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Clinical proteomics in lung diseases
ARTICLE IN PRESS
Pathology – Research and Practice 200 (2004) 147–154
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ORIGINAL ARTICLE
Clinical proteomics in lung diseases d . Nadine Waldburga, Thilo K.ahneb, Anita Reisenauerc, Christoph Rocken , a c, . Tobias Welte , Frank Buhling * a
Division of Pneumology and Critical Care, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany Institute of Experimental Internal Medicine, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany c Institute of Immunology, Otto-von-Guericke-University Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany d Institute of Pathology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany b
Received 23 December 2003; accepted 2 February 2004
Abstract Proteomics is a relatively new approach for understanding the pathology and pathogenesis of various diseases. It has also been used for characterizing the modifications in protein expression during the development of interstitial lung diseases, in lung tumors, or following exposure to exogenous stress signals. We compared the protein composition of primary human lung fibroblasts derived from patients with lung fibrosis and control fibroblasts of unaffected lung tissues. We found a predominant modulation in proteins related to the cytoskeleton, including decreased expression of vimentin and lamin A/C, and increased expression of moesin. Furthermore, we observed lower levels of components of the antioxidative system, such as omega class glutathione S-transferase and an up-regulation of an intracellular chloride channel. In fibroblasts obtained from fibrotic lungs, the expression of a major histocompatibility complex class I C was decreased, and so was the expression of tripeptidyl-peptidase-I-precursor, a collagen-degrading exopeptidase. Our results and the studies reviewed in this paper represent just the beginning of detailed studies that should unravel the relevance and the functional consequences of differential protein expressions in the diseased lung. r 2004 Elsevier GmbH. All rights reserved. Keywords: Lung; Proteomics; Disease
Introduction A new fundamental concept called proteome (PROTEin complement to a genOME) has recently emerged that should help to understand the biochemical and physiological mechanisms of complex diseases at the functional and molecular levels. This new discipline, proteomics, complements physical genomic research. Proteomics can be defined as the qualitative and quantitative comparison of proteomes under different conditions to further unravel biological processes. *Corresponding author. Tel.: 391-6713311; fax: 391-67190466. E-mail address: [email protected] (F. Buhling). . 0344-0338/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2004.02.006
Although the genome of an organism is the same in all somatic cells, the proteome is diverse in different tissues, cell types or under different physiological and pathological conditions [37]. In contrast to the study of mRNA expression (transcriptomics), the additional advantage of proteomics is the detection of post-translational modifications, which are important for the subcellular localization and function of proteins. Pathologic processes are often associated with changes in protein expression, modification, and cellular sorting. Proteomics is a research area that helps to understand the pathology of inflammation, malignant tumors, and fibrosis [1], also including that affecting the lung. In proteomics, proteins are commonly separated by two-dimensional gel electrophoresis (2D-PAGE) and
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visualized by silver or fluorescence staining. The protein spots of interest are excised and identified by mass spectrometry and database search [25] (for more detailed information, see further articles in this issue). A number of studies have analyzed changes in the proteome of tissues or isolated cells obtained from patients with lung diseases. Most of these studies have focused on interstitial lung diseases.
Proteomics in interstitial lung diseases Homeostasis of deposition and recycling of extracellular matrix (ECM) proteins are of general importance for the physiologic function of almost all organs, and particularly for that of the lung. The lung provides an excellent example of the hazardous effects of two opposing extremes: predominant matrix degradation leading to emphysema, and predominant matrix deposition leading to lung fibrosis [19,39,41]. Fibrosis of the lung is a final common pathway of many different lung diseases, such as idiopathic interstitial pneumonitis (idiopathic pulmonary fibrosis—IPF) and granulomatous diseases (sarcoidosis) [8,18,19]. Fibrotic areas are also commonly found in patients suffering from chronic obstructive pulmonary disease [31]; therefore, lung fibrosis is an urgent clinical problem. Fibrosis is characterized by the accumulation of matrix proteins produced by activated fibroblasts [19]. Depending on the localization and amount of fibrosis, organ integrity and function can be seriously disrupted [12]. A continuous ECM turnover exists under physiological conditions [17]. The dynamic equilibrium between synthesis and degradation maintains the physiological balance and is tightly controlled by de novo synthesis and deposition of ECM, and proteolytic degradation of existing ECM [6]. Under pathological conditions, the balance between ECM production and degradation may deteriorate, resulting in an increased amount of ECM, which then progressively impairs organ function. Whereas the signals leading to an increased secretion of ECM proteins are well understood [47,54], the molecular mechanisms limiting matrix deposition are poorly defined. Various attempts have been made to understand the underlying diseases and molecular pathways leading to lung fibrosis by using proteomics studies. However, critical for the ongoing analyses is the choice and preparation of the material under investigation. Samples obtained from the lung are either bronchoalveolar lavage fluid (BALF), tissue extracts, pleural effusions or isolated cells, and each of these starting materials cause specific problems. A number of studies used BALF. To analyze the protein composition of BALF, it was necessary to overcome some methodological problems, especially
