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Am J Physiol Lung Cell Mol Physiol 294: L34-L45, 2008. First published October 12, 2007; doi:10.1152/ajplung.00007.2007
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Functional role and species-specific contribution of arginases in pulmonary fibrosis

Kamila Kitowska, Dariusz Zakrzewicz, Melanie Königshoff, Izabella Chrobak, Friedrich Grimminger, Werner Seeger, Patrick Bulau, and Oliver Eickelberg

Department of Medicine, University of Giessen Lung Center, University of Giessen School of Medicine, Giessen, Germany

Submitted 5 January 2007 ; accepted in final form 3 October 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lung fibrosis is characterized by increased deposition of ECM, especially collagens, and enhanced proliferation of fibroblasts. L-arginine is a key precursor of nitric oxide, asymmetric dimethylarginine, and proline, an amino acid enriched in collagen. We hypothesized that L-arginine metabolism is altered in pulmonary fibrosis, ultimately affecting collagen synthesis. Expression analysis of key enzymes in the arginine pathway, protein arginine methyltransferases (Prmt), arginine transporters, and arginases by quantitative (q) RT-PCR and Western blot revealed significant upregulation of arginase-1 and -2, but not Prmt or arginine transporters, during bleomycin-induced pulmonary fibrosis in mice. HPLC revealed a concomitant, time-dependent decrease in pulmonary L-arginine levels. Arginase-1 and -2 mRNA and protein expression was increased in primary fibroblasts isolated from bleomycin-treated mice, compared with controls, and assessed by qRT-PCR and Western blot analysis. TGF-β1, a key profibrotic mediator, induced arginase-1 and -2 mRNA expression in primary and NIH/3T3 fibroblasts. Treatment of fibroblasts with the arginase inhibitor, NG-hydroxy-L-arginine, attenuated TGF-β1-stimulated collagen deposition, but not collagen mRNA expression or Smad signaling, in fibroblasts. In human lungs derived from patients with idiopathic pulmonary fibrosis, arginase activity was unchanged, but arginase-1 expression significantly decreased when compared with donor lungs. Our results thus demonstrate that arginase-1 is expressed and functionally important for collagen deposition in lung fibroblasts. TGF-β1-induced upregulation of arginase-1 suggests an interplay between profibrotic agents and L-arginine metabolism during the course of lung fibrosis in the mouse, whereas species-specific regulatory mechanisms may account for the differences observed in mouse and human.

lung fibrosis; fibroblasts; collagen

IDIOPATHIC PULMONARY FIBROSIS (IPF) is a progressive and fatal lung disease of unknown origin. It is characterized by alveolar epithelial cell (AEC) damage, increased deposition of ECM in the lung interstitium, enhanced fibroblast/myofibroblast proliferation and activation, and, ultimately, distortion of normal lung architecture and loss of respiratory function (15, 21, 37, 40). The hallmark lesions of IPF are fibroblast foci, sites featuring activated myofibroblasts that synthesize and deposit a collagen-rich ECM in the lung interstitium. The pathogenesis of IPF includes inappropriate alveolar regeneration in response to repetitive epithelial injuries, perpetuated by dysregulated wound repair in response to local inflammation. Whereas the initial trigger of this disease is most likely represented by an epithelial injury, the interstitial fibroblast/myofibroblast represents the key effector cell responsible for the increased ECM deposition characteristic for IPF (18, 22, 38).

Histologically, IPF is characterized by a heterogeneous picture of fibroblastic foci, often adjacent to injured and/or hyperplastic AEC, varying degrees of interstitial inflammation, and emphysematous areas (called honeycombing; Ref. 17). Some of these characteristics are recapitulated in the bleomycin-induced mouse model of lung fibrosis, which has led to widespread use of this animal model in pathogenetic studies of lung fibrosis (2). In mouse and human, fibroblasts represent key participants of repair and regeneration in the lung, mostly because of their central role in regulating ECM composition, which serves as a scaffold for tissue homeostasis and repair. Fibroblasts secrete large amounts of fibrillar collagens, which are significantly increased in IPF (13, 20, 22, 27, 37).

The most abundant amino acids in fibrillar collagens are glycine and L-proline. Posttranslational hydroxylation of L-proline occurs in an ascorbic acid-dependent manner and is essential for collagen stability (3). L-proline is sequentially generated from L-ornithine and L-arginine via ornithine aminotransferase (Oat) and arginase, respectively. The abundance of L-arginine, as well as Oat and arginase expression, therefore constitute essential determinants of intracellular L-proline levels (29, 43). L-arginine is also a key precursor for nitric oxide (NO), NG-monomethyl-L-arginine (L-NMMA), NGNG (asymmetric) dimethylarginine (ADMA), and NGN'G (symmetric) dimethylarginine (SDMA), mediators that control cell proliferation, differentiation, and apoptosis. L-arginine metabolism is highly controlled by the following sets of enzymes: 1) NO synthases (NOS), generating NO; 2) arginases, generating L-ornithine, further metabolized to L-proline; and 3) protein arginine methyltransferases (Prmt), generating L-NMMA, ADMA, or SDMA (5, 30, 41).

