of February 19, 2018.This information is current as

Cyclooxygenase-2 Expression-InducedαActivation Is Involved in TNF-

by Protein Kinase C-Dependent c-Src β/αB Kinase κTyrosine Phosphorylation of I-

Ching-Chow ChenWei-Chien Huang, Jun-Jie Chen, Hiroyasu Inoue and

http://www.jimmunol.org/content/170/9/4767doi: 10.4049/jimmunol.170.9.4767

2003; 170:4767-4775; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/170/9/4767.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2003 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Tyrosine Phosphorylation of I-�B Kinase �/� by ProteinKinase C-Dependent c-Src Activation Is Involved inTNF-�-Induced Cyclooxygenase-2 Expression1

Wei-Chien Huang,* Jun-Jie Chen,* Hiroyasu Inoue,† and Ching-Chow Chen2*

The signaling pathway involved in TNF-�-induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or aSrc kinase inhibitor (PP2) attenuated TNF-�- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity.TNF-�- or TPA-induced I-�B kinase (IKK) activation was also blocked by these inhibitors, which reversed I-�B� degradation.Activation of c-Src and Lyn kinases, two Src family members, was inhibited by the PKC, tyrosine kinase, or Src kinase inhibitors.The dominant-negative c-Src (KM) mutant inhibited induction of COX-2 promoter activity by TNF- � or TPA. Overexpression ofthe constitutively active PKC� (PKC� A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects wereinhibited by the dominant-negative c-Src (KM), NF-�B-inducing kinase (NIK) (KA), or IKK � (KM) mutant. The dominant-negative PKC� (K/R) or c-Src (KM) mutant failed to block induction of COX-2 promoter activity caused by wild-type NIKoverexpression. In coimmunoprecipitation experiments, IKK�/� was found to be associated with c-Src and to be phosphorylatedon its tyrosine residues after TNF-� or TPA treatment. Two tyrosine residues, Tyr188 and Tyr199, near the activation loop ofIKK �, were identified to be crucial for NF-�B activation. Substitution of these residues with phenylalanines attenuated COX-2promoter activity and c-Src-dependent phosphorylation of IKK� induced by TNF-� or TPA. These data suggest that, in additionto activating NIK, TNF- � also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK andconverge at IKK�/�, and go on to activate NF-�B, via serine phosphorylation and degradation of I�B-�, and, finally, to initiateCOX-2 expression. The Journal of Immunology, 2003, 170: 4767–4775.

P rostaglandins play important roles in many biological pro-cesses, including cell division, blood pressure regulation,immune responses, ovulation, bone development, and wa-

ter balance. Altered prostanoid production is associated with a va-riety of illnesses, including acute and chronic inflammation, car-diovascular disease, colon cancer, and allergic diseases (1, 2).Cyclooxygenase (COX),3 also known as PG synthase, is the keyenzyme in PG, prostacyclin, and thromboxane synthesis from ar-achidonic acid (1). COX converts arachidonic acid, released frommembrane phospholipid stores by phospholipases, to PGH2, thecommon precursor of all prostanoids. Two identified COX iso-forms, COX-1 and COX-2, are encoded by separated genes (3–5).COX-1, constitutively expressed in most human tissues (6), ap-pears to be responsible for the production of PGs that mediatenormal physiological functions, such as maintenance of integrityof gastric mucosa and regulation of renal blood flow (7, 8). Incontrast, COX-2 is induced by a wide range of mitogenic andinflammatory stimuli in many distinct cell types, such as activated

marcrophages, monocytes, endothelial cells, fibroblasts, and ovar-ian follicles (9–12), and has been identified in chronic inflammatoryconditions in vivo (13). It is implicated in physiological processes,such as ovulation and delivery (14), and in pathological states, such ascolorectal cancer, Alzheimer’s disease, heart failure, and even hyper-tension (15–18). Much evidence suggests that COX-2 is an importanttherapeutic target for prevention and treatment of arthritis and cancer.Reducing the levels of COX-2 will be an effective strategy for re-pressing inflammation and carcinogenesis (19, 20); to develop an ef-fective approach, however, it is important to define the signalingmechanism that governs COX-2 expression.

The induction of COX-2 expression requires de novo mRNAand protein synthesis (21), indicating regulation at the transcrip-tional level. The promoter region of human COX-2 gene has beencloned and sequenced, and shown to contain putative recognitionsequences for a variety of transcriptional factors, including NF-�B,NF-IL-6, and cAMP response element (22). Of these, NF-�B fam-ily proteins are the essential components for the enhanced COX-2gene expression seen on exposure to cytokines in human alveolarepithelial cells (23, 24). The rationale to study COX-2 gene ex-pression and the accompanying signaling pathway in alveolar ep-ithelial cells is that these cells play an active role in inflammationby producing various cytokines that are involved in the late asth-matic response, as previously stated (25). The intracellular signal-ing pathways by which TNF-� causes COX-2 mRNA and proteinexpression in human alveolar epithelial cells have been explored,including sequential activation of protein kinase C (PKC) �, tyrosinekinase, NF-�B-inducing kinase (NIK), and I-�B kinase (IKK) �/�(23, 25). The role of tyrosine kinase has been further investigated inthe present study. Using an immunocomplex kinase assay and site-directed mutagenesis, we have demonstrated that c-Src is involvedin TNF-�-inducing NF-�B transcriptional activation, and that, in

*Department of Pharmacology, College of Medicine, National Taiwan University,Taipei, Taiwan; and †Department of Pharmacology, National Cardiovascular CenterResearch Institute, Osaka, Japan

Received for publication October 29, 2002. Accepted for publication March 6, 2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a research grant from the National Science Council ofTaiwan.2 Address correspondence and reprint requests to Dr. Ching-Chow Chen, Departmentof Pharmacology, College of Medicine, National Taiwan University No.1, Jen-AiRoad, 1st Section Taipei 10018, Taiwan. E-mail address: ccchen@ha.mc.ntu.edu.tw3 Abbreviations used in this paper: COX, cyclooxygenase; IKK, I-�B kinase; NIK,NF-�B-inducing kinase; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRAF, TNFR-associated factor; wt, wild type.