when 2D-PAGE is to be applied. The high salt concentration of BALF has to be lowered, which results from the common use of phosphate buffered saline, for the lavage. Different approaches, for example, dialysis against water, gel filtration, trichloracetic acid precipitation, ultrafiltration and ion-exchange chromatography, may help to reduce the high salt concentration [21,22,24,38,48]. Another problem lies in the presence of large amounts of certain proteins, such as albumin, transferrin, alpha1-antitrypsin, and immunoglobulin A [22], which hinders the detection of low-abundance proteins. Nonlinear immobilized pH gradient (IPG) strips and, more recently, narrow-range IPG strips provide a significantly higher resolution and thus allow for the detection of more protein spots [24,49]. This methodological progress in the separation of BALF proteins led to the detection of more than 1000 different protein spots. Wattiez et al. [33] compiled a database containing all proteins identified so far: see http:// w3.umh.ac.be/Bbiochim/proteomic.htm. The same group has studied the protein content and composition of BALF obtained from healthy individuals, patients with sarcoidosis, IPF, or hypersensitivity pneumonitis (HP). Interestingly enough, they found a significantly decreased concentration of surfactant protein A (SP-A) in patients suffering from IPF and HP. SP-A is the most abundant surfactant protein secreted by lung epithelial cells. It has important functions in host defence [16]. Therefore, this finding may explain the increased susceptibility of patients with IPF to infectious agents [49]. In addition, they found significantly increased amounts of acidic low-molecularweight proteins, including cathepsin D, which until then had been unknown to be present in BALF. This ubiquitously expressed aspartic protease is normally localized in the endosomal and lysosomal compartments of macrophages, in bronchial epithelial cells and, to a lesser extent, in type-I pneumocytes. We and others have previously shown that there is no cathepsin D in type-II pneumocytes [5]. Thus, the high concentration of cathepsin D in BALF could result from cell lysis or increased secretion. Magi et al. characterized the protein composition of BALF derived from patients suffering from two different forms of interstitial lung diseases: sarcoidosis and IPF. Thirty-eight previously unknown proteins or protein fragments were found, and all showed statistically significant variations between sarcoidosis and IPF. In sarcoidosis, BALF contains significantly increased amounts of several plasma proteins, mostly with a molecular weight above 40 kDa. These results are in accordance with those of Wattiez et al. [49], and may be due to altered alveolar membrane integrity during alveolitis in sarcoidosis. In IPF, BALF contains increased amounts of low-molecular-weight proteins, such as MIF, calgranulin A and B, which are mostly
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secretory products of local cells or obtained by cell damage [23,26,49]. To gain a deeper knowledge about the pathophysiology of lung fibrosis, we applied proteomics to samples obtained from patients with and without lung fibrosis. In contrast to the studies described above, we compared isolated primary cell cultures of human fibroblasts from patients with lung fibrosis and fibroblasts derived from unaffected lung tissues of control patients, who underwent lobectomy for lung cancer. Cell lysates were separated by 2D-PAGE, and protein spots with different expression levels were excised and analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) (Fig. 1). We found more than 60 differentially expressed protein spots. Among them, proteins were related to the cytoskeleton, the antioxidative system, proteins of the major histocompatibility complex, and a protease involved in collagen degradation (Table 1). We found that altered expression of cytoskeletal proteins predominated. For example, the expression of vimentin was significantly reduced in fibroblasts of fibrotic lungs. This finding contrasts with that of . et al. [28], who used vimentin as a houseMalmstrom keeping protein. Vimentin is a filament polypeptide present in mesenchymal cells, including fibroblasts [20]. It is responsible for cell elongation, cell attachment, and signal transduction [34]. Lung fibrosis is associated with an increased apoptosis of mesenchymal cells and leads to the production of antivimentin antibodies [15]. This and the differentiation of fibroblasts during the pathogenesis of lung fibrosis may explain the lower vimentin levels in our samples. Furthermore, fibroblasts of fibrotic lungs expressed significantly less lamin A/C in our series. The lamins are components of the nuclear lamina that are organized in a fibrous layer on the nucleoplasmic side of the inner nuclear membrane. This layer was suggested to provide a framework for the nuclear envelope and to interact with chromatin. Lamins are expressed during mouse organogenesis [46]. Primary lung cancers also show altered expression of lamins A/C [30]. Moesin belongs to the ERM (ezrin/radixin/moesin) protein family that is proposed to function as a membrane-cytoskeletal linker in non-neoplastic alveolar and bronchiolar epithelial cells, and vascular endothelial cells. We found that its expression was up-regulated in fibroblasts obtained from fibrotic lungs. The ERM proteins bind to actin filaments at their carboxyterminal domain and are involved in a variety of cellular functions, such as cell adhesion, migration, and the organization of cell surface structures. Immunohistochemical studies confirmed that cancer cells express very little moesin [44]. Differentiation of cells during fibrosis is associated with modifications of the cellular metabolism. Accord-