Owing to the common use of L-arginine as a substrate, NOS, arginases, and Prmt can limit substrate availability for each other and also influence each other's activity and expression. In this respect, it has been demonstrated that arginases can regulate constitutive and inducible NOS activity (8, 39). Upregulation of arginase expression has also been shown to contribute to the pathophysiology of diseases, which exhibit dysregulated NO signaling, including hypertension, atherosclerosis, or asthma (26, 44, 47). Increased arginase activity was demonstrated in patients with cystic fibrosis (14), which is characterized by chronic airway inflammation and infection-associated pulmonary exacerbations.

As such, L-arginine metabolism is at the center of tissue remodeling, giving rise to mediators controlling vascular (ADMA, NO, L-NMMA) and interstitial (L-proline, NO) remodeling. We therefore hypothesized that L-arginine metabolizing enzymes are altered in pulmonary fibrosis and sought to characterize their expression in the mouse model of bleomycin-induced lung fibrosis and patients with IPF. We demonstrate arginase-1 and -2 upregulation in fibrotic mouse lungs, which correlated with decreased levels of pulmonary L-arginine. Both arginases localized to fibrotic lesions and were highly expressed in primary lung fibroblasts derived from bleomycin-treated mice. We further demonstrate that TGF-β1, a key profibrotic mediator, potently induced arginase-1 expression in primary lung fibroblasts. The arginase inhibitor, NG-hydroxy-L-arginine (NOHA), potently inhibited TGF-β1-induced collagen deposition by fibroblasts via posttranslational mechanisms while leaving collagen mRNA expression and Smad signaling unaffected. Lung specimen derived from IPF patients, however, did not exhibit higher arginase expression or activity, which suggests a species-specific expression profile and activity of arginases in the lung.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. NOHA monoacetate salt was purchased from Calbiochem/EMD Biosciences (San Diego, CA), and TGF-β1 ligand was obtained from R&D Systems (Minneapolis, MN). DMEM and FCS were purchased from GIBCO/Invitrogen (Carlsbad, CA) and PAA Laboratories (Pasching, Austria), respectively.

Animal model of fibrosis. All animal studies were performed according to the guidelines of the University of Giessen and approved by the local authorities (Regierungspraesidium Giessen, no. II25.319c20-15; GI20/10-Nr.22/2000). Specific pathogen-free female C57BL/6N mice weighing 18–20 g (supplied by Charles River, Sulzfeld, Germany) were used throughout this study. Bleomycin was dissolved in 0.9% (wt/vol) PBS and administered as a single dose of 0.08 mg in 200 µl of PBS per animal via microsprayer application on day 0. Control animals received only PBS instillations. The lungs from mice that were killed after 3, 5, 7, 14, or 21 days of bleomycin exposure were harvested and processed for embedding and sectioning, reserved for the isolation of primary fibroblasts, or immediately snap-frozen in liquid nitrogen.

Human tissues. Lung tissue biopsies were obtained from six patients with IPF (usual interstitial pneumonia pattern; mean age 51.3 ± 11.4 yr; 2 females, 4 males) and six control subjects (organ donors, mean age 47.5 ± 13.9 yr; 2 females, 4 males). The study protocol was approved by the Ethics Committee of the Justus-Liebig-University School of Medicine (AZ 31/93). Informed consent was obtained from each subject for the study protocol.

Cell culture. The NIH/3T3 murine fibroblast cell line (German Collection of Microorganisms and Cell Cultures; DSMZ, Braunschweig, Germany) as well as primary lung fibroblasts from bleomycin- or saline-treated mice were maintained in DMEM containing 10% FCS (10). For experiments described herein, cells were plated at 30,000 cells/well in six-well plates or 100-mm plates, depending on the experiment. Cells were treated with TGF-β1 (2 ng/ml) with or without the arginase inhibitor, NOHA monoacetate salt. After stimulation, cells were harvested at the indicated time points for the determination of mRNA, protein levels, or collagen production.

RNA isolation and analysis. Total RNA from lung tissue or cells was isolated using the total RNA purification system (Roti Quick Kit; Carl Roth, Karlsruhe, Germany; Ref. 46). For RT-PCR, 0.5 µg of RNA was reverse-transcribed to cDNA using ImProm-II Reverse Transcriptase (Promega, Madison, WI). One microliter of cDNA was then amplified using GoTaq Flexi DNA Polymerase (Promega) using the primers indicated in Table 1. The PCR reactions were optimized such that reactions were stopped within the exponential phase of amplification. For the detection of PCR products, 20 µl of each reaction mixture was electrophoresed on a 2% agarose gel and stained with ethidium bromide. Gapdh served as a loading control, as indicated. Real-time quantitative (q) RT-PCR was performed using fluorogenic SYBR Green and the Sequence Detection System 7700 (Applied Biosystems, Foster City, CA). Pbgd, a ubiquitously and equally expressed gene that is free of pseudogenes, was used as the reference gene in all qRT-PCR reactions. PCRs were performed using the primers listed in Table 1, used at a final concentration of 200 nM. Relative transcript abundance of a gene is expressed in cycle threshold ({Delta}Ct) values ({Delta}Ct = Ctreference – Cttarget). Relative changes in transcript levels compared with controls are expressed as {Delta}{Delta}Ct values ({Delta}{Delta}Ct = {Delta}Cttreated – {Delta}Ctcontrol). All {Delta}{Delta}Ct values correspond approximately to the binary logarithm of the fold change.