The Journal of Immunology

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00

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addition to serine phosphorylation of IKK�/� by NIK, Tyr188 andTyr199 phosphorylations of IKK� by PKC-dependent c-Src acti-vation also contribute to TNF-�-induced COX-2 expression.

Materials and MethodsMaterials

Rabbit polyclonal Abs specific for I-�B�, IKK�, c-Src, and Lyn werepurchased from Santa Cruz Biotechnology (Santa Cruz, CA), and rabbitpolyclonal anti-phosphotyrosine Ab was purchased from Upstate Biotech-nology (Lake Placid, NY). Human rTNF-� was purchased from R&D Sys-tems (Minneapolis, MN). The 12-O-tetradecanoylphorbol-13-acetate(TPA) was purchased from L.C. Service (Worburn, MA). RPMI 1640 me-dium, FCS, penicillin, and streptomycin were obtained from Life Technol-ogies (Gaithersburg, MD). Staurosporine, glutathione-agarose beads, andprotein A-Sepharose were obtained from Sigma-Aldrich (St. Louis, MO).Herbimycin A and PP2 were obtained from Calbiochem (San Diego, CA).HRP-labeled donkey anti-rabbit second Ab and the ECL detecting reagentwere obtained from Pharmacia Biotech (Uppsala, Sweden). [�-32P]ATP(3000 Ci/mmol) was obtained from DuPont-New England Nuclear (Bos-ton, MA). The luciferase assay kit was obtained from Promega (Madison,MA). SuperFect and plasmid purification and DNA recovery kits wereobtained from Qiagen (Chatsworth, CA). The Quickchange mutagenesiskit was obtained from Stratagene (La Jolla, CA). EcoRI, XboI, and SalIrestriction enzymes and T4 DNA ligase were obtained from NEB(Beverly, MA).

Cell culture

The human alveolar epithelial cell carcinoma cell line, NCI-H292, wasobtained from the American Type Culture Collection (Manassas, VA) andcultured in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin,and 100 �g/ml streptomycin in six-well plates for transfection experiments,in 6-cm dishes for IKK, c-Src, or Lyn kinase activity measurements andWestern blot analysis, or in 10-cm dishes for coimmunoprecipitation tests.

Plasmids

The COX-2 promoter construct pGS459 (�459/�9) was a generous giftfrom L. H. Wang (University of Texas, Houston, TX). The �B-luc plasmidwas from Stratagene. The PKC-� constitutively active (PKC-�/AE) ordominant-negative mutant (PKC�/KR) was provided by A. Altman (LaJolla Institute for Allergy and Immunology, San Diego, CA). The wild-type(wt) and dominant-negative mutants of NIK and IKK� (NIK wt and mutantKA; IKK� wt and mutant KM) were gifts from Signal Pharmaceuticals(San Diego, CA). The dominant-negative mutant of IKK� (AA) was fromM. Karin (University of California, San Diego, CA). pGEX-I-�B� (1–100)was a gift from H. Nakano (University of Juntendo, Tokyo, Japan). pGEX-IKK� (132–206) was a gift from M. Nakanishi (University of Nagoya,Nagoya, Japan).

Immunoprecipitation and kinase activity assay

Following treatment with TNF-� or TPA, with or without 30-min pretreatmentwith PKC, tyrosine kinase, or Src kinase inhibitors, the cells were rapidlywashed with PBS and lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH7.4, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 5 �g/mlof leupeptin, 20 �g/ml of aprotinin, 1 mM NaF, and 1 mM Na3VO4), thenIKK, c-Src, or Lyn was immunoprecipitated. For the in vitro kinase assay,100 �g of total cell extract was incubated for 1 h at 4°C with 0.5 �g ofrabbit anti-IKK�, anti-c-Src, or anti-Lyn Ab, then protein A-SepharoseCL-4B beads (Sigma-Aldrich) were added to the mixture, and incubationwas continued for 4 h at 4°C. The immunoprecipitates were collected bycentrifugation, washed three times with lysis buffer without Triton X-100,then incubated for 30 min at 30°C in 20 �l of kinase reaction mixture (20mM HEPES, pH 7.4, 5 mM MgCl2, 5 mM MnCl2, 0.1 mM Na3VO4, 1 mMDTT) containing 10 �M [�-32P]ATP and either 1 �g of bacterially ex-pressed GST-I-�B� (1–100) as IKK substrate, or 1 �g of acidic denaturedenolase as c-Src or Lyn substrate, or 6 �g of bacterially expressed GST-IKK� (132–206), GST-IKK� (132–206) (Y188F), GST-IKK� (132–206)(Y199F), or GST-IKK� (132–206) (Y188F; Y199F) as c-Src substrate.The reaction was stopped by addition of an equal volume of Laemmlibuffer, the proteins were separated by electrophoresis on 10% SDS poly-acrylamide gels, and phosphorylated GST-I-�B� (1–100), phosphorylatedGST-IKK� (132–206), or phosphorylated enolase visualized by autora-diography. Quantitative data were obtained using a densitometer with Im-ageQuant software and normalized by the protein expression.

Western blot analysis

Following treatment with TNF-� or TPA, total or immunoprecipitated celllysates were prepared and subjected to SDS-PAGE using 7.5% runninggels, as described previously (23). The proteins were transferred to a ni-trocellulose membrane, which was then incubated successively at roomtemperature for 1 h with 0.1% milk in TTBS (50 mM Tris-HCl, pH 7.5,0.15 M NaCl, and 0.05% Tween 20) for 1 h with rabbit Ab specific forIKK�, IKK�, I-�B�, c-Src, or Lyn, and for 30 min with HRP-labeledanti-rabbit Ab. After each incubation, the membrane was washed exten-sively with TTBS. The immunoreactive bands were detected using ECLdetection reagent and Hyperfilm-ECL (Amersham, Arlington Heights, IL).Quantitative data were obtained using a computing densitometer and Im-ageQuant software (Molecular Dynamics, Sunnyvale, CA).

