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Home , Cathepsin K

Clinic Rev Miner Metab (2011) 9:83–93 DOI 10.1007/s12018-011-9101-y

ORIGINAL PAPER

Cysteine and the Skeleton

Dieter Bro¨mme

Published online: 23 June 2011 Ó Springer Science+Business Media, LLC 2011

Abstract Cysteine cathepsins are lysosomal Keywords Arthritis Á Cathepsins Á K Á with housekeeping as well as tissue-specific functions. One Cysteine inhibitors Á Collagenase Á Á of their specialized functions includes the degradation of extracellular matrix proteins and the regulation of the immune response. In recent years, this protease family received increasing attention as drug targets for bone and Introduction -related diseases. Cathepsin K is identified as the major protease with a potent and unique colla- The adult human skeleton has 206 , and many of genase activity. The protease is responsible for the bulk of them are connected by joints allowing locomotion and degradation in bone remodeling and plays a sig- various movements. The skeleton has three main functions: nificant role in the regulation of osteoclast activity. Major (1) providing a framework for the whole body, (2) pro- efforts in the pharmaceutical industry led to the develop- tecting various organs, and (3) allowing for motion. The ment of highly potent and specific cathepsin K inhibitors two main skeletal disorders are osteoporosis and various for clinical trials in osteoporosis. Odanacatib, a nitrile- forms arthritides. Osteoporosis leads to a general loss of based non-lysosomotropic inhibitor is presently in phase III bone structure and mass, which will finally lead to trials. The compound is well tolerated and showed efficacy mechanical failure such as a fracture. Arthritides can be a regarding the increase of bone mineral density, and wear and tear problem of joints such as primarily in reduction of serum and urinary markers of . osteoarthritis or an inflammatory destruction of joints as in Moreover, cathepsin inhibitors do less or not interfere with rheumatoid arthritis. Other problems include bone cancers, the bone formation process. Cathepsin K is also a drug which can locally destroy bones but are also highly meta- target in arthritic diseases where it is likely involved in static, and various hereditary diseases such as defects in type II collagen and glycoprotein degradation. , bone formation and resorption. All pathological pheno- which lacks the collagenase activity, is involved in MHC types are accompanied by a destruction of the organic class II antigen presentation in inflammatory and auto- matrix of bones and joints in one way or another. immune related joint diseases and its inhibitors have the Bone consists of a mineral, mostly hydroxyapatite, and potential to represent a new class of anti-inflammatory an organic matrix component. The organic matrix is up to drugs. 90% type I collagen. The remainder is formed by bone sialoprotein, SPARC (secreted protein acidic and rich in cysteine), osteopontin, osteonectin, osteocalcin, growth factors, and glycosaminoglycans. Type I collagen, by far the most abundant protein in vertebrates (5–35% of total D. Bro¨mme (&) body protein), is synthesized in bones by osteoblasts and Department of Oral Biological and Medical Sciences, University consists of two a1 chains and one a2 chain, called tropo- of British Columbia, 2350 Health Science Mall, Rm 4558, Vancouver, BC V6T1Z3, Canada collagen. They form left-handed helices, which are twisted e-mail: [email protected] into a right-handed triple helix. The triple helices forms 123 84 Clinic Rev Bone Miner Metab (2011) 9:83–93 crystalline-like microfibril and macrofibers which form the mechanistic classes: cysteine, serine, and aspartic prote- scaffold of bones and are responsible for the tensile ases. In this review, only -like cysteine cathepsins strength [1, 2]. Type II collagen is the main collagen of the will be discussed which belong to the C1 subfamily of the cartilage and consists of a triple helix of three identical a1 clan CA. chains [1]. It is primarily synthesized by chondrocytes and The expresses 11 human thiol-dependent forms together with the proteoglycan, aggrecan, the bulk of cathepsins (cathepsins B, L, K, S, V, F, W, H, X, C, and O). the organic matrix in subchondral and articular cartilage. The mouse genome contains ten of the human orthologues Type I and II have a rather repetitive amino acid and eight additional cathepsins in the placenta [14]. All sequences following either a Gly-Pro-X or Gly-X-Hyp papain-like cysteine proteases are synthesized as prepro- pattern. The proline and hydroxyproline