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ingly, we found differences in proteins associated with antioxidative protection, collagen metabolism and antigen presentation. For example, we observed a lower level of glutathione S-transferase (GST), especially of the human isoenzyme GSTO (omega class GST), which plays a significant role in the cellular response to oxidative stress [45,53]. GSTs have been shown to be overexpressed in a wide variety of tumors and might be involved in asthma and some neurodegenerative diseases, such as multiple sclerosis [2,29,43]. Interestingly, recent studies have predicted structural and possibly functional similarities to a family of intracellular chloride channels (CLIC) [11]; GSTO may thus function in intracellular ion transport. In our study, CLIC was found to be up-regulated. Therefore, one might speculate that the up-regulation of CLIC compensates the down-regulation of GSTO. Fibrosis was also associated with decreased expression of a major histocompatibility complex class I C protein. This may protect fibroblasts from cell lysis mediated by cytotoxic T cells, when it comes to the presentation of altered self-proteins. By contrast, it makes fibroblasts more susceptible to cell lysis by natural killer cells. Activation of natural killer cells and secretion of interferon-gamma by natural killer cells may activate macrophages and amplify the inflammatory reaction in the lung [7]. The expression of tripeptidyl-peptidase-I-precursor (TPP-1) was also reduced in lung fibrosis. TPP-1 is a collagen-degrading exopeptidase previously described in lung tissues [9,10]. Under physiological conditions, the majority of collagen produced by fibroblasts is degraded intracellularly. This process is catalyzed by cytosolic proteases, e.g TPP-1. Decreased expression of TPP-1 in fibroblasts may contribute to increased secretion of collagen, thereby enhancing matrix deposition by fibroblasts. . Similar to our study, Westerngreen and Malmstrom [28] used lung fibroblasts for proteomics studies. To understand the pathogenesis of peribronchial fibrosis in asthma, they used primary cell cultures of human fibroblasts obtained from central bronchial biopsies of healthy volunteers and patients suffering from asthma. Human fetal lung fibroblasts (HFL-1) were used for comparison. The morphology and the proliferative response of the various fibroblasts to epidermal growth factor were similar. From this finding, the authors concluded that HFL-1 cells represent a suitable model for studies of the effects of transforming growth factorb1 (TGF-b1) on the protein profile of lung fibroblasts. Fibroblasts obtained from asthmatic patients were surrounded by more connective tissue characteristic of an increased matrix production. During inflammation, the amount of matrix molecules in connective tissue is higher, making it easier for inflammatory cells to invade the tissue. The composition of proteoglycans secreted
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Fig. 1. Proteome analysis of fibroblasts derived from fibrotic (A,C) and non-fibrotic lung tissues (B,D). Proteins are differentially expressed in fibroblasts (red—up-regulation, green—down-regulation in fibroblasts obtained from fibrotic lung tissue). Fibroblasts were harvested and washed in PBS, lysed at a concentration of 1 Mio cells/100 ml, and diluted in rehydration buffer. These samples were used to rehydrate 24 cm IPG-strips, pH 4.5–5.5 or 5.5–6.7. After isoelectric focusing on a Multiphor-Electrophoresis apparatus (Amersham Biosciences, Freiburg), the strips were equilibrated in 50 mM TRIS/HCl pH 8.8 and transferred to the top of SDSgradient gels (10%–16%). Gels for further comparison were run simultaneously in a Hoefer IsoDalt electrophoresis unit. After running the second dimension, the gels were silver-stained according to the modified method of Blum et al. [36]. Protein spots of interest were excised from the gels after computer-aided comparison (Image Master Software, Amersham). In-gel digestion was performed in an adapted manner according to Shevchenko et al. [40]. The peptides were extracted from the gel by repeated addition of NH4HCO3 and acetonitrile. All extracts were pooled, dried in a vacuum centrifuge, redissolved in water/TFA, purified on a 200 nl reversed-phase (C18)-nanocolumn, and subsequently co-crystallized with 8-cyano-4-hydroxycinnamic acid (20 mg/ml) in 70% acetonitrile on a SCOUT 384–600 mm anchor-Target. Mass spectrometry was performed on a MALDI-TOF-MS (Reflex III, Bruker Daltonics, Germany) in reflector mode with external calibration. Annotation of the tryptic fragments was done using the XMASS and BioTools 2.0 software (Bruker Daltonics, Germany). For database search, the ProFound-Software (http://www.prowl.rockefeller.edu) was used.
into the extracellular space was further characterized by ion exchange chromatography and SDS-PAGE. An increased amount of perlecan, a proteoglycan rich in heparan sulfate side chains, was found in the fibrotic ECM. This could be important for binding cytokines and proteases, and may have direct effects on fibroblasts [13,27]. Furthermore, the amount of the constitutive ECM proteins biglycan and collagen was increased, whereas the amount of decorin was decreased. Decorin is considered as one component of the collagen fibrils.
Typically, increased amounts of collagen and decreased amounts of decorin lead to the formation of unorganized collagen fibrils [32]. The subepithelial fibrosis seen in asthmatic patients is proposed to be promoted by TGF-b1, which induces the transformation of fibroblasts to myofibroblasts [14]. Furthermore, TGF-b1 induces the expression of several connective tissue components, for example, collagens, fibronectin, and hyaluronan. Studying the influence of TGF-b1 on the protein expression profile of lung fibroblasts, HFL-1
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Table 1. Differences in the protein expression between fibroblasts obtained from fibrotic and non-fibrotic lung tissue Protein
Database accession numbera
Expressionb
Function
Vimentin
XM 042950, M14144
Decreased
Lamin A/C
NM 005572
Decreased
Moesin
NM 002444
Increased
Glutathion-S-transferase omega
NM 004832
Decreased
Chloride intracellular channel I Major histocompatibility complex, class I, C Tripeptidyl aminopeptidase I
NM 001288 XM 044285
Increased Decreased
Component of the cellular cytoskeleton Component of the cellular cytoskeleton Component of the cellular cytoskeleton Anti-oxidative protection, intracellular ion transport Intracellular ion transport Antigen presentation
O14773
Decreased
a b
Intracellular collagen degradation
According to the NCBI database. Protein expression in fibroblasts obtained from fibrotic and non-fibrotic lung tissue was compared.
cells were treated with TGF-b1 prior to 2D-PAGE and proteome analysis [14]. This resulted in the up-regulation of actin and three different isoforms of tropomyosin by a factor of three–five for actin and six–ten for topomyosin [51]. These proteins are important for the formation of contractile fibers of myofibroblasts. In comparison, vimentin and dynacin were not differentially expressed. a-Enolase, myosin, and tubulin were equally expressed in the stimulated and unstimulated samples [28].
