Cysteine cathepsins and cystatins: from ancillary tasks to prominent status in lung diseases.

Biol. Chem. 2015; 396(2): 111–130

Review Gilles Lalmanach*, Ahlame Saidi, Sylvain Marchand-Adam, Fabien Lecaille and Mariana Kasabova

Cysteine cathepsins and cystatins: from ancillary tasks to prominent status in lung diseases Abstract: Human cysteine cathepsins (family C1, clan CA) have long been regarded as ubiquitous household enzymes, primarily involved in the recycling and degradation of proteins in lysosomes. This opinion has changed considerably during recent decades, however, with the demonstration of their involvement in various physiological processes. A growing body of evidence supports the theory that cathepsins play specific functions in lung homeostasis and pathophysiological events such as asthma, lung fibrosis (including idiopathic pulmonary fibrosis), chronic obstructive pulmonary disease (embracing emphysema and chronic bronchitis), silicosis, bronchopulmonary dysplasia or tumor invasion. The objective of this review is to provide an update on the current knowledge of the role of these enzymes in the lung. Particular attention has been paid to the understanding of the role of these proteases and their natural inhibitors, cystatins (family I25, clan IH), in TGF-β1-driven fibrotic processes with an emphasis on lung fibrosis. Keywords: asthma; chronic obstructive pulmonary disease; fibrosis; inflammation; protease; protease inhibitor. DOI 10.1515/hsz-2014-0210 Received June 6, 2014; accepted August 26, 2014; previously published online August 28, 2014

*Corresponding author: Gilles Lalmanach, INSERM U1100 ‘Pathologies Pulmonaires: Protéolyse et Aérosolthérapie’, Equipe Mécanismes Protéolytiques dans l’Inflammation/Centre d’Etude des Pathologies Respiratoires, Université François Rabelais, Faculté de Médecine, 10 Boulevard Tonnellé, F-37032 Tours cedex, France, e-mail: [email protected] Ahlame Saidi, Sylvain Marchand-Adam, Fabien Lecaille and Mariana Kasabova: INSERM U1100 ‘Pathologies Pulmonaires: Protéolyse et Aérosolthérapie’, Equipe Mécanismes Protéolytiques dans l’Inflammation/Centre d’Etude des Pathologies Respiratoires, Université François Rabelais, Faculté de Médecine, 10 Boulevard Tonnellé, F-37032 Tours cedex, France

Introduction Proteases (570 genes in humans) are classified according to their catalytic mechanism into six main categories: serine, acid (aspartate and glutamate), cysteine, metalloand threonine proteases (López-Otín and Bond, 2008). Among them, the archetypal model of cysteine cathepsins is papain, a plant enzyme that has been extensively studied. These enzymes, of which there are 11 in humans [B, H, L, S, C, K (O2), O, F, V, X (Z/P) and W], belong to the C1A (clan CA) family (Rawlings et al., 2012; see MEROPS, the peptidase database: http://merops.sanger.ac.uk). Cathepsin (Cat) C is an aminopeptidase, Cat X is a carboxypeptidase, and cathepsins F, K, L, O, S, V and W are endopeptidases; whereas Cat B is both a carboxypeptidase and an endopeptidase and Cat H is both an aminopeptidase and an endopeptidase (Lecaille et al., 2002; Turk et al., 2012). Cathepsins may also be classified into two main groups based on the propeptide sequence. The first group (cathepsins L, K, S, H and V) called ‘L-like’ is characterized by a proregion of about 100 residues and two consensus motifs: ERF(X)NIN and GxNxFxD. In the second ‘B-like’ group (including cathepsins B, C, O and X) the proregion that consists of around 60 residues has a single conserved motif: GxNxFxD (Karrer et al., 1993). Based on phylogenetic analyses showing that cathepsins F and W propeptides share a specific pattern, ERFNAQ, a third ‘F-like’ group, was proposed (Wex et al., 1999). Cathepsins have long been regarded as ubiquitous household enzymes, primarily involved in the recycling and degradation of proteins in lysosomes. This opinion has changed considerably during recent decades, however, with the demonstration of their involvement in various physiological processes, such as the presentation of antigenic peptides, and the maturation of thyroid hormones or neuropeptides (see for review Reiser et al., 2010). In addition to their natural endogenous inhibitors (see the ‘Specific natural inhibitors, cystatins’ section), the proteolytic activity of cathepsins is regulated by their propeptide, pH and ionic strength, and glycosaminoglycans. Cathepsins, because

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112 G. Lalmanach et al.: Cysteine cathepsins and cystatins in lung diseases of the high reactivity of the nucleophilic cysteine of the active site, are very sensitive to the action of reactive oxygen species, reactive nitrogen species, variations of redox potential, and also to Zn2+ and some metal ions (Lockwood, 2013). We will not discuss the regulation, the genomic organization, folding and structural features, catalytic mechanism or substrate specificity of cysteine cathepsins here, since outstanding reviews describe these aspects elsewhere (e.g., Lecaille et al., 2002; Turk et al., 2012).

Cellular localization of cysteine cathepsins Targeting to lysosomes Cathepsins are synthesized as inactive precursor proteins (pre-pro enzymes). They are then directed to the lumen of the endoplasmic reticulum by a signal peptide, which is released by ‘signal peptidases’. After correct folding, procathepsins are transported at the Golgi apparatus and glycosylated (Brix et al., 2008; Reiser et al., 2010). Following the addition of mannose-6-phosphate (M-6-P) groups to glycosylated chains, the inactive proforms are addressed via the M-6-P receptors to late endosomes where they are proteolytically maturated, releasing mono- or bi-catenary active forms. Maturation is provided by autocatalytic activation or by other proteases present in these acidic compartments such as pepsin, aspartic Cat D and legumain (e.g., Mach et al., 1994; Cygler and Mort, 1997). Cathepsins C and X, however, require an endopeptidase such as cathepsins L or S for their activation (Nägler et al., 1999; Dahl et al., 2001).

and tumor cells (Mohamed and Sloane, 2006). Independent M-6-P transport is observed in macrophages and fibroblasts due to a defect of the M-6-P receptor or due to the lack of M-6-P signals. This results in a constitutive secretion of proforms via classical secretory pathways (Brix et al., 2008). An unusual location was also observed in the mitochondria (Müntener et al., 2004) or in the nucleus (Goulet et al., 2004) for cathepsins lacking the signal peptide. Consequently, cathepsins may participate in the initiation of apoptosis or cell cycle regulation. For instance, nuclear Cat L may activate a transcription factor (CDP/ Cux) accelerating the passage of S-phase cell cycle (Goulet et al., 2004).

Secretory lysosomes Numerous cells store their secretion products in special compartments, called secretory granules. These granules may fuse with the plasma membrane and thereby release their contents into the extracellular medium. Interestingly, apart from the conventional lysosomes, some cells possess modified lysosomes that function as secretory compartments and are called secretory lysosomes with both secretory and lysosomal markers (Stinchcombe and Griffiths, 1999). Natural killer cells and mast cells use secretory lysosomes for the release of Cat C, while macrophages in turn release cathepsins B, L, K and S (Reddy et al., 1995). Smooth muscle cells are also capable of synthesizing and secreting Cat S after stimulation with interferon-γ (IFN-γ) (Shi et al., 1999).

