ HeN, but relatively weak in the BMDM of C3H ⁄ HeJ (Fig. 1C). The data suggest that TLR4 is involved in LPS-induced Nox1 expression. LPS phosphorylates GSK3b via TLR4 in macrophages LPS stimulation of innate immune cells has been shown to promote GSK3b inactivation via phosphorylation of serine 9 (S9) [26]. Therefore, we tested changes in GSK3b phosphorylation, using LPS stimulation, in J S. Kim et al. GSK3b-b-catenin is involved in Nox1 expression FEBS Journal 277 (2010) 2830–2837 ª 2010 The Authors Journal compilation ª 2010 FEBS 2831 Raw264.7 cells. LPS stimulation induced GSK3b phos- phorylation (Fig. 2A,B). To assess whether TLR4 is required for LPS to induce GSK3b phosphorylation, TLR4 wild-type and mutant macrophages were com- pared for their abilities to phosphorylate GSK3b upon LPS stimulation. GSK3b phosphorylation increased in the wild-type, but was expressed relatively weakly in the mutant type (Fig. 2C). LPS induces b-catenin accumulation and nuclear translocation Because LPS induced phosphorylation of GSK3b,we investigated whether b-catenin accumulation was affected by LPS stimulation. LPS induced b-catenin accumulation of macrophages and showed maximum effects at 45 min with dose dependency (Fig. 3A,B). To confirm the subcellular localization of b-catenin, we fractionated cells by cytosol and nuclear fraction after LPS stimulation. The time course of subcellular localization for b-catenin showed a progressive increase in the cytosolic fraction and a concomitant significant increase in the nuclear fraction (Fig. 3C), suggesting that b-catenin was translocated into the nucleus. These results were also supported by fluores- cent microscopy. In untreated cells, b-catenin was mainly localized in the cytosol at a low level. After 45 min of treatment with LPS, the distribution of b-catenin in the subcellular compartments was altered, as shown by the nuclear translocation (Fig. 3D). This shows that LPS is able to induce b-catenin accumula- tion and subsequent translocation into the nucleus. C Nox1 -actin 0 0.5 1 2 4 6 0 0.5 1 2 4 6 LPS (h) C3H/HeN (TLR4 WT) C3H/HeJ (TLR4 Mut) A Nox1 β β β -actin 0 1 2 3 4 6 LPS (h) B 0 0.4 0.8 1.2 0 60 120 180 240 300 LPS Ti me (min) ROS production (RLU) Con LPS Fig. 1. Induction of Nox1 mRNA and production of ROS by LPS in macrophages. (A) Raw264.7 cells were stimulated with LPS (100 ngÆmL )1 ) for the indicated times. Nox1 mRNA expression was determined by RT-PCR and was normalized to b-actin. (B) Raw264.7 cells were cultured in 96-well plates in a CO 2 incubator for 1 h. The cells were changed to NaCl ⁄ P i containing lucigenin (100 l M) and NADPH (200 lM) and treated with LPS (100 ngÆmL )1 ) for 300 min. Chemiluminescence was measured in relative light units (RLU) every 10 min over a period of 300 min. (C) Primary BMDMs were isolated from C3H ⁄ HeN (TLR4 wild-type) or C3H ⁄ HeJ (TLR4 mutant) mice. BMDMs were differentiated for 5–7 days in media containing M-CSF and stimulated with LPS (100 ngÆmL )1 ) for the indicated times. Nox1 mRNA expression was determined by RT-PCR and was normalized to b-actin. The data are representative of three independent experiments. 