Species differences between human and rat in the substrate specificity of cathepsin K

Cathepsin K is known to play an important role in bone resorption, and it has the P2 specificity for proline. Rat cathepsin K has 88% identity with the human enzyme. However, it has been reported that its enzymatic activity for a Cbz-Leu-Arg-MCA substrate is lower than that of human cathepsin K, and that the rat enzyme is not well inhibited by human cathepsin K inhibitors. For this study, we prepared recombinant enzyme to investigate the substrate specificity of rat cathepsin K. Cleavage experiments using the fragment of type I collagen and peptidic libraries demonstrated that rat cathepsin K preferentially hydrolyses the substrates at the P2 Hyp position. Comparison of the S2 site between rat and human cathepsin K sequences indicated that two S2 residues at Ser134 and Val160 in rat are varied to Ala and Leu, respectively, in the human enzyme. Cleavage experiments using two single mutants, S134A and V160L, and one double mutant, S134A/V160L, of rat cathepsin K showed that all the rat mutants lost the P2 Hyp specificity. The information obtained from our comparative studies on rat and human cathepsin K should make a significant impact on developing specific inhibitors of human cathepsin K since rat is usually used as test species.     

Serum cathepsin K levels of patients with longstanding rheumatoid arthritis: correlation with radiological destruction

Cathepsin K is a cysteine protease that plays an essential role in osteoclast function and in the degradation of protein components of the bone matrix by cleaving proteins such as collagen type I, collagen type II and osteonectin. Cathepsin K therefore plays a role in bone remodelling and resorption in diseases such as osteoporosis, osteolytic bone metastasis and rheumatoid arthritis. We examined cathepsin K in the serum of 100 patients with active longstanding rheumatoid arthritis. We found increased levels of cathepsin K compared with a healthy control group and found a significant correlation with radiological destruction, measured by the Larsen score. Inhibition of cathepsin K may therefore be a new target for preventing bone erosion and joint destruction in rheumatoid arthritis. However, further studies have to be performed to prove that cathepsin K is a valuable parameter for bone metabolism in patients with early rheumatoid arthritis.     

Serum cathepsin K levels of patients with longstanding rheumatoid arthritis: correlation with radiological destruction

Cathepsin K is a cysteine protease that plays an essential role in osteoclast function and in the degradation of protein components of the bone matrix by cleaving proteins such as collagen type I, collagen type II and osteonectin. Cathepsin K therefore plays a role in bone remodelling and resorption in diseases such as osteoporosis, osteolytic bone metastasis and rheumatoid arthritis. We examined cathepsin K in the serum of 100 patients with active longstanding rheumatoid arthritis. We found increased levels of cathepsin K compared with a healthy control group and found a significant correlation with radiological destruction, measured by the Larsen score. Inhibition of cathepsin K may therefore be a new target for preventing bone erosion and joint destruction in rheumatoid arthritis. However, further studies have to be performed to prove that cathepsin K is a valuable parameter for bone metabolism in patients with early rheumatoid arthritis.     

Conformational studies on the specific cleavage site of Type I collagen (alpha-1) fragment (157-192) by cathepsins K and L by proton NMR spectroscopy

Cathepsins K and L are cysteine proteinases which are considered to play an important role in bone resorption. Type I collagen is the most abundant component of the extracellular matrix of bone and regarded as an endogenous substrate for the cysteine proteinases in osteoclastic bone resorption. We have synthesized a fragment of Type I collagen (alpha-1) (157-192) as a substrate for the cathepsins and found that cathepsins K and L cleave the fragment at different specific sites. The major cleavage sites for cathepsin K were Met159-Gly160, Ser162-Gly163 and Arg165-Gly166, while those for cathepsin L were Gly166-Leu167 and Gln180-Gly181. The structure of the fragment was analyzed in aqueous solution by circular dichroism and proton NMR spectroscopy and the difference in the molecular recognition of collagen by cathepsins K and L was discussed from the structural aspect.     

Localization of cathepsins B,D, and L in the rat osteoclast by immuno-light and -electron microscopy

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and-electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border. Cathepsin L may be more effective in the degradation of bone matrix than cathepsin B.     