Physiological functions of cysteine cathepsins

Alternative trafficking pathways

Apoptosis

Despite the fact that cathepsins are primary found in late endosomes, alternative locations are also reported for these proteases (for review see Brix et al., 2008). Cathepsins may initially be directed in cellular compartments other than late endosomes/lysosomes or secreted in the extracellular environment. In epithelial cells, cathepsins may be redirected from endo/lysosomal compartments to the basal or apical surfaces before their secretion as active forms in the extracellular environment (Linke et al., 2002). Also, secreted cathepsins may facilitate the invasiveness of transformed cells at the initial stage of tumor formation. Additionally, extracellular Cat B binds to membrane caveolae and remains proteolytically active in endothelial

In addition to their traditional housekeeping role, cathepsins are involved in various physiological processes. Several studies indicate that these proteases have proapoptotic functions (for review see Repnik et al., 2012). For instance in tumor necrosis factor-α (TNF-α)-treated hepatocytes, the initiator caspase 8 induces an increase in the concentration of cytoplasmic Cat B, leading to apoptosis. This event is significantly decreased in Cat B-deficient mice (Guicciardi et al., 2000). In addition to the well-established apoptotic substrate Bid, cathepsins were found to degrade the anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2 and X-linked inhibitor of apoptosis protein), consequently triggering the release of mitochondrial cytochrome c (Turk


G. Lalmanach et al.: Cysteine cathepsins and cystatins in lung diseases 113

and Turk, 2009). Cat C also participates in apoptosis via activation of granzyme B, which in turn activates effector caspase 3 (Pham and Ley, 1999).

Maturation and/or activation of protein precursors Cathepsins are key players in the maturation/activation of pro-hormones and pro-enzymes. Cathepsins B, K, L and S hydrolyze thyroglobulin and release a thyroid hormone, thyroxin (Jordans et al., 2009). Cat B also participates in the maturation of pro-β-galactosidase, trypsinogen and pro-renin (for review see Reiser et al., 2010). In neuronal vesicles Cat L is involved in the maturation of neuropeptides like enkephalin, β-endorphin and dynorphin (Funkelstein and Hook, 2011). Cat L may also liberate bradykinin, a proinflammatory peptide (Veillard et al., 2008) and participates in the N-terminal proteolysis of histone H3, which has an important role in the development and differentiation of embryonic stem cells (Duncan et al., 2008). Unlike Cat L, Cat K inactivates hypotensive bradykinin, thus reducing blood pressure (Godat et al., 2004; Lecaille et al., 2007). Also, Cat X is involved in the activation of β2 integrin receptors, macrophage antigen-1 and lymphocyte function-associated antigen-1 and hence could regulate the migration of T lymphocytes (Obermajer et al., 2008).

(ECM) and basement membrane, such as collagens (Brömme and Lecaille, 2009). As a consequence of deregulation of the protease/anti-protease homeostasis, this lively mechanism of controlled proteolysis can be turned off and associated with a wide spectrum of pathologies such as osteoporosis, atherosclerosis and cancers. Since this subsection on the role and consequences of matrix remodeling by cathepsins was the main topic of a recent and comprehensive review (Fonović and Turk, 2014a), it will not be further analyzed here. Interestingly, a study has suggested the existence of a novel mode of regulation of cathepsins: indeed Cat S may proteolytically inactivate Cat K and thus would control its collagenolytic or elastinolytic potential (Barry and Platt, 2012).

Specific natural inhibitors: cystatins Family I25 (clan IH)

Cathepsins play a critical role in adaptive immunity. They are involved in both the release of antigenic peptides and in the maturation of MHC class II molecules (Honey and Rudensky, 2003). Cathepsins B and L participate in the degradation of antigens into antigenic peptides in association with other lysosomal proteases (Cat D, legumain) (Katunuma et al., 1998), whereas Cat S is essential for the generation of the invariant chain (Honey and Rudensky, 2003). Nevertheless, Cat S is not responsible for the generation of class II-associated invariant chain peptide in each antigen presenting cell type (Nakagawa et al., 1999). For instance murine Cat L participates in the generation of class II-associated invariant chain peptide in thymus cells, whereas it would be Cat V in humans (Tolosa et al., 2003).

The best-characterized inhibitors of cysteine cathepsins are the cystatins that belong to the MEROPS family I25 (clan IH) (for review see Abrahamson et al., 2003; Turk et al., 2008; Rawlings et al., 2012). Their members have structural and functional similarities and are classified into three main subfamilies: stefins (type 1), cystatins (type 2) and kininogens (type 3). They are potent, reversible and competitive inhibitors acting in intracellular compartments and in the extracellular environment (Bode and Huber, 1992). Members of the three families share another common property unrelated to their function of cysteine proteinase inhibitors: they act as potent immunomodulatory molecules (Kopitar-Jerala, 2006). Cystatins induce the synthesis of TNF-α and interleukin 10; in turn, these two cytokines up-regulate the production of nitric oxide by IFN-γ-activated macrophages (Verdot et al., 1996, 1999). Besides cystatins, there are also endogenous nonspecific inhibitors of cysteine cathepsins: α-2 macroglobulins (Barrett and Starkey, 1973), thyropins (Lenarcic and Turk, 1999) and some serpins (serine proteinase inhibitors) such as Squamous Cell Carcinoma Antigen 1, hurpin and endopin 2C (for review see Turk et al., 2002).

Remodeling of the extracellular matrix

Stefins

Cathepsins are essential participants in the remodeling and recycling of components of the extracellular matrix

Both stefins A and B (a.k.a. cystatins A and B) are found in humans and belong to the MEROPS subfamily I25A.

Antigen presentation


114 G. Lalmanach et al.: Cysteine cathepsins and cystatins in lung diseases Stefins are non-glycosylated single-chain proteins (∼100 amino acids, 11 kDa). Although they are primarily intracellular, stefins that are highly stable in a wide pH range are likely present in certain body fluids (Abrahamson et al., 1986). Human stefin A is heavily expressed in skin epithelial cells and blood cells, suggesting a role in defense against cysteine proteases produced by pathogens (Henskens et al., 1996). Moreover, in the tumor cells of squamous carcinomas stefin A is found in the environment of Cat L, thus advocating a protective role against uncontrolled proteolysis (Palungwachira et al., 2002). The expression of stefin B, in contrast, is wider and essentially cytoplasmic. Stefin B is also located in the nucleus, where it interacts with histones and may regulate the proteolytic activity of Cat L (Ceru et al., 2010). Stefin B-deficient mice develop a neurodegenerative disease called myoclonic epilepsy (Pennacchio et al., 1998). Stefin B has been shown to interact with amyloid-beta peptide of Alzheimer’s disease, and human stefins A and B may form amyloid fibrils (Zerovnik et al., 2010).