residues help to polypeptides consisting of a signal peptide, a propeptide, and stabilize the triple helix. Lysine and hydroxylysine residues a catalytic domain. (Fig 1a). Signal peptides are between 10 are used for cross-linking within the tropocollagen mole- and 20 amino acids long and are removed from the poly- cules and between different triple helices [2]. The tight peptides after translocation into the endoplasmic reticulum. winded helix structure and the cross-links render collagens The remaining procathepsin is transported to the endosomal- into proteolytically highly resistant macromolecules. Only lysosomal compartment. Propeptides have significantly a small number of proteases have developed, which are different lengths reaching from only 36 amino acids in capable to cleave within the triple helical structure of cathepsin X to 251 amino acids in . Propeptides collagens. These include members of the metalloproteinase are required for the folding of the catalytic domain, for the class such as mammalian matrix metalloproteinases transport into lysosomes, and they act as high-affinity MMP1, 8, 13, and MT-MMP1, Zn-dependent bacterial reversible inhibitors preventing the premature activation of collagenases [3, 4], and the cysteine protease, cathepsin K the catalytic domain [15]. Propeptides are removed either (previously also known as cathepsin O2) [5]. Matrix autocatalytically or with the help of other proteases resident metalloproteinases cleave collagens into distinctive 3/4 and in the acidified endosomal-lysosomal compartment. 1/4 fragments [3] whereas bacterial collagenases and The catalytic domains of human cathepsins are between cathepsin K cleave at multiple sites [6, 7]. 214 and 260 amino acids in length and contain a highly Prior to the cleavage of bone collagen, the matrix needs conserved consisting of a cysteine, histidine, and to be demineralized, which exposes the collagen fibers to asparagine residue. The cysteine residue (Cys25 based on proteolysis. This process is mediated by specialized bone papain numbering) and the histidine residue (His159) form a cells called . Multinucleated osteoclasts demin- catalytic thiolate-imidazolium ion pair [16, 17] where the eralize and degrade the bone matrix under acidic condi- cysteine thiolate acts as a nucleophile for the attack of the tions. This has mostly ruled out matrix metalloproteinases carbonyl carbon atom of the scissile peptide bond. as key bone-degrading , as they act optimally at The 3-dimensional structures of cathepsins have highly neutral pH. Acidic proteases can attack collagen, but limit similar folds consisting of an L domain harboring the their action to cleavages in the non-helical telopeptide catalytic cysteine residue at the end of an a-helix and an region of the matrix protein. Cysteine cathepsins such as R-domain holding the histidine residue (Fig. 1b). With the cathepsins B and L have been characterized as telopeptide exception of those of cathepsins O and W all other human removing proteases [8–10] and have been implicated in cathepsin 3-dimensional structures have been solved bone resorption. However, neither of them proved to have ( [18], [19], cathepsin K [20], the main bone-resorptive activity. Only in 1994 and fol- cathepsin S [21], [22], cathepsin F [23], lowing years, cathepsin K was discovered [11] and defined [24], cathepsin X [25], [26]. The as potent collagenase and target for pharmaceutical inter- propeptide is less structured and runs in inverse orientation vention for osteoporosis [5, 12]. through the substrate-binding cleft and thus blocks the This review will focus on cysteine cathepsins and will catalytic site of the parent cathepsin (Fig. 1b). discuss their role in matrix degradation and inflammation Cathepsins L, S, K, V, and F are strict endopeptidases, of the skeletal system. i.e., they cleave anywhere within a polypeptide amino acid sequence. Cathepsins B, C, X, and H are either strict exo- peptidases or exhibit mixed activities. Cathepsins X and B Cysteine Cathepsins are carboxypeptidases with cathepsin X cleaving single amino acids and cathepsin B dipeptides from the C-terminus Cathepsins were originally described as acidic proteases of peptide substrates. Cathepsins H and C are the appro- and the term ‘‘cathepsin’’, coined by Willstaetter and priate amino and dipeptidyl peptidases. Cathepsin B can Baman [13], means ‘‘to digest’’ in Greek. The term also act as an endopeptidase. The activities of cathepsins O ‘‘cathepsin’’ comprises proteases from three different and W have not yet been characterized. 123 Clinic Rev Bone Miner Metab (2011) 9:83–93 85