Site-directed mutagenesis

Using a Quickchange site-directed mutagenesis kit, according to the man-ufacturer’s manual, lysine (K) 295 in the mouse c-Src cloned in the pBlue-script vector was substituted with methionine (M) by changing the tripletsfrom AAG to ATG. Tyrosine (Y) 199, tyrosine 188, or both sites in thehuman IKK� cloned in the pcDNA3.1 vector, or in the human GST-IKK�(132–206) cloned in the pGEX vector was substituted with phenylalanine(F) by changing the triplet from TAC to TTC. The mutated primers usedwere primer 1 (5�-CGAGGGTTGCCATCATGACTCTGAAGCCAGGCA-3�) and primer 2 (3�-GCTCCCAACGGTAGTACTGAGACTTCGGTCCGT-5�) for c-Src (K295 M) mutation, primer 3 (5�-GGGGACCCTGCAGTTCCTGGCCCCAGAGC-3�), primer 4 (3�-CCCCTGGGACGTCAAGGACCGGGGTCTCG-5�) for IKK� (Y188F) mutation, primer 5 (5�-GGAGCAGCAGAAGTTCACAGTGACCGTCG-3�), and primer 6 (3�-CCTCGTCGTCTTCAAGTGTCACTGGCAGC-5�) for IKK� (Y199F) mutation, asdescribed previously (26).

Transient transfection and luciferase assay

NCI-H292 cells, grown to 60% confluent in six-well plates, were trans-fected with the human COX-2 pGS �459/�9, �327/�59, or KBM/firelyluciferase (Luc) plasmid using SuperFect (Qiagen), according to the man-ufacturer’s recommendations. Briefly, reporter DNA (1 �g) and �-galac-tosidase DNA (0.5 �g; plasmid pRK containing the �-galactosidase genedriven by the constitutively active SV40 promoter, used to normalize thetransfection efficiency) were mixed with 0.75 �l of SuperFect in 0.9 ml ofserum-free RPMI 1640. After 10- to 15-min incubation at room temperature,the mixture was applied to the cells, then 8 h later, 0.1 ml of FCS was added.Twenty-four hours after transfection, the cells were treated with inhibitors (asindicated) for 30 min, then TNF-� or TPA was added for 6 h. Cell extractswere then prepared, and luciferase and �-galactosidase activities were mea-sured, the luciferase activity being normalized to the �-galactosidase activity.In experiments using dominant-negative mutants, cells were cotransfectedwith reporter (0.5 �g) and �-galactosidase (0.25 �g) and either the dominant-negative NIK, IKK�, or c-Src mutant or the empty vector (1.0 �g).

In experiments using wt plasmids, cells were cotransfected with 0.5 �gof reporter plasmid, 0.25 �g of �-galactosidase plasmid, 1 �g of the con-stitutively active PKC� (A/E) plasmid, wt c-Src or NIK plasmid or emptyvector, and 1.5 �g of the dominant-negative NIK, IKK�, or c-Src mutantor empty vector.

Coimmunoprecipitation assay

Cell lysates containing 1 mg of protein were incubated for 1 h at 4°C with2 �g of anti-IKK�, anti-IKK�, or anti-c-Src Ab, or with 4 �g of anti-phosphotyrosine Ab, then 50 �l of 50% protein A-agarose beads wereadded and mixed for 16 h at 4°C. The immunoprecipitates were collectedand washed three times with lysis buffer without Triton X-100, then Lae-mmli buffer was added and the samples were subjected to electrophoresison 10% SDS polyacrylamide gels. Western blot analysis was performed, asdescribed above, using Abs against phosphotyrosine, IKK�, IKK�, or c-Src.

ResultsEffect of inhibitors of PKC, tyrosine kinase, or Src kinase on theinduction of COX-2 promoter activity by TNF-� or TPA in NCI-H292 cells

To further confirm NF-�B in the regulation of COX-2 expression(23), the COX-2 promoter-luciferase construct, �327/�59 or �Bsite (�223/�214) deletion mutant (KBM) (27), was transfectedinto NCI-H292 cells. As shown in Fig. 1A, TNF-� and TPA in-duced 3.6- and 4.3-fold increase, respectively, in COX-2 promoteractivity using �326/�59 construct. These effects were abolished

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using KBM plasmid, indicating the requirement of NF-�B in theregulation of COX-2 expression. In our previous study (23), wefound that PKC and tyrosine kinase were involved in TNF-�-in-duced COX-2 expression. Transient transfection using the COX-2promoter-luciferase construct, pGS459 (�459/�9), was per-formed to elucidate the signaling pathway mediated by these ki-nases. The pGS459 construct contains both upstream (�447/�438) and downstream (�223/�214) NF-�B site responsible formediating the induction of COX-2 promoter activity by TNF-� orTPA (23) (Fig. 1A). As shown in Fig. 1B, TNF-� led to a 3.1-foldincrease in COX-2 promoter activity. When cells were pretreatedwith inhibitors of PKC (staurosporine), tyrosine kinases (genistein orherbimycin A), or Src kinases (PP2), the TNF-�-induced increase wasinhibited by 72, 90, 94, or 89%, respectively. TPA, a direct PKCactivator, resulted in a 5.3-fold increase in COX-2 promoter activity,and this effect was inhibited by genistein, herbimycin A, or PP2 by 58,86, or 95%, respectively. None of these inhibitors alone affected thebasal luciferase activity (data not shown).

