The role of Bcl-2 family proteins in pulmonary fibrosis

European Journal of Pharmacology 741 (2014) 281–289

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Review

The role of Bcl-2 family proteins in pulmonary fibrosis Leila Safaeian a,n, Alireza Abed a, Golnaz Vaseghi b a Department of Pharmacology and Toxicology, Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Hezar Jarib Avenue, Isfahan, Iran b Applied Physiology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

art ic l e i nf o

a b s t r a c t

Article history: Received 24 May 2014 Received in revised form 14 July 2014 Accepted 14 July 2014 Available online 21 July 2014

Pulmonary fibrosis is characterized by epithelial injury, abnormal tissue repair, fibroproliferation and loss of pulmonary function as a result of a complex interaction of multiple cellular and molecular processes. There is accumulating evidence in support of a role for apoptosis in the pathogenesis of interstitial lung diseases. The Bcl-2 (B-cell lymphoma-2) family of proteins, which consists of antiapoptotic and pro-apoptotic members, is a critical regulator for apoptosis and development of pulmonary fibrosis. The association between Bcl-2 family members and various pathways and mediators has been also described in the pulmonary fibrosis. This article reviews the recent advances regarding the roles of Bcl-2 family as the apoptosis-regulatory factors in pulmonary fibrosis from human tissue studies, animal models, ex vivo and in vitro studies. Further understanding of apoptosis signaling regulation through Bcl-2 family proteins in the lung tissue may lead to better design of new therapeutic interventions for pulmonary fibrosis. & 2014 Elsevier B.V. All rights reserved.

Keywords: Pulmonary fibrosis Apoptosis Bcl-2 family proteins

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 Apoptosis overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 2.1. Bcl-2 family proteins in apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 3. Role of apoptosis in pulmonary fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282 3.1. Bcl-2 family proteins in pulmonary fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 3.2. Bcl-2 family proteins in human pulmonary fibrosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 3.3. Bcl-2 family proteins in animal models of pulmonary fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 3.4. Bcl-2 family proteins in ex vivo and in vitro studies of pulmonary fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 3.5. Relationship between apoptosis regulation through Bcl-2 family proteins and other mechanisms in pulmonary fibrosis . . . . . . . . . . . 286 3.5.1. Relationship between Bcl-2 family proteins and oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 3.5.2. Relationship between Bcl-2 family proteins and hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 3.5.3. Relationship between Bcl-2 family proteins and mechanotransduction pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 3.5.4. Relationship between Bcl-2 family proteins and cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 3.5.5. Relationship between Bcl-2 family proteins and some specific cellular processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 4. Perspective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

1. Introduction

n

Corresponding author. Tel.: þ 98 3117922596; fax: þ 98 3116680011. E-mail address: [email protected] (L. Safaeian).

http://dx.doi.org/10.1016/j.ejphar.2014.07.029 0014-2999/& 2014 Elsevier B.V. All rights reserved.

Pulmonary fibrosis is a progressive and life-threatening pathologic process resulting in organ failure as a response to lung tissue injuries. This fibroproliferative disease is the final common

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pathway of a various group of lung disorders with similar clinical, radiographic and pathophysiologic characteristics known as interstitial lung diseases (Pardo and Selman, 2002). Pulmonary fibrosis involves a severe scarring of the respiratory membrane, abnormal tissue repair and continuous damage of lung tissue, fibroproliferation and deposition of extracellular matrix, resulting in gradual replacement of normal lung parenchyma with fibrotic tissue. The fibrous scar tissue in the interstitial and alveolar spaces leads to irreparable loss of pulmonary function and reduction in oxygen diffusion capacity Crystal et al., 2002). Pulmonary fibrosis may result from known causes including autoimmune disorders, viral infections, environmental and occupational factors, genetic predisposition, some drugs and radiation. However, two-thirds maybe also appeared without any known cause such as idiopathic pulmonary fibrosis (Raghu et al., 2004; Kropski et al., 2013). Although the exact cellular and molecular mechanisms involved in lung fibrosis remain unknown, various pathways including inflammation, proteolytic/antiproteolytic imbalance, coagulation, angiogenesis, oxidative stress and apoptosis might be involved in the pathogenesis of pulmonary fibrosis (Antoniou et al., 2007). There is a growing body of evidence which points to the role of apoptosis in interstitial lung diseases (Safaeian et al., 2013; Uhal, 2002). Apoptosis or programmed cell death could be triggered by various physiological and pathological death signals. Bcl-2 (B-cell lymphoma-2) family proteins exist in upstream of apoptotic pathways and have a pivotal role in the regulation of apoptosis (Gross et al., 1999). This review highlights the participation of Bcl-2 family as the apoptosis-regulatory factors in pulmonary fibrosis and their targeting for potentially new therapeutic opportunities.

