Search

Pycnodysostosis, a rare autosomal recessive skeletal dysplasia, is caused by a deficiency of cathepsin K. Patients have impaired bone resorption in the presence of normal or increased numbers of multinucleated, but dysfunctional, osteoclasts. Cathepsin K degrades collagen type I and generates N-telopeptide (NTX) and the C-telopeptide (CTX) that can be quantified. Levels of these telopeptides are increased in lactating women and are associated with increased bone resorption. Nothing is known about the consequences of cathepsin K deficiency in lactating women. Here we present for the first time normalized blood and CTX measurements in a patient with pycnodysostosis, exclusively related to the lactation period. In vitro studies using osteoclasts derived from blood monocytes during lactation and after weaning further show consistent bone resorption before and after lactation. Increased expression of cathepsins L and S in osteoclasts derived from the lactating patient suggests that other proteinases could compensate for the lack of cathepsin K during the lactation period of pycnodysostosis patients.

Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.

Objective: Cathepsin L (CatL), cathepsin S (CatS), and arteriosclerosis adhesion molecules such as monocyte chemotactic protein-1 (MCP-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and soluble E-selectin (sE-selectin) are potent elastases implicated in human arterial wall remodeling. In this study, we aimed to evaluate the effects of intermittent exercise on the plasma concentrations of these cathepsins and arteriosclerosis adhesion molecules in night shift workers., Methods: Thirty male participants who were night shift workers (experimental group, n = 15; control group, n = 15) were included in this study. The experimental group performed an intermittent exercise at 10-min bouts (30 min per day), three days a week during 10 weeks. Body composition, blood pressure, and cardiovascular disease risk factors were measured., Results: After intermittent exercise, significant group time interactions for body weight (p < .01) and body fat percentage (p < .01) were found. With regard to cardiovascular disease risk factors, group time interactions for CatL (p < .01), CatS (p < .01), MCP-1 (p < .05), sE-selectin (p < .01), and sVCAM-1 (p < .01) were significant., Conclusions: This study provides preliminary evidence to suggest that intermittent exercise may represent an effective intervention strategy for preventing atherosclerosis, thus leading to improved cardiovascular health in night shift workers., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

Background: Cathepsin L (CatL), cathepsin K (CatK), and cathepsin V (CatV) are potent elastases implicated in human arterial wall remodeling. Whether plasma levels of these cathepsins are altered in patients with abdominal aortic aneurysms (AAAs) remains unknown., Methods and Results: Plasma samples were collected from 476 male AAA patients and 200 age-matched male controls to determine CatL, CatK, and CatV levels by ELISA. Student's t-test demonstrated significantly higher plasma CatL levels in AAA patients than in controls (P < 0.0001), whereas CatK and CatV levels were lower in AAA patients than in controls (P = 0.052, P = 0.025). ROC curve analysis confirmed higher plasma CatL levels in AAA patients than in controls (P < 0.001). As potential confounders, current smoking and use of angiotensin-converting enzyme (ACE) inhibitors, aspirin, clopidogrel, and statins associated with significantly increased plasma CatL. Pearson's correlation test demonstrated that plasma CatL associated positively with CatS (r = 0.43, P < 0.0001), body-mass index (BMI) (r = 0.07, P = 0.047) and maximal aortic diameter (r = 0.29, P < 0.001), and negatively with lowest measured ankle-brachial index (ABI) (r = -0.22, P < 0.001). Plasma CatL remained associated positively with CatS (r = 0.43, P < 0.0001) and aortic diameter (r = 0.212, P < 0.001) and negatively with ABI (r = -0.10, P = 0.011) after adjusting for the aforementioned potential confounders in a partial correlation analysis. Multivariate logistic regression analysis indicated that plasma CatL was a risk factor of AAA before (odds ratio [OR] = 3.04, P < 0.001) and after (OR = 2.42, P < 0.001) the same confounder adjustment., Conclusions: Correlation of plasma CatL levels with aortic diameter and the lowest ABI suggest that this cysteinyl protease plays a detrimental role in the pathogenesis of human peripheral arterial diseases and AAAs., (© 2013 Elsevier Ireland Ltd. All rights reserved.)

The receptor activator of NFkappaB ligand (RANKL) is a critical mediator of osteoclastogenesis and regulates cathepsin K (CTSK) expression, which is essential for normal bone resorption. RANKL acts, in part, via the Ca(2+)/calmodulin/calcineurin signaling pathway, which in turn, activates NFATc1 (nuclear factor of activated T-cells) and downstream gene expression. We investigated the signals and promoter elements that regulate CTSK gene expression in RAW 264.7 cells, which can be differentiated to osteoclasts by RANKL. Disrupting Ca(2+) signaling, by blocking Ca(2+) channels, thus inhibiting calcineurin or chelation of intracellular Ca(2+), prevented the stimulation of CTSK expression by RANKL. Both RANKL treatment and overexpression of NFATc1 dramatically enhanced CTSK promoter activity, but not in an identical manner. NFATc1 regulates CTSK promoter activity, but the motifs have not been explicitly identified. We found that as few as 238 bp of the CTSK promoter were sufficient to elicit a marked response to both RANKL and NFATc1, truncations of the CTSK promoter illustrated differences in regional responsiveness. Transfection analysis of CTSK promoter-luciferase plasmids revealed that NFATc1 binding sites at 85, 289 and 345 bp upstream of the transcriptional start site mediated responses to RANKL and NFATc1. Deletion of a 4-bp core element from the site at -85 bp dramatically reduced the response of the CTSK promoter to both RANKL and NFATc1, whereas a similar deletion at -345 bp decreased NFATc1- but not RANKL-mediated responses. Mutation of the site at -289 bp did not affect NFAT-mediated stimulation of CTSK on its own, but did decrease responsiveness in combination with either or both of the other two deletions. Electrophoretic mobility shift assays demonstrated NFATc1 binding to oligonucleotides containing the -85-bp and -345-bp sites, while chromatin immunoprecipitation assays demonstrated enhanced in situ binding by NFATc1 to two analogous sites in the mouse CTSK promoter in response to RANKL treatment. Therefore, proximal NFAT binding sites play a significant role in the NFATc1-mediated stimulation of CTSK gene expression by RANKL.

Cathepsin K (Cat K) degrades bone type I collagen and is a target for the pharmacological treatment of osteoporosis. Further roles for Cat K have been recently described, some of which are supported by the use of purportedly selective Cat K inhibitors in human and rodent cell-based assays. Twelve commercial and non-commercial Cat K inhibitors were profiled against a panel of purified human, rat, and mouse cysteine cathepsins and in two cell-based enzyme occupancy assays for activity against Cat K, B, and L. Ten inhibitors, including the carbohydrazide Cat K inhibitor II (Boc-Phe-Leu-NHNH-CO-NHNH-Leu-Z), the non-covalent K4b, and the epoxide NC-2300, have either little Cat K selectivity, or appear poorly cell penetrant. The amino-acetonitrile-containing inhibitors L-873724 and odanacatib show greater than 100-fold human Cat K enzyme selectivity and have similar IC(50) values against each cathepsin in cell-based and enzyme assays. The basic inhibitor balicatib has greater cellular potencies than expected on the basis of purified enzyme assays. The accumulation of [(14)C]-balicatib in fibroblasts is blocked by prior treatment of the cells with NH(4)Cl, consistent with balicatib having lysosomotropic properties. These results support the use of L-873724 and odanacatib as tools to identify novel roles for Cat K using human cell-based systems, but suggest using caution in the interpretation of studies employing the other compounds.

