Role of Ras in metal-induced EGF receptor signaling and NF-kappa B activation in human airway epithelial cells

Weidong Wu1, Ilona Jaspers1, Wenli Zhang1, Lee M. Graves2, and James M. Samet3

1 Center for Environmental Medicine and Lung Biology and 2 Department of Pharmacology, University of North Carolina, Chapel Hill 27599; and 3 Human Studies Division, National Health Effects and Environmental Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, North Carolina 27711


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We showed previously that epithelial growth factor (EGF) receptor (EGFR) signaling is triggered by metallic compounds associated with ambient air particles. Specifically, we demonstrated that As, Zn, and V activated the EGFR tyrosine kinase and the downstream kinases MEK1/2 and ERK1/2. In this study, we examined the role of Ras in EGFR signaling and the nuclear factor-kappa B (NF-kappa B) activation pathway and the possible interaction between these two signaling pathways in a human airway epithelial cell line (BEAS-2B) exposed to As, V, or Zn ions. Each metal significantly increased Ras activity, and this effect was inhibited by the EGFR tyrosine kinase activity inhibitor PD-153035. Adenoviral-mediated overexpression of a dominant-negative mutant form of Ras(N17) significantly blocked MEK1/2 or ERK1/2 phosphorylation in As-, Zn-, or V-exposed BEAS-2B cells but caused little inhibition of V-, Zn- or EGF-induced EGFR tyrosine phosphorylation. This confirmed Ras as an important intermediate effector in EGFR signaling. Interestingly, V, but not As, Zn, or EGF, induced Ikappa Balpha serine phosphorylation, Ikappa Balpha breakdown, and NF-kappa B DNA binding. Moreover, PD-153035 and overexpression of Ras(N17) each significantly blocked V-induced Ikappa Balpha breakdown and NF-kappa B activation, while inhibition of MEK activity with PD-98059 failed to do so. In summary, exposure to As, Zn, and V initiated EGFR signaling and Ras-dependent activation of MEK1/2 and ERK1/2, but only V induced Ras-dependent NF-kappa B nuclear translocation. EGFR signaling appears to cross talk with NF-kappa B signaling at the level of Ras, but additional signals appear necessary for NF-kappa B activation. Together, these data suggest that, in V-treated BEAS-2B cells, Ras-dependent signaling is essential, but not sufficient, for activation of NF-kappa B.

G proteins; epidermal growth factor receptor; nuclear factor-kappa B; mitogen-activated protein kinases; air pollution


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE RAS PROTEINS are the 21-kDa products of the Ras family of genes, which were first identified in retroviruses that trigger sarcoma-type tumors in rats (37). The importance of Ras proteins in growth regulation was recognized in the early 1980s, when it was discovered that ~30% of all human tumors contain an activating mutation in Ras (69). Ras activity is regulated through a GDP-GTP binding cycle, in which guanine nucleotide exchange factors (GEFs) promote the formation of active, GTP-bound Ras, while GTPase-activating proteins accelerate the formation of inactive, GDP-bound Ras (8). Increasing evidence has shown that Ras proteins act as a crucial switching station in intracellular signaling networks that regulate cell growth and differentiation. Ras activation is initiated by activated receptors with intrinsic or associated tyrosine kinase activity. Ras can also be activated through G protein-coupled receptors. Once activated, Ras proteins transmit signals to multiple downstream effectors, which mediate gene expression, remodeling of actin cytoskeleton, cell proliferation, survival, and transformation (69).

A major intracellular signaling pathway mediated by Ras is that initiated by the epidermal growth factor (EGF) receptor (EGFR). Altered regulation of EGFR signaling can lead to pleiotropic cellular responses, including mitogenesis or apoptosis, proliferation, oncogenic transformation, enhanced motility, protein secretion, and differentiation or dedifferentiation (62, 71). Generally, EGFR signaling begins with ligand binding, which leads to dimerization of EGFR monomers, activation of EGFR tyrosine kinase activity, autophosphorylation of EGFR tyrosine residues, and phosphorylation of downstream effectors. The best understood effector pathway is activation of Ras by the recruitment of son-of-sevenless (SOS) to the cell membrane via the adapter proteins Shc and/or Grb2. GTP-bound Ras is capable of interacting with downstream effectors such as the kinase Raf, which ultimately leads to the activation of mitogen-activated protein kinase (MAPK) and the corresponding cellular responses mentioned above (25). Our previous studies demonstrated that certain metals (As, Cu, V, or Zn) are able to activate the EGFR tyrosine kinase, MAPK kinase (MEK), and MAPK [extracellular signal-related kinase (ERK)] in human airway epithelial cells (58, 74). In preliminary studies, overexpression of dominant-negative Ras(N17) was observed to suppress the expression of interleukin (IL)-8 , IL-6, and cyclooxygenase-2 genes in BEAS-2B cells exposed to sodium metavanadate (+5) or vanadyl sulfate (+4) (unpublished observation). Because GTP-Ras is the putative activator of Raf-1 in the presence of certain cofactors (25), we have examined the role of active Ras in EGFR signaling induced by these metals.

As with signaling of Ras, nuclear factor-kappa B (NF-kappa B) has been found to play an important role in metal-induced gene expression of proinflammatory cytokines such as IL-6 and IL-8 in human airway epithelial cells (34, 51). NF-kappa B is a mammalian transcriptional activator that mediates the inducible expression of a wide variety of genes, including products involved in proinflammatory responses (15, 63). In most unstimulated cells, NF-kappa B exists in the cytoplasm mainly as a heterodimer composed of two subunits of 50 and 65 kDa and stays in an inactive form associated with inhibitory proteins termed Ikappa Bs, with Ikappa Balpha being the predominant form (4, 50, 52, 72). NF-kappa B activation is induced by a variety of extracellular stimuli, which activate signal transduction pathways that target the NF-kappa B-Ikappa B complex for disruption (28, 50). Degradation of Ikappa B by the proteasome allows NF-kappa B to translocate into the nucleus, where it binds to specific response elements on the promoter region of certain genes and regulates their transcription (5, 28, 46). Reactive oxygen species (ROS) have been shown to activate NF-kappa B in rat lung epithelial cells in a Ras-dependent manner (32).

