Vanadium-induced HB-EGF expression in human lung fibroblasts is oxidant dependent and requires MAP kinases

Jennifer L. Ingram1, Annette B. Rice1, Janine Santos2, Bennett Van Houten2, and James C. Bonner1

Laboratories of 1 Pulmonary Pathobiology and 2 Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709


    ABSTRACT
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ABSTRACT
INTRODUCTION
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Vanadium pentoxide (V2O5) is a transition metal derived from the burning of petrochemicals that causes airway fibrosis and remodeling. Vanadium compounds activate many intracellular signaling pathways via the generation of hydrogen peroxide (H2O2) or other reactive oxygen species. In this study, we investigated the regulation of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in human lung fibroblasts after V2O5 treatment. V2O5-induced HB-EGF mRNA expression was abolished by N-acetyl-L-cysteine, suggesting an oxidant-mediated effect. Exogenous H2O2 (>10 µM) mimicked the effect of V2O5 in upregulating HB-EGF expression. Fibroblasts spontaneously released low levels of H2O2 (1-2 µM), and the addition of V2O5 depleted the endogenous H2O2 pool within minutes. V2O5 caused a subsequent increase of H2O2 into the culture medium at 12 h. However, the burst of V2O5-induced H2O2 occurred after V2O5-induced HB-EGF mRNA expression at 3 h, indicating that the V2O5-stimulated H2O2 burst did not mediate HB-EGF expression. Either V2O5 or H2O2 activated ERK-1/2 and p38 MAP kinase. Inhibitors of the ERK-1/2 pathway (PD-98059) or p38 MAP kinase (SB-203580) significantly reduced either V2O5- or H2O2-induced HB-EGF expression. These data indicate that vanadium upregulates HB-EGF via ERK and p38 MAP kinases. The induction of HB-EGF is not related to a burst of H2O2 in V2O5 treated cells, yet the action of V2O5 in upregulating HB-EGF is oxidant dependent and could be due to the reaction of V2O5 with endogenous H2O2.

pulmonary fibrosis; fibroblast; growth factors; reactive oxygen species; heparin-binding epidermal growth factor-like growth factor


    INTRODUCTION
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INTRODUCTION
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THE PROLIFERATION OF LUNG FIBROBLASTS and their subsequent deposition of collagen are central features in the development of airway fibrosis in asthma or bronchitis (2, 3). Inflammation following lung injury by environmental factors that cause fibrosis has been shown to be associated with the generation of reactive oxygen species (ROS) such as superoxide anion, hydroxyl radical (·OH), and H2O2 (30). ROS have a profound influence on fibroblast function by serving as intermediates in intracellular signaling cascades (1), as well as contributing to apoptosis (4) and the regulation of gene expression (19). Genes that can be induced by ROS include proinflammatory cytokines such as interleukin (IL)-6, IL-8, and macrophage inflammatory protein (MIP)-2 (8, 33), as well as growth factors that stimulate fibroblast proliferation, including transforming growth factor-alpha (25, 40) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (27, 40). In particular, we recently reported that HB-EGF is the major mitogen for human lung fibroblasts in the conditioned medium from cultured normal human bronchial epithelial cells (43).

HB-EGF is a 19-23-kDa glycoprotein member of the EGF family. Like other members of the EGF family, HB-EGF is synthesized as a membrane-anchored growth factor, which binds to and phosphorylates the EGF receptor (erbB1), as well as erbB4 (10, 15). On processing by metalloproteinases, 12-O-tetradecanoylphorbol-13-acetate, or G protein-mediated signaling, the extracellular portion of the HB-EGF protein is cleaved from the transmembrane domain, yielding a mature, soluble form that is shed extracellularly (13, 14, 33). The soluble form of HB-EGF has been shown to stimulate mitogenesis in several different cell types, including fibroblasts (20). HB-EGF is expressed in a variety of tissues and cultured cells, including smooth muscle cells, vascular endothelial cells, tumor cells, and epithelial cells (29). Upregulation of HB-EGF mRNA expression occurs in response to a variety of endogenous factors (e.g., TNF-alpha , IL-1beta , and PDGF) as well as in response to external stimuli (e.g., bacterial lipopolysaccharide) (11, 29).