Background: Pycnodysostosis is a rare autosomal recessive skeletal dysplasia characterized by short stature, osteosclerosis, acro-osteolysis, frequent fractures and skull deformities. Mutations in the gene encoding cathepsin K (CTSK), a lysosomal cysteine protease, have been found to be responsible for this disease., Objectives: To identify pathogenic mutation in a consanguineous Pakistani family with 3 affected individuals demonstrating autosomal recessive pycnodysostosis., Methods: Genotyping of 10 members of the family, including three affected and seven unaffected individuals was carried out by using polymorphic markers D1S442, D1S498, and D1S305, which are closely linked to the CTSK gene on chromosome 1q21. To screen for mutations in the CTSK gene, all of its exons and splice junctions were PCR amplified from genomic DNA and sequenced directly in an ABI Prism 310 automated sequencer., Results: Genotyping results showed linkage of the pycnodysostosis Pakistani family to the CTSK locus. Sequence analysis of the CTSK gene revealed homozygosity for a missense mutation (A277V) in the affected individuals., Conclusion: We describe a missense mutation in the CTSK gene in a Pakistani family affected with autosomal recessive pycnodysostosis. Our study strengthens the role of this particular mutation in the pathogenesis of pycnodysostosis and suggests its prevalence in Pakistani patients.

The microphthalmia transcription factor/transcription factor E (TFE)-family translocation renal cell carcinomas bear specific translocations that result in overexpression of TFE3 or TFEB. TFE3 fusion gene product overexpression occurs as consequence of different translocations involving chromosome Xp11.2, whereas TFEB overexpression is the result of the specific translocation t(6;11)(p21;q12), which fuses the Alpha gene to TFEB. Both TFE3 and TFEB are closely related members of the microphthalmia transcription factor/TFE-family, which also includes TFEC and microphthalmia transcription factor. These transcription factors have overlapping transcriptional targets. Overexpression of microphthalmia transcription factor has been shown to mediate the expression of cathepsin-K in osteoclasts. We hypothesize that the overexpression of the related TFE3 fusion proteins and TFEB in translocation renal cell carcinomas may have the same effect. We studied cathepsin-K in 17 cytogenetically confirmed microphthalmia transcription factor/TFE-family translocation renal cell carcinomas. Seven cases showed a t(6;11)(p21;q12), ten cases showed translocations involving Xp11.2; five cases t(X;1)(p11;q21) resulting in a PRCC-TFE3 gene fusion; three cases t(X;1)(p11;p34) resulting in a PSF-TFE3 gene fusion, one t(X;17)(p11;q25) resulting in an ASPL-TFE3 gene fusion, and one t(X;3)(p11;q23) with an unknown TFE3 gene fusion. As control we analyzed cathepsin-K in 210 clear cell, 40 papillary, 25 chromophobe renal cell carcinomas and 30 oncocytomas. All seven TFEB translocation renal cell carcinomas were labeled for cathepsin-K. Among the cytogenetically confirmed TFE3 translocation renal cell carcinomas, 6 out of 10 were positive. None of the other renal neoplasms expressed cathepsin-K. We conclude the following: (1) cathepsin-K is consistently and strongly expressed in TFEB translocation renal cell carcinomas and in 6 of 10 TFE3 translocation renal cell carcinomas. (2) Cathepsin-K immunolabeling in both TFE3 and TFEB translocation renal cell carcinomas distinguishes these neoplasms from the more common adult renal cell carcinomas, and may be a specific marker of these neoplasms. (3) These results further support the concept that the overexpression of TFE3 or TFEB in these neoplasms activates the expression of genes normally regulated by microphthalmia transcription factor in other cell types.

Objective: The hypothesis of this study was that synovial factors playing a pivotal role in the pathogenesis of osteoarthritis (OA) and thus gene expression in the synovium would be altered at the initial stage of OA. The aims of this study were to identify the candidate genes in synovium related to OA initiation, to evaluate cartilage degeneration after knockdown of the gene using small interfering RNA (siRNA) gene silencing in the knee joints at the initial stage of OA, and to determine the potential role of the knocked-down gene in OA initiation., Methods: Genes overexpressed in synovium at the initial stage of disease in a rabbit model of anterior cruciate ligament transection (ACLT)-induced OA were identified using the suppression subtractive hybridization technique and differential screening. Candidate gene expression in the synovium of the knees of rabbits with OA was manipulated with electroporation-assisted siRNA transduction 4 times before and after operation. Four weeks after surgery, histologic analysis was performed., Results: Cathepsin K gene and protein expression was significantly up-regulated in synovium at the initial stage of OA in rabbits. Down-regulation of cathepsin K in synovium at the initial stage of OA significantly accelerated cartilage degeneration., Conclusion: These results indicate that cathepsin K plays a protective role in cartilage degeneration at the initial stage of OA. We believe that the current results obtained from models of the early phase of OA will provide useful information for developing a novel strategy to prevent disease progression.

Cathepsin X, a cysteine protease, has been shown to regulate an immune response by activating beta-2 integrin receptors. In this study we demonstrate its role in regulating the immune response to infection with H. pylori. The level of cathepsin X was determined in THP-1 monocyte cells primed with H. pylori antigens isolated from subjects suffering from gastritis, who had either eradicated or not the disease after the antibiotic therapy. We show that the specific clinical outcome of H. pylori eradication therapy correlates strongly with the membrane expression of cathepsin X in stimulated THP-1 cells, being significantly higher after stimulation with H. pylori strains from those subjects who did not respond to antibiotic therapy. The same antigens elicit a more vigorous immune response, increased expression of MHC II, however trigger inadequate cytokine profile (IFN-gamma and IL-4) to eradicate the pathogen. We propose that cathepsin X mediated activation of beta-2 integrin receptor Mac-1 suppresses the stimulatory signal in the form of cytokines. Cathepsin X co-localizes on the membrane of THP-1 cells with Mac-1 integrin receptor and its inhibition increases homotypic aggregation and mononuclear cell proliferation, events that are associated with low Mac-1 activity. Our study highlights the diversity of the innate immune response to H. pylori antigens leading to either successful eradication of the infection or maintenance of chronic inflammation, revealing cathepsin X location and activity as a regulator of the effectiveness of H. pylori eradication.

