Comparative Proteomic Analysis of Lung Tissue ... - ACS Publications

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Comparative Proteomic Analysis of Lung Tissue from Patients with Idiopathic Pulmonary Fibrosis (IPF) and Lung Transplant Donor Lungs Martina Korfei,† Sigrid Schmitt,‡ Clemens Ruppert,† Ingrid Henneke,† Philipp Markart,† Benjamin Loeh,† Poornima Mahavadi,† Malgorzata Wygrecka,‡ Walter Klepetko,^ Ludger Fink,† Philippe Bonniaud,z Klaus T. Preissner,‡ G€unter Lochnit,‡ Liliana Schaefer,§ Werner Seeger,† and Andreas Guenther*,†,# †

University of Giessen Lung Center (UGLC), Department of Internal Medicine II, Klinikstrasse 36, Justus-Liebig-University Giessen, Germany ‡ Institute of Biochemistry, Friedrichstrasse 24, Giessen, Germany ^ Department of Thoracic Surgery, Vienna General Hospital, Vienna, Austria z Service de Pneumologie et Reanimation Respiratoire, Centre Hospitalier Universitaire du Bocage, Dijon, France § Department of Pharmacology and Toxicology, Goethe University of Frankfurt am Main, Frankfurt, Germany # Lung Clinic Waldhof-Elgershausen, Greifenstein, Germany

bS Supporting Information ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease for which no effective therapy exists to date. To identify the molecular mechanisms underlying IPF, we performed comparative proteome analysis of lung tissue from patients with sporadic IPF (n = 14) and human donor lungs (controls, n = 10) using two-dimensional gel electrophoresis and MALDI-TOF-MS. Eighty-nine differentially expressed proteins were identified, from which 51 were up-regulated and 38 down-regulated in IPF. Increased expression of markers for the unfolded protein response (UPR), heat-shock proteins, and DNA damage stress markers indicated a chronic cell stressresponse in IPF lungs. By means of immunohistochemistry, induction of UPR markers was encountered in type-II alveolar epithelial cells of IPF but not of control lungs. In contrast, up-regulation of heat-shock protein 27 (Hsp27) was exclusively observed in proliferating bronchiolar basal cells and associated with aberrant re-epithelialization at the bronchiolo-alveolar junctions. Among the down-regulated proteins in IPF were antioxidants, members of the annexin family, and structural epithelial proteins. In summary, our results indicate that IPF is characterized by epithelial cell injury, apoptosis, and aberrant epithelial proliferation. KEYWORDS: idiopathic pulmonary fibrosis, usual interstitial pneumonia, proteomics, two-dimensional gel electrophoresis, unfolded protein response, valosin-containing protein, heat shock 27 kDa protein, epithelial injury, aberrant re-epithelialization, bronchiolization

’ INTRODUCTION Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and ultimately fatal parenchymal lung disease, with a median survival of less than 3 years following diagnosis.1 It is the most common and severe form of the idiopathic interstitial pneumonias (IIPs), with a prevalence of 13-20 per 100 000 people in the general population.1,2 IPF, for which no medical treatment exists to date, is characterized by a progressive distortion of the alveolar architecture and replacement by fibrotic tissue and extracellular matrix (ECM) deposition, resulting in progressive dyspnea and loss of respiratory function. The definite diagnosis of IPF requires proof of an usual interstitial pneumonia (UIP) pattern in a surgical lung biopsy.1 The UIP pattern is characterized by a spatial and temporal heterogeneity, with areas of normal appearing lung being directly adjacent to areas of fully established fibrosis, microscopic honeycombing, and areas of evolving fibrosis containing actively proliferating and collagen-producing fibroblasts/ r 2011 American Chemical Society

myofibroblasts, the so-called “fibroblast foci” (FF). These FF, the number of which represents a prognostic marker in IPF,3 are usually covered by the frequently entitled “hyperplastic epithelium”.4 These overlying epithelial cells concomitantly show signs of apoptosis as well as proliferation and they are therefore regarded as the primary site of ongoing epithelial injury and aberrant wound repair.4,5 The current hypothesis regarding the development of IPF conceptualizes ongoing multiple, small, focal, and isolated episodes of epithelial cell injury leading to apoptosis followed by aberrant fibrotic repair, excessive fibroblast proliferation and inappropriate re-epithelialization of damaged epithelium.6 This concept is supported by the previous description of gene mutations in the Surfactant Protein SP-C (SFTPC) gene, a protein exclusively expressed, synthesized and secreted by type-II Received: September 14, 2010 Published: February 14, 2011 2185

dx.doi.org/10.1021/pr1009355 | J. Proteome Res. 2011, 10, 2185–2205

Journal of Proteome Research alveolar epithelial cells (AECII), in some kindreds with familial IPF.7,8 In detail, the mutated proSP-C protein cannot be folded and processed correctly in the ER, leading to accumulation of the abnormal protein in the AECII and resulting in ER stress and apoptosis.7-9 More recently, gene mutations in the Surfactant Protein SP-A2 (SFTPA2) have also been described in two families with IPF and were also found to result in retention of the mutated SP-A in the ER.10 In addition, our group recently disclosed markers of profound ER stress and apoptosis in AECII of sporadic forms of IPF.11 It is thus presumable that ER stress leading to apoptosis in AECII represents a common pathomechanistic principle in IPF. However, the precise molecular mechanisms underlying the pro-apoptotic ER stress-response in AECII in sporadic cases of IPF as well as the molecular mechanisms responsible for the severe tissue remodelling, including the formation of bronchiolized honeycomb cysts, remain unknown. Proteomics comprises a set of tools for the large-scale study of gene expression at the protein level, thereby allowing identification of protein alterations responsible for the development of diseases. Compared to microarray based analysis of gene expression patterns, which has already been undertaken in IPF,12-15 proteomic profiling offers distinct advantages. In particular, proteomic technologies can provide information on post-translational modifications of proteins, for example, proteolytical processing, phosphorylation or ubiquitination. To identify candidate proteins involved in the pathomechanism of IPF, we therefore for the first time compared lung proteome profiles from patients with sporadic IPF to that of control subjects applying two-dimensional gel electrophoresis (2-DE) and MALDITOF-MS.

’ MATERIALS AND METHODS For detailed information, see Supporting Information. Human Lungs

The study was approved by the local research ethics committee, and written consent was obtained from all participants (no. 31/93, 84/93, 29/01). Explanted lungs from patients with sporadic IPF (n = 14) and lungs or lobes from organ donors (controls, n = 10), that were not utilized because of size incompatibilities or acute infection of the putative recipient, were obtained from the Dept. of Thoracic Surgery, Vienna (W. Klepetko). Already at the surgical theater, these lungs were flushed, placed on ice in a sterile manner, and shipped to our institute immediately. Upon arrival, lungs or lung lobes were sectioned under the hood on ice according to a predefined algorithm. All patients fulfilled the diagnostic criteria for IPF as defined by the American Thoracic Society/European Respiratory Society Consensus Conference.1 In particular, retrospective expert pathologist (L. Fink) evaluation of explanted lungs forwarded a typical UIP pattern in each IPF case. All controls obtained from donor lungs or lobes fulfilling transplantation criteria were also routinely evaluated by the expert pathologist and did not show signs of inflammation. Clinical information on patients and controls is summarized in Supplemental Table S1 (Supporting Information). The proteomic analysis was performed by use of peripheral lung tissue from the lower lobe, from the subpleural region of the lung (see Supplemental Figure SI for anatomical location). Sample Preparation for Two-Dimensional Gel Analysis

Frozen lung tissue samples (size 1 cm3) from IPF patients and controls were individually processed by grinding with a mortar

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and pestle cooled with liquid nitrogen. Frozen powders from each individual tissue block were thawed by adding 3 mL of cold lysis buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, 1% (w/v) Triton X-100, 0.5% (w/v) sodium-deoxycholate, 5 mmol/L EDTA, and 2 mmol/L PMSF), and incubated on ice for 1 h. Crude lung extracts were centrifuged at 10 000g for 10 min at 4 C to remove cell debris. The protein concentration in each supernatant lung homogenate was determined according to the BCA protein assay from Perbio Science. Thereafter, equal masses of total protein (0.5 mg) of each individual IPF- or control homogenate were pooled, respectively. For isoelectric focusing (IEF), proteins were then precipitated with acetone (80% final concentration). After brief air drying in the hood, precipitated proteins of the IPF- and control pool were resolubilized using a rehydration buffer containing 8 mol/L urea, 2 mol/L thiourea, 2% (v/v) Pharmalyte buffer for commercial pH 3-10 linear and nonlinear IPG strips (both GE Healthcare, Uppsala, Sweden), 4% (w/v) CHAPS, 30 mmol/L DTT, 20 mmol/L Tris, and 0.05% (w/v) bromphenol blue. The concentration was adjusted for both to 5 mg/mL. Again, the protein concentration in each stock was determined by the 2-D Quant Kit (GE Healthcare). The remaining lung homogenates were divided into aliquots and frozen at -80 C until used. Performance of Two-Dimensional Gel Electrophoresis (2-DE)

Each proteome (IPF- and control pool) was analyzed in triplicate. For first-dimension separation (IEF), 400 μg of sample protein was diluted in a final volume of 230 μL rehydration buffer and loaded by the in-gel rehydration method onto an immobilized pH gradient (IPG) gel strip (11 cm, pH 3-10 linear, Amersham Biosciences). The IEF electrophoresis of 400 μg protein per strip was carried out in a Multiphor chamber (Amersham Biosciences) at 20 C overnight applying the following conditions: phase 1, gradient from 0 to 100 V, 0.25 kVh; phase 2, gradient from 100 to 3500 V for 10.8 kVh; phase 3, 3500 V for 21 kVh; with limitation of current to 0.2 mA per strip. After focusing, the IEF-strips were either stored at -80 C or immediately equilibrated for 10 min in 2 mL equilibration stock solution [ESS; 6 mmol/L urea, 0.1 mmol/L EDTA, 0.01% (w/v) bromphenol blue, 50 mmol/L Tris-HCl pH 6.8, 30% (v/v) glycerol], followed by reduction for 15 min in 2 mL of ESS I [10 mL of ESS containing 200 mg of SDS, 100 mg of DTT], followed by alkylation for 15 min in ESS II [10 mL of ESS containing 200 mg of SDS, 480 mg of iodoacetamide]. Focused IPG gel strips were then loaded on top of 10%, 12.5% or 15% SDS-polyacrylamid gels (PAGE), and the second-dimension electrophoresis was carried out using a Hoefer600 14.5 14.5 cm vertical gel electrophoresis “Tetra Cell” chamber (Amersham Biosciences) according to the method described by Gorg et al.16 The proteomes of the IPF- (two SDS-PAGEs) and the control pools (two SDS-PAGEs) were separated simultaneously in one chamber. In the next run, the remaining SDSPAGEs (one for each group) were performed. In the second approach, focused pH 3-10 linear IPG gel strips were subjected to a 15% SDS-PAGE in the same manner as just described. In the third approach, focused pH 3-10 nonlinear IPG gel strips were subjected to a 12.5% SDS-PAGE resulting in a 2D-gel with greater resolution. After SDS-PAGE, gels were stained with the visible Coomassie Brilliant Blue stain. Coomassie stained protein spots were then digitized by scanning the 2D-gels on an image scanner (Amersham Biosciences). Apparent molecular masses were determined by running standard protein markers covering the range of 10-220 kDa (Invitrogen, 2186

