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Published ahead of print on August 11, 2005, doi:10.1164/rccm.200502-286OC
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American Journal of Respiratory and Critical Care Medicine Vol 172. pp. 1399-1411, (2005)
© 2005 American Thoracic Society
doi: 10.1164/rccm.200502-286OC

Original Article

Gene Expression Changes during the Development of Acute Lung Injury Role of Transforming Growth Factor {beta}

Scott C. Wesselkamper, Lisa M. Case, Lisa N. Henning, Michael T. Borchers, Jay W. Tichelaar, John M. Mason, Nadine Dragin, Mario Medvedovic, Maureen A. Sartor, Craig R. Tomlinson and George D. Leikauf

Department of Environmental Health, Center for Environmental Genetics, and Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Cincinnati Medical Center, University of Cincinnati, Cincinnati, Ohio

Correspondence and requests for reprints should be addressed to George D. Leikauf, Ph.D., Department of Environmental Health, P.O. Box 670056, University of Cincinnati, Cincinnati, OH 45267-0056. E-mail: george.leikauf{at}uc.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Acute lung injury can occur from multiple causes, resulting in high mortality. The pathophysiology of nickel-induced acute lung injury in mice is remarkably complex, and the molecular mechanisms are uncertain.

Objectives: To integrate molecular pathways and investigate the role of transforming growth factor {beta} (TGF-{beta}) in acute lung injury in mice.

Methods: cDNA microarray analyses were used to identify lung gene expression changes after nickel exposure. MAPPFinder analysis of the microarray data was used to determine significantly altered molecular pathways. TGF-{beta}1 protein in bronchoalveolar lavage fluid, as well as the effect of inhibition of TGF-{beta}, was assessed in nickel-exposed mice. The effect of TGF-{beta} on surfactant-associated protein B (Sftpb) promoter activity was measured in mouse lung epithelial cells.

Measurements and Main Results: Genes that decreased the most after nickel exposure play important roles in lung fluid absorption or surfactant and phospholipid synthesis, and genes that increased the most were involved in TGF-{beta} signaling. MAPPFinder analysis further established TGF-{beta} signaling to be significantly altered. TGF-{beta}–inducible genes involved in the regulation of extracellular matrix function and fibrinolysis were significantly increased after nickel exposure, and TGF-{beta}1 protein was also increased in the lavage fluid. Pharmacologic inhibition of TGF-{beta} attenuated nickel-induced protein in bronchoalveolar lavage. In addition, treatment with TGF-{beta}1 dose-dependently repressed Sftpb promoter activity in vitro, and a novel TGF-{beta}–responsive region in the Sftpb promoter was identified.

Conclusions: These data suggest that TGF-{beta} acts as a central mediator of acute lung injury through the alteration of several different molecular pathways.

Key Words: microarray • surfactant • fibrinolysis • extracellular matrix

Acute lung injury is common in critically ill patients and results from a variety of factors, including pneumonia, sepsis, trauma, and inhaled irritants (1). Acute lung injury involves epithelial and endothelial damage that subsequently leads to impairment of alveolar fluid clearance (edema), alveolar hemorrhage, disruption of surfactant homeostasis, degeneration of the alveolar capillary–epithelial barrier, and inadequate gas exchange (2). The prognosis for survival from acute lung injury is poor, with mortality at 20 to 40%; and, if resolution occurs, persistent pulmonary complications develop, including interstitial fibrosis and diminished lung compliance.

Transforming growth factor {beta} (TGF-{beta}) exists in three isoforms and is a member of a family of growth factors that is able to modulate several cellular processes in multiple organ systems (3). Of these isoforms, TGF-{beta}1 is generated in greatest abundance subsequent to tissue damage (4). TGF-{beta} has been widely studied for its vital role in the development of fibrosis after injury to the lung (5, 6). A broader function for TGF-{beta} is suggested from evidence that elevated levels of TGF-{beta}1 (as well as TGF-{beta}–inducible genes, such as procollagen, type III, {alpha}1) have been demonstrated in the lungs of patients with acute respiratory distress syndrome (7–9). Furthermore, in mouse models of bleomycin-induced lung injury, TGF-{beta}–inducible genes are increased within 48 h (10). Although the underlying mechanisms of TGF-{beta} mediation of acute lung injury are uncertain, studies have shown that TGF-{beta} may augment pulmonary injury through increased endothelial (11) and epithelial permeability (12) and decreased ion and fluid transport (13–15). These data suggest that TGF-{beta} may have a role in the development of acute lung injury in addition to the regulation of fibroproliferation.

Prior studies from our laboratory have assessed aspects of the molecular mechanisms involved in the pathogenesis of acute lung injury using inhaled nickel in mice (16–18). The purpose of our study was to use oligonucleotide microarray analysis to identify novel intersecting molecular pathways that lead to the development of acute lung injury. The TGF-{beta} signaling pathway was identified as being significantly altered in our mouse model of nickel-induced acute lung injury by means of initial application of general microarray statistical analysis, as well as the MAPPFinder analytic tool that creates a global gene expression profile at the level of biological processes (19). Consistent with this, TGF-{beta}1 protein levels were significantly increased in the bronchoalveolar lavage fluid (BALF) of mice after 72 h of nickel exposure. We also identified expression changes in TGF-{beta}–responsive genes involved in extracellular matrix changes, fibrinolysis, and surfactant synthesis. Pharmacologic inhibition of TGF-{beta} with soluble chimeric TGF-{beta} type II receptor–IgG-Fc (TGF{beta}RII-Fc) attenuated nickel-induced protein in BAL. Using promoter deletion constructs transiently transfected into mouse lung epithelial cells, we identified a novel TGF-{beta} regulatory region in the mouse surfactant-associated protein B (Sftpb) promoter. Taken together, these data suggest TGF-{beta} is a central mediator of acute lung injury.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and Exposure Protocol
Female 129S1/SvImJ mice (age, 7–10 wk) were purchased from the Jackson Laboratory (Bar Harbor, ME), and housed in our animal facilities 1 wk or more before exposure. Nickel aerosol was generated from a 50-mM solution of NiSO4 · 6 H2O (Sigma, St. Louis, MO) and monitored as described previously (16). Mice were exposed for 3 to 72 h to 150 ± 15 µg Ni2+/m3 in a 0.32-m3 stainless steel inhalation chamber. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Cincinnati Medical Center.