In this study, we explored the role of Ras in EGFR signaling and activation of NF-kappa B and the possible interaction between these two signaling pathways during exposure of human airway epithelial cells to the combustion-derived metals As, V, and Zn. We observed that all three metals significantly increased Ras activity and that Ras proved to be a crucial intermediate in signaling from the EGFR tyrosine kinase to the downstream kinases MEK1/2 and ERK1/2. Interestingly, of these three metals, only V caused Ikappa Balpha degradation and NF-kappa B DNA binding, which was significantly suppressed by the EGFR tyrosine kinase activity inhibitor PD-153035 and overexpression of dominant-negative Ras(N17) (11). However, inhibition of MEK activity with PD-98059 failed to inhibit V-induced Ikappa Balpha degradation and NF-kappa B activation, suggesting that V-induced activation of NF-kappa B is EGFR tyrosine kinase and Ras dependent, but MEK independent.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. American Chemical Society-grade metal salts were obtained from Alfa (Ward Hill, MA) or Sigma (St. Louis, MO); tissue culture medium, supplements, and supplies from Clonetics (San Diego, CA); SDS-PAGE supplies, such as molecular-mass standards, polyacrylamide, ready gels, and buffers, from Bio-Rad (Richmond, CA); [gamma -32P]ATP (6,000 Ci/mmol) from Amersham Pharmacia Biotech; specific anti-phospho MEK1/2 (Ser217/221) and anti-Ikappa Balpha (Ser32) antibody from New England Biolabs (Boston, MA); protein A-Sepharose from Pharmacia Biotech (Uppsala, Sweden); specific anti-phospho ERK (Tyr204) antibody, anti-Ikappa Balpha antibody, agarose-conjugated anti-EGFR antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody p-Tyr HRP antibody from Santa Cruz Biotechnology (Santa Cruz, CA); and MEK activity inhibitor PD-98059 (3), EGFR kinase inhibitor PD-153035 (27, 29), and proteasome inhibitor MG-132 (17) from Calbiochem-Novabiochem (La Jolla, CA).

Cell culture and exposure. BEAS-2B (subclone S6) cells were obtained from Drs. Curtis Harris and John Lechner (National Institutes of Health). The BEAS-2B cell line was derived by transforming human bronchial cells with an adenovirus 12-simian virus 40 construct (53). BEAS-2B cells (passages 70-80) were grown to 90-100% confluence on tissue culture-treated Costar 6- or 12-well plates in keratinocyte basal medium (KBM) supplemented with 30 µg/ml bovine pituitary extract, 5 ng/ml human EGF, 500 ng/ml hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, and 5 ng/ml insulin, as described previously (18, 49). Cells were placed in KBM (without supplements) for 20-22 h before treatment with metals. In some experiments, BEAS-2B cells were pretreated with 1 µM PD-153035 for 2 h or 20 µM MG-132 for 30 min before metal exposure.

A suspension of 100 mM sodium arsenite, vanadyl sulfate, and zinc sulfate was prepared in water and used as a stock for dilution into KBM, as described previously (58).

Western blotting. BEAS-2B cells treated with metals were lysed in RIPA buffer (1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS in PBS, pH 7.4) containing 0.1 mM vanadyl sulfate and protease inhibitors (0.5 mg/ml aprotinin, 0.5 mg/ml E-64, 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 mg/ml leupeptin) (58). After normalization for protein content, cell extracts were subjected to SDS-PAGE on 11% gradient polyacrylamide gels or 4-15% Tris · HCl ready gels (Bio-Rad) with a Tris-glycine-SDS buffer. Proteins were electroblotted onto a nitrocellulose membrane. The blots were blocked with 3% nonfat milk, washed briefly, incubated with phospho-specific MEK1/2, ERK1/2, or Ikappa Balpha antibodies in 3% BSA at 4°C overnight, and then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Bands were detected using chemiluminescence reagents and hypersensitive films, as described previously (58).

Immunoprecipitation. Confluent BEAS-2B cells were challenged with metals and lysed with RIPA buffer. The lysate supernatants were precleared with protein A-Sepharose and immunoprecipitated by incubation with agarose-conjugated anti-EGFR antibody for 1 h at 4°C. Immune complexes were washed twice with RIPA buffer and once with cold PBS. The immunoprecipitates were suspended with 25 µl of 4°C sample loading buffer (62.5 mM Tris · HCl, pH 6.8, 10% glycerol, 2% SDS, 0.7 M beta -mercaptoethanol, and 0.05% bromphenol blue) and boiled for 5 min before separation on 4-15% Tris · HCl ready gels. The EGFR tyrosine phosphorylation bands were detected as described previously (74).

Ras activation assay. The Ras activation assay kit was purchased from Upstate Biotechnology (Lake Placid, NY). Ras activity was determined according to the supplier's instruction. Briefly, BEAS-2B cells treated with metals were lysed with 5× Mg2+ lysis/wash buffer (MLB: 125 mM HEPES, pH 7.5, 750 mM NaCl, 5% Igepal CA-630, 50 mM MgCl2, 5 mM EDTA, and 10% glycerol). The lysates were precleared with glutathione agarose. Five microliters of a 50% slurry of Raf-1 Ras binding domain-agarose were incubated with 500-1,000 µg of cell lysate at 4°C for 30 min. The agarose was collected by centrifugation and washed with MLB three times and once with cold PBS and then boiled in 25 µl of reducing sample loading buffer. GTP-bound Ras protein was resolved by electrophoresis, transferred to nitrocellulose, and probed with a mouse monoclonal anti-Ras (clone Ras-10) antibody (1 µg/ml). Protein bands were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a enhanced chemiluminescence detection system (74).

Infection with adenovirus. Ras(N17), a GDP-bound dominant-negative mutant, is used widely as an interfering mutant to assess Ras function in vivo (11, 66). Confluent BEAS-2B cells were infected with a multiplicity of infection of 100 plaque-forming units/cell nonrecombinant control adenovirus ad5CMV3 or recombinant inactive Ras(N17) (obtained from Dr. Craig Logsdon, University of Michigan) for 24 h (48). The infection medium was removed and replaced with KBM for another 24 h before challenge with metals. BEAS-2B cells were lysed, and the lysates were subjected to Western blotting or electrophoretic mobility shift assay (EMSA).

Separation of cytoplasmic and nuclear proteins. BEAS-2B cells treated with metals were lysed with cytoplasmic extract buffer [10 mM Tris · HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT)] containing protease inhibitors (34). Nonidet P-40 (0.1%) was added to the cell lysate, and the lysate was vortexed for 15 s. After centrifugation at 14,000 rpm for 30 s, the supernatant (cytoplasmic fraction) was transferred to another tube and boiled with sample loading buffer to detect the degradation of Ikappa Balpha through Western blotting using rabbit kappa Balpha antibody (Santa Cruz Biotechnology). The nuclear pellets were resuspended with 100 µl of nuclear extract buffer (20 mM Tris · HCl, pH 8.0, 400 mM NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, 25% glycerol, and 1 mM DTT) containing protease inhibitors. After brief centrifugation, the supernatants containing the nuclear fraction were subjected to EMSA.