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Journal of Proteome Research Karlsruhe, Germany), and pI (isoelectrica point) values of the protein spots were calculated as given by the supplier of the IPG strips (Amersham Biosciences). Image Analysis of Two-Dimensional Gels

Representative example gels for the three different IPF proteomes are depicted in Figure 1A-C, and these for the three different control proteomes in Figure 1D-F. Among these gels, Figure 1A and 1D, Figure 1B and 1E, and Figure 1C and 1F belong to the same 2-DE separation setting. Computer-assisted analysis of digitized 2D-gels A, B, C [IPF] and D, E, F [control] (triplicate determination per group and 2-DE setting) was performed with Proteomweaver software version 4.0.0 (BioRad, Munich, Germany). Spot detection, matching of protein/peptide spots, background subtraction and normalization were carried out by the software using standard settings. Individual spot intensities were calculated by the software. A full comparison of all spots visualized was performed in the IPF and control group of the same 2-DE separation setting (A versus D, each n = 3; B versus E, each n = 3; C versus F, each n = 3). Only those spots with a 2-fold or greater change in expression levels between the IPF and the control group (regulation factor [RF] g 2 for 2-fold or more up-regulation, RF e 0.5 for 2-fold or more down-regulation) were considered as being differentially regulated. The Student’s t test (provided by the software) with significance set at p < 0.05 was used to filter out statistically insignificant variations in spot intensities. For a better overview, proteins up-regulated in IPF (RF g 2, p < 0.05) are indicated with circles in the IPF lung proteome maps (Figure 1A-C), and proteins down-regulated in IPF (RF e 0.5, p < 0.05) are depicted in the control lung proteome maps (Figure 1D-F). In-Gel Trypsin Digestion

Differently expressed protein spots between the two groups were excised manually from stained 2D-gels, transferred to a 1.5 mL Eppendorf tube, and rinsed once with ultrapure water and twice alternately with (1:1) mixture of 50 mmol/L (NH4)HCO3 and acetonitrile, and 100% acetonitrile. Excised gel spots were rehydrated in a minimal volume of a 10 ng/μL trypsin solution (Trypsin Gold, MS grade, Promega) in 25 mmol/L (NH4) HCO3 and incubated for 16 h at 37 C for digestion. Peptides were extracted with 5 μL of 1% (v/v) trifluoroacetic acid (TFA) containing 5 mmol/L n-octyl-β-D-glucopyranoside. MALDI-TOF-Analysis and Database Searching

Aliquots (2 μL) of the peptide solution were mixed with 0.5 μL of 2,5-dihydrobenzoic acid [DHB (20 mg/mL) in 50% acetonitrile/0.85% phosphoric acid] on a stainless steel target (Bruker Daltonik, Bremen, Germany) using the dried-droplet method. Peptide mass fingerprints (PMF) of tryptic digests were obtained by MALDI-TOF-MS using an Ultraflex TOF/TOF mass spectrometer (Bruker Daltonics). Peptide mass standards (Bruker Daltonics) were used for external calibration of the mass spectra. Mass spectra were acquired in reflector mode using FlexControl 2.4 software (Bruker Daltonics), and analyzed by the FlexAnalysis software 3.0. The Swiss-Prot database employing the MASCOT 2.0 program (http://www.matrixscience. com) was used for the search of peptide masses to identify proteins. The versions used were Swissprot 57.1 (04/14/2009, 462 764 sequences and 163 773 385 residues) and Swissprot 57.2 (05/05/2009, 466 739 sequences and 165 389 953 residues); Swissprot 57.5 (07/07/2009, 471 472 sequences and 167 326 533 residues) was used in four cases. The taxonomic category was

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“Homo sapiens (human)” (57.1, 20 405 sequences; 57.2, 20 402 sequences; 57.5, 20 399 sequences), because human tissue specimens were analyzed. The other main search parameters for all PMF searches were set as follows: enzyme specificity, trypsin; one missed cleavage permitted; fixed modification, carbamidomethylation of cysteine; variable modification, methionine oxidation; mass values, monoisotopic; protein mass, unrestricted; peptide mass tolerance (mass accuracy), (100 ppm or (50 ppm. Protein matches were assessed on the basis of the number of peptides matched to the target protein, the intensity of these peptides expressed as a percentage of all submitted peptides and MOWSE (molecular weight search) score. Search results that yielded a MOWSE score greater than 56 (p < 0.05) for MS data were regarded positive for protein identifications. The score threshold to achieve p < 0.05 is set by Mascot algorithm and is based on the size of the database used in the search. Only proteins with the best score (and >56) and the highest number of matching peptides in each Mascot search were accepted as successful identifications. The PMF mass spectra and protein identification results of the proteins identified in this study are shown in Supplemental Figures S1 to S89 (up-regulated proteins S1 to S51; down-regulated proteins S52 to S89), including MS-peak list, MOWSE score, the number of peptides matched to the identified protein from the total of peptides submitted, the sequence coverage, theoretical MW and pI of the identified protein, and the mass accuracy of matched peptides. In the case of a protein being either identified in multiple spots in one and the same gel or redundantly due to performance of three different 2-DE-systems for each condition, one example mass spectrum/protein identification result is shown. Summarized information of these raw data are provided in Table 1 for the up-regulated proteins and in Table 2 for the down-regulated proteins in IPF. In two cases, two PMF’s with distinguishable but similar scores were identified in one spot, presumably due to the similar MW and pI of both proteins (Grp78/PDIA4; ACTB/ACTA, indicated in Table 1). In the case of a protein being redundantly identified, the protein identification results are listed up maximal three times. Quantitative Western Blot Analysis and Statistics

For one-dimensional SDS-PAGE, equal amounts of lung homogenates (50 μg extracted protein per sample) were transferred to SDS-sample buffer (final concentration 2% (w/v) SDS, 2.5% (v/v) β-mercaptoethanol, 10% (v/v) glycerol, 12.5 mmol/L Tris-HCl [pH 6.8], 0.1% (w/v) bromophenol blue) and immersed in boiling water for 10 min. Proteins (50 μg/lane) from the individual lung lysates as used for the above 2-DE were then separated by 8%, 9%, 10%, 12%, 15% SDS-PAGE. Thereafter, the separated proteins were transferred to a PVDF membrane (GE Healthcare) in a semidry blotting chamber according to the manufacturer’s protocol (Bio-Rad, Munich, Germany), followed by immunostaining for Bip/Grp78, VCP, PPIB, Hsp90-alpha, ATF-6R, ATF-4/ CREB-2, CHOP, caspase 3, ubiquitin, DDB1, Hsp27, p-Hsp27, PGK1, Annexin A1, -A2 and A3, cathepsin D, catalase, peroxiredoxin 2 and β-actin. Blot membranes were developed with the ECL Plus chemiluminescent detection system and band intensity of exposed film was analyzed by densitometric scanning and quantified using AlphaEaseFC Imaging System (San Leandro, CA). The immunostaining procedure as well as the sources and dilutions of primary antibodies used are outlined in the Supporting Information. 2187


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Figure 1. Representative 2-DE maps of 400 μg of proteins extracted from lungs of IPF patients and control lungs. (A and D) Separation of proteins extracted from IPF (A) or control lungs (D) was performed on linear strips with a pH range of 3-10 followed by 10% SDS-PAGE. (B and E) Separation of proteins extracted from IPF (B) or control lungs (E) was performed on linear strips with a pH range of 3-10 followed by 15% SDS-PAGE. (C and F) Separation of proteins extracted from IPF (C) or control lungs (F) was performed on nonlinear strips with a pH range 3-10 followed by 12.5% SDS-PAGE. Each 2-DE was performed in triplicate, and gels were stained with Coomassie. The 2D-gels were scanned, and differences in protein abundance (represented as an average of the total number of gels) were detected using Proteomweaver software. Proteins that differed more than or equal to 2-fold are marked with circles, and identified proteins are indicated in representative 2-DE maps. Proteins up-regulated in IPF (RF g 2, p < 0.05) are depicted in the IPF lung proteome maps (A-C); proteins down-regulated in IPF (RF e 0.5, p < 0.05) are depicted in the control lung proteome maps (D-F). Differentially expressed protein spots which were not analyzed or which could not be successfully identified are indicated with n.d. (= not determined). In detail, 2-DE separation setting in panels A (IPF) and D (control) revealed 46 differentially regulated protein spots, from which 28 were up-regulated (being visible in A) and 18 down-regulated (being visible in D) in IPF. The second 2-DE separation setting shown in panels B (IPF) and E (control) revealed 78 differentially regulated protein spots, from which 37 were upregulated (being visible in B) and 41 down-regulated (being visible in E) in IPF. The third 2-DE separation setting shown in panels C (IPF) and F (control) revealed 79 differentially regulated protein spots, from which 41 were up-regulated (being visible in C) and 38 down-regulated (being visible in F) in IPF.

For the statistical analysis of differences between IPF (n = 9) and controls (n = 5) in quantitative immunoblot analysis, we used the nonparametric Mann-Whitney test provided by the software GraphPad Prism version 4.0

(GraphPad Software, Inc., La Jolla, CA). Results were considered to be statistically significant at p < 0.05 and are reported as (*p < 0.05), (**p < 0.01), (***p < 0.001) for IPF versus controls. 2188



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Table 1. Proteins Significantly Up-Regulated in IPF Lungs Relative to Control Lung Tissuea UniProt/ _HUMAN

protein name

score

matched

seq. cov.

theor. MW

exp. MW

RF

found in

(>56)

peptides

(%)

[kDa]/pI

[kDa]/pI

(g2)

2D-PAGE

UPR/ER Stress-Response P55072/TERA_

Transitional endoplasmic

247

34

52

89.9/5.14

98.4/5.49

3.3

A

78 kDa glucose-regulated protein Grp78 (Bip)

116

20

41

72.4/5.07

72.4/5.10

4.6

C*

P13667/PDIA4_

Protein disulfide-isomerase A4

104

22

40

73.3/4.96

72.4/5.10

4.6

C*

P23284/PPIB_

Peptidyl-prolyl cis-trans isomerase B

165

17

56

23.8/9.33

18.2/9.21

3.8

B

reticulum ATPase, TER ATPase, valosin-containing protein (VCP) P11021/GRP78_

precursor (PPIase) Proteasome/Degradation P25788/PSA3_

Proteasome subunit alpha type 3

74

11

37

28.6/5.19

28.3/5.39

2.7

A

109

16

59

29.8/7.57

28.4/7.82

2.0

C

29 36

128.1/5.14 85.0/4.94

130.1/5.33 96.9/4.69

4.4 2.3

A A

83.6/4.97

(Proteasome component C8) P25789/PSA4_

Proteasome subunit alpha type 4 (Proteasome component C9)

Cell Stress-Response Q16531/DDB1_ P07900/HS90A_

DNA damage-binding protein 1 Heat shock protein HSP90-alpha (HSP86)