2. Apoptosis overview Apoptosis is the process of cell suicide that plays an important regulatory role in the maintenance of cellular homeostasis. Apoptosis cascade may be triggered by intrinsic or extrinsic signals resulting in cellular changes including chromatin condensation, DNA fragmentation and cytoplasmic budding leading to cell death (Yan and Shi, 2005). The extrinsic death pathway induces apoptosis via cell surface death receptors and ligands like TNF (tumor necrosis factor) or Fas which result in the activation of caspases as the major executioners of apoptosis. The intrinsic death pathway is started in the mitochondria in response to DNA injury induced by various stress such as heat, radiation or oxidative stress. Mitochondrial outer membrane permeabilization is a critical event in the intrinsic pathway which precipitates to cell death through release of pro-apoptotic proteins and loss of mitochondrial functions (Landes and Martinou, 2011). The mitochondrial pathway also initiates a caspase cascade leading to apoptosis (Jin and ElDeiry, 2005). Complex regulatory systems participate in apoptosis adjustment. Abnormal regulation of this important biological phenomenon has been implicated in the pathogenesis of the various diseases (Meier et al., 2000). One of the important regulator molecules of the apoptosis process is Bcl-2 family proteins (Dewson and Kluck, 2010).

family consists of a hydrophobic helix and amphipathic helices. Many of them have transmembrane domains (Burlacu, 2003). Bcl-2 family members have at least one of the four conserved homology domains named the Bcl-2 homology (BH) domains (BH1–BH4) and can be divided into three main groups (Gross et al., 1999; Chipuk et al., 2010). The first group are anti-apoptotic proteins including Bcl-2, Bcl-xL (long isoform), Mcl-1 (myeloid cell leukemia-1), Bcl-w, A1 (Bcl-2-related gene A1) and Bcl-2-related gene which have generally all four BH domains. These proteins as inhibitors of apoptosis (IAP) promote cell survival by inhibiting pro-apoptotic proteins, directly binding and blocking activation of caspases by cytochrome C and preserving integrity of mitochondrial outer membrane against apoptotic stimuli (Llambi and Green, 2011; Konopleva et al., 1999). The second group are pro-apoptotic proteins including Bax (Bcl-2-associated  protein), Bak (Bcl-2 antagonist killer 1), and Bok (Bcl-2-related ovarian killer) which contain three domains (BH1–BH3). Activation of these proteins during intrinsic pathway which is triggered by intracellular stress signals, such as DNA damage or starvation, promotes mitochondrial outer membrane permeabilization leads to cellular death (Kim et al., 2009). The third group is BH3-only pro-apoptotic proteins including Bid (Bcl-2-interacting domain death agonist), Bim (Bcl-2-interacting mediator of cell death), Puma (p53 upregulated modulator of apoptosis), Noxa, Bad (Bcl-2 antagonist of cell death), Bmf (Bcl-2modifying factor), Hrk (Activator of apoptosis harakiri), and Bik (Bcl-2-interacting killer) share only the BH3 domain. The last group interacts with anti-apoptotic bcl-2 members and also induces apoptosis in response to various cellular stresses (Willis et al., 2007). Bid is a physiologic target of extrinsic pathway and its activation stimulates the intrinsic pathway. Therefore a crosstalk between the extrinsic and intrinsic pathways can also arise at upstream level (Thannickal and Horowitz, 2006). During apoptosis from loss of cell adhesion which is called anoikis, changes in cell shape and actin/microtubular cytoskeleton restructuring cause activation of Bim and Bmf and trigger the intrinsic pathway (Valentijn et al., 2007). Lately, new Bcl-2 family members with differing degrees of sequence homology have been recognized. Complex interactions between these different proteins regulate apoptosis through mitochondrial pathway. Following permeabilization of mitochondrial outer membrane due to apoptotic stimuli, diffusion of cytochrome c into the cytosol occurs and leads to oligomerization of apoptotic protease activating factor-1 into the apoptosome multimeric complex. The apoptosome triggers activation of caspase-9 which then motivates caspases-3 and -7 and finally results in the apoptosis (Riedl and Salvesen, 2007). Activated caspases are counter-regulated by IAP family of proteins. The antiapopotic activities of IAP proteins are also negatively regulated by the second mitochondria-derived activator of caspase (Salvesen and Duckett, 2002). Rho proteins also have an important role in Bcl-2, Bid and Mcl-1 activation. Inhibition of Rho proteins reduce the expression of antiapoptotic Bcl-2 and Mcl-1 proteins and induces pro-apoptotic protein such as Bid but had no effects on Bax or Fas-like interleukin-1converting enzyme-inhibitory protein levels. Rho inhibition increases apoptosis of some cells like cultured human endothelial cells (Dubreuil et al., 2003).

2.1. Bcl-2 family proteins in apoptosis

3. Role of apoptosis in pulmonary fibrosis

The Bcl-2 family proteins play an essential role in the regulation of the mitochondrial pathway of apoptosis through controlling the outer mitochondrial membrane integrity (Dewson and Kluck, 2010). This family contains more than 20 members with diverse proapoptotic or anti-apoptotic functions (Lindsay et al., 2011). Bcl-2

Apoptosis plays a critical role in wound repair and in the pulmonary epithelial injuries leading to fibrosis. Markedly increased alveolar epithelial cell (AEC) apoptosis along with decreased fibroblast/myofibroblasts apoptosis have been well-known in promotion of lung tissue damage into fibrosis (Uhal, 2002, 2008; Willis et al., 2007).