EMSA. EMSA was conducted as described previously (34). Briefly, an oligonucleotide probe (sequence from 5' to 3': GGCTGGGGATTCCCCATCT) for NF-kappa B binding site on myosin heavy chain class II gene was labeled by incubating 15 U T4 polynucleotide kinase (New England Biolabs, Beverly, MA), 100 ng double-stranded probe, and 100 µCi [gamma -32P]ATP at 37°C for 30 min. Unincorporated 32P was removed using a desalting column (Nuc Trap, Stratagene, San Diego, CA), and DNA-protein binding reactions were performed for 10 min at room temperature in a mixture containing 2 µg nuclear extract, 1 µl labeled probe, 10 µl running buffer (10 mM Tris · HCl, pH 7.5, 50 mM NaCl, 2 mM EDTA, 1 mM DTT, and 5% glycerol), and 2 µg poly(dI-dC) (Roche Molecular Biochemicals). Samples were separated by electrophoresis through 4.5% nondenaturing polyacrylamide gels containing 0.5× Tris-borate-EDTA. Gels were dried, and radiolabeled species were autoradiographed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of Ras in BEAS-2B cells exposed to metals. Ras has been reported to mediate its effects in part through activation of a cascade of kinases: Raf (c-Raf-1, A-Raf, and B-Raf), MEK (MAPK/ERK kinases 1 and 2), and ERK1/2 (21, 22). We previously showed that exposure to As, V, and Zn ions activates the MAPK cascade (MEK/ERK) in BEAS-2B cells (74). We therefore examined whether Ras plays a role in metal-induced activation of the MAPK cascade in BEAS-2B cells.

BEAS-2B cells grown to confluence were deprived of growth factors for 20-22 h and then challenged with 100 µM As, V, or Zn for 1 h. Ras activity was determined using a commercially available Ras activation assay kit, which is based on affinity precipitation of GTP-bound Ras with agarose-conjugated Raf-1-glutathione S-transferase. Western blotting using specific anti-Ras antibody showed that As, V, or Zn exposure induced a marked increase in levels of GTP-bound Ras compared with controls. As expected, EGF strongly activated Ras in BEAS-2B cells (Fig. 1).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Ras activation in BEAS-2B cells exposed to As, V, or Zn. Confluent BEAS-2B cells were depleted of growth factors for 24 h before stimulation with 100 µM As, Cu, V, or Zn for 60 min [epidermal growth factor (EGF) at 100 ng/ml was used as a positive control (Ct)] and lysed. Cell lysates were immunoprecipitated with Raf-1 Ras binding domain-conjugated agarose. Immunoprecipitates were subjected to 4-15% Tris · HCl ready gels. Membranes were blotted with anti-Ras IgG2ak overnight and then incubated with anti-mouse horseradish peroxidase-conjugated rabbit IgG. Results represent a summary of 3 separate experiments.

Ras mediates metal-induced signaling from EGFR tyrosine kinase to MEK1/2. To determine whether Ras activation by metal exposure is dependent on EGFR signaling, we pretreated BEAS-2B cells for 2 h with vehicle alone or 1 µM PD-153035, a specific EGFR tyrosine kinase inhibitor (40), before exposure to As, V, or Zn and measured levels of GTP-bound Ras. As, V, Zn, and EGF each induced an increase in the active GTP-bound Ras formation in BEAS-2B cells (Fig. 2). PD-150335 markedly suppressed Ras activation in BEAS-2B cells exposed to As, V, or Zn, with no detectable change in Ras protein expression. PD-153035 also significantly blocked EGF-induced Ras activation. This implicated EGFR tyrosine kinase activity as a requirement for metal-induced Ras activation in BEAS-2B cells.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   The EGF receptor (EGFR) tyrosine kinase inhibitor PD-153035 blocked Ras activation in BEAS-2B cells exposed to As, V, or Zn. BEAS-2B cells pretreated with 1 µM PD-153035 for 2 h were challenged with metals at 100 µM for 60 min and lysed. Cell lysates were subjected to immunoprecipitation using Raf-1 Ras binding domain-conjugated agarose for detection of active Ras and 10% SDS-PAGE for detection of total Ras protein. GTP-bound Ras was detected as described in MATERIALS AND METHODS. Autoradiograph is representative of 3 separate experiments.

Our previous work suggested that EGFR tyrosine kinase is necessary for metal-induced MEK/ERK activation in BEAS-2B cells (74). Therefore, we next determined whether active Ras is a necessary intermediate in the transduction of signals initiated by exposure to As, V, or Zn through the EGFR and leading downstream to MEK/ERK. With the use of an adenoviral vector for dominant-negative Ras(N17), we were able to determine the relationship between Ras and the MAPK cascade in these cells. To ensure that overexpression of recombinant inactive Ras(N17) takes place in infected BEAS-2B cells, the cells were infected with different multiplicities of infection of nonrecombinant control adenovirus ad5CMV3 or recombinant inactive Ras(N17). Expression of Ras protein was determined using a specific anti-Ras antibody. Similar to a previous report (48), Ras protein was overexpressed in BEAS-2B cells infected with adenovirus encoding a dominant-negative mutated form of Ras [Ras(N17)] compared with the cells infected with control adenovirus (Fig. 3A). Infected BEAS-2B cells were treated with 100 µM As, V, or Zn for 1 h. Phosphorylation of MEK1/2 or ERK1/2 was detected using phospho-specific MEK1/2 or ERK1/2 antibodies. Consistent with our previous reports (58, 74), Western blotting results showed that As, V, or Zn, as well as EGF, the potent inducer of MAPK phosphorylation, induced significant increases in MEK1/2 and ERK1/2 phosphorylation in BEAS-2B cells infected with the nonrecombinant control vector (Fig. 3, B and C). In contrast, in cells overexpressing Ras(N17), the phosphorylation of MEK1/2 and ERK1/2 induced by As, V, or Zn was significantly blocked. This was true of EGF-induced MEK1/2 and ERK1/2 phosphorylation as well. These data suggested that active Ras plays a pivotal role in metal-induced MEK/ERK activation in BEAS-2B cells.


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 3.   Overexpression of dominant-negative Ras(N17) inhibited MEK and ERK, but not EGFR, tyrosine phosphorylation in BEAS cells exposed to As, V, or Zn. A: dose-dependent overexpression of Ras(N17) in BEAS-2B cells. BEAS-2B cells infected with 100 plaque-forming units/cell of Ras(N17) adenovirus were treated with 100 µM As, V, or Zn for 60 min (EGF at 100 ng/ml was used as a positive control). Cell lysates or immunoprecipitates were subjected to 4-15% Tris · HCl ready gels. Phospho MEK (p-MEK, B), ERK (p-ERK, C), or EGFR tyrosine (p-EGFR, D) was probed with corresponding phospho-specific antibodies, and then the membranes were stripped and blotted with anti-MEK, anti-ERK, or anti-EGFR antibodies, respectively. Autoradiographs are representative of 3 separate experiments.

Ras is believed to be downstream of the EGFR; therefore, Ras inhibition should have no inhibitory effect on metal- or EGF-induced EGFR tyrosine phosphorylation. To determine the specificity of the Ras(N17) blockage of MEK/ERK phosphorylation in BEAS-2B cells and whether active Ras is an important transducer from the EGFR to the MAPK cascade, the effect of Ras(N17) overexpression on EGFR tyrosine kinase phosphorylation was measured in cells treated with As, V, Zn, or EGF. As shown in Fig. 3D, As, V, and Zn ions or recombinant EGF induced pronounced phosphorylation of EGFR tyrosine kinase, which was not affected by Ras(N17) overexpression.