P08238/HS90B_

Heat shock protein HSP90-beta (HSP84)

P31948/STIP1_

Stress-induced phosphoprotein 1,

116 128

26 26

248

33

39

154

25

35

260

43

52

63.2/6.40 61.2/5.70

99.2/5.18

4.8

A

103.2/5.01

2.7

C

62.8/6.42

2.3

C

Hsc70/Hsp90-organizing protein (Hop) P10809/CH60_ P21796/VDAC1_

60 kDa heat shock protein,

107

20

39

mitochondrial, (Hsp60)

116

16

37

63.4/5.39;

2.2

A

67.1/5.58

2.8

Voltage-dependent anion-selective channel

154

15

66

B

30.9/8.62

32.7/8.61

2.2

B

132

14

68

22.8/5.98

27.4/6.32

2.7

B

55

56.5/8.03

57.1/6.74

2.8

B

52.2/8.27

protein 1 (VDAC-1) P04792/HSPB1_

Heat shock protein beta-1 (HspB1), heat shock 27 kDa protein (HSP27)

Protein Processing P28838/AMPL_

Cytosol aminopeptidase, Leucine aminopeptidase (LAP)

Q01518/CAP1_

Adenylyl cyclase-associated protein 1

198

25

Signaling molecules

P43034/LIS1_

Platelet-activating factor

122

16

41

98

17

40

100

17

34

59.3/6.86

2.5

A

63.0/7.38

2.6

B

47.8/7.12

2.9

C

285.2/5.22 116.93/5.2 105.2/5.27 102.3/5.16 62.8/6.81 62.6/6.47

2.2 2.1 4.4

C C A

64.4/6.85 58.3/6.86

2.3 3.0

B B

48.8/5.12 49.2/5.23

3.8 4.0

A B

47.2/6.97

acetylhydrolase IB subunit alpha, Lissencephaly-1 protein Cytoskeleton/Cellular Organization Q13813/SPTA2_ Q43707/ACTN4_ P01876/PGM5_

Spectrin alpha chain, brain (fragm.) Alpha-actinin-4 Phosphoglucomutase-like protein 5

210 277 195

37 40 28

13 50 48

Q16658/FSCN1_

Fascin (Singed-like protein), 55 kDa

157 137

21 19

40 40

314 325

41 40

65 61

55.1/6.84

actin bundling protein P08670/VIME_

Vimentin

P05787/K2C8_

Keratin, type II cytoskeletal 8 (cytokeratin-8)

331 175

42 37

63 46

53.7/5.52

44.9/4.80 42.1/5.38

2.3 3.9

C A

P08727/K1C19_

Keratin, type I cytoskeletal 19 (cytokeratin-19)

245 202

33 27

48 63

44.1/5.04

41.9/5.31 40.0/5.12

2.9 4.2

B A

111

17

46

40.0/5.08

3.8

B

174

24

54

38.6/4.76

2.9

C

2189

53.7/5.06



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Table 1. Continued UniProt/ _HUMAN

protein name

score

matched

seq. cov.

theor. MW

exp. MW

RF

found in

(>56)

peptides

(%)

[kDa]/pI

[kDa]/pI

(g2)

2D-PAGE

P09493/TPM1_ P07951/TPM2_

Tropomyosin alpha-1 chain, tropomyosin 1 Tropomyosin beta chain, tropomyosin 2

227 100

30 16

65 37

32.7/4.69 32.9/4.66

34.3/4.80 38.7/5.00

5.3 3.9

C C

P62736/ACTA_

Actin, aortic smooth muscle, alpha-actin-2

159

26

56

42.4/5.23

42.5/5.10

2.2

C†

P60709/ACTB_

Actin, cytoplasmic 1, beta-actin

210

28

70

42.1/5.29

42.5/5.10

2.2

C†

P02545/LMNA_

Lamin-A/C (70 kDa lamin) (fragm.)

136

29

38

74.4/6.57

47.9/6.08

2.8

B

Q9NVA2/SEP11_

Septin-11

59

11

28

50.0/6.36

53.4/6.20

2.8

A

P00558/PGK1_

Phosphoglycerate kinase 1

161

24

55

44.9/8.30

47.0/7.21

2.4

A

P04406/G3P_

(primer recognition protein 2, PRP 2) Glyceraldehyde-3-phosphate

168

25

59

36.2/8.57

dehydrogenase (GAPDH)

128

17

54

Triosephosphate isomerase

163

17

82

201

18

86

Cell Cycle

Glycolysis/Gluconeogenesis

P60174/TPIS_

26.9/6.45

37.2/9.08

2.2

B

29.9/10.3

9.1

C

27.2/6.49

3.8

A

27.3/6.87

4.7

B C

193

18

87

26.0/7.32

2.3

P00338/LDHA_

L-lactate dehydrogenase A chain (LDH-A)

183

22

51

37.0/8.44

38.2/9.18

3.9

B

P18669/PGAM1_

Phosphoglycerate mutase 1 (PGAM1)

103

12

55

28.9/6.67

27.5/6.39

3.5

C

176

13

60

27.56.60

3.2

14

32

69.9/5.80

72.1/6.91

2.5

61.7/7.66

Inflammation P09960/LKHA4_

Leukotriene A-4 hydrolase

P00367/DHE3_

Glutamate dehydrogenase 1,

74

B

Mitochondria

P40926/MDHM_

81

16

29

mitochondrial, (GDH)

189 204

29 33

52 48

56.1/7.18

3.0

A

59.1/7.26; 55.4/7.34

2.5 3.3

B C

Malate dehydrogenase, mitochondrial,

283

32

76

36.0/8.92

34.3/9.21

6.7

C

9

37

24.6/7.01

26.5/7.63

6.8

A

12

28

62.8/6.15

65.3/6.52

3.8

B

26.9/5.30

2.1

B

(MDH) Nucleocytoplasmic Protein Transport P62826/RAN_

GTP-binding nuclear protein Ran,

88

GTPase Ran Macrophages P23141/EST1_

Liver carboxylesterase 1,

69

Monocyte/macrophage serine esterase General/Other Metabolism O00299/CLIC1_

Chloride intracellular channel protein 1,

123

13

68

27.2/5.09

Q16851/UGPA_

nuclear chloride ion channel 27 UTP-glucose-1-phosphate

113

20

40

57.1/8.16

59

9

27

60 126

11 15

21 68

uridylyltransferase

29.8/6.86

56.0/7.55;

3.1

A

58.2/7.61;

4.5

B

52.5/7.42 29.6/7.42

3.5 4.8

C B

P07451/CAH3_

Carbonic anhydrase 3

Q6NV95/PAB1L_

Putative protein PABPC1-like

65

11

37

30.3/8.88

28.1/8.40

3.6

C

P50995/ANX11_

Annexin A11 (fragm.),

59

10

11

54.7/7.53

14.1/5.60

2.2

C

413 364

45 42

70 69

71.3/5.92

73.2/5.82 69.4/6.00

2.5 4.1

A B

280

35

59

71.6/5.70

2.7

C

196

29

46

87.3/6.78

2.2

B

555

48

59

87.6/6.92

2.2

B

387

43

56

80.3/6.40

2.1

C

90.6/4.80

4.2

C

Calcyclin-associated annexin 50 P02768/ALBU_

Serum albumin

P02787/TRFE_

Serotransferrin, Siderophilin

79.3/6.81

Immune Response P01833/PIGR_

Polymeric immunoglobulin receptor

231

28

38

84.4/5.58

(Poly-Ig receptor) 2190



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Table 1. Continued UniProt/ _HUMAN P01876/IGHA1_ P01857/IGHG1_

protein name Ig alpha-1 chain C region Ig gamma-1 chain C region

score

matched

seq. cov.

theor. MW

exp. MW

RF

found in

(>56)

peptides

(%)

[kDa]/pI

[kDa]/pI

(g2)

2D-PAGE

38.5/6.08

110

11

42

180

11

44

3.1

B

4.8

C

128

12

42

122

9

44

112

11

50

52.5/8.37

3.5

88 84

5 6

80 86

11.8/5.58

27.8/7.43 27.2/5.71

3.9 2.3

B C

26

52.5/4.58

69.7/4.40

3.5

C

65.1/6.27

P01834/IGKC_

Ig kappa chain C region

Q9UBX5/FBLN5_

Fibulin-5 (FIBL-5)

P31939/PUR9_

Bifunctional purine biosynthesis

136

18

40

protein PURH, IMP cyclohydrolase

189

30

53

36.6/8.46

69.5/5.99 55.7/5.63 58.5/9.26

3.6

B

58.3/9.45

5.2

C

Extracellular Matrix (ECM) 86

16

Purin Metabolism 72.6/6.78

2.7

B

65.2/6.30

2.8

C

129.5/7.85

2.5

A

Transcription Factor Q9C0A1/ZFHX2_

Zinc finger homeobox protein 2 (fragm.)

60

14

13

120.6/6.00

a

Definition of abbreviations: Uniprot/_HUMAN = accession number as in Uniprot Knowledgebase (Swiss-Prot); score = MASCOT probability-based MOWSE score calculated for PMF results. Significance (p < 0.05) is reached at scores >56 when searching the Swiss-Prot database versions 57.1, 57.2 and 57.5; seq. cov. (%) = sequence coverage in %; theor. MW [kDa]/pI = theoretical molecular weight in kDa/isoelectric point; exp. MW [kDa]/pI = experimental molecular weight in kDa/isoelectric point; RF = regulation factor; Symbols (* and †) = both proteins were found to be coincident in one protein spot.

Immunohistochemistry

Standard methodology employing the ZytoChem-Plus AP Kit or ZytoChem-Plus HRP Kit, Broad Spectrum (Zytomed Systems, Berlin, Germany) was used for immunohistochemical localization of proSP-C, Bip/Grp78, VCP, PPIB, Hsp90β, DDB1, Hsp27, p-Hsp27 (phospho S78), PGK1, KRT5, p63 and Annexin A1,A2 and -A3 in formalin fixed, paraffin-embedded tissue sections from IPF- and control lungs, as described in detail in the Supporting Information. Lung tissue sections were scanned with a Mirax Desk slide scanning device (Mirax Desk, Zeiss, G€ottingen, Germany), and examined histopathologically at 100 , 200 and 400 original magnification. Immunohistochemistry for mentioned antibodies was undertaken in 10 IPF and 10 Control lung samples.

