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Published ahead of print on January 31, 2003, doi:10.1165/rcmb.2002-0261OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 762-769, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0261OC

Ras and Mitogen-Activated Protein Kinase Kinase Kinase-1 Coregulate Activator Protein-1– and Nuclear Factor-{kappa}B–Mediated Gene Expression in Airway Epithelial Cells

Limei Zhou, Alan Tan, Svetlana Iasvovskaia, Jing Li, Anning Lin and Marc B. Hershenson

Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan; and Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois

Address correspondence to: Marc B. Hershenson, M.D., University of Michigan, 1500 E. Medical Center Dr., L2211 Women's Hospital, Box 0212, Ann Arbor, MI 48109-0212. E mail: mhershen{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In 16HBE14o- human bronchial epithelial cells, maximal tumor necrosis factor (TNF)-{alpha}–induced interleukin (IL)-8 expression depends on the activation of two distinct signaling pathways, one constituted in part by activator protein (AP)-1 and the other by nuclear factor (NF)-{kappa}B. We examined the upstream signaling intermediates responsible for IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in this system, hypothesizing that p21 Ras and mitogen-activated protein kinase/extracellular signal–regulated kinase kinase kinase (MEKK)-1 function as common upstream activators of both the AP-1 and NF-{kappa}B pathways. TNF-{alpha} treatment induced both Ras and MEKK1 activation. Dominant-negative forms of Ras (N17Ras) and MEKK1 (MEKK1-KM) each inhibited TNF-{alpha}–induced transcription from IL-8 and GM-CSF promoters. Ras was required for maximal activation of extracellular signal–regulated kinase (ERK) and Jun amino terminal kinase (JNK) as well as AP-1 and NF-{kappa}B transcriptional activities, but not for activation of I{kappa}B kinase (IKK)-ß, an upstream activator of NF-{kappa}B. MEKK1 was required for maximal activation of ERK, JNK, and IKK, as well as for maximal AP-1 and NF-{kappa}B transcriptional activities. We conclude that Ras regulates TNF-{alpha}–induced chemokine expression by activating the AP-1 pathway and enhancing transcriptional function of NF-{kappa}B, whereas MEKK1 activates both the AP-1 and NF-{kappa}B pathways.

Abbreviations: activator protein-1, AP-1 • enzyme-linked immunosorbent assay, ELISA • extracellular signal regulated kinase, ERK • fetal bovine serum, FBS • granulocyte macrophage colony-stimulating factor, GM-CSF • hemagglutinin, HA • I{kappa}B kinase, IKK • interleukin-8, IL-8 • mitogen-activated protein kinase, MAP kinase • MAP kinase/ERK kinase, MEK • MEK kinase, MEKK • minimum essential medium, MEM • nuclear factor-{kappa}B, NF-{kappa}B • NF-{kappa}B–inducing kinase, NIK • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Airway epithelial cells synthesize a number of chemokines, including interleukin (IL)-8 (1, 2) and granulocyte-macrophage colony stimulating factor (GM-CSF) (3, 4), both of which are increased in the airways of patients with asthma (5–7). We have shown in human bronchial epithelial cells that maximal tumor necrosis factor (TNF)-{alpha}–induced IL-8 expression depends on the activation of two distinct signaling pathways, one constituted in part by activator protein (AP)-1 and the other by nuclear factor (NF)-{kappa}B (8). Chemical inhibitors of NF-{kappa}B block rhinovirus-induced GM-CSF expression in BEAS-2B human airway epithelial cells (9), and more detailed analyses in other systems demonstrate GM-CSF expression to be regulated by both NF-{kappa}B and AP-1 cis-acting promoter elements (10–13). However, the signaling intermediates leading to TNF-{alpha}–induced NF-{kappa}B and AP-1–mediated chemokine expression in human bronchial epithelial cells have not been studied.

The 21-kD GTPase Ras has been shown to participate in the activation of both AP-1 (14–16) and NF-{kappa}B transcription factor complexes (15, 17–19). Ras-induced activation of extracellular signal regulated kinase (ERK), a precursor of AP-1 transactivation (20–22), has been well studied (23–25). Ras may also activate NF-{kappa}B signaling via mitogen-activated protein (MAP) kinase/extracellular signal regulated kinase kinase kinase (MEKK), a downstream effector (26–28) that has been demonstrated to activate I{kappa}B kinase (IKK). IKK, which shares structural elements with MAP kinase kinases (29), mediates phosphorylation and degradation of I{kappa}B, allowing translocation of NF-{kappa}B to the nucleus. Ras has also been shown to enhance the transcriptional function of NF-{kappa}B complexes, independently of its effect on NF-{kappa}B translocation (17, 19, 30).