L. Safaeian et al. / European Journal of Pharmacology 741 (2014) 281–289

Abnormal fibroblasts phenotypes with apoptotic activity against AECs have been detected in fibrotic lung tissues (Uhal et al., 1995). Furthermore decreased apoptosis of unwanted cells such as inflammatory cells and parenchymal cells involves in prolonged inflammation which injures the lung and promote fibrogenesis (Polnovsky et al., 1993). Apoptosis of neutrophil and infiltrating leukocytes and clearance of apoptotic inflammatory cells by macrophages may play a significant role in declining proinflammatory cytokine production and resolution of inflammation (Kuwano et al., 2004). There are many inflammatory and death factors which mediate epithelial cell damage and apoptosis in fibrotic disease (Wynn, 2007). Both intrinsic and extrinsic apoptotic pathways have also been involved in pulmonary fibrosis (Kuwano et al., 2002; Maeyama et al., 2001). It is well recognized that vulnerability to apoptosis induced by death receptors or other apoptosis stimuli differs according to the cell type and regulation of apoptosis is cell specific (Iwai-Kanai et al., 1999). During pulmonary fibrosis, increasing expression of proapoptotic proteins and decreasing expression of antiapoptotic proteins within different cell types has also been reported (Plataki et al., 2005). Different apoptosis regulatory mechanisms may play critical role during lung fibrosis and clarification of these mechanisms could help to understand the underlying causes and may lead to the development of novel therapeutic strategies. In this study, the role of Bcl-2 family as one of the important apoptosis-regulatory molecules has been reviewed in the process of pulmonary fibrogenesis. 3.1. Bcl-2 family proteins in pulmonary fibrosis There is a growing body of evidence that suggests a crucial role for Bcl-2 family members in the pathogenesis of inflammation, apoptosis and fibrosis induced by various factors in the interstitial lung diseases. Deregulation of Bcl-2 family proteins and imbalance between pro- and anti-apoptotic activities may determine the cellular susceptibility to apoptosis (Oltvai et al., 1999). The role of these regulatory members was investigated in human pulmonary fibrosis and also in animal models, in vitro and ex vivo studies. 3.2. Bcl-2 family proteins in human pulmonary fibrosis The early study in 1995 provided evidence for expression of Bc1-2 protein in T lymphocytes infiltrating the alveolar interstitium in patients with idiopathic interstitial pneumonia. However, this finding was not correlated with pathological scores of fibrosis (Nambu et al., 1995). Another study showed positive Bcl-2 immunostaining in lymphoid cells of lung tissue in patients with idiopathic pulmonary fibrosis (IPF) though the expression was less than that of lung tissue in patients with lymphocytic interstitial pneumonia (Kaan et al., 1997). Immunohistochemistry investigation by Kazufumi et al. also showed Bc1-2 expression on mononuclear cells in the mantle zone of lymphoid follicules and smooth muscle cells, but not on other parenchymal cells in patients with IPF (Kazufumi et al., 1997). However, Guinee and coworkers reported Bax overexpression in pneumocytes in acute lung injury. Their surprising finding was focal Bcl-2 staining in interstitial myofibroblasts (Guinee et al., 1997). Bax protein which is a pro-apoptotic member can bind to anti-apoptotic protein Bcl-2 and forms bax/bcl-2 heterodimers. The imbalance between Bax/Bcl-2 expressions defines the susceptibility of a cell to apoptosis (Safaeian et al., 2009). They suggested that absence of Bcl-2 expression and overexpression of Bax which is involved in apoptosis of alveolar epithelial cells, in consistent with higher levels of Bcl-2 relative to Bax in interstitial myofibroblasts may contribute to the development of fibroproliferative phase in some patients. They also found overexpression of Bax within bronchiolar epithelium and alveolar macrophages in healthy persons (Guinee et al., 1997).