’ RESULTS Comparative Proteomic Analysis of IPF versus Control Lung Tissues

A full comparison of all protein spots visualized in three different proteome maps of IPF and controls was performed, revealing 203 protein spots that appeared to differ more than or equal to 2-fold (p < 0.05). Of these 203 spots, the identities for 164 proteins could be established by MALDI-TOF-MS of in-gel digested peptides and, after correction of redundantly identified spots, resulted in a total of 89 differentially regulated proteins. The three proteome maps are depicted in Figure 1, alongside with the 51 up-regulated (Figure 1A-C) and the 38 downregulated (Figure 1D-F) spots in IPF. Classification of these differentially regulated proteins was undertaken in terms of their biological/cellular function (based on information of the Swiss-Prot database) into the categories indicated in Table 1 for the up-regulated, and in Table 2 for the down-regulated proteins in IPF. Additionally, both tables provide information about the identification of these differentially

expressed protein spots, as well as the magnitude of difference with the control group (indicated as RF). Interestingly, a large part of the proteins we identified as being up-regulated in the IPF group fall into the related categories Unfolded Protein Response (UPR)/ER stress, proteasome/ degradation and general cell stress-response (Table 1). Up-Regulation of the Unfolded Protein Response (UPR) and Evidence of ER Stress-Induced Apoptosis in IPF-Lungs

The up-regulation of the UPR/ER stress markers 78 kDa glucose-regulated protein (Grp78), valosin-containing protein (VCP/TERA) and peptidly prolyl cis-trans isomerase B (PPIB), as well as of the cytosolic heat-shock protein member Hsp90R (HS90A) (Table 1) was further validated by Western blot analysis of “individual” lung homogenates prepared from IPF(n = 9) and control lungs (n = 5). Representative immunoblots (Figure 2A) and densitometric quantitation of normalized immunoblots revealed significant up-regulation of Grp78 (Figure 2B, p < 0.001), VCP (Figure 2C, p < 0.001), PPIB (Figure 2D, p < 0.01) and Hsp90R (Figure 2E, p < 0.01) in IPF compared to controls. Because of their overall low abundance, the ER stress sensor transmembrane proteins p90ATF-6, PERK and IRE-1 as well as their transcriptional enhancers p50ATF-6, ATF-4 and spliced XBP-1(s) were not detected on Coomassie stained 2D-gels. But, as published before,11 we could detect cleaved p50ATF-6 and induced expression levels of ATF-4, and of the proapoptotic transcription factor CHOP, as well as caspase-3 activation in every individual IPF but not in any control lung homogenate (Supplemental Figure SII). In addition to these observations, we were able to detect nondegraded polyubiquitinated substrates (Supplemental Figure SIII) in four (out of six) IPF-, but in none of the control lung homogenates, probably indicating proteasomal dysfunction. Next, the up-regulation of the expression of VCP, PPIB, Grp78 and of the Hsp90-complex was further confirmed by immunohistochemistry (IHC) in order to identify the cellular 2191



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Table 2. Proteins Significantly Down-Regulated in IPF Lungs Relative to Control Lung Tissuea UniProt/ _HUMAN

protein name

score

matched seq. cov. theor. MW

exp. MW

(>56)

peptides

(%)

[kDa]/pI

[kDa]/pI (e0.5)

RF found in 2D-

38.9/6.57

PAGE

Anti-Inflammation P04083/ANXA1_ Annexin-1, Lipocortin I, Calpactin II,

216

27

76

38.9/6.36

0.4

D

Chromobindin-9, p35, Phospholipase

170

21

65

38.8/6.70

0.5

E

A2 inhibitory protein

315

29

73

38.2/6.47

0.4

E

292

34

76

318

36

80

312 173

38 19

82 61

269

25

289

Fibrinolysis P07355/ANXA2_ Annexin-2, Lipocortin II, Calpactin I heavy chain, Chromobindin-8, p36, Placental anticoagulant protein IV P12429/ANXA3_ Annexin-3, Lipocortin III, 35-alpha calcimedin; Placental anticoagulant protein III

38.8/7.57

37.9/7.61

0.4

D

38.1/7.80

0.4

E

36.7/7.54 34.8/5.70

0.4 0.3

F D

73

36.1/5.90

0.3

E

38

47

99.6/5.34 106.7/5.50

0.5

F

83

12

40

38.0/5.60

34.8/5.46

0.4

F

73 73

11 10

45 41

23.3/5.02

28.5/5.43 28.9/5.19

0.4 0.4

D E

85

12

49

27.1/5.07

0.4

F

36.5/5.63

Signaling Molecules Q14764/MVP_

Major vault protein; Lung resistancerelated protein

P62879/GBB2_

Guanine nucleotide-binding protein G(I)/G(S)/G(T), Transducin beta chain 2, G protein subunit beta-2

P52565/GDIR1_ Rho GDP-dissociation inhibitor 1 (Rho-GDI alpha)

Antioxidant Function P04040/CATA_

Catalase

316

32

54

59.9/6.90

60.1/6.80

0.4

F

Q99536/VAT1_

Synaptic vesicle membrane protein

170

26

53

42.1/5.88

45.4/5.85

0.4

F

166

11

51

22.0/5.66

22.2/5.50

0.3

F

67.9/6.08

VAT-1 homologue P32119/PRDX2_ Peroxiredoxin-2, Thioredoxin peroxidase 1, Thiol-specific antioxidant protein Cytoskeleton/Cellular Organization P26038/MOES_ Moesin (Membrane-organizing extension spike protein) P13796/PLSL_

Plastin-2, L-Plastin; Lymphocyte

304

40

53

234

36

52

266

39

53

415

47

66

70.8/5.20

198

23

60

53.2/5.66

79.1/6.20

0.2

D

80.4/6.51

0.2

E

77.3/6.15

0.2

F

67.9/5.18

0.4

F

58.6/6.11

0.3

E

D

cytosolic protein 1 (LCP-1) Protease Q96KP4/CNDP2_Cytosolic nonspecific dipeptidase (CNDP dipeptidase 2), Peptidase A Protein Biosynthesis P13639/EF2_

Elongation factor 2 (EF-2)

209

36

36

96.2/6.41 102.3/6.59

0.3

181

25

42

102.2/6.67

0.5

P68104/EF1A1_ Elongation factor 1-alpha 1 (eEF1A-1),

128

14

29

Elongation factor Tu

68

12

23

19

47

285

37

127 255 276 243

50.5/9.10

49.0/8.42

0.1

49.0/8.65

0.2

D

62.7/5.95

63.8/6.27

0.3

E

67

58.5/7.95

63.1/7.56

0.4

D

16 29 31

44 66 71

47.5/7.01 37.2/6.54

51.9/7.58 39.3/6.48 40.2/6.56

0.4 0.5 0.3

E D E

27

60

36.8/6.54

0.2

F

Developmental Protein Q16555/DPYL2_ Dihydropyrimidinase-related protein 2 (DRP-2),

119

Collapsin response mediator protein 2 Glycolysis/Gluconeogenesis P14618/KPYM_ Pyruvate kinase isozymes M1/M2, pyruvate kinase muscle isozyme P06733/ENOA_ Alpha-enolase, non-neural enolase P09467/F16P1_ Fructose-1,6-bisphosphatase 1 (FBPase1)

2192



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Table 2. Continued UniProt/ _HUMAN

protein name

score

matched seq. cov. theor. MW

exp. MW

(>56)

peptides

RF found in 2D-

(%)

[kDa]/pI

[kDa]/pI (e0.5)

PAGE

Pentose Phosphate Pathway P29401/TKT_

Transketolase

136

23

42

68.5/7.58

67.5/7.26

0.4

F

P37837/TALDO_ Transaldolase

177

25

51

37.7/6.36

36.8/6.48

0.4

F

10

83

10.9/6.51

11.6/7.08

0.4

E

13.3/5.71

Inflammation P05109/S10A8_

Protein S100-A8, S100 calcium-binding protein A8,

133

Calgranulin-A, Cystic fibrosis antigen P06702/S10A9_

S100 calcium-binding protein A9, Calgranulin B

59

10

63

92

10

63

25

48

57.1/5.98

45.0/6.10

15.0/6.12

0.3

E

13.8/5.92

0.5

E

64.3/5.98

0.4

E

UPR/ER Stress Response P30101/PDIA3_ Protein disulfide-isomerase A3, ERp60,

196

58 kDa microsomal protein Lysosomal Degradation P07339/CATD_ Cathepsin D, cleaved into 2 chains: Cathepsin D light chain, 160 Cathepsin D heavy chain (28 kDa)

19

40

173

20

47

199

24

59

72

10

14

45.1/7.52

109

19

45

56.9/6.63

21.0/5.12

Q13510/ASAH1_ Acid ceramidase (AC), heterodimer: subunit alpha

31.6/6.05

0.3

E

28.9/5.54

0.4

F

28.9/6.01

0.2

F

14.9/5.83

0.1

E

55.3/5.80

0.4

F

(13 kDa, detected) and beta (32 kDa) Mitochondria P05091/ALDH2_ Aldehyde dehydrogenase, mitochondrial (ALDH class2)

Secondary Metabolite Metabolism Q04760/LGUL_ Lactoylglutathione lyase, Methylglyoxalase

84

12

50

86

11

40

25.6/5.35

0.2

E

25.6/5.49

0.2

E

43

28

188.6/6.02

68.0/6.92

0.4

F

19

42

42.8/5.90

40.1/5.86

0.3

F

7

50

22.4/5.21

34.6/5.05

0.4

E

Immune Response P01024/CO3_

Complement C3, cleaved into 10 chains: C3 alpha chain,

232

C3 beta chain (detected), C3d fragment,... Protease Inhibitors P30740/ILEU_

Leukocyte elastase inhibitor, Monocyte/neutrophil elastase,

123

Serpin B1 Calcium Binding P37235/HPCL1_ Hippocalcin-like protein 1, Calcium-binding protein BDR-1

66

General/Other Metabolism P00325/ADH1B_ Alcohol dehydrogenase 1B (ADH1B)

249

25

63

40.7/8.63

42.2/9.27

0.4

E

P21695/GPDA_ Glycerol-3-phosphate dehydrogenase [NADþ],

302

19

50

38.2/5.81

36.2/5.68

0.3

F

Carbonic anhydrase 1 (CA-I)

155

13

64

28.9/6.59

28.1/6.76

0.4

F

Q01469/FABP5_ Fatty acid-binding protein, epidermal (E-FABP)

180

15

74

15.5/6.60

15.7/6.52

0.2

E

93

14

70

14.8/6.08

0.4

F

99 408

16 34

39 85

46.8/4.69 52.9/5.93

38.1/5.90 52.4/5.79

0.3 0.4

E F

15.3/8.72

cytoplasmic (GPDH-C) P00915/CAH1_

Q9NZA1/CLIC5_ Chloride intracellular channel protein 5 (CLIC5) Q13228/SBP1_ Selenium-binding protein 1 P69905/HBA_ P68871/HBB_

Hemoglobin subunit alpha Hemoglobin subunit beta, cleaved into chain LVV-hemorphin

113

10

88

122

9

77

13.6/7.82

0.2

E

13.3/8.60

0.2

139

11

80

E

14.3/6.75

0.2

87

8

E

74

14.2/6.94

0.4

E

88

12

88

13.9/5.71

0.4

E

16.1/6.75

a

Definition of abbreviations: Uniprot/_HUMAN = accession number as in Uniprot Knowledgebase (Swiss-Prot); score = MASCOT probability-based MOWSE score calculated for PMF results. Significance (p < 0.05) is reached at scores >56 when searching the Swiss-Prot database versions 57.1, 57.2 and 57.5; seq. cov. (%) = sequence coverage in %; theor. MW [kDa]/pI = theoretical molecular weight in kDa/isoelectric point; exp. MW [kDa]/pI = experimental molecular weight in kDa/isoelectric point; RF = regulation factor.

distribution of UPR markers and cytosolic chaperones in IPF and control lung tissues. In addition, we performed staining for proSP-C

of sections serial to the ones used to identify UPR markers, in order to designate AECII in these lung tissues (Figure 3A,C,E,G,H). 2193



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Figure 2. Induction of the Unfolded Protein Response (UPR) and ER stress-induced apoptosis in IPF. (A) Representative immunoblots and (B-E) quantitative immunoblot analysis of peripheral lung tissue from patients with sporadic IPF (n = 9) and explanted donor lungs (controls, n = 5) using specific antibodies against Grp78 (B), VCP (C), PPIB (D), Hsp90R (E), and β-actin as loading control. Intensity of bands was densitometrically quantified and normalized to β-actin (mean ( SEM). ***p < 0.001, **p < 0.01, *p < 0.05 for IPF versus controls.