Collagenase activity of cathepsin K depends on complex formation with chondroitin sulfate

Bone resorption in balance with bone formation is vital for the maintenance of the skeleton and is mediated by osteoclasts. Cathepsin K is the predominant protease in osteoclasts that degrades the bulk of the major bone forming organic component, type I collagen. Although the potent collagenase activity of cathepsin K is well known, its mechanism of action remains elusive. Here, we report a cathepsin K-specific complex with chondroitin sulfate, which is essential for the collagenolytic activity of the enzyme. The complex is an oligomer consisting of five cathepsin K and five chondroitin sulfate molecules. Only the complex exhibits potent triple helical collagen-degrading activity, whereas monomeric cathepsin K has no collagenase activity. The primary substrate specificity of cathepsin K is not altered by complex formation, suggesting that the protease-chondroitin sulfate complex primarily facilitates the destabilization and/or the specific binding of the triple helical collagen structure. Inhibition of complex formation leads to the loss of collagenolytic activity but does not impair the proteolytic activity of cathepsin K toward noncollagenous substrates. The physiological relevance of cathepsin K complexes is supported by the findings that (i) the content of chondroitin sulfate present in bone and accessible to cathepsin K activity is sufficient for complex formation and (ii) Y212C, a cathepsin K mutant that causes pycnodysostosis (a bone sclerosing disorder) and that has no collagenase activity but remains potent as a gelatinase, is unable to form complexes. These findings reveal a novel mechanism of bone collagen degradation and suggest that targeting cathepsin K complex formation would be an effective and specific treatment for diseases with excessive bone resorption such as osteoporosis.     

Collagenolytic activity of cathepsin K is specifically modulated by cartilage-resident chondroitin sulfates

Cathepsin K is the predominant cysteine protease in osteoclast-mediated bone remodeling, and the protease is thought to be involved in the pathogenesis of diseases with excessive bone and cartilage resorption. Osteoclastic matrix degradation occurs in the extracellular resorption lacuna and upon phagocytosis within the cell's lysosomal-endosomal compartment. Since glycosaminoglycans (GAGs) are abundant in extracellular matrixes of cartilage and growing bone, we have analyzed the effect of GAGs on the activity of bone and cartilage-resident cathepsins K and L and MMP-1. GAGs, in particular chondroitin sulfates, specifically and selectively increased the stability of cathepsin K but had no effect on cathepsin L and MMP-1. GAGs strongly enhanced the stability and, to a lesser extent, the catalytic activity of cathepsin K. To combine the activity and stability parameters, we defined a novel kinetic term, named cumulative activity (CA), which reflects the total substrate turnover during the life span of the enzyme. In the presence of chondroitin-4-sulfate (C-4S), the CA value increased 200-fold for cathepsin K but only 25-fold with chondroitin-6-sulfate (C-6S). C-4S dramatically increased the hydrolysis of soluble as well insoluble type I and II collagens, whereas the effects of C-6S and hyaluronic acid were less pronounced. C-4S acts in a concentration-dependent manner but reaches saturation at approximately 0.1%, a concentration similar to that found in the synovial fluid of arthritis patients. C-4S increased the cathepsin K-mediated release of hydroxyproline from insoluble type I collagen 10-fold but had only a less than 2-fold enhancing effect on the hydrolysis of intact cartilage. The relatively small increase in the hydrolysis of cartilage by C-4S was attributed to the endogenous chondroitin sulfate content present in the cartilage. Although C-4S increased the pH stability at neutral pH, a significant increase in the collagenolytic activity of cathepsin K at this pH was not observed, thus suggesting that the unique collagenolytic activity of cathepsin K at acidic pH is mechanistically determined and not by the enzyme's instability at neutral pH. The selective and significant stabilization and activation of cathepsin K activity by C-4S may provide a rationale for a novel mechanism to regulate the enzyme's activity during bone growth and aging, two processes known for significant changes in the GAG content.     