Recent investigations have concluded that HB-EGF protein and gene expression levels are upregulated in response to either exogenous or accumulated intracellular ROS in epithelial and smooth muscle cell types (20, 27, 32). For example, Miyazaki et al. (26, 27) demonstrated that exogenously added H2O2 stimulates HB-EGF mRNA and cellularly secreted protein expression in rat gastric epithelial cells via tyrosine phosphorylation of the EGF receptor. In addition, several studies have indicated a role for HB-EGF in protecting cells from oxidative stress-induced apoptosis (12, 21, 36). These studies suggest that HB-EGF could play an important role in the inflammatory and fibroproliferative responses in the lung mediated by ROS.

We previously reported that vanadium pentoxide (V2O5), a transition metal derived from the burning of petrochemicals, causes human airway epithelial cells to secrete HB-EGF (43). Furthermore, the conditioned media from these human airway epithelial cells stimulate the mitogenesis of normal human lung fibroblasts, and an HB-EGF-neutralizing antibody blocks this effect. However, it is currently not known whether or not V2O5 mediates HB-EGF expression via the production of ROS. Moreover, there is currently no information on the induction of HB-EGF in lung cells by ROS. In the present study, we investigated the mechanisms of HB-EGF production in normal human lung fibroblasts in response to V2O5 or H2O2. We show that V2O5- and H2O2-induced HB-EGF requires the activation of ERK and p38 MAPKs. We also provide evidence that V2O5-induced HB-EGF expression is oxidant dependent yet does not require a major burst of H2O2 in vanadium-treated cells that occurs after HB-EGF expression.


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Reagents. V2O5 and TiO2 were purchased from Aldrich Chemical (Milwaukee, WI). H2O2 and N-acetyl-L-cysteine (NAC) were obtained from Sigma (St. Louis, MO). Recombinant human HB-EGF and anti-human HB-EGF antibodies were purchased from R&D Systems (Minneapolis, MN). Phospho-p38 and p38 MAPK antibodies were purchased from Cell Signaling Technology (Beverly, MA). The p38 MAPK inhibitor (SB-203580) was obtained from Calbiochem (La Jolla, CA).

Northern blot analysis. Normal adult human lung fibroblasts (ATCC 16 Lu) were purchased from American Type Culture Collection (Rockville, MD). Fibroblasts were seeded into 175-cm2 plastic culture dishes and grown to confluence in 20% fetal bovine serum/Dulbecco's modified Eagle's medium, then trypsinized, and seeded into 150-mm dishes. Confluent cell monolayers were rendered quiescent in serum-free defined media (SFDM) for 24 h before treatment for 3 h with V2O5, H2O2, or SFDM alone (control). Cells were pretreated for 1 h with 50 mM NAC or 10 µM SB-203580, a p38 MAPK inhibitor, before exposure to V2O5, H2O2, or SFDM alone. Total RNA was isolated using the Qiagen Rneasy Midiprep kit (Valencia, CA). A total of 20 µg of each sample were separated in 1% agarose/glyoxal gels and capillary transferred onto BrightStar-Plus positively charged nylon membranes (Ambion, Austin, TX). Dr. Judith Abraham (Scios, Sunnyvale, CA) kindly provided a human HB-EGF cDNA probe. We labeled the probe with [alpha 32P]deoxycytidine triphosphate using a DECAprime II DNA labeling kit (Ambion). We performed the hybridization and washing procedures for blotting using the Northern Max-Gly kit according to the manufacturer's protocol (Ambion).

Western blot analysis. Human lung fibroblasts were grown to confluence in 100-mm dishes and growth arrested in SFDM for 24 h. The cells were treated with 500 µM H2O2 for a time course. We collected cell lysates by washing the cells once with phosphate-buffered saline (PBS) on ice, and 200 µl of lysis buffer (50 mM Tris · HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.25% sodium deoxycholate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM sodium vanadate, and 1 mM sodium fluoride) were added to the cells. The cells were scraped from the dish in the lysis buffer, and the lysates were sonicated for 1 min and spun in a microfuge at maximum speed for 5 min to fractionate insoluble DNA and chromatin proteins from the soluble cellular proteins in the lysis. The samples were separated by SDS-PAGE, transferred to nitrocellulose membrane, and blocked for 1 h in 0.5% nonfat milk in Tris-buffered saline (20 mM Tris, 137 mM NaCl, and 0.1% Tween 20). The blots were then incubated at 4°C overnight in a 1:1,000 dilution of either anti-phospho p38 MAPK, anti-p38 MAPK, or anti-human HB-EGF antibody followed by incubation for 90 min in a 1:2,000 dilution of appropriate horseradish peroxidase-conjugated secondary antibody. The immunoblot signal was detected and visualized through enhanced chemiluminescence.