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  		Table 1. Primer list

 
Protein isolation and analysis. Lung tissues were homogenized in liquid nitrogen followed by addition of ice-cold cell lysis buffer [20 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (vol/vol) Triton X-100, 2 mM Na3VO4]. Homogenized tissues were then incubated on ice (30 min) and centrifuged at 16,000 g (15 min). The resulting supernatants were used as tissue extracts and stored at –20°C for further experiments. Protein concentrations were determined using the Quick Start Bradford Dye Reagent and a SmartSpec 3000 Spectrophotometer (both from Bio-Rad, Hercules, CA). For Western blot analysis, equal amounts of tissue extracts (30 µg) were separated on 10% SDS-PAGE gels and transferred to PVDF-Plus membranes (GE Osmonics, Trevose, PA; Ref. 46). Western blots were performed with antibodies against arginase-1 (1:1,000; BD Transduction Laboratories, Lexington, KY) and arginase-2 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), Prmt1 (1:2,000), Prmt3 (1:2,000), Prmt4 (1:1,000), and Prmt5 (1:2,000) (all from Upstate/Millipore, Dundee, United Kingdom), Prmt2 (1:1,000; Abcam, Cambridge, United Kingdom), or Prmt6 (1:500; Imgenex, San Diego, CA). After incubation with the respective secondary antibodies, specific bands were visualized by autoradiography using enhanced chemiluminescence according to the manufacturer's instructions (Lumigen; Amersham, Buckinghamshire, United Kingdom). Densitometric analysis of autoradiographies was performed using a GS-800 Calibrated Densitometer and Quantity One 1-D Analysis Software (both from Bio-Rad).

Isolation of basic amino acids and derivatization. All procedures were performed as previously described (6, 7, 46). Tissue extracts were subjected to crude fractionation on Oasis MCX solid-phase extraction (SPE) cartridges (30 mg, 1 ml; Waters, Milford, MA). One hundred microliters of each sample was then adjusted to a final volume of 1 ml with PBS. All conditioning, washing, and elution steps were performed on a vacuum-manifold with a capacity for 20 columns (Waters) at a flow rate of 0.5 ml/min. The SPE cartridges were conditioned with 2 ml of methanol/water/ammonia (50:45:5, vol/vol/vol), followed by 2 ml of PBS before sample application. Samples were passed through SPE cartridges and contaminating components removed with 2 ml of 0.1 M HCl, followed by 2 ml of methanol. Basic compounds were eluted with 1 ml of methanol/water/ammonia (50:45:5, vol/vol/vol). Samples were dried in a vacuum centrifuge and used immediately or stored at –20°C until analyzed. Eluates were redissolved in 230 µl of distilled water and centrifuged at 14,000 g for 2 min to remove particulates before derivatization for HPLC.

Derivatization and chromatographic separation. All procedures were performed as previously described (6, 7). The ortho-phthaldialdehyde (OPA) reagent was freshly prepared in potassium borate buffer (both Grom, Rottenburg-Hailfingen, Germany) according to the manufacturer's instructions. Samples (30 µl) were combined with 62.5 µl of OPA reagent, immediately transferred to the auto sampler, and injected exactly after 2 min. Quantification of amino acids was performed on a HPLC system consisting of an ASI-100 auto sampler, a model P680 gradient pump, a model RF-2000 fluorescence detector, and a data acquisition system (Chromeleon 6.60; Dionex, Sunnyvale, CA). Separation was performed according to a method previously described (6, 7). Fluorescent amino acid derivatives were separated on a SunFire C18 column (4.6 x 150 mm; 3.5-µm particle size; 100 Å pore size) with a µBondapak C18 guard column at 30°C and a flow rate of 1.1 ml/min (all from Waters). After sample injection (125 µl), separation was performed under isocratic conditions with 8.8% (vol/vol) acetonitrile in 25 mM potassium phosphate buffer (pH 6.8) as solvent. The isocratic conditions were maintained for 12 min. To elute strongly bound compounds, the column was flushed with acetonitrile/water (50:50, vol/vol) for 15 min and reequilibrated under isocratic conditions for 25 min before the next injection. Fluorescent derivatives were detected at excitation and emission wavelengths of 330 and 450 nm, respectively. Calibration was performed by external calibration with L-arginine standard. Statistical analysis was performed using Student's t-test.

Immunohistochemistry. Expression and localization of arginase-1 and -2 was assessed in paraffin-embedded tissue sections using the Histostain-Plus Kit from Zymed. To do so, 3-µm sections mounted on poly-L-lysine-coated slides were dewaxed and rehydrated by immersion in ethanol (100%, 95%, and 70%) and PBS. After antigen retrieval, endogenous peroxidase activity was blocked with 3% H2O2 for 20 min. Negative controls for immunostaining were performed on each run with species-matched preimmune serum.