Ras(N17) blocked V-induced NF-kappa B DNA binding activity and Ikappa Balpha degradation. Because previous studies showed that some metal ions can activate NF-kappa B, we next studied the role of Ras in NF-kappa B DNA binding activity in cells exposed to As, V, or Zn ions. The possible role of active Ras in metal-induced NF-kappa B DNA binding activity was tested in this study. BEAS-2B cells were infected with control or Ras(N17) adenovirus and challenged with metals. The cells were lysed, and nuclear proteins were extracted for EMSA. Interestingly, among As, V, and Zn, only V induced NF-kappa B DNA binding in BEAS-2B cells, while As, Zn, or EGF treatment did not induce an increase of NF-kappa B DNA binding activity relative to the vehicle control (Fig. 4A), which is consistent with our previous observations in primary human bronchial cells (34). Moreover, V-induced NF-kappa B DNA binding activity was suppressed by the Ras(N17) overexpression (Fig. 4A). The positive control tumor necrosis factor significantly enhanced NF-kappa B DNA binding activity (data not shown). Similar to our previous observations in primary human airway epithelial cells (33), supershift and lysis using specific antibodies against the p50, p65, and c-Rel compounds of the NF-kappa B binding complex identified the V-induced NF-kappa B DNA binding complex in BEAS-2B cells as the p65/p50 heterodimer (Fig. 4B). This suggested that Ras is required for V-induced NF-kappa B nuclear translocation in BEAS-2B cells.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 4.   Overexpression of dominant-negative Ras(N17) blocked nuclear factor-kappa B (NF-kappa B) DNA binding and Ikappa B breakdown in BEAS-2B cells exposed to As, V, or Zn. BEAS cells infected with Ras(N17) adenovirus were treated with 100 µM As, V, or Zn for 60 min (EGF at 100 ng/ml was used as a positive control). A: nuclear proteins were analyzed for NF-kappa B DNA binding using electrophoretic mobility shift assay (EMSA). B: V-induced NF-kappa B DNA-binding complex was further identified by addition of 100-fold excess unlabeled wild-type and mutated NF-kappa B oligonucleotides and antibodies against p65, p50, and c-Rel. C: cytoplasmic proteins were analyzed for Ikappa B breakdown by Western blotting. D: BEAS-2B cells were pretreated with MG-132 for 30 min and then stimulated with 100 µM As, V, or Zn for 60 min. Cytoplasmic protein fraction was separated by SDS-PAGE. Phosphorylated form of Ikappa Balpha (p-Ikappa B) at Ser32 was detected with phospho-specific antibody. Autoradiographs are representative of 3 separate experiments.

Ikappa Balpha degradation is an event that precedes NF-kappa B nuclear translocation (46, 47). To confirm the above observation on NF-kappa B activation, Ikappa Balpha levels were determined in BEAS-2B cells exposed to V. Western blotting using an anti-Ikappa Balpha antibody showed only V-induced Ikappa Balpha degradation in BEAS-2B cells, with no effect in cells exposed to As, Zn, or EGF. In addition, Ras(N17) overexpression significantly blocked V-induced Ikappa Balpha degradation (Fig. 4C). The Ikappa Balpha degradation process consists of a series of well-characterized steps (18). Inducible phosphorylation of Ikappa B at Ser32 or Ser36 leads to the immediate recognition of the NH2 terminus of Ikappa Balpha by an E3 ubiquitin-protein ligase complex (39, 43, 75), which results in the polyubiquitinylation of Ikappa B, leading to rapid degradation of Ikappa Balpha by the 26S proteasome (17, 61). BEAS-2B cells were pretreated with the proteasome inhibitor MG-132, which prevents Ikappa Balpha degradation via the 26S proteasome and, thereby, permits the accumulation of phosphorylated Ikappa Balpha in stimulated cells (17). Pretreated cells were stimulated with As, V, or Zn, and the cytoplasmic protein fractions were separated by SDS-PAGE and immunoblotted with a phospho-specific antibody against the Ser32-phosphorylated form of Ikappa Balpha . As shown in Fig. 4D, only V induced increased phosphorylation of Ikappa Balpha in BEAS-2B cells, which was consistent with the above observation that only V significantly induces Ikappa Balpha degradation and NF-kappa B DNA binding activity.

Effect of an EGFR tyrosine kinase inhibitor, PD-153035, or an MEK activity inhibitor, PD-98059, on V-induced NF-kappa B activation in BEAS-2B cells. NF-kappa B DNA binding activity and Ikappa Balpha phosphorylation and degradation suggested that only V induced NF-kappa B activation in BEAS-2B cells. Moreover, Ras is necessary for V-induced NF-kappa B activation. The EGFR is required for V-induced Ras activation. Therefore, the EGFR tyrosine kinase activity should also be necessary in V-induced NF-kappa B activation. We observed that the selective EGFR tyrosine kinase inhibitor PD-153035 suppressed V-induced Ikappa Balpha degradation and NF-kappa B DNA binding activity in BEAS-2B cells (Fig. 5, top panels). Next, we tested whether MEK, a downstream kinase of Ras, could also contribute to V-induced NF-kappa B nuclear translocation in BEAS-2B cells. Confluent BEAS-2B cells were pretreated with the MEK activity inhibitor PD-98059 and then stimulated with V. Extracted nuclear proteins were then subjected to EMSA for NF-kappa B activation. V induced Ikappa Balpha degradation and NF-kappa B DNA binding. PD-98059 had no inhibitory effect on V-induced Ikappa Balpha degradation and NF-kappa B DNA binding (Fig. 5, bottom panels), suggesting that MEK1/2 does not participate in V-induced NF-kappa B activation in BEAS-2B cells.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5.   EGFR tyrosine kinase activity inhibitor PD-153035, but not MEK activity inhibitor PD-98059, inhibited V-induced NF-kappa B DNA binding and Ikappa Balpha breakdown in BEAS-2B cells. BEAS-2B cells pretreated with 1 µM PD-153035 for 2 h or 50 µM PD-98059 for 30 min were stimulated with 100 µM vanadyl sulfate or 100 ng/ml EGF for 60 min. A: nuclear proteins were analyzed for NF-kappa B DNA binding using EMSA. B: cytoplasmic proteins were analyzed for Ikappa B breakdown by Western blotting using Ikappa B antibody. Autoradiographs are representative of 3 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ras is an important component of multiple signal transduction cascades (69). The data shown here demonstrate that combustion-derived metals such as As, V, and Zn induce Ras activation in human airway epithelial cells. Active Ras not only mediates signaling from the EGFR to the MAPK cascade in response to exposure to As, V, or Zn, but it also plays a pivotal role in V-induced NF-kappa B DNA binding. Although As, V, and Zn can each induce the activation of Ras, only V increases NF-kappa B activity, and it does so in a Ras-dependent manner, suggesting that, in BEAS-2B cells, Ras may be essential, but not sufficient, to induce NF-kappa B activity. Moreover, the MEK activity inhibitor PD-98059 failed to attenuate V-induced NF-kappa B DNA binding activity. These data suggest different mechanisms for signaling initiated by As, V, and Zn in human airway epithelial cells. The proposed mechanisms for metallic ion-induced intracellular signaling in BEAS-2B cells are depicted in Fig. 6.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   Proposed mechanism for metal ion-induced EGFR signaling and NF-kappa B activation in BEAS-2B cells. As, V, and Zn ions each induce activation of EGFR, Ras, MEK, and ERK. Only V induces NF-kappa B activation, an event that bifurcates from MEK activation at the level of Ras.