HB-EGF ELISA. To quantitate the HB-EGF protein that remained bound within the cell membranes, we performed an ELISA on lysed human lung fibroblasts. Cells were grown to confluence in 100-mm dishes and growth arrested in SFDM for 24 h. The cells were treated with 500 µM H2O2 for a time course. We collected cell lysates by washing the cells once with PBS on ice, and 200 µl of lysis buffer (50 mM Tris · HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 0.25% sodium deoxycholate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM sodium vanadate, and 1 mM sodium fluoride) were added to the cells. The cells were scraped from the dish in the lysis buffer. The lysed cells were sonicated for 1 min and spun in a microfuge at maximum speed for 5 min to fractionate insoluble DNA and chromatin proteins from the soluble cellular proteins in the lysis. A serial dilution of human recombinant HB-EGF (0.03-128 ng/ml) was added 60 µl/well in 96-well Immulon-4 plates (Dynatech Laboratories, Chantilly, VA) and incubated overnight at 4°C. We emptied the plates by tapping them over a paper towel, and blocking buffer [3% bovine serum albumin (BSA) in PBS-Tween with 0.1% sodium azide] 200 µl/well was added. The plates were then incubated for 1.5 h at 37°C. After washing the plates four times in PBS-Tween, we added 50 µl/well of 1 µg/ml of goat anti-human HB-EGF (R&D Systems), and the plates were incubated at 4°C overnight. The plates were washed again four times in PBS-Tween, and 50 µl/well of 1:50,000 anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) in dilution buffer (1% BSA-PBS-Tween with 0.1% sodium azide) were added. The plates were incubated for 1 h at 25°C. The plates were again washed in PBS-Tween four times, and 50 ul/well of 1:1,000 streptavidin-alkaline phosphatase (Jackson ImmunoResearch) in dilution buffer were added. The plates were incubated at 25°C for 1 h. The plates were again washed four times in PBS-Tween. The ELISA was developed with an alkaline-phosphatase substrate kit (Bio-Rad, Hercules, CA). The reactions were stopped with the addition of 50 µl/well of 2 N NaOH, and absorbance at 405 nm was read with a Dynatech MR 5000 microplate reader.

Assay of H2O2 in cell supernatants. The Amplex Red H2O2 kit (Molecular Probes, catalog no. A12212) was used to measure H2O2 released by the cells into the culture medium. The Amplex Red assay is based on the detection of H2O2 using 10-acetyl-7-hydroxiphenoxazine, a highly stable and specific probe for H2O2 (45). In the presence of horseradish peroxidase (HRP), Amplex Red reacts with H2O2 with a 1:1 stoichiometry, producing a highly fluorescent product, resofurin. The Amplex Red assay is more reliable than a commonly used 2',7'-dichlorofluorescein fluorometric assay, which is dependent on endogenous peroxidase and subject to artifacts of autooxidation (31). For the Amplex Red assay, confluent pulmonary myofibroblasts were rendered quiescent in SFDM for 24 h before treatment with V2O5 in fresh SFDM. Aliquots of the medium were collected at various times post-V2O5 exposure. H2O2 was detected by loading 100 µl of the cell supernatant in a microtiter plate and incubated for 30 min at 25°C (protected from light) in the presence of phosphate buffer containing 400 µM of Amplex Red reagent and 2 U/ml of HRP. We read fluorescence at 590 nm in a 96-well format using an FL600 Microtiter Plate Fluorescence Reader (Bio-Tek) with an excitation wavelength of 530 nm. We corrected background fluorescence by subtracting the values derived from medium alone. Because reducing agents present in the medium could lead to some background fluorescence, catalase was added to parallel wells in all experiments to ensure that the fluorescence detected was due to the presence of H2O2. In all cases, the readings decreased in >95% with the addition of catalase. All measurements were made in duplicate dishes at each time point, and three different aliquots collected from the same dish were examined at once. The mean of these determinations was used to estimate H2O2 concentration for each sample. For statistical purposes, the mean of three biological experiments was used and evaluated with Student's t-test.

Cytotoxicity assay. We measured the cytotoxic effects of V2O5 or H2O2 on human lung fibroblasts using a CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI). This kit detects the release of lactate dehydrogenase (LDH) from cells into the culture medium.

Statistical analysis. Statistical analysis was performed by analysis of variance and two-sample t-tests. A P value of <0.05 was considered to be significant.