Cystatins Cystatins C, D, E/M, F, G, S, SA and SN belong to the MEROPS subfamily I25B (Rawlings et al., 2012). They consist of about 120 amino acids (∼13 kDa) and are synthesized with a signal peptide, which supports a primarily extracellular localization (Abrahamson et al., 2003). It does, however, have to be noticed that in human epithelial or neuroblastoma cells, a significant uptake of extracellular cystatin C has been reported (Ekström et al., 2008; Wallin et al., 2010). Accordingly, the conventional denotation of the type 2 cystatins as extracellular inhibitors might be questioned, since internalization of cystatin C could have potentially important physiological consequences (Wallin et al., 2010). Most of cystatins are not glycosylated in humans and their main characteristic is the presence of two C-terminal disulfide bonds (Turk et al., 2008). Cystatins are present in all biological body fluids (Abrahamson et al., 1986). Cystatin SN is primarily found in saliva and tears, whereas cystatins S and SA have retrieved from saliva and seminal fluids (Abrahamson et al., 2003). Cystatin F, which is a glycosylated protein, has an additional third disulfide bond and is mainly expressed by leukocytes and the spleen (Ni et al., 1998). High concentrations of cystatin E/M are found in the amniotic fluid, suggesting a possible role in fetal development (Ni et al., 1997). Cystatin G is mainly expressed in the seminal fluids and cystatin D in the parotid glands (Wallin et al., 2010).

The best-described inhibitor of this subfamily is cystatin C, which has a broad distribution in the body with particularly high concentrations in the cerebrospinal fluid and seminal plasma (Abrahamson et al., 1986). Readers are encouraged to refer to former publications for complementary information on the three-dimensional structure of cystatin C (e.g., Ekiel et al., 1997; Janowski et al., 2001). A single mutation in the gene encoding cystatin C (located on chromosome 20) is the cause of frequent hereditary amyloid angiopathy in Iceland (Palsdottir et al., 1988) characterized by the accumulation of amyloid fibers in the cerebral arteries causing bleeding, paralysis, dementia and premature death (Jensson et al., 1987). This substitution (Leu68Gln) is responsible for the aggregation of cystatin C, thereby forming dimer or tetramer proteins (Sanders et al., 2004). Cystatin C also has a neuroprotective role, inducing neuronal autophagy via the inhibition of the mammalian target of rapamycin pathway (Tizon et al., 2010). In addition to being a highly potent inhibitor of Cats K, L and S (Ki: pM range), cystatin C is also an effective inhibitor of legumain (MEROPS family C13) (AlvarezFernandez et al., 1999). Cystatin C secreted by vascular smooth muscle cells may have a beneficial role in atherosclerosis by inhibiting the elastinolytic activity of Cats K and S (Shi et al., 1999). Previous studies have supported the hypothesis that cystatin C has a protective role in the proteolytic dysregulation that is a characteristic of some lung diseases, such as silicosis and proteinosis (Lalmanach et al., 2006), or during tumor progression (Coulibaly et al., 1999). Recently, Liaudet-Coopman and colleagues have reported that cleavage of cystatin C by secreted acidic Cat D enhances the extracellular proteolytic activity of the cathepsins of breast cancer cells and favors tumor progression (Laurent-Matha et al., 2012).

Kininogens Kininogens (I25C subfamily), first known as the precursors of pro-inflammatory kinins and actors of the coagulation cascade (for review see Bhoola et al., 1992), were later identified as protease inhibitors (Barrett et al., 1986). The gene encoding human kininogens is located on chromosome 3. Resulting from an alternative splicing of messenger RNA, two kininogens are present in humans: high molecular weight kininogen (HMWK), with an apparent molecular mass of 90–120 kDa and low molecular weight kininogen (LMWK), with an apparent molecular mass of 50–70 kDa (Kitamura et al., 1985). Structurally, kininogens consist of an N-terminal heavy chain and a C-terminal light chain linked by a disulfide bridge. The heavy chains



of LMWK and HMWK are identical and encompass three tandemly repeated cystatin-like domains (D1, D2, and D3) linked to a 9-mer peptide (D4) corresponding to bradykinin, a proinflammatory hormone (for review see Lalmanach et al., 2010). The light chain of LMWK consists of a single domain (D5) while that of HMWK has two domains (D5, and D6) (Kitamura et al., 1985). Kininogens, via their D2 and D3 domains (Salvesen et al., 1986), are effective inhibitors of cysteine cathepsins (Ki: pm–nm range), with the exception of Cat B which cleaves HMWK in proteolysis-sensitive inter-domain regions (Naudin et al., 2010). One can note, however, that a single mutation (His110Ala) located at the occluding loop of Cat B restors a tight-binding inhibition (Ki = 8 pm) (Naudin et al., 2010). Kininogens, which are synthesized primarily by hepatocytes, are present at high concentrations in blood plasma, urine or sperm (Bhoola et al., 1992). Pharmacokinetics studies have demonstrated that native HMWK is predominantly concentrated in the lung, while cleaved HMWK was mostly found in the kidney (Schmaier et al., 1998). Kininogens were also found in the liver, spleen and skin, and to a lesser extent in white blood cells (Figueroa et al., 1992). Interestingly, it could be mentioned that cleaved forms of HMWK (i.e., HKa, kininostatin) have compelling anti-angiogenic properties (Guo and Colman, 2005). Moreover, several degradation products of HMWK as well specific HMWK-derived peptides display potent and broad-spectrum microbicidal properties (Frick et al., 2006; Oehmcke et al., 2009).

Lung fibrosis Etiologic characteristics Lung fibrosis, also referred as interstitial pneumonia, embraces a heterogeneous group of pulmonary disorders that are characterized by the emergence of scar tissues and formation of fibrotic areas. Various factors cause interstitial pneumonia (El-Kersh et al., 2013): smoking, prolonged exposure to particles (e.g., asbestos, animal/ plant dusts), physical agents (ionizing radiations), viral infections or autoimmune diseases such as rheumatoid arthritis, scleroderma and systemic lupus erythematosus (Noble et al., 2012). These interstitial lung diseases are characterized by both acute and chronic inflammation coupled with a progressive and irreversible fibrotic process. Fibrosis occurs in the alveolar spaces and interstitium and is characterized by a widespread accumulation of differentiated fibroblasts (i.e., myofibroblasts) and

ECM components (collagens, elastin, and fibronectin). Consequently, an aberrant repair causes the destruction of alveolar architecture and leads to a relentless decline in pulmonary function (Wynn, 2011). Fibrotic disorders are associated with the continuous production of growth factors and cytokines. Proteolytic enzymes also favor the excessive deposition of altered ECM components leading to matrix remodeling and alteration of the normal tissue architecture. The prevalence and incidence of lung fibrosis are usually underestimated, and to date there is no efficient therapeutic option to reverse fibrosis (Chakraborty et al., 2013). Interstitial pneumonia may also have an unknown etiology. Idiopathic interstitial pneumonia comprises seven chronic distinct disorders, defined on histological criteria (Demedts and Costabel, 2002). Among them, idiopathic pulmonary fibrosis (IPF) is the most common (∼60%) and severe form (Johnston et al., 1997). The prognosis of people suffering from IPF is poor, with an average survival of 3–5 years after diagnosis (Raghu et al., 2011). While the pathogenesis of IPF remains poorly understood, the condition is thought to result from repeated episodes of alveolar injury in individuals with dysfunctional alveolar wound-healing mechanisms (Selman et al., 2001). IPF may also be related to a predisposing genetic background or to the aging-related accumulation of metabolic alterations (pollutants, gastro-esophageal reflux, occupational exposures, viral infections and mechanical stress) (Maher et al., 2007). Based on the current hypothesis, IPF begins with one or more persistent external aggressions leading to the excessive secretion of cytokines and growth factors promoting the activation, proliferation and differentiation of interstitial fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts. The excessive ECM deposition implies an aberrant re-epithelialization, followed by alveolar scarring and irreversible loss of respiratory function (Wilson and Wynn, 2009).