Figure 3. Up-regulation of markers for the Unfolded Protein Response (UPR) in type-II alveolar epithelial cells (AECII) in IPF lungs. Representative immunohistochemistry for proSP-C (A, C, G, and H), VCP (B and D), PPIB (I and J) and Grp78 (K and L) in serial sections of IPF lung tissues, and for proSP-C (E and M), VCP (F) and PPIB (N) in control lungs. (B and D) In IPF, strong staining for VCP was predominantly observed in AECII surrounded by fibrotic tissue. (F) In control lungs, a basal level of VCP expression was observed in AECII. (G-L) Co-localization of proSP-C (G and H), PPIB (I and J) and Grp78 (K and L) in AECII overlying fibroblast foci (FF) in IPF lungs. AECII are indicated by arrows in panels A-N. Original magnification of photomicrographs A-F, H, J, L, M and N: 400 (bar = 50 μm); original magnification of photomicrographs G, I, and K: 200 (bar =100 μm).

A prominent immunostaining for VCP (Figure 3B,D) was observed in AECII near areas of dense fibrotic remodelling, but not in fibrotic tissue itself of affected lungs from patients with sporadic IPF. In contrast, AECII in control lungs with normal histological appearance (Figure 3E) showed a basal, lower level of expression of VCP (Figure 3F). Similarly, the recently characterized ATF-6/XBP-1 target UPR gene PPIB17 was also found to be predominantly expressed in AECII adjacent to underlying fibroblast foci and areas of mature fibrosis (Figure 3I,J), and co-localized with Grp78 (Figure 3K,L). Of note, fibroblast foci were negative for PPIB staining, and no or minimal staining was observed in the interstitium

of older fibrotic tissue. AECII of control lungs (Figure 3M, proSP-C staining) showed a lower level of expression of PPIB (Figure 3N) and Grp78 (not shown). Finally, the cytosolic heat shock protein member Hsp90β (HS90B) was predominantly found in AECII both in the control and IPF lungs, thereby revealing a robust expression in AECII overlying dense zones of fibrosis in IPF (Supplemental Figure SIV). Other Up-Regulated Stress-Induced Genes in IPF and Their Localization in Lung Epithelium

Other stress-induced genes found to be upregulated in IPF were DNA damage-binding protein 1 (DDB1), heat shock 27-kDa protein (Hsp27) and the glycolytic enzyme phosphoglycerate 2194



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Figure 4. Increased expression of stress-induced genes DDB1, Hsp27 and PGK1 in IPF. (A) Representative immunoblots and (B-D) quantitative immunoblot analysis of peripheral lung tissue from patients with sporadic IPF (n = 9) and explanted donor lungs (controls, n = 5) using specific antibodies against DDB1 (B), Hsp27 (C), PGK1 (D), and β-actin as loading control. Hsp27 protein appeared as a 22-kDa band and a higher molecular weight band of about 25 kDa that was presumably due to phosphorylation. Both bands for Hsp27 revealed up-regulation in IPF lungs. The same phenomenon was observed for PGK1, which revealed detection of a 44-kDa and 47-kDa band, respectively. Both bands for PGK1 revealed up-regulation in IPF lungs. Intensity of DDB1 protein bands for (B) and upper phosphorylated bands of p-Hsp27 and PGK1 for (C and D) was densitometrically quantified and normalized to β-actin (mean ( SEM). ***p < 0.001, **p < 0.01, *p < 0.05 for IPF versus controls.

Figure 5. Induction of DDB1 in type-II alveolar epithelial cells (AECII) and metaplastic epithelial cells of IPF lungs. Representative immunohistochemistry for proSP-C in IPF (A, B, H, and I) and control (C) lung tissues to indicate AECII, and for DDB1 in serial sections of IPF (E, F, J, and K) and control (G) lung tissues. (E, F, J, and K) In IPF, DDB1 revealed immunostaining of AECII overlying fibroblast foci (E and F) and mature areas of fibrosis (J and K), and also of squamous metaplastic epithelial cells in honeycombed regions of IPF lungs (J and K). (G) Positive staining for DDB1 was not seen in any cell and in any of the control lung tissues. AECII are indicated by arrows in panels B, C, F, G, I, and K, squamous metaplasia by hashmarks in panels J and K. Original magnification of photomicrographs A, E, H, and J: 200 (bar = 100 μm); original magnification of photomicrographs B, C, F, G, I, and K: 400 (bar =50 μm).

kinase 1 (PGK1) (Table 1). Western blot analysis revealed markedly increased protein levels of DDB1, and two bands of elevated protein expression corresponding to Hsp27 and PGK1 in IPF-, but not in control lung tissues (Figure 4A). The upper bands of both proteins revealed a 2-3-kDa higher molecular weight (MW) in comparison to the unprocessed precursors of respective proteins, most likely due to phosphorylation. Finally, densitometric quantification demonstrated significantly increased levels of DDB1 (Figure 4B, p < 0.001), phosphorylated Hsp27 [p-Hsp27] (Figure 4C, p < 0.01) and in addition PGK1 [p-PGK1] (Figure 4D, p < 0.001) in IPF, as compared to control lungs. The up-regulation of DDB1, Hsp27/p-Hsp27 and PGK1 was further validated by IHC. DDB1 was found to be localized in IPF lung tissues in AECII overlying FF and dense zones of fibrosis, but never in fibrotic tissue itself (Figure 5E,F,J,K, indicated by arrows; Figure 5A,B,H,I, respective proSP-C staining). Expression of DDB1 was also found in metaplastic epithelial cells of “squamous metaplasia” in the honeycombed regions (Figure 5J,K), an epithelial abnormality occurring frequently in UIP/IPF.18,19 Of

note, no staining for DDB1 was observed in any cell or in any of the control lung tissues (Figure 5G). With regard to Hsp27, AECII overlying dense zones of fibrosis and FF (Figure 6A-D, and I and J, proSP-C staining) were apparently negative for phosphorylated (p)-Hsp27 (arrows in Figure 6E-H and K). Instead, an epithelial cell population adjacent to FF exhibited strong immunoreactivity for p-Hsp27 (Figure 6E-H, K,L,M,O). Interestingly, the p-Hsp27 positive epithelial cell sheets overlying FF were obviously located between negative luminal layers of epithelial cells (indicated by dashed arrows) and negative fibroblasts/myofibroblasts (Figure 6E-H, K,L,M,O), imposing as “sandwich-FF”.20 The p-Hsp27 positive epithelial cells overlying FF were immunophenotypically characterized as bronchiolar basal cells using antibodies against cytokeratin-5/KRT5 (Figure 6M,O, p-Hsp27; N,Q, KRT5) and p63 (Figure 6P) as well-established markers for this cell type. Importantly, the sandwich-FF we often found in close proximity to AECII, at the bronchiolo-alveolar junctions (Figure 6A-H), and also frequently in areas of bronchiolized epithelia (Figure 6I-L,M,N). Of note, only bronchiolar basal cells near 2195



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Figure 6. Induction and phosphorylation of Hsp27 in close proximity to fibroblast foci in IPF lungs. Representative immunohistochemistry for proSP-C (A-D, I, and J), p-Hsp27 (E-H, K-M, and O), cytokeratin-5/KRT5 (N and Q) and p63 (P) in serial sections of IPF lung tissues. (E-H, K-M, and O) In IPF, the antibody for phosphorylated Hsp27 (p-Hsp27) revealed strong staining of bronchiolar basal cell sheets (indicated by hashmark) located between negative luminal sheets of bronchiolar epithelial cells (indicated by dashed arrows) and negative fibroblast foci (FF); this lesion pattern has been termed “sandwich-FF”.20 AECII did not show any immunoreactivity for p-Hsp27 antibodies (indicated by arrows), which were marked by proSP-C staining in serial sections (A-D, I, and J). (A-H, I, and K) p-Hsp27 positive bronchiolar basal cells overlying FF were often detected in close proximity to AECII. (L) A fibroblast focus underlying p-Hsp27 positive basal cells in areas of bronchiolized epithelia in the honeycombed regions of an IPF lung is shown. In this area, no AECII was observed as proven by proSP-C staining in a serial section (J). (M-Q) p-Hsp27 positive cells (M and O) were immunophenotypically characterized as bronchiolar basal cells by cytoplasmic KRT5- (N and Q) and nuclear p63 staining (P). Original magnification of photomicrographs A and E, I-N: 200 (bar = 100 μm); original magnification of photomicrographs B-D, F-H, and O-Q: 400 (bar =50 μm).

sites of “active” fibrosis revealed immunostaining for p-Hsp27 and was not observed in the general basal cell population of bronchi and bronchioles in IPF-lung tissues. Interestingly, the smooth muscle present in the walls of blood vessels (vascular smooth muscle cells, VSMC) stained also positive for p-Hsp27 in IPF- and control lung tissues (Supplemental Figures SV and SVI). No notable staining for p-Hsp27 was observed in any other cell of the control lungs (Supplemental Figure SV). Use of an antibody directed against (nonphosphorylated) full-length Hsp27 revealed the same results as observed for the antip-Hsp27 antibody (not shown). Finally, immunostaining for PGK1 revealed a pronounced nuclear staining of this glycolytic enzyme in bronchiolar basal cells near active sites of fibrosis which showed in serial sections in part p-Hsp27 immunoreactivity (Supplemental Figure SVIIA-H). Nuclear staining for PGK1 was also observed in the basal proportion of hyperplastic bronchioles (not shown), and to some extent in the FF (Supplemental Figure SVIA,E). In contrast, AECII of IPF lungs mostly showed a cytoplasmic PGK1 staining (Supplemental Figure SVII,J). Biochemical Characterization of Down-Regulated Proteins in IPF

With regard to the down-regulated proteins in IPF (Table 2), a significantly reduced expression of annexin A1, A2 and A3 (ANXA1,