Proteomics in lung cancer research Lung cancer is a challenging clinical problem worldwide [35]. The incidence of lung cancer and lung cancerassociated deaths is increasing in men and women. The current therapies do not improve patient outcome significantly. Therefore, a better understanding of tumor biology, as well as the search for better therapies, is one of the foremost research targets. Apart from knowledgebased approaches that investigate the function and expression patterns of known cellular components, several techniques, including proteomics, have been developed over the past few years, allowing for the expression profiling of large amounts of cellular proteins. These approaches do not depend on the prediction of the functional importance of individual proteins, but permit the detection of unknown changes in the protein composition. The molecular mechanisms behind these changes can then be studied in more detail. Proteomics studies can be used for the classification of surgically resected lung tumors in meaningful groups and prognostic subsets. For example, a number of
studies have been performed using cDNA microarrays for profiling mRNA expression. The groups of Beer and Bhattaracharjee used cDNA arrays to correlate the gene expression of adenocarcinomas with the survival of patients. In addition, it was possible to discriminate between primary lung carcinomas and metastases of extrapulmonary origin [3,4]. The major drawback of these studies, with respect to tumor biology, is the poor correlation between gene transcription, protein expression, and protein function. Protein expression is often regulated post-transcriptionally. The post-translational modification and metabolism of proteins, including proteolytic processing, phosphorylation, and glycosylation, are important in determining protein function. These processes are not covered by transcriptomics. Yanagisawa et al. [42] used the newly developed imaging mass spectrometry for expression profiling of lung tumor samples at the protein level. Regions of single frozen tissue sections (1 mm3) from surgically resected tissues were used for direct mass spectrometric analyses and MALDI-TOF, without protein extraction and separation by 2D-PAGE. Finally, mass spectra of more than 1600 different protein species were detected in lung tumor samples. An artificial intelligence software was developed to handle the large amounts of data points, which were obtained from the analyses of 50 tissue samples. Finally, a class prediction model was developed that correctly classified different lung cancer histologies, distinguished primary tumors from metastases, allowed for the allocation of patients into good or poor prognostic groups, and predicted the risk of nodal involvement [52]. In a second step, they selected significant proteins based on statistical tests, with known prognostic features of these patients, and determined their identity. For example, they found that
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the small ubiquitin-like protein modifier-2 and ubiquitin were up-regulated in NSCLC, which emphasizes the importance of post-translational protein modification in tumorigenesis.
Proteomics in animal models related to lung diseases Proteomics studies can also be applied to animal models. This provides a specific opportunity to identify proteins involved in particular metabolic and pathophysiologic pathways. Mouse models are used to study disease processes in homogenous genomic backgrounds. Wattiez et al. [50] used the BALF of O3-sensitive C57Bl/6 and the O3-resistant C3H/HeJ mice for the identification of proteins involved in the regulation of susceptibility to oxidative stress. They found significant strain-specific differences in the expression of two isoforms of the antioxidant protein 2 (AOP2). C3H/ HeJ mice expressed only AOP2a, whereas AOP2b was found only in C57Bl/6 mice. In addition, the levels of the anti-inflammatory Clara cell protein 16 (CC16) were higher in the resistant C3H/HeJ mice. Thus, AOP2 and CC16 might participate in pathways protecting the respiratory tract from oxidative injury. Ray et al. [37] applied another approach to studying the mechanisms of oxidative lung injury in mice. Proteins present in BALF of mice exposed to room air were compared with those exposed to hyperoxia (100% oxygen). Both the amounts of thioether S-methyltransferase and the amounts of 1-cysteine peroxiredoxin were decreased after hyperoxia, indicating their involvement in the pathogenesis of oxidative stress. Thioether Smethyltransferase is usually abundantly expressed in murine lungs and is important for methylation of thioethers to more water-soluble ions suitable for urinary excretion. 1-cysteine peroxiredoxin, also abundantly expressed in lung tissue, is an antioxidant. Genetically modified mice are of particular interest for studying the impact of the overexpression or lack of single proteins on the proteome of cells and biofluids. In the future, interesting results can be expected from proteomics of lungs obtained from transgenic and knockout mice [37].
Conclusions Different groups and our studies have shown that proteomics is an extremely valuable tool that contributes to the search for new diagnostic and therapeutic targets of chronic lung diseases and lung cancer. However, these studies represent just the beginning. Future studies will unravel the relevance and functional
consequences of the changes in protein concentration. The combination of proteomics of lung tissues and BALF with functional in vitro studies, including the proteome analysis of isolated cells after defined treatments and the use of defined and relevant animal models, will allow us to gain a deeper insight into the signalling cascades causing these changes.
