TNFα Activates c-Jun Amino Terminal Kinase through p47phox

Experimental Cell Research 272, 62–74 (2002) doi:10.1006/excr.2001.5404, available online at http://www.idealibrary.com on

TNF␣ Activates c-Jun Amino Terminal Kinase through p47 phox Ying Gu, You Cheng Xu, Ru Feng Wu, Rhonda F. Souza, Fiemu E. Nwariaku, and Lance S. Terada 1 University of Texas Southwestern and Dallas VA Medical Center, Dallas, Texas

generation of reactive oxidants in various cell types. For example, O 2⫺•-trapped adducts are formed following treatment of fibroblasts with TNF␣ [2], and antioxidants prevent TNF␣-induced JNK activation in chondrocytes [3]. Antioxidants also decrease JNK activation resulting from TRAF2 but not MEKK1 overexpression, consistent with at least one redox-sensitive switch proximal to the level of MAP kinase kinase kinases (MAPKKKs) but distal to TRAF2 [4]. Likewise, antioxidants suppress activation of another MAPKKK upstream of JNK, apoptosis signal-regulating kinase 1 (ASK1) [5], and overexpression of Mn-SOD decreases downstream effects of TNF␣ such as JNK activation, AP-1 activation and translocation, and transactivation of IL-1 gene expression [6, 7]. Less clear is the enzymatic source of signaling oxidants. An agonist-stimulated NADPH oxidase exists in professional phagocytes, the chief function of which is the timely generation of large amounts of bactericidal oxidants. The participation of a homologous oxidase complex in parenchymal cell signaling has been implicated in part based on the use of diphenylene iodonium (DPI), an avid but nonselective flavoprotein inhibitor. DPI, for instance, blocks MAP kinase activation following stimulation with TNF␣, lactosylceramide, or constitutively active p21ras [3, 8, 9]. Other suggestive studies note that constitutively active forms of Rac1, a Rho family protein involved in activation of the phagocyte oxidase, increases oxidant production in HeLa cells [10], whereas dominant negative N17Rac1 inhibits integrin ligation and Ras-induced oxidant production [9, 11]. Homologs of the membrane-associated subunits of NADPH oxidase have been cloned in parenchymal cells. Rat vascular smooth muscle cells express a p22 phox homolog which appears to mediate angiotensin II-stimulated O 2⫺• production [12]. In addition, Mox1 and Renox (NOX1 and NOX4) are two recently identified epithelial/vascular smooth muscle homologs of gp91 phox which appear to mediate mitogenic and hypoxia-stimulated events, respectively [13, 14]. However, homologs of the cytosolic phagocyte oxidase subunits have not been cloned from parenchymal cells. In addition, studies investigating cell death from TNF␣ stimulation report that inhibitors of other oxidant generators such as 5⬘ lipoxygenase or mitochondrial res-

Reactive oxygen intermediates have been implicated in the transduction of TNF␣ signals, although the source of such oxidants has not been established. We found that activation of ECV-304 cells by TNF␣ was accompanied by a transient burst of oxidants and activation of JNK, both of which were suppressed by two distinct inhibitors of the phagocyte NADPH oxidase and the thiol antioxidant N-acetyl cysteine (NAC). We cloned partial and full-length cDNA sequences from ECV-304 cells and human umbilical vein endothelial cells (HUVEC), respectively, for p47 phox, demonstrating that these nonphagocytic cells express this adapter protein known to specifically initiate assembly of the NADPH oxidase in professional phagocytes. A mutant p47 phox, defective in the first Src homology 3 (SH3) domain (p47W(193)R), diminished JNK activation by TNF␣. Surprisingly, p47 phox resided entirely in the particulate, not cytosolic, fraction of cells. Immunostaining suggested partial colocalization with cytoskeletal elements, and cytoskeletal disrupters decreased both oxidant production and JNK activation by TNF␣. A p47-GFP fusion protein localized to the cortical cytoskeleton in living cells; further, stimulation of cells with TNF␣ caused a marked concentration of p47-GFP in membrane ruffles, actin-rich structures associated with intense respiratory burst activity in stimulated neutrophils. We conclude that nonphagocytic cells express p47 phox, which appears to localize to the cytoskeleton and participate in TNF␣ signaling. We speculate that this physical targeting may prove important in conferring signal specificity and enhancing signaling efficiency of unstable oxidants. © 2001 Elsevier Science Key Words: free radicals; cytokines; inflammation; protein kinases.

INTRODUCTION

During the inflammatory response, the activation of JNK by TNF␣ appears to initiate important cellular events such as the expression of adhesion proteins [1]. This JNK activation is thought to proceed through the 1 To whom correspondence and reprint requests should be addressed at Dallas VAMC, MC 151, 4500 S. Lancaster, Dallas, TX 75216. Fax: 214-857-0340. E-mail: [email protected].

0014-4827/01 $35.00 © 2001 Elsevier Science All rights reserved.