0 10 50 100 500 LPS (ng·mL –1 ) pGSK3β β GSK3 β B A pGSK3 β GSK3 β 0 153045 60 LPS(min) 1 1.6 1.9 1.4 0.7 (fold) C pGSK3 β GSK3 β 0 30 45 60 0 30 45 60 LPS (min) HeN (TLR4 WT) HeJ (TLR4 mut) 1 1.8 1.3 0.7 0.5 0.9 0.7 0.6 (fold) Fig. 2. Inactivation of GSK3b by LPS in macrophages. (A,B) Raw264.7 cells were incubated with LPS (100 ngÆmL )1 ) for different times or doses. To assess phospho-GSK3b (S9), total cell lysates were resolved on SDS ⁄ PAGE, immunoblotted with anti-(phospho-GSK3b) or anti-GSK3b sera, and developed using enhanced chemiluminescence. (C) Wild-type (C3H ⁄ HeN) and TLR4 mutant (C3H ⁄ HeJ) BMDMs were stimulated with LPS (100 ngÆmL )1 ) for the indicated times. Phosphorylated GSK3b expression was determined by western blot using anti-(phospho-GSK3b) serum and was normalized to GSK3b expression. The data are represen- tative of three independent experiments. GSK3b-b-catenin is involved in Nox1 expression J S. Kim et al. 2832 FEBS Journal 277 (2010) 2830–2837 ª 2010 The Authors Journal compilation ª 2010 FEBS GSK3b negatively controls Nox1 expression by LPS-stimulated macrophages Because LPS induced GSK3b phosphorylation and b-catenin accumulation, we investigated whether this pathway played a role in Nox1 gene expression. LiCl is a well-known pharmacologic GSK3b inhibitor. GSK3b-inactivated macrophages stimulated with LPS produced significantly more b-catenin protein which was also translocated more into the nucleus, than cells stimulated with LPS alone (Fig. 4A,B). Also, Nox1 expression, using LPS plus LiCl, showed a greater increase than LPS alone (Fig. 4C). The function of GSK3b was confirmed in Nox1 expression using a constitutively active GSK3b (S9A) mutant. Green fluorescent protein-tagged GSK3b (S9A) mutant was successfully overexpressed and the accumulation of b-catenin and translocation to the nucleus by LPS decreased more than in cells transfected with only the vector (Fig. 4D–F). Taken together, these results dem- onstrate that GSK3b plays a fundamental role in con- trolling the expression of Nox1 by TLR4-stimulated macrophages. Discussion ROS are known to act as a signaling molecule in vari- ous physiological processes because of their regulated production by ligands, the existence of catabolic metabolism to terminate their signaling and their redox-dependent reversible modification of target pro- teins [29]. ROS are also considered important in mac- rophage activation, because this process is significantly related to the pathogenesis of inflammatory diseases such as atherosclerosis or metabolic syndrome. The ROS production system of macrophages is vari- able and complicated, and Nox and its function have become current issues. Seven types of Nox have been found. The most studied is Nox2; however, other types, especially Nox1 and Nox4, are also active areas of research. ROS produced by Nox has a general downstream physiological role. ROS produced by Nox2 is required in the respiratory burst that occurs in phagocytes [30]. It has been suggested that other types of Nox are needed in host defense. For example, Nox1 is important in the colon and Duox-1 and -2 are important in the lung [31]. Nox1 also contributes sig- nificantly to gastrointestinal inflammation, hyperten- sion and restenosis after angioplasty development [8,18]. ROS production from Nox1 activity is primarily controlled by p22phox and the Nox1 regulators NoxA1 and NoxO1 [5], but an increase in Nox1 gene expression is also essential. Angiotensin II and plate- let-derived growth factor lead to increased Nox1 mRNA levels and contribute to vascular pathology [18,20]. The TLR agonists LPS, flagellin and CpG ODN have been shown to be Nox1 gene-inducing fac- tors, leading to the verification of Nox1 function in immune responses and the development of atheroscle- rosis [17]. Therefore, the regulation of Nox1 mRNA expression may be a potential therapeutic target for C β-catenin (short) β -catenin (long) β -tubulin 0 15 30 45 60 0 15 30 45 60 LPS (min) Cytosol Nucleus D DAPI FITC Merge Con LPS B 0 10 50 100 500 LPS (ng·mL –1 ) β β -catenin β -actin A 0 15 30 45 60 LPS (min) β -catenin β -actin Fig. 3. Changes in the