MEKK, a MAP kinase kinase kinase, was originally described as an activator of the MAP kinase/ERK kinase (MEK), the upstream activator of ERK (27, 31). Later studies suggested that MEKK1 only induces modest ERK activation in cells, and instead functions as a stress-activated protein kinase kinase kinase (32–35), thereby activating Jun amino terminal kinase (JNK) and p38 MAP kinase. JNK, in turn, phosphorylates c-Jun (36, 37), a constituent of the AP-1 transcription factor complex. As noted above, MEKK1 has also been demonstrated to activate IKK. Together, these reports suggest that MEKK1, like Ras, may function as an upstream activator of both AP-1 and NF-{kappa}B–dependent pathways mediating chemokine expression.

Ras and MEKK1 have been demonstrated to mediate TNF-{alpha} responses in other systems. Expression of a dominant-negative Ras (N17Ras) inhibits TNF-{alpha}–induced apoptosis in H-ras–transformed fibroblasts (38). In Kym-1 rhabdomyosarcoma cells, TNF-{alpha} treatment induces the association of Ras with the serine-threonine kinase Raf-1 (39), an indicator of Ras activation. In endothelial cells, expression of N17Ras has been demonstrated to attenuate TNF-{alpha}–induced RelA transcriptional activity (30) as well as expression of E-selectin and vascular cell adhesion molecule-1 (40). In rat2 fibroblasts, N17Ras blocks TNF-{alpha}–induced activation of phosphatidylinositol 3-kinase (41). MEKK1 has been shown to mediate TNF-{alpha}–induced responses in a diverse group of cell lines, including RLE alveolar type II cells (27, 32, 42, 43). Taken together, these reports suggest that Ras and MEKK1 may function as key regulators of TNF-{alpha}–stimulated chemokine expression in human bronchial epithelial cells.

We therefore examined the upstream signaling intermediates responsible for IL-8 and GM-CSF expression in human bronchial epithelial cells, hypothesizing that Ras and MEKK1 function as common upstream activators of both the AP-1 and NF-{kappa}B pathways. We found that Ras regulates TNF-{alpha}–induced chemokine expression by activating the AP-1 pathway and enhancing transcriptional function of NF-{kappa}B, whereas MEKK1 activates both the AP-1 and NF-{kappa}B pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
A derivative of 16HBE14o- human bronchial epithelial cells, provided by S. White (University of Chicago, Chicago, IL) was studied. This cell line was originally established from bronchial epithelial tissue by transfection with pSVori-, which contains the origin-defective SV40 genome (44). Unlike the parental line, these cells do not grow in distinct clusters and demonstrate improved transfection efficiency. Cultures show specific immunostaining with pan-cytokeratin c11 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), bind galactose or galactosamine-specific lectins particular to basal epithelial cells, and express {alpha}1-, ß2-, ß3-, and ß6-integrin subunits on their cell surface (8). The cells were grown on coated plates (fibronectin, 10 µg/ml; collagen, 30 µg/ml; bovine serum albumin, 100 µg/ml) in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 200 mM of L-glutamine.

Plasmid Vectors
The –162/+44 fragment from the full-length human IL-8 promoter was cloned into a luciferase reporter plasmid (-162/+44 hIL-8/Luc). The reporter activities of this fragment have been shown to be identical to the full-length promoter in response to respiratory syncytial virus infection (45), and this fragment contains the NF-{kappa}B, nuclear factor for IL-6 (NF-IL6) and AP-1 binding sites required for maximal TNF{alpha} responses (46). NF-{kappa}B and AP-1 reporter plasmids (NF-{kappa}B-TATA/Luc and AP-1-TATA/Luc, respectively) were purchased from Stratagene (La Jolla, CA). Plasmid DNAs encoding dominant-negative (N17Ras), HA-tagged JNK1, and ß-galactosidase were provided by M. Rosner (University of Chicago). cDNAs encoding constitutively active Ras (v-H-Ras) and constitutively active ({Delta}MEKK) (31) were provided by D. Templeton (Case Western Reserve University, Cleveland, OH). Construction of cDNAs encoding HA-tagged IKKß, MEKK1-KM (K432M), NF-{kappa}B–inducing kinase (NIK)-AA (KK429/AA430), and IKKß-AA (SS177/181AA), has been described elsewhere (29). A hemagglutinin (HA)-tagged ERK2 was constructed by fusion a DNA fragment encoding the seven amino acid influenza HA epitope to the 5' end of murine ERK2 (47). A bacterial expression vector encoding recombinant GST-I{kappa}B{alpha} was provided by M. Karin (University of California, San Diego, CA) (48). GST-Jun (1–79) was obtained from J. Posada (University of Vermont, Burlington, VT) (49).

