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Nickel compounds are environmental and occupational hazards that pose serious health problems and are causative factors of acute lung injury. The c-jun N-terminal kinases (JNKs) are regulated through a mitogen-activated protein (MAP) 3 kinase-MAP2 kinase cascade and have been implicated in nickel toxicity. In this study, we used genetically modified cells and mice to investigate the involvement of two upstream MAP3Ks, MAP3K1 and 2, in nickel-induced JNK activation and acute lung injury. In mouse embryonic fibroblasts, levels of JNK activation and cytotoxicity induced by nickel were similar in the Map3k2-null and wild-type cells but were much lower in the Map3k1/Map3k2 double-null cells. Conversely, the levels of JNK activation and cytotoxicity were unexpectedly much higher in the Map3k1-null cells. In adult mouse tissue, MAP3K1 was widely distributed but was abundantly expressed in the bronchiole epithelium of the lung. Accordingly, MAP3K1 ablation in mice resulted in severe nickel-induced acute lung injury and reduced survival. Based on these findings, we propose a role for MAP3K1 in reducing JNK activation and protecting the mice from nickel-induced acute lung injury.
Nickel compounds, having extensive usage in industry, are present in welding fumes and are components of ambient particulate matter released from coal-fired power plants (Lippmann et al., 2006). Welders and other workers exposed to nickel-containing materials can develop a variety of adverse health problems, particularly of the respiratory system (Antonini et al., 2003; Speizer, 2000). Nickel inhalation is associated with acute lung injury in humans that can lead to respiratory failure, clinically characterized as diffuse alveolar damage, inflammatory cell influx and activation, insufficient gas exchange, and ultimately death (Scott et al., 2002). Although a large number of people develop acute lung injury, only a fraction succumb to respiratory failure, suggesting that genetic variability may determine the patient's risk and subsequent outcome of acute lung injury.
Acute lung injury is caused by respiratory epithelial cell damage and loss of function. The initial tissue injury triggers the production of growth factors, extracellular matrices, and cytokines, which, through the activation of a series of intracellular signaling pathways, stimulate inflammatory responses and migration and proliferation of pulmonary epithelial cells (Geiser et al., 2000; Kheradmand et al., 1994). Signaling pathway activation, on the one hand, promotes re-epithelialization of the damaged epithelium thereby restoring normal barrier function, while on the other hand, contributes to pathological damage, such as inflammation, disruption of surfactant homeostasis, and fibrosis. Thus, gene-environment interactions affecting the efficiency of signal transduction may influence the development and severity of acute lung injury.
The mitogen-activated protein kinases (MAPKs), including the c-jun N-terminal kinase (JNK) (also known as MAPK8), the extracellular signal-regulated kinase (ERK1/2 also known as MAPK1/3), and the p38s (also known as MAPK14), play a pivotal role in the transmission of extracellular signals through the cytosol to the nucleus. The MAPK activation is mediated by a cascade involving a mitogen-activated kinase kinase kinase (MAP3K)-MAPK kinase (MAP2K) module, where extracellular signals first activate the MAP3K leading to the phosphorylation of MAP2K, which in turn phosphorylates and activates MAPKs (Davis, 2000). Once activated, the MAPKs can translocate to the nucleus, where they phosphorylate transcription factors, therefore converting transient biochemical signals to permanent changes of gene expression. Through this paradigm, MAPKs participate in numerous physiological and pathological processes, such as immunity, neuronal development, and cancer (Chang et al., 2003; Chen et al., 2001; Dong et al., 1998; Han et al., 2002; Kuan et al., 1999; Sabapathy et al., 2001).
The role of JNK activation in lung injury is controversial and yet to be fully understood. In vitro, lung injury inducers, including metals, mechanical ventilation, and inflammatory cytokines, can activate the JNK MAPK, specifically in human dendritic cells and in airway and alveolar epithelial cells (Boisleve et al., 2004; Sakashita et al., 2007; Timblin et al., 1998). The JNK activation in turn is connected to physiopathological responses in the lung, such as epithelial cell proliferation and tumorigenesis (Timblin et al., 1998), cell apoptosis, neutrophil chemotaxis (Li et al., 2004; Sakashita et al., 2007), and inflammatory gene expression (Han et al., 2005). In vivo, pharmacologic inhibition of JNK by SP600125 reduces ischema/reperfusion injury in the rat lung and prolongs survival in rats after smoke inhalation injury (Ishii et al., 2004; Syrkina et al., 2007).
