a novel therapeutic target in various lung diseases: airway proteases and protease-activated...

Pharmacology & Therapeutics 115 (2007) 70–83www.elsevier.com/locate/pharmthera

Associate editor: C.E. Müller

A novel therapeutic target in various lung diseases: Airwayproteases and protease-activated receptors

Elena Sokolova, Georg Reiser ⁎

Otto-von-Guericke-Universität Magdeburg, Medizinische Fakultät, Zentrum für Biochemie und Molekularbiologie,Institut für Neurobiochemie, Leipziger Strasse 44, D-39120, Magdeburg, Germany

Abstract

Protease-activated receptors (PAR), which are G protein-coupled receptors, have 4 members, PAR-1 to PAR-4. PARs are activated byproteolysis of a peptide bond at the N-terminal domain of the receptor. PARs are widely distributed throughout the airways. Their activity ismodulated by airway proteases of endogenous and exogenous origin, which can either activate or disable the receptors. The regulation of PARactivity by proteases is important under pathological conditions when the activity of proteases is increased. Moreover, various inflammatorymediators, such as cytokines, growth factors, or prostanoids, alter the PAR expression level. Elevated PAR levels are observed in various lungdisorders, and their significance in the development of pathological situations in the lung is currently intensively investigated. Consequences ofPAR activation can be either beneficial or deleterious, depending on the PAR subtype. PAR-1 has been shown to be an important player in thedevelopment of pulmonary fibrosis. Thus, PAR-1 represents an exciting target for clinical intervention in fibrotic diseases. PAR-2 contributes toallergic airway inflammation. However, the question whether the impact of PAR-2 is beneficial or deleterious is still under intensive discussion.Therefore, precise information concerning the participation of PAR-2 in various lesions is required. Moreover, it is necessary to generate selectivePAR- and organ-targeted approaches for treating the diseases. A thorough understanding of PAR-induced cellular events and the consequences ofreceptor blockade may help in the development of novel therapeutic strategies targeted to prevent lung destruction and to avoid deterioration ofconditions of patients with inflammatory or fibrotic lung diseases.© 2007 Elsevier Inc. All rights reserved.

Keywords: Inflammation; Prostaglandin E2; Protease-activated receptor; Pulmonary fibrosis; Serine protease

Abbreviations: APC, activated protein C; ASM, airway smooth muscle; CTGF, connective tissue growth factor; EGFR, epidermal growth factor receptor; HAT,human airway trypsin-like protease; HPAEC, human pulmonary artery endothelial cells; IL, interleukin; LPS, lipopolysaccharide; MMP, matrix matelloproteinase;OVA, ovalbumin; PAR, protease-activated receptor; PDGF, platelet-derived growth factor; PG, prostaglandin; TGF, transforming growth factor; TNF, tumournecrosis factor.

⁎ Corresponding aE-mail address:

0163-7258/$ - see fdoi:10.1016/j.pharm

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712. General aspects of protease-activated receptor activation . . . . . . . . . . . . . . . . . . . . . 713. Expression of protease-activated receptors in human lung tissue . . . . . . . . . . . . . . . . . 72

3.1. Expression of protease-activated receptors in human lung tissue and in cultured airwaycells under normal conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723.2. The influence of pathological conditions on the expression of protease-activated receptorsin the human lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

4. Role of protease-activated receptors in pathological situations in the airways . . . . . . . . . . 735. Protease-activated receptors in proliferative disorders . . . . . . . . . . . . . . . . . . . . . . . 74

uthor. Tel.: +49 391 6713088; fax: +49 391 [email protected] (G. Reiser).

ront matter © 2007 Elsevier Inc. All rights reserved.thera.2007.04.002

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http://dx.doi.org/10.1016/j.pharmthera.2007.04.002

71E. Sokolova, G. Reiser / Pharmacology & Therapeutics 115 (2007) 70–83

6. Regulation of protease-activated receptor expression and activity . . . . . . . . . . . . . . . . . 756.1. Regulation of protease-activated receptor expression by inflammatory mediators. . . . . . 756.2. Control of protease-activated receptor activity by proteases . . . . . . . . . . . . . . . . . 75

6.2.1. Endogenous proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 756.2.2. Exogenous proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766.2.3. Specific features of protease action on protease-activated receptors . . . . . . . . 77

7. Scenario of protease-activated receptor activity in airway cells. . . . . . . . . . . . . . . . . . . . . 778. Protease-activated receptors in pharmacological applications and protease-activated receptors as

possible drug targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80Aknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Fig. 1. Activation of protease-activated receptors by proteases which are active inthe respiratory tract. The receptor comprises an extracellular N-terminus (N), 7transmembrane domains and an intracellular C-terminus (C). Proteases ofexogenous origin (from airborn allergens) and endogenous origin (belonging tothe coagulation system, released from inflammatory cells, extrapancreatictrypsin) cleave the N-terminal part of PARs and unmask the tethered ligand(shown as a black box). Binding of this tethered ligand to the second extracellularloop of the receptor results in receptor activation and induction of cell signaling.

1. Introduction

Tissue repair with accompanied inflammation in the injuredlung is characterized by increased concentrations of serineproteases belonging to the coagulation system. Moreover, in theinflamed airways elevated activity of serine proteases, which arereleased from cells of the immune system recruited to the lung, isobserved. Bacterial infections also contribute to elevated levelsof proteases in airways by secretion of their own enzymes intothe environment. All of these proteases participate in tissueremodeling, development of inflammation, and hyperreactivityin the lung. Importantly, these proteases not only cleaveextracellular substrates but also directly induce cellular effectsvia binding to cell receptors. Some receptors become activatedafter binding of proteases in a “classic”manner without cleavageof the receptor [e.g., receptors for factor Xa, activated protein C(APC), or urokinase]. The so-called protease-activated receptors(PAR), however, are activated by limited proteolysis as ex-plained below (Section 2). Therefore, PARs are an importantfactor for lung tissue functioning.

The functional activity of the receptor depends on its level ofexpression, and “silent” receptors may gain an important role underpathological conditionswhen receptor expression is upregulated bydifferent stimuli as discussed below. Therefore, the modulation ofthe receptor expression levels represents an important regulatorymechanism of PAR activity. Another mechanism of regulation ofPAR activity involves activation/inactivation of the receptors byproteases.A number of proteases, such as those fromhematopoieticinflammatory cells or from bacteria, exhibit incapacitation pro-perties. They cleave PARs downstream of the activation site.Moreover, the same receptor may mediate different or even oppo-site cellular responses, depending on the activating protease or thecell type. In this review, we summarize the regulation of PARexpression by inflammatory mediators and the control of PARactivity by airway proteases and describe possible implications ofPARs in some lung diseases.