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Up-regulation of Bax and down-regulation of Bcl-2 in epithelial cells from IPF patients were also detected in other studies (Plataki et al., 2005; Nakashima et al., 2005). Evaluation of epithelial remodeling revealed Bcl-2 expression changes in epithelial cells in different areas of the parenchymal deterioration including alveolar collapse, fibroblastic foci, and honeycomb change areas (Baptista et al., 2006). There is a diverse report about the expression of Bcl-2 family members in lung fibroblasts by Emblom-Callahan et al. They found up-regulation of Bax gene in comparing the genomic phenotype of non-cultured pulmonary fibroblasts from advanced IPF patients' with normal lungs and (Emblom-Callahan et al., 2010). Mermigkis et al. has been observed overexpression of Bcl-2 in neutrophils and eosinophils in bronchoalveolar lavage fluid (BALF) of patients with IPF which may be participated in the pathophysiology of pulmonary inflammation and fibrosis (Mermigkis et al., 2001). In spite of decreased apoptosis rate in alveolar macrophages of IPF patients, there was not any difference in the Bcl-2 and Bax expression in alveolar macrophages of BALF between IPF and normal subjects (Drakopanagiotakis et al., 2012). Further studies showed correlation between Bcl-2 overexpression in alveolar lymphocytes in interstitial lung diseases such as IPF and sarcoidosis with expression of insulin-like growth factor-1 (IGF-1) as an antiapoptotic cytokine (Kopiński et al., 2006). However other factors such as tobacco consumption could also affect Bcl-2 expression in these patients. Increased apoptosis and decreased Bcl-2 expression were found in smokers' alveolar lymphocytes and there was positive correlation between TNFα levels and Bcl-2 expression in nonsmokers (Kopiński et al., 2012). A recent study has suggested the role of hepatocyte growth factor and increased expression of Bcl-xL in epithelial repair in lung fibrosis (Skwarna et al., 2011). The results of human studies about the role of Bcl-2 members were summarized in Table 1. 3.3. Bcl-2 family proteins in animal models of pulmonary fibrosis Different experimental models have been developed for understanding the cellular and molecular mechanisms of pulmonary apoptosis and fibrosis. Data from various animal models including bleomycin, asbestos, silica, paraquate, chemokine and radiation model of lung fibrosis support the role of bcl-2 family proteins in the regulation of apoptosis through fibrosis development (Table 2). The bleomycin model of pulmonary fibrosis is the best characterized murine model. Alveolar epithelial cell apoptosis has a critical role in the pathogenesis of bleomycin-induced lung injury and fibrosis hence beomycin has been used in several studies for identification of signaling mechanisms involved during the apoptosis process (Li et al., 2003). Like human studies, excessive apoptosis of bronchiolar and alveolar epithelial cells through the up-regulation of Bax protein and decreased apoptosis of lymphocytes and macrophages due to Bcl-xL protein up-regulation has been reported in bleomycininduced pulmonary fibrosis (Kuwano et al., 2000). The influence of proapoptotic Bid as a requirement for the progress of lung fibrosis after bleomycin administration was reported by Budinger et al. (2006). The role of strain variation has been evidenced in murine bleomycin-induced pulmonary fibrosis however there are limited data about the genetic susceptibility to lung apoptosis (JafarianDehkordi et al., 2007). The results of our study showed some differences in expression of Bcl-2 and Bax in myofibroblasts, neutrophils and lymphocytes between two different strains of mice suggesting some genetic variation in the expression of apoptotic regulatory genes in bleomycin model of lung fibrosis (Safaeian et al., 2008). Our results in consistent with other studies showed high expression of the anti-apoptotic gene, Bcl-2, and low expression of the pro-apoptotic gene, Bax in preventing apoptosis in lung

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Table 1 Involvement of Bcl-2 family proteins in pulmonary fibrosis in human studies. IPF: idiopathic pulmonary fibrosis; BAL: bronchoalveolar lavage; and ILD: interstitial lung diseases. Reference

Samples

Nambu et al. (1995)

Lung specimens from patients Open lung biopsy with idiopathic interstitial pneumonia Lung tissues from IPF patients Medical history

Kaan et al. (1997)

Evidence of pulmonary fibrosis

Kazufumi et al. Lung specimens from IPF (1997) patients Guinee et al. (1997) Lung tissues from patients with diffuse alveolar damage Plataki et al. (2005) Lung specimens from IPF patients Nakashima et al. Lung specimens from patients (2005) with IPF and non-specific interstitial pneumonia Baptista et al. Lung specimens from patients (2006) with IPF and usual interstitial pneumonia Emblom-Callahan et al. (2010) Mermigkis et al. (2001) Kopiński et al. (2006) Kopiński et al. (2012) Drakopanagiotakis et al. (2012) Skwarna et al. (2011)

Advanced IPF patients BAL from IPF patients BAL from ILD patients

Open lung biopsy Open lung biopsy, Diagnostic criteria for diffuse alveolar damage Lung biopsy

Controls

Involvement of Bcl-2 family proteins

Patients with collagen vascular diseases

Positive Bcl-2 immunostaining in T lymphocytes

Necropsy cases without pulmonary Positive Bcl-2 immunostaining in lymphoid cells disease Healthy subjects Positive Bcl-2 immunostaining in mononuclear cells and smooth muscle cells Uninvolved portions of lung from Bax↑ in pneumocytes, Bcl-2↑ in interstitial surgical resections of tumors myofibroblasts Control subjects

Apoptosis↑, Bax↑ and Bcl-2↓ in epithelial cells

Lung biopsy

Normal subjects

Bax↑ in epithelial cells

Open lung biopsy, Diagnostic criteria for usual interstitial pneumonia Medical history

Normal lung tissues from nonpneumonia and emphysematous areas from died individuals

Bcl-2↓ in epithelial cells from alveolar collapse and fibroblastic foci areas, Bcl-2↑ in epithelial cells from honeycomb change areas

Normal lungs

Bax↑ in fibroblasts

Normal subjects

Bcl-2↑ in neutrophils and eosinophils

Normal subjects

Apoptosis↓, Bcl-2↑ in alveolar lymphocytes Apoptosis↑, Bcl-2↓ in smokers' alveolar lymphocytes, no difference between groups in expression of Bcl-xL or Bak Apoptosis↓, no difference between groups in bcl2 and bax expression in alveolar macrophages Bcl-xL↑ in hyperplastic alveolar type II cells and in bronchial epithelial cells

Fiberoptic bronchoscopy/ CT Medical history

BAL from smoker and nonsmoker ILD patients

Medical history

Healthy smoker and nonsmoker volunteers

BAL from IPF patients

Surgical biopsy

Normal subjects

Lung homogenates from IPF subjects

Medical history

Healthy donors

Table 2 Involvement of Bcl-2 family proteins in pulmonary fibrosis in animal models. ROCK: Rho/Rho kinase; PEGylated: polyethylene glycol-modified; TGF-beta-1: transforming growth factor beta-1; IL-2: interleukin-1; PPAR-β/δ: peroxisome proliferator-activated receptor β/δ; and ERK: extracellular signal-regulated kinase. Reference