Background: Ankylosing spondylitis (AS) is a common, largely genetically determined, rheumatic disease that is characterised by spinal inflammation and new bone formation. However, the exact pathogenesis and pathology are still not clear., Objective: To analyse tissue obtained at spinal surgery by immunohistochemistry and compare the specimen of patients with AS to those with degenerative disc disease (DDD)., Methods: Bony and soft tissue specimens of 30 patients with AS and 20 with DDD were obtained during spinal osteotomy from different anatomic regions including articular and spinous processes, interspinous ligaments and intervertebral disks. Immunohistolochemistry was performed with established markers for cathepsin K, matrix metalloproteinase (MMP)1, MMP3 and receptor activator for nuclear factor kappaB (RANK) ligand., Results: Cathepsin K and MMP1-positive cells were only observed in AS specimens. Cathepsin K-positive multinucleated cells were detected at articular processes adjacent to fibrous tissues. MMP1 was expressed in smaller mononuclear cells attached to bone. Invasion of bone by MMP1 cells was seen at entheseal sites. In the intervertebral disks, most mononuclear cells were cathepsin K-positive. Isolated cells expressing these matrix-degrading enzymes found in DDD never showed signs of invasion. No differences were found for MMP3 between AS and DDD. Clear expression of RANK ligand was only detected in one patient with AS., Conclusions: Cathepsin K is strongly expressed in different regions of the spine in AS. Cathepsin K was mainly expressed by mononuclear cells, fibroblast-like cells and cells attached to bone and at sites of bone remodelling, suggestive of high osteoclastic activity. This supports the role of persistent inflammation in the pathogenesis of AS. How these changes relate to osteoproliferation remains to be determined.

The cysteine carboxypeptidase cathepsin X has been recognized as an important player in degenerative processes during normal aging and in pathological conditions. In this study we identify isozymes alpha- and gamma-enolases as targets for cathepsin X. Cathepsin X sequentially cleaves C-terminal amino acids of both isozymes, abolishing their neurotrophic activity. Neuronal cell survival and neuritogenesis are, in this way, regulated, as shown on pheochromocytoma cell line PC12. Inhibition of cathepsin X activity increases generation of plasmin, essential for neuronal differentiation and changes the length distribution of neurites, especially in the early phase of neurite outgrowth. Moreover, cathepsin X inhibition increases neuronal survival and reduces serum deprivation induced apoptosis, particularly in the absence of nerve growth factor. On the other hand, the proliferation of cells is decreased, indicating induction of differentiation. Our study reveals enolase isozymes as crucial neurotrophic factors that are regulated by the proteolytic activity of cathepsin X.

Inhibition of cathepsin K (CatK) is a potential new treatment for osteoporosis. In two double-blind, randomized, placebo-controlled phase I studies, postmenopausal female subjects received odanacatib (ODN), an orally active, potent, and selective CatK inhibitor, once weekly for 3 weeks or once daily for 21 days. Bone turnover biomarkers, safety monitoring, and plasma ODN concentrations were assessed. These studies showed ODN to be well tolerated. Pharmacokinetic (PK) analysis revealed a long half-life (t(1/2); 66-93 h) consistent with once-weekly dosing. Pronounced reductions in C-terminal telopeptide of type I collagen (approximately 62%) and N-terminal telopeptide of type I collagen normalized to creatinine (NTx/Cr) (approximately 62%) at trough (C(168 h)) were seen following weekly administration. Robust reductions in CTx (up to 81%) and NTx/Cr (up to 81%) were seen following daily administration. ODN exhibits robust and sustained suppression of bone resorption biomarkers (CTx and NTx/Cr) at weekly doses > or = 25 mg and daily doses > or = 2.5 mg.

Cathepsin K (catK) is a lysosomal cysteine protease with strong collagenolytic activity well known to mediate bone resorption in osteoclasts. Recently, catK has also been reported to be expressed in other tissues. In the dermis, it is expressed only under certain circumstances such as scarring or inflammation. We therefore investigated the expression and regulation of this protease in dermal fibroblasts using immunoblotting and immunostaining. Cultured skin fibroblasts were found to strongly express catK in lysosomes. Internalization of collagen I and IV to lysosomes of fibroblasts indicates a role of catK in intracellular collagen degradation after endocytosis, a process that is different from the metalloproteinase-mediated collagen degradation in the extracellular space. In fibroblasts, interleukin-1alpha and cellular confluence upregulate catK expression and transforming growth factor-beta1 inhibits confluence-induced catK upregulation in skin fibroblasts. RANKL (ligand of receptor activator of NF-kappaB) did not alter catK expression. These regulators of catK expression are likely to play a role in the as-needed upregulation in certain skin conditions, where the prominent matrix-degrading properties of catK are thought to require tight regulation to maintain the homeostasis of the extracellular matrix.

Cathepsin K (EC 3.4.22.38) is expressed by osteoclasts and synovial fibroblasts and its proteolytic activity is hypothesized to play a role in the pathology of rheumatoid arthritis. This study explored the effects of the cathepsin K inhibitor N-(1-{[(Cyanomethyl)amino]carbonyl}cyclohexyl)-4-[2-(4-methylpiperazin-1-yl)-1,3-thiazol-4-yl]benzamide (L-006235) in murine collagen-induced arthritis. L-006235 is a potent inhibitor of recombinant human and murine cathepsin K, enzymes (K(i):0.073 nM and IC(50): 2.4 nM, respectively) and at the cellular level in human osteoclasts (IC(50): 28 nM) with ~1000-fold selectivity against cathepsin S. L-006235 did not result in splenic invariant chain p10 accumulation, a specific marker of cathepsin S inhibition. L-006235 was dosed daily (25 mg/kg, p.o.), either prophylactically (days 0-42) or therapeutically (14 days post onset of disease) to DBA/1J mice subjected to collagen-induced arthritis. Disease severity was scored during the course of the study. Histological evaluation of cartilage and bone degradation together with related biomarkers namely, deoxypyridinoline, cartilage oligomeric matrix protein and C-terminal telopeptide degradation product of type I collagen (CTX-I) were analyzed after the study. After prophylactic or therapeutic administration, L-006235 significantly reduced biomarkers reflecting bone and cartilage degradation. Pathological changes at the histological level were significantly reduced after prophylactic treatment (P<0.01), but not after therapeutic treatment. Prophylactic treatment with L-006235 delayed disease onset (P<0.01) and reduced the disease severity score (P<0.05). Inhibition of cathepsin K activity exerts beneficial effects on collagen-induced arthritis in mice and thus warrants further investigation as a therapeutic intervention in human rheumatoid arthritis.

Recent studies associate cathepsin K with schizophrenia. The endogenous substrates of this protease, however, remain to be identified. We show here that cathepsin K is capable of liberating met-enkephalin from beta-endorphin (beta-EP) in vitro. To verify if this process might possibly contribute in the pathogenesis of schizophrenia post mortem brains of patients suffering from this disease were analysed immunohistochemically for the presence and co-localization of cathepsin K and beta-EP. In support of a functional role of the observed formation of met-enkephalin on the expense of beta-EP increased numbers of cathepsin K immunoreactive cells, but diminished numbers of both beta-EP-positive cells and double-positive (cathepsin K/beta-EP) cells were found in left and right arcuate nucleus of schizophrenics. In addition a reduced density of beta-EP-immunoreactive neuropil (fibres, nerve terminals) was estimated in the left and right paraventricular nucleus (PVN) of individuals with schizophrenia. Our results imply that cathepsin K, which becomes up-regulated in its cerebral expression by neuroleptic treatment, might significantly contribute to altered opioid levels in brains of schizophrenics, which have previously been reported by us and others, and might reinforce the interest in the putative roles of endorphin and enkephalins in neuropsychiatric disorders.

