Role In Osteoclast-mediated Bone Resorption Research Articles

Evaluation of the IDS-iSYS Bone Alkaline Phosphatase (BAP) assay

Evaluation of the IDS-iSYS Bone Alkaline Phosphatase (BAP) assay

Efficacy of ONO-5334, a cathepsin K inhibitor, on bone mass and strength in ovariectomized cynomolgus monkeys

Efficacy of ONO-5334, a cathepsin K inhibitor, on bone mass and strength in ovariectomized cynomolgus monkeys

Effect of nonsurgical periodontal therapy on crevicular fluid levels of Cathepsin K in periodontitis

Objectives Cathepsin K (CTSK), predominantly expressed in osteoclasts, is a potent extracellular matrix degrading enzyme that plays a critical role in osteoclast-mediated bone resorption. Its increased gingival crevicular fluid (GCF) levels in periodontal disease have been reported in a previous study. The present study has been carried out to assess the role of CTSK in periodontal disease and to determine the effect of periodontal treatment on CTSK concentration in GCF. Design 60 subjects were divided into three groups ( n = 20) based on gingival index (GI), probing pocket depth (PPD) and clinical attachment loss (CAL): healthy (group I), gingivitis (group II) and chronic periodontitis (group III). A fourth group (group IV) consisted of 20 subjects from group III, 6–8 weeks after nonsurgical periodontal therapy (scaling and root planing). GCF samples collected from each patient were quantified for CTSK using ELISA. Results The mean CTSK concentration in GCF was found to be the highest in group III, i.e. 55.55 pmol/l. The mean CTSK concentration in GCF in group I and group II was 5.95 pmol/l and 6.90 pmol/l respectively. The mean CTSK concentration in GCF in group IV decreased to 11.15 pmol/l, slightly more than that in groups I and II. Conclusions GCF CTSK levels increased in periodontitis and correlated negatively with clinical parameters like GI, PPD and CAL. CTSK levels decreased after nonsurgical treatment of periodontitis. Thus, CTSK can be considered as a ‘marker of osteoclastic activity’ in periodontal disease and also deserves further consideration as a therapeutic target.

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-kappaB ligand in cooperation with macrophage colony-stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb-/- mice were similarly affected. In vivo studies showed that Ckb-/- mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.

Increased numbers of osteoclasts expressing cysteine proteinase cathepsin K in patients with infected osteoradionecrosis and bisphosphonate-associated osteonecrosis—a paradoxical observation?

Erosion of the bone is a common problem in patients suffering from infected osteoradionecrosis (IORN) or bisphosphonate-associated osteonecrosis (BON). Besides inflammatory mechanisms and infectious agents, osteoclasts were recently suggested to be specifically involved in the osteolytic mechanisms. We therefore investigated the immunohistochemical labeling of the cysteine proteinase cathepsin K, which plays a central role in osteoclast-mediated bone resorption, in tissue specimens of patients with BON (n=9; four male, five female; mean age=69.2 years) and IORN (n=10; nine male, one female; mean age=60.5 years), and compared the results with control specimens (n=8; six male, two female; mean age=60 years). In all cases, osteoclasts were the predominant cell type expressing cathepsin K. For semiquantitative analysis, we therefore defined osteoclasts as multinuclear giant cells attached to the bone and expressing cathepsin K. Significantly higher numbers of osteoclasts were found for both types of osteonecrosis when compared with the control group (BON+IORN vs controls: P=0.0000036). Within the pathological lesions, IORN cases exhibited significantly less osteoclasts than BON (P=0.00097). Our study verified increased numbers of osteoclasts in patients suffering from BON and IORN. Although it is known that bisphosphonates (and to a lesser extent, irradiation, too) decrease osteoclast function, these findings suggest a critical involvement of osteoclasts in the mechanisms of bone destruction in the respective lesions.

A cytochemical assay for osteoclast cathepsin K activity.