2. General aspects of protease-activated receptor activation

PARs are G protein-coupled receptors which require, fortheir activation, proteolytic cleavage of a certain peptide bond(activation site) of the receptor protein, which is located in theN-terminal extracellular domain. The PAR family consists of 4

subtypes, PAR-1 to PAR-4, which are defined by different geneproducts. Each PAR subtype has a unique activation site whichis recognized by specific proteases, and its cleavage results inunmasking a new N-terminus, which serves as an intramolec-ular tethered ligand (Macfarlane et al., 2001). Fig. 1 presents ascheme for the mechanism of PAR activation by proteolysis. Theintact 7-transmembrane domain receptor (depicted on the leftside) undergoes transformation by cleavage through a specificprotease. Proteolytic activity generates the new N-terminus.Binding of this newly formed tethered ligand to the extracellularloop 2, as shown on the right side, causes subsequent receptoractivation and triggering of cellular responses. Some of theactivating proteases, which are active in the respiratory tract, arealso indicated besides the cleavage site in Fig. 1.

PAR activation until recently was ascribed exclusively to thefamily of serine proteases, like thrombin and trypsin. However,recent findings have broadened the spectrum of activatingproteases to matrix metalloproteinases (MMP), namely MMP-1(Boire et al., 2005; Goerge et al., 2006). Among the activatingproteases are enzymes of the coagulation system, trypsin ofpancreatic and extrapancreatic origin and mast cell tryptase(Macfarlane et al., 2001; Grishina et al., 2005).

72 E. Sokolova, G. Reiser / Pharmacology & Therapeutics 115 (2007) 70–83

PARs signal via stimulation of G proteins, members of Gi,Gq/11, and G12/13 families. Interaction of PARs with different Gprotein α-subunits leads to the activation of phosphoinositide 3kinase, phospholipase C isoforms, small G protein Rho, anincrease of cytosolic Ca2+ concentration, activation of mitogen-activated protein kinase cascade that finally leads to variouscellular responses. The signaling events triggered by PARactivation are unique for cell types and for PAR subtypes andare excellently reviewed in (Steinhoff et al., 2005).

In some cases PARs exert their effects not directly, but likeother G protein-coupled receptors, via transactivation of otherreceptors. For instance, PAR-1 and PAR-2 transactivate epidermalgrowth factor receptor (EGFR) that results in cell proliferation ormigration. Such mechanism of control of cell growth was shownfor both cancer and normal cells (Kalmes et al., 2000; Sabri et al.,2002; Darmoul et al., 2004a,b; Bergmann et al., 2006; Carusoet al., 2006). This indirect mitogenic effect of PARs can be a resultof induction of intrinsic signalling pathways that involve Src-dependent EGFR phosphorylation (Sabri et al., 2002; Carusoet al., 2006). Alternatively, the indirect effect can also be due toextracellular activation of EGFR after the release of EGFRligands. EGFR ligands, including epidermal growth factor,transforming growth factor (TGF)-α, amphiregulin, are expressedas precursors with transmembrane domains anchored in theplasma membrane. In response to an appropriate stimulus, thegrowth factors are released from the cell surface followingproteolytic shedding of the extracellular domain (ectodomainshedding) by zinc-dependent metalloproteinases (Gee & Knowl-den, 2003). Works based on broad-spectrum inhibitor studiesshowed the potential involvement of metalloproteases of theADAM (a disintegrin and metalloprotease) or MMP families inthe PAR-induced release of EGFR ligands (Darmoul et al., 2004a;Bergmann et al., 2006). There is convincing evidence that EGFRligand shedding upon PAR activation is mediated by tumournecrosis factor (TNF)-α-converting enzyme, a member of theADAM family (Darmoul et al., 2004a; Chokki et al., 2005;Heijink et al., 2007).

However, EGFR is not the only receptor for growth factorswhich mediates PAR-induced proliferation. PAR-1 can transacti-vate fibroblast growth factor receptor and induce release of basicfibroblast growth factor without affecting the EGFR system(Rauch et al., 2004). Platelet-derived growth factor (PDGF) andits receptor is also involved in PAR-induced cell proliferation(Blanc-Brude et al., 2001). It should be noted that transactivationof growth factor receptors is not the prerequisite for PAR-triggered mitogenesis. For several cell types the lack of in-volvement of EGFR trans-activation in cell proliferation wasshown (Sabri et al., 2002;Wang et al., 2002b; Neaud et al., 2004).

3. Expression ofprotease-activated receptors in human lung tissue

3.1. Expression of protease-activated receptors in humanlung tissue and in cultured airway cells under normal conditions

All 4 PARs have been detected immunohistochemically in thenormal human lung, however, with different pattern of distri-

bution in airway structures. PAR-1 to PAR-4 are expressed inepithelium and airway smooth muscle (ASM; D'Andrea et al.,1998; Knight et al., 2001; Miotto et al., 2002). In bronchialepithelium the immunoreactivity for PAR-2 and PAR-4 waslower than for PAR-1 and PAR-3 (Knight et al., 2001). Expres-sion of PAR-1, PAR-2, and PAR-4 was demonstrated on endo-thelium, PAR-4 was shown to be more abundant in microvesselsthan in large vessels (Knight et al., 2001; Schmidlin et al., 2001;Miotto et al., 2002; Fujiwara et al., 2005). Tissue-resident mac-rophages expressed PAR-1 and PAR-2, whereas PAR-3 immu-noreactivity was not detected (Knight et al., 2001; Howell et al.,2005). PAR-2 is present in bronchial glands and vascular smoothmuscle (Miotto et al., 2002). PAR-1 and PAR-2 expression isalso attributed to migratory cells (mast cells and polymorpho-nuclear cells; Lan et al., 2002).