Model

Intervention

Involvement of Bcl-2 family proteins

Kuwano et al. (2004) Budinger et al. (2006) Safaeian et al. (2013) Zhou et al. (2013) Machtay et al. (2006)

Mice

Bleomycin

Bax↑ in epithelial and inflammatory cells, Bcl-xL↑ in lymphocytes and macrophages

Bid(  /  ) mice

Bleomycin

Pulmonary fibrosis↓

C57BL/6 and NMRI mice Female mice

Bleomycin

Bcl-2↑ in myofibroblasts and neutrophils of NMRI mice, Bax↑ in alveolar epithelial cells of both strains, Bax↓ in myofibroblasts of C57BL/6 mice and lymphocytes of both strains Apoptosis↓ and Bcl-2↓ in myofibroblast

Huang et al. (2011) Kang et al. (2007) Safaeian et al. (2008) Satomi et al. (2007) TorresGonzález et al. (2013) Segel et al. (2005) Galuppo et al. (2010) Zhang et al. (2007) Galuppo et al. (2010)

Bleomycin þ ROCK inhibitor

Radiation þPEGylated superoxide dismutase and catalase Sprague-Dawley rats Paraquat þLysine acetylsalicylate Wild type, Bax and Bid TGF-beta1 (  /  ) mice Female NMRI mice Bleomycin þ Losartan C57/bl6 mice

Apoptosis↓ and Bax↓

Bcl-2↑, Bax↓, Bcl-2/Bax↑ Bax↑ and Bid↑

Rats

Paraquat

Bax/Bcl-2↓ in alveolar epithelial cells, lymphocytes, macrophages and interstitial myofibroblasts, Bcl-2↓ in neutrophils Altered expression of Bad

C57BL/6 mice

γherpesvirus 68 infection

Apoptosis↑, Bim↑

Mice

Bleomycin þ IL-2-Bax

Lymphocytic infiltration↓

Mice

Bleomycin þ PPAR-β/δ agonist

Apoptosis↓, Bax/Bcl-2↓

Mice lacking inhibitor of differentiation 1 Mice

Bleomycin

Bcl-2↓ in endothelial cell

Bleomycin þ Inhibitor of ERK

Apoptosis↓, Bax/Bcl-2↓

L. Safaeian et al. / European Journal of Pharmacology 741 (2014) 281–289

fibroblasts after bleomycin instillation as a key feature of chronic fibrotic diseases (Safaeian et al., 2008; Zhou et al., 2013). Network between apoptosis and other mechanisms finding from animal studies has been reviewed in the following sections. 3.4. Bcl-2 family proteins in ex vivo and in vitro studies of pulmonary fibrosis The result from ex vivo and in vivo studies also support the role of bcl-2 family proteins in the regulation of apoptosis during fibrosis progression (Table 3). In 1995, Hamilton and co-workers found that bleomycin resulted in marked apoptosis in human alveolar macrophages in vitro. However they did not find any alteration in intracellular bcl-2 levels (Hamilton et al., 1995). Like animal and human studies, higher expression of Bax and lower expression of Bcl-2 were observed in alveolar epithelial cell after intratracheally instillation of bleomycin (Kong et al., 2007). Further studies for finding the molecular mechanism of apoptosis during lung fibrosis revealed resistance to cell death in alveolar type II cells overexpressing Bcl-xL and in fibroblast cells deficient in Bax and Bak, and also suggested the role of Januskinase-dependent mitochondrial death pathway in alveolar epithelial cell apoptosis induced by bleomycin (Lee et al., 2005).

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As in human studies, the results of in vitro researches of pulmonary fibrosis revealed the protective role of Bcl-xL overexpression in epithelial repair and attenuation of apoptosis (Kopiński et al., 2012; Kamp et al., 2013). In primary pulmonary endothelial cells, exposure to bleomycin has induced the expression of Bcl-2 and Bcl-xL (Mungunsukh et al., 2010). Although some studies showed overexpression of Bcl-2, and low expression of Bax in lung fibroblasts after bleomycin administration (Safaeian et al., 2008; Zhou et al., 2013). Tanaka et al. found no changes in Bcl-2 and Bcl-xL in primary lung fibroblasts during Fas-mediated apoptosis (Tanaka et al., 2002). Increased apoptosis and alteration in the expression of Bcl-2 family members have been also reported in other models of pulmonary fibrosis. Asbestos as another cause of lung toxicity and fibrosis activates Bax in alveolar epithelial cells (Panduri et al., 2006). Inhalation of cadmium as a xenobiotic toxicant has been implicated in the development of pulmonary fibrosis. After cadmium exposure, various genes including Bax gene has been induced in lung fibroblast cell line (Shin et al., 2003). In age-related susceptibility to lung fibrosis, apoptosis of type II alveolar epithelial cells has been also associated with expression of the proapoptotitc molecule, Bcl-2 interacting mediator (Bim) and up-regulation of endoplasmic reticulum stress marker (Sorescu et al., 2012; Torres-González et al., 2013).