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www.elsevier.de/prp
ORIGINAL ARTICLE
Clinical proteomics in lung diseases d . Nadine Waldburga, Thilo K.ahneb, Anita Reisenauerc, Christoph Rocken , a c, . Tobias Welte , Frank Buhling * a
Division of Pneumology and Critical Care, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany Institute of Experimental Internal Medicine, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany c Institute of Immunology, Otto-von-Guericke-University Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany d Institute of Pathology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany b
Received 23 December 2003; accepted 2 February 2004
Abstract Proteomics is a relatively new approach for understanding the pathology and pathogenesis of various diseases. It has also been used for characterizing the modifications in protein expression during the development of interstitial lung diseases, in lung tumors, or following exposure to exogenous stress signals. We compared the protein composition of primary human lung fibroblasts derived from patients with lung fibrosis and control fibroblasts of unaffected lung tissues. We found a predominant modulation in proteins related to the cytoskeleton, including decreased expression of vimentin and lamin A/C, and increased expression of moesin. Furthermore, we observed lower levels of components of the antioxidative system, such as omega class glutathione S-transferase and an up-regulation of an intracellular chloride channel. In fibroblasts obtained from fibrotic lungs, the expression of a major histocompatibility complex class I C was decreased, and so was the expression of tripeptidyl-peptidase-I-precursor, a collagen-degrading exopeptidase. Our results and the studies reviewed in this paper represent just the beginning of detailed studies that should unravel the relevance and the functional consequences of differential protein expressions in the diseased lung. r 2004 Elsevier GmbH. All rights reserved. Keywords: Lung; Proteomics; Disease
Introduction A new fundamental concept called proteome (PROTEin complement to a genOME) has recently emerged that should help to understand the biochemical and physiological mechanisms of complex diseases at the functional and molecular levels. This new discipline, proteomics, complements physical genomic research. Proteomics can be defined as the qualitative and quantitative comparison of proteomes under different conditions to further unravel biological processes. *Corresponding author. Tel.: 391-6713311; fax: 391-67190466. E-mail address: [email protected] (F. Buhling). . 0344-0338/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2004.02.006
Although the genome of an organism is the same in all somatic cells, the proteome is diverse in different tissues, cell types or under different physiological and pathological conditions [37]. In contrast to the study of mRNA expression (transcriptomics), the additional advantage of proteomics is the detection of post-translational modifications, which are important for the subcellular localization and function of proteins. Pathologic processes are often associated with changes in protein expression, modification, and cellular sorting. Proteomics is a research area that helps to understand the pathology of inflammation, malignant tumors, and fibrosis [1], also including that affecting the lung. In proteomics, proteins are commonly separated by two-dimensional gel electrophoresis (2D-PAGE) and
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visualized by silver or fluorescence staining. The protein spots of interest are excised and identified by mass spectrometry and database search [25] (for more detailed information, see further articles in this issue). A number of studies have analyzed changes in the proteome of tissues or isolated cells obtained from patients with lung diseases. Most of these studies have focused on interstitial lung diseases.
Proteomics in interstitial lung diseases Homeostasis of deposition and recycling of extracellular matrix (ECM) proteins are of general importance for the physiologic function of almost all organs, and particularly for that of the lung. The lung provides an excellent example of the hazardous effects of two opposing extremes: predominant matrix degradation leading to emphysema, and predominant matrix deposition leading to lung fibrosis [19,39,41]. Fibrosis of the lung is a final common pathway of many different lung diseases, such as idiopathic interstitial pneumonitis (idiopathic pulmonary fibrosis—IPF) and granulomatous diseases (sarcoidosis) [8,18,19]. Fibrotic areas are also commonly found in patients suffering from chronic obstructive pulmonary disease [31]; therefore, lung fibrosis is an urgent clinical problem. Fibrosis is characterized by the accumulation of matrix proteins produced by activated fibroblasts [19]. Depending on the localization and amount of fibrosis, organ integrity and function can be seriously disrupted [12]. A continuous ECM turnover exists under physiological conditions [17]. The dynamic equilibrium between synthesis and degradation maintains the physiological balance and is tightly controlled by de novo synthesis and deposition of ECM, and proteolytic degradation of existing ECM [6]. Under pathological conditions, the balance between ECM production and degradation may deteriorate, resulting in an increased amount of ECM, which then progressively impairs organ function. Whereas the signals leading to an increased secretion of ECM proteins are well understood [47,54], the molecular mechanisms limiting matrix deposition are poorly defined. Various attempts have been made to understand the underlying diseases and molecular pathways leading to lung fibrosis by using proteomics studies. However, critical for the ongoing analyses is the choice and preparation of the material under investigation. Samples obtained from the lung are either bronchoalveolar lavage fluid (BALF), tissue extracts, pleural effusions or isolated cells, and each of these starting materials cause specific problems. A number of studies used BALF. To analyze the protein composition of BALF, it was necessary to overcome some methodological problems, especially