Pro-fibrotic factors The injured epithelium is an important source of cytokines and growth factors that promote chemotaxis and the deposit of ECM components. Several cytokines are known to induce the differentiation of fibroblasts into myofibroblasts during lung repair, the most powerful and best known being transforming growth factor-β1 (TGF-β1) (Fernandez and Eickelberg, 2012). Other mediators include platelet-derived growth factor (PDGF) or TNF-α, which induces the proliferation, differentiation and secretion of ECM proteins (Kapanci et al., 1995). Overexpression of


116 G. Lalmanach et al.: Cysteine cathepsins and cystatins in lung diseases TNF-α leads to fibrosis in a murine experimental model. TNF-α-deficient mice have an increased resistance to fibrosis (Allen and Spiteri, 2002) whereas inhibition of PDGF by 5-methyl-1-phenylpyridin-2-one (i.e., pirfenidone) attenuates the development of fibrosis in an experimental rodent model (Gurujeyalakshmi et al., 1999). Also endothelin-1, a vasoactive peptide, induces myofibrogenesis (Swigris and Brown, 2010), and bronchoalveolar lavage fluids (BALFs) of fibrotic patients contain significantly higher concentrations of endothelin-1 than those of healthy people (Reichenberger et al., 2001). Damaged or inflammatory cells release other cytokines and growth factors during fibrosis, including interleukins (IL-4, IL-13, IL-1β, and IL-17A), insulin growth factor-1), and chemokines (CCL18, CCL2 and CCL12) (for review see Wynn, 2011). Mammalian TGF-β, which has three isoforms called β1, β2 and β3, respectively, is involved in many cellular processes such as proliferation, differentiation, motility, adhesion and apoptosis (Massagué, 1998). Disruption of its signaling pathways is associated with developmental disorders and illnesses such as cancer, autoimmune and cardiovascular diseases and fibrosis (Horbelt et al., 2012). While TGF-β2 and TGF-β3 are constitutively expressed in the lung, TGF-β1 is overexpressed in patients with fibrosis and this overexpression has been correlated with disease chronicity (Khalil et al., 1996). TGF-β1 is secreted by macrophages, epithelial cells and fibroblasts, and is a pivotal mediator of myodifferentiation, acting via both autocrine and paracrine mechanisms (Hinz et al., 2007). TGF-β1 is synthesized as an inactive pre-pro-peptide of 390 amino acids with an N-terminal signal peptide (30 residues). The propeptide latency associated peptide has three N-glycosylation sites. Two of them possess M-6-P residues allowing targeting to the endosomal/lysosomal compartments (Purchio et al., 1988). Different proteases (e.g., plasmin and furin) are involved in the release of mature and active TGF-β1 (Lyons et al., 1990; Dubois et al., 1995). Indirectly, Cat B may participate in this process via the activation of urokinase plasminogen activator (Guo et al., 2002). The maturation pathways of TGF-β1 remain in part a controversial subject; thereby, pro-TGF-β1 could be matured in the trans-Golgi (Dubois et al., 1995). Other studies indicate that pro-TGF-β1 is targeted to acid compartments, given the presence of M-6-P residues (Purchio et al., 1988). Pro-TGF-β1 may also be secreted directly before its activation by extracellular proteases (Taipale et al., 1994; Massagué, 1998). Finally, extracellular proTGF-β1 can associate transiently to latent TGF-β-binding protein, an ECM glycoprotein, to form a dormant protein complex (Miyazono et al., 1988).

TGF-β1 binds to a membrane serine/threonine kinase receptor, TGF-β1 Receptor II (TβRII), which in turn forms a heterodimer with TGF-β1 Receptor I and induces its phosphorylation. Besides the conventional signaling pathway of TGF-β1 (so called the canonical Smad pathway) (Gauldie et al., 2006), non-canonical pathways could contribute to the activation of genes involved in the fibrotic process (p38/ mitogen-activated protein kinase, extracellular signal-regulated kinase) and stress-associated protein kinase/c-Jun amino terminal kinase) (Derynck and Zhang, 2003).

Cysteine cathepsins and cystatin C in fibrotic diseases Cysteine cathepsins and rodent models of lung fibrosis Most of the studies describing the putative role of cathepsins during lung fibrosis depend on a murine model of experimental fibrosis induced by an anti-cancer agent, bleomycin. In this model bleomycin induces fibrosis following its administration by intratracheal instillation. This results in an accumulation of ECM components, an increase in cytokines and growth factors, apoptosis of type I pneumocytes, and activation of type II pneumocytes. Although this well-characterized model is sometimes questioned, it remains a paradigm of clinical relevance (Moore and Hogaboam, 2008). Bleomycintreated Cat K-deficient mice develop more severe fibrosis than wild-type mice, while primary Cat K-/- fibroblasts show a decrease in collagenolytic activity (∼40%) in agreement with the central role of Cat K in the degradation of collagen (Bühling et al., 2004). The expression of Cat K, however, increases both at the transcriptional and translational levels in normal murine fibroblasts following bleomycin administration. Similarly, primary fibroblasts from patients with lung fibrosis exhibit a greater transcriptional expression as well proteolytic activity of Cat K than those from non-fibrotic patients. This overexpression may be a foremost anti-fibrotic response following a profibrotic challenge sustaining that Cat K has a pivotal role in pulmonary homeostasis (Bühling et al., 2004). Although the molecular mechanisms involved in these processes are still not well understood, similar studies have confirmed a protective role of lung Cat K (Srivastava et al., 2008). Also, mice deficient in Cat K display a thicker epithelium and an increase in the expression of α-SMA and vimentin. After treatment with TGF-β1, murine



lung fibroblasts proliferated faster and an increase in secretion of ECM components was observed, emphasizing the involvement of Cat K in the structural integrity of airways (Zhang et al., 2011a). It is of interest to note that an imbalance of cathepsins/inhibitors ratio to the detriment of enzymes was found in a rat model of bleomycininduced fibrosis. No peptidase activity of cathepsins B, L, H, and S was detected in the BALFs of rats (day 7), before the balance was partly restored during the later phase of tissue remodeling (day 14) (Koslowski et al., 2003).