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V2O5 induces HB-EGF mRNA expression in human lung fibroblasts in a concentration- and time-dependent manner. We used Northern analysis to determine whether vanadium stimulated HB-EGF gene expression in normal human lung fibroblasts. Confluent, quiescent cultures of human lung fibroblasts were treated with increasing concentrations of V2O5 for 3 h before total RNA extraction. To determine the kinetics of HB-EGF mRNA induction by V2O5, we performed time-course experiments in which fibroblasts were treated with 10 µg/cm2 of V2O5. Northern analysis demonstrated that V2O5 induced HB-EGF mRNA expression 3 h after treatment, yet expression returned to basal levels by 6 h (Fig. 1A). Treatment of 10 µg/cm2 V2O5 was sufficient to induce HB-EGF mRNA expression (Fig. 1B). TiO2, an inert metal, did not upregulate HB-EGF mRNA expression (data not shown). To investigate the hypothesis that V2O5 treatment exerts an oxidative stress, which results in the stimulation of HB-EGF gene expression, we pretreated human lung fibroblasts with 50 mM NAC for 1 h before treatment with 10 µg/cm2 V2O5 for 3 h. Northern analysis showed that V2O5-induced HB-EGF mRNA expression was abolished by NAC (Fig. 1C). NAC also completely inhibited H2O2-induced HB-EGF mRNA expression (data not shown).


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Fig. 1.   A: time-dependent increase of HB-EGF mRNA in human lung fibroblasts exposed to vanadium pentoxide (V2O5). Confluent, quiescent cultures of fibroblasts were exposed to 10 µg/cm2 V2O5 for the indicated time points before the extraction of total RNA. B: concentration-dependent increase in heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA levels by V2O5 in normal human lung fibroblasts. We incubated confluent, quiescent fibroblast cultures with the indicated concentrations of V2O5 for 3 h before total RNA extraction and subsequent Northern blot analysis using a radiolabeled human HB-EGF cDNA probe. The HB-EGF mRNA was induced on 3-h exposure to 10 µg/cm2 V2O5. C: V2O5-induced HB-EGF is mediated via oxidative stress. Cells were treated with 50 mM N-acetyl-L-cysteine (NAC) for 1 h before exposure to 10 µg/cm2 V2O5 for 3 h before RNA extraction and Northern analysis. NAC blocked V2O5-induced HB-EGF expression, indicating an oxidant-mediated effect.

H2O2 induces HB-EGF mRNA expression in human lung fibroblasts. The inhibition of V2O5-induced HB-EGF expression by NAC indicated an oxidant-dependent mechanism. Therefore, we explored the effect of H2O2 on HB-EGF mRNA and protein expression. Northern analysis demonstrated that H2O2 (500 µM) stimulated HB-EGF mRNA expression within 1 h posttreatment and returned to control levels by 6 h posttreatment (Fig. 2A). Concentrations of H2O2 as high as 500 µM H2O2 were not cytotoxic as determined by LDH assay (data not shown). To determine whether HB-EGF protein expression occurred after H2O2 treatment, we performed Western blot analysis and ELISA. Because HB-EGF is expressed as a transmembrane protein and then cleaved by a variety of factors [e.g., matrix metalloproteinase (MMP)-3, phorbol esters] to produce a soluble form of the protein (14, 34), both cell lysates and fibroblast-conditioned media were analyzed for HB-EGF protein expression. Western blot analysis demonstrated that an increase in HB-EGF protein was maximal between 3 and 6 h after exposure to 500 µM H2O2 (Fig. 2B). Three apparent molecular weight masses of HB-EGF, between 17 and 22 kDa, were detected under nonreducing conditions and corresponded to the molecular masses of recombinant human HB-EGF. ELISAs confirmed that HB-EGF protein was increased in cell lysates following H2O2 treatment (untreated 0.5 ± 0.2 ng/mg protein vs. treated 2.8 ± 0.45 ng/mg protein), whereas HB-EGF was not detected in the conditioned media from cell cultures.


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Fig. 2.   Time-dependent increase in HB-EGF protein levels by H2O2 in normal human lung fibroblasts. Confluent, quiescent fibroblast cultures were incubated with 500 µM H2O2 for the indicated time points before the collection of cell lysates. A: HB-EGF mRNA expression detected by Northern blot analysis peaked at 1 h post-H2O2 treatment and declined by 6 h. B: HB-EGF protein in cell lysates was measured by Western blot analysis and peaked 6 h post-H2O2 treatment. Arrows (a, b, and c) indicate 3 apparent molecular weight masses between 17 and 22 kDa were detected under nonreducing conditions that corresponded to the molecular masses of human recombinant (r) HB-EGF. C: relationship of HB-EGF protein expression from B to HB-EGF mRNA expression from A.