Table 3 Involvement of Bcl-2 family proteins in pulmonary fibrosis in ex vivo and in vitro studies. TGF-beta-1: transforming growth factor beta-1; TNF: tumor necrosis factor; IGF-1: insulin-like growth factor-1; and IL-6: interleukin-6. Reference

Model

Intervention

Involvement of Bcl-2 family proteins

Hamilton et al. (1995) Kong et al. (2007) Lee et al. (2005)

Human alveolar macrophage

Bleomycin

Apoptosis↑, no changes in Bcl-2

Sprague-Dawley rat alveolar type II cells Mouse and rat alveolar epithelial cells, fibroblast cells Alveolar epithelial cells isolated from Bid(  /  ) mice Primary pulmonary endothelial cells

Bleomycin

Bax↑, Bcl-2↓

Bleomycin Bleomycin þ TGF-beta1

Apoptosis↓ in alveolar cells overexpressing Bcl-xL and in fibroblasts deficient in Bax and Bak TGF-beta1-induced apoptosis↓

Bleomycin

Bcl-2↑, Bcl-xL↑, Apoptosis↑ through TNF↑

MLE-12 cells

Fas

Bcl-xL↓

Human A549, rat primary alveolar type II cells Human diploid fibroblasts

Asbestosþ overexpression of Bcl-xL

Apoptosis↓ in alveolar epithelial cells

Exogenous H2O2

Resistance to apoptosis, Bcl-2↑, Bax↓

Alveolar epithelial cells

Asbestos

Bax mitochondrial translocation↑

Normal human lung fibroblast cell

Cadmium

Bax↑

Human embryonic lung epithelial cells Rat alveolar epithelial typeII cells

Glyoxal

Bax↑, Bcl-2↓

Hypoxia

Bnip3L↑

Rat lung fibroblasts

TGF-beta1þ IL-1beta

Apoptosis↓, Prevention of a decline in Bcl-2

Rat alveolar macrophages

Asbestos

Bcl-2↑ and Bcl-xL↑ in alveolar macrophages

Airway epithelial cells

IGF-1

Bcl-2↑

Budinger et al. (2006) Mungunsukh et al. (2010) Skwarna et al. (2011) Kamp et al. (2013) Sanders et al. (2013) Panduri et al. (2006) Shin et al. (2003) Kasper et al. (2000) Krick et al. (2005) Zhang and Phan (1999) Nishimura et al. (2007) Chand et al. (2012) Lee et al. (2010) Kim and Day (2012) Tanaka et al. (2002) Moodley et al. (2003) Bridges et al. (2009) Ricci et al. (2013)

Primary lung endothelial cells Angiotensin II Primary lung artery endothelial cells Angiotensin II

Bcl-xL↓ by inhibition of nucleolin binding to Bcl-xL mRNA Bim promoter activation↑

WI-38 lung fibroblast cells, Primary Fas lung fibroblasts Human primary lung fibroblasts Fas, IL-6

No changes in Bcl-2 and Bcl-xL

Rat lung fibroblasts Human primary lung fibroblasts

Profibrotic growth factors, Twist1 gene expression profiling of IPF lungs Cisplatin

Apoptosis↑ and Bax↑ in normal fibroblasts, Apoptosis↓ and Bcl-2↑ in IPF fibroblasts Apoptosis↑, Bim↑ and PUMA↑ during Twist1 depletion Bcl-2↓ in human IPF fibroblasts

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3.5. Relationship between apoptosis regulation through Bcl-2 family proteins and other mechanisms in pulmonary fibrosis The association between apoptosis and various cellular and molecular pathways has been demonstrated in pulmonary fibrosis. It is noteworthy that complex agent-dependent and tissuesspecific mechanisms are involved in the regulation of apoptosis event (Jehle et al., 2012). Various death mediators and signals such as Fas ligand, p53, reactive oxygen species (ROS), nitrogen species, pro-inflammatory cytokines and chemokines like transforming growth factor (TGF)-β1, TNF-α, angiotensin II, and others are involved in inflammatory process and may interfere with apoptosis signaling in different pulmonary cells and result in a profibrotic circumstance in the damaged lung tissue (Plataki et al., 2005; Wang et al., 1999; Bühling et al., 2005; Nakashima et al., 2005; Lee et al., 2006; Wang et al., 2000). In the following sections, network between apoptosis and different mechanisms has been reviewed in fibrotic lung diseases. Identifying and understanding the mechanisms of the crosstalk between apoptosis and other pathways would be helpful in the development of new approaches for treatment of pulmonary fibrosis. 3.5.1. Relationship between Bcl-2 family proteins and oxidative stress Oxidative stress has been found to play an essential role in the pathogenesis of lung fibrosis. Alveolar inflammatory cells and activated myofibroblasts release significant concentrations of ROS which lead to injury and apoptosis of epithelial cells (Strausz et al., 1990; Waghray et al., 2005). However the role of oxidative stress is not fully studied in the function of Bcl-2 family members. Oxidative stress may regulate the expression of some Bcl-2 family members such as Bcl-2, Bax or Bak (de Brito and Scorrano, 2010; Meunier and Hayashi, 2010). The results of investigation on asbestos-induced pulmonary fibrosis have shown the role of oxidative stress in AECs apoptosis through mitochondrial pathway. Administration of free-radical scavengers and also overexpression of Bcl-xL have been able to block apoptosis in this model of lung fibrosis (Panduri et al., 2003). Treatment with systemic polyethylene glycol-modified superoxide dismutase and catalase mixture has attenuated the increased apoptosis and marked elevation of Bax in lung tissue of mice in radiation-induced pulmonary fibrosis (Machtay et al., 2006). Treatment of lung epithelial cell with glyoxal, a reactive alpha-oxoaldehyde and a physiologic metabolite of oxidative degradation of glucose, has been associated with increased apoptosis through enhancement of Bax and decreasing Bcl-2 expression (Kasper et al., 2000). In paraquat-induced pulmonary fibrosis, treatment with lysine acetylsalicylate had potentially protective effects through antioxidant effects by increasing superoxide dismutase, glutathione peroxidase and catalase activities and also anti-apoptosis effects by reduction of Bax expression, increasing Bcl-2 expression and increasing the Bcl-2/Bax ratio of lung (Huang et al., 2011). 3.5.2. Relationship between Bcl-2 family proteins and hypoxia Hypoxia enhances alveolar epithelial cell apoptosis and has been implicated in pulmonary fibrosis. Increased expression of proapoptotic protein Bnip3L belonging to the Bcl-2 family, has been reported in hypoxic conditions. Bnip3L is known to be one of the hypoxia inducible-factor-1-dependent target genes (Krick et al., 2005). 3.5.3. Relationship between Bcl-2 family proteins and mechanotransduction pathway The role of a specific mechanotransduction pathway in the regulation of myofibroblast differentiation and survival has been shown