when 2D-PAGE is to be applied. The high salt concentration of BALF has to be lowered, which results from the common use of phosphate buffered saline, for the lavage. Different approaches, for example, dialysis against water, gel filtration, trichloracetic acid precipitation, ultrafiltration and ion-exchange chromatography, may help to reduce the high salt concentration [21,22,24,38,48]. Another problem lies in the presence of large amounts of certain proteins, such as albumin, transferrin, alpha1-antitrypsin, and immunoglobulin A [22], which hinders the detection of low-abundance proteins. Nonlinear immobilized pH gradient (IPG) strips and, more recently, narrow-range IPG strips provide a significantly higher resolution and thus allow for the detection of more protein spots [24,49]. This methodological progress in the separation of BALF proteins led to the detection of more than 1000 different protein spots. Wattiez et al. [33] compiled a database containing all proteins identified so far: see http:// w3.umh.ac.be/Bbiochim/proteomic.htm. The same group has studied the protein content and composition of BALF obtained from healthy individuals, patients with sarcoidosis, IPF, or hypersensitivity pneumonitis (HP). Interestingly enough, they found a significantly decreased concentration of surfactant protein A (SP-A) in patients suffering from IPF and HP. SP-A is the most abundant surfactant protein secreted by lung epithelial cells. It has important functions in host defence [16]. Therefore, this finding may explain the increased susceptibility of patients with IPF to infectious agents [49]. In addition, they found significantly increased amounts of acidic low-molecularweight proteins, including cathepsin D, which until then had been unknown to be present in BALF. This ubiquitously expressed aspartic protease is normally localized in the endosomal and lysosomal compartments of macrophages, in bronchial epithelial cells and, to a lesser extent, in type-I pneumocytes. We and others have previously shown that there is no cathepsin D in type-II pneumocytes [5]. Thus, the high concentration of cathepsin D in BALF could result from cell lysis or increased secretion. Magi et al. characterized the protein composition of BALF derived from patients suffering from two different forms of interstitial lung diseases: sarcoidosis and IPF. Thirty-eight previously unknown proteins or protein fragments were found, and all showed statistically significant variations between sarcoidosis and IPF. In sarcoidosis, BALF contains significantly increased amounts of several plasma proteins, mostly with a molecular weight above 40 kDa. These results are in accordance with those of Wattiez et al. [49], and may be due to altered alveolar membrane integrity during alveolitis in sarcoidosis. In IPF, BALF contains increased amounts of low-molecular-weight proteins, such as MIF, calgranulin A and B, which are mostly
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secretory products of local cells or obtained by cell damage [23,26,49]. To gain a deeper knowledge about the pathophysiology of lung fibrosis, we applied proteomics to samples obtained from patients with and without lung fibrosis. In contrast to the studies described above, we compared isolated primary cell cultures of human fibroblasts from patients with lung fibrosis and fibroblasts derived from unaffected lung tissues of control patients, who underwent lobectomy for lung cancer. Cell lysates were separated by 2D-PAGE, and protein spots with different expression levels were excised and analyzed using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) (Fig. 1). We found more than 60 differentially expressed protein spots. Among them, proteins were related to the cytoskeleton, the antioxidative system, proteins of the major histocompatibility complex, and a protease involved in collagen degradation (Table 1). We found that altered expression of cytoskeletal proteins predominated. For example, the expression of vimentin was significantly reduced in fibroblasts of fibrotic lungs. This finding contrasts with that of . et al. [28], who used vimentin as a houseMalmstrom keeping protein. Vimentin is a filament polypeptide present in mesenchymal cells, including fibroblasts [20]. It is responsible for cell elongation, cell attachment, and signal transduction [34]. Lung fibrosis is associated with an increased apoptosis of mesenchymal cells and leads to the production of antivimentin antibodies [15]. This and the differentiation of fibroblasts during the pathogenesis of lung fibrosis may explain the lower vimentin levels in our samples. Furthermore, fibroblasts of fibrotic lungs expressed significantly less lamin A/C in our series. The lamins are components of the nuclear lamina that are organized in a fibrous layer on the nucleoplasmic side of the inner nuclear membrane. This layer was suggested to provide a framework for the nuclear envelope and to interact with chromatin. Lamins are expressed during mouse organogenesis [46]. Primary lung cancers also show altered expression of lamins A/C [30]. Moesin belongs to the ERM (ezrin/radixin/moesin) protein family that is proposed to function as a membrane-cytoskeletal linker in non-neoplastic alveolar and bronchiolar epithelial cells, and vascular endothelial cells. We found that its expression was up-regulated in fibroblasts obtained from fibrotic lungs. The ERM proteins bind to actin filaments at their carboxyterminal domain and are involved in a variety of cellular functions, such as cell adhesion, migration, and the organization of cell surface structures. Immunohistochemical studies confirmed that cancer cells express very little moesin [44]. Differentiation of cells during fibrosis is associated with modifications of the cellular metabolism. Accord-
