Janice R. Connor

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.

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.

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.

EXPRESSION OF CATHEPSIN K MESSENGER RNA IN GIANT CELLS AND THEIR PRECURSORS IN HUMAN OSTEOARTHRITIC SYNOVIAL TISSUES

OBJECTIVE To investigate the expression of cathepsin K messenger RNA (mRNA) in the giant cells found in human osteoarthritic (OA) synovium and associated reparative connective tissues, and to compare this with mRNA expression of cathepsins B, L, and S, which are cysteine proteases known to be highly expressed by cells of the monocyte/macrophage lineage. METHODS Sections of human OA synovium were processed for in situ hybridization and probed for cathepsins K, B, L, and S. Serial sections were reacted for tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase (NSE) activity, which are selective markers for the osteoclast and cells of the macrophage/monocyte lineage, respectively. RESULTS At 3 sites of monocyte infiltration/giant cell formation (granulation tissue, the intimal and subintimal synovial layers, and deep stroma extending to the periphery of osteophytic tissue), both TRAP-positive mono- and multinucleated cells and TRAP-negative, NSE-positive mononuclear precursors were identified. Cells containing both enzyme activities were also found, potentially indicating an intermediate stage of differentiation. The TRAP-positive mononuclear/giant cells, and the occasional NSE-positive precursor, expressed an intense signal for cathepsin K mRNA, but did not express cathepsins B, L, and S. In contrast, the deep zone of phagocytic-like cells adjacent to sites of ossification expressed high levels of mRNA for cathepsins L, B, and S as well as cathepsin K mRNA. CONCLUSION Giant cells that form within OA synovial tissue express high levels of cathepsin K mRNA. It appears that cathepsin K acts principally to digest the bone (and cartilage) fragments sheered from the joint surface during OA. The high TRAP activity and the undetectable expression of the macrophage-associated degradative proteases (cathepsins B, L, and S) by synovial giant cells strengthens the hypothesis that cathepsin K is the primary protease involved in bone degradation. At sites of synovial osteogenesis, a population of phagocytic-like cells expressed TRAP and cathepsins B, L, S, and K, and may represent blood-derived macrophages pushed toward an osteoclast phenotype.

Cathepsin K mRNA Detection Is Restricted to Osteoclasts During Fetal Mouse Development

We recently identified a novel cysteine protease, cathepsin K, by random sequencing of an osteoclast cDNA library, and in situ hybridization studies in adult human tissues demonstrated high and specific expression in osteoclasts. To determine whether the expression of cathepsin K mRNA during mouse embryogenesis was more widespread, cryostat sections of early (day 11–13) and late (day 15–17) mouse fetuses were analyzed by in situ hybridization. Serial cross‐sections were collected through each fetus, and co‐reacted for tartrate‐resistant acid phosphatase (TRAP) and nonspecific esterase (NSE), selective markers for the osteoclast, and precursor cells derived from the macrophage/monocyte lineage, respectively. In the 11–13 day fetuses, cathepsin K mRNA was not expressed in any extraskeletal tissue; at this stage of embryogenesis, no osteoclasts are present. However, in the 15–17 day fetuses, a distinctive, developmental stage‐dependent pattern of cathepsin K expression was observed in osteoclasts and preosteoclasts at sites of cartilage and bone modeling. Cathepsin K positive osteoclasts differentiated within a peripheral zone of the osteogenic stacked cell layer of the cartilage rudiments (prior to ossification), migrated and/or resorbed the bone collar, and invaded the cartilage core. Furthermore, following the invasive penetration of vasculature into the degenerating cartilage core, the calcified cartilage was resorbed by cathepsin K positive mononuclear osteoclast precursors (NSE+ve, negligible TRAP); cells positive for both enzymes were identified indicative of osteoclast differentiation. The deposition of bone by osteoblasts onto the cartilage remnants is followed by mononucleated and multinucleated osteoclastic resorption; these osteoclasts demonstrated intense cathepsin K expression. Similar expression patterns were observed at sites of intramembranous ossification. No expression was observed in chondrocytes, osteoblasts, marrow, or in any other nonskeletal tissue at these time points. These data indicated that cathepsin K expression during embryogenesis occurred only following the onset of osteoclast differentiation.

Cloning and Characterization of a Novel Integrin β3Subunit

We have identified a novel integrin β3 subunit, termed β3C, from a human osteoclast cDNA library. The COOH-terminal sequence and 3′-untranslated region of the β3C subunit differs from the previously reported β3A (platelet) and β3B (placenta) sequences, while the regions coding for the transmembrane and extracellular domains are identical. The β3C cytoplasmic domain contains 37 amino acids, the last 17 of which are encoded by a novel exon located about 6 kilobase pairs downstream of exon 14 of the β3A gene. HEK 293 cells were stably co-transfected with αV and either β3C (HEKβ3C) or β3A(HEKβ3A). The viability of HEKβ3C cells was lower than that of HEKβ3A cells, and HEKβ3Ccells in culture grew as clusters rather than as a monolayer. The novel cytoplasmic domain did not affect receptor binding affinity; both αVβ3A and αVβ3Cisoforms exhibited high affinity binding to 125I-echistatin and cyclic and linear RGD peptides. However, in contrast to HEKβ3A, HEKβ3C cells failed to adhere to osteopontin, an αVβ3 matrix protein. The data provide further support for the key role of the cytoplasmic domain of the β3 integrin in cell adhesion and suggest a potential role for the β3C integrin subunit in modulating cell-matrix interactions.

The application of immunocytochemistry and in situ hybridization to cryostat sections of undecalcified bone

This chapter presents the in situ techniques for determining the temporal and spatial expression patterns of both mRNA (in situ hybridization) and protein (immuno-cytochemistry) in individual cells within sections of bone.