through the disruption of mechanotransduction signaling with the Rho/Rho kinase inhibitor which induced myofibroblast apoptosis through downregulation of Bcl-2 (Zhou et al., 2013). The global and locus-specific histone modifications of chromatin also regulate Bcl-2/ Bax gene expression in senescent fibroblasts and contribute in resistant to apoptosis in this phenotype (Sanders et al., 2013). 3.5.4. Relationship between Bcl-2 family proteins and cytokines Various ex vivo and in vivo studies have shown the relationship between bcl-2 family proteins and other factors such as cytokines in pulmonary fibrosis. Transforming growth factor beta-1 (TGF-beta1) is an important inductor of apoptosis in different cells through caspase activation, upregulation of p21, and downregulation of antiapoptotic Bcl-2 (Yanagisawa et al., 1998; Gressner et al., 1997). Bax, Bid, and matrix metalloproteinase-12 also play essential role in the pathogenesis of TGF-beta1-induced fibrosis and apoptosis in the lung tissue (Kang et al., 2007). TGF-beta1 is able to promote myofibroblast survival by prevention of myofibroblast apoptosis. Apoptosis of myofibroblasts induced by interleukin-1beta (IL-1beta) is inhibited by TGF-beta1 through suppression of inducible nitric oxide synthase expression and prevention of bcl-2 down- regulation (Zhang and Phan, 1999). Increased levels of Bcl-2 and Bcl-xL in the alveolar macrophages surviving under the exposure to low doses of asbestos and production of TGF-beta1 by these cells have been also observed in asbestos model of lung fibrosis (Nishimura et al., 2007). Altered expression of pro-apoptotic gene Bad has been implicated as an important factor in paraquat-induced pulmonary apoptosis and fibrosis which was associated with markedly increased expression of TGFbeta3 (Satomi et al., 2007). Another growth factor involved in the modulation of Bcl-2 expression in airway epithelial cells is IGF-1. Intracellular IGF-1 induces Bcl-2 expression in epithelial cells by increasing Bcl-2 mRNA stability via IGF-1 receptor and epidermal growth factor receptor pathways (Chand et al., 2012). Angiotensin II is another potent inducer of apoptosis in various cells which contributes to the development of fibrotic response to tissue injury via its growth factor properties (Marshall et al., 2004). We found that angiotensin type 1 receptor blockade by losartan reduced the bax/bcl-2 expression ratio in the alveolar epithelial cells, lymphocytes, macrophages and interstitial myofibroblasts, and also prevented the bcl-2 upregulation in neutrophils in bleomycin model of pulmonary fibrosis (Safaeian et al., 2009). Lee and his colleagues provided evidence for molecular mechanisms of angiotensin-II-induced apoptosis in primary lung endothelial cells. They found that nucleolin which binds Bcl-xL mRNA and prevents its degradation, is a primary target of angiotensin II signaling (Lee et al., 2010). Further studies have shown that angiotensin II causes apoptosis in primary pulmonary artery endothelial cells through up-regulation of Bim via activation of AMP (adenosine monophosphate)-regulated protein kinaseβ1/2 and cyclin-dependent kinase4 (Kim and Day, 2012). Cytokines also play significant role in the apoptosis pathway. IL-6 is a profibrotic factor and its altered signaling has been associated with resistance of fibroblast to apoptosis through changes in Bax and Bcl-2 expression in IPF patients (Moodley et al., 2003). IL-2-Bax, a novel apoptosis-inducing interleukin-2receptor-targeted chimeric protein has reduced the lymphocytic infiltration of the lungs in response to bleomycin (Segel et al., 2005). 3.5.5. Relationship between Bcl-2 family proteins and some specific cellular processes Galuppo et al. showed the role of peroxisome proliferatoractivated receptor β/δ which belongs to a family of ligandactivated transcription factors in the treatment of pulmonary