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piration interfere with downstream TNF␣ events [15, 16], casting doubt on the necessity of an NADPH oxidase in TNF␣ signaling. In this study, we demonstrate expression by endothelial cells of p47 phox, an adapter protein necessary for the regulated activation of the phagocyte NADPH oxidase. Further, we find that disruption of the first SH3 binding site interferes with activation of JNK by TNF␣, implicating a homologous oxidase complex in this signal pathway. p47 phox appears to associate with cytoskeletal elements, suggesting that site direction of a functional oxidase may be one basis for the signal specificity of oxidants. MATERIALS AND METHODS Sources of reagents. TNF␣ (recombinant human) was obtained from PeproTech; goat anti-JNK1 (C17) and GST-c-Jun (amino acids 1–79) were from Santa Cruz Biotechnology. [␥- 32P]ATP was from NEN; jasplakinolide, rhodamine phalloidin, and 2⬘,7⬘-dichlorofluorescin diacetate (DCF) were from Molecular Probes. Apocynin (acetovanillone) was obtained from Aldrich Chemical; N-acetyl cysteine (NAC), DPI, cytochalasin B, paclitaxel, nocodazole, colchicine, and fibronectin were from Sigma Chemical. Rabbit antisera against p47 phox was a generous gift from Dr. Bernard Babior [17]. Anti-␤ tubulin (clone TUB 2.1) and secondary anti-rabbit FITC conjugate were from Sigma; anti-vimentin (clone V9), secondary anti-mouse rhodamine conjugate were from Immunotech; and anti-actin (clone C4) was from Boehringer Mannheim. Protein G–agarose was from Pharmacia. Oxidant production. Intracellular oxidant production was assessed using the oxidant-sensitive fluorochrome DCF. ECV-304 cells were grown in DMEM with 10% FBS and routinely used within 1 day of confluence. Cells were washed three times with Hanks’ buffered salt solution (HBSS) to remove any serum-associated esterases, then incubated with 80 ␮M DCF in HBSS for 20 min at 37°C, in the presence or absence of oxidase inhibitors. Cells were then washed and exposed to TNF␣ in Opti-MEM with 3.75% FBS. After a 10-min incubation at 37°C, cells were washed in Ca 2⫹/Mg 2⫹-free HBSS, dislodged and separated with trypsin/EDTA (GIBCO), and analyzed by flow cytometry (Becton Dickson) within 20 min. Mean fluorescence values of four separate experiments were compared using ANOVA with Student–Neuman–Keuls comparison test. In some studies, parallel measurements were obtained by assessing DCF fluorescence in situ by analyzing digitized images of intact monolayers. Fluorescence intensity of all cells in five random fields was quantified. Superoxide production was measured kinetically. ECV304 cells, IFN ␥/1,25 di-OH vitamin D 3-differentiated U937 cells, or purified human neutrophils [18] (10 6/ml) were stimulated with either TNF␣ (100 ng/ml) or PMA (100 nM), and the rate of SOD-inhibitable cytochrome c reduction was assessed [18]. Values were compared using ANOVA. Kinase activity. JNK activity was assessed by an IP kinase method. For inhibitor experiments, cells were exposed to inhibitors for 30 min prior to stimulation with TNF␣. Cells were then washed and lysed in cold RIPA (50 mM Tris, pH 7.2, 150 mM NaCl, 0.1% SDS, 0.5% Na deoxycholate, 1% Triton X-100, 10 mM Na pyrophosphate, 25 mM ␤-glycerophosphate, 2 mM sodium orthovanadate, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 1 mM PMSF) and drawn through a 23-G needle three times. Two 75-cm 2 flasks were pooled for each assay. Following centrifugation at 12,000g for 10 min at 4°C, supernatants were adjusted for protein content, precleared with Protein G–agarose (Pharmacia), immunoprecipitated with antiJNK1 (30 min at 4°C) and Protein G–agarose (90 min at 4°C), and