Cysteine cathepsins and fibrosis Fibrotic disorders are associated to a dysregulation of proteolytic activities. There are increasing indications that cathepsins might be involved, although their exact

functions remain to be clarified, which probably account for some apparently contradictory reports. For instance, Niemann-Pick disease is an inherited lysosomal storage disease caused by a shortfall in acid sphingomyelinase (ASMase), which is frequently associated with liver fibrosis, and may progress to cirrhosis. Interestingly, hepatic stellate cells (HSCs) from ASMase-null mice exhibit increased basal level and activity of Cat B, paralleling the enhanced expression of TGF-β1 and the fibrogenic marker α-SMA (Table 1). In addition to the liver, enhanced proteolytic processing of Cat B was observed in the lungs of ASMase-knock-out mice. Also, expression of Cat B is augmented during HSCs activation and matches the increase in TGF-β1 and α-SMA, supporting the hypothesis that Cat B may drive HSCs’ trans-differentiation and hence participate in fibrogenesis (Moles et al., 2009). Taken together, Mari and colleagues suggested that targeting Cat B might

Table 1 Cysteine cathepsins in fibrotic disorders. Disease

Cat

Study findings

Cardiac fibrosis

B L

S

– Deficiency attenuates cardiac dysfunction and fibrosis –D eficient mice develop dilated cardiomyopathy and interstitial fibrosis in the myocardium – Overexpression decreases cardiac hypertrophy, inflammation and fibrosis – Simvastatin-reduced cardiac hypertrophy and fibrosis is associated with a decreased expression – Deficiency enhances angiotensin-induced fibrosis – I nactivation suppresses fibroblast trans-differentiation, collagen deposition and extra domain-A fibronectin expression – Increased levels in the sera of patients –C at B inactivation reduces fibrogenesis during cholestasis in bile duck ligated mice – I nvolvement in hepatic stellate cells trans-differentiation and in fibrogenesis in vivo –N uclear Cat F regulates the transcriptional expression of α-smooth muscle actin and collagen I in hepatic stellate cells – I ts inhibition delays transforming growth factor (TGF)-β1-induced fibroblast differentiation and leads to the increased expression of intracellular pro-TGF-β1 –D eficiency is associated with severe fibrosis and decreased collagenolytic activity in fibroblasts –O verexpression leads to a decrease in collagen deposition and improved lung function – I nvolvement in the structural integrity of the lung airway and in TGF-β1 degradation –A nti-fibrotic effects of curcumin are associated with an increased expression of both proteases – Its inhibition decreases fibrosis in acidic sphingomyelinase-deficient mice

Liver cirrhosis Liver fibrosis

B, L B

Lung fibrosis

F

B

K

K, L

Niemann-Pick disease Skin fibrosis

B

B K

V

– Increased concentration in the sera of patients – Strong expression in the dermal fibroblasts of young surgical scars, in keloid and dermatofibroma scars –D ecreased expression in the sera of patients, in microvascular endothelial cells, vascular smooth muscle cells and keratinocytes


References Liu et al., 2013 Stypmann et al., 2002; Spira et al., 2007 Tang et al., 2009 Qin et al., 2010 Pan et al., 2012 Chen et al., 2013 Leto et al., 1997 Canbay et al., 2003 Moles et al., 2009, 2010 Maubach et al., 2008 Kasabova et al., 2014a

Bühling et al., 2004 Srivastava et al., 2008 Zhang et al., 2011a Zhang et al., 2011b Moles et al., 2012 Noda et al., 2012 Rünger et al., 2007 Noda et al., 2013

118 G. Lalmanach et al.: Cysteine cathepsins and cystatins in lung diseases be relevant in the treatment of liver fibrosis in patients with Niemann-Pick disease (Moles et al., 2012). Moreover, genetic inhibition of Cat B in a murine bile duct ligation model reduced hepatic inflammation, collagen deposition and fibrogenesis (Canbay et al., 2003). Similarly, Cat B contributes to lung fibroblast differentiation into myofibroblasts by triggering the TGF-β1-driven canonical Smad pathway (Kasabova et al., 2014a), supporting the theory that Cat B can act as signaling scissors (Figure 1A, B) (for review see Turk et al., 2012). Also, pharmacological inhibition and genetic silencing of Cat B diminished α-SMA expression, delayed fibroblast differentiation and led to an accumulation of intracellular pro-TGF-β1 (Kasabova

et al., 2014a). Similar statements were found to be true for primary IPF fibroblasts. Other members of the family, including cathepsins K, F, L, S and V, were reported to be biological markers or putative actors of cardiac and skin fibrotic disorders (as summarized in Table 1). Regarding lung fibrosis, Cat K has been proposed to be an essential effector in lung homeostasis since its deficiency exacerbates lung fibrosis (Bühling et al., 2004), Conversely, Cat K is able to inactivate TGF-β1 and restrict excessive ECM deposition (Zhang et al., 2011a). TGF-β1, in contrast, downregulates both Cat K expression in fibroblasts in a model of silica-induced fibrosis (Van den Brûle et al., 2005) and Cat L expression in lung epithelial cells (Gerber et al., 2001).

A

C

B TGF-β1 TGF-β1

CC TGF-β1

TGF-β1

B CC TβRII

TβRI P P Smad 2/3

P Smad 4 Smad 2/3

Cats

CC

P Smad 4 Smad 2/3 ECM components

B

pro-TGF-β1

Cat B α-SMA

CC

Cyst C

Figure 1 Role of cathepsin (Cat) B and cystatin C in the myodifferentiation of human lung fibroblasts. The hypothesis we propose is the following: (A) lysosomal Cat B may participate in the intracellular proteolytic activation of TGF-β1; (B) bioactive TGF-β1 binds to its specific receptors and triggers Smad 2/3 phosphorylation and differentiation of lung fibroblasts. (C) Otherwise, TGF-β1 up-regulates secretion of cystatin C. Consequently, the cystatin C-dependent inhibition of extracellular matrix-degrading cathepsins may favor collagen deposition and promote fibrosis. (A, B) Immunolabeling of α-SMA. (C) Immunolabeling of cystatin C. CCD-19Lu fibroblasts were cultured in the absence (A) or presence (B, C) of TGF-β1 (10 ng/ml) for 48 h. Immunolabelling was performed using a mouse anti-α-SMA antibody (Sigma-Aldrich, St Louis, MO, USA) (1:100) or a mouse anti-cystatin C antibody (R&D Systems, Minneapolis, MN, USA) (1:100). Nuclei were stained with Hoechst dye (1:300). Photomicrographs were acquired with a Leica DMR fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). (Adapted from Kasabova et al., 2014a.)