Cathepsin K Activity-dependent Regulation of Osteoclast Actin Ring Formation and Bone Resorption

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.Osteoclasts are monocyte-macrophage lineage-derived, large multinucleated cells. They are the major bone resorbing cells, essential for bone turnover and development. Active osteoclasts display characteristic membranes, including the ruffled border, attachment zone, and the basolateral secretory membrane. After attachment to bone, the ruffled border secretes enzymes and protons enabling the solubilization and digestion of the bone matrix. Osteoclasts express many proteases including cathepsins and matrix metalloproteases (MMPs)² (for review see Refs. 1-3). However, it is the general consensus that cathepsin K (catK) is the major bone-degrading enzyme (4-7).Rapid cytoskeletal reorganization is essential for osteoclast function and formation of the specialized membranes. Bone resorption occurs within the sealing zone, which is formed by an actin ring structure. This can be identified as a solid circular belt like formation and consists of an actin filament core surrounded by actin-binding proteins such as talin, α-actinin, and vinculin, which link matrix-recognizing integrins to the cytoskeleton (8). The ruffled border is contained within this structure. The actin ring is initiated by the formation of podosomes, which represent dot-like actin structures of small F-actin containing columns surrounded by proteins also found in focal adhesion such as vinculin and paxillin (9). It was previously thought that the sealing zone was formed by the fusion of podosomes after the osteoclast becomes activated (10, 11), but it has since been demonstrated that podosomes and the sealing zone are distinct structures (12, 13). It should be noted that bone resorption only occurs when the sealing zone is formed and the actin ring is present (14).Osteoclasts bind and interact with the bone surface through specific integrin receptors. The most abundant integrin present in osteoclasts is the αvß3 receptor also known as the vitronectin receptor (15, 16). This receptor attaches to RGD sequence containing components of the bone matrix, e.g. vitronectin, osteopontin, and type I collagen (17-19). This interaction enables the formation and regulation of the actin ring and therefore osteoclast activity (20-22). It has previously been shown that soluble RGD containing peptides added to cell supernatant are capable of inhibiting osteoclast binding and bone resorption (18, 22-24).This study investigates the effect of collagen degradation fragments on osteoclast activity. Soluble type I collagen and the bone powder of murine long bones were subjected to digestion reactions by the cysteine proteases, catK and catL, and the interstitial collagenase, MMP-1. The effect of these degradation products on osteoclasts was investigated by monitoring actin ring and resorption pit formation. We further investigated the role of cathepsins using catK- and catL-deficient mice. Finally, we looked in more detail at the effect of collagen, as a cell adhesion matrix, on osteoclast activity.     

Changes in Structural-Mechanical Properties and Degradability of Collagen during Aging-associated Modifications

During aging, changes occur in the collagen network that contribute to various pathological phenotypes in the skeletal, vascular, and pulmonary systems. The aim of this study was to investigate the consequences of age-related modifications on the mechanical stability and in vitro proteolytic degradation of type I collagen. Analyzing mouse tail and bovine bone collagen, we found that collagen at both fibril and fiber levels varies in rigidity and Young''s modulus due to different physiological changes, which correlate with changes in cathepsin K (CatK)-mediated degradation. A decreased susceptibility to CatK-mediated hydrolysis of fibrillar collagen was observed following mineralization and advanced glycation end product-associated modification. However, aging of bone increased CatK-mediated osteoclastic resorption by ∼27%, and negligible resorption was observed when osteoclasts were cultured on mineral-deficient bone. We observed significant differences in the excavations generated by osteoclasts and C-terminal telopeptide release during bone resorption under distinct conditions. Our data indicate that modification of collagen compromises its biomechanical integrity and affects CatK-mediated degradation both in bone and tissue, thus contributing to our understanding of extracellular matrix aging.     

Localization of rat cathepsin K in osteoclasts and resorption pits: inhibition of bone resorption and cathepsin K-activity by peptidyl vinyl sulfones.

We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.     