Transient Transfection and Reporter Assays
DNA transfections were performed using Lipofectamine, as described (8, 50). 16HBE14o- cells were grown on 6-well plates at 70–80% confluence and then cotransfected with 0.5 µg of the relevant reporter construct and 30–200 ng of the expression vector of interest or empty vector. Transfection efficiency was assessed by cotransfection with 30 ng of CMV-ß-galactosidase (pCMV-ß-Gal DNA). Twenty-four hours after transfection, cells were serum-starved for 8 h and treated with TNF-{alpha} (R&D Systems, Minneapolis, MN). Sixteen hours after treatment, cells were harvested and analyzed for luciferase and ß-galactosidase activities, as described (50).

In Vitro Kinase Assays
To examine activation of ERK, JNK, and IKK, 16HBE14o- cells were cotransfected with cDNA encoding a hemagglutinin-tagged form of ERK2, JNK1, or IKKß, respectively. In selected experiments, cells were cotransfected with either empty vector or cDNA encoding Ras or MEKK1, as described (25). Forty-eight hours after transfection, cells were serum-starved in MEM. The next day, cells were treated with TNF-{alpha} for 10 min. Cells were then lysed in 800 µl of lysis buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Triton, 40 mM ß-glycerophosphate, 0.2 mM Na3VO4, 1 mM PMSF, and 1% proteinase inhibitor cocktail (Sigma Chemical, St. Louis, MO). Lysates were centrifuged (5 min at 4°C) to remove cellular debris. After preclearance with protein A sepharose beads (1 h), the supernatant was immunoprecipitated with mouse monoclonal anti-HA antibody HA.11 (Covance, Princeton, NJ) precoupled to protein G sepharose (4°C for 3 h). Immunoprecipitates were washed three times with lysis buffer (4°C) and resuspended in 20 µl of kinase buffer containing 25 mM Tris (pH 7.5), 5 mM ß-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, 50 µM ATP, and 5 µCi of [{gamma}-32P]-ATP. For measurements of ERK, JNK, or IKK activation, GST-Elk1 (Cell Signaling Technology, Beverly, MA), GST-Jun, or GST-I{kappa}B, respectively, was added as a substrate. After incubation (30 min at 30°C), the reactions were stopped with 4x SDS-PAGE loading buffer. The reaction components were separated by 10% SDS-PAGE, transferred to nitrocellulose and exposed to film.

To confirm the expression level of HA-tagged kinase in bronchial epithelial cell immunoprecipitates, the membranes were probed with HA.11. Signals were amplified and visualized using peroxidase-linked rat anti-mouse {kappa} light chain IgG (Zymed Laboratories, South San Francisco, CA) and enhanced chemiluminescence.

To examine activation of MEKK1, cells were serum-starved in MEM for 24 h and treated with TNF-{alpha} (10 ng/ml). After preclearance with protein A sepharose beads, whole cell lysates were immunoprecipitated with a monoclonal antibody against MEKK1 (Santa Cruz Biotechnology) precoupled to protein G sepharose. After washing, immunoprecipitates were resuspended in kinase buffer containing [{gamma}-32P]-ATP and recombinant MEK1 (Santa Cruz Biotechnology). After incubation (30 min at 30°C), reaction components were separated by 10% SDS-PAGE, transferred to nitrocellulose, and exposed to film. To confirm equal levels of MEKK-1 in the immunoprecipitates, membranes were probed with anti-MEKK1.

Measurement of IL-8 and GM-CSF Protein
Cells at 75–80% confluence were serum-starved for 24 h and then treated with TNF-{alpha} overnight. Conditioned media were collected, centrifuged to remove cell debris (14,000 rpm for 10 min), and frozen at -80°C. IL-8 and GM-CSF proteins were measured by enzyme-linked immunosorbent assay (ELISA; Endogen, Woburn, MA).

Activated Ras Affinity Precipitation Assay
A Ras activation assay kit (Upstate Biotechnology, Lake Placid, NY) was used. Briefly, 16HBE14o- cells were grown at 75–80% confluence, serum-starved for 24 h, and treated with TNF-{alpha} (10 ng/ml). Cells were then lysed with Mg2+ lysis/wash buffer (MLB) (125 mM HEPES, pH7.5, 750 mM NaCl, 5% Igepal CA-630, 50 mM MgCl, 5 mM EDTA, and 10% glycerol). Lysates (500 µg each) were then affinity precipitated with 10 µg of GST-RBD (Ras-binding domain of Raf-1) bound to glutathinone-agarose (4°C for 30 min). The beads were then extensively washed with MLB. The eluted proteins were resolved on a 12% SDS-PAGE gel and transferred to nitrocellulose membrane. After staining with Ponceau Red to check protein loading, the membrane was probed with anti-Ras monoclonal antibody.

