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Tyrosine Phosphorylation of I-B Kinase / by Protein Kinase C-Dependent c-Src Activation Is Involved in TNF--Induced Cyclooxygenase-2 Expression¹

Wei-Chien Huang^*, Jun-Jie Chen^*, Hiroyasu Inoue and Ching-Chow Chen^2,*

^* Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan; and Department of Pharmacology, National Cardiovascular Center Research Institute, Osaka, Japan

	Abstract

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The signaling pathway involved in TNF--induced cyclooxygenase-2 (COX-2) expression was further studied in human NCI-H292 epithelial cells. A protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or a Src 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 of the constitutively active PKC (PKC A/E) or wild-type c-Src plasmids induced COX-2 promoter activity, and these effects were inhibited 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 NIK overexpression. In coimmunoprecipitation experiments, IKK/ was found to be associated with c-Src and to be phosphorylated on its tyrosine residues after TNF- or TPA treatment. Two tyrosine residues, Tyr¹⁸⁸ and Tyr¹⁹⁹, near the activation loop of IKK, were identified to be crucial for NF-B activation. Substitution of these residues with phenylalanines attenuated COX-2 promoter activity and c-Src-dependent phosphorylation of IKK induced by TNF- or TPA. These data suggest that, in addition to activating NIK, TNF- also activates PKC-dependent c-Src. These two pathways cross-link between c-Src and NIK and converge at IKK/, and go on to activate NF-B, via serine phosphorylation and degradation of IB-, and, finally, to initiate COX-2 expression.

	Introduction

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Prostaglandins play important roles in many biological processes, including cell division, blood pressure regulation, immune responses, ovulation, bone development, and water balance. Altered prostanoid production is associated with a variety of illnesses, including acute and chronic inflammation, cardiovascular disease, colon cancer, and allergic diseases (1, 2). Cyclooxygenase (COX),³ also known as PG synthase, is the key enzyme in PG, prostacyclin, and thromboxane synthesis from arachidonic acid (1). COX converts arachidonic acid, released from membrane phospholipid stores by phospholipases, to PGH2, the common precursor of all prostanoids. Two identified COX isoforms, COX-1 and COX-2, are encoded by separated genes (3, 4, 5). COX-1, constitutively expressed in most human tissues (6), appears to be responsible for the production of PGs that mediate normal physiological functions, such as maintenance of integrity of gastric mucosa and regulation of renal blood flow (7, 8). In contrast, COX-2 is induced by a wide range of mitogenic and inflammatory stimuli in many distinct cell types, such as activated marcrophages, monocytes, endothelial cells, fibroblasts, and ovarian follicles (9, 10, 11, 12), and has been identified in chronic inflammatory conditions in vivo (13). It is implicated in physiological processes, such as ovulation and delivery (14), and in pathological states, such as colorectal cancer, Alzheimer’s disease, heart failure, and even hypertension (15, 16, 17, 18). Much evidence suggests that COX-2 is an important therapeutic target for prevention and treatment of arthritis and cancer. Reducing the levels of COX-2 will be an effective strategy for repressing inflammation and carcinogenesis (19, 20); to develop an effective approach, however, it is important to define the signaling mechanism that governs COX-2 expression.

The induction of COX-2 expression requires de novo mRNA and protein synthesis (21), indicating regulation at the transcriptional level. The promoter region of human COX-2 gene has been cloned and sequenced, and shown to contain putative recognition sequences for a variety of transcriptional factors, including NF-B, NF-IL-6, and cAMP response element (22). Of these, NF-B family proteins are the essential components for the enhanced COX-2 gene expression seen on exposure to cytokines in human alveolar epithelial cells (23, 24). The rationale to study COX-2 gene expression and the accompanying signaling pathway in alveolar epithelial cells is that these cells play an active role in inflammation by producing various cytokines that are involved in the late asthmatic response, as previously stated (25). The intracellular signaling pathways by which TNF- causes COX-2 mRNA and protein expression in human alveolar epithelial cells have been explored, including sequential activation of protein kinase C (PKC) , tyrosine kinase, NF-B-inducing kinase (NIK), and I-B kinase (IKK) / (23, 25). The role of tyrosine kinase has been further investigated in the present study. Using an immunocomplex kinase assay and site-directed mutagenesis, we have demonstrated that c-Src is involved in TNF--inducing NF-B transcriptional activation, and that, in addition to serine phosphorylation of IKK/ by NIK, Tyr¹⁸⁸ and Tyr¹⁹⁹ phosphorylations of IKK by PKC-dependent c-Src activation also contribute to TNF--induced COX-2 expression.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

Rabbit polyclonal Abs specific for I-B, IKK, c-Src, and Lyn were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and rabbit polyclonal anti-phosphotyrosine Ab was purchased from Upstate Biotechnology (Lake Placid, NY). Human rTNF- was purchased from R&D Systems (Minneapolis, MN). The 12-O-tetradecanoylphorbol-13-acetate (TPA) was purchased from L.C. Service (Worburn, MA). RPMI 1640 medium, FCS, penicillin, and streptomycin were obtained from Life Technologies (Gaithersburg, MD). Staurosporine, glutathione-agarose beads, and protein 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 reagent were obtained from Pharmacia Biotech (Uppsala, Sweden). [-³²P]ATP (3000 Ci/mmol) was obtained from DuPont-New England Nuclear (Boston, MA). The luciferase assay kit was obtained from Promega (Madison, MA). SuperFect and plasmid purification and DNA recovery kits were obtained from Qiagen (Chatsworth, CA). The Quickchange mutagenesis kit was obtained from Stratagene (La Jolla, CA). EcoRI, XboI, and SalI restriction enzymes and T4 DNA ligase were obtained from NEB (Beverly, MA).