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washed extensively. The immunoprecipitated JNK1 was assayed for kinase activity in kinase buffer (25 mM Hepes, pH 7.6, 20 mM ␤-glycerophosphate, 20 mM MgCl 2, 2 mM DTT, 0.1 mM sodium orthovanadate, 20 mM p-nitrophenyl phosphate, 20 ␮M ATP) by addition of 5 ␮Ci [␥- 32P]ATP and 2 ␮g GST-Jun. After a 30-min incubation at 30°C, reactions were stopped by addition of Laemmli buffer, and samples resolved by SDS–PAGE. p47 cloning. ECV-304 total RNA was reverse-transcribed and amplified with 35 PCR cycles (Advantage proofreading polymerase, Clontech) using unique primers, based on the phagocyte p47 phox coding sequence (Accession No. M26193), spanning nucleotides 277–306 (S1, ending with a unique Met codon conserved in p47 phox over human, mouse, and bovine) and 816 –787 (A1, ending with a conserved unique Trp codon). A second overlapping product was produced using primers spanning nt 342–369 (S2) and 1163–1135 (A2). Both products were gel-purified, subcloned into pCR-TOPO 2.1 (Invitrogen), and sequenced in both directions. Total RNA from fourth-passage HUVEC was also reverse-transcribed and amplified using primers S2 and A2, subcloned, and sequenced bidirectionally. Based on the composite sequence data, p47 was amplified from a HUVEC library which was cloned directionally into Lambda ZAP-CMV XR (Stratagene), using 35 PCR cycles and Pfu polymerase (Stratagene). Three overlapping products were obtained using an upstream primer external to the library insert (primer CMV5⬘) and primer A1, primers CMV5⬘ and A2, and a third set of primers spanning nt 799 – 828 (S3, sense) and a downstream sequence external to the library insert (CMV3⬘, antisense). Each product was gel-purified, subcloned, and sequenced in both directions. Sequences have been entered in GenBank (see Table 2). Quantitative RT-PCR. A truncated mimic was created by removal of a 180-base BglII fragment from p47 phox. Total RNA was used in an RT-linked competitive PCR assay using primers flanking this deletion [19]. Plasmid construction and transfection. p47 phox cDNA from phagocytes, cloned into the EcoRI site of pBluescript SK⫹, was obtained from ATCC. A 3-base mutation (576-TTGG3 CCGC) was performed using PCR mutagenesis (QuickChange, Stratagene), introducing a new NotI site and the W(193)R mutation. Both p47W(193)R and wild-type p47 phox were subcloned into the inducible expression vector pTRE (Tet-On, Clontech) at the EcoRI site, resulting in pTRE-p47 and pTRE-p47W(193)R. ECV-304 cells were first stably transfected (Lipofectamine, GIBCO) with the tetracycline receptor-bearing pTet-on and selected with G418, and single-cell clones were then tested for receptor expression using pTRE-luciferase. A single ECV304 clone was then stably transfected with pTRE-p47, pTREp47W(193)R, or the empty pTRE vector and selected with hygromycin. Double stable transfectants were grown in media containing tetracycline-free FBS (Clontech) through at least one passage (four to five doublings) prior to each experiment. Transgenes were induced for 24 h with 1 ␮g/ml doxycycline. A p47-GFP fusion was constructed by mutating the p47 phox stop codon (TGA3 GAC), introducing a new SalI site which was used to ligate p47 phox to the amino terminus of GFP (pEGFP-N3, Clontech). Transient transfection of this plasmid was accomplished by electroporation. All mutations were confirmed by direct sequencing. Cell fractionation and Western immunoblotting. One-day postconfluent ECV-304 (5 ⫻ 10 8) cells were washed twice with PBS, mechanically harvested in relaxation buffer (3 mM NaCl, 100 mM KCl, 1.5 mM EGTA, 3.5 mM MgCl 2, 10 mM Pipes, pH 7.4, 1 mM PMSF, 50 ␮g/ml leupeptin, 25 ␮g/ml pepstatin A, 25 ␮g/ml aprotinin, and 1 mM ATP), and sonicated with two 5-s bursts at 50 W. Nuclei and intact cells were pelleted at 340g and the postnuclear fraction was separated by centrifugation (100,000g, 45 min at 4°C) into particulate and soluble fractions [17, 20]. The soluble fraction was precipitated with ice-cold acetone and resuspended in SDS sample buffer. The particulate fraction was washed twice with relaxation buffer before solubilization in SDS sample buffer with 4 M urea.

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Neutrophils (3 ⫻ 10 7 per condition) were purified from the blood of healthy adult humans and purified by density gradient centrifugation [18], incubated in HBSS with 1 mM PMSF, 10 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin (4°C for 15 min), sedimented, and resuspended in relaxation buffer. Particulate and soluble fractions were separated as above. Cytoskeletons were isolated as previously described [21]. Briefly, cells were washed in cold PBS, scraped, resuspended in extraction buffer (100 mM Hepes, pH 7.4, 10 mM EGTA, and 1% Triton X-100), and incubated at 4°C for 15 min. The detergent-insoluble pellet was recovered by centrifugation (13,000g for 15 min), washed twice with extraction buffer, and denatured with boiling SDS sample buffer (containing 2% SDS and 15% ␤-mercaptoethanol). The detergent soluble fraction was concentrated with 30-kDa cutoff filters prior to addition of SDS sample buffer. Membrane fractions were obtained by density gradient separation [22]. Cells were washed with PBS, scraped, resuspended in relaxation buffer, and sonicated with a single 5-s burst (50 W). The postnuclear supernatant was transferred onto a 15–50% discontinuous sucrose gradient and spun at 100,000g, 4°C, for 30 min. The membrane-enriched sucrose interface was washed twice in 0.25 M sucrose, resuspended in cytoskeletal buffer (20 mM Tris, pH 7.4, 3 mM MgCl 2, 8% sucrose, 5 mM EGTA, 0.5% Triton X-100, 1 mM PMSF, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, 10 ␮g/ml pepstatin A), and pelleted at 180,000g for 2 h. The detergent-insoluble membrane pellet was washed once in cytoskeletal buffer and denatured in SDS loading buffer; the detergent soluble supernatant was precipitated in ⫺20°C acetone overnight and resuspended in SDS loading buffer. After SDS–PAGE separation, all samples were transferred onto nitrocellulose and probed with anti-p47 phox antisera. Microscopy. For immunohistology, ECV-304 cells were plated onto fibronectin-coated chamber slides (Nunc) and allowed to reach confluence. Cells were washed twice with PBS, fixed with 3% formaldehyde in PBS for 5 min, washed twice, and permeabilized (50 mM NaCl, 3 mM MgCl 2, 200 mM sucrose, 10 mM Hepes, pH 7.4, 0.5% Triton X-100) for 5 min. After blocking with 3% BSA in PBS, cells were incubated with primary antibody (anti-p47, 1:1000; anti-␤tubulin, 1:50; anti-vimentin, 1:200) in 1% BSA at 25°C for 1 h. After three washes with 1% BSA, cells were incubated with secondary antibody (1:200) or rhodamine–phalloidin (5 U/ml) in 1% BSA for 30 min at 25°C, washed twice with PBS/1% BSA and twice with PBS, and then mounted with ProLong antifade (Molecular Probes). For in vivo imaging, GFP-p47-transfected cells were grown in dishes with glass coverslip bottoms (MatTek) which were coated with fibronectin. Imaging was acquired with a Zeiss Axiovert S100TV LSM 410 laser scanning system. Epifluorescence and DIC images were obtained using a Nikon TE300 system.

