Ian E. James

Osteoprotegerin Is a Receptor for the Cytotoxic Ligand TRAIL

TRAIL is a tumor necrosis factor-related ligand that induces apoptosis upon binding to its death domain-containing receptors, DR4 and DR5. Two additional TRAIL receptors, TRID/DcR1 and DcR2, lack functional death domains and function as decoy receptors for TRAIL. We have identified a fifth TRAIL receptor, namely osteoprotegerin (OPG), a secreted tumor necrosis factor receptor homologue that inhibits osteoclastogenesis and increases bone density in vivo. OPG-Fc binds TRAIL with an affinity of 3.0 nm, which is slightly weaker than the interaction of TRID-Fc or DR5-Fc with TRAIL. OPG inhibits TRAIL-induced apoptosis of Jurkat cells. Conversely, TRAIL blocks the anti-osteoclastogenic activity of OPG. These data suggest potential cross-regulatory mechanisms by OPG and TRAIL.

Cathepsin K, but Not Cathepsins B, L, or S, Is Abundantly Expressed in Human Osteoclasts

Random high throughput sequencing of a human osteoclast cDNA library was employed to identify novel osteoclast-expressed genes. Of the 5475 ESTs obtained, approximately 4% encoded cathepsin K, a novel cysteine protease homologous to cathepsins S and L; ESTs for other cathepsins were rare. In addition, ESTs for cathepsin K were absent or at low frequency in cDNA libraries from numerous other tissues and cells. In situ hybridization in osteoclastoma and osteophyte confirmed that cathepsin K mRNA was highly expressed selectively in osteoclasts; cathepsins S, L, and B were not detectable. Cathepsin K was not detected by in situ hybridization in a panel of other tissues. Western blot of human osteoclastoma or fetal rat humerus demonstrated bands of 38 and 27 kDa, consistent with sizes predicted for pro- and mature cathepsin K. Immunolocalization in osteoclastoma and osteophyte showed intense punctate staining of cathepsin K exclusively in osteoclasts, with a polar distribution that was more intense at the bone surface. The abundant expression of cathepsin K selectively in osteoclasts strongly suggests that it plays a specialized role in bone resorption. Furthermore, the data suggest that random sequencing of ESTs from cDNA libraries is a valuable approach for identifying novel cell-selective genes.

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. Cbz‐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.

Potent and Selective Inhibition of Human Cathepsin K Leads to Inhibition of Bone Resorption In Vivo in a Nonhuman Primate

Cathepsin K is a cysteine protease that plays an essential role in osteoclast‐mediated degradation of the organic matrix of bone. Knockout of the enzyme in mice, as well as lack of functional enzyme in the human condition pycnodysostosis, results in osteopetrosis. These results suggests that inhibition of the human enzyme may provide protection from bone loss in states of elevated bone turnover, such as postmenopausal osteoporosis. To test this theory, we have produced a small molecule inhibitor of human cathepsin K, SB‐357114, that potently and selectively inhibits this enzyme (Ki = 0.16 nM). This compound potently inhibited cathepsin activity in situ, in human osteoclasts (inhibitor concentration [IC]50 = 70 nM) as well as bone resorption mediated by human osteoclasts in vitro (IC50 = 29 nM). Using SB‐357114, we evaluated the effect of inhibition of cathepsin K on bone resorption in vivo using a nonhuman primate model of postmenopausal bone loss in which the active form of cathepsin K is identical to the human orthologue. A gonadotropin‐releasing hormone agonist (GnRHa) was used to render cynomolgus monkeys estrogen deficient, which led to an increase in bone turnover. Treatment with SB‐357114 (12 mg/kg subcutaneously) resulted in a significant reduction in serum markers of bone resorption relative to untreated controls. The effect was observed 1.5 h after the first dose and was maintained for 24 h. After 5 days of dosing, the reductions in N‐terminal telopeptides (NTx) and C‐terminal telopeptides (CTx) of type I collagen were 61% and 67%, respectively. A decrease in serum osteocalcin of 22% was also observed. These data show that inhibition of cathepsin K results in a significant reduction of bone resorption in vivo and provide further evidence that this may be a viable approach to the treatment of postmenopausal osteoporosis.