JNK has been implicated in nickel toxicity (Zhang et al., 2007); however, in vitro studies have not always been consistent (Samet et al., 1998; Tessier and Pascal, 2006). The signaling factors mediating nickel-induced JNK activation have remained elusive. At least 14 MAP3Ks, such as MAP3K1–4, are involved in mediating diverse upstream signals to the JNK pathway (Schlesinger et al., 1998), but it is not known which MAP3Ks are involved in transmitting the nickel signals. In the present work, we used genetically modified cells and mice to evaluate the role of two MAP3Ks, MAP3K1 and MAP3K2, in JNK activation and nickel-induced acute lung injury.
The Map3k1ΔKD/ΔKD, also known as Mekk1ΔKD/ΔKD, mice were described before (Zhang et al., 2003) and were backcrossed for at least seven generations into the C57/BL6 background. Intercrosses were carried out to generate Map3k1+/ΔKD and Map3k1ΔKD/ΔKD mice. The wild-type C57/BL6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). The Map3k2+/−, also known as Mekk2+/−, mice were described previously (Guo et al., 2002). All mice were maintained in the Experimental Animal Laboratory at the University of Cincinnati. Nickel aerosol (mass median aerodynamic diameter = 0.2 μm) was generated from 50mM NiSO4•6H2O (Sigma, St Louis, MO) and monitored as described previously (Wesselkamper et al., 2000). Mice were exposed to 150 ± 15 μg Ni2+/m3 in a 0.32-m3 stainless steel inhalation chamber continuously, and survival time was recorded. The experimental procedures were approved by the University of Cincinnati Animal Care and Use Committee.
Mouse embryonic fibroblasts (MEFs) were prepared from E13.5 wild-type or mutant fetuses as described (Giroux et al., 1999) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. All cell culture reagents were from Invitrogen (Carlsbad, CA). Nickel sulfate was from Sigma. Phospho-JNK antibody and X-gal were from Promega (Fitchburg, WI); antibodies for phospho-ERK and phospho-p38 were from Cell Signaling Technology (Danvers, MA); antibodies for β-actin, JNK, p38, and ERK were from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-MAP3K1 was described previously (Xia et al., 2000).
Whole-mount X-gal staining of wild-type, Map3k1+/ΔKD, and Map3k2+/− mice lung was performed as described previously (Zhang et al., 2003). In brief, whole lung was inflated with 4% paraformaldehyde (PFA) and isolated from mice, washed with cold phosphate buffer, and placed in PFA at 4°C for 20 min. Tissue was washed with cold phosphate buffer for three times, 5 min/each time, followed by washing in staining buffer containing 5mM K3Fe(CN)6, 5mM K4Fe(CN)6, 0.02% nonidet p40, 100mM NaHPO4, pH 7.3, and 2mM MgCl2 for 15 min. For staining, 1 mg/ml of X-gal was added to fresh staining buffer and incubated with tissue in the dark at 30°C overnight. Tissue was postfixed in 4% PFA overnight. For whole-mount examination, tissues were cleared by incubating serially in solutions containing 20, 50, 80, and 100% glycerol (vol/vol) brought to the final volume by 1% KOH (w/v). Each incubation step was carried out at 30°C and lasted 4–7 days. Pictures were taken using a Leica MZ16 F stereomicroscope with a ×0.5 plan objective. For histology, tissues were fixed in 4% PFA, dehydrated with a graded ethanol series, and embedded in paraffin. Tissues were serially sectioned at 4 μm and counterstained with eosin. Pictures were taken at various magnifications under the microscope.