Lung Tissue Preparation and RNA Isolation
After exposure, mice were killed with an intraperitoneal injection of 50 mg/kg of sodium pentobarbital followed by exsanguination. Lungs were removed, placed in liquid nitrogen, and stored at –80°C. Total cellular RNA was isolated from frozen lung tissue with Trizol (Invitrogen, Carlsbad, CA), and quantity was assessed by A260/A280 spectrophotometric absorbance (SmartSpec 3000; Bio-Rad, Hercules, CA).

For gross pathologic analysis, the trachea was ligated, and the entire heart-lung block was removed from the chest cavity, washed with phosphate-buffered saline, and digitally photographed. To examine lung pathology, the diaphragm was punctured, a cannula was inserted into the trachea, and the lungs were instilled in situ with phosphate-buffered formalin (~ 900 µl, pH 7.1; Fisher Scientific, Fairlawn, NJ). After fixation, lung tissue was dehydrated, processed into paraffin blocks, sagittally sectioned (5 µm), and stained with hematoxylin and eosin.

RNA Expression Analysis by Oligonucleotide Microarray and Reverse Transcriptase–Polymerase Chain Reaction
Lung RNA quality was assessed by separation with a denaturing formaldehyde/agarose/ethidium bromide gel, and quantified by analysis with an Agilent Bioanalyzer (Quantum Analytics, Inc., Foster City, CA). To examine differential gene expression of 13,443 70-mer oligonucleotides, a microarray was fabricated by the Genomic and Microarray Laboratory, Center for Environmental Genetics, University of Cincinnati (http://microarray.uc.edu/), using a commercial library (Qiagen-Operon, Alameda, CA). Clones (70-mers) from the Operon Library were amplified by polymerase chain reaction (PCR), printed onto glass slides (Omnigrid Microarrayer; GeneMachines, San Carlos, CA), and consisted of 8,077 known genes: 5,017 RIKEN cDNAs, 210 nonannotated sequences/segments/IMAGE clones, and 139 genes for hypothetical proteins. Exposure groups consisted of nine mice. RNA from three mice per group were pooled for each microarray, and three separate microarrays per exposure group were compared with nonexposed control animals using 20 µg total RNA per array. Each sample of mRNA was reverse transcribed and randomly reciprocally tagged with fluorescent cyanine 3 (Cy3) or cyanine 5 (Cy5; e.g., Cy3 for control and Cy5 for 72-h exposure). Cy3 and Cy5 samples were cohybridized with the printed 70-mers. After hybridization, slides were washed and scanned at 635 (Cy5) and 532 (Cy3) nm (GenePix 4000B; Axon Instruments, Inc., Union City, CA). Data normalization was performed in three steps for each microarray as described previously (20). Consistency of fluorescence measurements in different hybridizations was assessed by calculating pairwise correlation coefficients between measurements for the same mouse in different hybridizations. Pairwise Pearson's correlation coefficients were above 0.9, indicating the reproducibility of measurements.

To further assess the expression changes of genes within the TGF-{beta} and fibrinolysis molecular pathways in lungs of mice exposed to nickel for 24, 48, and 72 h, as well as nonexposed control animals, reverse transcriptase–PCR (RT-PCR) was performed (SingleGene PCR kits; SuperArray, Frederick, MD). Initially, reverse transcription reactions were performed with 2.5 µg of total RNA (First Strand cDNA synthesis kit; SuperArray), and the reactions were stored at –20°C. PCR reactions used manufacturer-supplied primer pairs to measure the following: integrin {alpha}V (Itgav; cat. no. SPM-0796A, 24 cycles, 559-bp product); secreted phosphoprotein 1 (Spp1; cat. no. SPM-0782A, 24 cycles, 567-bp product); TGF-{beta}1 (Tgfb1; cat. no. SPM-0112A, 24 cycles, 463-bp product); thrombospondin 1 (Thbs1; cat. no. SPM-0221A, 32 cycles, 443-bp product); tenascin C (Tnc; cat. no. SPM-0945A, 30 cycles, 480-bp product); and serine (or cysteine) proteinase inhibitor, clade E, member 1 (Serpine1; cat. no. SPM-0216A, 24 cycles, 470-bp product). The expression levels of these genes were normalized to the expression of the ribosomal protein S2 (Rps2; cat. no. SPM-0479A, 24 cycles, 423-bp product). Data acquisition was performed by electrophoresis of 10 µl of each reaction on a 2% agarose gel containing 0.5 µg/ml ethidium bromide, and band intensities were analyzed with a Typhoon 8600 imager and ImageQuant software (Amersham Biosciences, Piscataway, NJ).

BAL and TGF-{beta}1 Immunoassay
To measure TGF-{beta} protein levels in the lung, BALF was collected from mice (n = 5) exposed for 72 h and compared with nonexposed control animals (n = 5). Mice were anesthetized, the diaphragm punctured, and the lungs were lavaged with two 1-ml aliquots of Hanks' balanced salt solution without Ca2+ and Mg2+ (pH 7.2, 37°C; Invitrogen). Recovered BALF samples were centrifuged (500 x g, 5 min, 4°C), and the cell-free supernatants were decanted and stored at –80°C until concentrated with centrifugal filter devices (Microcon YM-3; Millipore, Bedford, MA). To determine the concentration of TGF-{beta}1 in BAL supernatants, an ELISA assay (Quantikine, cat. no. MB100; R&D Systems, Minneapolis, MN) was used according to the manufacturer's protocol. The lower limit of detection for TGF-{beta}1 was 2.9 pg/ml.