Fig. 1 Domain organization and 3-D structure of cathepsin K. a Schematic presentation of the protein domain organization in a signal peptide, a propeptide, and catalytic peptide region. The total length of the full length protein is 329 amino acids, of the proenzyme is 314 amino acids, of the pro-region is 99 amino acids and of the catalytically active protease is 215 amino acids. b Structure of procathepsin K with the propetide blocking the substrate-binding cleft (PDB: 1BY8) left panel and the two domain structure of human cathepsin K with E64 bound in the active site of the protease (PDB: 1ATK) right panel

The biological substrate specificity of cathepsins is also cysteine proteases are known to be lysosomal proteases and determined by their site of expression. Cathepsins B, L, H mostly active at acidic pH. If secreted in cell cultures, are found in most if not in all cell types and tissues and inactive procathepsins were found [10]. Moreover, the have been attributed to non-specific protein degradation in extracellular matrix is oxidative and would easily oxidize lysosomes and acting mostly as house-keeping enzymes. the catalytic cysteine residue and inactivate the cathepsin. On the other hand, cathepsins S, K, V, F, C, and W are Thus, cathepsins were considered as less suitable for more selectively expressed. Cathepsins S, F, and V are extracellular matrix degradation. However, more recent found in macrophages, dendritic cells and/or thymic cor- research has altered this view. First, extracellular matrix tical epithelial cells and are involved in antigen processing including the bone and cartilage matrices, are degraded to a and presentation [27–29]. Cathepsins C and W have been significant amount intracellularly after phagocytosis. described as T-cell resident proteases. Cathepsin C (also Macrophages, fibroblasts, foreign body giant cells, and known as DPPI) cleaves various precursor proteins osteoclasts are well-known phagocytosing cells and all including zymogens of granzymes [30]. is express various cathepsins at high levels [41]. The role of specifically expressed in CD8 and natural killer cells [31, cathepsins in intracellular matrix degradation became 32] and has been described to be highly apparent when cysteine protease inhibitors led to an expressed in colon cancer cells [33]. intracellular accumulation of undigested collagen fibers Cathepsins K is predominantly expressed in osteoclasts within cells. Collagen fibrils accumulated in osteoclasts but reveals significant expression levels in macrophages, when cells were treated with either leupeptin or E64, both synovial fibroblasts, chondrocytes, lung, and thyroid epi- potent cathepsin inhibitors [42]. The same observation was thelial cells [34–40]. Its main biologically relevant sub- made with a pan-cathepsin inhibitor, Mu-Leu-HomoPhe- strates are triple helical collagens. vinylsulfone, which led to an accumulation of type II collagen fibrils in fibroblasts when cultured on cartilage [36]. Extra and Intracellular Activities of Cathepsins Extracellular activities of cathepsins are supported by the following findings. (1) Not all cathepsins are neutral pH Extracellular matrix degradation in bone and cartilage as labile. Cathepsin S has a pH optimum at 6.5 pH and it well as other sites is traditionally considered an extracel- retains a potent matrix degrading activity at neutral to lular event. Therefore, proteases either secreted or local- slightly alkaline pH values [43, 44]. (2) The pericellular ized on the outer side of the plasma membrane of cells and environment at sites of inflammation is acidic. pH values as active at neutral pH have been considered as culprit low as pH 5 have been measured in arthritic cartilage [45]. enzymes. These include the matrix metalloproteases as Moreover, pericellular acidic pH values seem to increase well as some secreted serine proteases. On the other hand, the redistribution of lysosomes to the cell surface and the