Identification and Cloning of a Connective Tissue Growth Factor-like cDNA from Human Osteoblasts Encoding a Novel Regulator of Osteoblast Functions

We have identified and cloned a novel connective tissue growth factor-like (CTGF-L) cDNA from primary human osteoblast cells encoding a 250-amino acid single chain polypeptide. Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was obtained from a murine lung cDNA library. CTGF-L protein bears significant identity (∼60%) to the CCN (CTGF, Cef10/Cyr61, Nov) family of proteins. CTGF-L is composed of three distinct domains, an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain implicated in dimerization and heparin binding. CTGF-L mRNA (∼1.3 kilobases) is expressed in primary human osteoblasts, fibroblasts, ovary, testes, and heart, and a ∼26-kDa protein is secreted from primary human osteoblasts and fibroblasts. In situ hybridization indicates high expression in osteoblasts forming bone, discrete alkaline phosphatase positive bone marrow cells, and chondrocytes. Specific binding of125I-labeled insulin-like growth factors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant human CTGF-L promotes the adhesion of osteoblast cells and inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros 17/2.8 cells. Taken together, these results suggest that CTGF-L may play an important role in modulating bone turnover.

A Potent Small Molecule, Nonpeptide Inhibitor of Cathepsin K (SB 331750) Prevents Bone Matrix Resorption in the Ovariectomized Rat

Inhibition of the cyteine proteinase, cathepsin K (E.C. 3.4.22.38) has been postulated as a means to control osteoclast-mediated bone resorption. The preferred animal models for evaluation of antiresorptive activity are in the rat. However, the development of compounds that inhibit rat cathepsin K has proven difficult because the human and rat enzymes differ in key residues in the active site. In this study, a potent, nonpeptide inhibitor of rat cathepsin K (K(i) = 4.7 nmol/L), 5-(2-morpholin-4-yl-ethoxy)-benzofuran-2-carboxylic acid ((S)-3-methyl-1-(3-oxo-1-[2-(3-pyridin-2-yl-phenyl)-ethenoyl]-azepan-4-ylcarbanoyl)-butyl)-amide (SB 331750), is described, which is efficacious in rat models of bone resorption. SB 331750 potently inhibited human cathepsin K activity in vitro (K(i) = 0.0048 nmol/L) and was selective for human cathepsin K vs. cathepsins B (K(i) = 100 nmol/L), L (0.48 nmol/L), or S (K(i) = 14.3 nmol/L). In an in situ enzyme assay, SB 331750 inhibited osteoclast-associated cathepsin activity in tissue sections containing human osteoclasts (IC(50) approximately 60 nmol/L) and this translated into potent inhibition of human osteoclast-mediated bone resorption in vitro (IC(50) approximately 30 nmol/L). In vitro, SB 331750 partially, but dose-dependently, prevented the parathyroid hormone-induced hypercalcemia in an acute rat model of bone resorption. To evaluate the ability of SB 331750 to inhibit bone matrix degradation in vivo, it was administered for 4 weeks at 3, 10, or 30 mg/kg, intraperitoneally (i.p.), u.i.d. in the ovariectomized (ovx) rat. Both 10 and 30 mg/kg doses of compound prevented the ovx-induced elevation in urinary deoxypyridinoline and prevented the ovx-induced increase in percent eroded perimeter. Histological evaluation of the bones from compound-treated animals indicated that SB 331750 retarded bone matrix degradation in vivo at all three doses. The inhibition of bone resorption at the 10 and 30 mg/kg doses resulted in prevention of the ovx-induced reduction in percent trabecular area, trabecular number, and increase in trabecular spacing. These effects on bone resorption were also reflected in inhibition of the ovx-induced loss in trabecular bone volume as assessed using microcomputerized tomography (microCT; approximately 60% at 30 mg/kg). Together, these data indicate that the cathepsin K inhibitor, SB 331750, prevented bone resorption in vivo and this inhibition resulted in prevention of ovariectomy-induced loss in trabecular structure.

Mechanical injury potentiates proteoglycan catabolism induced by interleukin‐6 with soluble interleukin‐6 receptor and tumor necrosis factor α in immature bovine and adult human articular cartilage

OBJECTIVE Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha. METHODS Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. RESULTS In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation. CONCLUSION Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab.