Studies of cultured primary airway cells and cell linesconfirmed that different cell types represent different PARexpression profiles. This reflects selective responses to a varietyof proteases acting in the airways. Primary adult lung fibroblastsexpress PAR-1, PAR-2, and PAR-3 with predominance of PAR-1 (Sokolova et al., 2005). Expression of the 3 PARs, PAR-1 toPAR-3, was detected in ASM cells by RT-PCR, and thepresence of PAR-1 and PAR-2 was confirmed by immunocy-tochemistry (Hauck et al., 1999; Schmidlin et al., 2001).However, the detection of PAR-4 on ASM cells has not beenperformed so far. Primary bronchial epithelial cells express 4PARs (PAR-1–PAR-4; Shimizu et al., 2000; Asokananthanet al., 2002a; Suzuki et al., 2005), consistent with immuno-histochemical data. However, epithelial cell lines display adifferent PAR expression pattern, depending on their age (pro-pagation number), which may reflect the unstable genetic stateof immortalized cells. We have found recently that airwayepithelial A549 cells (alveolar carcinoma cell line) and HBEcells (bronchial epithelial cells transformed with human pa-pilloma virus 18 E6 and E7 genes) express PAR-1 to PAR-3with predominant expression of PAR-2 (Grishina et al., 2005).In addition, PAR-4 was also detected (Shimizu et al., 2000;Asokananthan et al., 2002a). In human pulmonary arteryendothelial cells (HPAEC) PAR-1, PAR-2, and PAR-4 mRNAexpression with predominance of PAR-1 was shown (Hamiltonet al., 2001; Fujiwara et al., 2004). All 3 receptors were found tobe functionally active (Fujiwara et al., 2004, 2005). Unfor-tunately, the analysis of PAR-3 in HPAEC has not beenperformed. Eosinophils express PAR-1 and PAR-2 but onlyPAR-2 activation results in cellular effects such as generationof reactive oxygen species and shape change (Bolton et al.,2003).

3.2. The influence of pathological conditions on theexpression of protease-activated receptors in the human lung

PARs, especially PAR-1 and PAR-2, are suggested to play animportant role in inflammatory and fibroproliferative processes.One of the supportive pieces of evidence for this hypothesis isthe fact that their expression is up-regulated during the devel-opment of pulmonary diseases, which are coupled to inflam-mation, such as pulmonary fibrosis, asthma, and bronchitis. An


increased protein level of PAR-1 was detected by immunohis-tochemistry in lung tissues of patients with pulmonary fibrosis(Howell et al., 2005) and in early stages of pulmonary fibrosisassociated with scleroderma (systemic sclerosis) (Bogatkevichet al., 2005). In both cases the overexpression of PAR-1 wasattributed to resident alveolar macrophages and to fibroproli-ferative and inflammatory foci. It should be noted that there is adissociation between gene and protein expression of PAR-1,since its mRNA level, unlike protein level, was not altered inprimary fibroblasts from patients with established pulmonaryfibrosis compared to the cells from healthy control subjects(Sokolova et al., 2005). A comparable discrepancy betweenmRNA and protein level was observed by Roche et al. (2003b),where increased PAR-1 protein staining was detected in alveolarmacrophages from chronic smokers compared to healthysubjects, whereas the mRNA level was reduced. Interestingly,macrophages from asthmatic patients and healthy subjectsexpressed similar levels of PAR-1 (Roche et al., 2003b).

Airway diseases associated with inflammation are often, butnot always, accompanied by increased levels of PAR-2 mainlyon cells of epithelial origin. For example, an increased expres-sion of PAR-2 was detected in bronchial epithelium of asthmaticpatients (Knight et al., 2001). However, no differences in PAR-2expression were observed in central airways from smoking andnonsmoking patients (Miotto et al., 2002) and in pleural tissuein patients with and without pleural diseases (pleuritis,emphysema) (Lee et al., 2005). There is increasing evidencethat PAR-2 may participate not only in allergic or inflammatoryrespiratory diseases but may also be involved in fibroprolifera-tive processes. Increased levels of PAR-2 mRNAwere detectedin lung fibroblasts from fibrotic tissues (Sokolova et al., 2005),and increase of PAR-2 protein was seen in myofibroblasts offibrotic alveolar walls, in addition to bronchial epithelium inlung samples from preterm infants with bronchopulmonarydysplasia, which is characterized by inflammation and subse-quent interstitial fibrosis (Cederqvist et al., 2005).

4. Role of protease-activatedreceptors in pathological situations in the airways

Here, we will focus mostly on recent data concerningpulmonary functions of PARs, because they have already beenreviewed before (Lan et al., 2002; Chambers, 2003; Reed &Kita, 2004; Kawabata & Kawao, 2005). So far, in vivo studiesconcerning the role of PARs in the airway function have beendone using mainly animal models of diseases and knockoutmice. The model of bleomycin-induced fibrosis demonstratedthat PAR-1 actively participates in acute inflammation and thechronic fibrotic phase of lung injury. PAR-1 was shown to beinvolved in inflammatory cell recruitment in response tobleomycin injury, collagen accumulation, and expression ofprofibrotic factors connective tissue growth factor (CTGF) andTGFβ1 (Howell et al., 2005). Moreover, PAR-1 activation inhuman lung fibroblasts protects the cells from apoptosisinduced by several apoptotic stimuli, and elevated levels ofPAR-1 in sclerodermal fibroblasts were shown to be coupled toresistance of the cells to apoptosis (Bogatkevich et al., 2005).

These changes together with the ability of PAR-1 to transformfibroblasts into the myofibroblast phenotype (Bogatkevichet al., 2001) may provide the explanation for the persistenceof myofibroblasts in fibrotic lesions. These studies furtherbroadened our knowledge about the function of PAR-1 indriving inflammation and abnormal tissue remodeling. There-fore, blocking of PAR-1 function will provide a possibility oftreatment of fibrotic lung diseases.

There is evidence that PAR-2 can also contribute to thepathogenesis of lung fibrosis and enhance profibrotic effects ofPAR-1. In favour of this suggestion are the findings that PAR-2expression is increased in fibrotic lesion (Cederqvist et al.,2005), and it can be induced by profibrotic growth factors(Gruber et al., 2004).