Cell culture

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

Plasmids

The COX-2 promoter construct pGS459 (-459/+9) was a generous gift from L. H. Wang (University of Texas, Houston, TX). The B-luc plasmid was from Stratagene. The PKC- constitutively active (PKC-/AE) or dominant-negative mutant (PKC/KR) was provided by A. Altman (La Jolla Institute for Allergy and Immunology, San Diego, CA). The wild-type (wt) and dominant-negative mutants of NIK and IKK (NIK wt and mutant KA; IKK wt and mutant KM) were gifts from Signal Pharmaceuticals (San Diego, CA). The dominant-negative mutant of IKK (AA) was from M. 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 pretreatment with PKC, tyrosine kinase, or Src kinase inhibitors, the cells were rapidly washed with PBS and lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 5 µg/ml of leupeptin, 20 µg/ml of aprotinin, 1 mM NaF, and 1 mM Na₃VO₄), then IKK, c-Src, or Lyn was immunoprecipitated. For the in vitro kinase assay, 100 µg of total cell extract was incubated for 1 h at 4°C with 0.5 µg of rabbit anti-IKK, anti-c-Src, or anti-Lyn Ab, then protein A-Sepharose CL-4B beads (Sigma-Aldrich) were added to the mixture, and incubation was continued for 4 h at 4°C. The immunoprecipitates were collected by centrifugation, washed three times with lysis buffer without Triton X-100, then incubated for 30 min at 30°C in 20 µl of kinase reaction mixture (20 mM HEPES, pH 7.4, 5 mM MgCl₂, 5 mM MnCl₂, 0.1 mM Na₃VO₄, 1 mM DTT) containing 10 µM [-³²P]ATP and either 1 µg of bacterially expressed GST-I-B (1–100) as IKK substrate, or 1 µg of acidic denatured enolase as c-Src or Lyn substrate, or 6 µg 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 Laemmli buffer, the proteins were separated by electrophoresis on 10% SDS polyacrylamide gels, and phosphorylated GST-I-B (1–100), phosphorylated GST-IKK (132–206), or phosphorylated enolase visualized by autoradiography. Quantitative data were obtained using a densitometer with ImageQuant software and normalized by the protein expression.

Western blot analysis

Following treatment with TNF- or TPA, total or immunoprecipitated cell lysates were prepared and subjected to SDS-PAGE using 7.5% running gels, as described previously (23). The proteins were transferred to a nitrocellulose membrane, which was then incubated successively at room temperature 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 for IKK, IKK, I-B, c-Src, or Lyn, and for 30 min with HRP-labeled anti-rabbit Ab. After each incubation, the membrane was washed extensively with TTBS. The immunoreactive bands were detected using ECL detection reagent and Hyperfilm-ECL (Amersham, Arlington Heights, IL). Quantitative data were obtained using a computing densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Site-directed mutagenesis

Using a Quickchange site-directed mutagenesis kit, according to the manufacturer’s manual, lysine (K) 295 in the mouse c-Src cloned in the pBluescript vector was substituted with methionine (M) by changing the triplets from AAG to ATG. Tyrosine (Y) 199, tyrosine 188, or both sites in the human 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 used were 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, as described previously (26).

Transient transfection and luciferase assay

NCI-H292 cells, grown to 60% confluent in six-well plates, were transfected with the human COX-2 pGS -459/+9, -327/+59, or KBM/firely luciferase (Luc) plasmid using SuperFect (Qiagen), according to the manufacturer’s recommendations. Briefly, reporter DNA (1 µg) and -galactosidase DNA (0.5 µg; plasmid pRK containing the -galactosidase gene driven by the constitutively active SV40 promoter, used to normalize the transfection efficiency) were mixed with 0.75 µl of SuperFect in 0.9 ml of serum-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 (as indicated) for 30 min, then TNF- or TPA was added for 6 h. Cell extracts were then prepared, and luciferase and -galactosidase activities were measured, the luciferase activity being normalized to the -galactosidase activity. In experiments using dominant-negative mutants, cells were cotransfected with reporter (0.5 µg) and -galactosidase (0.25 µg) and either the dominant-negative NIK, IKK, or c-Src mutant or the empty vector (1.0 µg).

In experiments using wt plasmids, cells were cotransfected with 0.5 µg of reporter plasmid, 0.25 µg of -galactosidase plasmid, 1 µg of the constitutively active PKC (A/E) plasmid, wt c-Src or NIK plasmid or empty vector, and 1.5 µg of the dominant-negative NIK, IKK, or c-Src mutant or empty vector.