An increase in H2O2 by V2O5-treated human lung fibroblasts occurs after V2O5-induced HB-EGF mRNA expression. Because both V2O5 and H2O2 upregulated HB-EGF expression, we next determined whether V2O5 caused an increase in H2O2 when added to quiescent human lung fibroblasts. We measured H2O2 released into the cell culture medium using the Amplex Red assay. Quiescent human lung fibroblasts spontaneously generated H2O2 in a time-dependent manner, and levels of H2O2 increased to as high as 2 µM by 12 h and remained elevated for as long as 24 h (Fig. 3A). The addition of V2O5 (10 µg/cm2) to cell cultures reduced H2O2 rapidly (within 5 min), and H2O2 levels remained suppressed for as long as 6 h post-V2O5 treatment. However, V2O5 stimulated a peak of H2O2 in cell culture supernatants as high as 5 µM at 12 h posttreatment (Fig. 3A). This peak of V2O5-stimulated H2O2 at 12 h occurred later than the peak of HB-EGF mRNA expression at 3 h (Fig. 1). Moreover, the minimal amount of H2O2 required to induce HB-EGF expression was 10 µM (Fig. 3B).


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Fig. 3.   Generation of H2O2 by V2O5-exposed human lung fibroblasts. A: H2O2 generation, as measured by an Amplex Red assay, showed that human lung fibroblasts spontaneously generated H2O2 into the culture medium over time, and the addition of V2O5 (10 µg/cm2) initially depleted cell-generated H2O2, which was followed by a peak of H2O2 generation at 12 h post-V2O5. The arrow indicates the temporal peak of HB-EGF mRNA expression caused by V2O5 treatment from Fig. 1 and shows that V2O5-induced HB-EGF expression precedes V2O5-induced H2O2 generation. **P < 0.01 compared with control. Data are the means ± SE of 3 separate experiments. B: Northern blot showing that the minimum concentration of H2O2 required to induced HB-EGF expression was 10 µM.

H2O2 and V2O5-induced HB-EGF expression requires ERK and p38 MAPK. We previously reported that vanadium-induced HB-EGF in normal human bronchial epithelial cells required activation of ERK and p38 MAPK (44). Western blot showed that 500 µM H2O2 or 10 µg/cm2 V2O5 activated ERK and p38 MAPK in a time-dependent manner (Fig. 4). To determine whether ERK or p38 MAPK played a role in mediating HB-EGF gene expression, we pretreated fibroblasts with the p38 MAPK inhibitor (SB-203580) or a MEK inhibitor (PD-98059) 1 h before treatment with V2O5 or H2O2 for 3 h. Dose-response experiments with these inhibitors demonstrated that SB-203580 blocked phosphorylation of its downstream substrate, activating transcription factor (ATF)-2, whereas PD-98059 blocked phosphorylation of ERK (Fig. 5, A and B). Northern analysis indicated that 40 µM PD-98059 almost completely blocked V2O5- or H2O2-induced HB-EGF mRNA expression, whereas 10 µM SB-203580 partially reduced HB-EGF mRNA expression following either V2O5 or H2O2 treatment (Fig. 5C).


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Fig. 4.   Time-dependent phosphorylation of ERK and p38 MAPK by H2O2 or V2O5. Confluent, quiescent cultures of normal human lung fibroblasts were exposed to 500 µM H2O2 or 10 µg/cm2 V2O5 for the indicated time points before the collection of cell lysates. Western blot analysis using antibodies selective for the phosphorylated form of ERK-1/2 (p-ERK) or total ERK protein (ERK, A) or antibodies selective for phosphorylated p38 MAPK (p-p38) or total p38 protein (p38, B).