Induction of IKK activation and I-�B� degradation by TNF-�and TPA, and the inhibitory effect of inhibitors of PKC, tyrosinekinase, or Src kinase

Because TNF-�- and TPA-induced COX-2 promoter activity inNCI-H292 cells is inhibited by the dominant-negative IKK� and

IKK� mutants (23), endogenous IKK activity was measured byimmunoprecipitation with anti-IKK� Ab. When cells were treatedwith 30 ng/ml of TNF-� for 5, 10, 30, or 60 min, maximal IKKactivity was seen after 10 min (Fig. 2A), which was paralled withmaximal degradation of I-�B� after 10 min. I-�B� level was re-stored to the resting level after 1 h of treatment (Fig. 2B). In TPA-treated cells, maximal IKK activity was seen after 5 min of treat-ment and sustained for 60 min (Fig. 2A), and maximal I-�B�degradation was seen after 60 min (Fig. 2B). The TNF-�-in-duced IKK activation was inhibited by PKC, tyrosine kinase, orSrc kinase inhibitors by 78, 99, or 74%, respectively, while stau-rosporine, herbimycin A, or PP2 suppressed TPA-induced IKK

FIGURE 2. Kinetics of TNF-�-induced IKK activation and I-�B� deg-radation. NCI-H292 cells were treated with 30 ng/ml of TNF-� or 1 �MTPA for 5, 10, 30, or 60 min, then cell lysates were prepared. A, Celllysates were immunoprecipitated with anti-IKK� Ab, then the kinase assayand autoradiography for phosphorylated GST-I-�B� (1–100) were per-formed on the precipitates, as described in Materials and Methods. Levelsof immunoprecipitated IKK� protein were estimated by Western blotting(W.B.) using anti-IKK� Ab. B, Cytosolic levels of I-�B� were measuredusing anti-I-�B� Ab, as described in Materials and Methods.

FIGURE 3. Effect of various inhibitors on TNF-�- or TPA-inducedIKK activity and I-�B� degradation in NCI-H292 cells. Cells were pre-treated for 30 min with 300 nM staurosporine, 1 �M herbimycin A, or 10�M PP2 before incubation with 30 ng/ml of TNF-� for 10 min or 1 �MTPA for 30 min, then whole cell lysates were prepared. A, Whole celllysates were immunoprecipitated with anti-IKK� Ab, and the kinase assayand autoradiography for phosphorylated GST-I�B� (1–100) were per-formed on the precipitates, as described in Materials and Methods. Levelsof immunoprecipitated IKK� were estimated by Western blotting (W.B.)using anti-IKK� Ab. B, Cytosolic levels of I-�B� were measured by West-ern blotting using anti-I-�B� Ab, as described in Materials and Methods.

FIGURE 1. Effect of various inhibitors on TNF-�- or TPA-inducedCOX-2 promoter activity in NCI-H292 cells. A, Cells were transfected with�327/�59 or KBM luciferase expression vector, then treated with 30ng/ml of TNF-� or 1 �M TPA for 6 h. B, Cells were transfected withpGS459 luciferase expression vector, then pretreated for 30 min with ve-hicle, 300 nM staurosporine, 30 �M genistein, 1 �M herbimycin A, or 10�M PP2 before incubation for 6 h with 30 ng/ml of TNF-� or 1 �M TPA.Luciferase activity was then measured, as described in Materials and Meth-ods, normalized to the �-galactosidase activity, and expressed as themean � SEM of three independent experiments performed in triplicate. �,p � 0.05; ��, p � 0.01 compared with TNF-� or TPA alone.

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activation by 85, 78, or 89%, respectively (Fig. 3A). The I-�B�degradation induced by TNF-� or TPA was reversed by PKC,tyrosine kinase, or Src kinase inhibitors (Fig. 3B).

Induction of c-Src and Lyn activation by TNF-� and TPA, andthe inhibitory effect of inhibitors of PKC, tyrosine kinase, or Srckinase

TNF-�- or TPA-induced IKK activation was inhibited by PKC,tyrosine kinase, and Src kinase inhibitors, indicating the in-volvement of tyrosine kinase, or at least the Src family, down-stream of PKC in the induction of IKK activation. Our previousdata showed that in contrast to other members of Src family,c-Src and Lyn were abundantly expressed in NCI-H292 cell andanother human alveolar epithelial A549 cell (26). c-Src and Lynin NCI-H292 cells were therefore isolated by immunoprecipi-tation using anti-c-Src or anti-Lyn Ab, and their in vitro kinaseactivity was measured using enolase as substrate. As shown inFig. 4A, when NCI-H292 cells were treated with 30 ng/ml ofTNF-� for 10, 30, or 60 min, maximal c-Src and Lyn activity(enolase phosphorylation) was seen after 10 min and began tobe declined after 60 or 30 min, respectively. The TNF-�- andTPA-induced activations of c-Src and Lyn were inhibited bystaurosporine, herbimycin A, and PP2 (Fig. 4B).

FIGURE 4. Effect of various inhibitors on TNF-�- or TPA-induced c-Srcor Lyn activation in NCI-H292 epithelial cells. Cells were treated with 30ng/ml of TNF-� for 10, 30, or 60 min (A) or pretreated for 30 min with vehicle,300 nM staurosporine, 1 �M herbimycin A, or 10 �M PP2 before incubationfor 10 min with 30 ng/ml of TNF-� or 30 min with 1 �M TPA (B). Whole celllysates were prepared and immunoprecipitated with anti-c-Src or anti-Lyn Ab.The kinase assay and autoradiography for phosphorylated enolase were per-formed on the precipitates, as described in Materials and Methods. Levels ofimmunoprecipitated c-Src or Lyn were estimated by Western blotting (W.B.)using anti-c-Src or anti-Lyn Ab, respectively.