Immunofluorescence analyses. Cells were plated on eight-well chamber slides, rinsed once with PBS, and fixed with ice-cold methanol for 5 min at room temperature. After blocking unspecific binding with 5% (vol/vol) FBS at room temperature for 1 h, slides were incubated with primary antibodies overnight at 4°C. After washing, slides were incubated with secondary antibodies coupled to FITC (Zymed), whereas nuclei were visualized by 4,6-diamidino-2-phenylindole staining (Roche Diagnostics, Basel, Switzerland). Staining was analyzed with a Leica AS MDW deconvolution fluorescence microscope (Bensheim, Germany).

Collagen assay. Total collagen content in cell lysates was determined using the Sircol Collagen Assay kit (Biocolor, Belfast, Northern Ireland). Equal amounts of cell lysates were added to 1 ml of Sircol dye reagent, followed by 30 min of mixing. After centrifugation at 10,000 g for 10 min, the supernatant was carefully aspirated, and 1 ml of alkali reagent was added. Samples and collagen standards were then read at 540 nm on a spectrophotometer (Bio-Rad). Collagen concentrations were calculated using a standard curve with acid-soluble type 1 collagen.

Luciferase assay. NIH/3T3 cells were plated on 48-well plates at 15,000 cells/well. At 24 h before transfection, 0.2 µg/well of luciferase reporter plasmid pCAGA12-luc or pGL3-Control (Promega) were used for transfection with Lipofectamine (Invitrogen) at a 1:3 (wt/wt) ratio. The cells were then treated with TGF-β1 (2 ng/ml) for 24 h with or without NOHA at the indicated concentrations. TGF-β1-stimulated luciferase activity was determined as described previously (11).

Statistical analysis. All {Delta}Ct values obtained from qRT-PCR were analyzed for normal distribution using the Wilk-Shapiro test, with the assignment of a normal distribution with P > 0.05. Normality of data was confirmed using quantile-quantile plots. Compliance values were analyzed using the two-tailed, two-sample t-test. All {Delta}{Delta}Ct values were analyzed using the two-tailed, one-sample t-test. Intergroup differences of {Delta}Ct values from patients and bleomycin-treated mice were derived using a one-tailed, two-sample t-test. All P values obtained from multiple tests were adjusted using the procedure from Benjamini and Hochberg. Results were considered statistically significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression analysis of Prmt, arginine transporters, and arginases during bleomycin-induced lung fibrosis. First, we analyzed the expression of key arginine-metabolizing enzymes in lung homogenates of mice subjected to bleomycin-induced lung fibrosis for up to 21 days. RNA and protein expression analysis by semiquantitative RT-PCR and Western blot, respectively, revealed only little differences in Prmt expression comparing lung tissues from mice subjected to bleomycin treatment for 7, 14, or 21 days with control mice (Fig. 1, A and B). We observed a downregulation of Prmt6 protein expression 7 days after bleomycin treatment, which recovered after 14 days of treatment. Similarly, we detected no differences in arginine transporter Slc7a1–7 (Fig. 1C), Nos3, Oat, or ornithine decarboxylase (Odc) (Fig. 1D) mRNA expression. In contrast, we observed significant differences in arginase-1 and -2 and, to a lesser extent, Nos2 mRNA expression (Fig. 1D). Whereas the expression of both arginases was significantly increased as early as 7 days after bleomycin treatment, Nos2 expression gradually increased up to 21 days after treatment.


Figure 1
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  		Fig. 1. A–D: expression of protein arginine methyltransferase (Prmt) isoforms, arginine transporters, and arginine-metabolizing enzymes in lung homogenates of control or bleomycin-treated mice. Semiquantitative RT-PCR analysis of Prmt1-7 expression (A), Western blot analysis of Prmt1-6 expression (B), and semiquantitative RT-PCR analysis of arginine transporter Slc7a1-7 (C) and arginase-1 (Arg1) or -2 (Arg2), Nos2 or -3, ornithine aminotransferase (Oat), or ornithine decarboxylase (Odc) (D) expression were performed in lung homogenates from control or bleomycin-treated mice. Expression of Gapdh or β-actin served as loading controls for semiquantitative RT-PCR and Western blot, respectively, as indicated. All data are representative of 3 independent experiments using at least 9 different mouse lung tissues for every time point.

 
Next, we quantified arginase expression in lung homogenates using real-time qRT-PCR and Western blot analysis (Fig. 2). Arginase-1 and -2 mRNAs were equally abundant in the healthy mouse lung, but arginase-1 expression increased to higher levels than arginase-2 7 days after bleomycin application (72-fold induction vs. 5.1-fold induction for arginase-1 and -2, respectively; Fig. 2A). Arginase-1 and -2 protein expression also significantly increased in lung homogenates of bleomycin-treated mice, peaking 7 days after bleomycin application, as quantified by densitometric analysis (Fig. 2, B and C). Interestingly, arginase-1 protein was undetectable in the healthy mouse lung using our conditions (Fig. 2B), suggesting different translational control of the two arginases in the lung.