Data Analysis
Each experiment was performed at least three times. Statistical significance was assessed by ANOVA. Differences identified by ANOVA were pinpointed by Student Neuman-Keuls' multiple range test. For reporter assays, changes in promoter activity were calculated as luciferase arbitrary light units/ß-galactosidase calorimetric units/h. Results were then reported as fold increase over the empty vector/untreated control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF-{alpha} Treatment of Human Bronchial Epithelial Cells Induces IL-8 and GM-CSF Expression
16HBE14o- human bronchial epithelial cells were co-transfected with DNA fragments encoding the human IL-8 or GM-CSF promoters subcloned into a luciferase reporter plasmid, as well as ß-galactosidase. After serum starvation, cells were treated with TNF-{alpha} (10 ng/ml) overnight. Sixteen hours after TNF-{alpha} treatment, cells were lysed and luciferase, and ß-galactosidase activities were measured by luminometer and colorimetric assay. TNF-{alpha} increased transcription from both the IL-8 and GM-CSF promoters (Figures 1A and 1B). To determine whether changes in promoter activity were reflected in protein expression, cells were incubated with TNF-{alpha} (10 ng/ml) for overnight. Aliquots of conditioned medium were examined for IL-8 and GM-CSF protein abundance by ELISA. TNF-{alpha} treatment significantly increased IL-8 and GM-CSF protein expression (Figure 1C).



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  		Figure 1. TNF-{alpha} treatment of human bronchial epithelial cells induces IL-8 and GM-CSF expression: effect of dominant-negative Ras. (A, B) 16HBE14o- human bronchial epithelial cells were transfected with DNA fragments encoding the human IL-8 or GM-CSF promoters subcloned into a luciferase reporter plasmid, as well as ß-galactosidase. Selected cultures were also transfected with empty vector or a cDNA encoding a dominant-negative Ras (N17Ras). After serum starvation, cells were treated with TNF-{alpha} (10 ng/ml) overnight. TNF-{alpha} significantly increased transcription from both the IL-8 and GM-CSF promoters (n = 6, **different from untreated, P < 0.05, ANOVA). Expression of N17Ras significantly attenuated promoter activity (n = 6, *different from TNF-{alpha}, P < 0.05, ANOVA). (C) Differences in promoter activity were reflected by changes in protein abundance, as measured by ELISA (n = 3–4, **different from untreated, P < 0.05, ANOVA).

 
Ras and MEKK1 Are Required for Transcription from the IL-8 and GM-CSF Promoters
We tested whether TNF-{alpha} treatment activates Ras using a commerically available affinity precipitation assay. TNF-{alpha} induced binding of Ras to beads conjugated to the Ras-binding domain (RBD) of Raf, indicative of Ras activation (Figure 2A). To determine whether TNF-{alpha} treatment activates MEKK1, cell lysates were immunoprecipitated with an antibody against MEKK1, and MEKK1 activation assessed by in vitro phosphorylation assay using MEK1 as a substrate (Figure 2B). TNF-{alpha} treatment induced activation of MEKK1, as evidenced by MEK1 phosphorylation.



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  		Figure 2. TNF-{alpha} induces both Ras and MEKK1 activation. (A) Ras activation was assessed using a commerically-available affinity precipitation assay. (B) MEKK1 activation was assessed by immunoprecipitation with an antibody against MEKK1, followed by in vitro phosphorylation assay using MEK1 as a substrate. Representative results are shown. Each experiment was repeated twice. (C, D) Cells were transfected with DNA fragments encoding the human IL-8 or GM-CSF promoters subcloned into a luciferase reporter plasmid, ß-galactosidase and either empty vector or a cDNA encoding a dominant-negative MEKK1 (MEKK1-KM) (n = 3–6, *different from TNF-{alpha}, **different from control, P < 0.05, ANOVA).

 
We examined the requirements of Ras and MEKK1 for transcription from the IL-8 and GM-CSF promoters by cotransfecting cells with N17Ras, kinase-inactive MEKK1 (MEKK1-KM) or empty vector, and the appropriate reporter plasmid. Inhibition of Ras and MEKK1 each significantly attenuated TNF-{alpha}–induced IL-8 and GM-CSF promoter activities (Figures 1A, 1B, 2C, and 2D).

Requirements of Ras and MEKK1 for AP-1 and NF-{kappa}B Transactivation
Given the importance of NF-{kappa}B and AP-1 transcription factors in chemokine expression, we examined the requirement of Ras and MEKK1 for AP-1 and NF-{kappa}B transactivation. Cells were transfected with dominant-negative forms of Ras, MEKK1 or empty vector, and the appropriate reporter plasmid. Inhibition of either Ras or MEKK1 significantly attenuated TNF-{alpha}–induced AP-1 and NF-{kappa}B transactivation (Figures 3A–3D).