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Fig. 5.   Inhibitors of ERK and p38 MAPK reduce V2O5-induced HB-EGF expression. A: Western blot showing that 10 µM of the p38 MAPK inhibitor SB-203580 was effective in blocking V2O5- or H2O2-induced activating transcription factor (ATF)-2 phosphorylation (p-ATF-2). B: Western blot demonstrating that 40 µM PD-98059 blocked V2O5- or H2O2-induced ERK phosphorylation (p-ERK). C: Northern blot analysis showing that SB-203580 (SB) or PD-98059 (PD) significantly reduced HB-EGF mRNA expression. Confluent, quiescent fibroblast cultures were pretreated with 10 µM SB-203580 for 1 h before exposure to 10 µg/cm2 V2O5 or 500 µM H2O2 for 3 h. Scanning densitometry was used to normalize the HB-EGF signal against GAPDH to demonstrate that SB-203580 or PD-98059 treatment significantly reduced V2O5- or H2O2-induced HB-EGF mRNA expression. *P < 0.05; **P < 0.01. Data are the means ± SE of 3 separate experiments.


    DISCUSSION
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ROS play a primary role in the initiation of the inflammatory response following lung injury after exposure to a wide variety of environmental agents, including transition metals. This response includes the activation of intracellular signaling proteins as well as the production of cytokines and growth factors involved in inflammation and airway remodeling. In this study, we found that V2O5, a transition metal released during the industrial burning of fuel oil that causes occupational bronchitis (23), upregulates the expression of HB-EGF, a polypeptide growth factor that promotes the proliferation and homoeostasis of airway epithelial cells and fibroblasts.

H2O2 also upregulated HB-EGF expression and mimicked the time course of V2O5-induced HB-EGF mRNA expression (Figs. 1 and 2). Moreover, V2O5-induced HB-EGF expression was blocked by the antioxidant NAC (Fig. 1C), suggesting that ROS generated by V2O5 mediated the upregulation of HB-EGF. We did find that V2O5 caused an increase in cell-generated H2O2, yet two major observations led us to conclude that V2O5-stimulated H2O2 did not mediate induction of HB-EGF. Most convincing was that the peak of V2O5-stimulated H2O2 at 12 h occurred well after the peak of HB-EGF mRNA expression at 3 h (Fig. 3). Also, the concentration of H2O2 detected in the medium from fibroblast cultures was ~5 µM, and we found that 10 µM was the minimal concentration of H2O2 required to activate HB-EGF mRNA expression (Fig. 3).

Nevertheless, endogenous levels of H2O2 could be important in mediating V2O5-induced HB-EGF production. We observed that quiescent human lung fibroblasts spontaneously generated 1-2 µM of H2O2 in the absence of V2O5, and the addition of V2O5 to these cultures depleted H2O2 levels in cell culture supernatants within 5 min, and H2O2 remained suppressed for as long as 6 h (Fig. 3). Liochev and Fridovich (24) demonstrated that vanadyl decomposes H2O2 to yield HO- and · OH. Therefore, · OH generated by the reaction of V2O5 with endogenous H2O2 could activate signal transduction pathways leading to the upregulation of HB-EGF gene expression. The reaction of vanadium with H2O2 also forms pervanadate, a peroxovanadium compound that irreversibly oxidizes protein tyrosine phosphatases (17), such as those associated with the EGF receptor. We previously showed that V2O5 phosphorylates the EGF receptor and the downstream ERK pathway (41) and that an inhibitor of the EGF receptor tyrosine kinase blocks V2O5-induced HB-EGF mRNA expression in bronchial epithelial cells (43). Moreover, Wang and Bonner (41) demonstrated that NAC blocks ERK activation by V2O5. Therefore, we postulate that relatively low levels of endogenous H2O2 react with V2O5 to form · OH and/or peroxovanadium derivatives that could initiate signal transduction pathways leading to HB-EGF expression. A hypothetical model that illustrates our concept of V2O5-induced HB-EGF is presented in Fig. 6.


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Fig. 6.   Hypothetical model illustrating V2O5- and H2O2-induced HB-EGF expression in human lung fibroblasts. V2O5 is taken up by cells and depletes endogenous H2O2 (<2 µM) within minutes, presumably forming peroxovanadium derivatives or free radicals (e.g., · OH) that serve as protein tyrosine phosphatase (PTP) inhibitors. PTP inhibition then leads to downstream MAPK (ERK) activation and transcriptional activation leading to HB-EGF gene expression. p38 MAPK is also required, yet the mechanism of upstream activation is unknown. V2O5 treatment also causes a late (12 h) burst of H2O2 (~5 µM released into culture medium) that follows HB-EGF mRNA expression. The addition of relatively high concentrations of exogenous H2O2 to cells (>10 µM) also causes HB-EGF expression via PTP inhibition and MAPK activation. RTK, receptor tyrosine kinase.