RESULTS

TNF␣ activates JNK1 through an oxidase. As in most cells reported, TNF␣ strongly and rapidly activated JNK1 in ECV-304 cells, with peak activation occurring within 15 min and diminishing by 30 – 60 min (data not shown). We found that TNF␣-induced JNK1 activation was diminished by two structurally unrelated inhibitors of the phagocyte NADPH oxidase (Fig. 1a). DPI, a flavoprotein inhibitor which targets cytochrome b 558 , and apocynin, which appears to block assembly of the NADPH oxidase subunits on the phagocyte membrane [23], both decreased TNF␣ signaling. In addition, TNF␣-induced JNK1 activation was also blocked by the sulfhydryl antioxidant N-acetyl cysteine, consistent with its effects in mono-

cytic U937 cells [24]. In contrast, however, inhibitors of xanthine oxidase (allopurinol), nitric oxide synthase (N ␻-nitro-L-arginine), or mitochondrial respiration (rotenone) did not diminish JNK1 activation (not shown). We also found a rapid, parallel increase in intracellular oxidant levels following stimulation of ECV-304 cells with TNF␣ (Fig. 1b). This oxidant burst activity was decreased by DPI, apocynin, and N-acetyl cysteine, suggesting that endogenous production of oxidants by a DPI and apocynin-inhibitable oxidase was necessary for JNK activation signals. Apocynin itself appears to be an autofluorescent compound, which may explain the increase in fluorescent signal with higher doses of the compound. The absolute levels of superoxide production by stimulated ECV-304 cells was low relative to PMA-stimulated phagocytic cells such as differentiated U937 cells or mature neutrophils (Table 1). Unstimulated ECV-304 cells did not release detectable levels of superoxide. Endothelial cells express p47 phox. Because the JNK activation inhibitor profile suggested participation of a phagocyte-like oxidase, we sought validation for the expression of a vascular cell homolog of p47 phox, a key phosphorylated adapter protein responsible for receiving activation signals which culminate in oxidase assembly in phagocytes [17, 25]. By RT-PCR, we found evidence that both ECV-304 cells and HUVEC express p47 phox. Sequence information from three overlapping HUVEC library products (spanning bases ⫺10 –786, ⫺10 –1134, and 829 –1330) yielded the entire open reading frame, which was nearly identical with the published sequence of myeloid cell-derived p47 phox (Table 2). A single base change (A3 G at 387) has been reported as a polymorphism of p47 phox in differentiated HL-60 myeloid cells [26, 27]. In addition, the HUVEC library cDNA suggests a relatively short 5⬘ UTR, as is the case with p47 phox [26 –28]. Importantly, the HUVEC cDNA excluded the possibility of an expressed p47 phox pseudogene, which bears a frame-shifting GT deletion of bases 73–74 and a premature stop codon [29]. To further ensure that the three HUVEC library PCR products reflected a single transcript, primers were designed from the extreme 5⬘ (5⬘-CCACCCAGTCATGGGGGACAC) and 3⬘ (5⬘-CCACTCCAAGCAACATTTATTGAGGGTGGC) ends of the HUVEC sequence (initiation codon and polyadenylation signal underlined) and used to amplify the expected 1.34-kb transcript from the HUVEC library (data not shown). Relative expression of p47 phox mRNA was found to be lower in endothelial cells than phagocytic cells by quantitative RT-PCR (3.9 ⫻ 10 ⫺7 fmol cDNA/RT reaction in ECV-304 cells versus 16 ⫻ 10 ⫺7 fmol cDNA/RT reaction in differentiated U937 cells). p47 phox participates in JNK activation. p47 phox contains tandem SH3 binding domains which are critical