Inhibition of TGF-{beta} In Vivo
Murine soluble chimeric TGF{beta}RII-Fc (Biogen Idec, Cambridge, MA; 50 µg in 50 µl sterile saline) or saline alone was intravenously administered to 129S1/SvImJ mice immediately before and 6 and 30 h after a 24-h exposure to nickel (n = 5 mice/group). Nonexposed control mice were concurrently injected with saline (n = 5). All mice were lavaged 72 h after the initiation of nickel exposure as previously described. Aliquots (200 µl) of BALF were cytocentrifuged (Cytospin; Shandon Scientific Ltd., Runcorn, United Kingdom), and the cells were stained with Hemacolor (EM Science, Gibbstown, NJ) for differential cell analysis. Differential cell counts were performed according to standard cytologic procedures by identifying at least 300 cells (21). Total cell counts were performed with a hemacytometer using trypan blue (Invitrogen). Total BAL protein was measured in cell-free supernatants using a bicinchoninic acid assay with bovine serum albumin used as the standard (Pierce, Rockford, IL).

Mouse Lung Epithelial Cell Culture, Transfections, and Reporter Gene Assay
To investigate the effect of TGF-{beta} on Sftpb transcription, mouse lung epithelial (MLE)-15 cells were grown in HITES (hydrocortisone, insulin, transferrin, estradiol, and sodium selenite) medium containing 4% fetal bovine serum (Sigma), as previously described (22), and transfected with Sftpb promoter constructs. These constructs were cloned in pGL3-basic luciferase reporter vector (Promega, Madison, WI) and sequenced. The initial promoter Sftpb template was a DNA sequence that contained 653 bp (–653) 5' and 35 bp (+35) 3' to the transcriptional start site. Using the –653/+35 (wild-type) Sftpb construct as a template, a {Delta}(–616/–198) promoter deletion construct was generated using PvuII and Bpu10I restriction endonuclease sites followed by blunt-end ligations. Transient transfections were conducted using FuGENE 6 transfection reagent (3 µl/well; Roche Applied Science, Indianapolis, IN), as per the manufacturer's instructions, in six-well Costar plates in duplicate or triplicate samples on at least two occasions. The Sftpb promoter constructs (750 ng/well) were cotransfected with cytomegalovirus promotor–{beta}-galactosidase (250 ng/well) as an internal transfection control. Transfected MLE-15 cells were treated with 0, 1.0, 10, and 30 ng/ml TGF-{beta}1 (R&D Systems; cat. no. 240-B) for 24 h. Cell extracts were prepared with one freeze–thaw cycle in 200 µl Reporter lysis buffer (Promega). Luciferase assays were performed using the Luciferase Assay System (Promega), and relative luciferase units for each sample were measured in a single-tube luminometer (Sirius; Berthold Detection Systems, Oak Ridge, TN). {beta}-Galactosidase activity for each sample was measured in 96-well Falcon plates using the {beta}-galactosidase Enzyme Assay System (Promega) and a Spectramax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA). All relative luciferase unit measurements were normalized to {beta}-galactosidase values.

Statistical Analysis and Assessment of Microarray Data
Statistical analysis of microarray data was performed by fitting the following mixed-effects linear model for each gene separately: Yijk = m + Ai + Sj + Ck+ eijk, where the Yijk corresponds to the normalized log-intensity on the ith array (i = 1,...,48), labeled with the kth dye (k = 1 for Cy5 and k = 2 for Cy3) and for the jth treatment condition, and m is the overall mean log-intensity, Ai is the effect of the ith array, Sj is the effect of the jth treatment condition, and Ck is the effect of the kth dye. Assumptions about model parameters were the same as described by Wolfinger and colleagues (23), with array effects assumed to be random, and treatment and dye effects assumed to be fixed. This model was fitted for each gene, and statistical significance of the differential expression between exposure groups after adjusting for the array and dye effects was assessed by calculating p values for corresponding linear contrasts. Multiple hypotheses testing adjustment was performed by calculating a false discovery rate (FDR) (24), and comparisons of the effect of various stringencies for statistical significance were performed. The data normalization and the statistical analysis were performed using SAS statistical software package (SAS Institute, Inc., Cary, NC). Each treatment comparison was performed in triplicate, and the data discussed in this article were analyzed as part of a larger experiment that involved two earlier time points of nickel exposure, as well as equivalent time series in two different mouse strains.

To further analyze the microarray dataset, we used the MAPPFinder program to dynamically link microarray data to the Gene Ontology (GO) hierarchy database as illustrated previously (19). MAPPFinder generates a gene expression profile at the level of biological processes, cellular components, and molecular functions that allows for identification of specific biological pathways that merit further investigation. The results, calculated using Fisher's exact test, are expressed as a "z score" for a particular pathway, and values greater than 2.0 were considered to be significant. GenMAPP was also used to view and analyze microarray data on biological pathways (25).