There is still conflicting data concerning the role of PAR-2 inairway inflammatory response. Protective antiinflammatoryeffects of PAR-2 activation in mice were documented (Moffattet al., 2002; De Campo & Henry, 2005). Administration ofPAR-2-activating peptide inhibited the influx of leukocytes inlipopolysaccharide (LPS)- or ovalbumin (OVA)-challengedmice. PAR-2 activation also inhibited OVA-induced hyperre-sponsiveness via a cyclooxygenase-dependent pathway. Simi-larly, Morello et al. (2005) showed a protective relaxant effect ofPAR-2 in LPS-treated rat bronchi via release of PGE2 fromepithelium. On the contrary, the proinflammatory role of PAR-2was observed in the airways of mouse, guinea pig and human(Chambers et al., 2001; Barrios et al., 2003; Ebeling et al., 2005)and confirmed by studies with PAR-2-deficient mice (Su et al.,2005; Takizawa et al., 2005). In those studies, PAR-2 wasshown to be involved in airway hyperresponsiveness and OVA-induced allergic inflammation by producing eotaxin andsubsequent recruitment of eosinophils and mononuclear cellsinto airways. However, PAR-2 itself does not initiate inflam-matory cell influx but enhances airway deleterious effects inOVA-challenged mice (Ebeling et al., 2005). Moreover,proinflammatory and contractile effects of PAR-2 are mediatedby neurogenic mechanisms via release of neuropeptides, such assubstance P and calcitonin gene-related peptide from afferent Cfibres (Barrios et al., 2003; Su et al., 2005). In lung diseases,such as asthma, which are characterized by damaged epitheli-um, the proinflammatory and contractile component of PAR-2action may overbalance its relaxant component due to loss ofability of the epithelial layer to produce the relaxant factorPGE2. Taken together, PAR-2 evidently plays an important rolein lung defence mechanisms, although the precise role of PAR-2activation needs further investigation.

Table 1 provides an overview of the participation of PARs inseveral lung disorders. Under these pathological conditions thelevels of PARs and proteases which can modulate PAR activityare increased. Some consequences of PAR activation during thedevelopment of the disorders are shown. Interestingly, through-out the body in various organs and tissues elevated levels ofPARs and PAR-activating proteases are detected at differentpathological situations. The involvement of PARs in thedevelopment of diseases in other organs, like the cardiovascular,digestive and the nervous system is also briefly summarized inTable 1. Thus, PARs have been documented to be unequivocally


involved in the development of inflammation. Therefore,further studies of PARs and PAR-protease interactions are ofimportance for understanding the mechanisms involved in thedevelopment of pathological conditions.

5. Protease-activated receptors in proliferative disorders

Although the 4 members of the PAR family are expressed inthe lung, so far the role of only PAR-1 in the development of

Table 1Involvement of protease-activated receptors in disorders of the airways and of other

Pathologic state Change in PAR and protease levels

LungPulmonary fibrosis Increased PAR-1 on alveolar macrophages

and in fibroproliferative fociIncreased PAR-2 on fibroblasts and myofibroIncreased activity of thrombin and proteasesthe coagulation cascade, MMP-1, -2, -9

Chronic obstructive pulmonarydisease (chronic bronchitis)

Increased PAR-2 on epitheliumIncreased levels of neutrophil proteases,trypsin, MMP-1, -8, -9

Asthma Increased PAR-2 on epitheliumIncreased levels of tryptase MMP-2;MMP-9 or elastase depending oninflammatory phenotype of asthma

Neoplasia Increased PAR-1 and -2 inneoplastic cellsIncreased expression oftrypsin in carcinoma, increasedactivity of MMP-1

IntestineInflammatory bowel diseases(Crohn disease, ulcerative colitis)

Increased PAR-1 and -2 (detected atmRNA level)Increased activity of trypsin, tryptase

Irritable bowel syndrome Increased activity of trypsin, tryptase

SkinAtopic dermatitis Increased PAR-2 in nerve fibers,

keratinocytesIncreased activity of tryptase

Cardiovascular systemVascular injury Increased PAR-2 in endothelial cells

Increased activity of thrombinand proteases of the coagulation cascade

Heart ischemia/infarction Increased activity of proteases of thecoagulation cascade, tryptase, MMP-9

BrainParkinson's disease Increased PAR-1 in astrocytes

Increased activity of thrombinHIV encephalitis Increased PAR-1 in astrocytes

Increased activity of thrombinIschemia Increased PAR-2 in neurons

Increased PAR-1, -2, -3 in rathippocampus

proliferative disorders, such as pulmonary fibrosis was studiedin detail. Pulmonary fibrosis is characterized by rapid fibroblastproliferation and overproduction of extracellular matrix pro-teins. In vitro studies showed that PAR-1 and PAR-2 induceproliferation of lung fibroblast. Other profibrotic effects, such asextracellular matrix production, cell migration, secretion ofprofibrotic growth factors, and cytokines, have been describedexclusively for PAR-1. A strong impact of PAR-1 in thedevelopment of lung fibrosis was shown in PAR-1 deficient

organs

Consequences of PAR activation

Profibrotic effects of PAR-1, enhancementof inflammation and abnormal tissue remodeling(Chambers, 2003; Howell et al., 2005)blasts

of

No data

Proinflammatory effects of PAR-2. Involvement ineosinophil recruitment (Ebeling et al., 2005; Su et al., 2005;Takizawa et al., 2005)

PAR-1 contributes to the invasive and metastatic process of cancer,

PAR-1 and PAR-2 involvement in angiogenesis (Jin et al., 2003)

Proinflammatory effects of PAR-1 and PAR-2. Involvementin epithelial damage and increased intestinal permeability,granulocyte recruitment, edema formation (Cenac et al., 2002;Vergnolle et al., 2004)Pronociceptive effect of PAR-2; involvement in activation ofsensory neurons, generation of hyperalgesia and visceralhypersensitivity (Cenac et al., 2007)

PAR-2 contributes to development of cutaneous inflammationand itch enhancement (Steinhoff et al., 2003)

PAR-1 and -2 contribute to vascular remodeling and reparativeangiogenesis (Steinberg, 2005)

Cardioprotective effects of PAR-2, coronary vasodilation,improvement of myocardial functional recovery (Steinberg, 2005)

Neuroprotective effects of PAR-1 against neuronal degenerationand cell death (Ishida et al., 2006)PAR-1 contributes to brain inflammation and neuronal damage(Boven et al., 2003)Neurodegenerative role of PAR-1 (decreased infarct volumeat PAR-1 deficiency in mice; Olson et al., 2004); protectiverole of PAR-1 in rats (Striggow et al., 2000)Protective effect of PAR-2 (increased infarct volume at PAR-2deficiency; Jin et al., 2005)


mice, when fibrosis was induced by intratracheal bleomycininstillation. PAR-1 is responsible, although not fully, for thedestruction of alveolar units, collagen accumulation andrecruitment of inflammatory cells into the lung (Howell et al.,2005). Interestingly, the activator of PAR-1 thrombin, whoseactivity in bronchoalveolar lavage fluid is elevated in pul-monary fibrosis, is responsible only for some of the PAR-1-mediated profibrotic effects in the rat model of fibrosis (Howellet al., 2001). Therefore, other PAR-1 activators are relevant inthe development of this disorder.