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FIG. 1. TNF␣ activates JNK and increases oxidant production by ECV-304 cells. (a) Cells were exposed to TNF␣ (100 ng/ml) for 15 min and JNK1 activity was assessed by an IP kinase method. TNF␣ increased JNK1 activity 14-fold; pretreatment with apocynin, DPI, or NAC at the doses indicated diminished JNK1 activation by TNF␣. Western blot of total JNK1 protein is shown below. Histogram represents the mean of three to six individual experiments. (b) ECV-304 cells were loaded with DCF-diacetate and stimulated with TNF␣ (100 ng/ml) for 15 min. Intracellular oxidant production was measured by DCF fluorescence. Pretreatment with apocynin, DPI, or NAC at the doses indicated decreased TNF␣-stimulated oxidant production. Values are the mean ⫾ SEM of four individual experiments.

to the assembly of the NADPH oxidase in vitro and in intact myeloid cells [25]. Specifically, upon activation in phagocytes, the first SH3 domain of p47 phox appears to target a proline-rich motif on the cytochrome-associated p22 phox, causing translocation of the cytosolic subunits to the phagocyte membrane [30]. Detailed studies of the first SH3 domain of p47 phox have demonstrated that mutation of Trp193, a strictly conserved residue bridging two of three hydrophobic binding pockets on SH3 surfaces, completely disrupts p47 phox binding to p22 phox in vitro and translocation of p47 phox and assembly of a functional oxidase in intact cells [31]. We therefore attempted to interrupt p47 phox-dependent signaling by expression of a comparable SH3-

TABLE 1 Superoxide Production by ECV-304 Cells, U937 Cells, and Neutrophils

Stimulated Unstimulated

ECV-304

U937

Neutrophil

0.37 ⫾ 0.21 ND

2.27 ⫾ 0.69* ND

2.23 ⫾ 0.10* 0.07 ⫾ 0.04

Note. Superoxide production is given as mean ⫾ SEM of six determinations in fmol/cell/min. ND, none detected. ECV-304 cells were stimulated with TNF␣ (100 ng/ml); U937 cells and neutrophils were stimulated with PMA (100 nM). *P ⬍ 0.05 compared with ECV-304 cells.

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TABLE 2 Coding Region Sequence Differences in p47 from ECV-304, HUVEC, and Myeloid Cells Position: ECV-304 HUVEC RT-PCR HUVEC library myeloid p47 phox Predicted a.a. changes

387

598

645

772

804

G G G A T(129)T

A G G G A(200)T

T C T T S(215)S

A G A A K(258)Q

G A A A K(268)K

Note. Sequences of p47 from RT-PCR products derived from ECV-304 (bases 307–1134, GenBank Accession No. AF330625) and HUVEC RNA (bases 370 –1134, Accession No. AF330626), from a HUVEC library (bases ⫺10 –1330. Accession No. AF 330627), and from differentiated HL-60 cell libraries (from [26] and [28], Accession No. M26193) differ as shown.

defective mutant in ECV-304 cells. Induction of the p47W(193)R mutant blocked TNF␣-stimulated oxidant production (TNF␣ caused a nonsignificant 6 ⫾ 3% decrease in DCF fluorescence in p47W(193)R-expressing cells versus a 242 ⫾ 49% increase in control cells) and significantly decreased activation of JNK1 by TNF␣ (Fig. 2a). JNK activation was not altered by overexpression of the wild-type p47 phox or transfection of the empty vector (Fig. 2a), or by uninduced p47W(193)R transfectants (Fig. 2b). p47 phox is a cytoskeletal protein. Surprisingly, in ECV-304 cells we found that an immunoreactive protein of MW ⬃48 – 49 kDa, consistent with p47 phox, was recovered entirely in the particulate fraction, independent of cell activation by TNF␣ (Fig. 3a). p47 phox was not recovered from the soluble fraction even under resting conditions. In contrast, p47 phox exists as a cytosolic complex in resting neutrophils. Upon activation of the neutrophil oxidase, a fraction of p47 phox translocates from the soluble to the particulate (largely membranous) fraction (Fig. 3b). Separation of ECV-304 cells into Triton X-100-soluble and -insoluble fractions further demonstrated the association of endothelial p47 phox with the cytoskeletal-associated insoluble fraction (Fig. 3c), again independent of TNF␣ stimulation. The appearance of a doublet in the detergent-insoluble fraction is at present unexplained, although the upper band may represent a more heavily phosphorylated form of p47 phox. Immunofluorescent images demonstrated decoration of submembranous and reticular cytosolic structures and nuclei with antibodies to p47 phox (Figs. 4b, 4e, and 4h). Secondary FITC-labeled antibodies did not crossreact with these structures (data not shown). Costaining with anti-␤-tubulin revealed partial colocalization of p47 phox with tubulin-containing structures (Figs. 4a– 4c). Interestingly, p47 phox also partially colocalized with actin microfilament-binding rhodamine phalloidin, particularly at submembranous cortical actin sites (Figs. 4d– 4f). In contrast, p47 phox did not specifically stain vimentin-containing intermediate filaments (Figs. 4g– 4i). Apparent colocalization of several in-