RT-PCR quantitation data, TGF-{beta}1 immunoassay data, BAL data, and Sftpb reporter construct data are presented as means ± SEM. Significant differences among groups were identified by analysis of variance. Individual comparisons between groups were confirmed by the two-tailed Student's t test. A p value less than 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Transcriptional Changes during Acute Lung Injury
After exposure to nickel, lung mRNA expression of 129S1/SvImJ mice was analyzed using oligonucleotide microarrays at various times. Of the 13,664 genes that were present on the array, 77% or more were detected in each array. For the purposes of this analysis, we only considered values significantly different from control values when p was less than 0.5 times 10–6, expected false-positive factor was less than 0.05, and the FDR was less than 0.0003, with an average intensity of at least 300, ensuring a high degree of confidence in the identified genes. Genes with the greatest decreases in expression are presented in Table 1, and are sorted by their 72-h mean expression value. Twenty-one of the 26 genes listed in Table 1 are localized to the lung, and eight are expressed in alveolar type II cells. The molecular functions of these genes as classified by GO are varied. Several of these genes with significantly decreased expression play important roles in fluid absorption and the synthesis, trafficking, or reutilization of surfactant proteins and phospholipids. These genes include ATPase, Na+/K+ transporting, {beta}1 polypeptide (Atp1b1), solute carrier family 34 (sodium phosphate), member 2 (Slc34a2), carbonyl reductase 2 (Cbr2), fatty acid synthase (Fasn), napsin A aspartic peptidase (Napsa), stearoyl-coenzyme A desaturase 2 (Scd2), and phospholipid transfer protein (Pltp). Expression of a related gene, sodium channel, nonvoltage-gated 1{beta} (Scnn1b), was also decreased –1.9 ± 0.4-fold, and although likely significant (FDR = 0.01; p = 0.0008), it did not reach the twofold threshold for inclusion. Thus, downregulation of these genes may result in disruption of essential lung surfactant function and alveolar fluid clearance in our model of nickel-induced acute lung injury.


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  		TABLE 1. SIGNIFICANTLY DECREASED GENES DETERMINED BY OLIGONUCLEOTIDE MICROARRAY ANALYSIS IN 129S1/SVIMJ MOUSE LUNGS AFTER EXPOSURE TO NICKEL FOR 24, 48, OR 72 H

 
Genes with the greatest increases in expression during nickel-induced acute lung injury are presented in Table 2, and are also sorted by their 72-h mean expression value. Of the 32 genes listed in Table 2, 28 were previously detected in lung, and a subset of these 28 included 16 that were detected in alveolar type II cells. The GO molecular functions of the genes with increasing expression were again wideranging. Notably, a number of the genes with significantly increased expression are known to be regulated by TGF-{beta} signaling, including Thbs1, S100 calcium-binding protein A9 (calgranulin B; S100a9), Tnc, Spp1(also known as osteopontin), Serpine1 (also known as plasminogen activator inhibitor-1 [PAI-1]), and matrix metalloproteinase 14 (membrane-inserted; Mmp14). These changes suggest that activation of the TGF-{beta} pathway could be an important molecular event in the development of nickel-induced acute lung injury.


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  		TABLE 2. SIGNIFICANTLY INCREASED GENES DETERMINED BY OLIGONUCLEOTIDE MICROARRAY ANALYSIS IN 129S1/SVIMJ MOUSE LUNGS AFTER EXPOSURE TO NICKEL FOR 24, 48, OR 72 H

 
To further evaluate our microarray data, GenMAPP and MAPPFinder were used to organize gene expression data into MAPPs (microarray pathway profiles) that represent specific biological pathways and functionally grouped genes. We focused on microarray gene expression data from mice exposed for 72 h. Using gene-association files from the GO Consortium, MAPPFinder assigns the thousands of genes in the 72-h expression dataset to numerous GO terms. Several significant (i.e., z score > 2.0) functional MAPPs were revealed with MAPPFinder and are presented in Table 3. Consistent with our initial assessment above, the TGF-{beta} signaling pathway was significantly changed at 72 h (z score = 2.8), reinforcing the importance of this pathway in our model of acute lung injury. Gene pathways that are involved in processes of the extracellular matrix (e.g., proteolysis, fibrinolysis; z score = 3.7) and cytokine and chemokine production (z scores = 2.8 and 2.3, respectively) are also significantly changed.


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  		TABLE 3. IDENTIFICATION OF SIGNIFICANTLY CHANGED MOLECULAR PATHWAYS BY MAPPFINDER ANALYSIS OF AN OLIGONUCLEOTIDE MICROARRAY DATASET FROM LUNGS OF 129S1/SVIMJ MICE EXPOSED TO NICKEL FOR 72 H

 
Activation of the TGF-{beta} Signaling Pathway during Acute Lung Injury
The amount of TGF-{beta}1 protein released in the lung during nickel-induced acute lung injury was assessed in the BALF by an immunoassay for TGF-{beta}1. TGF-{beta}1 increased in 72-h nickel-exposed mice as compared with control animals (Figure 1). The transcript levels of TGFB1 also were increased significantly over nonexposed control values after 72 h as measured by RT-PCR (1.9 ± 0.2-fold, p < 0.05). These results agree with the increased gene expression of genes known to activate TGF-{beta} (i.e., Itgav and Thbs1) shown in Figure 2B.



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  		Figure 1. Increased transforming growth factor (TGF)-{beta} protein in lungs of mice exposed to nickel for 72 h. TGF-{beta}1 in bronchoalveolar lavage fluid (BALF) was measured by ELISA. Data are presented as means ± SEM (n = 5 mice/group). * Denotes significant difference from nonexposed control group, p < 0.05.