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ingly, we found differences in proteins associated with antioxidative protection, collagen metabolism and antigen presentation. For example, we observed a lower level of glutathione S-transferase (GST), especially of the human isoenzyme GSTO (omega class GST), which plays a significant role in the cellular response to oxidative stress [45,53]. GSTs have been shown to be overexpressed in a wide variety of tumors and might be involved in asthma and some neurodegenerative diseases, such as multiple sclerosis [2,29,43]. Interestingly, recent studies have predicted structural and possibly functional similarities to a family of intracellular chloride channels (CLIC) [11]; GSTO may thus function in intracellular ion transport. In our study, CLIC was found to be up-regulated. Therefore, one might speculate that the up-regulation of CLIC compensates the down-regulation of GSTO. Fibrosis was also associated with decreased expression of a major histocompatibility complex class I C protein. This may protect fibroblasts from cell lysis mediated by cytotoxic T cells, when it comes to the presentation of altered self-proteins. By contrast, it makes fibroblasts more susceptible to cell lysis by natural killer cells. Activation of natural killer cells and secretion of interferon-gamma by natural killer cells may activate macrophages and amplify the inflammatory reaction in the lung [7]. The expression of tripeptidyl-peptidase-I-precursor (TPP-1) was also reduced in lung fibrosis. TPP-1 is a collagen-degrading exopeptidase previously described in lung tissues [9,10]. Under physiological conditions, the majority of collagen produced by fibroblasts is degraded intracellularly. This process is catalyzed by cytosolic proteases, e.g TPP-1. Decreased expression of TPP-1 in fibroblasts may contribute to increased secretion of collagen, thereby enhancing matrix deposition by fibroblasts. . Similar to our study, Westerngreen and Malmstrom [28] used lung fibroblasts for proteomics studies. To understand the pathogenesis of peribronchial fibrosis in asthma, they used primary cell cultures of human fibroblasts obtained from central bronchial biopsies of healthy volunteers and patients suffering from asthma. Human fetal lung fibroblasts (HFL-1) were used for comparison. The morphology and the proliferative response of the various fibroblasts to epidermal growth factor were similar. From this finding, the authors concluded that HFL-1 cells represent a suitable model for studies of the effects of transforming growth factorb1 (TGF-b1) on the protein profile of lung fibroblasts. Fibroblasts obtained from asthmatic patients were surrounded by more connective tissue characteristic of an increased matrix production. During inflammation, the amount of matrix molecules in connective tissue is higher, making it easier for inflammatory cells to invade the tissue. The composition of proteoglycans secreted
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Fig. 1. Proteome analysis of fibroblasts derived from fibrotic (A,C) and non-fibrotic lung tissues (B,D). Proteins are differentially expressed in fibroblasts (red—up-regulation, green—down-regulation in fibroblasts obtained from fibrotic lung tissue). Fibroblasts were harvested and washed in PBS, lysed at a concentration of 1 Mio cells/100 ml, and diluted in rehydration buffer. These samples were used to rehydrate 24 cm IPG-strips, pH 4.5–5.5 or 5.5–6.7. After isoelectric focusing on a Multiphor-Electrophoresis apparatus (Amersham Biosciences, Freiburg), the strips were equilibrated in 50 mM TRIS/HCl pH 8.8 and transferred to the top of SDSgradient gels (10%–16%). Gels for further comparison were run simultaneously in a Hoefer IsoDalt electrophoresis unit. After running the second dimension, the gels were silver-stained according to the modified method of Blum et al. [36]. Protein spots of interest were excised from the gels after computer-aided comparison (Image Master Software, Amersham). In-gel digestion was performed in an adapted manner according to Shevchenko et al. [40]. The peptides were extracted from the gel by repeated addition of NH4HCO3 and acetonitrile. All extracts were pooled, dried in a vacuum centrifuge, redissolved in water/TFA, purified on a 200 nl reversed-phase (C18)-nanocolumn, and subsequently co-crystallized with 8-cyano-4-hydroxycinnamic acid (20 mg/ml) in 70% acetonitrile on a SCOUT 384–600 mm anchor-Target. Mass spectrometry was performed on a MALDI-TOF-MS (Reflex III, Bruker Daltonics, Germany) in reflector mode with external calibration. Annotation of the tryptic fragments was done using the XMASS and BioTools 2.0 software (Bruker Daltonics, Germany). For database search, the ProFound-Software (http://www.prowl.rockefeller.edu) was used.
into the extracellular space was further characterized by ion exchange chromatography and SDS-PAGE. An increased amount of perlecan, a proteoglycan rich in heparan sulfate side chains, was found in the fibrotic ECM. This could be important for binding cytokines and proteases, and may have direct effects on fibroblasts [13,27]. Furthermore, the amount of the constitutive ECM proteins biglycan and collagen was increased, whereas the amount of decorin was decreased. Decorin is considered as one component of the collagen fibrils.
Typically, increased amounts of collagen and decreased amounts of decorin lead to the formation of unorganized collagen fibrils [32]. The subepithelial fibrosis seen in asthmatic patients is proposed to be promoted by TGF-b1, which induces the transformation of fibroblasts to myofibroblasts [14]. Furthermore, TGF-b1 induces the expression of several connective tissue components, for example, collagens, fibronectin, and hyaluronan. Studying the influence of TGF-b1 on the protein expression profile of lung fibroblasts, HFL-1
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Table 1. Differences in the protein expression between fibroblasts obtained from fibrotic and non-fibrotic lung tissue Protein
Database accession numbera
Expressionb
Function
Vimentin
XM 042950, M14144
Decreased
Lamin A/C
NM 005572
Decreased
Moesin
NM 002444
Increased
Glutathion-S-transferase omega
NM 004832
Decreased
Chloride intracellular channel I Major histocompatibility complex, class I, C Tripeptidyl aminopeptidase I
NM 001288 XM 044285
Increased Decreased
Component of the cellular cytoskeleton Component of the cellular cytoskeleton Component of the cellular cytoskeleton Anti-oxidative protection, intracellular ion transport Intracellular ion transport Antigen presentation
O14773
Decreased
a b
Intracellular collagen degradation
According to the NCBI database. Protein expression in fibroblasts obtained from fibrotic and non-fibrotic lung tissue was compared.
cells were treated with TGF-b1 prior to 2D-PAGE and proteome analysis [14]. This resulted in the up-regulation of actin and three different isoforms of tropomyosin by a factor of three–five for actin and six–ten for topomyosin [51]. These proteins are important for the formation of contractile fibers of myofibroblasts. In comparison, vimentin and dynacin were not differentially expressed. a-Enolase, myosin, and tubulin were equally expressed in the stimulated and unstimulated samples [28].