V ions are known inhibitors of protein tyrosine phosphatases, including those that dephosphorylate signaling kinases (23). Zn (10, 70) and, to a lesser extent, As (14) ions are also reported inhibitors of certain tyrosine phosphatases. Part of the impetus for the work undertaken in this study is the aim to identify the initiating event(s) in metal ion-induced signaling. Our previous studies have suggested that dysregulation of protein tyrosine phosphate metabolism and inhibition of tyrosine phosphatases may be pivotal effects in this process (59, 60). We have shown that exposure to V and Zn, but not As, can cause profound inhibition of multiple tyrosine phosphatases in human airway epithelial cells (59). Thus the possibility exists that the effects of As, V, and Zn ions on signaling to NF-kappa B and/or MEK/ERK are secondary to an inhibitory effect on phosphatases that keeps these proteins in an inactive state in resting cells. In principle, the effect of phosphatase inhibition could occur at any or all levels in a signaling cascade. However, the data we present in this study, showing that As, V, and Zn each signal to MEK/ERK in a Ras-dependent manner, suggest that inhibition to tyrosine phosphatases, if required in the signaling induced by these metal ions, occurs upstream of Ras.

An EGFR tyrosine kinase inhibitor significantly blocked metal-induced Ras activation in human airway epithelial cells, and dominant-negative Ras(N17) posed no detectable effect on EGFR tyrosine kinase activity. These data suggest that EGFR tyrosine kinase is required for Ras activation in BEAS-2B cells exposed to combustion-derived metals. The events involved in the signaling from EGFR to Ras were not examined in this study. It has been documented that Ras activation by the EGFR occurs through adapter proteins and GEFs (8). Furthermore, activation of Ras is responsible for metal-induced MEK and ERK phosphorylation in BEAS-2B cells exposed to metals. One question raised by these findings concerns the mechanism through which Ras transmits the signal from the EGFR to MEK and ERK. Our previous study demonstrated that MEK/ERK phosphorylation induced by As, V, or Zn occurred in a Raf-1-independent manner (74). Observations from mammalian cells indicate that the events downstream of Ras are more complex than simply activating the Raf kinases. Multiple Ras effectors have been recognized, such as p120Ras GAP, PI3KRIN1, AF6, and RalGDS (24, 30, 38, 55, 76). Additionally, MEK kinase (MEKK1), a large (196-kDa) protein, has been shown to be activated by low-molecular-weight GTP-binding proteins (Ras, Rac, and Cdc42) and activates MEK/ERK, JNK, and Ikappa B kinases (44, 57, 78).

Interestingly, in addition to the pivotal role of Ras in the EGFR signaling in BEAS-2B cells exposed to combustion-derived metals, this study also shows that active Ras is necessary for V-induced NF-kB activation. As described previously (33), the crucial step in NF-kappa B activation is the signal-induced proteolytic degradation of Ikappa B in the cytoplasm (47). Ikappa B breakdown is initiated after the phosphorylation of its NH2-terminal serine residues by Ikappa B kinases such as IKK1 and IKK2 (7, 41, 47). V has been shown to induce IKK activity and increase Ikappa Balpha phosphorylation and degradation and NF-kappa B nuclear translocation in different cell types, including human airway epithelial cells (16, 31, 33, 34). Several studies have suggested that IKK1 and IKK2 are themselves phosphorylated and activated by one or more upstream activating kinases such as MEKK1 and NF-kappa B-inducing kinase (6, 19, 42, 54, 67, 73, 79, 80), with NF-kappa B-inducing kinase displaying a preference for IKK1 and MEKK1 a preference for IKK2 (47). In addition, IKK2 activity was also found to be significantly elevated by V in mouse macrophages (16). Therefore, MEKK1, one of the downstream kinases of Ras, could be involved in V-induced IKK2 activation (16), further leading to Ikappa Balpha breakdown and NF-kappa B nuclear translocation. Moreover, the MEK activity inhibitor PD-98059 had no detectable inhibitory effect on V-induced NF-kappa B activation in BEAS-2B cells, which implies that the upstream kinases of MEK, but not MEK itself, are responsible for V-induced NF-kappa B nuclear translocation. Transfection of transformed rat lung epithelial cells with a constitutively active MEKK1 construct or dominant-negative MEKK1 can significantly modulate oxidant-induced NF-kappa B activation (32). It is possible that MEKK1 was involved in the V-induced NF-kappa B activation in BEAS-2B cells. MEKK1 may therefore act as a bifurcation point in V-induced MEK/ERK activation and NF-kappa B nuclear translocation.

Ras-dependent NF-kappa B activation induced by diverse stimuli has been reported (32, 33, 40). It is intriguing that, although As, V, and Zn are able to induce Ras activation in BEAS-2B cells, only V induces NF-kappa B activation. In one study, Ras(N17) failed to block tumor necrosis factor-alpha -induced NF-kappa B activation in rat lung epithelial cells (32). Together, these observations suggest that active Ras is not sufficient for V-induced NF-kappa B DNA binding activity in BEAS-2B cells and that input from other signaling cascades or cofactors is necessary to activate NF-kappa B. In preliminary work, As and Zn were unable to induce oxidative stress in BEAS-2B cells. However, vanadyl sulfate (+4)- or vanadate (+5)-induced NF-kappa B nuclear translocation could be blocked by overexpression of catalase or the antioxidant N-acetyl-L-cysteine, respectively (33, 77), suggesting a crucial role for ROS in V-induced NF-kappa B activation. Additional evidence has shown that oxidant-induced NF-kappa B activation is Ras dependent in transformed rat lung epithelial cells (32). Therefore, it is possible that generation of ROS, which are induced by V, but not by As or Zn, contributes to the additional signals required for NF-kappa B activation (7). The mechanisms for synergism of Ras with ROS in V-induced NF-kappa B DNA binding in BEAS cells are not known. However, it has recently been demonstrated that the role of ROS in NF-kappa B activation is cell type or stimulus specific, and its role may be facilitatory, rather than causal (9).