 



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  		Figure 2. Expression changes in genes of the TGF-{beta} signaling pathway in lungs of mice exposed to nickel as measured by oligonucleotide microarray and reverse transcriptase–polymerase chain reaction (RT-PCR). (A) Changes in gene expression are expressed using the GenMAPP format. Genes that were increased atleast 1.5-fold over control values and had a significance value of p <= 0.05 are labeled in red. Those genes that were decreased atleast 1.5-fold from control values and had a significance value of p <= 0.05 are labeled in green. Those genes that did not meet the aforementioned criteria are labeled in gray, and those genes that were not on the array are labeled in white. (B) RT-PCR analysis of mRNA for genes that are activators of TGF-{beta} after 24, 48, or 72 h of exposure. Data are presented as fold changes over control values (n = 5 mice/group, means ± SEM) and are normalized to ribosomal protein S2 (Rps2) expression. * Denotes significant difference from nonexposed control group, p < 0.05. ITGAV = integrin {alpha}V; THBS1 = thrombospondin 1. (C) RT-PCR analysis of mRNA for genes that are activated by TGF-{beta} after 24, 48, or 72 h of exposure. Data are presented as fold changes over control values (n = 5 mice/group, means ± SEM) and are normalized to Rps2 expression. * Denotes significant difference from nonexposed control group, p < 0.05. SPP1 = secreted phosphoprotein 1; TIMP1 = tissue inhibitor of metalloproteinase 1; TNC = tenascin C.

 
An overview of expression changes in the TGF-{beta} pathway are presented in Figure 2A for 129S1/SvImJ mice exposed to nickel for 72 h. To visually analyze the TGF-{beta} signaling pathway, GenMAPP was used to characterize the response at 72 h. Increases and decreases in gene expression are presented as fold changes as compared with nonexposed control mice. Of the 38 genes examined in the TGF-{beta} pathway, 21 were significantly altered, with 16 increased (shown in red) and 5 decreased (shown in green). The increases in expression were mostly in genes known to stimulate the pathway, and the decreases were in genes known to inhibit the pathway. Thus, these changes are consistent with activation of TGF-{beta} signaling.

RT-PCR was performed using SuperArray SingleGene PCR kits to confirm the expression changes of key genes in this pathway. Increases in expression of genes that are activators of TGF-{beta} (Itgav and Thbs1; Figure 2B), and genes that are activated by TGF-{beta} (Spp1, tissue inhibitor of metalloproteinase 1 [Timp1], and Tnc; Figure 2C) after 24, 48, and 72 h of nickel exposure were confirmed.

Modulation of the Fibrinolysis Cascade during Acute Lung Injury
The involvement of fibrinolysis and coagulation in our model of nickel-induced acute lung injury was also evaluated on gross pathologic and histologic levels. Photographs of lungs from mice exposed to nickel for 48 and 72 h and nonexposed control animals are shown in Figures 3A–3C. As acute lung injury progressed, the lung surface appeared red in color, indicative of hemorrhage and coagulation. Histologic analysis of lung sections from nickel-exposed mice as compared with control animals revealed progressive pulmonary edema marked by perivascular swelling and interstitial thickening (Figures 3D–3I).





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  		Figure 3. Gross pathology (A–C) and light microscope histology (D–I) of lungs of mice exposed to nickel for 48 and 72 h, and nonexposed control animals. (A) Nonexposed control lung, (B) 48-h exposed lung, (C) 72-h exposed lung, (D) non-exposed control (original magnification x 40), (E) 48-h exposed (original magnification x 40), (F) 72-h exposed (original magnification x 40), (G) nonexposed control (original magnification x 400), (H) 48-h exposed (original magnification x 400), (I) 72-h exposed (original magnification x 400). The gross pathologic progression of acute lung injury is apparent, with the lung surface appearing in red from hemorrhage and coagulation as exposure progressed as compared with nonexposed control lungs (A–C). Histologic analysis of lung sections from nickel-exposed mice compared with control animals showed a progressive increase in perivascular swelling (arrows, E and F) and alveolar wall thickening (arrows, H and I).

 
The increased expression of genes involved in fibrinolysis as measured by microarray are presented in Figure 4A. RT-PCR confirmed SERPINE1 transcript levels increased at 24, 48, and 72 h (Figure 4B). These RT-PCR data support increased gene expression observed by microarray (Table 2).




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  		Figure 4. Expression changes in genes of the fibrinolysis signaling pathway in lungs of mice exposed to nickel for 72 h as measured by oligonucleotide microarray and RT-PCR. (A) Changes in gene expression are expressed using the GenMAPP format. Genes that were increased at least 1.5-fold over control values and had a significance value of p <= 0.05 are labeled in red. Those genes that were decreased at least 1.5-fold from control values and had a significance value of p <= 0.05 are labeled in green. Those genes that did not meet the aforementioned criteria are labeled in gray, and those genes that were not on the array are labeled in white. (B) RT-PCR analysis of serine (or cysteine) proteinase inhibitor, clade E, member 1 (Serpine1) mRNA after 24, 48, or 72 h of exposure. Data are presented as fold changes over control values (n = 5 mice/group, means ± SEM) and are normalized to Rps2 expression. * Denotes significant difference from nonexposed control group, p < 0.05.

 
Effect of TGF-{beta} Inhibition In Vivo
Total BAL protein (measured at 72 h after the initiation of exposure to nickel) was significantly lower in 129S1/SvImJ mice administered murine soluble chimeric TGF{beta}RII-Fc just before and 6 and 30 h after a 24-h exposure to nickel as compared with saline-injected mice (Figure 5). Both TGF{beta}RII-Fc– and saline-treated mice had significantly greater BAL protein after nickel exposure compared with saline-treated nonexposed control animals. Total cells, neutrophils, and macrophages in BAL were not different between all treatment/exposure groups (Table 4).



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  		Figure 5. Soluble chimeric TGF-{beta} type II receptor (TGF{beta}RII-Fc) attenuates nickel-induced BAL protein in 129S1/SvImJ mice. Mice were intravenously administered TGF{beta}RII-Fc (50 µg in 50 µl sterile saline) or saline alone immediately before and 6 and 30 h after a 24-h exposure to nickel. Nonexposed control mice were also concurrently injected with saline. All mice were lavaged 72 h after the initiation of nickel-exposure. Data are presented as means ± SEM. * Denotes significant difference from nonexposed saline-treated control group, p < 0.05; +denotes significant difference from nickel-exposed saline-treated group, p < 0.05.