123 86 Clinic Rev Bone Miner Metab (2011) 9:83–93 subsequent secretion of lysosomal proteases as it has been demonstrated for cathepsin B [46]. (3) Specialized acidic extracellular compartments such as the resorption lacunae are formed by bone-degrading osteoclasts. Vacuolar ATPase driven acidification leads to the demineralization of the bone matrix and thus to the exposure of the type I collagen scaffold. This acidification provides optimal pH conditions for collagen degradation in the resorption lacunae by cathepsin K, the predominant collagenase of osteoclasts [5, 47]. Cathepsin K was found extracellularly in the resorption lacunae [47]. (4) Secreted procathepsins can undergo autoactivation or activation by other proteases under acidic conditions [48, 49]. (5) Secretion of mature cathepsins is stimulated in the presence of extracellular matrix protein as demonstrated for cathepsins B, L, and S [50]. (6) Cathepsins can retain significant catalytic activi- Fig. 2 Type I collagen degradation by cathepsin K and the binding of ties under oxidative stress. Thyroglobulin was degraded by chondroitin 4-sulfate (C4-S) to cathepsin K. There is only minimal degradation of the triple helical collagen in the absence of C4-S. The cathepsins B, L, K, and S at pH 7.4 under oxidative con- addition of C4-S leads to a complete degradation of the c, b, and ditions [51]. Moreover, it has been demonstrated that oxi- a-chains. b 3-D structure of cathepsin K and C4-S (PDB: 3C9E). C4S dation of cathepsins is partially reversible. For example, binds remote from the active site of cathepsin K (E64 binding) and about 30% of cathepsin K activity could be restored by does not interfere with the classical substrate- dithiothreitol after the exposure to H2O2 [52]. All these observations are highly supportive of extracellular activi- ties of cathepsins. However, the total cathepsin activity studies identified specific interaction sites on the backside involved in cartilage and bone degradation is certainly of cathepsin K (Fig. 2b) [61]. Mutations in the amino acids distributed at both sites. No quantitative analysis to deter- interacting with chondroitin sulfate significantly reduced mine the intra and extracellular contributions of cathepsins the collagenase activity of the mutant proteins without in bone and cartilage is known. affecting its gelatinase activity or kinetic parameters toward synthetic peptide substrates [62]. It is speculated that the glycosaminoglycan-cathepsin K complex acts as a helicase to unfold triple helical collagens. The collagenase Regulation of Cathepsin Activities by Bone activity depends on the concentration of the glycosamino- and Cartilage Glycosaminoglycans and Collagen glycans. Excess chondroitin sulfate or other glycosamino- Degradation Products glycans completely abrogates the collagenase activity of cathepsin K without affecting the general protease activity. The regulation of cathepsin expression and secretion in This is of importance as mucopolysaccharidosis are char- response to various are well documented [53–57]. acterized by severe bone deformities and problems in The regulation of their activity by extracellular matrix bone development [63]. We have shown that cathepsin components such as its degradation products and glycosa- K-mediated type II collagen degradation is impaired in minglycans is less well known. We have previously dem- mucopolysaccharidosis of type I mice. Using a specific onstrated that glycosaminoglycans can have a stimulatory antibody, which recognizes a cathepsin K cleavage site in effect on the collagenase activity of cathepsin K without type II collagen, no activity in osteoclasts was observed. This interfering with the general protease function [58]. Gel led to a lack of ossification in the growth plate area [64]. filtration experiments led to the identification of high Proteolytic products from extracellular matrix proteins molecular complexes of cathepsin K and chondroitin sul- are also able to regulate osteoclast activity. RGD peptides fate [59] and that these complexes are unique for cathepsin are known to inhibit osteoclast activity. Type I collagen K. The same complex was observed when purified chon- contains 7 RGD peptides in its a-chains. Cathepsin K droitin sulfate was replaced by bone powder or aggrecan predigested type I collagen or bone powder, when added to ([59] unpublished DB). Neither cathepsins B, L, F, and S osteoclast cultures, terminated their activity as demon- formed similar complexes [60]. The removal of glycos- strated by the loss of actin rings. Inhibitory RGD peptide aminoglycan contaminations in collagen preparations concentrations were only achieved by predigesting the completely prevented the degradation of type I collagen by extracellular matrix with cathepsin K but not with recombinant cathepsin K (Fig. 2a) [59]. Crystallographic cathepsin L or MMP1 [65].