-2 and -3), the lysosomal protease cathepsin D (CATD), as well as of the antioxidant proteins catalase and peroxiredoxin 2 (PRDX2) were confirmed by quantitative immunoblotting (Figure 7A-G). Employing IHC, an apical staining pattern of ANXA1 was encountered in ciliated bronchial epithelial cells of IPF and control lungs, supporting secretion of this annexin (Figure 8, F-J, indicated by hashmark). ANXA1 was also expressed by few inflammatory interstitial cells in IPF and control lungs (Figure 8F-J, indicated by arrows). In contrast, AECII of IPF (Figure 8A, indicated by asterisks) and control lungs (Figure 8D,E) showed no immunoreactivity for ANXA1 on respective serial sections (Figure 8F,I,J, see asterisks). In contrast to ANXA1, IHC for ANXA3 revealed expression of this annexin in AECII of IPF (Figure 8K-M, O-Q) and control lungs (Figure 8N,R). Bronchial epithelial cells of IPF and control lung tissues revealed also immunoreactivity for ANXA3, but no secretion (not shown). The presence of ANXA2 in AECII and its participation as a plasma membrane located protein in surfactant secretion has been reported in various studies.21-23 In IPF lungs, however, we observed no immunostaining for ANXA2 in AECII near dense zones of fibrosis, which were indicated in respective serial sections by proSP-C staining (Figure 9A-H,I,L, indicated by asterisks). Instead, strong ANXA2-staining was encountered in the endothelium of vessels (Figure 9E,F, indicated by hashmarks), 2196



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Figure 7. Down-regulation of annexin A1, -A2, and -A3, as well as of cathepsin D, catalase and peroxiredoxin 2 in lungs from patients with IPF. (A) Representative immunoblots and (B-G) quantitative immunoblot analysis of peripheral lung tissue from patients with sporadic IPF (n = 9) and explanted donor lungs (controls, n = 5) using specific antibodies against annexin A1 [ANXA1] (B), annexin A2 [ANXA2] (C), annexin A3 [ANXA3] (D), cathepsin D [CATD] (E), catalase (F), peroxiredoxin 2 [PRDX2] (G), and β-actin as loading control. Intensity of bands was densitometrically quantified and normalized to β-actin (mean ( SEM). ***p < 0.001, **p < 0.01, *p < 0.05 for IPF versus controls.

as well as on the surface of “proliferative” bronchiolar basal cells overlying FF or mature areas of fibrosis in IPF lungs (Figure 9G,H, indicated by arrows), as immunophenotypically characterized by KRT5 staining (Supplemental Figure SVIII). Robust ANXA2 immunoreactivity of multilayered basal cell sheets was observed in bronchiolar abnormalities such as “bronchiolar hyperplasia” or “basal cell hyperplasia” (Figure 9I-K). Minimal staining for ANXA2 was observed in the interstitium of IPF lungs (Figure 9E-I). Of note, control lungs revealed a strong surface staining of alveolar septae (Figure 9M,N), which are usually covered with very thin, flattened type-I alveolar epithelial cells (AECI) and with AECII (Figure 9O,P). Vascular and capillary endothelium of donor lungs was also stained by anti-ANXA2 antibodies (Figure 9N).

’ DISCUSSION Unfolded Protein Response (UPR) and Chronic Endoplasmic Reticulum (ER)-Stress in the Epithelium of IPF Lungs

The Unfolded Protein Response in IPF. The ER is the principal organelle for proteins destined for the secretory pathway and is also the site of biosynthesis for steroids and many lipids. Any perturbation of the ER microenvironment, such as altered Ca2þ homeostasis, protein overflow, viral infection, oxidative stress, deprivation of glucose or other sugars, altered glycosylation, and the folding of mutant proteins, leads to accumulation of misfolded proteins and causes ER stress.24 As a part of this ER stress response, genes involved in protein folding (such as chaperons), protein degradation and attenuation of protein translation are activated within the so-called Unfolded Protein Response (UPR).25 If the accumulation of unfolded or misfolded proteins cannot be overcome by the UPR, ER stressmediated apoptosis pathways are induced to enforce the death of the concerned cell.24,26-28

Extending a previous report from our group disclosing upregulation of ER stress-sensor proteins, our proteomic approach now disclosed that, in lungs of patients with sporadic IPF, more precisely in the type-II alveolar epithelial cells (AECII), several proteins involved in the UPR were significantly up-regulated, including ER- (Grp78, PPIB) and cytosolic chaperones (HS90A, HS90B), an ER disulfide-isomerase (PDIA4), the multifunctional AAA-ATPase VCP, subunits of the 20S proteasome (PSA3, PSA4), as well as a cytosolic leucine aminopeptidase (AMPL), being involved in protein processing and trimming of peptides (Table 1). Among the down-regulated proteins in IPF (Table 2), we identified elongation factor 2 (EF-2) and EF1A1, consistent with the suppression of general translation by the UPR,24 as well as the ER resident chaperone protein disulfide-isomerase A3 (PDIA3, ERp57), a chaperone not interacting with Grp78, PDIA4, and PPIB in the large chaperone multiprotein complex. Additionally, an unresolved ER stress response has also been directly linked to mitochondrial dysfunction, through the induction of the proapoptotic transcription factor CHOP, which is coupling death signals of the stressed ER to the mitochondrial apoptosis pathway.24,26 This might explain why mitochondrial Hsp60 (CH60) and VDAC1 were found to be increased in our IPF patients (Table 1). VDAC1 is localized at the outer mitochondrial membrane and, in response to pro-apoptotic stimuli, participates in the formation of the permeability transition pore complex which is responsible for the release of mitochondrial products triggering apoptosis.29 Unequivocally, our data indicate full activation of the ER stress-response in AECII in IPF, alongside with a significant disturbance in the protein synthesis and degradation machinery. Ultimately, such unopposed ER-stress induced apoptosis of the AECII, at least partly through CHOP. The underlying reasons of 2197



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Figure 8. Expression and localization of annexin A1 and annexin A3 in lung tissues of IPF patients and controls. (A-J) Representative immunohistochemistry for proSP-C in IPF (A-C) and control (D and E) lung tissues to indicate AECII, and for annexin A1 [ANXA1] in serial sections of IPF (F-H) and control (I and J) lung tissues, revealing an apical staining pattern of ANXA1 in ciliated bronchial epithelial cells (indicated by hashmarks) as well as ANXA1 expression in interstitial inflammatory cells (indicated by arrows); AECII did not express ANXA1 (indicated by asterisks). K-R) Representative immunohistochemistry for proSP-C in IPF (K-M) and control (N) lung tissues to indicate AECII, and for annexin A3 [ANXA3] in serial sections of IPF (O-Q) and control (R) lung tissues, revealing ANXA3 expression in AECII (indicated by arrows). Original magnification of photomicrographs A, B, D, F, G, I, K and O: 200 (bar = 100 μm); original magnification of photomicrographs C, E, H, J, L-N, P-R: 400 (bar = 50 μm).

this ER stress pattern are unknown and deserve further exploitation. It is, however, an intriguing finding that in familial forms of IPF due to surfactant protein C (SFTPC) gene mutations, the pathomechanistic sequelae starts with misfolding of proSP-C, induction of ER-stress and proteasome dysfunction and apoptosis of AECII,8,9 hence, the same cellular reaction pattern as observed in our sporadic IPF cases without any SFTPC mutation. Grp78, the Key Chaperone Responsible for Induction of the Unfolded Protein Response. Grp78 is believed to be the key regulatory molecule in the ER, because it usually complexes with the three resident ER stress sensor proteins PERK, ATF-6 and IRE-1, keeping them in an inactive state. Occurrence of unfolded and misfolded proteins in the ER lumen causes dissociation of Grp78 from the above-mentioned sensors, thereby allowing them to become activated.30 In addition to its function as the key ERresident chaperone, Grp78 counterbalances ER stress-induced apoptosis.31,32 Overexpression of Grp78 in vitro resulted in resistance to etoposide-induced apoptosis through inactivation of caspase-7.32 Finally, several studies suggest that Grp78 may also protect cells against cell death by suppressing oxyradical accumulation and stabilizing mitochondrial function.33,34 With regard to IPF, however, it appears that the observed upregulation of Grp78 in AECII of IPF lungs appears insufficient to counterbalance the ER stress-induced apoptosis. Nevertheless, it appears an intriguing thought that a further therapeutic raise in intracellular Grp78 levels may be helpful to reduce AECII apoptosis rate and hence lung fibrosis.

Valosin-Containing Peptide (VCP), a Double-Edged Sword of the UPR?. VCP is a member of the AAA (ATPases associated with diverse cellular activities) family of ATP-binding, homo-oligomeric ATPase proteins, and participates in numerous cellular processes including protein folding, ER associated degradation (ERAD) of misfolded proteins via ubiquitin-proteasome pathway, and apoptosis.35 In detail, VCP forms a ternary complex with the ERAD proteins Ufd1 and Npl4 to facilitate polyubiquitination of misfolded substrate proteins through interaction with E3 ubiquitin ligase gp78 and to accelerate export of unfolded proteins from the ER to the cytoplasm, where they are degraded by the proteasome.36,37 Therefore, the increase in VCP levels in AECII of IPF lungs might reflect the ER dysfunction due to an increasing number of unfolded and misfolded proteins requiring ubiquitination and degradation in this highly secretory cell type. However, VCP may also associate with polyubiquitinated, misfolded substrate proteins and interact with ataxin 3 and cytosolic histone deacetylase HDAC6, resulting in formation of abnormal multiprotein aggregates called “aggresomes”.38-40 Such intracellular inclusion bodies may deposit and cannot then be removed by the proteasome anymore.38,40,41 In addition, despite its essential functions in the cytoprotective ERAD pathway, VCP has also been referred as a required mediator of ER stress induced apoptosis through multimerization with the “apoptosis-linked gene 2 protein” (ALG-2) in presence of ATP and Ca2þ, leading to the activation of caspase-7, -12, -9, and -3.42,43 Co-immunoprecipitation studies have suggested that both VCP and ALG-2 may function as part of a large 2198



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Figure 9. Expression and localization of annexin A2 in lung tissues from IPF patients and controls. Representative immunohistochemistry for proSP-C in IPF (A-D, and L) and control (O and P) lung tissues to indicate AECII, for annexin A2 [ANXA2] in serial sections of IPF (E-H, and I) and control (M and N) lung tissues, and for cytokeratin-5/KRT5 (J) and p63 (K) in serial sections of IPF lung tissues. (E-H) In IPF, expression of ANXA2 was observed in vascular endothelium (E and F, indicated by hashmarks), as well as in the cytoplasm and on the cell-surface of “proliferating” bronchiolar cells (G and H) overlying dense areas of fibrosis. In contrast, AECII surrounded by dense areas of fibrosis, which were indicated in serial sections by proSP-C staining (A-D, and L), did not show ANXA2 expression (indicated by asterisks in A-H, I, and L). (I-L) ANXA2 expression was observed in bronchiolar basal cell layers of “hyperplastic” bronchioles in IPF lung tissues (I, indicated by arrows), which were immunophenotypically characterized in serial sections by KRT5- (J) and nuclear p63 expression (K); AECII surrounded by dense fibrotic tissue did not express ANXA2 (I and L, indicated by asterisks). (M-P) In control lungs with normal histological appearance, ANXA2 antibodies stained strongly the surface of alveolar septae, and endothelial cells of the vascular lumen (M and N); AECII are indicated by proSP-C staining in respective serial sections (O and P). Original magnification of photomicrographs A and E: 200 (bar = 100 μm); original magnification of photomicrographs B-D, F-H, I-L, and M-P: 400 (bar = 50 μm).