We hypothesized that the anabolic action of parathyroid hormone (PTH) with the anti-catabolic agents cathepsin K inhibitor and alendronate differs depending on the remodeling status in the bone. C57/BL/6J mice, 8 weeks of age, were subjected to ovariectomized (OVX) or sham surgery. At 6 weeks after surgery, the mice were treated with cathepsin K inhibitor, alendronate, or a vehicle (daily, for 8 weeks), with or without PTH (1-34) (5 times/week, for the last 4 weeks). We assessed the bone chemical markers of the serum and urine, bone mineral density (BMD), histomorphomery in the primary and secondary spongiosa of the proximal tibia after fluorescence labeling, primary cell culture, and mRNA expressions in bone marrow cells. Cathepsin K inhibitor and alendronate significantly increased the BMD and the bone volume of the primary and secondary spongiosa, with a reduction of the urinary C-telopeptide of type I collagen that was increased by OVX, respectively. Cathepsin K inhibitor augmented the anabolic action of PTH on the BMD and bone volume at both the primary and secondary spongiosa, while alendronate had the same effect on the BMD and bone volume only at the primary spongiosa. Cathepsin K inhibitor did not decrease serum osteocalcin with or without PTH, while alendronate did decrease it. Cathepsin K inhibitor did not decrease the values of osteoclast number or bone formation rate with or without PTH, while alendronate decreased those values and increased osteoclast apoptosis. The combination of PTH and cathepsin K inhibitor increased alkaline phosphatase-positive CFU-f formation and c-fos, osterix, and osteocalcin mRNA expressions of bone marrow cells as well as PTH alone, while the combination of PTH and alendronate decreased those values. This study demonstrated that alendronate enhances the anabolic action of PTH at the primary spongiosa, but blunts it in the remodeling trabecular bone, while cathepsin K inhibitor enhances the action at both sites in OVX mice. In conclusion, the anabolic action of intermittent PTH in combination with cathepsin K inhibitor or alendronate differs depending on the remodeling status of bone in OVX mice.

It is well known that the collagenolytic cathepsins play an important role in the degradation of bone matrix. Therefore, the purpose of this study was to clarify the prevention effect of bone resorption by milk components. Using double-layer reverse zymography, we found a 20 kDa protein in milk which inhibited cysteine proteases. This inhibitory protein was identified as beta-lactoglobulin B. The inhibitory activity of beta-lactoglobulin B against cathepsin K was stronger than that of beta-lactoglobulin A. Beta-lactoglobulin B specifically inhibited papain type cysteine proteases such as cathepsins K and L, but not serine proteases, aspartic proteases or metallo proteases. Beta-lactoglobulin B inhibited cathepsin K non-competitively and the Ki value was 10(-5) M. The formation of osteoclastic pits in the culture system was effectively inhibited by 10(-4)-10(-5) M beta-lactoglobulin B in vitro. Furthermore, we demonstrated that beta-lactoglobulin B inhibited degradation of type I-collagen by collagenolytic cathepsins. Using the everted-sac method in rat small intestines, it was found that beta-lactoglobulin was more easily absorbed from the intestines of young rats (5 wk-old) than from those of older rats (more than 20 wk-old). The digested products of beta-lactoglobulin B with lysyl-endopeptidase showed a similar inhibitory activity against cathepsin K to the intact beta-lactoglobulin B did. Therefore, peroral intake of beta-lactoglobulin in milk and its digested peptides are expected to help protect osteoclastic bone resorption via inhibition of collagenolytic cathepsins K and L.

Background: Mixed CLA isomers variably affect bone resorption in animals and decrease osteoclast formation and activity in murine osteoclasts. These variable effects may be due to the different isomers present in commercial preparations of CLA, and the effects of the predominant individual isomers, 9cis,11trans (9,11) and 10trans,12cis (10,12) CLA are not clear. The objectives of this study were to determine the effects of the individual CLA isomers on osteoclast formation and activity from human CD14+ monocytes, and to determine whether any changes are accompanied by changes in cathepsin K, matrix metalloproteinase-9 (MMP-9), receptor activator of NF-kappaB (RANK) and tumour necrosis factor alpha (TNFalpha) gene expression. Osteoclasts were identified as TRAP+ multinucleated cells. Osteoclast activity was quantified by the amount of TRAP in the cultured media., Results: At 50 microM, 9,11 CLA inhibited osteoclast formation by approximately 70%, and both 9,11 and 10,12 CLA decreased osteoclast activity by approximately 85-90%. Both isomers inhibited cathepsin K (50 microM 9,11 by approximately 60%; 10,12 by approximately 50%) and RANK (50 microM 9,11 by approximately 85%; 50 microM 10,12 by approximately 65%) expression, but had no effect on MMP-9 or TNFalpha expression., Conclusion: 9,11 CLA inhibits osteoclast formation and activity from human cells, suggesting that this isomer may prevent bone resorption in humans. Although 10,12 CLA did not significantly reduce osteoclast formation, it reduced osteoclast activity and cathepsin K and RANK expression, suggesting that this isomer may also affect bone resorption.

Purpose: Patient survival after resection of non-small cell lung cancer (NSCLC) strongly correlated with the occurrence of distant metastasis. Cathepsins are members of the lysosomal cysteine proteases family and can support the metastatic process by degrading the extracellular matrix. The purpose of this study was to identify members of the Cathepsin family that correlate with recurrence-free and overall survival of NSCLC patients., Patients and Methods: The expression of 13 Cathepsins was examined using DNA-microarray technology in tumor tissues of 89 surgically treated NSCLC patients. All NSCLC samples were classified according to median Cathepsin expression value into either a high or a low expression group. All Cathepsin expression groups were subjected to clinical prognostic analyses regarding survival and local as well as distant recurrences., Results: Patients with high Cathepsin C tumor expression showed higher tumor recurrence rate compared to patients with low Cathepsin C expression (p = 0.02). The tumor expression of Cathepsins K and B significantly correlated with recurrence-free and overall survival as determined by multivariate analysis. A high expression of Cathepsin B or K was associated with a considerable reduction of recurrence-free as well as overall survival. NSCLC patients with a high expression of both Cathepsin B and K had a significantly (p = 0.001) poorer outcome (5-year survival rate: 13%) than patients with low expression of both genes (5-year survival rate: 75%)., Conclusions: The combined expression level of Cathepsins B and K identifies high-risk NSCLC patients. A selection of gene expression panels is theoretically superior to established clinical and pathological criteria.