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  		Figure 3. Regulation of AP-1 and NF-{kappa}B transactivation by Ras and MEKK1. Cells were transfected with AP-1 and NF-{kappa}B reporter plasmids and either empty vector, N17Ras, v-H-Ras, MEKK1-KM or {Delta}MEKK. TNF-{alpha} significantly induced AP-1 and NF-{kappa}B transactivation, whereas inhibition of either Ras or MEKK1 significantly attenuated the TNF-{alpha} signal (A–D, n = 3–12, **different from control, *different from TNF-{alpha}, P < 0.05, ANOVA). Active Ras and MEKK1 were each sufficient to induce AP-1 promoter activity (A and C, n = 6, **different from control, P < 0.05, ANOVA). As a positive control, the response to bryostatin 1 was also tested (C). Neither active Ras nor MEKK1 was sufficient to induce a level of NF-{kappa}B transactivation similar to that observed following TNF-{alpha} stimulation (B and D).

 
To learn more about these pathways, cells were also transfected with v-H-ras, {Delta}MEKK, or empty vector, and AP-1 and NF-{kappa}B reporter activities assessed. Although overexpression of active forms of Ras and MEKK1 may be superphysiologic, the sufficiency of Ras or MEKK1 for AP-1 and NF-{kappa}B transactivation would provide further evidence that these signaling intermediates are important mediators of TNF-{alpha}–activated responses. Active Ras and MEKK1 were each sufficient to induce AP-1 promoter activity (Figures 3A and 3C). However, neither active Ras nor MEKK1 was sufficient to induce a level of NF-{kappa}B transactivation similar to that observed following TNF-{alpha} stimulation (Figures 3B and 3D).

Although statistically significant, the AP-1 response to TNF-{alpha} appeared small relative to active Ras or MEKK1. To examine this further, we examined AP-1 transactivation following treatment with bryostatin 1, a selective activator of novel protein kinase C isoforms which we have found to be a potent stimulus of IL-8 expression (M. Hershenson, unpublished data). The AP-1 response to bryostatin 1 was similar to that induced by active Ras or MEKK1 (Figure 3C).

Ras Is Required for Activation of ERK and JNK but Not IKK
We have previously shown that TNF-{alpha} treatment induces activation of both ERK and JNK in human bronchial epithelial cells (8). We tested whether Ras regulates TNF-{alpha}–mediated MAP kinase activation. Cells were cotransfected with cDNAs encoding HA-tagged ERK or JNK, and the appropriate Ras mutant or empty vector. ERK and JNK activities were assessed by immunoprecipitation of the epitope tag followed by in vitro kinase assay using recombinant Elk-1 or Jun as a substrate. Expression of N17Ras attenuated activation of ERK (Figure 4A) and JNK (Figure 4B). In contrast, selective activation of Ras was sufficient for ERK but not JNK activation.



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  		Figure 4. Ras is required for activation of ERK and JNK, but not IKK. Cells were transiently transfected with cDNAs encoding HA-tagged forms of ERK2, JNK1, or IKKß and either empty vector, N17-Ras or v-H-Ras. ERK, JNK, and IKK activities were assessed by immunoprecipitation of the epitope tag followed by in vitro kinase assay using recombinant Elk-1, c-Jun, or I{kappa}B{alpha} as a substrate, respectively. Expression of N17Ras attenuated activation of ERK (A) and JNK (B), but not IKKß (C). Activation of Ras was sufficient for ERK but not JNK or IKK activation. Typical results are shown; each experiment was repeated at least twice.

 
We tested the effects of mutant Ras proteins on the activation of IKKß. Cells were cotransfected with cDNAs encoding HA-tagged IKKß and the appropriate Ras mutant or empty vector. IKKß activity was assessed by immunoprecipitation of the epitope tag followed by in vitro kinase assay using recombinant I{kappa}B as a substrate. TNF-{alpha} treatment induced substantial activation of IKKß, as reflected by I{kappa}B phosphorylation (Figure 4C). IKKß activation was not attenuated by expression of N17Ras, however. Although expression of v-H-Ras appeared to slightly increase I{kappa}B{alpha} phosphorylation, this was accompanied by an increase in HA-IKKß expression. Densitometric analysis showed that active Ras increased normalized IKK activity by an average of only 24% (data not shown), confirming that selective activation of Ras was insufficient for IKK activity.