Coimmunoprecipitation assay

Cell lysates containing 1 mg of protein were incubated for 1 h at 4°C with 2 µg of anti-IKK, anti-IKK, or anti-c-Src Ab, or with 4 µg of anti-phosphotyrosine Ab, then 50 µl of 50% protein A-agarose beads were added and mixed for 16 h at 4°C. The immunoprecipitates were collected and washed three times with lysis buffer without Triton X-100, then Laemmli buffer was added and the samples were subjected to electrophoresis on 10% SDS polyacrylamide gels. Western blot analysis was performed, as described above, using Abs against phosphotyrosine, IKK, IKK, or c-Src.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of inhibitors of PKC, tyrosine kinase, or Src kinase on the induction 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 B site (-223/-214) deletion mutant (KBM) (27), was transfected into NCI-H292 cells. As shown in Fig. 1A, TNF- and TPA induced 3.6- and 4.3-fold increase, respectively, in COX-2 promoter activity using -326/+59 construct. These effects were abolished using KBM plasmid, indicating the requirement of NF-B in the regulation of COX-2 expression. In our previous study (23), we found that PKC and tyrosine kinase were involved in TNF--induced COX-2 expression. Transient transfection using the COX-2 promoter-luciferase construct, pGS459 (-459/+9), was performed to elucidate the signaling pathway mediated by these kinases. The pGS459 construct contains both upstream (-447/-438) and downstream (-223/-214) NF-B site responsible for mediating the induction of COX-2 promoter activity by TNF- or TPA (23) (Fig. 1A). As shown in Fig. 1B, TNF- led to a 3.1-fold increase in COX-2 promoter activity. When cells were pretreated with inhibitors of PKC (staurosporine), tyrosine kinases (genistein or herbimycin A), or Src kinases (PP2), the TNF--induced increase was inhibited by 72, 90, 94, or 89%, respectively. TPA, a direct PKC activator, 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 the basal luciferase activity (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Effect of various inhibitors on TNF-- or TPA-induced COX-2 promoter activity in NCI-H292 cells. A, Cells were transfected with -327/+59 or KBM luciferase expression vector, then treated with 30 ng/ml of TNF- or 1 µM TPA for 6 h. B, Cells were transfected with pGS459 luciferase expression vector, then pretreated for 30 min with vehicle, 300 nM staurosporine, 30 µM genistein, 1 µM herbimycin A, or 10 µM PP2 before incubation for 6 h with 30 ng/ml of TNF- or 1 µM TPA. Luciferase activity was then measured, as described in Materials and Methods, normalized to the -galactosidase activity, and expressed as the mean ± SEM of three independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01 compared with TNF- or TPA alone.

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

Because TNF-- and TPA-induced COX-2 promoter activity in NCI-H292 cells is inhibited by the dominant-negative IKK and IKK mutants (23), endogenous IKK activity was measured by immunoprecipitation with anti-IKK Ab. When cells were treated with 30 ng/ml of TNF- for 5, 10, 30, or 60 min, maximal IKK activity was seen after 10 min (Fig. 2A), which was paralled with maximal degradation of I-B after 10 min. I-B level was restored to the resting level after 1 h of treatment (Fig. 2B). In TPA-treated cells, maximal IKK activity was seen after 5 min of treatment and sustained for 60 min (Fig. 2A), and maximal I-B degradation was seen after 60 min (Fig. 2B). The TNF--induced IKK activation was inhibited by PKC, tyrosine kinase, or Src kinase inhibitors by 78, 99, or 74%, respectively, while staurosporine, herbimycin A, or PP2 suppressed TPA-induced IKK 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).

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Kinetics of TNF--induced IKK activation and I-B degradation. NCI-H292 cells were treated with 30 ng/ml of TNF- or 1 µM TPA for 5, 10, 30, or 60 min, then cell lysates were prepared. A, Cell lysates were immunoprecipitated with anti-IKK Ab, then the kinase assay and autoradiography for phosphorylated GST-I-B (1–100) were performed on the precipitates, as described in Materials and Methods. Levels of immunoprecipitated IKK protein were estimated by Western blotting (W.B.) using anti-IKK Ab. B, Cytosolic levels of I-B were measured using anti-I-B Ab, as described in Materials and Methods.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Effect of various inhibitors on TNF-- or TPA-induced IKK activity and I-B degradation in NCI-H292 cells. Cells were pretreated for 30 min with 300 nM staurosporine, 1 µM herbimycin A, or 10 µM PP2 before incubation with 30 ng/ml of TNF- for 10 min or 1 µM TPA for 30 min, then whole cell lysates were prepared. A, Whole cell lysates were immunoprecipitated with anti-IKK Ab, and the kinase assay and autoradiography for phosphorylated GST-IB (1–100) were performed on the precipitates, as described in Materials and Methods. Levels of immunoprecipitated IKK were estimated by Western blotting (W.B.) using anti-IKK Ab. B, Cytosolic levels of I-B were measured by Western blotting using anti-I-B Ab, as described in Materials and Methods.