Figure 2
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  		Fig. 2. A–C: expression of arginase isoforms in lung homogenates of control or bleomycin-treated mice. Quantitative (q) real-time RT-PCR (A) and Western blot (B) analysis of arginase-1 and -2 expression were performed using lung homogenates from control or bleomycin-treated mice 3, 5, 7, 14, or 21 days after bleomycin administration. The expression of Pbgd, a ubiquitously and equally expressed gene that is free of pseudogenes, and β-actin served as loading controls for qRT-PCR and Western blot, respectively, as indicated. The qRT-PCR values are represented as means ± SE, n = 6. *P < 0.05. {Delta}{Delta}Ct = {Delta}Cttreated – {Delta}Ctcontrol. C: relative quantification of arginase protein expression by densitometry. Values are represented as means ± SE in optical density (OD), n = 6. *P < 0.05.

 
Analysis of lung tissue L-arginine levels during bleomycin-induced lung fibrosis. We next investigated whether the dramatic increase in arginase-1 levels resulted in altered free pulmonary L-arginine levels in bleomycin-induced lung injury. To do so, we measured the concentration of free cellular arginine in lung tissue homogenates, derived from bleomycin-treated or control mice. We detected decreased pulmonary L-arginine levels in lungs of mice subjected to bleomycin treatment (Fig. 3). This decrease was most evident 7 days after bleomycin treatment, at the time of the highest arginase-1 expression in the lung (104.4 ± 7.5 pmol of L-arginine per milligram of protein vs. 965.9 ± 208.8 pmol of L-arginine per milligram of protein, respectively). L-arginine levels gradually increased after day 7 but remained decreased even 21 days after bleomycin treatment compared with controls (Fig. 3B).


Figure 3
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  		Fig. 3. A and B: free cellular L-arginine levels in mouse lung homogenates of control or bleomycin-treated mice. A: representative HPLC chromatograms showing free L-arginine in lung homogenates from control and bleomycin-treated mice (3 and 7 days after bleomycin administration, as indicated). B: concentration of free L-arginine in lung homogenates is represented as means ± SE, n = 6. *P < 0.05.

 
Analysis of arginase localization and expression in bleomycin-induced lung injury. Arginases have been reported to be highly expressed in alveolar macrophages and immune cells during lung inflammation (8, 47). To exert a direct effect on collagen metabolism during lung fibrosis, however, arginase expression should be detectable in interstitial fibroblasts, the key cell type responsible for increased collagen deposition in fibrosis. We therefore analyzed arginase expression in the mouse lung, as well as in primary lung fibroblasts isolated from bleomycin-treated and control mice, by immunohistochemistry, immunofluorescence, qRT-PCR, and Western blot analysis. In mouse lungs, both arginases localized to macrophages and bronchial epithelial cells but also to interstitial fibroblasts, specifically in lesions exhibiting fibrosis (Fig. 4A). Fibroblast staining was particularly observed in the lungs of mice subjected to bleomycin treatment for 14 days and, to a lesser extent, 7 days (Fig. 4A). To confirm these findings, immunofluorescence analysis of arginase expression was performed using cultured primary fibroblasts isolated from mouse lungs, demonstrating strong cytosolic staining for arginase-1 and -2 (Fig. 4B).


Figure 4
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  		Fig. 4. A and B: expression and localization of arginase isoforms in lung fibroblasts in vivo and in vitro. A: immunohistochemical analysis of marker {alpha}-smooth muscle actin ({alpha}SMA) and arginase-1 and -2 was performed in paraffin-embedded lung specimens from control or bleomycin-treated mice (7 and 14 days after application, as indicated). Stainings are representative of 3 independent experiments using at least 3 different mouse lungs per experiment. B: immunofluorescent analysis of arginase-1 and -2 expression in primary mouse lung fibroblasts (magnification, x40). Nuclei are visualized by 4,6-diamidino-2-phenylindole (DAPI) staining (blue). Data are representative of at least 3 independent experiments.

 
We went on to quantify arginase mRNA expression in primary lung fibroblasts cultured from control or bleomycin-treated animals for up to 21 days. Both arginase-1 and -2 mRNAs were expressed in normal lung fibroblasts and significantly increased on the mRNA (Fig. 5, A and B) and protein levels (Fig. 5, C and D) in fibroblasts isolated from bleomycin-treated mice. Interestingly, arginase-1 expression was significantly enriched in fibroblasts, compared with lung homogenates. This was evident on the mRNA level [compare the {Delta}Ct levels of 4 (Fig. 5B) and 1 (Fig. 2A) in fibroblasts and lung homogenates, respectively] as well as on the protein level, suggesting an important functional role of arginase-1 in interstitial fibroblasts.


Figure 5
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  		Fig. 5. A–D: expression of arginase mRNAs in primary mouse lung fibroblasts isolated from control or bleomycin-treated mice. A: semiquantitative RT-PCR. B: qRT-PCR. Rel., relative. {Delta}Ct = Ctreference – Cttarget. C: Western blot analysis of arginase-1 and -2 expression in primary mouse lung fibroblasts. Expression of Gapdh, Pbgd, or β-actin served as loading controls for RT-PCR, qRT-PCR, and Western blot, respectively. The qRT-PCR values are represented as means ± SE, n = 6. *P < 0.05. D: relative quantification of arginase protein expression by densitometry. Values are represented as means ± SE in OD, n = 6. *P < 0.05.