Bone resorption is a multistep process including osteoclast attachment, cytoskeletal reorganization, formation of four distinct plasma membrane domains, and matrix demineralization and degradation followed by cell detachment. The present study describes the intracellular mechanisms by which overexpression of cathepsin K in osteoclasts results in enhanced bone resorption. Osteoclasts and bone marrow-derived osteoclast and osteoblast precursors were isolated from mice homozygous (UTU17(+/+)) and negative for the transgene locus. Cells cultured on bovine cortical bone slices were analyzed by fluorescence and confocal laser scanning microscopy, and bone resorption was studied by measurements of biochemical resorption markers, morphometry, and FESEM. Excessive cathepsin K protein and enzyme activity were microscopically observed in various intracellular vesicles and in the resorption lacunae of cathepsin K-overexpressing osteoclasts. The number of cathepsin K-containing vesicles in UTU17(+/+) osteoclasts was highly increased, and co-localization with markers for the biosynthetic and transcytotic pathways was observed throughout the cytoplasm. As a functional consequence of cathepsin K overexpression, biochemical resorption markers were increased in culture media of UTU17(+/+) osteoclasts. Detailed morphometrical analysis of the erosion in bone slices indicated that the increased biosynthesis of cathepsin K was sufficient to accelerate the osteoclastic bone resorption cycle. Cathepsin K overexpression also enhanced osteogenesis and induced the formation of exceptionally small, actively resorbing osteoclasts from their bone marrow precursors in vitro. The present study describes for the first time how enhancement in one phase of the osteoclastic resorption cycle also stimulates its other phases and further demonstrate that tight control and temporal coupling of mesenchymal and hematopoietic bone cells in this multistep process.

Most of the strategies developed in bone tissue engineering in the past three decades have been aimed to repair/regenerate the tissue with forming elements such as osteoblasts and bone morphogenetic proteins. All these materials are selected as they are known to induce bone formation. Since it is known that bone turnover in basic multicellular units (BMUs) is at equilibrium, inducing an imbalance in this process via molecules known to resorb and transmit resorption signals and therefore initiate activation in bone forming cells from adjacent tissue may offer a radical approach to bone regeneration. Possible targets for such an approach may include resorbing molecules such as tartrate resistant acid phosphatase (TRAP) and cathepsin K. Delivering these enzymes (TRAP and cathepsin K) and/or other molecules involved in bone resorption into bone defects and thus obtaining a concentration difference in the levels of these materials may induce bone forming cells to balance bone turnover, therefore inducing bone regeneration.

Cathepsin X is a lysosomal cysteine protease, found predominantly in cells of monocyte/macrophage lineage. It acts as a monocarboxypepidase and has a strict positional and narrower substrate specificity relative to the other human cathepsins. In our recent studies we identified-beta(2) subunit of integrin receptors and alpha and gamma enolase as possible substrates for cathepsin X carboxypeptidase activity. In both cases cathepsin X is capable to cleave regulatory motifs at C-terminus affecting the function of targeted molecules. We demonstrated that via activation of beta(2) integrin receptor Mac-1 (CD11b/CD18) active cathepsin X enhances adhesion of monocytes/macrophages to fibrinogen and regulates the phagocytosis. By activation of Mac-1 receptor cathepsin X may regulate also the maturation of dendritic cells, a process, which is crucial in the initiation of adaptive immunity. Cathepsin X activates also the other beta(2) integrin receptor, LFA-1 (CD11a/CD18) which is involved in the proliferation of T lymphocytes. By modulating the activity of LFA-1 cathepsin X causes cytoskeletal rearrangements and morphological changes of T lymphocytes enhancing ameboid-like migration in 2-D and 3-D barriers and increasing homotypic aggregation. The cleavage of C-terminal amino acids of alpha and gamma enolase by cathepsin X abolishes their neurotrophic activity affecting neuronal cell survival and neuritogenesis.

Cathepsin K is a member of the papain family of cysteine proteases and is highly expressed in osteoclasts that mediate bone resorption. In this review, some of the known features of cathepsin K such as structure, function in bone resorption, gene regulation and its roles in physiological or pathophysiological processes are highlighted.

A series of pyrimidine nitrile inhibitors of Cathepsin K with reduced glutathione reactivity has been identified and Molecular Core Matching (MoCoM) has been used to quantify the effect of an amino substituent at C5.

Cathepsin X (CTSX) is strongly up-regulated in Helicobacter pylori-infected gastric mucosa and intestinal-type gastric cancer. The overexpression of CTSX is mediated predominantly by associated macrophages; depends on a functional type IV-secretion system; and leads to increased migration of gastric epithelial cells. In the present study, we analysed the role of CagA in CTSX overexpression and identified H. pylori-induced inflammatory factors and signalling pathways required for stimulating CTSX expression by H. pylori. Gastric epithelial cells were co-cultured with macrophages in Transwell chambers of 0.4 microm pore size, enabling exchange of fluids but retracting H. pylori. N87 gastric epithelial cells were infected with H. pylori P1 wild-type strain in the presence of inhibitors for p38, JNK, and ERK1/2 signal transduction pathways. Furthermore, cytokines and growth factors were tested for their regulatory function using inhibitory antibodies, and their gene expression was studied by quantitative RT-PCRs and western blots. CTSX is strongly up-regulated at both the mRNA and the protein levels by TNF-alpha, IL-1beta, IL-6, and IL-8, depending on cell type. All these cytokines were found to be increased by five- to ten-fold in macrophages by H. pylori infection of co-cultured N87 gastric epithelial cells. In macrophages, H. pylori up-regulated CTSX via ERK1/2 signalling pathways, and in N87 cells via JNK irrespective of p38 signalling. Our results suggest that H. pylori induced overexpression of CTSX in macrophages and epithelium through specific cytokines that are initiated by CagA-dependent pathways in a cell type-dependent manner.

Cathepsin K deficiency in humans causes pycnodysostosis, which is characterized by dwarfism and osteosclerosis. Earlier studies of 10-week-old male cathepsin K-deficient (knockout, KO) mice showed their bones were mechanically more brittle, while histomorphometry showed that both osteoclasts and osteoblasts had impaired activity relative to the wild type (WT). Here, we report detailed mineral and matrix analyses of the tibia of these animals based on Fourier transform infrared microspectroscopy and imaging. At 10 weeks, there was significant hypercalcification of the calcified cartilage and cortices in the KO. Carbonate content was elevated in the KO calcified cartilage as well as cortical and cancellous bone areas. These data suggest that cathepsin K does not affect mineral deposition but has a significant effect on mineralized tissue remodeling. Since growth plate abnormalities were extensive despite reported low levels of cathepsin K expression in the calcified cartilage, we used a differentiating chick limb-bud mesenchymal cell system that mimics endochondral ossification but does not contain osteoclasts, to show that cathepsin K inhibition during initial stages of mineral deposition retards the mineralization process while general inhibition of cathepsins can increase mineralization. These data suggest that the hypercalcification of the cathepsin K-deficient growth plate is due to persistence of calcified cartilage and point to a role of cathepsin K in bone tissue development as well as skeletal remodeling.

Membrane nanotubes were recently described as a new principle of cell-cell communication enabling complex and specific messaging to distant cells. Calcium fluxes, vesicles, and cell-surface components can all traffic between cells connected by nanotubes. Here we report for the first time the mechanism of membrane nanotube formation in T cells through LFA-1 (CD11a/CD18; alpha(L)beta(2)) integrin activation by the cysteine protease cathepsin X. Cathepsin X is shown to induce persistent LFA-1 activation. Cathepsin X-upregulated T cells exhibit increased homotypic aggregation and polarized, migration-associated morphology in 2D and 3D models, respectively. In these cells, extended uropods are frequently formed, which subsequently elongate to nanotubes connecting T lymphocytes. Our results demonstrate that LFA-1 activation with subsequent cytoskeletal reorganization induces signal transmission through a physically connected network of T lymphocytes for better coordination of their action at various stages of the immune response.