FIGURE 5. Effect of dominant-negative mutants on TNF-�- or TPA-induced COX-2 promoter activity in NCI-H292 cells. Cells were cotrans-fected with pGS459 and the dominant-negative c-Src (KM) or NIK (KA)mutant or empty vector, then treated for 6 h with 30 ng/ml of TNF-� or 1�M TPA. Luciferase activity was then measured, as described in Materialsand Methods, and the results were normalized to the �-galactosidase ac-tivity and expressed as the mean � SEM of three independent experimentsperformed in triplicate. ��, p � 0.05 compared with TNF-� or TPA alone.

FIGURE 6. Effect of various dominant-negative mutants on wt plas-mid-induced COX-2 promoter activity. A, NCI-H292 cells were co-transfected with pGS459 and the constitutively active form of PKC�(A/E), wt c-Src, NIK, or IKK� or empty vector. B, NCI-H292 cellswere cotransfected for 24 h with PKC� (A/E), wt c-Src or NIK andPKC� (K/R), c-Src (K295 M), IKK� (KM), or NIK (KA). Luciferaseactivity was then assayed, as described in Materials and Methods, andthe results were normalized to the �-galactosidase activity and ex-pressed as the mean � SEM of three independent experiments per-formed in triplicate. �, p � 0.05; ��, p � 0.01 compared with thecontrol vector.

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Induction of COX-2 promoter activity by overexpression ofPKC� or c-Src and the inhibitory effect of dominant-negativemutants of NIK, c-Src, or IKK�

Because the TNF-�- or TPA-induced activation of c-Src and Lynwas inhibited by PKC, tyrosine kinase, or Src kinase inhibitors,this indicated that PKC-dependent c-Src and Lyn activation wasrequired to induce IKK and NF-�B activation in NCI-H292 cells.To further examine the involvement of c-Src, overexpression ofc-Src (KM) attenuated the TNF-�- or TPA-induced COX-2 pro-moter activity (Fig. 5). The TNF-�- or TPA-induced COX-2 pro-moter activity was also inhibited by the dominant-negative NIK(KA) mutant (Fig. 5), as previously reported (23). To characterizethe relationship between PKC, c-Src, NIK, and IKK�/�, overex-pression of the constitutively active form of PKC� (A/E) or wt ofc-Src, NIK, or IKK� was performed. Overexpression of PKC�(A/E) or wt c-Src, NIK, or IKK� significantly increased COX-2promoter activity by 11-, 9.4-, 9.9-, or 5.6-fold, respectively (Fig.6A). The COX-2 promoter activity induced by overexpression ofPKC� (A/E) or c-Src wt was inhibited by the dominant-negativec-Src (KM), NIK (KA), or IKK� (KM) mutant; however, thatinduced by wt c-Src was not attenuated by the dominant-negativePKC� (K/R) mutant. The dominant-negative IKK� (KM) mutant,but not the PKC� (K/R) or c-Src (KM) mutant, attenuated thepromoter activity induced by overexpression of wt NIK (Fig. 6B).These results indicate that PKC/c-Src/IKK�, NIK/IKK�, and

PKC�/c-Src/NIK/IKK� pathways were involved in TNF-�-induced COX-2 expression in NCI-H292 cells.

Induction by TNF-� or TPA of tyrosine phosphorylation ofIKK�/� and of the c-Src and IKK�/� association, and theinhibitory effect of PP2

Because c-Src-dependent IKK activation was shown to be in-volved, coimmunoprecipitation of c-Src and IKK�/� was per-formed to examine whether c-Src directly regulates IKK activitythrough phosphorylation of tyrosine residues. When cells weretreated with TNF-� for 5, 10, or 15 min, IKK� was tyrosine phos-phorylated in a time-dependent manner, the maximal effect beingseen at 15 min; a similar effect was seen after 30-min treatmentwith TPA (Fig. 7A). Both effects were inhibited by PP2 (Fig. 7A).To demonstrate that c-Src associated with IKK�/� and phosphor-ylated their tyrosine residues, cell lysates were immunoprecipi-tated with anti-IKK� or anti-IKK� Abs, then the immunoprecipi-tates were separated by SDS-PAGE, transferred to membranes,and blotted with anti-phosphotyrosine Abs. As shown in Fig. 7, Band C, tyrosine phosphorylation of IKK� or IKK� was seen afterTNF-� or TPA treatment, the effect being maximal at 10 or 30min, respectively, and inhibited by PP2. These results indicate thatc-Src can directly/indirectly associate with IKK�/� and phosphor-ylate their tyrosine residues after TNF-� or TPA stimulation. Thedirect/indirect association between c-Src and IKK�/� was furtherexamined. Anti-IKK� or anti-IKK� Ab was used to precipitateIKK from NCI-H292 cells, then the immunoprecipitated proteinswere subjected to Western blotting using anti-c-Src Ab. As shownin Fig. 8, A and B, an increased amount of c-Src coprecipitated

FIGURE 7. Tyrosine phosphorylation of IKK� or IKK� induced byTNF-� or TPA and the inhibitory effect of PP2. Control cells or cellspretreated for 30 min with 10 �M PP2 were stimulated with TNF-� for 5,10, or 15 min or with TPA for 10 or 30 min. A, Crude lysates were pre-pared. B and C, Equal amounts (1 mg) of cell lysate were immunoprecipi-tated (IP) with anti-IKK� (A) or anti-IKK� (B) Abs. Crude lysates andimmunoprecipitated proteins were separated by SDS-PAGE on a 10% gel,immunoblotted (WB) with anti-phosphotyrosine (PY) (A, B, and C), andreprobed with anti-IKK� (A, B) or anti-IKK� (C) Abs.