 
Regulation of arginase expression by TGF-β1 in fibroblasts. To elucidate the regulatory mechanisms of arginase expression in lung fibrosis, we investigated whether treatment of fibroblasts with TGF-β1, the key profibrotic growth factor operative in lung fibrosis, altered arginase expression. Primary lung fibroblasts were stimulated with TGF-β1 (2 ng/ml) for up to 24 h, and mRNA expression was investigated by qRT-PCR. TGF-β1 rapidly increased arginase-1 mRNA expression as early as 2 h after treatment (Fig. 6, A and B). Arginase-1 and -2 expression was increased by up to 10-fold and twofold, respectively, in response to TGF-β1, in primary mouse fibroblasts (Fig. 6B).


Figure 6
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  		Fig. 6. A–D: regulation of arginase mRNA levels by TGF-β1 in primary lung fibroblasts. Semiquantitative RT-PCR (A) and qRT-PCR (B) of arginase-1 and -2 expression in primary lung fibroblasts 2, 6, 8, 12, or 24 h after TGF-β1 stimulation (2 ng/ml) are indicated. The qRT-PCR values are represented as means ± SE, n = 3. *P < 0.05. The collagen content (C) and mRNA levels of type 1 collagen-{alpha}1 (Col1a1) and {alpha}SMA (D) were determined by Sircol Collagen Assay and qRTPCR, respectively, in primary fibroblasts treated with TGF-β1 (0.2–2 ng/ml) in the presence or absence of NG-hydroxy-L-arginine (NOHA), an arginase inhibitor. D: the qRT-PCR values are represented as means ± SE, n = 3. *P < 0.05 vs. control and **P < 0.05 vs. TGF-β1 stimulation in the absence of NOHA.

 
Effect of arginase inhibition on TGF-β1-induced collagen deposition and signaling. Increased collagen deposition, largely mediated by enhanced TGF-β1 signaling, represents the pathological hallmark of fibrosis (37). We therefore investigated collagen mRNA expression, deposition, and TGF-β1 signaling in the presence of arginase inhibition in fibroblasts. TGF-β1 treatment led to a significant increase in the mRNA levels of type 1 collagen-{alpha}1 (Col1a1) and the fibroblast activation marker {alpha}-smooth muscle actin (SMA) in primary lung fibroblasts (Fig. 6D). Similarly, TGF-β1 led to a dose-dependent increase in the cellular collagen content of primary lung fibroblasts and NIH/3T3 fibroblasts (Figs. 6C and 7B, respectively). Interestingly, the TGF-β1-induced increase in collagen content was completely attenuated by arginase inhibition, using NOHA at concentrations from 100 to 500 µM, in both primary lung fibroblasts and NIH/3T3 fibroblasts (Figs. 6C and 7B, respectively). This inhibition of collagen production by arginase inhibition was achieved in a posttranslational manner, as the mRNA levels of Col1a1 or SMA were increased by TGF-β1, but this increase was not affected by NOHA (Fig. 6D). Similarly, this inhibitory effect was also not due to a general blockade of TGF-β1 signaling by NOHA, as the SMAD-driven reporter gene expression of pCAGA12-luc remained unchanged in the presence of NOHA (Fig. 7, C and D).


Figure 7
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  		Fig. 7. A–D: effect of arginase inhibition on collagen deposition and TGF-β1 signaling in NIH/3T3 fibroblasts. A: immunofluorescent analysis of arginase-1 and -2 expression in NIH/3T3 fibroblasts (magnification, x40). Nuclei are visualized by DAPI staining (blue). B: the collagen content was determined by Sircol Collagen Assay in NIH/3T3 fibroblasts treated with TGF-β1 (2 ng/ml) in the presence or absence of the indicated concentrations of NOHA, an arginase inhibitor. Values are represented as means ± SE, n = 3. *P < 0.05 vs. control and **P < 0.05 vs. TGF-β1 stimulation. C and D: the effect of NOHA, at the indicated concentrations, on baseline (C) and TGF-β1-induced (2 ng/ml; D) luciferase reporter plasmid pCAGA12-luc expression was assessed. Luciferase expression is plotted in relative luciferase units, corrected for transfection efficiencies (n = 6).