tensely antivimentin staining areas appeared to be artifactual bleed-through of red into green. Because of the apparent colocalization of ECV-304 cell p47 phox with the submembranous cytoskeleton, we probed ECV-304 membrane preparations for the protein. Again, p47 phox was not recovered in the high-speed supernatant (largely cytosolic) fraction, or the Triton X-100-soluble membrane fraction (Fig. 5a). Instead, p47 phox appeared in the Triton X-100-resistant membrane pellet, enriched for membranous cytoskeletal elements [22]. This association with the detergent-insoluble membrane fraction persisted despite extraction in 200 mM NaCl and 5% Triton X-100 (not shown). An additional demonstration of this membrane cortex targeting was evident in the localization of GFP-tagged p47 phox. To avoid microscopic artifacts from fixation and permeabilization, we viewed ECV-304 cells transiently expressing the p47-GFP fusion protein in situ in viable, intact cells. Transfection efficiencies were 10 –15%. Again, confocal images demonstrated focal membrane association of the protein (Fig. 5c). Lamellar protrusions were common (10 –20% of unstimulated cells), and displayed focal accumulation of p47-GFP in transduced cells. In these images, the overexpressed fusion protein stained both central cytosol and nucleus intensely and diffusely. It is not clear why such a diffuse pattern predominated, although this may in part reflect the distribution of the GFP protein (Fig. 5b, GFP control) or overexpression of p47-GFP with saturation of native binding sites. When stimulated with TNF␣, 10 – 60% of cells displayed prominent membrane ruffles, dynamic curtainlike membrane structures containing a scaffold of polymerized actin. p47-GFP localized strongly to membrane ruffles in all transduced, TNF␣-responding cells examined (Fig. 6). These p47-GFP-staining ruffles could be observed as vertical structures which appeared, moved, and disappeared within a 5- to 10-min time frame. Ruffling was not observed in the absence of TNF␣ stimulation. TNF␣ signaling is cytoskeleton-dependent. Given the association of p47 phox with the cytoskeleton in ECV-

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FIG. 2. Effect of p47W(193)R on JNK activation. (a) ECV-304 cells stably transfected with pTet-on and either empty vector (pTRE), wild-type p47 phox, or p47W(193)R were induced with doxycycline (1 ␮g/ml for 24 h) and treated with TNF␣ (100 ng/ml) for 15 min, and then JNK activity was assessed by IP kinase. Transfection with the wild-type p47 phox had minimal effect on TNF␣-stimulated JNK1 activation compared with empty vector or untransfected (not shown), whereas transfection with p47W(193)R decreased JNK1 activation approximately two- to threefold. Western blot for total JNK1 in the immunoprecipitates is shown below. Histograms represent the mean of four individual experiments. (b) Double stable transfected ECV-304 cells were treated as in (a) except that recombinant proteins were not induced with doxycycline prior to TNF␣ stimulation. TNF␣ activated JNK1 to comparable levels in cells transfected with the empty vector, wild-type p47 phox, and p47W(193)R. Histograms represent the mean of three individual experiments.

304 cells, one might expect disruption of such cytoskeletal elements to interfere with JNK activation. Indeed, cytochalasin B, which arrests new F-actin assembly, greatly diminished TNF␣-induced JNK1 activation (Fig. 7a). In addition, nocodazole and colchicine, which facilitate microtubule disassembly, decreased TNF␣induced JNK1 activation. Although colchicine only decreased JNK1 activation by ⬃35%, the effect was especially striking given the propensity for this agent to cause significant activation of JNK1 by itself (Fig. 7b). The microtubule stabilizer paclitaxel and the microfilament stabilizer jasplakinolide had minimal effects on

TNF␣-induced JNK1 activation. In addition, the cytoskeletal disrupters cytochalasin B, nocodozole, and cochicine abolished TNF␣-stimulated oxidant production (Fig. 7a). In contrast, paclitaxel had no effect and jasplakinolide significantly increased TNF␣-stimulated oxidant production. DISCUSSION

An oxidase similar to the bactericidal phagocyte NADPH oxidase has been postulated to exist in parenchymal cells based on several observations. Roughly