Eighty percent confluent monolayers of MEFs were deprived of growth factors for 16–24 h to minimize the basal levels of MAPK phosphorylation. The cells were treated with serum-free DMEM in the presence or absence of various doses of nickel sulfate for 4 h. By the end of the treatment, cells were washed with ice-cold PBS twice and lysed on ice with “egg lysis buffer” (50mM Tris, pH 7.5, 0.1% nonidet p40, 120mM NaCl, 1mM EDTA, 6mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 20mM NaF, 1mM sodium pyrophosphate, 30mM 4-nitrophenyl phosphate, 1mM benzamidine, one tablet of EDTA-free protease inhibitor cocktail per 25 ml of buffer). Cell lysates were incubated on ice for 10 min and frozen at −80°C until use. After thaw on ice, cell lysates were centrifuged at 13,500 rpm for 15 min, and total cellular proteins were collected in the supernatant. In total, 100 μg of cell and tissue lysates was resolved on SDS-polyacrylamide gel electrophoresis followed by Western blot detection using appropriate antibodies.
Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cytotoxicity assay, using CellTiter 96 AQueous One Solution (Promega, Madison, WI). Briefly, cells were seeded into 96-well plates at 1.5 × 104 cells per well and were cultured overnight. The cells were treated with NiSO4 at different concentrations in serum-free medium for 10 h. CellTiter 96 AQueous One Solution (20 μl per 100 μl medium) was added into each well, and cells were incubated at 37°C for 1–4 h. The absorbance (490 nm) was measured, and cell survival ratios were calculated as the ratio of absorbance in treated cells versus that in untreated cells. At least four wells were tested on each treatment condition in a single experiment, and the data from three independent experiments were used for statistical analyses.
MEFs were seeded at 2 × 103 cells per 6-cm tissue culture dishes and were maintained in the growth medium containing 1μM nickel sulfate. On day 7, the cells were washed with PBS and the colonies were stained for 2 min by 0.3% crystal violet (in 25% ethanol). The colonies on each plate were counted; the average numbers in the total of 10 plates under each treatment condition were calculated and were used for statistical analyses.
To investigate the potential involvement of the MAP3Ks in MAPK activation, we examined nickel-induced MAPK phosphorylation in wild-type MEFs and MEFs deficient in MAP3K1, MAP3K2, or both. When exposed to increasing concentrations (1–50mM) of nickel, there was a dose-dependent induction in the phosphorylation of JNK, ERK, and p38 in all the cells examined (Fig. 1A). The overall pattern of MAPK phosphorylation did not appear to be overtly altered by MAP3K1 and MAP3K2 deficiency; however, quantification showed that the levels of phospho-JNK induced by nickel were quite different in the various gene ablation cells. In comparison to wild-type cells, phospho-JNK was more abundant in Map3k1ΔKD/ΔKD, unchanged in Map3k2−/−, and markedly reduced in Map3k1ΔKD/ΔKD/Map3k2−/− cells (Fig. 1B). The levels of phospho-p38 and phospho-ERK were not obviously affected by MAP3K1 and MAP3K2 ablation, and nickel exposure had no effect on the levels of JNK or β-actin expression (Figs. 1A and C and data not shown).
To determine the involvement of MAP3K1 and MAP3K2 in nickel cytotoxicity, we exposed wild-type, Map3k1ΔKD/ΔKD, Map3k2−/−, and Map3k1ΔKD/ΔKDMap3k2−/− MEFs to various concentrations of nickel for 10 h and measured cell survival by MTS cytotoxicity assay. Survival decreased more in Map3k1ΔKD/ΔKD cells than in wild-type and Map3k2−/− cells. Conversely, Map3k1ΔKD/ΔKD/Map3k2−/− MEFs were more resistant to nickel cytotoxicity (Fig. 2A). By colony formation assay, we evaluated cytotoxicity by chronic exposure to low concentration (1μM) of nickel. Similar to its acute toxicity, nickel caused a marked reduction of the Map3k1ΔKD/ΔKD colonies, while the Map3k2−/− and Map3k1ΔKD/ΔKD/Map3k2−/− cells were similar to the wild-type MEFs in colony formation. Taken together, our observations reveal a role for MAP3K1 in suppressing JNK activation and preventing nickel cytotoxicity; on the other hand, MAP3K1 and MAP3K2 together potentiate JNK activation and nickel cytotoxicity. In either case, there is a good correlation between the levels of JNK activation and cytotoxicity caused by nickel.