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fibrosis through decreasing inflammation and apoptosis evaluated by Bax and Bcl-2 balance (Galuppo et al., 2010). The inhibition of extracellular signal-regulated kinase cascade, as an important pathway in the activation of various cellular processes, has shown therapeutic effects on pulmonary damage by decreasing many inflammatory and apoptotic parameters, such as Bax and Bcl-2 balance (Galuppo et al., 2011). The inhibitor of differentiation type 1 encodes negative regulators of some transcription factors and plays essential role in promoting endothelial survival during bleomycin-induced pulmonary fibrosis. Increased vascular permeability and endothelial apoptosis in the lungs has been reported in mice lacking Id1 which was associated with decrease in the level of Bcl-2 (Zhang et al., 2007). Twist1 is a basic helix–loop–helix protein that acts as an antiapoptotic factor and stimulates accumulation of fibroblasts during growth factors signaling. It has been detected that growth factors increase fibroblasts apoptosis during Twist1 depletion which is partly mediated by overexpression of Bim and PUMA (proapoptotic Bcl-2 family members) (Bridges et al., 2009). Interconnection between autophagy and apoptosis has been also suggested in the regulation of cell fate during stress. Expression of Beclin 1 as a key regulator of autophagy has been modified in consistent with Bcl-2 proteins alteration in human IPF fibroblasts (Ricci et al., 2013).

4. Perspective Various studies provided insight into the involvement of apoptosis and Bcl-2 family in the pathogenesis of pulmonary fibrosis. Alveolar epithelial cell damage is a typical feature of IPF. Abnormal wound healing leads to significant loss of type I pneumocytes and hyperplasia of type II pneumocytes. In the severely damaged area, death of both types of AECs and replacement of abundant fibroblasts and smooth muscle cells occurs (Kuwno et al., 2001a). Data from most of human and experimental studies have shown the role of decreased expression of Bcl-2 and overexpression of Bax in increased epithelial cell death and the role of higher levels of Bcl-2 relative to Bax in resistance to apoptosis in interstitial myofibroblasts and inflammatory cells. A schematic drawing of the role of changes in the well-known Bcl-2 family members in the pathogenesis of pulmonary fibrosis is shown in Fig. 1. Limited findings have been so far reported regarding the role of other members of Bcl-2 family especially newer ones. However, some members such as Bcl-xL have been found to contribute as an anti-apoptotic in epithelial repair and Bid as a pro-apoptotic in lung fibrosis pathogenesis. Therefore modifying apoptosis by blockade of epithelial cell apoptosis and promotion of apoptosis in myofibroblast and inflammatory cells through Bcl-2 pathway would be anticipated as potential therapeutic intervention for lung fibrosis. Several Pharmacological studies have revealed inhibition of apoptosis and fibrosis using broad-spectrum caspase inhibitors and blockers of the angiotensin pathways in animal models of pulmonary fibrosis (Kuwno et al., 2001b; Li et al., 2003). Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase have been able to induce apoptosis in fibrotic lung fibroblasts due to their ability to inhibit the expression of some growth factors (Nadrous et al., 2004). Some therapeutic strategies targeting Bcl-2 proteins have been developed for the treatment of particular neoplastic diseases (Kang and Reynolds, 2009) but studies on the lung fibrosis are scarce. Pulmonary fibrosis is a progressive disorder with different stages in the severity of lung fibrotic changes (Sugino et al., 2010). Several processes might be implicated in the different stages of pulmonary

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Fig. 1. A schematic drawing of the role of changes in the well-known Bcl-2 family members in the pathogenesis of pulmonary fibrosis.

fibrosis development. Therefore changes of apoptosis and Bcl-2 family members should be studied according to the stages of disorder. Although the Bcl-2 expression has been investigated in different stages of some fibrotic diseases such as liver fibrosis (Tsamandas et al., 2003), unfortunately there is no study about the changes of Bcl-2 family proteins in various stages of pulmonary fibrosis to explore their correlation with the severity of this disease. More studies are needed in future to exactly clarify the importance of bcl-2 family in diagnosis, grading and treatment of pulmonary fibrosis. However, it is known that targeting apoptosis signaling and accelerating the tissue repair at an early stage of the disease could be more effective approach against fibrosis development compared to management at a severely damaged stage. The reports on the genetic predisposition to the lung apoptosis are also limited. More investigations on the role of polymorphisms in Bcl-2 or some other genes associated with apoptosis would be valuable. According to the close network between regulation of apoptosis by Bcl-2 proteins and other pathways and mediators over fibrosis development and on the basis of identification of detailed molecular signaling mechanisms, some interventions such as reducing oxidative stress, inhibition of some kinase cascades or angiotensin type 1 receptor have been developed which have been associated with beneficial effects on pulmonary fibrosis. Further investigations need to be carried out for improvement of our knowledge about the details of these correlations and for development of therapeutic interventions through inhibition of these pathways and mediators in a cell type-specific manner.

5. Conclusion The increasing body of evidence described the involvement of Bcl-2 family members as the critical regulators in the development of pulmonary fibrosis. However, there are still significant gaps in our knowledge regarding the details of these regulatory processes causing lung fibrotic changes. Progress in understanding the apoptotic regulation through Bcl-2 family proteins in the lung tissue and cells may lead to the identification of novel intervention strategies for the treatment of pulmonary fibrosis. Because of diverse roles of apoptosis in various cells and specific expression of apoptotic regulatory Bcl-2 genes in each cell types, targeted

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