ER stress-induced caspase-activating complex, which has been referred to an ER-based apoptosome or “eraptosome”.42,43 Moreover, the above-mentioned aggresomes, being resistant to proteolytic degradation, have been reported to disrupt cellular function and to also induce cell death.38,41 In line with this notion, we could detect high molecular weight-polyubiquitinated substrates in four IPF lung homogenates, indicative of an impaired degradative capacity of the proteasome. With regard to our previous observation of caspase-3 activation and execution of apoptosis in AECII near dense zones of fibrosis in IPF,11 the herein presented data may suggest that VCP up-regulation in AECII of IPF lungs contributes to ER stress mediated apoptosis independent of CHOP and the mitochondrial apoptosis pathway. Other Cellular Stress Markers in IPF

Other (non-ER) stress-induced genes found to be up-regulated in IPF were DNA damage-binding protein 1 (DDB1) and heat shock 27-kDa protein (Hsp27). DDB-1, the up-regulation of which is reported here for the first time in IPF, has been described to be induced as a part of the DNA damage-response due to UV irradiation44 or viral infection.45 Under these conditions, DDB1 forms a complex with E3-cullin4A ubiquitin ligase for ubiquitinating histone 2A in order to modify the chromatin structure at the sites of DNA lesions to promote efficient nucleotide excision repair.44 Interestingly, several viruses

deregulate DNA damage response signaling in order to facilitate their propagation and persistent infection, because infected host cells recognize viral replication as a DNA damage stress. For example, paramyxoviruses target the Cul4A/DDB1 complex for degradation of STAT1 to avoid host immune attack,45 and other viruses such as herpes simplex virus type I (HSV-1) and human cytomegalovirus can activate and exploit a cellular DNA damage response, which aids viral replication.46 In line with this notion, infection with herpesviruses like Epstein-Barr are frequently found in the epithelium of IPF lungs47 and have been claimed to be associated with the progression of disease, also due to their ability to trigger ER stress.48 Taken together, the observed induction of DDB1 in epithelial cells of IPF lungs in our study might be linked to DNA damage or to reported viral infection in this disease, possibly due its contributory role in persistent infection. Hsp27 is a multifunctional stress-inducible protein involved in stress resistance, actin organization, as well as cell proliferation, cell motility and migration, which seems to require phosphorylation of Hsp27.49,50 Moreover, phosphorylated Hsp27 can mediate resistance against cell death induced by oxidative stress and differentiation, and is therefore related to cell survival.51-53 Interestingly, in our IPF population, intensive immunoreactivity for p-Hsp27 was observed in bronchiolar basal cells overlying FF, which were themselves underlying areas of p-Hsp27 negative luminal (bronchiolar) epithelial cells, thereby forming a structure 2199


Journal of Proteome Research recently recognized as “sandwich fibroblast foci (FF)”.20 In contrast, the epithelial cells overlying the “non-sandwich-FF” in UIP lungs exhibited the phenotype of AECII, which never demonstrated immunoreactivity for Hsp27 or p-Hsp27. Hence, since Hsp27/p-Hsp27 was only observed at sites of abnormal reepithelialization at the bronchiolo-alveolar junctions, characterized by abnormal proliferation of bronchiolar basal cells adjacent to underlying myofibroblasts in sandwich-FF, induction and phosphorylation of Hsp27 may reflect a feature of bronchiolization, the process of exaggerated bronchiolar proliferation and progressive colonization of alveolar spaces through migrating bronchiolar basal cells, leading to alveolar loss and honeycomb formation. Additionally, IPF lungs revealed pronounced up-regulation of the glycolytic enzyme PGK1. PGK1 not only functions in glycolysis, but also as a nuclear protein in DNA replication, and has been therefore related to cell proliferation.54-57 In IPF, nuclear PGK1 expression was observed in bronchiolar basal cells of hyperplastic bronchioles, as well as in p-Hsp27 positive basal cells near active sites of fibrosis. Thus, up-regulation and nuclear translocation of PGK1 highlighted also bronchiolar basal cell proliferation and the process of bronchiolization in IPF. Markers of Altered Cytoskeleton and Matrix Turnover

In line with enhanced tissue fibrosis in IPF, a variety of cytoskeletal proteins associated with the function of myofibroblasts and fibroblasts were found to be up-regulated, such as phosphoglucomutase-like protein 5 (PGM5), vimentin (VIME), tropomyosin 1 and 2 (TPM1 and -2), as well as R-SMA (ACTA) (Table 1).58 In 2-DE, R-SMA was comigrating with β-actin (ACTB) in one protein spot due to the similar MW and pI of both proteins (see Figure 1C). Although ACTB was identified with the higher MOWSE score in this spot (Table 1), it seems possible that this “actin spot” was increased due to elevated expression of R-SMA in IPF lungs. R-SMA is the key marker of myofibroblast differentiation, the key effector cell in fibrogenesis; these spindle-shaped cells are highly synthetic for collagen, have a contractile phenotype, and localize to FF and other sites of active fibrosis.59 In accordance with excessive ECM production in UIP/ IPF, biosynthesis of the ECM-compound fibulin-5 (FBLN5) (Table 1) was found to be up-regulated in IPF lung tissues. Epithelial Markers

The epithelial cell marker proteins cytokeratin-8 (K2C8) and -19 (K1C19) were found to be increasingly expressed in IPF (Table 1). K2C8 and K1C19 are specific cytoskeletal structure proteins for simple epithelia, including bronchial and alveolar epithelial cells (BAEC), in which they are abundantly found. In previous reports, increased levels of K1C19 in sera and BALF of IPF patients were suggested to reflect ongoing epithelial injury and aberrant re-epithelialization60,61 and this, obviously, represents also a reasonable explanation for the herein described increase in K2C8 and K1C19 on tissue level. In line with abnormal epithelial repair and observed bronchiolization in IPF, fascin (FSCN1), a cytoskeleton actin-bundling protein involved in motility and cell migration of epithelial cells,62 was up-regulated in IPF lung tissues (Table 1). Much to our surprise the annexins A1, -A2 and -A3 were greatly down-regulated in IPF. These proteins belong to the multigene annexin family of Ca2þ-regulated phospholipid-binding and membrane-binding proteins, with several physiological functions to be assigned to the different annexins: Annexin A1 (ANXA1) is involved in many biological functions such as calcium channel regulation, cell differentiation and

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control of inflammation.63 Strong support for the anti-inflammatory action of ANXA1 stems from knockout mice, displaying higher acute and chronic inflammatory responses characterized by a higher degree of neutrophil trafficking and extravasation.64 ANXA1 was also reported to be degraded in BAL fluids from patients with cystic fibrosis,65 sarcoidosis and IPF.66,67 As a result, a truncated form of ANXA1 was detected, that exhibited catalytic properties distinct from those of the full-length protein.65,66 ANXA1 has been primarily described to inhibit the activity of cytosolic phospholipase A2 (cPLA2). Given that cPLA2 plays a key role in the release of arachidonic acid for the production of lipid inflammatory mediators such as leukotrienes, down-regulation of ANXA1 activity has been shown to result in increased cPLA2 activitiy and LTB4 production in inflammatory lung disease.65,68,69 In the bleomycin model of lung fibrosis, knock out of cPLA2 resulted in a significant reduction in leukotrienes and an attenuation of pulmonary fibrosis,70 thus, underscoring the relevance of leukotrienes in this disease. More recently, antibodies directed against ANXA1 have been described in serum and BALF from roughly 50% of IPF patients with acute exacerbations67 and the authors concluded that truncated ANXA1 or ANXA1 fragments represent potential autoantigens causing acute exacerbations in IPF. Taken together, the herein observed down-regulation of ANXA1 expression in IPF lungs may be linked to reported proteolytic degradation of this annexin in this disease. However, we could not detect ANXA1 fragments in IPF lung tissues by means of immunoblotting (Figure 7A) and proteomic analysis did not reveal the occurrence of a truncated ANXA1 protein. Instead, we detected a 14-kDa fragment of ANXA11 in the IPF proteome (Figure 1C; Table 1), which was not seen in the control lung proteome. Annexin A2 (ANXA2) has originally been described as an endothelial cell-surface receptor for plasminogen and tissue-type plasminogen activator (tPA), thereby acting as a positive modulator in the fibrinolytic cascade.71 Accordingly, ANXA2 deficiency is associated with a functionally significant hypofibrinolytic phenotype. This has been reported in studies of homozygous ANXA2(-/-) knockout mice, which showed a marked decrease in tPA-dependent plasmin generation at the endothelial cell surface and incomplete clearance of injury-induced arterial thrombi, as well as displayed deposition of fibrin in the microvasculature, and also within pulmonary alveolar capillaries and renal glomeruli in kidneys.71 Moreover, ANXA2 has been implicated in surfactant secretion in the AECII by allowing fusion of lamellar bodies with the cell membrane through the membrane-located ANXA2 heterotetramer.21-23 It is currently unknown if the evident down-regulation of ANXA2 in alveolar epithelium of IPF lungs observed herein may partially explain the marked abnormalities of the alveolar surfactant system that have been previously described for IPF patients.72,73 Finally, ANXA2 seems to play a role in regulation of the actin cytoskeleton and has been implicated in motility and cell migration. Accordingly, invasive neoplasms such as ovarian and renal cell carcinoma show increased ANXA2 expression.74,75 Additionally, in an in vitro model of wound healing, it has been recently reported that migrating epithelial cells revealed increased expression of ANXA2, and RNAi mediated knockdown of ANXA2 expression resulted in a significantly reduced epithelial cell spreading and wound closure.76 In line with such notion, expression of ANXA2 was observed in “regenerative” bronchiolar basal cells overlying areas of active fibrosis, as well as in multilayered 2200


Journal of Proteome Research basal cell sheets of “hyperplastic bronchioles” in IPF lungs, and seemed to be associated with bronchiolization of terminal airspaces in IPF. In contrast to ANXA2, the role and function of the anticoagulant protein annexin A3 (ANXA3) in the lung has yet not been disclosed. Suppression of ANXA3 expression by RNA interference in primary cultured rat hepatocytes has been reported to result in decreased DNA synthesis and significant inhibition of hepatocyte growth, indicating a crucial function for this annexin in cell proliferation.77 In line with such notion, ANXA3 is induced in proliferating hepatocytes after carbon tetrachloride-induced rat liver damage via an hepatocyte growth factor (HGF)-mediated pathway.78 Increased expression of ANXA3 has also been observed in approximately two-thirds of colorectal tumors,79 and has been more recently reported as a novel biomarker for lymph node metastasis (LNM) in lung adenocarcinoma.80 In our study, significant down-regulated expression of ANXA3 was encountered in IPF lung tissues in comparison to controls by comparative proteome analysis and immunoblotting. Since we observed ANXA3 expression in AECII and bronchial epithelial cells of IPF- and control lungs, we suggest that ANXA3 may be involved in proliferation and growth of epithelial cells, but may be down-regulated in IPF due to increased apoptosis of alveolar epithelial cells.5,11,15 Other interesting, down-regulated proteins in IPF possibly reflecting epithelial cell injury in this disease were the structural protein Moesin (MOES) and the multifunctional major vault protein (MVP) (Table 2). Moesin, the expression of which is restricted to the alveolar epithelium of the distal lung, has been recently suggested to play a key role in the preservation of alveolar structure and lung homeostasis.81 This notion is supported by a study about heterozygous moesin deficient mice, which imposed with marked enlargement of the distal air space and, when treated with bleomycin, developed exaggerated lung injury and fibrosis and reduced survival as compared to the wildtype mice.81 Thus, the observed moesin deficiency in IPF lung tissues may add to the extensive fibrotic reaction in IPF. MVP, a unique and evolutionary highly conserved, also called “lung resistance-related protein”, is the main component of ubiquitous, large cellular ribonucleoprotein particles termed vaults, which are implicated in the regulation of several cellular processes including transport mechanisms, signal transduction, drug resistance and immune responses.82 Because of its functions, MVP is highly expressed in lung epithelial cells with secretory and excretory functions,82 such as the AECII and bronchial cells. In the airway epithelium, MVP has been described to be rapidly recruited to lipid rafts when lung epithelial cells were infected with Pseudomonas aeruginosa, and appeared to be essential for raft recruitment and optimal epithelial cell internalization and clearance of this pathogen.83 MVP represents a critical mediator of host resistance to lung infection. Down-regulation of MVP in UIP lungs may thus offer as one explanation for the increased susceptibility of IPF patients toward infection.84 Finally, our study indicated significant depletion of three crucial antioxidant enzymes, VAT-1 (an oxidoreductase), catalase and PRDX2 in IPF lung tissues. These enzymatic antioxidants have been described to be predominantly expressed in bronchial and alveolar epithelial cells.85-87 The lack of these antioxidants indicated the described redox imbalance in IPF lungs88 which presumably contributes to the loss of (alveolar) epithelial integrity and function, and thus to progression of this fatal lung disease.