PARs can display different and sometimes opposite effectsdepending on their tissue and organ distribution (see Table 1).Nevertheless, it is interesting to note that PARs in brain,similarly to lung, participate in proliferative dysregulation,namely in astrogliosis. Astrogliosis is a feature of acute andchronic neurodegenerative diseases and is characterized byastrocyte proliferation, their phenotypic changes and cellularhypertrophy. PAR-1 and PAR-2 induce proliferation of culturedastrocytes from rat and mouse (Wang et al., 2002a,b; Nicoleet al., 2005). PAR-1 deficiency prevents astrocyte proliferationafter brain injury, which implies that PAR-1 plays a role in theprocess of gliotic scar formation (Nicole et al., 2005).

6. Regulation ofprotease-activated receptor expression and activity

6.1. Regulation of protease-activatedreceptor expression by inflammatory mediators

The regulation of PAR activity can be realized at the genetranscriptional level by a variety of inflammatory mediators.These mediators may induce or suppress both the receptor geneexpression and the presentation of the receptor protein on thecell surface. The regulation can also occur at the level offunctional activity as a result of activity of endogenous andexogenous proteases that can either activate or inactivate PARs.There are numerous observations of modulation of PARexpression under pathological conditions in vivo (see above).However, there is limited information about particular media-tors responsible for this effect. Studies with cultured cellsrevealed several potential modulators of PAR expression invitro. In endothelial cells, PAR-1 and PAR-2 gene expressionwas enhanced by macrophage migration inhibitory factor, aproinflammatory cytokine participating in the inflammatoryphase of the wound healing process (Shimizu et al., 2004). Theproinflammatory and asthma-associated cytokine interleukin(IL)-1β stimulated the expression of PAR-2 in human ASMcells (Freund-Michel & Frossard, 2006). In fibroblasts PAR-2expression can be stimulated by the profibrotic growth factors,TGFβ1 and PDGF (Gruber et al., 2004), and by proinflamma-tory mediators LPS and TNF-α (Ramachandran et al., 2007).Interestingly, LPS and TNF-α in bronchial fibroblasts inducedfunctional expression of PAR-4 which is not expressed underresting conditions (Ramachandran et al., 2007).

It is of great interest to note that the action of regulatorymolecules is cell type- and PAR subtype-specific. For example,stimulation of HPAEC with TNF-α resulted in increase in PAR-

2 level, whereas the PAR-1 expression level remainedunchanged (Fujiwara et al., 2004). Similarly, LPS up-regulatedthe PAR-2 expression level in human alveolar epithelial cellsA549 without affecting the PAR-1 level (Ostrowska and Reiser,unpublished data). The cytokine IL-4, one of the key contri-butors to the pathogenesis of asthma with profibrotic effects(Saito et al., 2003), was shown to actively down-regulate PAR-1to PAR-3 in macrophages both at mRNA and protein levels(Colognato et al., 2003). However, in human lung fibroblaststhe cytokine failed to influence the PAR expression (Sokolovaand Reiser, unpublished data).

Recent work on human lung fibroblasts performed in ourlaboratory revealed another potential negative regulator of PARexpression, prostaglandin (PG) E2. The mediator PGE2 actedvia its EP2 receptor with subsequent elevation of cAMP(Sokolova et al., 2005). The effect of PGE2 seems to be celltype-specific, since the PAR expression on human airwayepithelial cells (A549, HBE) was hardly influenced by PGE2 orforskolin (Ostrowska and Reiser, unpublished data). Moreover,cAMP-dependent down-regulation of PAR expression infibroblasts is probably restricted mostly to the action of PGE2

but not to ligands of other receptors coupled to elevatingintracellular cAMP levels (e.g., β2-adrenergic receptors), sincethe effect of a well-known β2-adrenergic receptor agonist,isoproterenol, was much smaller than that of PGE2 (Sokolovaand Reiser, unpublished data).

Interestingly, persistent PAR activation can also influence thePAR expression. In A549 cells, continuous activation of bothPAR-1 and PAR-2 caused up-regulation of expression of thesereceptors. It was mostly the expression of PAR-2, which wasaffected. The 3-fold up-regulation of the PAR-2 expression levelwas reached by cell stimulation with PAR-1 or PAR-2 activatingpeptides. The effect of PAR activation persisted up to 24 hr(Ostrowska and Reiser, unpublished data). On the contrary,PAR-1 activation resulted in transient down-regulation of PARsin lung fibroblasts (Sokolova et al., 2005).

6.2. Control of protease-activated receptor activity by proteases

6.2.1. Endogenous proteasesAn intricate level of regulation of PAR activity is activation/

inactivation of the receptors by specific proteases (reviewed inLan et al., 2002). Fig. 2 shows cleavage sites of the N-terminalparts of human PAR-1 (A) and PAR-2 (B) by proteases ofendogenous and exogenous origin, which display activity in theairways. Cleavage at the activation site (arrow Nr 1 in Fig. 2)reveals the tethered ligand that results in PAR activation.Cleavage at positions located C-terminally from the activationsite removes the tethered ligand. That process preventssubsequent PAR activation and thus disables PARs. Thepotential scissile bonds for leukocyte proteases and elastasefrom Pseudomonas aeruginosa were identified by cleavage ofsynthetic peptides or the recombinant N-terminal domain ofPAR-1 and PAR-2 (Renesto et al., 1997; Loew et al., 2000;Dulon et al., 2005).

Table 2 summarises the available data describing the actionof the airway proteases on PARs in cultured lung cells

Fig. 2. Potential cleavage sites of the N-terminal exodomain of human PARs byairway proteases. Partial sequences (amino acids 31–77 in single-letter code) ofhuman PAR-1 (A) and human PAR-2 (B) are shown. The tethered ligandsequence is depicted in bold underlined letters. The locations of cleavage sitesare indicated by numbered arrows. (A) The activation of PAR-1 by cleavage atthe activation site (number “1”) is mediated by thrombin, trypsin, factor Xa,activated protein C, cathepsin G, and plasmin. Disabling cleavage by cathepsinG occurs at positions 3–5, 9; leukocyte elastase 2, 7, 8; proteinase-3 2,7; plasmin9. (B) The activation of PAR-2 by cleavage at activation site (number “1”) ismediated by trypsin, factor Xa, tryptase, and house dust mite proteases Der p3and Der p9. Disabling cleavage by cathepsin G occurs at positions 4, 5;proteinase-3 and leukocyte elastase 6; P. aeruginosa elastase 2, 3.