123 Clinic Rev Bone Miner Metab (2011) 9:83–93 87

Cathepsins in Osteoclastic Bone Resorption disorder, [68]. Pycnodysostosis is char- acterized by short stature, generalized , dys- The human skeleton is maintained by the balance between morphic appearance, and pathologic fractures on the bone resorption and formation. This process is called bone clinical level [69]. On the cellular/tissue level, it exhibits remodeling or bone turnover. It is estimated that the entire disordered growth plates in long bones and the accumula- skeleton is renewed every 7–10 years. The bone remodel- tion of undigested collagen fibrils within osteoclasts and ing cycle is initiated by the activation of bone-resorbing fibroblasts [70, 71]. Cathepsin K-deficient mice showed osteoclasts at prospective repair sites. After forming a similar defects: osteopetrotic bones, hypercalcified carti- sealing zone, the bone surface facing membrane, called lage in growth plates, and impaired osteoclast activity ruffled border, secretes protons via a V-ATPase, which [72–75]. On the other hand, cathepsin K deficiency in mice acidifies the area below the osteoclast. This leads to the had an overall milder phenotype when compared to the demineralization of the matrix and to the exposure of human phenotype as neither dwarfism nor intracellular collagen fibrils. Exposed collagen fibrils will be removed collagen fibril accumulation were observed. This may by the secretion of proteolytic activities into the resorption indicate a partial redundancy in collagen degrading activ- lacunae or phagocytosed by the osteoclasts. After com- ities in mice [76]. It is also interesting that osteoclasts in pletion of the resorption process, the osteoclast moves calvarial bones appear to use different proteases than long away or undergoes apoptosis and the remaining resorption bone osteoclasts. Here, matrix metalloproteinases may play pit will be occupied by osteoblasts, which are responsible a significant role. MMP inhibitors specifically inhibited for refilling the pit with fresh bone matrix. The resorption calvarial osteoclast-dependent bone resorption but not that and the refilling of the pit takes about 3 weeks [41, 66]. in long bones [77]. Moreover, matrix metalloproteinases The number of osteoclasts and osteoblasts interacting at a compensated in long bones for cathepsin K deficiency in specific repair site is called basic multicellular unit (BMU) mice [78], whereas in calvarial bones also cathepsin L (Fig. 3). played a role. Stimulants of bone resorption such as IL-1a The resorption process of the organic matrix in the and TNF-a, increased the contribution of cathepsin L to resorption lacunae is mostly mediated by cathepsin K. calvarial bone resorption [56]. On the other hand, overex- Cathepsin K represents 4% of random EST clones pression of cathepsin K led to accelerated resorption of sequenced of the osteoclast genome and about 98% of total trabecular bone [79]. cathepsin ESTs [35]. Northern blot analyses revealed very Highly relevant to osteoporosis is the regulation of low expression levels for cathepsins L and S in osteoclasts cathepsin K expression in osteoclasts by estrogen. Estrogen [34]. The characterization of the recombinant protease downregulates the expression of cathepsin K and thus revealed that cathepsin K is a potent collagenase, which in correlates with increased bone resorption rates under contrast to MMPs, cleaves at multiple sites with triple estrogen deficiency as usually observed in postmenopausal helical collagens of type I [7, 67]. This suggests that woman. Estrogen decreased cathepsin K mRNA expression cathepsin K is the only relevant cathepsin and the most in mature osteoclasts by 50% [80] and caused a reduction abundant collagenolytic activity in osteoclasts. in the depth of osteoclast-mediated resorption pits [81]. The pivotal role of cathepsin K in bone resorption was Ovariectomized mice showed an increase in cathepsin K supported by the finding that cathepsin K-deficiency causes protein expression by 200% when compared to sham- a severe bone sclerosing autosomal recessive bone operated littermates. Treatment with estradiol-17S (E2)