Cystatin C and fibrosis Besides its up-regulation in profibrogenic stellate cells, a high serum concentration of cystatin C, closely related to the progression of liver disorder (hepatic fibrosis or cirrhosis) is detected in patients with stable serum creatinine (Table 2). Likewise, the expression of cystatin C is significantly increased at both transcriptional and protein levels in patients with oral submucous fibrosis, a disease associated with betel (areca) nut chewing as well chronic exposure of the buccal mucosa to chili and pepper (Chung-Hung et al., 2007). After an early stage of stomatitis, oral submucous fibrosis is characterized by an epithelial atrophy and a progressive accumulation of collagens in the submucosal tissues (lamina propria and deeper connective tissues). Cystatin C as well Cat S are currently being evaluated as putative biomarkers in multiple sclerosis (Haves-Zburof et al., 2011). Dosage of serum creatinine is the most widely used technique for measuring glomerular filtration rate (GFR) (Swedko et al., 2003). Although less broadly used in the clinical routine, cystatin C has been also identified as a promising and valuable biomarker of GFR (sometimes reported as ‘The Lund Model’) (for review see Grubb, 2010) because its dosage is less sensitive to interference due to factors such as age, muscle mass or patients’ diet (Filler et al., 2005). There is evidence now to combine the dosage of both serum creatinine and cystatin C for better diagnosis of kidney disease (Levey et al., 2014). Recently, a notably enhanced protein level of cystatin C in the BALFs of IPF patients was reported (Kasabova et al., 2014b). The increased concentration of alveolar cystatin C is significant for each IPF grade (stage I to stage III). No noteworthy variations in renal clearance and serum creatinine levels were observed, upholding the hypothesis that the

increased concentration of cystatin C in IPF BALFs did not rely on kidney damage. Since patients with chronic renal failure do not have a greater risk of developing IPF, data support the fact that increase in cystatin C relates directly to the pathogenesis of fibrosis and that cystatin C could be a specific and valuable biomarker of lung fibrosis. Furthermore, secretion of cystatin C increases during TGF-β1-induced differentiation of IPF fibroblasts. Gene silencing of cystatin C restored the proteolytic activities of extracellular cathepsins, supporting the theory that TGFβ1 promotes fibrosis by driving the cystatin C-dependent inhibition of cathepsins (Figure 1C) (Kasabova et al., 2014a). Since ECM remodeling is the result of a subtle balance between synthesis and degradation, increased secretion of cystatin C might favor the development of lung fibrosis by impairing the collagenolytic activity of ECM-degrading cathepsins. More mechanistic studies of molecular mechanisms involved in the pathogenesis of lung fibrosis have to be undertaken, however, to clarify putative roles of cystatin C both in vivo and in vitro.

Cysteine cathepsins in other lung diseases Silicosis Environmental or occupational extended exposure to crystalline silica particles is associated with the promotion of silicosis. Silica particles activate lung macrophages that produce reactive oxygen species, various cytokines and chemokines. In turn they stimulate fibroblasts, which result in the formation of nodular lesions and the enlargement into fibrotic lesions (Ding et al., 2002). The

Table 2 Cystatin C in fibrotic disorders. Disease

Study findings

References

Cardiac fibrosis

Xie et al., 2010

Idiopathic pulmonary fibrosis

– Increased expression in cardiomyocytes and plasma. Role in cardiac extracellular matrix remodeling Increase of immunoreactive cystatin C in the bronchoalveolar lavage fluids of idiopathic pulmonary fibrosis patients. Potential use as a biomarker. – Proposed as a more pertinent marker than serum creatinine for estimating glomerular filtration rate – Elevated concentrations in sera of patients.

Kasabova et al., 2014b

Rognant and Lemoine, 2014

Liver cirrhosis

Liver fibrosis

Oral submucous fibrosis

Potential use as a biomarker. – Transforming growth factor-β1 induces cystatin C secretion during hepatic stellate cell trans-differentiation – Strong immunostaining in fibroblasts, endothelial and inflammatory cells


Takeuchi et al., 2001; Chu et al., 2004; Ladero et al., 2012 Gressner et al., 2006

Chung-Hung et al., 2007

120 G. Lalmanach et al.: Cysteine cathepsins and cystatins in lung diseases associated inflammatory effects of crystalline silica could be mediated by the Nalp3 inflammasome (Cassel et al., 2008). Exposure of monkeys to quartz aerosols leads to an increased release of superoxide anion by bronchoalveolar macrophages, as well as high levels of α1-proteinase inhibitor, despite a slight but constant increase of free elastase-like activity (Hannothiaux et al., 1991). Also, an increase of MMP-2 (gelatinase A), MMP-9 (gelatinase B) and stromelysin was reported in alveolar macrophages from silica-treated rats contributing to ECM and basement membrane disruption (Scabilloni et al., 2005). Administration of silica particles to mice causes an up-regulation of Cat K transcripts as well as significantly higher activity in silicotic lung homogenates compared to control lungs. Lung fibroblasts and macrophages were identified as the main Cat K-producing cells. Expression of Cat K is inversely correlated to the level of TGF-β1, suggesting a protective role of Cat K during silicotic process (Van den Brûle et al., 2005). Mature active Cats B, H, K, L, and S were identified in the BALFs from patients suffering from silicosis. Among them, Cat H was the most abundant aminopeptidase; Cats B and L were the most abundant thioldependant endoproteases; and the cathepsins/inhibitors ratio was dysregulated in favor of uncontrolled proteolysis (Perdereau et al., 2006). The overall concentration of active cathepsins is ∼10 times lower in silicotic BALFs compared to those from patients with acute inflammatory lung diseases (Lalmanach et al., 2006).

Cystic fibrosis The pathogenesis of cystic fibrosis (CF), a rare genetic disease, is associated with mutations of the cystic fibrosis transmembrane conductance regulator gene that leads to the synthesis of truncated or improperly addressed cystic fibrosis transmembrane conductance regulator protein. The disturbance in ion transport generates an increase of the mucus viscosity and its accumulation in the airways, creating a breeding ground for the multiplication of opportunistic pathogens as Pseudomonas aeruginosa, Staphylococcus aureus and Haemophilus influenzae. Recurrent exacerbations of inflammation and infection lead to a decline in lung function, respiratory failure, and ultimately death (Vankeerberghen et al., 2002). The deficiency of transmembrane conductance regulator and the subsequent accumulation of several anions (e.g., HCO3- and Cl-) induce an acidification of airway surface liquid. Consecutively, the decrease in pH causes a reduction in ciliary beat frequency and antimicrobial protein activities whereas acidic pH favors mucin viscosity and