FIGURE 8. c-Src coimmunoprecipitates with IKK� or IKK� afterTNF-� or TPA treatment. NCI-H292 cells were treated with TNF-� for 5,10, or 15 min or with TPA for 10 or 30 min. Equal amounts (1 mg) of celllysate were immunoprecipitated (IP) with anti-IKK� (A) or anti-IKK� (B)Abs. Immunoprecipitated proteins were separated by SDS-PAGE on a 10%gel and immunoblotted (WB) with anti-c-Src or reprobed with anti-IKK�(A) or IKK� (B) Abs. C, Subdomains VII and VIII of the kinase domainsof PKC�, Akt1, and IKK�/� are aligned.

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with IKK� or IKK� after TNF-� or TPA stimulation. These re-sults show a direct/indirect association between c-Src and IKK�/�and that IKK�/� tyrosine residues were phosphorylated.

Inhibitory effect of the dominant-negative mutants, IKK�(Y188F), IKK� (Y199F), or IKK� (FF), on TNF-�- and TPA-induced COX-2 promoter activity and on the PKC�- and c-Src-induced increase in NF-�B activity

The above experiments demonstrated that c-Src could directly as-sociate with IKK�/� and phosphorylate its tyrosine residues afterTNF-� or TPA stimulation. When the amino sequences of subdo-main VII and VIII in the kinase domain of PKC�, AKT1, andIKK�/� were aligned, the tyrosine residues were found to be con-served (Fig. 8C). Assuming that Tyr188 and/or Tyr199 of IKK�were the targets for c-Src phosphorylation after TNF-� or TPAstimulation, we used site-directed mutagenesis to generate thedominant-negative tyrosine mutants, IKK� (Y188F), IKK�(Y199F), and IKK� (Y188F, Y199F) (26). Overexpression ofthese mutants attenuated the TNF-�- or TPA-induced COX-2 pro-moter activity (Fig. 9A). The dominant-negative IKK� (KM) mu-tant, with Lys44 mutated to methionine, had a similar inhibitoryeffect to IKK� (Y188F) or IKK� (Y199F) on TNF-�- and TPA-induced COX-2 promoter activity, while IKK� (AA), with Ser177

and Ser181 mutated to alanine, was as effective as IKK� (Y188F)

or IKK� (Y199F) in inhibiting TNF-�-induced COX-2 promoteractivity, but had less inhibitory effect on TPA-induced COX-2 pro-moter activity (Fig. 9A).

To further confirm the involvement of tyrosine phosphorylationin PKC�/c-Src/IKK� pathway and serine phosphorylation in NIK/IKK� or PKC�/c-Src/NIK/IKK� pathway, the dominant-negativeIKK� mutants with either a tyrosine or serine mutation were co-transfected with PKC� (A/E), wt c-Src, or wt NIK to examine theirinhibitory effects on the constitutively active or wt plasmid-in-duced NF-�B activity. As shown in Fig. 9B, PKC� (A/E)- or wtc-Src-induced NF-�B activity was inhibited by all three tyrosinemutants, but less extent of inhibition by the double serine mutantwas seen. The wt NIK-induced NF-�B activity was inhibited bythe double serine mutant, but not by the three tyrosine mutants(Fig. 9B).

Because Tyr188 and Tyr199 in IKK� were found to be critical forthe PKC�/c-Src/IKK� pathway to elicit NF-�B activation, leadingto induction of TNF-�- or TPA-stimulated COX-2 promoter ac-tivity (Fig. 9), endogenous c-Src-depdendent phosphorylation ofTyr188 and Tyr199 in IKK� was further examined. c-Src was im-munoprecipitated using anti-c-Src Ab and its ability to phosphor-ylate IKK� measured using GST-IKK� (132–206) as an in vitrosubstrate. When cells were treated with TNF-�, IKK� was phos-phorylated by c-Src in a time-dependent manner. The maximal

FIGURE 9. Effect of the dominant-negativetyrosine mutants, IKK� (Y188F), IKK�(Y199F), and IKK� (FF), on TNF-�- or TPA-induced COX-2 promoter activity and on wtplasmid-induced NF-�B activity. In A, NCI-H292 cells were cotransfected with pGS-459plus one of the dominant-negative tyrosine mu-tants (IKK� (188F), IKK� (Y199F), or IKK�(FF)), dominant-negative mutant (IKK� (KM)),or dominant-negative serine mutant (IKK�(AA)), or the respective empty vector, thentreated with 30 ng/ml of TNF-� or 1 �M TPAfor 6 h. B, NCI-H292 cells were cotransfectedwith �B-luc and the constitutively active form ofPKC� (A/E), wt c-Src, or wt NIK, plus the dom-inant-negative mutants, IKK� (Y188F), IKK�(Y199F), IKK� (FF), or IKK� (AA), or the re-spective empty vector. Luciferase activity wasthen measured, as described in Materials andMethods, and the results were normalized to the�-galactosidase activity and expressed as themean � SEM for three independent experimentsperformed in triplicate. �, p � 0.05; ��, p � 0.01compared with TNF-� or TPA alone (A) or wtalone (B).

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effect was seen at 10-min treatment with TNF-�. Similar effect wasseen after 30-min treatment with TPA, and both effects were in-hibited by PP2 (Fig. 10A). The c-Src-dependent IKK� phosphor-ylation was specific for Tyr188/Tyr199, as it was not seen wheneither or both tyrosine residues were substituted with phenylala-nines (Fig. 10B).