It has been previously reported that H2O2 upregulates HB-EGF expression in gastric epithelial cells (27). In that study, the induction of HB-EGF expression occurred maximally 3 h after treatment, and NAC blocked H2O2-induced HB-EGF expression. These observations are consistent with the data presented in this investigation, which is the first report of HB-EGF induction by H2O2 in lung cells. In a previous study, we reported that V2O5 stimulates HB-EGF expression in normal human bronchial epithelial cells (43). However, in that study we did not investigate ROS as mediators of HB-EGF expression. Nevertheless, other studies have provided evidence to show that vanadium compounds upregulate gene expression of several growth factors and cytokines via ROS. Ye et al. (42) found that vanadate is a potent inducer of TNF-alpha in macrophages and airway epithelial cells. TNF-alpha production was enhanced by NADPH, which facilitates the generation of ROS in the presence of vanadate. Furthermore, NAC inhibited the vanadate-mediated stimulation of TNF-alpha gene expression. Dong et al. (8) demonstrated that V2O5 increases IL-8, MIP-2, and TNF-alpha gene expression and protein secretion by hepatocytes. The V2O5-induced secretion of these inflammatory cytokines was inhibited by the antioxidants, NAC, and 1,1,3,3-tetramethylthiourea. Further evidence points to the involvement of vanadium compounds in the activation of a variety of transcription factors such as NF-kappa B and activator protein-1 (6, 7, 9, 16), as well as cell cycle regulatory proteins (44). All of these vanadium-mediated proinflammatory events appear to be driven through a mechanism that involves the generation of ROS such as · OH or H2O2.

An important finding in this study is the requirement of ERK-1/2 and p38 MAPKs for V2O5- or H2O2-induced HB-EGF expression. We found that either H2O2 or V2O5 stimulates the phosphorylation of ERK-1/2 or p38 MAPKs (Fig. 4), and pretreatment with the MEK inhibitor (PD-98059) or the p38 MAPK inhibitor (SB-203580) effectively reduced the induction of HB-EGF mRNA by V2O5 or H2O2 (Fig. 5). These findings are similar to our previous observations that indicated a requirement of ERK-1/2 and p38 MAPK in V2O5-induced HB-EGF mRNA expression in human bronchial epithelial cells (43). Ellis et al. (11) showed that ERK-1/2 was involved in HB-EGF gene expression induced by scrape wounding epithelial cell monolayers. Recently, Tschumperlin and coworkers (37) reported that mechanical compression of bronchial epithelial cells induces HB-EGF expression via the activation of ERK-1/2. In their study, they reported that mechanical compression of the epithelium selectively activates ERK-1/2, but not JNK or p38 MAPK.

We found that V2O5 and H2O2 activated p38 MAPK as well as ERK-1/2. The activation of p38 MAPK has been shown to be an important component in the regulation of the gene expression of proinflammatory cytokines. Carter and coworkers (5) demonstrated that p38 MAPK is activated by endotoxin in human alveolar macrophages and that this activation is correlated with increased secretion of IL-6 and TNF-alpha . In that study, inhibitors of p38 MAPK also blocked gene expression of the cytokines. Similarly, we show in this study that H2O2 and V2O5 activate p38 MAPK in a time-dependent manner. Furthermore, we demonstrated that SB-203580, a specific inhibitor of p38 MAPK, reduces H2O2 and V2O5-induced HB-EGF gene expression. These results suggest that the upregulation of HB-EGF expression by oxidative stress is dependent in part on the p38 MAPK signaling pathway. A recent investigation by Sano et al. (33) showed that p38 MAPK is an important signaling intermediate in the upregulation of IL-6 in response to angiotensin II in cardiac fibroblasts. These authors suggested that angiotensin II stimulates the production of ROS, which in turn mediates activation of p38 and subsequent increase in IL-6 gene expression. Jaspers and coworkers (18) demonstrated that catalase, an antioxidant specific for peroxides, inhibits vanadyl sulfate-induced NF-kappa B-dependent transcription and that this repression is correlated with an inhibition of p38 MAPK activation in human airway epithelial cells. Together, these studies indicate that metal compounds such as vanadium generate ROS, which then activate the p38 MAPK signaling pathway, culminating in increased expression of cytokine and growth factor gene expression.