 
Analysis of arginase localization and expression in IPF. We next analyzed arginase expression in human lungs derived from healthy donors or patients with IPF by immunohistochemistry. Interestingly, the expression pattern of both arginases was different from mouse lungs. In human lungs, both arginases predominantly localized to bronchial epithelial cells and AEC (Fig. 8A). Arginase-1 protein was highly expressed in the cytosolic compartment and apical layer of bronchial epithelial cells, and its expression was less in lungs from patients with IPF. Similarly, arginase-2 protein was expressed in the cytosolic compartment, but not the apical layer, of bronchial epithelial cells and, in addition, in hyperplastic AEC in lungs from patients with IPF (Fig. 8A). Quantification of arginase mRNA expression by qRT-PCR revealed a significant downregulation of arginase-1 expression in IPF, whereas arginase-2 expression was unchanged (Fig. 8B). This did not translate into a significant difference in arginase activity, which was similar in donor and IPF samples (Fig. 8C). Finally, fibroblasts cultured from donor and IPF lungs did not exhibit differences in arginase expression, as analyzed by qRT-PCR, although they had higher levels of the fibroblast activation marker SMA (Fig. 8D). Interestingly, arginase expression was quite different in human lungs and fibroblasts, compared with mouse samples. Both arginase-1 and -2 were expressed at lower levels in human lungs compared with mouse lungs (Figs. 8B and 2A, respectively). Even more pronounced differences are observed in primary lung fibroblasts: in mouse fibroblasts, arginase-1 and -2 are highly expressed (Fig. 5B), whereas both are expressed at very low levels in human fibroblasts (Fig. 8D).


Figure 8
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  		Fig. 8. A–D: expression and activity of arginase isoforms in lung tissues of donor and idiopathic pulmonary fibrosis (IPF) patients. A: immunohistochemical staining of arginase-1 and -2 was performed on tissue sections of donor or IPF lungs, as indicated. Stainings are representative of 2 independent experiments using 6 different donor or IPF lung tissues (magnification as indicated). B: the mRNA levels of arginase-1 and -2 in lung homogenates from donor or IPF lung explants (n = 6) were assessed by qRT-PCR. Results are presented as means ± SE. *P < 0.05. C: arginase activity was assessed in lung homogenates from 6 donors and 6 IPF patients and presented as means ± SE. D: the mRNA levels of arginase-1 and -2, Col1a1, and SMA were assessed by qRT-PCR in primary human lung fibroblasts derived from donor or IPF lung explants. Results are presented as means ± SE, n = 3. *P < 0.05.

 

   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Recently, there has been an increased interest in arginase expression and activity in lung diseases, such as asthma (47) and cystic fibrosis (14), due to their potential for affecting L-arginine availability for NO synthesis (34). In both humans and rodents, limited NO production by constitutive NOS has been linked to airway hyperreactivity (48), which could be reversed by pharmacological inhibition of arginases (45). In addition to NO synthesis, arginase activity also directly influences collagen metabolism, as the arginase product L-ornithine is metabolized to polyamines and L-proline, which is highly enriched in collagen, by Odc and Oat, respectively. As such, arginase expression and activity can exert a significant impact on the following pathophysiological mechanisms operative in lung fibrosis: 1) increased collagen synthesis due to increased bioavailability of L-proline; 2) endothelial dysfunction of the pulmonary vascular bed due to decreased NO synthesis; and 3) increased cell proliferation due to enhanced bioavailability of polyamines.

In the present study, we therefore sought to analyze in detail the regulation of L-arginine metabolism in lung fibrosis, using the well-established model of bleomycin-induced fibrosis in mice as well as lung specimen from donor and IPF lungs. The salient findings of our investigations were as follows: a significant upregulation of arginase-1 and -2 expression could be documented in fibrotic mouse lungs, whereas other enzymes of arginine metabolism remained unchanged. This increased arginase expression was of functional relevance, as it correlated with decreased levels of free lung L-arginine levels. Both arginases were highly expressed in primary mouse lung fibroblasts, and their expression was increased in fibroblasts derived from bleomycin-treated mice compared with controls. We also report that a key fibrotic mediator in lung fibrosis, TGF-β1, potently induced arginase-1 expression in lung fibroblasts. The inhibition of arginase activity using NOHA significantly attenuated TGF-β1-induced collagen deposition, but not mRNA expression and Smad signaling, by fibroblasts. Surprisingly, we were unable to document similar changes in lungs from patients with IPF. Rather, arginase-1 expression was decreased in IPF, whereas arginase activity remained unchanged. Arginase localization was different in human lungs, exhibiting a predominantly epithelial cell expression pattern with significantly less expression in fibroblasts compared with mice.

This study addressed the expression and function of arginases in fibroblast biology in lung fibrosis in vitro and in vivo. In the pig lung, high arginase concentrations have already been determined back in 1964 by Kossmann et al. (19). Localization analysis by in situ hybridization and immunohistochemistry has further revealed that arginases are predominantly expressed in macrophages (8, 12, 28, 36, 47) and endothelial cells (28, 33, 44) in the lung. Their expression and role in interstitial fibroblasts and AEC, however, remained enigmatic. Here, we could document high basal expression levels of arginase-1 and, to a lesser extent, arginase-2 in mouse primary lung fibroblasts (Fig. 5).