Fig. 3 Bone remodeling cycle of a basic unit. a Osteoclast recog- initiate the deposition of fresh bone matrix. d Resorption pit is nizes site of repair and initiates its activation. b Demineralization and completely filled with new bone matrix. The whole process has a organic matrix resorption process leads to the formation of a duration of approximately 3 weeks resorption pit. c Resorption pit gets occupied by osteoblasts, which 123 88 Clinic Rev Bone Miner Metab (2011) 9:83–93 reduced cathepsin K expression to baseline levels of the A cathepsin B inhibitor was able to reduce proteoglycan sham-operated mice [82]. release from IL-1a treated cartilage explants [93]. Similar Cathepsin K might not only be critical for the bulk results were obtained when cathepsin L expression was degradation of collagen fibrils in the resorption lacunae but suppressed [94]. Cathepsin B expression correlated with also involved in the initial activation of the osteoclasts. disease progression in human osteoarthritis but its zonal Osteoclast activity is activated by the recognition of distribution was similar in healthy bones implying that it exposed RGD peptide motifs of bone surface collagen by may contribute to the maintenance of the pathological avb3 integrin on the osteoclast. Collagen–integrin inter- status but not to its initiation [87, 95]. Cathepsin B and L actions lead to the formation of actin rings which correlate expression was also increased in the synovial membrane of with the activity of osteoclasts [83]. We have shown that patients with early inflammatory arthritis [96] and their cathepsin K activity is required to expose cryptic RGD secretion was induced by cytokines [97]. Low pH values in motifs and to initiate actin ring formation. Cathepsin diseased cartilage provide an ideal environment for secre- K-deficient osteoclasts and cathepsin K inhibitor-treated ted cathepsins [45]. cells displayed significantly less actin rings than wild-type Mice with a type II collagen mutation which are prone or untreated cells. In contrast, a pretreatment of the colla- for early onset osteoarthritis showed increased cathepsin K gen matrix with recombinant cathepsin K but not with expression in chondrocytes [98] Moreover, the expression cathepsin L or MMP1 restored the formation of actin rings of cathepsins K, L, and B is regulated by the proteolytically in osteoclasts when grown on collagen. [65]. Thus, it may released N-terminal telopeptide of type II collagen via the not be necessary that a cathepsin K inhibitor blocks the activation of protein kinase C and p38 MAP kinase in intracellular and intra-resorption lacunae collagenase chondrocytes [99]. A direct proof that cathepsin K activity of the protease but only the initial and most likely degrades type II collagen in human and horse osteoarthritis extracellular digest of collagen fibrils to prevent osteoclast was obtained by the immunohistochemical detection of activation. These findings could be consequential for the cathepsin K-specific neoepitopes of cleaved type II colla- development of cathepsin K inhibitors. gen in cartilage. The staining pattern also correlated well with macroscopic changes in the arthritic cartilage [100, 101]. Under in vitro condition, cathepsin K is a potent type Cathepsins in Joint Erosion II collagenase and aggrecanase [36, 67, 91]. Strong support for a critical role of cathepsin K in osteoarthritis and Articular hyaline cartilage in synovial joints covers the inflammatory arthritis comes from the correlation between appropriate bone surfaces and allows for a smooth move- cathepsin K expression in osteoarthritic cartilage and ment, reduces frictions, and serves as shock absorber. In synovial tissues and disease severity [37, 45, 102, 103]. contrast to highly vascular bone, articular cartilage is Cathepsin K expression increased with disease severity. In avascular and thus has little turnover. Its half-life exceeds contrast, cathepsin S concentrations in the inflamed syno- human life [84]. There are two main organic components in vium did not correlate with disease severity [37]. Cathepsin articular cartilage: type II collagen and aggrecan. Both K expression was observed in mice with collagen-induced constituents are subject to local proteolysis in healthy joints arthritis in osteoclasts, the inflamed synovium and within [85] which is regulated by cytokines, growth factors, and the pannus forming on the articular cartilage (Fig. 4). matrix degradation products. Main proteases in chondro- Salminen–Mankonen and coworkers suggested that the cytes are members of the metalloprotease class and include main function of cathepsin K in osteoarthritis is the deg- matrix metalloproteinases such as MMP1 and members of radation of cartilage and bone fragments sheared from the the ADAMTS family (aggrecanases) [86]. Cathepsins joint surface during the progression of the disease [104]. In appear to be expressed at very low levels in healthy joints a dog osteoarthritis model, a potent inhibitor of cathepsin K and their spatial distribution seems to depend on age [87]. (SB-55348) decreased the overall tibial degeneration score Activity staining for cathepsin B only revealed low activity by 21% and the cartilage degradation score by 32% [105]. [88]. However, cathepsins have been implicated in patho- In contrast, overexpression of cathepsin K in mice showed logical cartilage turnover early on. Cathepsin B and L are increased cathepsin K expression in chondrocytes and led capable of cleaving type II collagen, though only in the to the spontaneous development of synovitis and cartilage telopeptide region [89], and cathepsin B may act as an degradation [98, 106]. However, the role of cathepsin K in alternative aggrecanase [90]. Aggrecan is effectively arthritic diseases is not undisputed. For example, cathepsin degraded by cathepsins S and K as well and cathepsin S K deficiency in TNF-a transgenic mice only partially pre- cleaves this proteoglycan also at neutral pH [91]. Proin- vented arthritic bone resorption [107]. Even more unex- flammatory cytokines such as IL-1a and TNF-a upregulate pected, a pycnodysostosis patient (human cathepsin K cathepsins B and L expression in chondrocytes [92]. deficiency) developed a severe erosive arthritis. Distal 123 Clinic Rev Bone Miner Metab (2011) 9:83–93 89