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

TNF-- or TPA-induced IKK activation was inhibited by PKC, tyrosine kinase, and Src kinase inhibitors, indicating the involvement of tyrosine kinase, or at least the Src family, downstream of PKC in the induction of IKK activation. Our previous data showed that in contrast to other members of Src family, c-Src and Lyn were abundantly expressed in NCI-H292 cell and another human alveolar epithelial A549 cell (26). c-Src and Lyn in NCI-H292 cells were therefore isolated by immunoprecipitation using anti-c-Src or anti-Lyn Ab, and their in vitro kinase activity was measured using enolase as substrate. As shown in Fig. 4A, when NCI-H292 cells were treated with 30 ng/ml of TNF- for 10, 30, or 60 min, maximal c-Src and Lyn activity (enolase phosphorylation) was seen after 10 min and began to be declined after 60 or 30 min, respectively. The TNF-- and TPA-induced activations of c-Src and Lyn were inhibited by staurosporine, herbimycin A, and PP2 (Fig. 4B).

View larger version (40K): [in this window] [in a new window]   FIGURE 4. Effect of various inhibitors on TNF-- or TPA-induced c-Src or Lyn activation in NCI-H292 epithelial cells. Cells were treated with 30 ng/ml of TNF- for 10, 30, or 60 min (A) or pretreated for 30 min with vehicle, 300 nM staurosporine, 1 µM herbimycin A, or 10 µM PP2 before incubation for 10 min with 30 ng/ml of TNF- or 30 min with 1 µM TPA (B). Whole cell lysates were prepared and immunoprecipitated with anti-c-Src or anti-Lyn Ab. The kinase assay and autoradiography for phosphorylated enolase were performed on the precipitates, as described in Materials and Methods. Levels of immunoprecipitated c-Src or Lyn were estimated by Western blotting (W.B.) using anti-c-Src or anti-Lyn Ab, respectively.

Induction of COX-2 promoter activity by overexpression of PKC or c-Src and the inhibitory effect of dominant-negative mutants of NIK, c-Src, or IKK

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

View larger version (31K): [in this window] [in a new window]   FIGURE 5. Effect of dominant-negative mutants on TNF-- or TPA-induced COX-2 promoter activity in NCI-H292 cells. Cells were cotransfected 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 µM TPA. Luciferase activity was then measured, as described in Materials and Methods, and the results were normalized to the -galactosidase activity and expressed as the mean ± SEM of three independent experiments performed in triplicate. **, p < 0.05 compared with TNF- or TPA alone.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. Effect of various dominant-negative mutants on wt plasmid-induced COX-2 promoter activity. A, NCI-H292 cells were cotransfected with pGS459 and the constitutively active form of PKC (A/E), wt c-Src, NIK, or IKK or empty vector. B, NCI-H292 cells were cotransfected for 24 h with PKC (A/E), wt c-Src or NIK and PKC (K/R), c-Src (K295 M), IKK (KM), or NIK (KA). Luciferase activity was then assayed, as described in Materials and Methods, and the results were normalized to the -galactosidase activity and expressed as the mean ± SEM of three independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01 compared with the control vector.

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

Because c-Src-dependent IKK activation was shown to be involved, coimmunoprecipitation of c-Src and IKK/ was performed to examine whether c-Src directly regulates IKK activity through phosphorylation of tyrosine residues. When cells were treated with TNF- for 5, 10, or 15 min, IKK was tyrosine phosphorylated in a time-dependent manner, the maximal effect being seen at 15 min; a similar effect was seen after 30-min treatment with TPA (Fig. 7A). Both effects were inhibited by PP2 (Fig. 7A). To demonstrate that c-Src associated with IKK/ and phosphorylated their tyrosine residues, cell lysates were immunoprecipitated with anti-IKK or anti-IKK Abs, then the immunoprecipitates were separated by SDS-PAGE, transferred to membranes, and blotted with anti-phosphotyrosine Abs. As shown in Fig. 7, B and C, tyrosine phosphorylation of IKK or IKK was seen after TNF- or TPA treatment, the effect being maximal at 10 or 30 min, respectively, and inhibited by PP2. These results indicate that c-Src can directly/indirectly associate with IKK/ and phosphorylate their tyrosine residues after TNF- or TPA stimulation. The direct/indirect association between c-Src and IKK/ was further examined. Anti-IKK or anti-IKK Ab was used to precipitate IKK from NCI-H292 cells, then the immunoprecipitated proteins were subjected to Western blotting using anti-c-Src Ab. As shown in Fig. 8, A and B, an increased amount of c-Src coprecipitated with IKK or IKK after TNF- or TPA stimulation. These results show a direct/indirect association between c-Src and IKK/ and that IKK/ tyrosine residues were phosphorylated.