We should consider not only controlling disease activity using DMARDs and biologics as a matter of course, but also preventing against joint destruction, in the treatment for rheumatoid arthritis. Although we can indirectly regulate bone erosion via controlling disease activity, the osteoclast-targeting therapy might be more effective to stop joint destruction. We are waiting for new drugs directly targeting osteoclasts, such as OPG which is the natural inhibitor of RANKL, or cathepsin K inhibitor which reduces degeneration of bone matrix.

Objective: The mechanisms leading to degeneration of articular cartilage in osteoarthritis (OA) are complex and not yet fully understood. Cathepsin K (CK) is a cysteine protease which can also cleave the triple helix of type II collagen. This exposes a neoepitope that can now be identified by specific antibodies. The aim of this study was to obtain evidence suggesting a role for CK in naturally occurring equine OA in both lesional and peri-lesional regions., Methods: Articular cartilages (n=12 horses; 5 healthy, 7 OA) were harvested from animals postmortem. A gross macroscopic examination, histologic (Safranin O-Fast Green and Picrosirius red staining) and immunohistochemical evaluation were performed. Samples were divided into normal appearing cartilage, peri-lesional and lesional cartilage. Cartilage degradation in the samples was graded histologically and immunohistochemically. CK and possible CK cleavage were detected immunohistochemically with specific anti-protein and anti-neoepitope antibodies, respectively. A comparison of CK neoepitope (C2K) production with the collagenase-generated neoepitope produced by matrix metalloproteinases (MMP)-1, 8 and 13 (C2C) was also assessed immunohistochemically., Results: CK and CK cleavage were significantly more abundant in OA cartilage (both peri-lesional and lesional) when compared to remote cartilage within the sample joint or cartilage from healthy joints. The immunohistochemical pattern observed for CK degradation (C2K) was similar to that of collagenase degradation (C2C). Macroscopic cartilage changes and histologic findings were significantly correlated with immunohistochemistry results., Conclusion: The data generated suggests that CK may be involved in cartilage collagen degradation in naturally occurring osteoarthritis.

The molecular events linking lipid accumulation in atherosclerotic plaques to complications such as aneurysm formation and plaque disruption are poorly understood. BALB/c-Apoe(-/-) mice bearing a null mutation in the Npc1 gene display prominent medial erosion and atherothrombosis, whereas their macrophages accumulate free cholesterol in late endosomes and show increased cathepsin K (Ctsk) expression. We now show increased cathepsin K immunostaining and increased cysteinyl proteinase activity using near infrared fluorescence imaging over proximal aortas of Apoe(-/-), Npc1(-/-) mice. In mechanistic studies, cholesterol loading of macrophage plasma membranes (cyclodextrin-cholesterol) or endosomal system (AcLDL+U18666A or Npc1 null mutation) activated Toll-like receptor (TLR) signaling, leading to sustained phosphorylation of p38 mitogen-activated protein kinase and induction of p38 targets, including Ctsk, S100a8, Mmp8, and Mmp14. Studies in macrophages from knockout mice showed major roles for TLR4, following plasma membrane cholesterol loading, and for TLR3, after late endosomal loading. TLR signaling via p38 led to phosphorylation and activation of the transcription factor Microphthalmia transcription factor, acting at E-box elements in the Ctsk promoter. These studies suggest that free cholesterol enrichment of either plasma or endosomal membranes in macrophages leads to activation of signaling via various TLRs, prolonged p38 mitogen-activated protein kinase activation, and induction of Mmps, Ctsk, and S100a8, potentially contributing to plaque complications.

An enantioselective synthesis of the Cathepsin K inhibitor odanacatib (MK-0822) 1 is described. The key step involves the novel stereospecific S(N)2 triflate displacement of a chiral alpha-trifluoromethylbenzyl triflate 9a with (S)-gamma-fluoroleucine ethyl ester 3 to generate the required alpha-trifluoromethylbenzyl amino stereocenter. The triflate displacement is achieved in high yield (95%) and minimal loss of stereochemistry. The overall synthesis of 1 is completed in 6 steps in 61% overall yield.

Cathepsin K (CatK) is a cysteine protease expressed predominantly in osteoclasts, that plays a prominent role in degrading Type I collagen. Growing CatK null mice have osteopetrosis associated with a reduced ability to degrade bone matrix. Bone strength and histomorphometric endpoints in young adult CatK null mice aged more than 10 weeks have not been studied. The purpose of this paper is to describe bone mass, strength, resorption, and formation in young adult CatK null mice. In male and female wild-type (WT), heterozygous, and homozygous CatK null mice (total N=50) aged 19 weeks, in-life double fluorochrome labeling was performed. Right femurs and lumbar vertebral bodies 1-3 (LV) were evaluated by dual-energy X-ray absorptiometry (DXA) for bone mineral content (BMC) and bone mineral density (BMD). The trabecular region of the femur and the cortical region of the tibia were evaluated by histomorphometry. The left femur and sixth lumbar vertebral body were tested biomechanically. CatK (-/-) mice show higher BMD at the central and distal femur. Central femur ultimate load was positively influenced by genotype, and was positively correlated with both cortical area and BMC. Lumbar vertebral body ultimate load was also positively correlated to BMC. Genotype did not influence the relationship of ultimate load to BMC in either the central femur or vertebral body. CatK (-/-) mice had less lamellar cortical bone than WT mice. Higher bone volume, trabecular thickness, and trabecular number were observed at the distal femur in CatK (-/-) mice. Smaller marrow cavities were also present at the central femur of CatK (-/-) mice. CatK (-/-) mice exhibited greater trabecular mineralizing surface, associated with normal volume-based formation of trabecular bone. Adult CatK (-/-) mice have higher bone mass in both cortical and cancellous regions than WT mice. Though no direct measures of bone resorption rate were made, the higher cortical bone quantity is associated with a smaller marrow cavity and increased retention of non-lamellar bone, signs of decreased endocortical resorption. The relationship of bone strength to BMC does not differ with genotype, indicating the presence of bone tissue of normal quality in the absence of CatK.

Aims: Cathepsin K (CatK), an established drug target for osteoporosis, has been reported to be upregulated in atherosclerotic lesions. Due to its proteolytic activity, CatK may influence the atherosclerotic lesion composition and stability. In this study, we investigated the potential role of leucocyte CatK in atherosclerotic plaque remodelling., Methods and Results: To assess the biological role of leucocyte CatK, we used the technique of bone marrow transplantation to selectively disrupt CatK in the haematopoietic system. Total bone marrow progenitor cells from CatK(+/+), CatK(+/-), and CatK(-/-) mice were transplanted into X-ray irradiated low-density lipoprotein receptor knockout (LDLr(-/-)) mice. The selective silencing of leucocyte CatK resulted in phenotypic changes in bone formation with an increased total bone mineral density in the CatK(-/-) chimeras and an effect of gene dosage. The absence of leucocyte CatK resulted in dramatically decreased collagen and increased macrophage content of the atherosclerotic lesions while lesion size was not affected. The atherosclerotic lesions also demonstrated less elastic lamina fragmentation and a significant increase in the apoptotic and necrotic area in plaques of mice transplanted with CatK(-/-) bone marrow., Conclusion: Leucocyte CatK is an important determinant of atherosclerotic plaque composition, vulnerability, and bone remodelling, rendering CatK an attractive target for pharmaceutical modulation in atherosclerosis and osteoporosis.