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In summary, the protein changes presented here were obtained by analysis of subpleural lung tissue from the lower lobe of IPF patients being transplanted. Importantly, we have expanded our analysis and determined additionally the expression levels of the ER stress marker proteins p50ATF6 and ATF4, as well as of two of the identified factors, namely, DDB1 and Hsp27, in lung samples originating from the pleural as well as hilar part of the upper and middle regions of three explanted IPF lungs, in comparison to the Control group. We observed that ER stressinduction as well as the changes in protein expression of these two identified factors, as obtained by comparative proteome analysis of subpleural lung tissue, were virtually identical in all other samples from the very same lung (see Supplementary Figure SIX). These results indicate that the proteomic signature was not restricted to the most concerned parts of IPF lungs, but may represent an UIP typical change, as we were also able to observe an UIP pattern in all samples regardless of origin. Bearing in mind the differences in age between the IPF and the donor population of our study, we also analyzed the expression of some of the markers studied herein (p50ATF6, ATF4, Grp78, Hsp90R, DDB1 and catalase) in dependency of age and did not observe any meaningful dependency between age and expression level within IPF subjects as well as donor lungs (see Supplemental Figure SX). Methodological Considerations

Lung proteome profiling is challenging, because of interference with high-abundance proteins such as serum albumin, immunoglobulins (IgGs) and transferrin, which are synthesized intracellularly in lung tissue. In particular, serum albumin is the very dominant protein in lung homogenates. Although prefractionation techniques such as depletion of serum albumin and IgGs may be useful procedures in proteome profiling studies, they may introduce bias as well. We did not deplete highabundance proteins, because prefractionation increases the risk of depletion of low-abundance proteins, as a result of their multiple protein-protein interactions with albumin, IgGs and transferrin. Besides, among the up-regulated proteins in IPF were serum albumin, transferrin and some IgGs (Table 1), indicating presumably secondary inflammatory reactions possibly due to respiratory infections in these IPF patients. Some proteins may have also escaped detection during our preanalytical procedures; this holds true especially for matrix proteins, mainly collagens. Although we observed collagen deposition by use of trichrome staining in every IPF lung, we could not detect up-regulation of collagen type I or III. It has, however, to be kept in mind that, due to the compact helix structure and fibrillike organization of collagen type I, it is very difficult to extract collagen from fibrotic lung tissue, even with use of our lysis buffer (containing two detergents) and mortar and pestle in combination with liquid nitrogen. One collagen type I triple helix consists of one alpha2 chain (129 kDa) and two alpha1 chains (139 kDa each). These chains are cross-linked, and these cross-links cannot be broken, even after reduction with DTT and SDS and boiling for SDS-PAGE. Also, if isolated from lung tissue, one would always have the dimeric (about 270 kDa) and trimeric form (about 400 kDa) of collagen type I. Consequently, it is hard to see the size of these large fragments on SDS-PAGE because the separation capacity of the gels used in our study as second dimension (10%, 12.5% and 15%) is not good enough in this range. So, in summary, we did not really expect to observe an increase in collagen in our study. 2201


Journal of Proteome Research Thus, this mentioned issue represents one limitation with respect to a transcriptome study which allows the analysis of the extracellular matrix protein/collagen protein profile of fibrotic lungs in an optimal way. In line with this notion, various transcriptome studies of IPF lungs revealed up-regulation of COL1A1 and COL1A2 as well as of many other collagen genes (COL3A1, COL5A2, COL6A3, COL14A1, COL18A1).12,14,15,89,90 It may also be asked about the value of proteomic versus transcriptomic studies in IPF. Our proteomic approach confirms some signatures recently observed via gene expression profiling in IPF lungs, such as aberrant epithelial proliferation and myofibroblast differentiation.12,14,15,90 In contrast, the herein described signature of ER stress has not been detected in these transcriptomic studies, maybe due to low transcript levels and the activation process of the ER stress components, which may not be reflected by the microarray approach. Limitations of traditional proteomics using 2-DE in respect to gene expression profiling include lower number of differentially regulated proteins obtained, which will be always higher in a gene expression array. Nevertheless, proteomics offers distinct advantages which cannot be fulfilled by transcriptomics: First, the level of transcription of a gene gives only a rough estimate of its expression on protein level. It is now known that mRNA is not always translated into protein:91,92 A mRNA produced in abundance may be degraded rapidly or translated inefficiently, resulting in a small amount of protein. Second, many transcripts give rise to more than one protein, through alternative splicing or through alternative post-translational modifications, such as proteolytical processing, phosphorylation, glycosylation, sumoylation, ubiquitination, and so on; and this is not reflected on transcriptomic level. Finally, transcriptomics cannot povide information about the protein degradation rate and half-life of proteins in a current physiological state of an organism. In summary, these issues might be the reason transcriptome profiles were found not to correlate with protein expression profiles.93,94 This also means that both approaches are complementary to each other and are both necessary to identify key regulatory molecules and to dissect the gene and protein networks that underlie disease.

’ CONCLUDING REMARKS In conclusion, we demonstrated for the first time a comparative proteome analysis of subpleural lung tissue from patients with sporadic IPF, with explanted donor lungs serving as controls. The underlying signature obtained from the IPF proteome is that of a chronic epithelial cell stress-response dominated by ER stress, resulting in epithelial instability and cell death, paralleled and confirmed by signatures of enhanced infection susceptibility and deterioration of alveolar structure. Additionally, signatures of exaggerated bronchiolar cell proliferation and mobility, fibroblast activation and matrix deposition were evident. Our results suggest that central molecular events in the pathogenesis of IPF are localized to the alveolar epithelium, particularly the type-II alveolar epithelial cell, and provide a list of novel targets that may be addressed in future studies. ’ ASSOCIATED CONTENT

bS

Supporting Information Additional Materials and Methods include detailed information about the methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*Andreas Guenther, M.D., University of Giessen Lung Center (UGLC), Department of Internal Medicine II, Klinikstrasse 36, 35392 Giessen, Germany. E-mail: Andreas.Guenther@innere. med.uni-giessen.de. Tel: þ49 (641) 99 42515. Fax: þ49 (641) 99 42508.

’ ACKNOWLEDGMENT We thank Michael Dreisbach, Christina Schmidt and HansG€unter Welker for their great technical assistance. We thank Dr. G€unter Lochnit for his valuable advice and discussion. This work has been funded by grants from the Deutsche Forschungsgemeinschaft (Clinical Research Group 118 “Lung Fibrosis” [DFG, KFO 118] and Excellence Cluster “Cardiopulmonary System”) as well as from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no. HEALTH-F2-2007-202224 eurIPFnet. None of the authors has any conflicts of interest. ’ REFERENCES (1) American Thoracic Society. American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. This joint statement of the American Thoracic Society (ATS), and the European Respiratory Society (ERS) was adopted by the ATS board of directors, June 2001 and by the ERS Executive Committee, June 2001. Am. J. Respir. Crit. Care Med. 2002, 165 (2), 277–304. (2) Coultas, D. B.; Zumwalt, R. E.; Black, W. C.; Sobonya, R. E. The epidemiology of interstitial lung diseases. Am. J. Respir. Crit. Care Med. 1994, 150 (4), 967–72. (3) King, T. E., Jr.; Schwarz, M. I.; Brown, K.; Tooze, J. A.; Colby, T. V.; Waldron, J. A., Jr.; Flint, A.; Thurlbeck, W.; Cherniack, R. M. Idiopathic pulmonary fibrosis: relationship between histopathologic features and mortality. Am. J. Respir. Crit. Care Med. 2001, 164 (6), 1025–32. (4) Katzenstein, A. L.; Myers, J. L. Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification. Am. J. Respir. Crit. Care Med. 1998, 157 (4 Pt. 1), 1301–15. (5) Uhal, B. D.; Joshi, I.; Hughes, W. F.; Ramos, C.; Pardo, A.; Selman, M. Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung. Am. J. Physiol. 1998, 275 (6 Pt. 1), L1192–9. (6) Selman, M.; Pardo, A. Idiopathic pulmonary fibrosis: an epithelial/fibroblastic cross-talk disorder. Respir. Res. 2002, 3, 3. (7) Nogee, L. M.; Dunbar, A. E., III; Wert, S. E.; Askin, F.; Hamvas, A.; Whitsett, J. A. A mutation in the surfactant protein C gene associated with familial interstitial lung disease. N. Engl. J. Med. 2001, 344 (8), 573–9. (8) Thomas, A. Q.; Lane, K.; Phillips, J., III; Prince, M.; Markin, C.; Speer, M.; Schwartz, D. A.; Gaddipati, R.; Marney, A.; Johnson, J.; Roberts, R.; Haines, J.; Stahlman, M.; Loyd, J. E. Heterozygosity for a surfactant protein C gene mutation associated with usual interstitial pneumonitis and cellular nonspecific interstitial pneumonitis in one kindred. Am. J. Respir. Crit. Care Med. 2002, 165 (9), 1322–8. (9) Wang, W. J.; Mulugeta, S.; Russo, S. J.; Beers, M. F. Deletion of exon 4 from human surfactant protein C results in aggresome formation and generation of a dominant negative. J. Cell Sci. 2003, 116 (Pt. 4), 683–92. (10) Wang, Y.; Kuan, P. J.; Xing, C.; Cronkhite, J. T.; Torres, F.; Rosenblatt, R. L.; DiMaio, J. M.; Kinch, L. N.; Grishin, N. V.; Garcia, C. K. Genetic defects in surfactant protein A2 are associated with pulmonary fibrosis and lung cancer. Am. J. Hum. Genet. 2009, 84 (1), 52–9. 2202


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