View larger version (39K): [in this window] [in a new window]   FIGURE 7. Tyrosine phosphorylation of IKK or IKK induced by TNF- or TPA and the inhibitory effect of PP2. Control cells or cells pretreated for 30 min with 10 µM PP2 were stimulated with TNF- for 5, 10, or 15 min or with TPA for 10 or 30 min. A, Crude lysates were prepared. B and C, Equal amounts (1 mg) of cell lysate were immunoprecipitated (IP) with anti-IKK (A) or anti-IKK (B) Abs. Crude lysates and immunoprecipitated proteins were separated by SDS-PAGE on a 10% gel, immunoblotted (WB) with anti-phosphotyrosine (PY) (A, B, and C), and reprobed with anti-IKK (A, B) or anti-IKK (C) Abs.

View larger version (41K): [in this window] [in a new window]   FIGURE 8. c-Src coimmunoprecipitates with IKK or IKK after TNF- 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 cell lysate 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 domains of PKC, Akt1, and IKK/ are aligned.

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 associate with IKK/ and phosphorylate its tyrosine residues after TNF- or TPA stimulation. When the amino sequences of subdomain VII and VIII in the kinase domain of PKC, AKT1, and IKK/ were aligned, the tyrosine residues were found to be conserved (Fig. 8C). Assuming that Tyr¹⁸⁸ and/or Tyr¹⁹⁹ of IKK were the targets for c-Src phosphorylation after TNF- or TPA stimulation, we used site-directed mutagenesis to generate the dominant-negative tyrosine mutants, IKK (Y188F), IKK (Y199F), and IKK (Y188F, Y199F) (26). Overexpression of these mutants attenuated the TNF-- or TPA-induced COX-2 promoter activity (Fig. 9A). The dominant-negative IKK (KM) mutant, with Lys⁴⁴ mutated to methionine, had a similar inhibitory effect 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 promoter activity, but had less inhibitory effect on TPA-induced COX-2 promoter activity (Fig. 9A).

View larger version (45K): [in this window] [in a new window]   FIGURE 9. Effect of the dominant-negative tyrosine mutants, IKK (Y188F), IKK (Y199F), and IKK (FF), on TNF-- or TPA-induced COX-2 promoter activity and on wt plasmid-induced NF-B activity. In A, NCI-H292 cells were cotransfected with pGS-459 plus one of the dominant-negative tyrosine mutants (IKK (188F), IKK (Y199F), or IKK (FF)), dominant-negative mutant (IKK (KM)), or dominant-negative serine mutant (IKK (AA)), or the respective empty vector, then treated with 30 ng/ml of TNF- or 1 µM TPA for 6 h. B, NCI-H292 cells were cotransfected with B-luc and the constitutively active form of PKC (A/E), wt c-Src, or wt NIK, plus the dominant-negative mutants, IKK (Y188F), IKK (Y199F), IKK (FF), or IKK (AA), or the respective empty vector. Luciferase activity was then measured, as described in Materials and Methods, and the results were normalized to the -galactosidase activity and expressed as the mean ± SEM for three independent experiments performed in triplicate. *, p < 0.05; **, p < 0.01 compared with TNF- or TPA alone (A) or wt alone (B).

Because Tyr¹⁸⁸ and Tyr¹⁹⁹ in IKK were found to be critical for the PKC/c-Src/IKK pathway to elicit NF-B activation, leading to induction of TNF-- or TPA-stimulated COX-2 promoter activity (Fig. 9), endogenous c-Src-depdendent phosphorylation of Tyr¹⁸⁸ and Tyr¹⁹⁹ in IKK was further examined. c-Src was immunoprecipitated using anti-c-Src Ab and its ability to phosphorylate IKK measured using GST-IKK (132–206) as an in vitro substrate. When cells were treated with TNF-, IKK was phosphorylated by c-Src in a time-dependent manner. The maximal effect was seen at 10-min treatment with TNF-. Similar effect was seen after 30-min treatment with TPA, and both effects were inhibited by PP2 (Fig. 10A). The c-Src-dependent IKK phosphorylation was specific for Tyr¹⁸⁸/Tyr¹⁹⁹, as it was not seen when either or both tyrosine residues were substituted with phenylalanines (Fig. 10B).