The MAP3Ks display tissue and cell-type specificity in signal transduction, partly due to differential tissue distribution patterns (Schlesinger et al., 1998). To determine in which adult tissues MAP3K1 may be functionally important, we examined MAP3K1 expression in lysates of various mouse tissues. The highest levels of MAP3K1 expression were seen in the olfactory bulb, thymus, spleen, skin, and lung, less MAP3K1 protein was detected in the cerebellum, heart, and testis, and none was observed in the kidney and liver (Fig. 3A). These results indicate that MAP3K1 protein is differentially expressed in mouse tissues and is highly expressed in the lung.
The Map3k1ΔKD mice contain a β-galactosidase gene knocked in at the Map3k1 locus such that β-gal expression is controlled by the endogenous Map3k1 promoter (Xia et al., 2000), and as such the location of β-gal reflects Map3k1 endogenous promoter activity. To further localize the MAP3K1-expressing cells, we performed whole-mount X-gal staining of the Map3k1+/ΔKD mouse tissues followed by glycerol clarification procedures to identify the endogenous localization of the β-gal–positive, i.e., MAP3K1-expressing, cells. While no β-gal–positive cells were seen in the wild type, a distinct X-gal staining pattern was observed in the Map3k1+/ΔKD mouse lung (Fig. 3B). Specifically, the β-gal–positive cells were located along the distal bronchi but not in the main stem bronchi or parenchyma of the lung. Further histological examination located the β-gal–positive cells to the bronchial epithelium and the wall of associated blood vessels, possibly in the smooth muscle cells (Fig. 3C). Very few, if any, MAP3K1-positive cells were present in the liver, consistent with nondetectable MAP3K1 proteins in this tissue by Western blotting (Fig. 3A). It was interesting to note that the Map3k2+/− mice, that also have a β-galactosidase gene knocked in (Guo et al., 2002), displayed β-gal–positive staining, i.e., Map3k2 promoter activity, in the trachea, main stem bronchi, and distal bronchi of the lung, a pattern partially overlapping with that of Map3k1 (Fig. 3B). The redundant and unique expression pattern of MAP3K1 and MAP3K2 suggests that these two MAP3Ks may play both common and cell-type–specific roles in the lung.
Because MAP3K1 is abundantly expressed in the airway and is functionally involved in nickel cytotoxicity, we hypothesize that MAP3K1 may be involved in nickel-induced acute lung injury. To test this hypothesis, we exposed wild-type, Map3k1+/ΔKD and Map3k1ΔKD/ΔKD mice to nickel and recorded survival time during exposure. Ablation of one functional Map3k1 allele in Map3k1+/ΔKD mice did not alter survival; however, Map3k1 homozygous mutants died significantly sooner than wild-type mice (Fig. 4A). The mean survival time of the Map3k1ΔKD/ΔKD mice was shortened by 20 h relative to wild-type or Map3k1+/ΔKD mice (Fig. 4B). During exposure, mice developed severe dyspnea, and gross pathology of the lung indicated air trapping and extensive hemorrhagic edema, supporting that lethality was a consequence of severe lung injury. We conclude that MAP3K1 expression and activity in the lung epithelium offer protection against nickel-induced lung injury.
Activation of signaling pathways is considered to be critical for acute lung injury; however, only a few in vivo studies have addressed which signaling factors are involved in the process. In previous studies, we have found that ablation of metallothionein-nitric oxide signaling (Wesselkamper et al., 2006) and macrophage stimulating 1 receptor (c-met-related tyrosine kinase) (McDowell et al., 2002) increases susceptibility to nickel-induced acute lung injury in mice. Conversely, transgenic expression of transforming growth factor α (Hardie et al., 2002) and keratinocyte growth factor 7 (Tichelaar et al., 2007) decreases susceptibility to nickel-induced acute lung injury in mice. Here, we have identified MAP3K1 as one of the targets that could be augmented to reduce acute lung injury because MAP3K1 ablation reduces mean survival time. MAP3K1 protein is expressed in mouse lung in the airway epithelium and blood vessel; its ablation therefore likely impairs the ability of the airway to counteract nickel toxicity.