Cathepsin L-like endopeptidases of the papain family are synthesized as proenzymes. N-terminal proregions are essential for folding and latency of the enzyme unit. While selectivity has been reported for the inhibitory function of papain-family propeptides, there is no systematic investigation of the selectivity of their chaperone-like function to date. The chaperone-like cross-reactivity between the cathepsins S, K, and L were thoroughly quantified in trans-experiments, i.e., with isolated propeptides and mature enzymes, and compared to the inhibitory cross-reactivity. The three endopeptidases have been chosen due to only minimal evolutionary distance and nearly identical X-ray structures of their zymogenes. The intramolecular chaperone function of the proregion was found to be more selective than the inhibitory activity and significant differences were found between the selectivity profiles, underlining the assumption that the inhibitory and the chaperone-like propeptide functions are autonomous. Considering the data published on the intramolecular chaperone-like propeptide function within other protease classes as well, our data suggest that intrinsically structured propeptides are more selective than intrinsically unstructured propeptides, i.e., those adopting tertiary structure elements only in complex with their maternal enzyme.

Osteoclasts degrade bone matrix by demineralization followed by degradation of type I collagen through secretion of the cysteine protease, cathepsin K. Current imaging modalities are insufficient for sensitive observation of osteoclast activity, and in vivo live imaging of osteoclast resorption of bone has yet to be demonstrated. Here, we describe a near-infrared fluorescence reporter probe whose activation by cathepsin K is shown in live osteoclast cells and in mouse models of development and osteoclast upregulation. Cathepsin K probe activity was monitored in live osteoclast cultures and correlates with cathepsin K gene expression. In ovariectomized mice, cathepsin K probe upregulation precedes detection of bone loss by micro-computed tomography. These results are the first to demonstrate non-invasive visualization of bone degrading enzymes in models of accelerated bone loss, and may provide a means for early diagnosis of upregulated resorption and rapid feedback on efficacy of treatment protocols prior to significant loss of bone in the patient.

Highly potent and selective 4-amidofuran-3-one inhibitors of cathepsin S are described. The synthesis and structure-activity relationship of a series of inhibitors with a sulfonamide moiety in the P3 position is presented. Several members of the series show sub-nanomolar inhibition of the target enzyme as well as an excellent selectivity profile and good cellular potency. Molecular modeling of the most interesting inhibitors describes interactions in the extended S3 pocket and explains the observed selectivity towards cathepsin K.

Lymphangioleiomyomatosis is a rare and progressive lung cystic disease, caused by the infiltration of lung parenchyma by mesenchymal cells characterized by co-expression of contractile proteins and melanocytic markers. The pathogenesis of lymphangioleiomyomatosis is determined by mutations affecting tuberous sclerosis complex (TSC) genes, with eventual deregulation of the Rheb/mTOR/p70S6K pathway, and the potential therapeutic activity of mTOR inhibitors is currently under investigation. To better understand the molecular mechanisms involved in the pathogenesis of lymphangioleiomyomatosis, we investigated the expression of cathepsin-k (a papain-like cysteine protease with high matrix-degrading activity). The rationale of this choice was based on the recent demonstration that mTOR inhibitors can regulate major functional activities of osteoclasts, including the expression of cathepsin-k. The immunohistochemical study included 12 cases of lymphangioleiomyomatosis. Twelve angiomyolipomas and several lung diseases (sarcoidosis, organizing pneumonia, usual interstitial pneumonia, emphysema) were investigated as controls. In all lymphangioleiomyomatosis cases, strong cathepsin-k immunoreactivity was demonstrated, restricted to lymphangioleiomyomatosis cells. Similar expression levels were observed in renal angiomyolipomas. These observations extend the knowledge regarding the immunophenotypic profile of lymphangioleiomyomatosis cells, and provide a useful new marker for diagnosis in difficult cases (eg, in small transbronchial biopsies). The strong expression of such a potent papain-like cysteine protease in lymphangioleiomyomatosis cells can significantly contribute to the progressive remodelling of lung parenchyma observed in this deadly disease, with eventual formation of lung cysts. It is possible to speculate that mTOR inhibitors may exert part of their action by limiting the destructive remodelling of lung structure.

Cathepsin K is responsible for the degradation of type I collagen in osteoclast-mediated bone resorption. Collagen fragments are known to be biologically active in a number of cell types. Here, we investigate their potential to regulate osteoclast activity. Mature murine osteoclasts were seeded on type I collagen for actin ring assays or dentine discs for resorption assays. Cells were treated with cathepsins K-, L-, or MMP-1-predigested type I collagen or soluble bone fragments for 24 h. The presence of actin rings was determined fluorescently by staining for actin. We found that the percentage of osteoclasts displaying actin rings and the area of resorbed dentine decreased significantly on addition of cathepsin K-digested type I collagen or bone fragments, but not with cathepsin L or MMP-1 digests. Counterintuitively, actin ring formation was found to decrease in the presence of the cysteine proteinase inhibitor LHVS and in cathepsin K-deficient osteoclasts. However, cathepsin L deficiency or the general MMP inhibitor GM6001 had no effect on the presence of actin rings. Predigestion of the collagen matrix with cathepsin K, but not by cathepsin L or MMP-1 resulted in an increased actin ring presence in cathepsin K-deficient osteoclasts. These studies suggest that cathepsin K interaction with type I collagen is required for 1) the release of cryptic Arg-Gly-Asp motifs during the initial attachment of osteoclasts and 2) termination of resorption via the creation of autocrine signals originating from type I collagen degradation.

This study investigated the relationship between a cathepsin K (CTSK) gene mutation and the pathogenesis of pycnodysostosis in a Chinese patient. A typical pycnodysostosis case and 30 healthy controls were enrolled into the study. Genomic DNA was extracted from blood samples taken from the patient and controls, and the encoding exons of CTSK were amplified and sequenced. Sequencing of the CTSK gene revealed homozygosity for a novel missense mutation in the pycnodysostosis patient, predicting the amino acid exchange from glutamine to proline at position 187 (Q187P). This point mutation in exon 5 of the CTSK gene results in the typical clinical features found in Chinese patients with pycnodysostosis. No similar changes in the CTSK gene sequences were found in the healthy controls.

We previously found that cathepsin K (CTSK) played an important role in adipocyte differentiation. However, the underlying molecular mechanism is not clear. Through the time window study, it was observed that CTSK activities were required mainly in the early phases of adipogenic process. At the same time, the expression of type I collagen disappeared. However, type I collagen can still be observed during the whole process when the CTSK inhibitor-E64 was added. The mRNA levels of peroxisome proliferator-activated receptor gamma (PPAR-gamma) and CCAAT/enhancer binding protein alpha (C/EBP-alpha) was also declining. These imply that CTSK may play a role in adipogenesis in early differentiation phases and produce an effect at least partly by degrading type I collagen, which may provides a basis for developing novel therapeutic approaches to treat obesity and the diseases associated with it.