View larger version (36K): [in this window] [in a new window]   FIGURE 10. c-Src-dependent phosphorylation of IKK at Y188 and Y199 is induced by TNF- or TPA and inhibited by PP2. A, NCI-H292 cells were treated with 30 ng/ml of TNF- for 5, 10, 30, or 60 min or 1 µM TPA for 30 min, or pretreated with 10 µM PP2 for 30 min before stimulation with TNF- for 10 min or TPA for 30 min. Whole cell lysates were prepared and immunoprecipitated with anti-c-Src Ab, then a kinase assay (KA) and autoradiography for phosphorylated GST-IKK (132–206) were performed, as described in Materials and Methods. The amount of immunoprecipitated 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 µM TPA for 30 min, and the whole cell lysates were immunoprecipitated with anti-c-Src Ab, followed by kinase assay (KA) and autoradiography for phosphorylated 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 Western blotting (WB) using anti-c-Src Ab. Amount of GST-IKK (132–206) was detected by Coomassie brilliant blue staining.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our previous results showed that TNF--induced increase in COX-2 promoter activity in NCI-H292 cells was inhibited by the dominant-negative NIK (KA), IKK (KM), and IKK (KM) mutants (23). The dominant-negative IKK (KM) mutant attenuated wt NIK-induced COX-2 promoter activity (Fig. 6B), indicating that NIK/IKK/ pathway is involved in TNF--induced COX-2 expression (23). To elucidate the relationship between the PKC/c-Src/IKK/ and NIK/IKK/ pathways in TNF--induced COX-2 expression, overexpression of a constitutively active PKC plasmid and wt of c-Src, NIK, and IKK plasmids was used. These plasmids all induced increased COX-2 promoter activity, and their effects were blocked by the dominant-negative IKK (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 promoter activity was also diminished by the dominant-negative NIK (KA), but not PKC (K/R) mutant; that of the wt NIK plasmid was not 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 NIK exists in the TNF--mediated induction of COX-2 expression. TNFR-associated factor 2 (TRAF2) has been reported to interact with NIK, thus linking I-B degradation and NF-B activation to the TNFR complex (28). In this study, Src inhibitor or dominant-negative c-Src mutant almost completely inhibited TNF--induced COX-2 promoter activity, indicating that TRAF2/NIK/IKK/ pathway may not be compensated if PKC/c-Src pathway is defective. It is probable that PKC/c-Src lies downstream of TRAF2, because the findings that c-Src acts downstream of TRAF in response to TNF-related activation-induced cytokine or IL-2 in osteoclasts, dendritic cells (29), or 293 cells (30) have been reported.

c-Src is involved in NF-B activation in bone marrow macrophages, U937 cells, and B cells (31, 32, 33). In bone marrow macrophages, TNF- induces activation of c-Src, which associates with I-B and phosphorylates Tyr⁴² of I-B, leading to NF-B activation and IL-6 release (31). In contrast to the rapid degradation of serine-phosphorylated I-B (34), tyrosine-phosphorylated I-B is not subject to rapid proteolysis (31, 35). In the present study of TNF--induced COX-2 expression, the downstream target of c-Src was IKK or IKK, and rapid degradation of I-B was seen (Fig. 2B). Involvement of a tyrosine kinase upstream of IKK activation has also been reported in CD23 signaling in U937 cells (32) and in Ag receptor stimulation in B cell (33). A similar PKC-dependent c-Src activation pathway has been found in human osteoblasts (36), in which fibroblast growth factor-2 increases N-cadherin expression; in A7r5 vascular smooth muscle cells (37), in which TPA induces Rho-dependent actin reorganization; and in SH-SY5Y neuroblastoma cells (38), in which TPA induces Cas-Crk complex formation. Furthermore, the PKC/c-Src/IKK pathway, in this study shown to be involved in induction of COX-2 expression, might be a common signaling pathway for inducible gene expression, as TNF--, IL-1-, or IFN--induced ICAM-1 expression in human alveolar epithelial cells also involves PKC-dependent activation of c-Src or Lyn (26, 39, 40, 41).

Because involvement of the PKC/c-Src/IKK/ pathway has been demonstrated, tyrosine phosphorylation of IKK/ by c-Src is further examined. Several lines of evidence show that this occurs. First, in both crude cell lysates and anti-IKK/-immunoprecipitates, IKK/ was found to be tyrosine phosphorylated after TNF- or TPA stimulation. Second, all these effects were inhibited by PP2. Third, a direct/indirect association between c-Src and IKK/ was shown to be increased after TNF- or TPA treatment using immunoprecipitation with either anti-IKK or anti-IKK Ab, followed by blotting with anti-c-Src Ab. Fourth, an in vitro kinase assay demonstrated that c-Src directly phosphorylated IKK at Tyr¹⁸⁸ and Tyr¹⁹⁹. IKK is a Thr/Ser kinase, and phosphorylation of Ser¹⁷⁷ and Ser¹⁸¹ in the kinase domain is necessary for its activation, because substitution of these two residues with alanines reduces IKK activity and leads to reduced Rel A nuclear translocation and NF-B-dependent gene expression (42, 43). Mitogen-activated protein kinase kinase kinase 1 and NIK are reported to phopshorylate these two serine residues (44). The present experiments further demonstrated Tyr¹⁸⁸ and Tyr¹⁹⁹ phosphorylation by c-Src via a PKC-dependent activation pathway. This tyrosine phosphorylation of IKK was essential for TNF--induced COX-2 expression in NCI-H292 cells, because the dominant-negative mutants, IKK (Y188F), IKK (Y199F), or IKK (FF), abrogated the effects of both TNF- and TPA. Tyrosine phosphorylation of Thr/Ser kinases, such as PKCs and Akt, has also been reported to be important for their activation (45, 46). Akt activation by extracellular stimuli is a multistep process involving translocation and phosphorylation. Two phosphorylation sites, Thr³⁰⁸ and Ser⁴⁷³, have been shown to be critical for growth factor-induced activation of Akt (47, 48, 49). In addition to the phosphorylation of these two sites, tyrosine phosphorylation plays an important role in regulation of Akt activity. Both the epidermal growth factor-induced tyrosine phosphorylation and kinase activity of Akt are blocked by PP2, and Tyr³¹⁵ and Tyr³²⁶ of Akt are phosphorylated by Src both in vivo and in vitro (45). It is noteworthy that these tyrosine residues are conserved in 50% of Ser/Thr kinases, and that phosphorylation of the corresponding residues, Tyr⁵¹² and Tyr⁵²³, in PKC is critical for PKC activation in response to H₂O₂ (46). Phosphorylation of these two conserved tyrosine residues in the kinase domain of Ser/Thr kinases may be a general mechanism by which Akt, PKC, and IKK/ are regulated (26, 45, 46 ; present study; Fig. 8C). The Src tyrosine kinase family therefore directly regulates IKK activity via phosphorylation at Tyr¹⁸⁸ and Tyr¹⁹⁹, rather than solely by NIK-mediated phosphorylation at Ser¹⁷⁷ and Ser¹⁸¹, as previously suggested (50). Two findings further support the notion that PKC/c-Src/IKK pathway induces tyrosine phosphorylation, while the NIK/IKK pathway induces serine phosphorylation. First, NF-B activity induced by PKC (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 by IKK (Y188F), IKK (Y199F), or IKK (FF) (Fig. 9B). In addition to demonstration above, cross talk of these two pathways between c-Src and NIK was further proved. First, PKC (A/E)- and c-Src-induced NF-B activation were inhibited by the IKK (AA), in which Ser¹⁷⁷ and Ser¹⁸¹ are mutated. Second, TPA-induced COX-2 promoter activity was inhibited by NIK (KA) and IKK (AA). However, the extent of inhibition caused by double serine mutant was less than that caused by tyrosine mutants. Our data therefore demonstrate that, in addition to phosphorylation of Ser¹⁷⁷ and Ser¹⁸¹, Tyr¹⁸⁸ and Tyr¹⁹⁹ phosphorylation of IKK is required for its full activation and biological functions.