Members of the EGF family of proteins, including HB-EGF, are synthesized as a membrane-anchored form, which is then cleaved and solubilized by various agents including MMPs, phorbol esters, or G protein ligands (14, 32, 34, 36, 38). In this study, both Western analyses and ELISA demonstrated the presence of HB-EGF protein in the cell membranes with no soluble protein detectable in conditioned media. Western blot analysis using an anti-HB-EGF antibody revealed three molecular weight masses between 17 and 22 kDa under nonreducing conditions that correspond to the molecular masses of human recombinant HB-EGF (Fig. 4). These molecular masses of HB-EGF are similar to those reported by Higashiyama and coworkers (15). Our in vitro system and experimental conditions did not include any cleavage reagents, which are required to process membrane-tethered HB-EGF protein to a soluble form. In vivo, these cleavage agents are hypothesized to play a role in the regulation of cell growth and differentiation by modulating the juxtacrine/paracrine/autocrine activities of HB-EGF. Umeda et al. (39) demonstrated the importance of HB-EGF processing by metalloproteinases in organ development. They showed that treatment of rudimentary mouse submandibular gland cells with an MMP inhibitor is sufficient to block HB-EGF-mediated morphogenesis. Similar results were observed in a study by Takemura and coworkers (35), in which the soluble and membrane-anchored forms of HB-EGF were found to play different roles during the morphogenesis of the collecting duct system in the developing kidney. Treatment of cells with phorbol ester to solubilized HB-EGF resulted in short tubules with many branches, whereas cells expressing only the membrane-anchored form of HB-EGF resulted in long tubules with few branches (35). An earlier study by this group reported that renal epithelial cells transfected with the membrane-anchored form of HB-EGF are protected from H2O2-induced apoptosis (36). Together, these data demonstrate the importance of both forms of HB-EGF in regulating cell growth either through juxtacrine interactions of the membrane-anchored form on adjacent cells or through paracrine and autocrine interactions involving the soluble form. In light of these observed effects of HB-EGF on cell differentiation and growth in other systems, HB-EGF appears to be a strong candidate for serving an important role in lung injury and repair. Evidence of a role for HB-EGF in airway remodeling was demonstrated in a study by Powell and coworkers (28). They found that HB-EGF mRNA levels are increased 100-fold in lung tissue from rats exposed to hyperoxia for 7 days compared with normal lung tissue. HB-EGF has been shown to act as a mitogen for alveolar type II cells, indicating that it may play a role in cell growth and differentiation in alveolar repair following lung injury (22). In this study, we have shown that HB-EGF gene expression is induced in human lung fibroblasts after oxidative stress. We previously reported that HB-EGF is produced by cultured human bronchial epithelial cells after vanadium-induced stress (43). Recently, HB-EGF was shown to be upregulated in bronchial epithelial cells in a model of compressive stress that mimics airway strain caused by smooth muscle constriction during an asthmatic attack (37). Therefore, induction of HB-EGF expression by inflammatory mediators, oxidative stress, or mechanical stress could contribute to the repair and injury responses in a variety of different lung diseases.

In summary, we have shown that HB-EGF mRNA expression is stimulated by V2O5 and that this upregulation of gene expression is oxidant dependent. H2O2 at concentrations of 10 µM or above mimicked the effect of V2O5 in stimulating HB-EGF expression. Although V2O5 treatment caused a peak of H2O2 generation by fibroblasts, this occurred 12 h post-V2O5 treatment, whereas the peak of V2O5-induced HB-EGF expression occurred at 3 h posttreatment. Moreover, the level of H2O2 generated by V2O5 treatment was <10 µM. Interestingly, we found that relatively low levels of H2O2 (1-2 µM) generated spontaneously by resting fibroblasts were depleted within minutes of V2O5 treatment, and we postulate that the reaction of H2O2 with V2O5 caused the formation of peroxovanadium derivatives and/or ROS that resulted in HB-EGF expression. Finally, HB-EGF gene expression in response to either V2O5 or H2O2 was dependent on the activition of ERK and p38 MAPKs. HB-EGF produced by human lung fibroblasts may contribute to airway remodeling and pulmonary fibrosis by acting in a autocrine or juxtaparacrine manner to stimulate growth and differentiation of airway epithelia, airway smooth muscle, or peribronchiolar fibroblasts.


    FOOTNOTES

Address for reprint requests and other correspondence: J. C. Bonner, NIEHS, PO Box 12233, Research Triangle Park, NC 27709 (E-mail: bonnerj{at}niehs.nih.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.

First published January 10, 2003;10.1152/ajplung.00189.2002

Received 15 June 2002; accepted in final form 8 January 2003.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 284(5):L774-L782