(fibroblasts, epithelial, endothelial, smooth muscle cells). PARactivation or disabling by particular proteases and cellularconsequences of PAR activation are shown. An important groupof endogenous activators of PARs are proteases of thecoagulation/fibrinolysis system. This includes thrombin, factorXa, transient ternary tissue factor–factor VIIa–factor Xacomplex, plasmin, and APC. Besides participating in homeo-stasis, these factors can directly regulate cellular behaviour viaactivation of PARs. The proteases can activate PAR-1 or PAR-2or both receptors, depending on the cell type (for review, seeRuf et al., 2003). These enzymes actively participate in normalwound healing, and under normal conditions their levels aremaintained in a tight balance. However, in acute lung injury orin chronic lung diseases (e.g., asthma), there is an increasedprocoagulant and decreased fibrinolytic activity (Gabazza et al.,1999; Idell, 2003). The procoagulant proteases contribute to thedevelopment of profibrotic events in the lung by activation ofPAR-1. Thus, they are important players in progression offibrotic diseases and tissue remodelling. Detailed informationabout the role of the coagulation cascade proteases in PAR-1-mediated responses in the lung can be found in the review byChambers (Chambers, 2003).

Lung epithelium is a source of potent PAR-2 activators,trypsin and human airway trypsin-like protease (HAT). Bothenzymes have been localized to normal human bronchialepithelium (Cocks et al., 1999; Takahashi et al., 2001). Increasedexpression of trypsin has been shown to be coupled to neoplasia(Jin et al., 2003). Active HAT is the predominant trypsin-like

protease in the sputum of patients with chronic airway diseases,such as chronic bronchitis and bronchial asthma (Matsushimaet al., 2006). HAT can probably be involved in the defence ofairways as well as in the pathogenesis of chronic airwaydiseases, considering its ability to mediate mucus productionfrom airway epithelial cells and to induce proliferation offibroblasts via PAR-2-dependent mechanisms (Chokki et al.,2004; Chokki et al., 2005; Matsushima et al., 2006).

Other endogenous proteases which possess the ability tomodulate PAR activity originate from hematopoietic cells (mastcell tryptase, neutrophil elastase, cathepsin G and proteinase 3).Tryptase is released from activated mast cells in airways duringallergen challenge, and its concentration is increased inasthmatic patients (Bousquet et al., 2000). The protease isconsidered to be an activator of PAR-2 but different tryptasesubtypes can elicit different cellular responses (for review, seeReed & Kita, 2004).

Neutrophil serine proteases, elastase, cathepsin G andproteinase 3 are stored in azurophil granules of polymorphonu-clear neutrophils and released into the environment upon cellactivation. They contribute to the pathogenesis of chronicpulmonary diseases, such as emphysema, chronic bronchitis andcystic fibrosis. Under normal conditions, the level of the activeproteases is rather low compared to inflammatory conditions,when lung is infiltrated by polymorphonuclear neutrophils.Thus, neutrophil proteases seem to play a significant role onlyduring inflammation but not in the healthy airways. Neutrophilelastase and cathepsin G disable PAR-1 and PAR-2 on lungepithelial cells and fibroblasts, respectively, and thus preventactivation of PARs by other proteases. As we described above,PAR activation on these cells contributes to the recruitment ofcells of the immune system into airways. Therefore, inactivationof PAR-2 by neutrophil proteases may serve as a regulatorymechanism for termination of influx of immune cells into theairway space and for resolution of inflammatory events.

6.2.2. Exogenous proteasesAirborne allergens (house dust mite, fungi, pollen) and

respiratory bacteria are a source of many proteases, which areinvolved in airway tissue remodelling, inflammation, andallergy. These proteases induce secretion of proinflammatorycytokines and chemokines, and increase vascular permeabilityand epithelial cell detachment (Kauffman, 2003b; Reed & Kita,2004). One of the targets for the allergen proteases can beairway PARs, as shown for some of the enzymes. For example,the serine proteases Der P3 and Der P9 from house dust miteDermatophagoides pteronyssinus have the potency to activatePAR-2 on epithelial cells with subsequent release of proin-flammatory cytokines and chemokines (IL-6, IL-8, thymus, andactivation-regulated chemokine/CCL17; Sun et al., 2001;Asokananthan et al., 2002b; Darmoul et al., 2004a; Chokkiet al., 2005; Heijink et al., 2007). The cysteine protease Der P1mediates cellular effects independently of PAR-2 activation(Adam et al., 2006) but this protease can inactivate PAR-1 onepithelial cells (Asokananthan et al., 2002b). Work withcockroach extract revealed that its serine proteases activatedPAR-2 but not PAR-1, in epithelial cells with subsequent IL-

Table 2Proteases acting in the airways and their effects on PARs in cultured lung cells

PARsubtype

Cell type Consequences ofPAR activation

References

Endogenous proteasesProteases of the coagulation/fibrinolysis systemThrombin PAR-1 (+) fibroblasts/endothelial

cells/epithelial cells⁎profibrotic/proinflammatoryeffects

Blanc-Brude et al., 2005

Factor Xa PAR-1 (+) fibroblasts profibrotic effect Blanc-Brude et al., 2005PAR-1 (+)/PAR-2 (+)

endothelial cells protective antiinflammatory effects Feistritzer et al., 2005

Plasmin PAR-1 (+) fibroblasts mitogenic effect Mandal et al., 2005APC PAR-1 (+) endothelial cells antiinflammatory effects Riewald and Ruf, 2005

Trypsin-like proteasesExtrapancreatic trypsin PAR-2 (+) epithelial cellsHAT PAR-2 (+) epithelial cells/fibroblasts mucus production/mitogenic effect Chokki et al., 2004, 2005;

Matsushima et al., 2006Proteases from cells of the immune systemTryptase PAR-2 (+) fibroblasts/ASM cells profibrotic effect Akers et al., 2000Elastase PAR-2 (−) epithelial cells induction of apoptosis Dulon et al., 2003; Suzuki

et al., 2005PAR-1⁎

Cathepsin G PAR-1 (−) fibroblasts Sokolova et al., 2005; Ramachandranet al., 2007PAR-2 (−)

PAR-4 (+)PAR-2 (−) epithelial cells Dulon et al., 2003PAR-1 (−) monocytes Roche et al., 2003a

Exogenous proteasesDust miteDer p3, Der p9 PAR-2 (+) epithelial cells proinflammatory effects Sun et al., 2001; Asokananthan

et al., 2002bDer p1 PAR-1 (−) epithelial cells Asokananthan et al., 2002bBacterialP. aeruginosa elastase PAR-2 (−) epithelial cells Dulon et al., 2005

(+), PAR activation; (−), PAR inactivation; ⁎, no evidence of direct PAR involvement.