In summary, the signaling pathways involved in TNF--induced COX-2 expression in NCI-H292 cells have been further explored. In addition to activating the NIK/IKK/ pathway, TNF- activates the PKC-dependent c-Src pathway. These two pathways cross-link between c-Src and NIK, and converge at IKK/, and are, respectively, responsible for phosphorylation of Ser¹⁷⁷/Ser¹⁸¹ and Tyr¹⁸⁸/Tyr¹⁹⁹ of IKK, then go on to activate NF-B, via serine phosphorylation and degradation of I-B, and, finally, initiate COX-2 expression. A schematic diagram showing the involvement of these two pathways in TNF--induced COX-2 expression in NCI-H292 epithelial cells is shown in Fig. 11.

View larger version (19K): [in this window] [in a new window]   FIGURE 11. Schematic representation of the signaling pathways involved in TNF--induced COX-2 expression in NCI-H292 epithelial cells. TNF- binds to TNFR1 and activates PI-PLC to induce PKC and c-Src activation, leading to tyrosine phosphorylation of IKK/. TNF- also activates TRAF2 to induce NIK activation, leading to serine phosphorylation of IKK/. These two pathways cross-link between c-Src and NIK, and converge at IKK/, resulting in phosphorylation and degradation of I-B, stimulation of NF-B in the COX-2 promoter, and, finally, initiation of COX-2 expression.

	Footnotes

 
¹ This work was supported by a research grant from the National Science Council of Taiwan.

² Address correspondence and reprint requests to Dr. Ching-Chow Chen, Department of Pharmacology, College of Medicine, National Taiwan University No.1, Jen-Ai Road, 1st Section Taipei 10018, Taiwan. E-mail address: ccchen{at}ha.mc.ntu.edu.tw

³ 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.