subcellular localization of b-catenin induced by LPS in macrophages. (A,B) Raw264.7 cells were incubated with LPS (100 ngÆmL )1 ) for different times or doses. To assess b-catenin, total cell lysates were resolved on SDS ⁄ PAGE, immunoblotted with an anti-(b-catenin) serum and developed using enhanced chemilumi- nescence. (C) Cells were treated with LPS (100 ngÆmL )1 ) for the indicated times and fractionated into cytosolic and nuclear extracts. Western blot on both extracts was determined by using anti- (b-catenin) serum. b-Tubulin served as loading control and nuclear marker. (D) Macrophages were treated with LPS (100 ngÆmL )1 ) for 40 min and stained with anti-b-catenin and DAPI at room tempera- ture for 10 min. The cells were washed three times with NaCl ⁄ P i . The images were acquired and analyzed using fluorescent micros- copy. The data is representative of five independent experiments. J S. Kim et al. GSK3b-b-catenin is involved in Nox1 expression FEBS Journal 277 (2010) 2830–2837 ª 2010 The Authors Journal compilation ª 2010 FEBS 2833 these diseases. However, more research into the mecha- nism controlling Nox1 mRNA expression is needed. We previously reported that various types of TLR agonists increase ROS production by inducing Nox1 mRNA expression, and the increased ROS convert macrophages to foam cells by low-density lipoprotein oxidation [14]. The aim of this study was to find the signaling transduction molecule contributing to Nox1 mRNA expression by LPS. Our results showed that GSK3b is a very important factor in b-catenin signal- ing. In other words, LPS inactivates GSK3b through phosphorylation, and the inactivated GSK3b inhibits the degradation of b-catenin, promoting translocation into the nucleus. It is hypothesized that the translocat- ed b-catenin forms a complex with a specific transcrip- tion factor, and thereby leads to an increase in Nox1 mRNA expression. Furthermore, experimental results, using GSK3b inhibitor and constitutively active GSK3b, have shown the importance of GSK3b in Nox1 mRNA regulation. Signaling molecules, such as protein kinase C-d (PKC-d) and calcium-independent phospholipase A 2 (iPLA 2 b), are known to regulate Nox1 mRNA [24,32]. We previously reported that Akt also regulates Nox1 mRNA expression [24]. Therefore, we investigated the correlation between Akt and GSk3b. The Akt inhibitor LY294002 inhibited GSK3b phosphorylation by LPS and also decreased Nox1 mRNA expression (Fig. S1). These results suggest that LPS induces Akt phosphory- lation and the activated Akt inactivates GSK3b, thereby regulating Nox1 gene expression. However, various signaling proteins that control Akt exist and it is believed that more proteins may participate in the process, showing the need for further research. Consequently, LPS induces Nox1 expression via TLR4. Phosphorylation of GSK3b by LPS inactivates GSK3b and increases translocation of b-catenin to the nucleus by inhibiting degradation. We suggest that translocated b-catenin will activate specific transcrip- tion factors and eventually increase Nox1 mRNA C – + – + LPS –– ++LiCl Nox1 -actin B Con MergeFITCDAPI LPS LiCl LPS LiCl A –+–+LPS ––++LiCl -catenin -actin D -catenin EGFP- GSK3 GSK3 –+ –+ LPS –+ –+ LPS Vec GSK3 S9A F -actin Nox-1 Vec GSK3 S9A DAPI TRITC Merge - LPS - LPS Vector GSK3 S9A E Fig. 4. The effect of GSK3b on LPS-induced b-catenin and Nox1 expression. (A–C) Raw264.7 cells were stimulated with LPS (100 ngÆmL )1 ) in the presence or absence of the GSK3b inhibitor, LiCl (5 l M). (A) The cell lysates were analyzed for b-catenin using western blotting. (B) Cells were fixed and stained with anti-(b-catenin) serum and DAPI