In addition to its ability to generate ROS, other factors may also explain the effect of V on NF-kappa B DNA binding relative to that of As or Zn exposure. Arsenite has been reported to directly inhibit the activity of IKK and, thereby, limit the phosphorylation and degradation of Ikappa Balpha (56). This is consistent with our data showing that As exposure of BEAS-2B cells does not lead to Ikappa B degradation (34). Furthermore, As and other thiol-reactive metals, including Zn, have been found to inhibit NF-kappa B DNA binding through interactions with critical protein sulfhydryls (64).

In addition to Ras, members of the Ras superfamily of GTP-binding proteins such as Rho and Rac, also regulate a variety of signal transduction pathways in eukaryotic cells (20). It has recently been reported that, in certain circumstances, Rac is required for NF-kappa B activation (12). However, in our preliminary study, we did not observe Rac activation in BEAS-2B cells exposed to As, V, or Zn (data not shown).

Metal ions can be abundant in airborne particulate matter (PM) (1, 2, 45). It has been argued that metals contribute to PM-induced pulmonary inflammatory gene expression (13, 26, 34-36, 65, 68). The role of Ras in metal-induced EGF signaling and NF-kappa B activation in human airway epithelial cells may provide an important link in the elucidation of the mechanism of toxicity of PM inhalation.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of L. Dailey and advice from Dr. W. Reed.


    FOOTNOTES

This work was funded in part by US Environmental Protection Agency Grant 824915.

The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names constitute endorsement or recommendation for use.

Address for reprint requests and other correspondence: J. M. Samet, Human Studies Div., National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711 (E-mail: Samet.jim{at}epa.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00390.2001

Received 13 March 2001; accepted in final form 26 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aastrup, P, Riget F, Dietz R, and Asmund G. Lead, zinc, cadmium, mercury, selenium and copper in Greenland caribou and reindeer (Rangifer tarandus). Sci Total Environ 245: 149-159, 2000[ISI][Medline].

2.   Adamson, IY, Prieditis H, Hedgecock C, and Vincent R. Zinc is the toxic factor in the lung response to an atmospheric particulate sample. Toxicol Appl Pharmacol 166: 111-119, 2000[ISI][Medline].

3.   Alessi, DR, Cuenda A, Cohen P, Dudley DT, and Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270: 27489-27494, 1995[Abstract/Free Full Text].

4.   Baeuerle, PA, and Baltimore D. NF-kappa B: ten years after. Cell 87: 13-20, 1996[ISI][Medline].

5.   Baldwin, AS, Jr. The NF-kappa B and Ikappa B proteins: new discoveries and insights. Annu Rev Immunol 14: 649-683, 1996[ISI][Medline].

6.   Bhullar, IS, Li YS, Miao H, Zandi E, Kim M, Shyy JY, and Chien S. Fluid shear stress activation of Ikappa B kinase is integrin-dependent. J Biol Chem 273: 30544-30549, 1998[Abstract/Free Full Text].

7.   Bonizzi, G, Piette J, Merville MP, and Bours V. Cell type-specific role for reactive oxygen species in nuclear factor-kappa B activation by interleukin-1. Biochem Pharmacol 59: 7-11, 2000[ISI][Medline].

8.   Bourne, HR, Sanders DA, and McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature 349: 117-127, 1991[ISI][Medline].

9.   Bowie, A, and O'Neill LA. Oxidative stress and nuclear factor-kappa B activation: a reassessment of the evidence in the light of recent discoveries. Biochem Pharmacol 59: 13-23, 2000[ISI][Medline].

10.   Brautigan, DL, Bornstein P, and Gallis B. Phosphotyrosyl-protein phosphatase. Specific inhibition by Zn. J Biol Chem 256: 6519-6522, 1981[Abstract/Free Full Text].

11.   Cai, H, and Cooper GM. Inducible expression of Ras N17 dominant inhibitory protein. Methods Enzymol 255: 230-237, 1995[ISI][Medline].

12.   Cammarano, MS, and Minden A. Dbl and the Rho GTPases activate NFkappa B by Ikappa B kinase (IKK)-dependent and IKK-independent pathways. J Biol Chem 276: 25876-25882, 2001[Abstract/Free Full Text].

13.   Carter, JD, Ghio AJ, Samet JM, and Devlin RB. Cytokine production by human airway epithelial cells after exposure to an air pollution particle is metal-dependent. Toxicol Appl Pharmacol 146: 180-188, 1997[ISI][Medline].

14.   Cavigelli, M, Li WW, Lin A, Su B, Yoshioka K, and Karin M. The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase. EMBO J 15: 6269-6279, 1996[Abstract].

15.   Chabot-Fletcher, M. A role for transcription factor NF-kappa B in inflammation. Inflamm Res 46: 1-2, 1997[ISI][Medline].

16.   Chen, F, Demers LM, Vallyathan V, Ding M, Lu Y, Castranova V, and Shi X. Vanadate induction of NF-kappa B involves Ikappa B kinase-beta and SAPK/ERK kinase 1 in macrophages. J Biol Chem 274: 20307-20312, 1999[Abstract/Free Full Text].

17.   Chen, Z, Hagler J, Palombella VJ, Melandri F, Scherer D, Ballard D, and Maniatis T. Signal-induced site-specific phosphorylation targets Ikappa Balpha to the ubiquitin-proteasome pathway. Genes Dev 9: 1586-1597, 1995[Abstract].

18.   Devlin, RB, McKinnon KP, Noah T, Becker S, and Koren HS. Ozone-induced release of cytokines and fibronectin by alveolar macrophages and airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 266: L612-L619, 1994[Abstract/Free Full Text].

19.   DiDonato, JA, Hayakawa M, Rothwarf DM, Zandi E, and Karin M. A cytokine-responsive Ikappa B kinase that activates the transcription factor NF-kappa B. Nature 388: 548-554, 1997[ISI][Medline].

20.   Diekmann, D, Abo A, Johnston C, Segal AW, and Hall A. Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity. Science 265: 531-533, 1994[ISI][Medline].

21.   Egan, SE, Giddings BW, Brooks MW, Buday L, Sizeland AM, and Weinberg RA. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature 363: 45-51, 1993[ISI][Medline].

22.   Egan, SE, and Weinberg RA. The pathway to signal achievement. Nature 365: 781-783, 1993[ISI][Medline].

23.   Fauman, EB, and Saper MA. Structure and function of the protein tyrosine phosphatases. Trends Biochem Sci 21: 413-417, 1996[ISI][Medline].

24.   Feig, LA, Urano T, and Cantor S. Evidence for a Ras/Ral signaling cascade. Trends Biochem Sci 21: 438-441, 1996[ISI][Medline].

25.   Force, T, and Bonventre JV. Growth factors and mitogen-activated protein kinases. Hypertension 31: 152-161, 1998[Abstract/Free Full Text].

26.   Frampton, MW, Ghio AJ, Samet JM, Carson JL, Carter JD, and Devlin RB. Effects of aqueous extracts of PM10 filters from the Utah valley on human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 277: L960-L967, 1999[Abstract/Free Full Text].