IKKß but Not NIK1 Is Required for TNF-{alpha}–Induced NF-{kappa}B Transactivation
As noted above, inhibition of MEKK1 significantly attenuated TNF-{alpha}–induced transcription from the IL-8 and GM-CSF promoters, as well as AP-1 and NF-{kappa}B transactivation. We examined the requirement of another IKK kinase, NF-{kappa}B–inducing kinase (NIK), for NF-{kappa}B transcriptional activity. NIK was originally identified as a protein that interacts with TNF receptor–associated factor-2 (TRAF2). Constitutive activation of NIK causes I{kappa}B{alpha} degradation and NF-{kappa}B activation (51), suggesting that NIK is involved in NF-{kappa}B signaling. However, functional dominant-negative NIK (52) had no effect on NF-{kappa}B transactivation in 16HBE14o- cells (Figure 5A). These experiments imply that, in the context of TNF-{alpha} stimulation, MEKK1, but not NIK, regulates TNF-{alpha}–induced chemokine expression.



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  		Figure 5. IKKß but not NIK1 is required for TNF-{alpha}–induced NF-{kappa}B transactivation. Cells were transfected with the NF-{kappa}B reporter plasmid and either empty vector, dominant-negative NIK (NIK-AA; A) or dominant-negative IKKß (IKK-AA; B). Expression of a dominant-negative NIK had no effect on NF-{kappa}B transactivation (n = 3). Expression of IKKß-AA significantly attenuated NF-{kappa}B transactivation (n = 3, *different from TNF-{alpha}, **different from control, P < 0.05, ANOVA).

 
We have previously shown in human bronchial epithelial cells that I{kappa}B phosphorylation and NF-{kappa}B transactivation are required for TNF-{alpha}–induced transcription from the IL-8 promoter (8). We tested whether IKKß is required for NF-{kappa}B transactivation in this system. Cells were cotransfected with the NF-{kappa}B reporter and either empty vector or a cDNA encoding a dominant-negative form of IKKß (IKKß-AA). Expression of IKKß-AA significantly attenuated NF-{kappa}B transactivation (Figure 5B).

MEKK1 Is Required for Activation of ERK, JNK, and IKK
We tested whether MEKK1 regulates TNF-{alpha}–mediated MAP kinase activation. Cells were co-transfected with cDNAs encoding HA-tagged ERK or JNK and the appropriate MEKK1 mutant or empty vector. ERK and JNK activities were assessed by immunoprecipitation of the epitope tag followed by in vitro kinase assay using recombinant Elk-1 or Jun as a substrate. Expression of MEKK1-KM attenuated activation of ERK and JNK (Figures 6A and 6B). Selective activation of MEKK1 was sufficient for the activation of both MAP kinases.



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  		Figure 6. MEKK1 is required for activation of ERK, JNK, and IKK. Cells were transiently transfected with cDNAs encoding HA-tagged forms of ERK2, JNK1, or IKKß and either empty vector, MEKK1-KM, or {Delta}MEKK. ERK, JNK, and IKK activities were assessed by immunoprecipitation of the epitope tag followed by in vitro kinase assay using recombinant Elk-1, c-Jun, or I{kappa}B{alpha} as a substrate, respectively. Expression of MEKK1-KM attenuated activation of ERK (A), JNK (B), and IKKß (C). Activation of MEKK1 was also sufficient for ERK, JNK, and IKK activation. Typical results are shown; each experiment was repeated at least twice.

 
We asked whether MEKK1 is an upstream activator of IKK. Cells were cotransfected with HA-IKKß and either empty vector, MEKK-KM, or {Delta}MEKK. IKKß activation was assessed by immunoprecipitation of the epitope tag followed by in vitro kinase assay using recombinant I{kappa}B{alpha} as a substrate. Expression of kinase-inactive MEKK1 attenuated TNF-{alpha}–induced IKKß activation, whereas expression of active MEKK1 was sufficient for IKKß activation (Figure 6C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously found that in 16HBE14o- human bronchial epithelial cells, maximal TNF-{alpha}–induced IL-8 expression depends on the activation of two distinct signaling pathways, the first constituted in part by AP-1 and the second by NF-{kappa}B (8). In the present study, we hypothesized that Ras and MEKK1 regulate the expression of two important airway chemokines, IL-8 and GM-CSF via both AP-1 and NF-{kappa}B pathways. Expression of N17Ras attenuated IL-8, GM-CSF, and AP-1 reporter activities. N17Ras also attenuated activation of ERK, a downstream kinase that regulates AP-1 transactivation. Expression of v-H-ras was also sufficient for ERK activation and AP-1 transcriptional activity. Together, these data suggest that Ras regulates TNF-{alpha}–induced chemokine expression by activating the ERK/AP-1 pathway.