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47- and 67-kDa proteins immunoreactive with antip47 phox and p67 phox antibodies have been reported in immortalized human chondrocytes [32], and appropriately sized RT-PCR products have been detected in HUVEC for p47 phox, p67 phox, p22 phox, and gp91 phox [33], although the identity of these products has not been established. Interestingly, redox difference spectra reveal a cytochrome b 558 , characteristic of the NADPH oxidase, in human fibroblasts, even those from a patient with an inherited deficiency of phagocyte cytochrome b 558 [34], suggesting a genetically distinct cytochrome subunit. Indeed, homologs of the two cytochrome subunits, p22 phox and gp91 phox, have been identified and shown to be expressed in smooth muscle and epithelial cells [12–14]. We found that HUVEC and ECV-304 cells express p47 phox, a gene product whose only known function is to facilitate activation of the NADPH oxidase in phagocytes. Elegant work from a number of laboratories has illuminated some of the initial steps involved in activation of this oxidase in whole or reconstituted phagocyte systems. One of the earliest events appears to be the serine phosphorylation of the C-terminal tail of p47 phox, an event which triggers translocation of the cytosolic complex to the membrane [17]. The proposed mechanism may involve a resting conformation of p47 phox in which its first SH3 domain is occupied by intramolecular binding to a proline-rich motif(s) on the carboxyl tail. Upon activation, this association is disrupted, allowing both the first and the second SH3 domains to bind to membrane-associated p22 phox [30, 31]. An additional arginine-rich region also participates in targeting p47 phox to the cytochrome [35]. Activation of the oxidase appears to occur as a result of the consequent recruitment of p47 phox-associated p67 phox, which may contain an NADPH binding site and/or an activation domain [36]. In phagocytes, p47 phox thus acts as a signal-receiving adapter protein which initiates assembly of the active oxidase. We utilized this phagocyte paradigm to potentially disrupt p47 phox-dependent events in ECV-304 cells by expression of the W(193)R mutant, which in a phagocyte system blocks reconstitution of an active oxidase

FIG. 3. p47 phox is recovered from the particulate fraction of ECV304 cells. (a) Unstimulated ECV-304 cells or cells stimulated with TNF␣ (100 ng/ml for 15 min) were lysed and sonicated, and the postnuclear fraction was separated into the 100,000g particulate (P) and soluble (S) fractions. Equal cell number equivalents were loaded for particulate and soluble fractions. A 48- to 49-kDa protein immunoreactive with antibodies to p47 phox was identified exclusively in the

particulate fractions of both unstimulated and TNF␣-stimulated cells. Representative of three experiments. (b) Human neutrophils were isolated and were either stimulated with PMA (100 nM for 30 min) or left untreated. Particulate (P) and soluble (S) fractions, prepared in parallel with the ECV-304 cell fractions in (a), were immunoblotted for p47 phox, demonstrating translocation of the protein from neutrophil cytosol to membrane fractions. Representative of two experiments. (c) ECV-304 cells were either treated with TNF␣ (100 ng/ml for 15 min) or left unstimulated, and then extracted with Triton X-100 into insoluble (CK) and soluble (S) fractions. A 48- to 50-kDa protein immunoreactive with antibodies to p47 phox was recovered entirely in the detergent-insoluble fraction of both stimulated and unstimulated cells. Representative of two experiments.

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FIG. 4. p47 phox colocalizes with specific cytoskeletal elements. ECV-304 cells were double-stained with antibodies to p47 phox (FITC, all rows) and microtubules (rhodamine anti-␤-tubulin (a– c)), microfilaments (rhodamine–phalloidin (d–f)), or intermediate filaments (rhodamine–antivimentin (g–i)), and confocal images were obtained. (a, d, g) Red channel demonstrating cytoskeletal (CK) elements. (b, e, h) Green channel demonstrating p47 phox localization. Antibodies to p47 phox displayed a reticular and sometimes punctate staining pattern within the cytosolic compartment (b, e, h), which partially colocalized with ␤-tubulin (a– c). In addition, anti-p47 phox also colocalized with the cortical actin-rich cytoskeleton (arrows, (e)). Staining patterns for p47 phox and vimentin differed from each other.

[31]. We found that this latter mutant decreased TNF␣stimulated JNK activation, providing evidence for p47phox in TNF␣ signaling and supporting our inhibitor studies which also implicate an NAD(P)H oxidase in JNK activation. These observations strengthen the notion that reactive oxidants constitute important endogenous signaling intermediates. The potential targets of such oxi-

dants have not been established, although it is noteworthy that O 2⫺• can reversibly inhibit protein tyrosine phosphatase 1B (PTP-1B) [37], thus facilitating tyrosine kinase-dependent signaling. The cytoskeleton-dependent tethering of a regulatory phosphatase to a kinase upstream of JNK would similarly explain our observations that destabilization of the cytoskeleton with nocodozole

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FIG. 5. p47 phox is associated with the membrane skeleton. (a) ECV-304 cells were fractionated into high-speed supernatant (cytosolic, Cyto) and membrane fractions. The membrane fraction was further subjected to Triton X-100 extraction to separate the membrane cortex (Memb cortex) and soluble membrane (Memb sol) fractions. p47 phox was recovered solely from the membrane cortex fraction. Representative of three experiments. (b, c) ECV-304 cells were transiently transfected with a p47-GFP fusion construct or the pEGFP plasmid alone. Images obtained in living cells show diffuse cytosolic and nuclear staining of the GFP control (b) and focal membrane-associated staining of p47-GFP (c).

or colchicine appeared to activate JNK. Another potential oxidant-sensitive target has been proposed to be ASK1. In addition to causing dimerization and consequent en-

hancement of ASK1 activity, oxidants also appear to oxidize thioredoxin, causing release of ASK1 in an active form [5, 38].