Two arginase isoforms, arginase-1 and -2, have been cloned and characterized in humans and rodents (9, 16, 42). Whereas arginase-1 is reported to be expressed in large amounts in the liver and red blood cells, arginase-2 is described to be present in the kidney, brain, lung, gastrointestinal tract, and prostate (1, 9, 30). Localization studies have demonstrated that arginase-1 is a cytosolic enzyme, whereas arginase-2 protein is enriched in mitochondria. In this respect, our studies suggest subtle differences in arginase isoform expression in the lung: in the healthy mouse lung, arginase-1 and -2 mRNAs are expressed at similar levels, but arginase-2 protein can be detected at higher levels than arginase-1 (Fig. 2). In contrast, arginase-1 mRNA is expressed at higher levels than arginase-2 in lung fibroblasts (Fig. 5) and is also regulated much more potently by bleomycin treatment in vivo (Fig. 5) and TGF-β1 in vitro (Fig. 6), as demonstrated by qRT-PCR and Western blot analysis. This presents strong evidence that arginase-1 is a key enzyme influencing collagen synthesis in fibroblasts, supported also by the fact that arginase inhibition attenuated TGF-β1-induced collagen synthesis in fibroblasts (Figs. 6 and 7B).

Much to our surprise, the scenario in IPF was different from the mouse model. In our view, the most likely explanation for the observed differences in arginase expression comparing mouse and human is species-specific transcriptional control, supported by our finding of different arginase expression levels at baseline, but also comparing the ratio of arginase-1 to-2. Whereas arginase inhibition decreased collagen content in lung fibroblasts irrespective of their origin (Figs. 6 and 7) and thus presents a valid approach for inhibiting collagen deposition at least in vitro, we could not find increased arginase expression in IPF samples compared with donors. This may indicate that arginases play an important role not during the fibroproliferative phase of fibrosis, but during the activation phase in lung injury, a disease the bleomycin model is also frequently used for.

Ongoing studies will address the in vivo potential of this approach in mice and humans, but a recent study by Mora et al. (28) has suggested that increased arginase expression is found in herpesvirus-induced lung fibrosis. This study described increased arginase-1 expression in alternatively activated macrophages in response to infection with the herpesvirus MHV68 but also in areas of pleural thickening and interstitial fibrosis in human lungs (28). Furthermore, our data confirmed results from an earlier study by Endo et al. (12), which described upregulation of arginase and Odc mRNA and arginase-1 and -2 protein in lung homogenates of bleomycin-exposed mice. Similar to data presented in our study (Fig. 3), arginase-1 protein expression was also undetectable in normal lung homogenates, but was present after 7 days of bleomycin treatment, in the study by Endo et al. (12). In contrast, we were able to detect arginase-2 protein in normal lungs, whereas this was not the case in the study by Endo et al. (12), probably due to exposure times or antibody sensitivity.

How could arginase-1 affect the process of lung fibrosis and collagen synthesis? Based on our results and on published data, it can be argued that arginine depletion by increased arginase expression can affect cell and lung functions in several ways. First, the dramatic reduction of free L-arginine leads to its decreased bioavailability for NOS, thus leading to decreased NO production, as has been demonstrated in detail in macrophages and epithelial cells (23–25, 31, 32). Although arginases have a significantly lower Km for L-arginine (2–20 mM range and 2–20 µM range for arginases and NOS, respectively), they exhibit a 1,000x higher Vmax compared with NOS, as such having the capacity to functionally inhibit NOS via substrate depletion (4, 43). Reduced NO levels, on the other hand, have been shown to lead to airway and vascular smooth muscle cell proliferation as well as enhanced susceptibility to pulmonary infections (34, 35), features occurring in pulmonary fibrosis. Second, and probably much more important, increased generation of L-ornithine by arginases increases the bioavailability of polyamines and L-proline, essential regulators of cell proliferation and collagen synthesis, respectively (29, 30). Since fibroblast proliferation and collagen accumulation are hallmarks of lung fibrosis (17, 37, 40), this pathway could present an attractive option for therapeutic intervention. To further test this possibility, we incubated fibroblasts with the arginase inhibitor NOHA and analyzed TGF-β1-dependent signaling and collagen synthesis. Interestingly, arginase inhibition was able to attenuate collagen content but not collagen mRNA expression or TGF-β1-dependent Smad signaling (Figs. 7 and 8). This documents that arginase inhibition by NOHA specifically prevents the posttranslational processing and deposition of collagen, as such exhibiting pathway specificity, and the perspective of modulating collagen metabolism, leaving the potent anti-inflammatory properties exerted by TGF-β1 unaffected.

In sum, we present strong evidence for a critical role of arginases in collagen synthesis in fibroblasts in vitro and a potential impact in lung fibrosis in vivo. The outlook of being able to selectively modulate collagen synthesis, in a specific cell type, may provide an incentive for ongoing research in this area and may add lung fibrosis or lung injury to the panel of diseases that may profit investigating the effect of arginase inhibition in vivo.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a German Research Foundation (DFG) Clinical Research Group 118 Pathomechanisms and Therapy of Lung Fibrosis Grant and a Sofja Kovalevskaja Award from the Alexander von Humboldt Foundation, both to O. Eickelberg.


   ACKNOWLEDGMENTS
 
We are grateful for stimulating discussions provided by all members of the Eickelberg Lab and Andreas Jahn for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. Eickelberg, Dept. of Medicine II, Univ. of Giessen Lung Center, Aulweg 123, D-35392 Giessen, Germany (e-mail: Oliver.Eickelberg{at}innere.med.uni-giessen.de)

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.


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