Cathepsin K is a member of the papain superfamily of cysteine proteases and plays a pivotal role in osteoclast-mediated bone resorption. This enzyme is an excellent target for antiresorptive therapies for osteopenic disorders such as osteoporosis.(1) Although isolated inhibitor studies on purified enzymes is required to discover potent and selective inhibitors of cathepsin K, a quantitative cytochemical assay(2) for cathepsin K would allow inhibitors to be tested on actual osteoclasts within sections of bone. Furthermore cathepsin K activity could be used to identify and analyse osteoclasts at definitive stages of their lifespan. A cytochemical assay is described that localizes osteoclast cathepsin K activity in unfixed, undecalcified cryostat sections of animal and human bone.

Human osteoclast cathepsin K is processed intracellularly prior to attachment and bone resorption.

Cathepsin K is a member of the papain superfamily of cysteine proteases and has been proposed to play a pivotal role in osteoclast-mediated bone resorption. We have developed a sensitive cytochemical assay to localize and quantify osteoclast cathepsin K activity in sections of osteoclastoma and human bone. In tissue sections, osteoclasts that are distant from bone express high levels of cathepsin K messenger RNA (mRNA) and protein. However, the majority of the cathepsin K in these cells is in an inactive zymogen form, as assessed using both the cytochemical assay and specific immunostaining. In contrast, osteoclasts that are closer to bone contain high levels of immunoreactive mature cathepsin K that codistributes with enzyme activity in a polarized fashion toward the bone surface. Polarization of active enzyme was clearly evident in osteoclasts in the vicinity of bone. The osteoclasts apposed to the bone surface were almost exclusively expressing the mature form of cathepsin K. These cells showed intense enzyme activity, which was polarized at the ruffled border. These results suggest that the in vivo activation of cathepsin K occurs intracellularly, before secretion into the resorption lacunae and the onset of bone resorption. The processing of procathepsin K to mature cathepsin K occurs as the osteoclast approaches bone, suggesting that local factors may regulate this process.

Tyrosine kinase src inhibitors: potential therapeutic applications.

More than 20 years after its discovery, tyrosine kinase Src is still the focus of intense research, in both academia and the pharmaceutical industry. A prototype for non-receptor tyrosine kinases and proto-oncogenes, Src also has a specific role in bone biology. Src's essential role in osteoclast-mediated bone resorption relies on its high expression in this cell type and on the presence of specific substrates, the identity of which is currently being unraveled. Recent identification of selective, potent and orally available Src family kinase inhibitors and their detailed characterization in cells and in animal models of bone loss has produced a new impetus and a novel tool in the field. Here we review the cellular and molecular mechanisms through which Src kinase inhibitors could find an application as therapeutics, particularly in the fields of bone and cancer-induced bone metastases.

Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo.

We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Chz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.

Genomic Organization and Chromosome Localization of the Human Cathepsin K Gene (CTSK)

Human cathepsin K is a recently described cysteine protease with high sequence homology to cathepsins S and L, members of the papain superfamily of cysteine proteases. Cathepsin K is abundantly and selectively expressed in osteoclasts and may perform a specialized role in osteoclast-mediated bone resorption. In the present study, the genomic organization and chromosomal localization of human cathepsin K (HGMW-approved symbolCTSK) were determined. Intron–exon boundaries were identified by PCR on human genomic DNA, and subsequently a P1 genomic clone containing the full-length gene was isolated. Cathepsin K spans approximately 12.1 kb of genomic DNA and is composed of eight exons and seven introns. The genomic organization of cathepsin K is similar to that of cathepsins S and L. The gene was mapped to chromosome 1q21 by fluorescencein situhybridization. Primer walking on the P1 genomic clone identified 1108 bp of 5′ flanking sequence and 459 bp of 3′ flanking sequence. Ribonuclease protection assay and 5′ RACE indicated a single transcriptional start site 49 bp upstream of the initiator Met codon. Analysis of the 5′ flanking region indicates that this gene lacks canonical TATA and CAAT boxes and contains multiple potential transcription regulatory sites. The characterization of the cathepsin K gene and its promoter may provide valuable insights not only into its osteoclast-selective expression, but also into the molecular mechanisms responsible for osteoclast activation.