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.

CKβ‐8 [CCL23], a novel CC chemokine, is chemotactic for human osteoclast precursors and is expressed in bone tissues

We have previously demonstrated that a tartrate‐resistant acid phosphatase (TRAP)‐positive subpopulation of mononuclear cells isolated from collagenase digests of human osteoclastoma tissue exhibits an osteoclast phenotype and can be induced to resorb bone. Using these osteoclast precursors as a model system, we have assessed the chemotactic potential of 16 chemokines. Three CC chemokines, the recently described CKβ‐8, RANTES, and MIP‐1α elicited significant chemotactic responses. In contrast, 10 other CC chemokines (MIP‐1β, MCP‐1, MCP‐2, MCP‐3, MCP‐4, HCC‐1, eotaxin‐2, PARC, SLC, ELC) and 3 CXC chemokines (IL‐8, GROα, SDF‐1) were inactive. None of these chemokines showed any chemotactic activity for either primary osteoblasts derived from human bone explants or the osteoblastic MG‐63 cell line. The identity of the osteoclast receptor that mediates the chemotactic response remains to be established. However, all three active chemokines have been reported to bind to CCR1 and cross‐desensitization studies demonstrate that RANTES and MIP‐1α can partially inhibit the chemotactic response elicited by CKβ‐8. CKβ‐8, the most potent of the active CC chemokines (ECmax 0.1–0.3 nM), was further characterized with regard to expression in human bone and cartilage. Although expression is not restricted to these tissues, CKβ‐8 mRNA was shown to be highly expressed in osteoblasts and chondrocytes in human fetal bone by in situ hybridization. In addition, CKβ‐8 protein was shown to be present in human osteophytic tissue by immunolocalization. These observations suggest that CKβ‐8, and perhaps other chemokines, may play a role in the recruitment of osteoclast precursors to sites of bone resorption. J. Cell. Physiol. 183:196–207, 2000.

Antagonism of the osteoclast vitronectin receptor with an orally active nonpeptide inhibitor prevents cancellous bone loss in the ovariectomized rat.

An orally active, nonpeptide Arg‐Gly‐Asp (RGD) mimetic αvβ3 antagonist, (S)‐3‐Oxo‐8‐[2‐[6‐(methylamino)pyridin‐2‐yl]‐1‐ethoxy]‐2‐(2,2,2‐trifluoroethyl)‐2,3,4,5‐tetrahydro‐1H‐2‐benzazepine‐4‐acetic acid (compound 1), has been generated, which prevented net bone loss and inhibited cancellous bone turnover in vivo. The compound binds αvβ3 and the closely related integrin αvβ5 with low nanomolar affinity but binds only weakly to the related integrins αIIbβ3, and α5β1. Compound 1 inhibited αvβ3‐mediated cell adhesion with an IC50 = 3 nM. More importantly, the compound inhibited human osteoclast‐mediated bone resorption in vitro with an IC50 = 11 nM. In vivo, compound 1 inhibited bone resorption in a dose‐dependent fashion, in the acute thyroparathyroidectomized (TPTX) rat model of bone resorption with a circulating EC50 ∼ 20 μM. When dosed orally at 30 mg/kg twice a day (b.i.d.) in the chronic ovariectomy (OVX)‐induced rat model of osteopenia, compound 1 also prevented bone loss. At doses ranging from 3 to 30 mg/kg b.i.d., compound 1 partially prevented the OVX‐induced increase in urinary deoxypyridinoline. In addition, the compound prevented the OVX‐induced reduction in cancellous bone volume (BV), trabecular number (Tb.N), and trabecular thickness (Tb.Th), as assessed by quantitative microcomputerized tomography (μCT) and static histomorphometry. Furthermore, both the 10‐mg/kg and 30‐mg/kg doses of compound prevented the OVX‐induced increase in bone turnover, as measured by percent osteoid perimeter (%O.Pm). Together, these data indicate that the αVβ3 antagonist compound 1 inhibits OVX‐induced bone loss. Mechanistically, compound 1 prevents bone loss in vivo by inhibiting osteoclast‐mediated bone resorption, ultimately preventing cancellous bone turnover.