Proteomics in lung cancer research Lung cancer is a challenging clinical problem worldwide [35]. The incidence of lung cancer and lung cancerassociated deaths is increasing in men and women. The current therapies do not improve patient outcome significantly. Therefore, a better understanding of tumor biology, as well as the search for better therapies, is one of the foremost research targets. Apart from knowledgebased approaches that investigate the function and expression patterns of known cellular components, several techniques, including proteomics, have been developed over the past few years, allowing for the expression profiling of large amounts of cellular proteins. These approaches do not depend on the prediction of the functional importance of individual proteins, but permit the detection of unknown changes in the protein composition. The molecular mechanisms behind these changes can then be studied in more detail. Proteomics studies can be used for the classification of surgically resected lung tumors in meaningful groups and prognostic subsets. For example, a number of
studies have been performed using cDNA microarrays for profiling mRNA expression. The groups of Beer and Bhattaracharjee used cDNA arrays to correlate the gene expression of adenocarcinomas with the survival of patients. In addition, it was possible to discriminate between primary lung carcinomas and metastases of extrapulmonary origin [3,4]. The major drawback of these studies, with respect to tumor biology, is the poor correlation between gene transcription, protein expression, and protein function. Protein expression is often regulated post-transcriptionally. The post-translational modification and metabolism of proteins, including proteolytic processing, phosphorylation, and glycosylation, are important in determining protein function. These processes are not covered by transcriptomics. Yanagisawa et al. [42] used the newly developed imaging mass spectrometry for expression profiling of lung tumor samples at the protein level. Regions of single frozen tissue sections (1 mm3) from surgically resected tissues were used for direct mass spectrometric analyses and MALDI-TOF, without protein extraction and separation by 2D-PAGE. Finally, mass spectra of more than 1600 different protein species were detected in lung tumor samples. An artificial intelligence software was developed to handle the large amounts of data points, which were obtained from the analyses of 50 tissue samples. Finally, a class prediction model was developed that correctly classified different lung cancer histologies, distinguished primary tumors from metastases, allowed for the allocation of patients into good or poor prognostic groups, and predicted the risk of nodal involvement [52]. In a second step, they selected significant proteins based on statistical tests, with known prognostic features of these patients, and determined their identity. For example, they found that
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the small ubiquitin-like protein modifier-2 and ubiquitin were up-regulated in NSCLC, which emphasizes the importance of post-translational protein modification in tumorigenesis.
Proteomics in animal models related to lung diseases Proteomics studies can also be applied to animal models. This provides a specific opportunity to identify proteins involved in particular metabolic and pathophysiologic pathways. Mouse models are used to study disease processes in homogenous genomic backgrounds. Wattiez et al. [50] used the BALF of O3-sensitive C57Bl/6 and the O3-resistant C3H/HeJ mice for the identification of proteins involved in the regulation of susceptibility to oxidative stress. They found significant strain-specific differences in the expression of two isoforms of the antioxidant protein 2 (AOP2). C3H/ HeJ mice expressed only AOP2a, whereas AOP2b was found only in C57Bl/6 mice. In addition, the levels of the anti-inflammatory Clara cell protein 16 (CC16) were higher in the resistant C3H/HeJ mice. Thus, AOP2 and CC16 might participate in pathways protecting the respiratory tract from oxidative injury. Ray et al. [37] applied another approach to studying the mechanisms of oxidative lung injury in mice. Proteins present in BALF of mice exposed to room air were compared with those exposed to hyperoxia (100% oxygen). Both the amounts of thioether S-methyltransferase and the amounts of 1-cysteine peroxiredoxin were decreased after hyperoxia, indicating their involvement in the pathogenesis of oxidative stress. Thioether Smethyltransferase is usually abundantly expressed in murine lungs and is important for methylation of thioethers to more water-soluble ions suitable for urinary excretion. 1-cysteine peroxiredoxin, also abundantly expressed in lung tissue, is an antioxidant. Genetically modified mice are of particular interest for studying the impact of the overexpression or lack of single proteins on the proteome of cells and biofluids. In the future, interesting results can be expected from proteomics of lungs obtained from transgenic and knockout mice [37].
Conclusions Different groups and our studies have shown that proteomics is an extremely valuable tool that contributes to the search for new diagnostic and therapeutic targets of chronic lung diseases and lung cancer. However, these studies represent just the beginning. Future studies will unravel the relevance and functional
consequences of the changes in protein concentration. The combination of proteomics of lung tissues and BALF with functional in vitro studies, including the proteome analysis of isolated cells after defined treatments and the use of defined and relevant animal models, will allow us to gain a deeper insight into the signalling cascades causing these changes.
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