Objective: Previous studies demonstrated increased levels of cysteine proteases cathepsins in serum and adipose tissues from obese patients. We now provide evidence from a mouse model of obesity to suggest a direct participation of cathepsin K (CatK) in mouse body weight gain and glucose metabolism., Methods and Results: Using real-time polymerase chain reaction, we detected 12-fold increase in CatK transcripts after adipogenesis of human preadipocytes. Using an immunohistology analysis, we consistently observed high levels of CatK expression in adipose tissues from obese humans and mice. Selective inhibition of CatK activity blocked the lipid accumulation in human and mouse preadipocytes. In mice, CatK deficiency reduced significantly diet-induced body weight gain and serum glucose and insulin levels. Similar results were obtained in diet-induced and genetically created (ob/ob) obese mice after animals were treated with a CatK-selective inhibitor. Mechanistic study demonstrated a role for CatK in degrading fibronectin, a matrix protein that controls adipogenesis. Deficiency or inhibition of CatK leads to fibronectin accumulation in muscle and adipose tissues., Conclusions: This study demonstrates an essential role of CatK in adipogenesis and mouse body weight gain, possibly via degradation of fibronectin, thus suggesting a novel therapeutic strategy for the control of obesity by regulating CatK activity.

The maturation status of dendritic cells (DCs) is crucial for effective antigen presentation and initiation of the primary immune response. Maturation stimuli cause the adhesion of immature DCs to the extracellular matrix, which is accompanied by recruitment of the CD11b/CD18 [macrophage antigen-1 (Mac-1)] integrin receptor, cytoskeleton reorganization, and podosome formation. Cathepsin X, a cysteine protease expressed in DCs and other APCs, is involved in Mac-1 activation. We have shown that during maturation, cathepsin X translocates to the plasma membrane of maturing DCs, enabling Mac-1 activation and consequently, cell adhesion. In mature DCs, cathepsin X redistributes from the membrane to the perinuclear region, which coincides with the de-adhesion of DCs, formation of cell clusters, and acquisition of the mature phenotype. Inhibition of cathepsin X activity during DC differentiation and maturation resulted in an altered phenotype and function of mature DCs. It reduced surface expression of costimulatory molecules, increased expression of inhibitory Ig-like transcripts 3 and 4 (ILT3 and ILT4), almost completely abolished cytokine production, diminished migration, and reduced the capacity of DCs to stimulate T lymphocytes. These results stress the importance of cathepsin X in regulating DC adhesion, a crucial event for their maturation and T cell activation.

Objective: The excellent poster painter Henri de Toulouse-Lautrec is the most famous patient with cathepsin K-deficient pycnodysostosis. Cathepsin K is believed to play a major role in osteoclast-driven bone resorption. In this study we explored the role of cathepsin K in bone resorption in a patient with a cathepsin K mutation causing pycnodysostosis in whom psoriatic arthritis also developed. We hypothesized that the patient would develop only inflammatory synovitis but would not develop bone erosions or other osteolytic changes., Methods: Monocytes from the patient with pycnodysostosis and normal control monocytes were isolated and stimulated to fuse and form multinuclear osteoclast-like cells, which were identified by evaluating messenger RNA expression of osteoclast markers. The ability to resorb bone was assessed by determining the extent of pit formation and levels of collagen degradation products generated by cathepsin K (C-terminal crosslinking telopeptide of type I collagen [CTX]) and matrix metalloproteinases (pyridinoline crosslinked C-terminal telopeptide of type I collagen). These experiments were also done in normal control cells after incubation with the cathepsin K inhibitor E64 during bone resorption., Results: In contrast to our a priori hypothesis, the patient developed a mutilating disease with extensive bony erosions associated with lysis of some of the distal phalanges of her hands and feet. After stimulation of monocytes from this patient, the cells formed multinuclear tartrate-resistant acid phosphatase-positive and calcitonin receptor-positive multikaryons, which, however, totally lacked cathepsin K. These multinuclear cells were able to resorb bone but, in contrast to normal control osteoclasts, did not produce CTX. The resorption pattern was abnormal in that, unlike normal control osteoclasts, both osteoclasts from the patient and E64-inhibited osteoclasts did not leave extensive osteoclast trails, but were relatively sessile., Conclusion: In this "experiment of nature" we observed that cathepsin K is not necessary for bone degradation. These findings may be pertinent to our understanding of the functions of cathepsin K inhibitors, which are currently being developed as drugs to treat metabolic bone diseases.

Cathepsin K is the major collagenolytic enzyme produced by bone-resorbing osteoclasts. We showed earlier that the unique triple-helical collagen-degrading activity of cathepsin K depends on the formation of complexes with bone-or cartilage-resident glycosaminoglycans, such as chondroitin 4-sulfate (C4-S). Here, we describe the crystal structure of a 1:n complex of cathepsin K:C4-S inhibited by E64 at a resolution of 1.8 A. The overall structure reveals an unusual "beads-on-a-string"-like organization. Multiple cathepsin K molecules bind specifically to a single cosine curve-shaped strand of C4-S with each cathepsin K molecule interacting with three disaccharide residues of C4-S. One of the more important sets of interactions comes from a single turn of helix close to the N terminus of the proteinase containing a basic amino acid triplet (Arg8-Lys9-Lys10) that forms multiple hydrogen bonds either to the caboxylate or to the 4-sulfate groups of C4-S. Altogether, the binding sites with C4-S are located in the R-domain of cathepsin K and are distant from its active site. This explains why the general proteolytic activity of cathepsin K is not affected by the binding of chondroitin sulfate. Biochemical analyses of cathepsin K and C4-S mixtures support the presence of a 1:n complex in solution; a dissociation constant, K(d), of about 10 nM was determined for the interaction between cathepsin K and C4-S.

Herein, we report on the identification of nonbasic, potent, and highly selective, nitrile-containing cathepsin K (Cat K) inhibitors that are built on our previously identified cyclohexanecarboxamide core structure. Subsequent to our initial investigations, we have found that incorporation of five-membered heterocycles as P2-P3 linkers allowed for the introduction of a methyl sulfone P3-substitutent that was not tolerated in inhibitors containing a six-membered aromatic P2-P3 linker. The combination of a five-membered N-methylpyrazole linker and a methyl sulfone in P3 yielded subnanomolar Cat K inhibitors that were minimally shifted (<10-fold) in our functional bone resorption assay. Issues that arose because of metabolic demethylation of the N-methylpyrazole were addressed through introduction of a 2,2,2-trifluoroethyl substituent. This culminated in the identification of 31 (MK-1256), a potent (Cat K IC 50 = 0.62 nM) and selective (>1100-fold selectivity vs Cat B, L, S, C, H, Z, and V, 110-fold vs Cat F) inhibitor of cathepsin K that is efficacious in a monkey model of osteoporosis.