cathepsin activity (for review see Berkebile and McCray, 2014). In addition to this, contrary to healthy volunteers, the epithelial lining fluids of CF patients are at a pH close to optimal conditions for cathepsins activity and stability (Tate et al., 2002). An early article McElvaney and collaborators reported the important enzymatic activity of human cathepsins B, H, L and S in CF BALFs (Taggart et al., 2001). Cathepsins B, L and S hydrolyze and inactivate antimicrobial peptides (e.g., β-defensin-2 and -3) at the respiratory epithelium of CF patients, leading to the loss of their antimicrobial properties toward P. aeruginosa (Taggart et al., 2003). Cathepsins B, L and S cleave both lactoferrin, an innate immunity protein that inhibits the formation of bacterial biofilms (Rogan et al., 2004), and secretory leucoprotease inhibitor, impairing its antimicrobial and antiinflammatory properties (Taggart et al., 2001). Moreover, Cat L inactivates human α1-proteinase inhibitor (Johnson et al., 1986) and thus favors uncontrolled proteolytic activity. Cathepsins B and S expressions were correlated with levels of human neutrophil elastase (HNE) and IL-8, and they were proposed as markers of CF airway inflammation (Martin et al., 2010). Nevertheless, concentrations of active cathepsins B, H, K, L and S were the same in P. aeruginosapositive and P. aeruginosa-negative samples, unlike those of HNE, which support the theory that cathepsins are not suitable biomarkers of bacterial infection in this disease (Naudin et al., 2011). Type II pneumocytes secrete pulmonary surfactant whose main function is to reduce the surface tension at the air/liquid interface (Garcia-Verdugo et al., 2010). The hydrophobic surfactant proteins SP-B and SP-C are mainly involved in the reduction of surface tension, whereas the hydrophilic proteins SP-A and SP-D have an important role in innate lung immunity (Frerking et al., 2001). Cat H is involved in the processing of SP-C in human pneumocytes II (Brasch et al., 2002) as well as in SP-B production, and has a beneficial role in mice pulmonary homeostasis (Bühling et al., 2011). Immunohistochemical studies have shown that both Cat S and SP-A have a matching distribution in distal bronchial and bronchiolar/alveolar epithelia (Figure 2). Interestingly, Cat S specifically hydrolyzes human SP-A within residues of the solvent-exposed loop of its carbohydrate recognition (C-type lectin) domain that allows binding to pathogens. Accordingly, Cat S decreases the aggregation properties of SP-A and impairs the agglutination of P. aeruginosa (Lecaille et al., 2013). It appears that cathepsins could display immuno-modulatory functions that finely tune the increase in bacterial infections during lung disorders by inactivating key proteins involved in the innate immunity response.



Asthma Asthma is a chronic inflammatory disease characterized by the activation of several cell types (mast cells, neutrophils and eosinophils) and the spread of Th2-cells, leading to excessive cytokine production and airway remodeling (Hamid and Tulic, 2009). Administration of E-64 (a broad-spectrum cathepsin inhibitor) to ovalbumin-challenged mice decreases lung weight, number of eosinophils, IL-5 levels and also T-cell migration into the lymph nodes (Layton et al., 2001). Cat S has been associated with asthma pathogenesis (Small et al., 2011), and the use of human Cat S as a biomarker reflecting the extent of the disease has been proposed (Cimerman et al., 2001). High levels of Cat S have also been detected in a mouse model of induced allergic inflammation (Fajardo et al., 2004), where its pharmacological inhibition blocks the infiltration of eosinophils, macrophages and neutrophils and reduces oxidative stress and inflammation (Williams et al., 2009). Likewise, prophylactic administration of a specific Cat S inhibitor to mice reduces ovalbumininduced inflammation (Deschamps et al., 2010). Besides Cat S, Cat F may modulate the excessive immune response associated with this pathology (Somoza et al., 2002). A recent paper reported that Cat K digests tumstatin, a potent anti-angiogenic collagen IV fragment whose levels are reduced in asthma subjects (Faiz et al., 2013).

Chronic obstructive pulmonary disease

Figure 2 Immunohistochemical analysis of lung cathepsin (Cat) S and SP-A. Formalin-fixed and paraffin-embedded tissue samples were obtained from the Department of Histopathology (University Hospital Center Trousseau, Tours, France). Serial sections of alveolar lung tissue were prepared from normal lung biopsies. (A) Immunodetection of thyroid transcription factor 1 (TTF-1), a nucleus marker of type II pneumocytes and Clara cells, was used as the control. (B) Immunostaining of SP-A. (C) Immunostaining of Cat S. The anti-TTF-1 antibody was from Dako France SAS (Trappes, France). Anti-human SP-A and anti-human Cat S antibodies were supplied by Abcam (Cambridge, UK). Staining was conducted using 3’3-diaminobenzidine chromogen (brown precipitate) according to the manufacturer

Chronic obstructive pulmonary disease (COPD) represents a major health problem that should become the fourth worldwide most common disability in 2030 (Ojo et al., 2014). As defined by the Global Initiative for Obstructive Lung Disease program, COPD is a ‘progressive and irreversible limitation of airflow, associated with an abnormal inflammatory response in the lungs to noxious particles and gases’ (Pauwels et al., 2001). The progressive airway obstruction may lead to death by respiratory failure. The main cause of COPD is tobacco, but other household pollutants or extended exposures to

(Dako). Microscope analysis was done using a Nikon E600 optical microscope (magnification: 20–1000). Images were captured (Olympus DP70 camera) and processed with the Olympus DP Controller software. M, macrophage; PII, type II pneumocytes. We acknowledge Prof Serge Guyetant (University Hospital Center Trousseau, Tours, France) for providing lung tissue samples. We are most grateful to Dr Agnès Petit-Courty (INSERM UMR 1100/CEPR, Tours, France) who carried out immunohistochemistry analysis.


122 G. Lalmanach et al.: Cysteine cathepsins and cystatins in lung diseases infectious agents may promote lung deterioration. COPD mainly refers to: i) chronic bronchitis that is characterized by excessive secretion of mucus and small airways obstruction; and ii) emphysema that is characterized by an enlargement of airspaces, a destruction of the lung parenchyma and the loss of lung elasticity (Tuder and Petrache, 2012). During inflammatory episodes, activated neutrophils and macrophages release substantial amounts of reactive oxygen species and proteases (Wada and Takizawa, 2013). Cathepsins B, S, L, H and K are overproduced and secreted during inflammation of the lungs of transgenic mice with emphysema in response to mediators such as IL-13 and IFN-γ (Wang et al., 2000; Zheng et al., 2000). In addition to TNF-α, IFN-γ and IL-6, mice exposed to ozone present an increased level of BALF Cat S; moreover, inhibition of Cat S reduces airway hyper-responsiveness and neutrophil recruitment, suggesting that the enzyme could be a potential target in the treatment of oxidative stress-induced inflammation (Williams et al., 2009). An amplified expression of Cat S was also observed in epithelial cells in a murine model of IFN-γ-induced emphysema (Zheng et al., 2005), while macrophages incubated with BALFs from patients with COPD showed an increased expression of Cat S, which was inhibited by IFN-γ-neutralizing antibodies (Geraghty et al., 2008). Cigarette smoke up-regulates IL-18 in macrophages in accordance with the overexpression of lung cathepsins B and S for patients with COPD who actively smoke (Kang et al., 2007). In a guinea pig model of emphysema, a significant decrease in collagens and elastin was associated with a three-fold spread of Cat K activity (Golovatch et al., 2009). Emphysematous patients also display an enlarged expression of lung Cat K compared to healthy subjects, which relates to the disruption of lung ECM. Lastly, the hypothesis of using Cat B as a ‘downstream biomarker’ of local HNE activity during COPD was evoked (Stockley, 2014).