Fig. 4 Cathepsin K expression in arthritic mouse joints. a Healthy joint with intact articular cartilage. Cathepsin K expression is limited to osteoclasts in growth plate area. b Diseased joint (collagen- induced arthritis, CIA). Cathepsin K is expressed in osteoclasts within the bone*, in pannus covering the articular cartilage by fibroblast-like cells**, and within the inflamed synovium (likely synovial fibroblasts and macrophages)***

phalanges of hands and feet revealed dramatic bony ero- depends on the initiation of an MHC class II mediated sions and partial lysis [108]. These findings may indicate a immune response toward an auto antigen. Cathepsin S has limited role of cathepsin K in arthritis and the involvement likely two functions: (1) the endosomal degradation of of other proteolytic activities. antigens and (2) the degradation of then invariant chain In inflammatory arthritis such as rheumatoid arthritis, which blocks the access of the antigenic peptide to the synovial fibroblasts and macrophages play a critical role in peptide binding groove of the MHC class II complex. It has cartilage erosion. Cathepsin K is expressed in both cell been well documented that cathepsin S is the major CLIP types whereas cathepsin S is only expressed in macro- peptide releasing factor in antigen presenting cells [27, phages. Immunostaining for cathepsin K revealed that 111, 112]. Cathepsin S-deficient mice revealed a reduced cathepsin K expressing cells infiltrated the cartilage tissue susceptibility toward collagen-induced arthritis [113]. or were found in the pannus attached to the articular car- However, no information was provided whether this tilage [36, 58]. Figure 4 shows a strong cathepsin K affected the cartilage erosion. A broad specificity inhibitor staining in pannus-like cell layer covering the articular of cathepsins K, L, and S (Mu-Leu-HomoPhe-vinylsulfone, cartilage, in subchondral osteoclasts, and within the syno- LHVS) was tested in an adjuvant-induced arthritis model in vium. Other major cell types of cathepsin K expression rats and revealed a significant decrease in inflammation and include giant multinucleated cells within the synovium in the amount of cysteine protease activity measured in the [103]. The destructive activity of synovial fibroblasts is joint tissue [114]. More detailed studies are necessary to limited to the articular cartilage and they do not contribute address a potentially joint protective effect of cathepsin S to bone erosion. RANKL knockout mice were protected inhibitors. against bone resorption but not against cartilage erosion [109]. On the other hand, synovial fibroblasts secrete RANKL and thus promote osteoclastogenesis and subse- Development of Specific Cathepsin K Inhibitors quently cathepsin K-mediated bone resorption in rheumatoid and Clinical Trials arthritis [110]. Bone resorption requires demineralization of the bone matrix prior to the hydrolysis of the collagen A multitude of cathepsin inhibitors have been synthesized fibers. Fibroblasts do not possess a ‘‘demineralization and tested under in vitro and preclinical conditions. The apparatus’’ consisting of V-ATPase, a ruffled border, and interested reader is referred to several review articles sealing zone. However, fibroblasts are potent phagocytos- summarizing cathepsin inhibitor development as thera- ing cells [41] and take up type II collagen fibrils form the peutics [115–119]. The only cathepsin inhibitors, which cartilage matrix which are degraded intracellularly by have extensively been tested in clinical trials are cathepsin lysosomal cathepsin K. Inhibition of cathepsin K leads to K inhibitors. These inhibitors were primarily tested in an accumulation of undigested collagen fibers inside the osteoporosis related trials. At least four cathepsin K fibroblast [36]. inhibitors entered clinical trials: balicatib is a nitrile-based Besides their roles in the joint erosion, cathepsins are inhibitor developed by Novartis with a high selectivity and also involved in the inflammatory response in arthritic potency for cathepsin K, relacatib, a peptidomimetic-based diseases. Here, cathepsin S plays a central role. Rheuma- inhibitor from GlaxoSmithKline with a surprisingly poor toid arthritis is considered an autoimmune disease, which specificity [120], odanacatib, a Merck designed nitrile