and observed using fluorescent microscopy. (C) Nox1 mRNA was analyzed by RT-PCR. (D–F) Raw264.7 cells were transfected with a pEGFP-C1 vector expressing a constitu- tively active form (GSK3b S9A) or vector alone. Gene-transfected cells were stimu- lated with or without LPS (100 ngÆmL )1 ). (D) Cell lysates were analyzed for GSK3b, b-catenin by western blotting. (E) Cells were fixed and stained with anti-(b-catenin) serum and DAPI and observed with fluorescent microscopy. (F) Nox1 mRNA was analyzed by RT-PCR. The data are representative of five independent experiments. GSK3b-b-catenin is involved in Nox1 expression J S. Kim et al. 2834 FEBS Journal 277 (2010) 2830–2837 ª 2010 The Authors Journal compilation ª 2010 FEBS expression. Our results showing the possibility of the Nox1 gene being regulated by GSK3b ⁄ b-catenin are ori- ginal and lead to the possibility that GSK3b ⁄ b-catenin may contribute to the development of atherosclerosis. Materials and methods Reagents Cell culture reagents, including fetal bovine serum, were obtained from Life Technologies (Grand Island, NY, USA). GSK3b, b-catenin and b-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and phospho-GSK3b, Akt, p-Akt antibodies were from Cell Signaling Technology (Danvers, MA, USA). Escherichia coli LPS (0111:B4, Cat. No: L3024, purified by ion-exchanged chromatography and containing > 1% protein and RNA), NADPH and lucigenin were from Sigma-Aldrich (St. Louis, MO, USA), the RT-PCR kit was from Takara Bio Inc (Otsu, Japan). TLR4 wild-type (C3H ⁄ HeN) and mutant (C3H ⁄ HeJ) mice were purchased from Central Lab Animal Inc (Seoul, Korea). 4¢,6-diamidino-2-phenylindole (DAPI) and Alexa fluor 488 goat anti-(rabbit IgG) were from Invitrogen (Carlsbad, CA, USA). Rhodamine (TRITC)-conjugated AffiniPure donkey anti-(rabbit IgG) was from Jackson ImmunoResearch Laboratories Inc (West Grove, PA, USA). Plasmids and transfection In order to make a GSK3b (S9A) plasmid construction, cDNA from Raw264.7 cells was amplified by PCR with mutation primer-1 (forward: 5¢-ACTCCACCCTTTTTCTC CTC-3¢, reverse: 5¢-GCTCTCCGCAAAGGCGGTGGT-3¢) and mutation primer-2 (forward: 5¢-CGACCGAGAACCA CCGCCTTTGC-3¢, reverse: 5¢-CGCGTCGACCTCCTGG GGGCTGTTCAG-3¢). Two PCR products were mixed and amplified by cloning primer (forward: 5¢-CGCAGATCTA TGTCGGGGCGACCGAGA-3¢, reverse: 5¢-CGCGTCGA CCTCCTGGGGGCTGTTCAG-3¢) to obtain insert cDNA. Insert cDNA was ligated with pEGFP-C1 vector (Invitrogen) and the ligated vector was transformed into DH5a cells. Nucleotide sequencing was performed after plasmid prepara- tion. Cells were transfected with pEGFP-C1–GSK3b (S9A) plasmid using Lipofectamine LTX reagent (Invitrogen) according to the manufacturer’s protocol and then incubated for 24 h before LPS stimulation. Cell culture and mouse BMDM preparation The Raw264.7 macrophage cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% antibi- otics (100 UÆmL )1 penicillin and 100 lgÆmL )1 streptomycin) at 37 °Cin5%CO 2 . Primary BMDMs were isolated from C3H ⁄ HeN or C3H ⁄ HeJ mice. BMDMs were differentiated for 5–7 days in media containing M-CSF. The culture med- ium consisted of Dulbecco’s modified Eagle’s medium sup- plemented with 10% L929 cell-conditioned medium (as a source of M-CSF). This study was conducted in accordance with the guidelines for the care and use of laboratory ani- mals provided by Yeungnam University and all experimen- tal protocols were approved by the Ethics Committee of Yeungnam University, South Korea. Lucigenin assay NADPH-dependent ROS generation was measured by monitoring lucigenin-derived chemiluminescence at room temperature using the Lmax II luminometer (Molecular