DiscussionThe involvement of NF-�B in TNF-�-induced COX-2 expressionwas further confirmed by transfection with NF-�B mutant plasmid(Fig. 1A). PKC-dependent tyrosine kinase activation is involved inTNF-�-induced NF-�B activation and COX-2 expression in NCI-H292 alveolar epithelial cells (23). The role and molecular identityof the tyrosine kinase involved have been further characterized inthe present study. TNF-�- and TPA-induced COX-2 promoter ac-tivity were both inhibited by the PKC, tyrosine kinase, and Srckinase inhibitors, indicating the possible involvement of the Srctyrosine kinase family downstream of PKC activation in the in-duction of COX-2 expression. Both IKK� and IKK� are involvedin the TNF-�- and TPA-induced COX-2 promoter activity (23),and stimulation of IKK activity and parallel degradation of I-�B�by these two agents were seen in the present study. The TNF-�-and TPA-induced IKK activity and I-�B� degradation were atten-uated by PKC, tyrosine kinase, and Src kinase inhibitors, indicat-ing that the Src tyrosine kinase family is involved downstream ofPKC in the induction of IKK�/� activation, leading to NF-�Bactivation and COX-2 expression in NCI-H292 cells. Western blotanalysis showed that c-Src and Lyn were abundantly expressed inNCI-H292 cells (26), and that TNF-� and TPA induced activation

of these two Src tyrosine kinases. The c-Src and Lyn activationinduced by either stimulus was also inhibited by the PKC, tyrosinekinase, and Src kinase inhibitors. These results demonstrate thatthe tyrosine kinase involved downstream of PKC is c-Src or Lyn.The involvement of PKC/c-Src/IKK�/� activation in TNF-�-in-duced COX-2 expression was confirmed by the finding that thedominant-negative c-Src (KM) mutant attenuated the TNF-�- andTPA-induced COX-2 promoter activity.

Our previous results showed that TNF-�-induced increase inCOX-2 promoter activity in NCI-H292 cells was inhibited by thedominant-negative NIK (KA), IKK� (KM), and IKK� (KM) mu-tants (23). The dominant-negative IKK� (KM) mutant attenuatedwt NIK-induced COX-2 promoter activity (Fig. 6B), indicatingthat NIK/IKK�/� pathway is involved in TNF-�-induced COX-2expression (23). To elucidate the relationship between the PKC/c-Src/IKK�/� and NIK/IKK�/� pathways in TNF-�-inducedCOX-2 expression, overexpression of a constitutively activePKC� plasmid and wt of c-Src, NIK, and IKK� plasmids wasused. These plasmids all induced increased COX-2 promoter ac-tivity, and their effects were blocked by the dominant-negativeIKK� (KM) mutant. The effect of the constitutively active PKC�(A/E) was blocked by the dominant-negative NIK (KA) or c-Src(KM) mutant. The effect of the wt c-Src plasmid on COX-2 pro-moter activity was also diminished by the dominant-negative NIK(KA), but not PKC� (K/R) mutant; that of the wt NIK plasmid wasnot affected by the dominant-negative PKC� (K/R) or c-Src (KM)mutant. These results show that the PKC/c-Src/IKK�/� and NIK/IKK�/� pathways function, and cross-link between c-Src and NIKexists in the TNF-�-mediated induction of COX-2 expression.TNFR-associated factor 2 (TRAF2) has been reported to interactwith NIK, thus linking I-�B degradation and NF-�B activation tothe TNFR complex (28). In this study, Src inhibitor or dominant-negative c-Src mutant almost completely inhibited TNF-�-inducedCOX-2 promoter activity, indicating that TRAF2/NIK/IKK�/�pathway may not be compensated if PKC/c-Src pathway is defec-tive. It is probable that PKC/c-Src lies downstream of TRAF2,because the findings that c-Src acts downstream of TRAF in re-sponse to TNF-related activation-induced cytokine or IL-2 in os-teoclasts, dendritic cells (29), or 293 cells (30) have been reported.

c-Src is involved in NF-�B activation in bone marrow macro-phages, U937 cells, and B cells (31–33). In bone marrow macro-phages, TNF-� induces activation of c-Src, which associates withI-�B� and phosphorylates Tyr42 of I-�B�, leading to NF-�B ac-tivation and IL-6 release (31). In contrast to the rapid degradationof serine-phosphorylated I-�B� (34), tyrosine-phosphorylatedI-�B� is not subject to rapid proteolysis (31, 35). In the presentstudy of TNF-�-induced COX-2 expression, the downstream tar-get of c-Src was IKK� or IKK�, and rapid degradation of I-�B�was seen (Fig. 2B). Involvement of a tyrosine kinase upstream ofIKK activation has also been reported in CD23 signaling in U937cells (32) and in Ag receptor stimulation in B cell (33). A similarPKC-dependent c-Src activation pathway has been found in humanosteoblasts (36), in which fibroblast growth factor-2 increasesN-cadherin expression; in A7r5 vascular smooth muscle cells (37),in which TPA induces Rho-dependent actin reorganization; and inSH-SY5Y neuroblastoma cells (38), in which TPA induces Cas-Crk complex formation. Furthermore, the PKC/c-Src/IKK path-way, in this study shown to be involved in induction of COX-2expression, might be a common signaling pathway for induciblegene expression, as TNF-�-, IL-1�-, or IFN-�-induced ICAM-1expression in human alveolar epithelial cells also involves PKC-dependent activation of c-Src or Lyn (26, 39–41).

Because involvement of the PKC/c-Src/IKK�/� pathway hasbeen demonstrated, tyrosine phosphorylation of IKK�/� by c-Src

FIGURE 10. c-Src-dependent phosphorylation of IKK� at Y188 andY199 is induced by TNF-� or TPA and inhibited by PP2. A, NCI-H292 cellswere treated with 30 ng/ml of TNF-� for 5, 10, 30, or 60 min or 1 �M TPAfor 30 min, or pretreated with 10 �M PP2 for 30 min before stimulationwith TNF-� for 10 min or TPA for 30 min. Whole cell lysates were pre-pared and immunoprecipitated with anti-c-Src Ab, then a kinase assay(KA) and autoradiography for phosphorylated GST-IKK� (132–206) wereperformed, as described in Materials and Methods. The amount of immu-noprecipitated c-Src was detected by Western blotting (WB) using anti-c-Src Ab. B, Cells were treated with 30 ng/ml of TNF-� for 10 min or 1 �MTPA for 30 min, and the whole cell lysates were immunoprecipitated withanti-c-Src Ab, followed by kinase assay (KA) and autoradiography forphosphorylated wt GST-IKK� (132–206), GST-IKK� (132–206) (Y188F),GST-IKK� (132–206) (Y199F), or GST-IKK� (132–206) (Y188F;Y199F). The amount of immunoprecipitated c-Src was detected by West-ern blotting (WB) using anti-c-Src Ab. Amount of GST-IKK� (132–206)was detected by Coomassie brilliant blue staining.