Bronchopulmonary dysplasia Bronchopulmonary dysplasia (BPD) is a common complication in prematurely born infants that regularly requires prolonged oxygen mechanical ventilation due to a deficiency of lung development. The main characteristics of BPD are chronic inflammation, alveolar hypoplasia and respiratory infections (Coalson, 2003; Madurga et al., 2013). An imbalance between proteases and their inhibitors is involved in the pathogenesis of BPD. A high elastase activity, associated with excessive degradation of the elastic fibers and reduced levels of endogenous

inhibitors, is often found in the lung secretions of BPD patients (Bruce et al., 1992). In a baboon model of BPD, a significant increase in mRNA and protein expression as well as in the proteolytic activities of cathepsins B, H, K, L and S was observed while the expression of stefin B and cystatin C remained constant (Altiok et al., 2006). Hyperoxic Cat S-deficient mice revealed enhanced alveolarization and macrophage influx, suggesting a deleterious role of Cat S in BPD (Hirakawa et al., 2007). Also, in tracheal aspirates from BPD newborns, a decreased expression of Cat K, probably associated with an increase of extracellular collagen, was detected (Knaapi et al., 2006). Whereas 20% of wild-type mice survived for 2 weeks in hyperoxia, all Cat K-deficient mice died within the first 9 postnatal days (Knaapi et al., 2011). Contrastingly, hyperoxia is better tolerated in transgenic mice overexpressing Cat K than in wild-type littermates, sustaining the theory of Cat K having a protective role in BPD (Knaapi et al., 2014).

Rare pulmonary diseases Lymphangioleiomyomatosis, a rare and progressive lung disease that usually affects women of premenopausal age, is characterized by the infiltration of smooth muscle cells that express contractile proteins such as α-SMA and desmin. Immunohistochemical studies have demonstrated a strong expression of Cat K that was restricted to lymphangioleiomyomatosis cells, and Cat K was proposed as a possible marker for diagnosis (Chilosi et al., 2009). Intense immunostaining for Cat K was also observed in epithelioid and giant cells in a variety of granulomatous diseases (e.g., sarcoidosis, Wegener’s granulomatosis, berylliosis and tuberculosis) (Reghellin et al., 2010). In a mouse model of sarcoidosis, the lack of Cat K increased the incidence of lung granulomas, collagen deposition and the number of multinucleated giant cells, while a lack of Cat L and Cat S activities prevented the formation and/or development of granulomas (Samokhin et al., 2011), suggesting that Cat inhibitors may be useful for the treatment of lung sarcoidosis. Increased Cat H expression was reported in BALFs from patients suffering from pulmonary proteinosis (Woischnik et al., 2008). Active cathepsins B, H, K, L and S were detected in BALFs and the corresponding zymogens autoactivate under acid conditions (ServeauAvesque et al., 2006). It may be hypothesized that proforms provide a reservoir of active enzymes that may further contribute to the imbalance between cathepsins and their endogenous inhibitors.



Lung cysteine cathepsins as biomarkers and therapeutic targets In addition to be relevant clinical targets, innovative proteomic-based methods have emerged for the identification of new physiological substrates of cathepsins. Their significance as prognostic and diagnostic biomarkers has been discussed elsewhere in a recent review (Fonović and Turk, 2014b). Their endogenous inhibitors, i.e., stefins and cystatins, however, have also been considered as potential valuable markers of various disorders (e.g., malignant tumors, fibrosis or renal failure) (Kos et al., 2000; Grubb, 2010; Kasabova et al., 2014b). Cat K has been validated for the treatment of osteoporosis and bone metastases (for reviews see Vasiljeva et al., 2007; Brömme and Lecaille, 2009; Clézardin, 2011). Since 2004, over 50 patents have been filed on Cat K inhibitors. Among them, odanacatib is currently in clinical phase III trials (Rachner et al., 2011). Another inhibitor of Cat K, ONO-5334, following a successful phase II study, is in a clinical phase III trial (the OCEAN study) for the treatment of postmenopausal osteoporosis (Engelke et al., 2014). Also, a promising small allosteric inhibitor of the collagenolytic activity of Cat K was identified by high-throughput computational docking methods (Novinec et al., 2014). Nevertheless, some Cat K inhibitors may have side effects. For instance, inhibition of Cat K in arthritis may lead to lung and skin fibrosis. Recently, a reversible Cat B inhibitor (VBY-376 from Virobay, Meno Park, CA, USA) has been tested for hepatic fibrosis. The initial results of the study (phase I clinical trial) look promising for the future. The development of Cat S inhibitors for the treatment of autoimmune diseases, psoriasis, multiple sclerosis and rheumatoid arthritis began at the same time as those of Cat K (for reviews see Palermo and Joyce, 2008; Kasabova et al., 2011; Small et al., 2011). Studies have progressed slowly, however, despite some of the inhibitors currently being in clinical trials (Lee-Dutra et al., 2011). While the use of Cat inhibitors is widely endorsed for disorders ranging from rheumatoid arthritis to bone diseases, it is still a prerequisite to learn more about the biological properties of lung cathepsins. Indeed, little is known about the network of proteolytic interactions and signaling pathways that modulate cathepsin activities in lung tissues. Deciphering such molecular events is a crucial step to avoid side-effects such as those observed some years ago with inhibitors of metalloproteases (Turk, 2006). Cat S could be a valuable target in the treatment of oxidative stress-induced airway hyper-responsiveness (Williams et al., 2009), while regulating the deleterious elastinolytic activity of Cat S could be helpful during

bronchopulmonary dysplasia and emphysema (Zheng et al., 2005; Hirakawa et al., 2007). Moreover, inhibition of Cat S in a prophylactic paradigm could reduce airway inflammation and be advantageous during asthma treatment. Hence some inhibitors are currently in preclinical trials for treating bronchial asthma (Vasiljeva et al., 2007). According to its ‘beneficial’ role in pulmonary homeostasis, Cat K does not appear to be a relevant target. Instead, Brömme and colleagues have made an attractive proposal, suggesting that the administration of Cat K-inducing drugs may be beneficial in the treatment of lung fibrosis (Zhang et al., 2011b). The over-expression of Cat K, and to a lesser extent that of Cat L, was accompanied by a decrease in the deposition of collagen and TGF-β1 expression. We recently reported that lung Cat B could play a crucial role in myofibrogenesis and the progression of pulmonary fibrosis. Taken together, both studies supporting the fact that Cat B drives activation of hepatic stellate cells, and hence participates in liver fibrogenesis, and promising phase I trials for the treatment of hepatic fibrosis (Cat B inhibitor, VBY-376), indicate that the use of Cat B inhibitors could be appropriate for therapy of lung fibrosis. Acknowledgments: We apologize for any references omitted due to space limitations. We thank la Région Centre for their financial support (FibroCat project). We acknowledge the Institut National de la Santé et de la Recherche Médicale for institutional funding. Dr Mariana Kasabova had former doctoral scholarship from Ministère de l’Education Nationale, de la Recherche et de la Technologie, France. The authors have declared no conflict of interest.

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