Devices, Sunnyvale, CA, USA). Briefly, cells were cultured in 96-well plates, pretreated with LiCl for 1 h, subsequently suspended in NaCl ⁄ P i and incubated with lucigenin (100 lm) and NADPH (200 lm). LPS was added exoge- nously to the suspended cells. Chemiluminescence was mea- sured in relative light units every 10 min over a period of 200–300 min. Fluorescent microscopy assay Cells were plated in 24-well plates containing embedded glass cover slips and pretreated with LiCl for 1 h before stimulation by 100 ngÆmL )1 LPS for 40 min. Cells were fixed and stained with anti-b-catenin serum and DAPI and observed using fluorescent microscopy. Fluorescent micros- copy images were acquired and analyzed using an Olympus BX51 fluorescent microscope and DP Manager Software (Olympus, Japan). Cytosolic and nuclear fractionation Cells were plated in a 100 mm diameter dish and pretreated with LiCl for 1 h before stimulation with 100 ngÆmL )1 LPS for the indicated times. Cytosolic and nuclear fractionation was performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA) according to manufacturer’s protocol. RT-PCR Total RNA was extracted from cells using Trizol reagent (Invitrogen). One microgram of total RNA was used as a template to make first-strand cDNA by oligo(dT) priming using a commercial reverse transcriptase system (Promega, Madison, WI, USA). The synthetic gene-specific primer sets used for PCR were Nox1 forward primer, 5¢-AAGTGGCT GTACTGGTTGG-3¢, and reverse primer, 5¢-GTGAGGA J S. Kim et al. GSK3b-b-catenin is involved in Nox1 expression FEBS Journal 277 (2010) 2830–2837 ª 2010 The Authors Journal compilation ª 2010 FEBS 2835 AGAGTCGGTAGTT-3¢, which amplified 238 bp of mouse Nox1 cDNA, and b-actin forward primer, 5¢-TCCTTCGT TGCCGGTCCACA-3¢, and reverse primer, 5¢-CGTCTCC GGAGTCCATCACA-3¢, which amplified 509 bp of mouse b-actin cDNA. Cycling conditions were 95 °C for 5 min, followed by 28 cycles of 95 °C for 1 min, 57 °C for 1 min and 72 °C for 1 min. For quantification, target genes were normalized against b-actin. Western blot analysis Macrophages were cultured in six-well plates and treated with LPS in the presence or absence of an inhibitor. Cell pellets were resuspended in lysis buffer (50 mm Tris ⁄ HCl, pH 8.0, 5 mm EDTA, 150 mm NaCl, 0.5% Nonidet P-40, 1mm phenylmethanesulfonyl fluoride, and protease inhibi- tor cocktail). Proteins were separated by 8% reducing SDS ⁄ PAGE and immunoblotted onto nitrocellulose mem- branes in 20% methanol, 25 mm Tris and 192 mm glycine. Membranes were then blocked with 5% non-fat dry milk and incubated with primary antibody overnight. The mem- branes were washed, incubated for 1 h with a secondary antibody conjugated to horseradish peroxidase, rewashed and developed using an enhanced chemiluminescence sys- tem (GE Healthcare, Chalfont St Giles, UK). Acknowledgement This work was supported by the Korean Science and Engineering Foundation via the Aging-associated Vas- cular Disease Research Center at Yeungnam University (R13-2005-005-02001-0). References 1 Takeya R & Sumimoto H (2003) Molecular mechanism for activation of superoxide-producing NADPH oxidas- es. 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GSK3b-b-catenin is involved in Nox1 expression FEBS Journal 277 (2010) 2830–2837 ª 2010 The Authors Journal compilation ª 2010 FEBS 2837 . Glycogen synthase kinase 3b and b-catenin pathway is involved in toll-like receptor 4-mediated NADPH oxidase 1 expression in macrophages Jin-Sik Kim 1 *,. IRAK -1 increases Nox1 expression to produce ROS [ 21] , and the increase in Nox1-mediated ROS production is involved in macro- phage differentiation into receptor