123 90 Clinic Rev Bone Miner Metab (2011) 9:83–93 compound with a high potency and selectivity for cathepsin reduction of incidence rates of radiographic spine and other K, and ONO-5334, a yet undisclosed compound from Ono bone fractures. These data will be available for evaluation Pharmaceuticals. Balicatib was withdrawn after phase II by July 2012. Odanacatib has been also evaluated in clinical trials due to potential adverse effects with sclero- intervention studies in breast and prostate cancer-associ- derma-like symptoms [121, 122]. Relacatib showed prob- ated bone tumors. Clinical trials showed promising results lems in phase I trial due to drug–drug interactions (GSK in reducing urinary NTX by 77% after 4 weeks of 5 mg Protocol summary PSB-462795/008). ONO-5334 was well oral per day in breast caner patients [129]. tolerated in phase II trials [123] and resulted in increased bone mineral density (BMD) similar to the , alendronate, in the lumbar spine and better improvements Conclusion in the femur. In contrast to , balicatib and ONO-5334 did not significantly affect bone formation Cysteine cathepsins have gained significant prominence as parameters. drug targets for skeletal diseases. They play a major role in Odanacatib, a non-basic und thus non-lysosomotropic the destructive part in these diseases as they exhibit highly compound advanced into phase III trials. The compound potent collagenase and proteoglycan degrading activities. showed a clear dose response regarding the inhibition of They are inducible by inflammatory cytokines and regu- bone resorption and similar to balicatib and ONO-5334 did lated by matrix degradation products and estrogen. not suppress bone formation [124]. In placebo-controlled Cathepsins are active intracellularly and extracellularly. phase I studies, healthy postmenopausal women received Cathepsin K is the main collagenase of osteoclasts and either daily (n = 49) or weekly (n = 30) varying doses of highly potent and selective inhibitors have been developed odanacatib for 3 weeks. At 50 mg per week, about 60% to target its activity. Cathepsin K inhibition reduces bone reductions in serum CTX and urinary NTX were observed. resorption in humans without negatively affecting bone Slightly larger reductions in both resorption markers were formation. Cathepsin S may play a major role in the recorded at 2.5 mg/day regimen [125]. Phase II trials inflammatory response in rheumatoid arthritis as its inhi- included a 12-month treatment of postmenopausal women bition leads to a reduction in inflammation. For diseases with low bone mineral density. Four different doses were such as rheumatoid arthritis, where joint destruction and tested in a weekly regimen (3, 10, 25, 50 mg). All, but the inflammation go hand in hand, inhibitors specific for lowest dose increased lumbar spine and femoral bone cathepsins K and S might be beneficial. mineral density in a dose-dependent manner. Urinary NTX was decreased by 60% from baseline after 12 months. Test Acknowledgments The work was supported by a Canada Research extension for additional 12 months resulted in similar Award and grants from the Canadian Institutes of Health Research (MOP 89974 and 86586). parameters. At 10 and 25 mg/week, bone formation factors such as PINP remained at the levels of the control placebo Conflict of interest The author has no conflict of interest. group after 6 months of treatment, whereas the 50 mg treatment group showed about a 30% decrease after 12 months and a 20% decrease after 24 months [126]. 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