 

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  		TABLE 4. TOTAL CELLS, NEUTROPHILS, AND MACROPHAGES RECOVERED IN BRONCHOALVEOLAR LAVAGE FLUID FROM 129S1/SVIMJ MICE ADMINISTERED SALINE OR TGF{beta}RII-FC AND EXPOSED TO NICKEL OR NONEXPOSED CONTROL ANIMALS

 
TGF-{beta} Represses Sftpb Transcription in Mouse Lung Epithelial Cells
Previously, we reported that Sftpb gene expression decreases in lungs of mice exposed to nickel (17). MLE-15 cells transfected with the –653/+35 (wild-type) promoter construct were treated with increasing doses of TGF-{beta}1 (0.1–30 ng/ml) for 24 h to determine the effect of TGF-{beta} on Sftpb transcription. Luciferase activities (normalized to {beta}-galactosidase) of the –653/+35 (wild-type) promoter were significantly decreased below untreated (control) values in a dose-dependent manner after treatment with TGF-{beta}1 (Figure 6A). This is consistent with previously noted repression of human SFTPB by TGF-{beta} in vitro (26, 27). In addition, the effect of TGF-{beta}1 on the repression of Sftpb transcription was reversed in cells transfected with the {Delta}(–616/–198) construct treated with 30 ng/ml TGF-{beta}1 (Figure 6B). Luciferase activity of the –653/+35 (wild-type; 43% of the nonexposed control value) was significantly decreased below that of the {Delta}(–616/–198) construct (69% of the nonexposed control value) after 30 ng/ml TGF-{beta}1 treatment. These data suggest that cis-acting elements between –616 to –198 in the Sftpb promoter are partially responsible for its repression by TGF-{beta}1.




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  		Figure 6. Repression of Sftpb promoter activity by TGF-{beta}1 in vitro. (A) Transient transfections of the –653/+35 (wild-type) mouse Sftpb luciferase promoter construct were done in MLE-15 cells (750 ng of construct/well and 250 ng of cytomegalovirus promoter–{beta}-galactosidase/well as an internal control) and treated with 0.1, 1.0, 10, and 30 ng/ml TGF-{beta}1 for 24 h. (B) Transient transfections of the –653/+35 (wild-type) and {Delta}(–616/–198) mouse Sftpb luciferase promoter constructs were done in MLE-15 cells (750 ng of construct/well and 250 ng of pCMV–{beta}-galactosidase/well as an internal control) and treated with 30 ng/ml TGF-{beta}1 for 24 h. For both panels, relative luciferase activities normalized to cotransfected {beta}-galactosidase are expressed as percentage of untreated control values. Values are means ± SEM (n = 5–11/group). * Denotes significant difference from untreated control, p < 0.05; + denotes significant difference from –653/+35 (wild-type) Sftpb promoter construct, p < 0.05. Striped boxes denote location of "CAGA" elements. Other promoter elements are as labeled in the figure. CREB = cydic adenosine monophosphate (CAMP)–responsive element-binding protein; HNF-3 = hepatocyte nuclear factor 3; TTF-1 = thyroid transcription factor 1.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Microarray Analyses
In the present study, molecular events of acute lung injury were identified using oligonucleotide microarray analysis to examine temporal transcript changes in the lungs of 129S1/SvImJ mice after exposure to nickel. The first level of analysis of the microarray data was a statistical analysis of genes that were significantly different from controls. This information was the starting point from which subsequent analyses were performed, and served as a foundation for investigations of the underlying biological pathways that control responses to inhaled nickel. Transcriptional analyses, however, are not trouble-free and can suffer from limitations. Microarray results are typically expressed as a fold increase or decrease. Although useful, without a context it may be difficult to ascribe biological meaning to this outcome measure. For example, large fold increases (i.e., 10- to 100-fold) may solely reflect tissue-specific (an absence of constitutive) expression in control tissue.

In the initial analysis of the microarray data, many of the genes that were decreased the most after 72 h of exposure to nickel play important roles in lung fluid absorption or surfactant and phospholipid synthesis (Table 1). Perturbation of these physiologic processes contributes to the development of acute lung injury. For example, Atp1b1 expression was notably decreased in our model of acute lung injury. Atp1b1 is a key element in the regulation of active Na+ transport in alveolar epithelium that keeps the airspaces free of excess fluid (28–30), which is a hallmark of patients with acute lung injury (31).

Genes involved in surfactant and phospholipid production, metabolism, and trafficking were also among those that were most significantly decreased. For instance, Pltp assists in the uptake of secreted surfactant lipids, and plays a role in surfactant lipid trafficking and reutilization in alveolar type II epithelial cells (32). Slc34a2 is a transporter that recycles intracellular phosphate, an essential component for surfactant phospholipid synthesis (33). Fatty acids are important constituents of surfactant, and can also alter surfactant synthesis. Fasn and Scd2 were significantly decreased by nickel exposure and function in the generation of fatty acid in alveolar epithelial cells (34). In addition, Napsa is involved in the proper processing of surfactant-associated protein B (35), and knockdown of Napsa in type II cells results in decreased levels of the mature SP (36). Taken together, these data and our previous data on decreased Sftpb expression in nickel-induced acute lung injury indicate that the transcript levels of several essential genes in the synthesis and function of pulmonary surfactant are significantly decreased, and these expression changes may cumulatively lead to surfactant disruption in nickel-induced acute lung injury.