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

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Dewitt, D. L.. 1991. Prostaglandin endoperoxide synthase: regulation of enzyme expression. Biochim. Biophys. Acta 1083:121.[Medline]
2. Smith, W. L., L. J. Marnett. 1991. Prostaglandin endoperoxide synthase: structure and catalysis. Biochim. Biophys. Acta 1083:1.[Medline]
3. Fletcher, B. S., D. A. Kujubu, D. M. Perrin, H. R. Herschman. 1992. Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase. J. Biol. Chem. 267:4338.[Abstract/Free Full Text]
4. Hla, T., K. Neilson. 1992. Human cyclooxygenase-2 cDNA. Proc. Natl. Acad. Sci. USA 89:7384.[Abstract/Free Full Text]
5. Kraemer, S. A., E. A. Meade, D. L. DeWitt. 1992. Prostaglandin endoperoxide synthase gene structure: identification of the transcriptional start site and 5'-flanking regulatory sequences. Arch. Biochem. Biophys. 293:391.[Medline]
6. O’Neill, G. P., A. W. Ford-Hutchinson. 1993. Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett. 330:156.[Medline]
7. Smith, W. L., E. M. Gararto, R. L. Dewitt. 1996. Prostaglandin endoperoxide H synthase (cyclooxygenase)-1 and -2. J. Biol. Chem. 271:33157.[Free Full Text]
8. Smith, W. L., D. L. DeWitt. 1995. Biochemistry of prostaglandin endoperoxide H synthase-1 and synthase-2 and their differential susceptibility to nonsteroidal anti-inflammatory drugs. Semin. Nephrol. 15:179.[Medline]
9. O’Sullivan, G. M., F. H. Chilton, E. M. Huggins, Jr., C. E. McCall. 1992. Lipopolysaccharide priming of alveolar macrophages for enhanced synthesis of prostanoids involves induction of a novel prostaglandin H synthase. J. Biol. Chem. 267:14547.[Abstract/Free Full Text]
10. Jones, D. A., D. P. Carlton, T. M. McIntyre, G. A. Zimmerman, S. M. Prescott. 1993. Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines. J. Biol. Chem. 268:9049.[Abstract/Free Full Text]
11. Evett, G. E., W. Xie, J. G. Chipman, D. L. Robertson, D. L. Simmons. 1993. Prostaglandin G/H synthase isoenzyme 2 expression in fibroblasts: regulation by dexamethasone, mitogens, and oncogenes. Arch. Biochem. Biophys. 306:169.[Medline]
12. Sirois, J., D. L. Simmons, J. Richards. 1992. Hormonal regulation of messenger ribonucleic acid encoding a novel isoform of prostaglandin endoperoxide H synthase in rat preovulatory follicles: induction in vivo and in vitro. J. Biol. Chem. 267:11586.[Abstract/Free Full Text]
13. Vane, J. R., J. A. Mitchell, I. Appleton, A. Tomlinson, D. Bishop-Bailey, J. Croxtall, D. A. Willoughby. 1994. Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Proc. Natl. Acad. Sci. USA 91:2046.[Abstract/Free Full Text]
14. Lim, H., B. C. Paria, S. K. Das, J. E. Dinchuk, R. Langenbach, J. M. Trzaskos, S. K. Dey. 1997. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 91:197.[Medline]
15. Levy, G. N.. 1997. Prostaglandin H synthases, nonsteroidal anti-inflammatory drugs, and colon cancer. FASEB J. 11:234.[Abstract]
16. Oka, A., S. Takashima. 1997. Induction of cyclooxygenase 2 in brains of patients with Down’s syndrome and dementia of Alzheimer type: specific localization in affected neurones and axons. NeuroReport 8:1161.[Medline]
17. Hartner, A., M. Goppelt-Struebe, K. F. Hilgers. 1998. Coordinate expression of cyclooxygenase-2 and renin in the rat kidney in renovascular hypertension. Hypertension 31:201.[Abstract/Free Full Text]
18. Wong, S. C., M. Fukuchi, P. Melnyk, I. Rodger, A. Giaid. 1998. Induction of cyclooxygenase-2 and activation of nuclear factor-B in myocardium of patients with congestive heart failure. Circulation 98:100.[Abstract/Free Full Text]
19. Anderson, G. D., S. D. Hauser, K. L. McGarity, M. E. Bremer, P. C. Isakson, S. A. Gregory. 1996. Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J. Clin. Invest. 97:2672.[Medline]
20. Kawamori, T., C. V. Rao, K. Seibert, B. S. Reddy. 1998. Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res. 58:409.[Abstract/Free Full Text]
21. Hempel, S. L., M. M. Monick, G. W. Hunninghake. 1994. Lipopolysaccharide induces prostaglandin H synthase-2 protein and mRNA in human alveolar macrophages and blood monocytes. J. Clin. Invest. 93:391.
22. Kosaka, T., A. Miyata, H. Ihara, S. Hara, T. Sugimoto, O. Takeda, E. Takahashi, T. Tanabe. 1994. Characterization of the human gene (PTGS2) encoding prostaglandin-endoperoxide synthase 2. Eur. J. Biochem. 221:889.[Medline]
23. Chen, C. C., Y. T. Sun, J. J. Chen, K. T. Chiu. 2000. TNF--induced cyclooxygenase-2 expression in human lung epithelial cells: involvement of the phospholipase C-2, protein kinase C-, tyrosine kinase, NF-B-inducing kinase, and I-B kinase 1/2 pathway. J. Immunol. 165:2719.[Abstract/Free Full Text]
24. Chen, C. C., Y. T. Sun, J. J. Chen, Y. J. Chang. 2001. Tumor necrosis factor--induced cyclooxygenase-2 expression via sequential activation of ceramide-dependent mitogen-activated protein kinases, and IB kinase 1/2 in human alveolar epithelial cells. Mol. Pharmacol. 59:493.[Abstract/Free Full Text]
25. Chen, C. C., K. T. Chiu, S. T. Chan, J. W. Chern. 2001. Conjugated polyhydroxybenzene derivatives block TNF--mediated NF-B activation and COX-2 gene transcription by targeting I kinase activity. Mol. Phamarcol. 60:1439.
26. Huang, W. C., J. J. Chen, C. C. Chen. 2003. c-Src-dependent tyrosine phosphorylation of IKK is involved in TNF--induced intercellular adhesion molecule-1 expression. J. Biol. Chem. 278:9944.[Abstract/Free Full Text]
27. Inoue, H., K. Umesono, T. Nishimori, Y. Hirata, T. Tanabe. 1999. Glucocorticoid-mediated suppression of the promoter activity of the cyclooxygenase-2 gene is modulated by expression of its receptor in vascular en