8 expression (Hong et al., 2004; Page et al., 2005). Elastasefrom P. aeruginosa, an opportunistic pathogen that is persistentin the respiratory tract in patients with cystic fibrosis, disarmsPAR-2 on epithelial cells, thus preventing its activation by otherproteases, which can be one of the reasons of altered regulationof inflammatory responses in cystic fibrosis (Dulon et al.,2005). Fungal allergens from Aspergillus fumigatus, possessingserine protease activity, induce release of IL-6, IL-8, andmonocyte chemotactic protein-1 from airway epithelial cells(Borger et al., 1999; Kauffman, 2003a). Moreover, proteolyticactivity from A. fumigatus extract was responsible for cellshrinking and desquamation (Kauffman et al., 2000). Theauthors proposed PAR-2-dependent mechanism of the chemo-kine production from epithelial cells at low concentrations offungal extract. They suggested PAR-2 inactivation by highconcentrations of fungal extract.

6.2.3. Specific features ofprotease action on protease-activated receptors

Interestingly, a protease can signal via different PAR subtypeson different cell types. For example, factor Xa in endothelial cellsmediates signaling mainly through PAR-2, whereas in fibroblaststhe enzyme acts via PAR-1, as shown in work with transgenicmice (Camerer et al., 2002). Another interesting feature ofprotease–PAR interaction is that activation of the same receptor

by different proteases can trigger different cellular responses,sometimes even opposite effects. The following example is quitestriking. Although thrombin and APC activate the same receptor(PAR-1) on endothelial cells, the 2 proteases induced theexpression of different groups of genes and had even oppositeeffects on the expression of proinflammatory and proapoptoticgenes in TNF-α-treated cells (Riewald & Ruf, 2005).

Proteases can also elicit cellular responses by activation ofother receptors than PARs. For example, in human ASM cells,in contrast to fibroblasts, catalytically active thrombin andtryptase caused proliferation independently of PAR-1 and PAR-2 activation, respectively, despite the functional presence of thereceptors on the cell surface (Brown et al., 2002; Tran &Stewart, 2003). The same non-PAR-2-mediated action oftryptase was observed in studies on histamine release fromhuman mast cells (He et al., 2004).

7. Scenario ofprotease-activated receptor activity in airway cells

Effects of PARs on some of the airway cells are schematicallysummarized in Fig. 3. For simplification 2 cell types, epithelialcells and fibroblasts, are depicted together with routes of PARactivation on these cells and pathophysiological consequences.The figure shows 3 major regulatory pathways: (1) Activation of


PARs (especially PAR-2) on cells of the epithelial layer both byexogenous proteases accessing epithelium from the lumenal sideand by endogenous proteases coming from resident or recruitedcells, results in secretion of proinflammatory cytokines (like IL-6), chemoattractants (IL-8, eotaxin), hematopoietic growth factorgranulocyte macrophage colony stimulating factor (GM-CSF),MMP-1 (marked as pathway 1 in Fig. 3). These mediatorsparticipate in the development or enhancement of inflammation,thus indicating the implication of PAR-2 in the inflammatoryresponse.

(2) On lung fibroblasts the main protease receptor is PAR-1,which mediates synthesis and release of extracellular matrixproteins (collagen, tenascin), growth factors (CTGF, PDGF),proinflammatory agents IL-6, IL-8 (marked as pathway 2 inFig. 3). PAR-1 activation results also in cell proliferation,transition into the myofibroblast phenotype and persistence ofthe cells by increased resistance to apoptosis. Apparently, underfibrotic conditions profibrotic and proinflammatory eventsmediated by PAR-1 are enhanced due to elevated levels ofPAR-1 expression (“up-regulation” on fibroblasts in Fig. 3) andalso due to the impact of PAR-2 activation, where the PAR-2expression level is increased in fibrotic diseases as well.

(3) On the other hand, both cell types, epithelial cells andfibroblasts, release the antiinflammatory and relaxant agent PGE2after PAR-2 and PAR-1 activation, respectively (marked aspathway 3 in Fig. 3). PGE2 possesses antifibrotic activity byinhibition of fibroblast proliferation and chemotaxis, myofibro-blast transition, and by downregulation of PAR-1 expression(“down-regulation” on fibroblasts in Fig. 3). However, this

Fig. 3. A scheme of PAR regulatory networks in the airways. Activation of PAR-2 onof different proinflammatory mediators and enhancement of inflammation (pathwproinflammatory mediators, and promotes lung fibrosis (pathway 2). Under fibrotic cother hand, activation of PAR-2 in epithelial cells and of PAR-1 in fibroblasts cause(pathway 3), which in autocrine or paracrine fashion can suppress fibroblast activity. MActivation of PARs is shown by short arrows, which point at the activation site and

beneficial effect of PGE2 may be reduced in fibrotic and inflamedlungs because of damaged epithelium or reduced capacity offibrotic fibroblasts to produce PGE2 (Wilborn et al., 1995).

8. Protease-activated receptorsin pharmacological applications andprotease-activated receptors as possible drug targets

Increasing evidence is being accumulated concerning theharmful role of PAR-1 in lung injury and fibrosis. Therefore,PAR-1 represents a promising target for interfering with thislesion. Blocking of PAR-1 or interfering with the signalingpathways triggered by PAR-1 activation is a promisingtherapeutic strategy. This seems to be better than inhibition ofthe activating proteases (thrombin, factor Xa). This will allow topreserve the role of the proteases in homeostasis.