MAPPFinder was used to further analyze the functional pathways altered by nickel exposure by performing pathway-based analysis of the microarray data. A major benefit of this type of analysis is that it generates a description of the biology that is occurring within the dataset, in contrast to hierarchic clustering or self-organizing maps that arrange genes according to similarity in pattern of gene expression (17, 37, 38). Pathways that had a large number of expression changes include extracellular matrix, cytokine, and chemokine. In another microarray analysis of TGF-{beta} signaling, Verrecchia and colleagues (39) identified genes (including TIMP1, THBS1, PAI-1, TNC, and MMP14) rapidly induced by TGF-{beta} in human dermal fibroblasts. Similarly, many of the identified genes (67% of those measured in both experiments) exhibited altered expression in mice with acute lung injury. Furthermore, MAPPFinder established that TGF-{beta} signaling was significantly altered in our mouse model, which supported the changes in genes of the TGF-{beta} signaling pathway in the initial analysis of the genes most increased in our microarray data (Table 2), and suggested that TGF-{beta} was a central mediator of nickel-induced acute lung injury.

Modulation of Extracellular Matrix and Fibrinolysis by TGF-{beta}
TGF-{beta} activation is known to play a role in the development of acute lung injury in humans and laboratory animals through alteration of fibroproliferative responses, lung permeability, and inflammatory cell influx (7, 12, 40, 41). However, the molecular mechanisms of TGF-{beta} in acute lung injury are not well understood. Our data suggest that increased TGF-{beta} altered multiple lung cell functions important to the initiation and progression of acute lung injury. The careful use of gene expression microarray analysis identified specific molecular alterations underlying the functional changes.

We found that TGF-{beta}1 protein increased in BAL after exposure to nickel. The extracellular concentration of TGF-{beta} protein is mainly controlled by the conversion of inactive TGF-{beta} (secreted as inactive complexes containing TGF-{beta}, the TGF-{beta} propeptide, and latent TGF-{beta} binding protein) to active TGF-{beta}. Activated TGF-{beta} is then able to induce lung cells to generate more total TGF-{beta} via autoinduction. There are two distinct potential mechanisms by which TGF-{beta} could be activated in our system. First, integrin {alpha}v{beta}6 is an in vivo activator of latent TGF-{beta}1 through the recognition of an arginine-glycine-aspartic acid site on the TGF-{beta} propeptide (42, 43). Second, activation of latent TGF-{beta} can be achieved by direct binding of thrombospondin 1 protein (44, 45). Transcripts for ITGAV and THBS1 were significantly increased after nickel exposure.

Increased TGF-{beta} levels are able induce Spp1 (46), Timp1 (47, 48), and Tnc (49) gene expression. SPP1 is a matricellular protein that possesses cytokine-like properties and can act as a chemoattractant for macrophages (50, 51). SPP1 also regulates fibrogenic signals in epithelial repair after bleomycin-induced lung injury (52). TIMP1 is a metalloproteinase inhibitor that can mediate inflammation and repair processes during acute lung injury through stabilization of matrix components (53). TNC is an extracellular matrix glycoprotein that may play a role similar to TIMP1 (54, 55). Interestingly, SPP1 (56) and TNC (57) contain an arginine-glycine-aspartic amino acid sequence that could activate TGF-{beta} in a positive feedback loop involving integrins (58). The expression of Spp1, Timp1, and Tnc was markedly increased after 72 h of nickel exposure, suggesting that the activation of the TGF-{beta} pathway has an effect on the expression of extracellular matrix–altering genes in our model of acute lung injury.

Coagulation and fibrinolysis are processes that form and dissolve fibrin, respectively. These processes are precisely regulated and serve to protect from excessive blood loss or undue fibrin deposition. Moreover, acute and chronic pulmonary diseases are characterized by impaired fibrinolytic activity within the lung (59, 60). Increased lung TGF-{beta} may play a role in the modulation of fibrinolysis through the induction of Serpine1 in our model of acute lung injury. PAI-1 (known as Serpine1 in mice) is the major inhibitor of fibrinolytic activity in the alveolar space (59), and is highly induced by TGF-{beta} (61). Previous studies by Andrew and coworkers (62, 63) have demonstrated that nickel is also able to transcriptionally induce PAI-1 and inhibit fibrinolytic activity in human airway epithelial cells (64). Transcript levels of SERPINE1 were significantly increased after nickel exposure (Figure 4), and pathologic and histologic analyses of the exposed mouse lungs confirmed alterations in fibrinolysis.

Attenuation of Nickel-induced BAL Protein by TGF{beta}RII-Fc In Vivo
Studies investigating the role of TGF-{beta} in lung injury have focused mainly on its function in the mediation of fibroproliferation and resolution of tissue injury. Inhibition of TGF-{beta} by a monoclonal antibody hindered the induction of proinflammatory cytokines and lung injury induced by hemorrhagic shock (65). More recently, Pitett and colleagues (12) found that administration of the soluble TGF{beta}RII-Fc chimera was an effective inhibitor of integrin {alpha}v{beta}6–mediated activation of TGF-{beta}, and also inhibits many TGF-{beta}–mediated processes in vitro and in vivo (66, 67). Moreover, these investigators have demonstrated that integrin {alpha}v{beta}6–mediated local activation of TGF-{beta} is important in the development of bleomycin-induced pulmonary edema using integrin {beta}6–/– mice. In this study, treatment of integrin {beta}6+/+ wild-type mice (129/terSVEMS strain) with TGF{beta}RII-Fc prevented increased lung permeability as assessed approximately 120 h after instillation of bleomycin or Escherichia coli endotoxin (12). These observations agree with our data in which administration of soluble TGF{beta}RII-Fc chimera attenuated nickel-induced BAL protein measured at 72 h after the initiation of nickel exposure. The attenuation of permeability we observed was less than that of Pittet and coworkers, which may be due to differences in the agonist (nickel vs. bleomycin vs. E. coli endotoxin) or the magnitude of injury. Moreover, TGF-{beta} has been found to decrease sodium channel–dependent fluid transport (13–15), and decreases in ATP1B1 and SCNN1B transcript levels noted here may have contributed to edema. Inhibition of TGF-{beta} did not lessen BAL neutrophils and macrophages after nickel exposure, which is also consistent with Pitett and colleagues' findings with bleomycin-treated integrin {beta}6–/– mice. Taken together, these findings support an important role for TGF-{beta} activation in the development of lung hyperpermeability and edema in acute lung injury.