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is further examined. Several lines of evidence show that this oc-curs. First, in both crude cell lysates and anti-IKK�/�-immuno-precipitates, IKK�/� was found to be tyrosine phosphorylated af-ter TNF-� or TPA stimulation. Second, all these effects wereinhibited by PP2. Third, a direct/indirect association between c-Srcand IKK�/� was shown to be increased after TNF-� or TPA treat-ment using immunoprecipitation with either anti-IKK� or anti-IKK� Ab, followed by blotting with anti-c-Src Ab. Fourth, an invitro kinase assay demonstrated that c-Src directly phosphorylatedIKK� at Tyr188 and Tyr199. IKK� is a Thr/Ser kinase, and phos-phorylation of Ser177 and Ser181 in the kinase domain is necessaryfor its activation, because substitution of these two residues withalanines reduces IKK� activity and leads to reduced Rel A nucleartranslocation and NF-�B-dependent gene expression (42, 43). Mi-togen-activated protein kinase kinase kinase 1 and NIK are re-ported to phopshorylate these two serine residues (44). The presentexperiments further demonstrated Tyr188 and Tyr199 phosphoryla-tion by c-Src via a PKC-dependent activation pathway. This ty-rosine phosphorylation of IKK� was essential for TNF-�-inducedCOX-2 expression in NCI-H292 cells, because the dominant-neg-ative mutants, IKK� (Y188F), IKK� (Y199F), or IKK� (FF), ab-rogated the effects of both TNF-� and TPA. Tyrosine phosphor-ylation of Thr/Ser kinases, such as PKCs and Akt, has also beenreported to be important for their activation (45, 46). Akt activa-tion by extracellular stimuli is a multistep process involving trans-location and phosphorylation. Two phosphorylation sites, Thr308

and Ser473, have been shown to be critical for growth factor-in-duced activation of Akt (47–49). In addition to the phosphoryla-tion of these two sites, tyrosine phosphorylation plays an importantrole in regulation of Akt activity. Both the epidermal growth fac-tor-induced tyrosine phosphorylation and kinase activity of Akt are

blocked by PP2, and Tyr315 and Tyr326 of Akt are phosphorylatedby Src both in vivo and in vitro (45). It is noteworthy that thesetyrosine residues are conserved in �50% of Ser/Thr kinases, andthat phosphorylation of the corresponding residues, Tyr512 andTyr523, in PKC� is critical for PKC� activation in response toH2O2 (46). Phosphorylation of these two conserved tyrosine res-idues in the kinase domain of Ser/Thr kinases may be a generalmechanism by which Akt, PKC�, and IKK�/� are regulated (26,45, 46; present study; Fig. 8C). The Src tyrosine kinase familytherefore directly regulates IKK� activity via phosphorylation atTyr188 and Tyr199, rather than solely by NIK-mediated phosphor-ylation at Ser177 and Ser181, as previously suggested (50). Twofindings further support the notion that PKC/c-Src/IKK� pathwayinduces tyrosine phosphorylation, while the NIK/IKK� pathwayinduces serine phosphorylation. First, NF-�B activity induced byPKC� (A/E) or wt c-Src was inhibited by the tyrosine mutants,IKK� (Y188F), IKK� (Y199F), or IKK� (FF). Second, wt NIK-induced NF-�B activity was inhibited by IKK� (AA), but not byIKK� (Y188F), IKK� (Y199F), or IKK� (FF) (Fig. 9B). In addi-tion to demonstration above, cross talk of these two pathways be-tween c-Src and NIK was further proved. First, PKC� (A/E)- andc-Src-induced NF-�B activation were inhibited by the IKK� (AA),in which Ser177 and Ser181 are mutated. Second, TPA-inducedCOX-2 promoter activity was inhibited by NIK (KA) and IKK�(AA). However, the extent of inhibition caused by double serinemutant was less than that caused by tyrosine mutants. Our datatherefore demonstrate that, in addition to phosphorylation of Ser177

and Ser181, Tyr188 and Tyr199 phosphorylation of IKK� is requiredfor its full activation and biological functions.

In summary, the signaling pathways involved in TNF-�-inducedCOX-2 expression in NCI-H292 cells have been further explored.In addition to activating the NIK/IKK�/� pathway, TNF-� acti-vates the PKC-dependent c-Src pathway. These two pathwayscross-link between c-Src and NIK, and converge at IKK�/�, andare, respectively, responsible for phosphorylation of Ser177/Ser181

and Tyr188/Tyr199 of IKK�, then go on to activate NF-�B, viaserine phosphorylation and degradation of I-�B�, and, finally, ini-tiate COX-2 expression. A schematic diagram showing the in-volvement of these two pathways in TNF-�-induced COX-2 ex-pression in NCI-H292 epithelial cells is shown in Fig. 11.

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FIGURE 11. Schematic representation of the signaling pathways in-volved in TNF-�-induced COX-2 expression in NCI-H292 epithelial cells.TNF-� binds to TNFR1 and activates PI-PLC� to induce PKC� and c-Srcactivation, leading to tyrosine phosphorylation of IKK�/�. TNF-� alsoactivates TRAF2 to induce NIK activation, leading to serine phosphoryla-tion of IKK�/�. These two pathways cross-link between c-Src and NIK,and converge at IKK�/�, resulting in phosphorylation and degradation ofI-�B�, stimulation of NF-�B in the COX-2 promoter, and, finally, initia-tion of COX-2 expression.

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