Expression of N17Ras also attenuated TNF-{alpha}–induced NF-{kappa}B transcriptional activity. However, Ras was not required or sufficient to activate IKK, an upstream activator of NF-{kappa}B signaling. Activation of Ras was also insufficient to induce NF-{kappa}B transactivation. These data are consistent with the notion that Ras enhances the transcriptional function of NF-{kappa}B, but is not itself sufficient for stimulation of the NF-{kappa}B signaling pathway. The importance of Ras for NF-{kappa}B transcriptional function has been previously demonstrated in fibroblasts and primary endothelial cells (17, 19, 30). In fibroblasts, oncogenic forms of Ha-Ras activate NF-{kappa}B not through induced nuclear translocation but rather through the activation of the transcriptional function of the NF-{kappa}B RelA/p65 subunit (17). Although we have recently shown in human bronchial epithelial cells that TNF{alpha} treatment induces binding of p65 Rel A and p50 NF-{kappa}B1 to DNA (8), we did not test the direct effects of Ras activation on NF-{kappa}B binding, owing to the relatively modest transfection efficiency of 16HBE14o- cells. Nevertheless, the inability of Ras to activate IKK suggests that Ras regulates airway epithelial cell chemokine expression at a point subsequent to Rel A binding to DNA.

The mechanism by which Ras enhances the transcriptional function of NF-{kappa}B is not completely known. In endothelial cells, the transcriptional activity of RelA is dependent on phosphorylation of the N-terminal Rel homology domain, and expression of dominant-negative mutants of either Ras or protein kinase C-{zeta} result in the loss of RelA transcriptional activity without interfering with DNA binding (30). In fibroblasts, Ras-induced RelA transcriptional activity is attenuated by coexpression of a dominant-negative MAP kinase kinase-4, an upstream activator of the JNK and p38 MAP kinases (19). Because Ras was insufficient for NF-{kappa}B transactivation in human bronchial epithelial cells, we could not test the requirement of JNK for Ras-induced NF-{kappa}B transcriptional activity. Nonetheless, we previously found that JNK activation is required for maximal TNF-{alpha}–induced NF-{kappa}B transactivation in this system (9), and now show that Ras is required for JNK activation. Together, these data are consistent with the notion that JNK may be involved in Ras-mediated enhancement of NF-{kappa}B transcriptional activity.

Recent studies have examined the role of Ras in lung epithelial cell responses. Expression of N17Ras blocks MUC2 gene expression in Pseudomonas aeruginosa–stimulated NCIH292 airway epithelial cells (53). In the latter study, chemical inhibition of NF-{kappa}B with caffeic acid phenethyl ester also attenuated MUC2 expression, suggesting that NF-{kappa}B functions as a downstream transcription factor target of Ras in this context. N17Ras inhibits vanadium-induced NF-{kappa}B DNA binding in BEAS-2B human airway epithelial cells (54). Expression of N17Ras blocked oxidant- but not TNF-{alpha}–induced NF-{kappa}B transactivation in RLE alveolar type II cells (43). In the present study, we confirmed that NF-{kappa}B may function as a downstream target of Ras activation in human bronchial epithelial cells, and have extended these studies by proposing a specific role in the regulation of NF-{kappa}B transcriptional function.

Ras has been demonstrated to mediate TNF-{alpha} responses in fibroblasts (38, 41) and endothelial cells (30, 40). At least two mechanisms for Ras activation have been identified. First, it has been shown that sphingomyelinase activation with consequent production of ceramide contributes to TNF-{alpha}–induced Ras activation (39, 41, 55). Second, TNF receptor type I has been demonstrated to bind with the cytosolic adaptor protein Grb2 via its C-terminal SH3 domain (56). Grb2, in turn, is found in a stable complex with the nucleotide exchange factor Son of sevenless (Sos), an upstream activator of Ras. Once activated, Ras may initiate a number of signaling intermediates by recruiting them to the cell membrane, including Raf, MEKK, PI 3-kinase and Rac1.

Studies utilizing a kinase-inactive mutant of MEKK1 demonstrated that, like Ras, MEKK1 is required for IL-8, GM-CSF, AP-1, and NF-{kappa}B reporter activities, as well as for ERK activation. However, unlike Ras, MEKK1 was required and sufficient for IKK activation. On the other hand, expression of a dominant-negative NIK had no effect on chemokine promoter activity. Taken together, these data suggest that MEKK1, not NIK, is the upstream activator of IKK in cytokine-treated 16HBE14o- cells. Further, because IKK is required for NF-{kappa}B transactivation, these data demonstrate that, in human bronchial epithelial cells, MEKK1 is a common upstream activator of both the AP-1 and NF-{kappa}B signaling pathways leading to chemokine expression. We are aware of one previous study examining the function of MEKK in respiratory epithelial cells. In RLE alveolar type II cells, MEKK1 is required for TNF-{alpha}– and oxidant-induced JNK activity and NF-{kappa}B transactivation (43). We now extend these data by elucidating the mechanism by which MEKK1 induces NF-{kappa}B transcriptional activity (i.e., IKK activation), as well as the importance of MEKK1 for AP-1–type responses.