One of the strategies is to apply specific receptor antagonists.Recently, heterocycle-based peptide-mimetic antagonists ofPAR-1, indole-based RWJ-56110 (Fig. 4A) and indazole-based RWJ-58259 (Fig. 4B), were designed (Andrade-Gordonet al., 2001; Zhang et al., 2001). These compounds showedselective PAR-1 antagonistic effects without affecting otherPARs. The selectivity was confirmed in studies with cellsderived from PAR-1 deficient mice which were transfected witheither human PAR-1, PAR-2, or PAR-4 (Andrade-Gordon et al.,2001; Zhang et al., 2001). Both compounds equipotentlyinhibited thrombin-induced platelet aggregation with IC50

values of 340 and 370 nM. In the guinea pig model of exvivo platelet aggregation, RWJ-58259 showed improved

epithelial cells by either exogenous or endogenous proteases results in the releaseay 1). Activation of fibroblast PAR-1 leads to profibrotic events, release ofonditions, the levels of PAR-1 and PAR-2 in fibroblasts are up-regulated. On thes synthesis and secretion of the anti-inflammatory and antifibrotic agent PGE2

oreover, PGE2 down-regulates PAR expression. For further details see the text.the tethered ligand (black box).


efficacy over RWJ-56110. RWJ-58259 completely inhibitedthrombin-induced platelet aggregation at a dose as low as0.3 mg/kg versus 1 mg/kg for RWJ-56110 (Zhang et al., 2001).RWJ-58259 displayed antithrombotic activity in a cynomolgusmonkey arterial injury model (Maryanoff et al., 2003).

Studies with another related indole-based compound (Fig.4C; IC50 value for thrombin-induced platelet aggregation of0.9 μM (Zhang et al., 2001) in a rat model of cirrhosis showedprotective effects of the PAR-1 antagonist against liver fibrosisdevelopment (Fiorucci et al., 2004). This substance at aconcentration 50 μM selectively inhibited PAR-1 withoutaffecting PAR-2 activity. It partially antagonized PAR-4. In invivo studies the maximal effect of the PAR-1 antagonist wasachieved at a dose of 1.5 mg/kg/day.

Recently, a new series of PAR-1 antagonists, derivatives ofhimbacine (tetracyclic piperidine alkaloid from Australian

Fig. 4. Low-molecular weight PAR-1 and PAR-2 antagonists. Antagonists of PAR-1: S(B) given in Zhang et al. (2001); himbacine derivative (D) given in ChackalamannilPAR-2 piperazine-based antagonist (F) from Kelso et al. (2006).

magnolia trees) was described (Chackalamannil et al., 2005).Among them, the m-(trifluoromethyl)phenyl derivative (Fig. 4D)was the most potent PAR-1 antagonist with an IC50 value of11 nM. In the radioligand binding assay, the antagonist showed aKi of 2.7 nM against PAR-1. This compound demonstrated goodoral bioavailability of 50% and a half-life of 12.4 h after in-travenous administration. Complete inhibition of platelet aggre-gation in the cynomolgus monkey model was achieved at a doseof 3 mg/kg. Hence, those antagonists may have a therapeuticpotential for the treatment of lung fibrotic disease. Their effect onthe injured lung is an important subject for future studies.

It should be mentioned that an antagonist can display anexcellent blocking activity when applied to certain types ofcells, whereas it can be toxic for others. This was seen for thepyrroloquinazoline derived PAR-1 antagonist SCH 79797(Fig. 4E). SCH 79797 inhibited thrombin-induced platelet

FLLR peptide mimetics with an indole template (A,C) and an indazole templateet al. (2005); pyrroloquinazoline derivative (E) presented in Ahn et al. (1999);


aggregation with an IC50 value of 3 μM (Ahn et al., 1999). Inthe radioligand binding assay, the antagonist showed an IC50

value of 70 nM (Ki =35 nM) (Ahn et al., 2000). SCH 79797 alsopotently prevented PAR-1 activation in vascular smooth musclecells, endothelial cells (Zania et al., 2006), and astrocytes(Wang et al., 2006). However, it has shown toxic properties onlung fibroblasts (Sokolova and Reiser, unpublished observa-tion). Therefore, thorough investigations and trials will benecessary for further clinical applications of PAR antagonists.

Another very promising direction aimed at blocking PAR-1might be the reduction of its expression level. PGE2 is known tohave antifibrotic, antiinflammatory and relaxant effects duringthe development of fibrosis and asthma. Moreover, PGE2 wasshown to be able to downregulate PAR-1 on fibroblasts. Thisprostanoid can be safely delivered into the lung by inhalation(Hartert et al., 2000). Therefore, it is of great relevance toexplore the possibility of the application of PGE2 (or relateddrugs) for the treatment of fibrosis.

Recently, PAR-2 antagonists, the piperazine derivativeENMD-1068 (Fig. 4F; Kelso et al., 2006) and a hexapeptidebased on the PAR-1 tethered ligand sequence with a reversesequence of the first 2 amino acids, FSLLRY-NH2 (Al-Ani et al.,2002), were developed. Such antagonists enable us to considerPAR-2 as a pharmacological target. ENMD-1068 was proven toblock selectively PAR-2 but not other PARs. Pharmacologicalspecificity was shown in in vitro studies using cultured mousecell lines and in an in vivo mouse model of joint inflammation.Although the compound requires high concentrations (up to5 mM) for blocking PAR-2, it can be used as a prototype forfuture development of therapeutically valuable agents. Thepeptide antagonist FSLLRY-NH2 prevented PAR-2 activationby trypsin in cell lines transfected with rat or human PAR-2 withan IC50 value about 50 μM. The antagonist blocked the relaxantaction of trypsin but not that of thrombin, in the rat aorta,implying a selective PAR-2-antagonizing effect.

9. Concluding remarks

The important regulatory role of PARs in both normal statesand inflammation of airways emphasizes the need to inves-tigate further the functioning of PARs and proteases whichmodulate the receptor activity. The studies of modulation ofPAR activity will help to define more clearly novel potentialtherapeutic approaches for treating airway diseases. This im-plies, firstly, effector molecules which control PAR expressionand activity and, secondly, agents that inhibit PAR activationin diseased states. Understanding the intricate network ofextracellular proteases and PAR on lung cells will unravelpromising pharmaceutical targets for the treatment of variouslung diseases.

Aknowledgments

The work was supported by grants from Deutsche For-schungsgemeinschaft (grant Re563/11) and from the Bundes-ministerium für Bildung und Forschung (01ZZ0407). We thankProf. Tobias Welte, Klinik für Pneumologie, Medizinische

Hochschule Hannover, Germany, for most valuable cooperationduring some of the work performed in the authors' laboratory.

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a novel therapeutic target in various lung diseases: airway proteases and protease-activated...

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