Modulation of Sftpb Promoter Activity by TGF-{beta} In Vitro
We have previously demonstrated that exposure to nickel decreases lung Sftpb gene expression in a time-dependent fashion in mice (17). The increase in TGF-{beta} in the lungs of mice after nickel exposure may have contributed to the decrease in Sftpb expression. Using a human fetal lung explant model, TGF-{beta} inhibited Sftpb expression and protein synthesis (26, 68). In the present study, treatment with TGF-{beta}1 dose-dependently repressed –653/+35 (wild-type) Sftpb promoter construct activity in MLE-15 (Figure 5A). Furthermore, the {Delta}(–616/–198) construct exhibited an attenuation of TGF-{beta}1 repression of Sftpb activity, providing evidence for a putative TGF-{beta}1–responsive cis-acting repressor element(s) between –653 to –198 in the mouse Sftpb promoter. The lack of Sftpb repression by TGF-{beta}1 with the {Delta}(–616/–198) construct is in contrast to a study by Kumar and coworkers (69) in which repression was observed in a similar human SFTPB construct that was used in National Cancer Institute H441 lung tumor cells. The dissimilarities between these data could be due to the collective differences in the reporter constructs used, the transfection conditions, the duration of TGF-{beta}1 exposure, and the cell types used in the experiments.

Transcriptional regulation by TGF-{beta} is regulated by a family of factors known as mothers against decapentaplegic homologs (SMAD), which act as intracellular effectors of TGF-{beta} signaling (70). One mechanism of TGF-{beta} inhibition of human SFTPB was found to be through SMAD3 protein–protein interactions with thyroid transcription factor-1 (TITF1) and hepatocyte nuclear factor transcription factors in H441 cells (27). TGF-{beta}–mediated gene expression changes can also occur via direct binding of SMAD proteins to promoter "CAGA" elements in target genes (71–74). In the –653/+35 (wild-type) promoter used in the present study, there are four CAGA motifs located between –653 to –198 that could be involved in SMAD-mediated TGF-{beta} inhibition of Sftpb.

Several additional transcription factor binding sites are located between –653 to –198 that could be involved in TGF-{beta} inhibition of Sftpb, including one activator protein (AP)-1 site, one nuclear factor (NF)-{kappa}B site, two NFI sites, and one TITF1 site. AP-1 binding sites in the promoters of several genes have been functionally associated with transcriptional regulation by TGF-{beta} (13, 75–77), and SMAD proteins act synergistically with AP-1 complexes in TGF-{beta}–responsive genes (78–81). Transcriptional regulation of the mouse Sftpb promoter can be regulated by AP-1 family members (82). Alterations in gene expression by TGF-{beta} can also be transcriptionally regulated by NF-{kappa}B (83). In addition, SMAD and NFI DNA binding domains are similar (84), and NFI and TITF-1 have been shown to modulate surfactant-associated protein C transcription (85). Further investigation is necessary to elucidate the transcriptional mechanism of TGF-{beta} suppression of Sftpb in our in vitro model.

The probable role of TGF-{beta} in our model of acute lung injury is schematically presented in Figure 7. Identification of the genes that changed the most using high stringencies for significance demonstrated that many genes that are involved in proper surfactant synthesis and trafficking and fluid absorption were significantly decreased, and several genes that are associated with the TGF-{beta} pathway were significantly increased. MAPPFinder analysis indicated that TGF-{beta} signaling is significantly altered in lungs of mice after exposure to nickel. Activation of TGF-{beta} can then regulate critical downstream events in the development of acute lung injury, such as extracellular matrix gene expression, fibrinolysis (through Serpine1), and Sftpb expression. Inhibition of TGF-{beta} with soluble chimeric TGF{beta}RII-Fc attenuated nickel-induced protein in BAL. Transient transfection assays with mouse Sftpb promoter constructs demonstrated that TGF-{beta} plays an important role in the regulation of Sftpb transcription in mouse lung epithelial cells. Due to the complex nature of the molecular mechanisms of acute lung injury and the innate limitations of any mouse model of disease, the implications of our results to acute lung injury in humans is an important question. In addition, we do not know how TGF-{beta} may be functioning at much earlier time points in our model of acute lung injury. However, our findings suggest that TGF-{beta} may act as a central mediator in the development of acute lung injury, and reinforce previous studies that have implicated TGF-{beta} in other models of acute lung injury. These data should stimulate further investigations on the role of TGF-{beta} in acute lung injury, novel therapeutic tactics, and additional mechanisms by which intersecting molecular pathways are able to regulate the pathogenesis of acute lung injury.



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  		Figure 7. Schematic diagram of the role of TGF-{beta} in acute lung injury. After exposure to nickel, TGF-{beta} is increased in the lungs of mice. TGF-{beta} and TGF-{beta}–inducible genes are able to modify lung permeability, epithelial ion transport, fibrinolysis, the extracellular matrix, and surfactant homeostasis. The integration of the changes in these molecular pathways by TGF-{beta} implicates it as a central mediator of acute lung injury.

 

   Acknowledgments
 
The authors thank Cindy J. Bachurski (Cincinnati Children's Hospital Medical Center) for the plasmid gift and Dean Sheppard (University of California–San Francisco) for critical reading of this manuscript.


   FOOTNOTES
 
Supported by National Institutes of Health grants ES10562, HL65612, ES06096, and ES07250.

Originally Published in Press as DOI: 10.1164/rccm.200502-286OC on August 11, 2005

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 22, 2005; accepted in final form August 9, 2005


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