As noted above, MEKK was originally described as an activator of MEK, the upstream activator of ERK (27, 31). Later studies suggested that MEKK1 only induces modest ERK activation in cells, and instead functions as an upstream activator of JNK and p38 (32–35). We were therefore surprised when inhibition of MEKK1 attenuated TNF-{alpha}–induced ERK activation. It is conceivable that overexpression of MEKK1-KM blocks ERK activation by non-specifically binding to MEK1, thereby preventing activation by other upstream activators such as Raf-1. On the other hand, TNF-{alpha} stimulates MEKK but not Raf-1 or B-Raf in mouse macrophages (42), suggesting that, in contrast to growth factor stimulation (32, 35), cytokine stimulation may induce a significant level of MEKK activity that is physiologically important for MEK activation.

While MEKK1 was required for NF-{kappa}B transactivation, expression of a dominant-negative inhibitor of NIK had no effect TNF-{alpha}–induced transcriptional activity. These data are consistent with studies demonstrating TNF-{alpha}–induced responses in NIK-deficient mice (57, 58). On the other hand, NIK has been shown to be required for TNF-{alpha}–induced signaling in cultured human epidermal keratinocytes (59) and HeLa cells (60), suggesting that this requirement may be species or cell type specific.

Our data suggest a model by which Ras and MEKK1 are both required for maximal TNF-{alpha}–induced AP-1 and NF-{kappa}B transactivation. Because Ras was neither required nor sufficient for IKK activation, MEKK1 must not function as a downstream target of Ras in this context (Figure 7). While as noted above MEKK1 may interact with Ras and function as a downstream effector (26–28), it has been shown in HEK293 cells that TNF{alpha} treatment enhances the binding of native TRAF2 to MEKK1, stimulating MEKK1 kinase activity in a Ras-independent manner (61). Laboratory studies examining the upstream activator of MEKK1 in human bronchial epithelial cells are ongoing. In addition, further studies will be needed to determine whether Ras and MEKK1 regulate NF-{kappa}B in bronchial epithelial cells following treatment with other stimuli. For example, in BEAS-2B cells, vanadate, arsenic, zinc and epidermal growth factor each activate Ras, but only vanadate induces activation of NF-{kappa}B (54), suggesting that the regulation of NF-{kappa}B by Ras may be stimulus-specific. Similarly, in RLE alveolar type II cells, Ras is required for oxidant but not TNF-{alpha}–induced NF-{kappa}B activation (43).



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  		Figure 7. Model of AP-1 and NF-{kappa}B transactivation by TNF-{alpha}. Ras and MEKK1 regulate each pathway via the activation of MAP kinases. MEKK1 is required for IKK activation (with subsequent translocation and binding of NF-{kappa}B family transcription factors), whereas Ras is required for maximal NF-{kappa}B transactivation, perhaps via a JNK-dependent pathway.

 
In our model, maximal TNF-{alpha}–induced chemokine expression depends on the activation of two distinct signaling pathways, one constituted in part by AP-1 and the other by NF-{kappa}B. It should be noted that the AP-1 response to TNF-{alpha} is relatively modest, however. Nevertheless, we believe the change in AP-1 activity is significant, for the following reasons. First, mutation of the IL-8 AP-1 site attenuates TNF-{alpha}–induced transcription from the IL-8 promoter (8). Also, TNF-{alpha} treatment induced binding of nuclear proteins to AP-1 sequences. Finally, inhibition of signaling intermediates leading to AP-1 activation (Ras and ERK) attenuates the IL-8 signal. Together, these data suggest that the increase in AP-1 transactivation following TNF-{alpha} treatment is indeed important.

In summary, we have shown that Ras and MEKK1 coregulate AP-1 and NF-{kappa}B-mediated chemokine expression in cultured human bronchial epithelial cells. We speculate that Ras and MEKK1 may represent important targets for therapeutic intervention in asthma.


   Acknowledgments
 
These studies were supported by grants from the National Institutes of Health (HL56399) and the Cystic Fibrosis Foundation. The authors sincerely thank Marsha Rosner, Alan Brasier, Dennis Templeton, Michael Karin, and James Posada for their gifts of plasmid vectors, and Steven White for his gift of 16HBE14o- human bronchial epithelial cells.

Received in original form November 19, 2002

Received in final form January 27, 2003


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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