MAP3K2, another MAP3K sharing similar activities with MAP3K1 in downstream pathway induction, is also highly expressed in the airway epithelium (Fanger et al., 1997; Schlesinger et al., 1998). It is possible that MAP3K1 and MAP3K2 are functionally redundant at least in some cells; therefore, lacking MAP3K1 causes only a 20% increase in lethality to the Map3k1ΔKD/ΔKD mice during nickel exposure. Studies using MEFs, however, suggest that MAP3K2 may be dispensable for cytotoxicity because Map3k2−/− cells do not differ from wild-type cells in response to nickel treatment. Interestingly, when both MAP3K1 and MAP3K2 are ablated, the Map3k1ΔKD/ΔKDMap3k2−/− compound mutant cells are resistant to cytotoxicity. It is worth noting that molecular responses taking place in fibroblasts may not be quite the same as those in lung epithelial cells; hence, the precise role of MEKK2 in nickel-induced acute lung injury may have to be evaluated in vivo.
In vitro, cell survival is inversely related to phospho-JNK levels in response to nickel; however, this relationship is complex. In comparison to wild-type cells, the Map3k1ΔKD/ΔKD cells display higher JNK activation and reduced survival, whereas the Map3k1ΔKD/ΔKDMap3k2−/− cells have lower JNK activation and are resistant to cytotoxicity. This observation is consistent with the notion that JNK activity is promoting cell death (Faris et al., 1998; Lin and Dibling, 2002). Such a role would be consistent with the increase in survival noted in other models of acute lung injury when JNK phosphorylation is inhibited pharmacologically (Ishii et al., 2004; Syrkina et al., 2007). In addition to its well-established roles in transcription factor activation (Davis, 2000), JNK has recently been shown to mediate epigenetic histone modification in response to nickel (Ke et al., 2008). It is hence reasonable to suggest that the JNK pathway activation by nickel may potentiate lung injury by the way of activating gene expression.
At this time, we cannot explain why the Map3k1ΔKD/ΔKD cells differ from the Map3k1ΔKD/ΔKDMap3k2−/− cells in regard to nickel-induced JNK activation. A likely scenario may be that nickel induces the JNK pathway through several MAP3Ks, including MAP3K1, MAP3K2, and others, and only when multiple members of the MAP3Ks are ablated, as in the Map3k1ΔKD/ΔKDMap3k2−/− cells, there will be an obvious suppression of JNK activation. Furthermore, MAP3K1 may have additional negative feedback roles in downregulation of the JNK pathway; thus, Map3k1ΔKD/ΔKD cells, lacking such negative inhibition, will display more abundant JNK activation during nickel exposure. A role of MAP3K1 in inhibiting its downstream pathways has been described before (Lu et al., 2002; Witowsky and Johnson, 2003); this function is mediated through the plant homeodomain domain located at the N-terminus of MAP3K1, which acts as an E3 ubiquitin ligase that catalyzes downstream signaling factors for degradation.
Taken together, our studies suggest that there is a complex molecular interplay between MAP3K1 and MAP3K2 in the signal transduction pathways responding to nickel exposure. The MAP3K1 exhibits an unexpected role in suppressing nickel-induced JNK activation and reducing cytotoxicity. Hence, pharmacological agents that increase MAP3K1 activity may be useful in the treatment of nickel-induced acute lung injury happening in an occupational setting.
NIH (ES11798 to Y.X., ES15675 and HL063700 to G.L.); NIEHS Center for Environmental Genetics P30 Grant (ES06096) (University of Cincinnati).
We thank Alvaro Puga for a critical reading of the manuscript.