27.   Fry, DW, Kraker AJ, McMichael A, Ambroso LA, Nelson JM, Leopold WR, Connors RW, and Bridges AJ. A specific inhibitor of the epidermal growth factor receptor tyrosine kinase. Science 265: 1093-1095, 1994[ISI][Medline].

28.   Ghosh, S, May MJ, and Kopp EB. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 16: 225-260, 1998[ISI][Medline].

29.   Hamilton, M, and Wolfman A. Oncogenic Ha-Ras-dependent mitogen-activated protein kinase activity requires signaling through the epidermal growth factor receptor. J Biol Chem 273: 28155-28162, 1998[Abstract/Free Full Text].

30.   Han, J, Luby-Phelps K, Das B, Shu X, Xia Y, Mosteller RD, Krishna UM, Falck JR, White MA, and Broek D. Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 279: 558-560, 1998[Abstract/Free Full Text].

31.   Huang, C, Ding M, Li J, Leonard SS, Rojanasakul Y, Castranova V, Vallyathan V, Ju G, and Shi X. Vanadium-induced NFAT activation through hydrogen peroxide. J Biol Chem 5: 5, 2001.

32.   Janssen-Heininger, YM, Macara I, and Mossman BT. Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-kappa B: requirement of Ras/mitogen-activated protein kinases in the activation of NF-kappa B by oxidants. Am J Respir Cell Mol Biol 20: 942-952, 1999[Abstract/Free Full Text].

33.   Jaspers, I, Samet JM, Erzurum S, and Reed W. Vanadium-induced kappa B-dependent transcription depends upon peroxide-induced activation of the p38 mitogen-activated protein kinase. Am J Respir Cell Mol Biol 23: 95-102, 2000[Abstract/Free Full Text].

34.   Jaspers, I, Samet JM, and Reed W. Arsenite exposure of cultured airway epithelial cells activates kappa B-dependent interleukin-8 gene expression in the absence of nuclear factor-kappa B nuclear translocation. J Biol Chem 274: 31025-31033, 1999[Abstract/Free Full Text].

35.   Jiang, N, Dreher KL, Dye JA, Li Y, Richards JH, Martin LD, and Adler KB. Residual oil fly ash induces cytotoxicity and mucin secretion by guinea pig tracheal epithelial cells via an oxidant-mediated mechanism. Toxicol Appl Pharmacol 163: 221-230, 2000[ISI][Medline].

36.   Kodavanti, UP, Hauser R, Christiani DC, Meng ZH, McGee J, Ledbetter A, Richards J, and Costa DL. Pulmonary responses to oil fly ash particles in the rat differ by virtue of their specific soluble metals. Toxicol Sci 43: 204-212, 1998[Abstract].

37.  Krauss G. Signal transmission via Ras proteins. In: Biochemistry of Signal Transduction and Regulation, translated by Schonbrunnner N and Cooper J. Weinheim, Germany: Wiley, 1999, p. 324-343.

38.   Kuriyama, M, Harada N, Kuroda S, Yamamoto T, Nakafuku M, Iwamatsu A, Yamamoto D, Prasad R, Croce C, Canaani E, and Kaibuchi K. Identification of AF-6 and canoe as putative targets for Ras. J Biol Chem 271: 607-610, 1996[Abstract/Free Full Text].

39.   Laney, JD, and Hochstrasser M. Substrate targeting in the ubiquitin system. Cell 97: 427-430, 1999[ISI][Medline].

40.   Li, JD, Feng W, Gallup M, Kim JH, Gum J, Kim Y, and Basbaum C. Activation of NF-kappa B via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells. Proc Natl Acad Sci USA 95: 5718-5723, 1998[Abstract/Free Full Text].

41.   Ling, L, Cao Z, and Goeddel DV. NF-kappa B-inducing kinase activates IKK-alpha by phosphorylation of Ser176. Proc Natl Acad Sci USA 95: 3792-3797, 1998[Abstract/Free Full Text].

42.   Malinin, NL, Boldin MP, Kovalenko AV, and Wallach D. MAP3K-related kinase involved in NF-kappa B induction by TNF, CD95 and IL-1. Nature 385: 540-544, 1997[ISI][Medline].

43.   Maniatis, T. A ubiquitin ligase complex essential for the NF-kappa B, Wnt/Wingless, and Hedgehog signaling pathways. Genes Dev 13: 505-510, 1999[Free Full Text].

44.   Marshall, MS. Ras target proteins in eukaryotic cells. FASEB J 9: 1311-1318, 1995[Abstract/Free Full Text].

45.   Martin, CJ, Le XC, Guidotti TL, Yalcin S, Chum E, Audette RJ, Liang C, Yuan B, Zhang X, and Wu J. Zinc exposure in Chinese foundry workers. Am J Ind Med 35: 574-580, 1999[ISI][Medline].

46.   May, MJ, and Ghosh S. Signal transduction through NF-kappa B. Immunol Today 19: 80-88, 1998[ISI][Medline].

47.   Mercurio, F, and Manning AM. Multiple signals converging on NF-kappa B. Curr Opin Cell Biol 11: 226-232, 1999[ISI][Medline].

48.   Nicke, B, Tseng MJ, Fenrich M, and Logsdon CD. Adenovirus-mediated gene transfer of RasN17 inhibits specific CCK actions on pancreatic acinar cells. Am J Physiol Gastrointest Liver Physiol 276: G499-G506, 1999[Abstract/Free Full Text].

49.   Noah, TL, Paradiso AM, Madden MC, McKinnon KP, and Devlin RB. The response of a human bronchial epithelial cell line to histamine: intracellular calcium changes and extracellular release of inflammatory mediators. Am J Respir Cell Mol Biol 5: 484-492, 1991[ISI][Medline].

50.   Norris, JL, and Baldwin AS, Jr. Oncogenic Ras enhances NF-kappa B transcriptional activity through Raf-dependent and Raf-independent mitogen-activated protein kinase signaling pathways. J Biol Chem 274: 13841-13846, 1999[Abstract/Free Full Text].

51.   Quay, JL, Reed W, Samet J, and Devlin RB. Air pollution particles induce IL-6 gene expression in human airway epithelial cells via NF-kappa B activation. Am J Respir Cell Mol Biol 19: 98-106, 1998[Abstract/Free Full Text].

52.   Rahman, I, and MacNee W. Role of transcription factors in inflammatory lung diseases. Thorax 53: 601-612, 1998[Free Full Text].

53.   Reddel, RR, Ke Y, Kaighn ME, Malan-Shibley L, Lechner JF, Rhim JS, and Harris CC. Human bronchial epithelial cells neoplastically transformed by v-Ki-ras: altered response to inducers of terminal squamous differentiation. Oncogene Res 3: 401-408, 1988[Medline].

54.   Regnier, CH, Song HY, Gao X, Goeddel DV, Cao Z, and Rothe M. Identification and characterization of an Ikappa B kinase. Cell 90: 373-383, 1997[ISI][Medline].