FIG. 6. Localization of p47-GFP to membrane ruffles. ECV-304 cells transfected with p47-GFP were stimulated with TNF␣ (100 ng/ml). (a, b) The same cell is imaged at apical (a) and basal (b) focal planes, demonstrating vertical nature of the targeted structures. (c) DIC image of a TNF␣-treated ECV-304 cell shows leading edge ruffles. (d) Epifluorescence of same field as (c) demonstrates targeting of p47-GFP to edge ruffles/lamellae.

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FIG. 7. Activation of JNK by TNF␣ is cytoskeleton-dependent. (a) JNK1 activity is shown. Pretreatment of ECV-304 cells for 60 min with cytochalasin B (CB, 10 ␮M), nocodazole (Noc, 10 ␮M), or colchicine (Col, 10 ␮M) decreased both oxidant production (upper histogram) and JNK1 activation (lower histogram) by TNF␣ (100 ng/ml for 15 min), whereas paclitaxel (Pac, 10 ␮M) and jasplakinolide (Jas, 5 ␮M) had minimal or no effects. (b) ECV-304 cells were treated with the cytoskeletal-modifying agents for 60 min as above, without TNF␣ stimulation. Nocodazole and colchicine alone increased JNK1 activity approximately three- to fourfold from baseline. Histograms represent the mean of two to three individual experiments.

Although phagocyte p47 phox is largely cytosolic and a minor fraction translocates to the membrane only upon stimulation, we found that native p47 phox in ECV-304 cells is exclusively associated with the particulate fraction. This finding suggests that the oxidase may exist as a cytoskeleton-associated complex, and is consistent with observations that NAD(P)H-driven O 2⫺• production is collected in the particulate fraction of aortic preparations [39, 40]. It is possible that the oxidase may instead be associated with Triton X-insoluble membrane microdomains such as caveolae; however, the colocalization studies suggest that at least p47 phox is associated with actin- and/or tubulin-binding proteins. Although ostensibly at odds with the phagocyte paradigm, there does appear to be a clear association of the active NADPH oxidase with the phagocyte cytoskele-

ton. Neutrophil p40 phox, for example, associates with the actin-binding protein coronin [41]. Further, upon stimulation of neutrophils the most heavily phosphorylated forms of p47 phox are recovered from the cortical cytoskeleton and are associated with active oxidase [22, 42]. Interestingly, phorbol ester-stimulated neutrophils develop ruffled membranes which contain large amounts of H 2O 2 [43]. Thus, our finding of p47 phox in the cortical cytoskeleton and membrane ruffles of stimulated ECV-304 cells may reflect a targeting of the oxidase in parenchymal cells similar to that in phagocytes. The mechanism of cytoskeletal association is not understood, although it is clear that p47 phox contains a number of functional binding sites, the partners of which have not been identified [44]. The localization of p47 phox to the cytoskeleton also

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emphasizes the potential of an associated oxidase to affect cell signaling events. An increasing number of signaling factors are being found to congregate around cytoskeletal elements, suggesting that they may act as a solid scaffolding matrix which increases both efficiency and specificity of signals. JNK itself, as well as factors upstream of JNK such as the guanyl nucleotide exchange factors Lfc and frabin, the MAPKK MKK4, and the MAPKKKs MEKK1 and MLK2, associate with both microtubules and microfilaments [45– 49]. Accordingly, we found that disruption of both microtubular and actin cytoskeletons disrupted JNK activation by TNF␣. In parallel, we also found that microfilament and microtubule disrupters abolished TNF␣-stimulated oxidant production, suggesting that the cytoskeleton may serve as a scaffold for oxidase assembly and/or is necessary for linkage of the oxidase with upstream signaling events initiated by ligation of the TNF␣ receptor(s). In support of the latter possibility, others have noted that the importance of the actin scaffold for the respiratory burst of phagocytes is stimulus-dependent: indeed, cytochalasin B inhibits TNF␣but augments fMLP-stimulated O 2⫺• production by mature neutrophils [50]. We conclude that nonphagocytic cells express p47 phox, an important adapter protein regulating oxidase activity, and that activation of JNK by TNF␣ is driven at least in part by this oxidase. A key biochemical property of oxidants is their transience, which makes them well suited to transmit rapid and self-limited signals. Specific local targeting of a signaling oxidase would seem paramount to promote signal efficiency and diminish potentially damaging effects of misdirected oxidants. Parenchymal cells may possess an oxidase which is both rapidly activatable and site-directed, two potentially important characteristics of a signaling oxidase. The authors acknowledge Drs. Paul Heyworth and Bernard Babior for helpful comments and for antibodies. This work was supported by the NIH (R29-HL52591, R01-HL61897), The American Heart Association, The Veterans Administration, and The Glaxo Foundation. Dr. Terada is an Established Investigator of the American Heart Association.

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