55.   Rodriguez-Viciana, P, Warne PH, Dhand R, Vanhaesebroeck B, Gout I, Fry MJ, Waterfield MD, and Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370: 527-532, 1994[ISI][Medline].

56.   Roussel, RR, and Barchowsky A. Arsenic inhibits NF-kappa B-mediated gene transcription by blocking Ikappa B kinase activity and Ikappa Balpha phosphorylation and degradation. Arch Biochem Biophys 377: 204-212, 2000[ISI][Medline].

57.   Russell, M, Lange-Carter CA, and Johnson GL. Direct interaction between Ras and the kinase domain of mitogen-activated protein kinase kinase kinase (MEKK1). J Biol Chem 270: 11757-11760, 1995[Abstract/Free Full Text].

58.   Samet, JM, Graves LM, Quay J, Dailey LA, Devlin RB, Ghio AJ, Wu W, Bromberg PA, and Reed W. Activation of MAPKs in human bronchial epithelial cells exposed to metals. Am J Physiol Lung Cell Mol Physiol 275: L551-L558, 1998[Abstract/Free Full Text].

59.   Samet, JM, Silbajoris R, Wu W, and Graves LM. Tyrosine phosphatases as targets in metal-induced signaling in human airway epithelial cells. Am J Respir Cell Mol Biol 21: 357-364, 1999[Abstract/Free Full Text].

60.   Samet, JM, Stonehuerner J, Reed W, Devlin RB, Dailey LA, Kennedy TP, Bromberg PA, and Ghio AJ. Disruption of protein tyrosine phosphate homeostasis in bronchial epithelial cells exposed to oil fly ash. Am J Physiol Lung Cell Mol Physiol 272: L426-L432, 1997[Abstract/Free Full Text].

61.   Scherer, DC, Brockman JA, Chen Z, Maniatis T, and Ballard DW. Signal-induced degradation of Ikappa Balpha requires site-specific ubiquitination. Proc Natl Acad Sci USA 92: 11259-11263, 1995[Abstract].

62.   Seger, R, and Krebs EG. The MAPK signaling cascade. FASEB J 9: 726-735, 1995[Abstract/Free Full Text].

63.   Sha, WC, Liou HC, Tuomanen EI, and Baltimore D. Targeted disruption of the p50 subunit of NF-kappa B leads to multifocal defects in immune responses. Cell 80: 321-330, 1995[ISI][Medline].

64.   Shumilla, JA, Wetterhahn KE, and Barchowsky A. Inhibition of NF-kappa B binding to DNA by chromium, cadmium, mercury, zinc, and arsenite in vitro: evidence of a thiol mechanism. Arch Biochem Biophys 349: 356-362, 1998[ISI][Medline].

65.   Silbajoris, R, Ghio AJ, Samet JM, Jaskot R, Dreher KL, and Brighton LE. In vivo and in vitro correlation of pulmonary MAP kinase activation following metallic exposure. Inhal Toxicol 12: 453-468, 2000[ISI][Medline].

66.   Stewart, S, and Guan KL. The dominant negative Ras mutant, N17Ras, can inhibit signaling independently of blocking Ras activation. J Biol Chem 275: 8854-8862, 2000[Abstract/Free Full Text].

67.   Uhlik, M, Good L, Xiao G, Harhaj EW, Zandi E, Karin M, and Sun SC. NF-kappa B-inducing kinase and Ikappa B kinase participate in human T-cell leukemia virus I Tax-mediated NF-kappa B activation. J Biol Chem 273: 21132-21136, 1998[Abstract/Free Full Text].

68.   Veronesi, B, Oortgiesen M, Carter JD, and Devlin RB. Particulate matter initiates inflammatory cytokine release by activation of capsaicin and acid receptors in a human bronchial epithelial cell line. Toxicol Appl Pharmacol 154: 106-115, 1999[ISI][Medline].

69.   Vojtek, AB, and Der CJ. Increasing complexity of the Ras signaling pathway. J Biol Chem 273: 19925-19928, 1998[Free Full Text].

70.   Wang, Y, and Pallen CJ. Expression and characterization of wild-type, truncated, and mutant forms of the intracellular region of the receptor-like protein tyrosine phosphatase HPTP-beta . J Biol Chem 267: 16696-16702, 1992[Abstract/Free Full Text].

71.   Wells, A. EGF receptor. Int J Biochem Cell Biol 31: 637-643, 1999[ISI][Medline].

72.   Whiteside, ST, and Israel A. Ikappa B proteins: structure, function and regulation. Semin Cancer Biol 8: 75-82, 1997[ISI][Medline].

73.   Woronicz, JD, Gao X, Cao Z, Rothe M, and Goeddel DV. Ikappa B kinase-beta : NF-kappa B activation and complex formation with Ikappa B kinase-alpha and NIK. Science 278: 866-869, 1997[Abstract/Free Full Text].

74.   Wu, W, Graves LM, Jaspers I, Devlin RB, Reed W, and Samet JM. Activation of the EGF receptor signaling pathway in human airway epithelial cells exposed to metals. Am J Physiol Lung Cell Mol Physiol 277: L924-L931, 1999[Abstract/Free Full Text].

75.   Yaron, A, Hatzubai A, Davis M, Lavon I, Amit S, Manning AM, Andersen JS, Mann M, Mercurio F, and Ben-Neriah Y. Identification of the receptor component of the Ikappa Balpha -ubiquitin ligase. Nature 396: 590-594, 1998[ISI][Medline].

76.   Yatani, A, Okabe K, Polakis P, Halenbeck R, McCormick F, and Brown AM. Ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K+ channels. Cell 61: 769-776, 1990[ISI][Medline].

77.   Ye, J, Ding M, Zhang X, Rojanasakul Y, Nedospasov S, Vallyathan V, Castranova V, and Shi X. Induction of TNF-alpha in macrophages by vanadate is dependent on activation of transcription factor NF-kappa B and free radical reactions. Mol Cell Biochem 198: 193-200, 1999[ISI][Medline].

78.   Yujiri, T, Fanger GR, Garrington TP, Schlesinger TK, Gibson S, and Johnson GL. MEK kinase 1 (MEKK1) transduces c-Jun NH2-terminal kinase activation in response to changes in the microtubule cytoskeleton. J Biol Chem 274: 12605-12610, 1999[Abstract/Free Full Text].

79.   Zandi, E, Chen Y, and Karin M. Direct phosphorylation of Ikappa B by IKKalpha and IKKbeta : discrimination between free and NF-kappa B-bound substrate. Science 281: 1360-1363, 1998[Abstract/Free Full Text].

80.   Zandi, E, Rothwarf DM, Delhase M, Hayakawa M, and Karin M. The Ikappa B kinase complex (IKK) contains two kinase subunits, IKKalpha and IKKbeta , necessary for Ikappa B phosphorylation and NF-kappa B activation. Cell 91: 243-252, 1997[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 282(5):L1040-L1048