Pycnodysostosis: role and regulation of cathepsin K in osteoclast function and human disease

Patients with pycnodysostosis, a rare skeletal dysplasia, present with bone abnormalities such as short stature, acroosteolysis of distal phalanges, and skull deformities. The disease is caused by a deficiency of the cysteine protease cathepsin K which is responsible for degradation of collagen type I and other bone proteins. Osteoclasts, bone cells of hematopoietic origin responsible for bone mineral as well as protein matrix degradation, are dysfunctional in patients with pycnodysostosis due to mutations in the cathepsin K gene. Cathepsin K deficient osteoclasts can demineralize bone but cannot degrade the protein matrix. Mutations in the cathepsin K gene disrupting wild type cathepsin K activity have been described in patients with pycnodysostosis. Animal models of cathepsin K deficiency have been created and provide a valuable tool to study osteoclast function and treatment for cathepsin K deficiency. Understanding the regulation and role of cathepsin K in osteoclast function is important for designing future therapies for pycnodysostosis. Cathepsin K inhibitors will be useful in pathological processes involving excess osteoclast activation and bone resorption such as osteoporosis, bone metastasis and multiple myeloma. This review will discuss the bone remodeling cycle, the human disease pycnodysostosis caused by cathepsin K deficiency and cathepsin K activity and regulation.     

Effect of TNF-alpha on human osteosarcoma cell line Saos2--TNF-alpha regulation of bone sialoprotein gene expression in Saos2 osteoblast-like cells

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of inflammatory response in many diseases. It inhibits bone formation and stimulates bone resorption. To determine the molecular mechanisms involved in the regulation of gene expression of osteoblast-like cells, we analyzed the effects of TNF-alpha on the human osteosarcoma cell line Saos2. We used RT-PCR to examine the effects of TNF-alpha on bone sialoprotein (BSP), core binding factor a1 (Cbfa1), osterix, alpha 1 (I) collagen, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), cathepsin B, cathepsin L and tissue inhibitors of metalloproteinase-1 (TIMP-1). TNF-alpha (10ng/ml) increased BSP, IL-6 and COX-2 mRNA levels after 3h, reaching maximal levels at 12 h. Cbfa1 mRNA levels increased after 3 h, but decreased by 24 h. Osterix, cathepsin B, cathepsin L and TIMP-1 mRNA levels did not change after stimulation with TNF-alpha. On the other hand, alpha 1 (I) collagen mRNA expression was suppressed by TNF-alpha at 24 h. Transient transfection analyses were performed using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. TNF-alpha (10 ng/ml) had no effect on the promoter activities of BSP transfected into Saos2 cells. The results of gel mobility shift assays using radiolabeled double-stranded cAMP response element (CRE) and FGF2 response element (FRE) oligonucleotides in the proximal promoter of the rat BSP gene showed increased binding of nuclear proteins at 6 h. Gel mobility shift assays with radiolabelled COX-2-CRE and COX-2-NF kappa B oligonucleotides revealed an increase in the binding of nuclear proteins from TNF-alpha-stimulated Saos2 cells. These studies, therefore, showed that TNF-alpha indirectly increased BSP expression, and that it could be mediated through COX-2 and Cbfa1 expression in Saos2 osteoblast-like cells.     

Potential synergies between matrix proteins and soluble factors on resorption and proteinase activities of rabbit bone cells

Human growth hormone (GH) has recently been found to stimulate osteoclastic resorption, cysteine-proteinase and metalloproteinase activities (MMP-2 and MMP-9) in vitro via insulin-like growth factor-I (IGF-I) produced by stromal cells. The present study investigated the effects of two extracellular matrix components (vitronectin and type-I collagen) on hGH- and hIGF-1-stimulated osteoclastic resorption and proteinase activities in a rabbit bone cell model. After 4 days of rabbit bone cell culture on dentin slices with vitronectin coating, hGH and hIGF-1 stimulated bone resorption and hIGF-1 upmodulated cysteine-proteinase activities. MMP-2 expression (but not resorption, cathepsin or MMP-9 activities) was upmodulated by hGH and hIGF-1 on dentin slices coated with type I collagen as compared to those without coating. Then, vitronectin was synergistic with hIGF-1 in the regulation of cysteine-proteinase production whereas collagen showed synergy with hGH and hIGF-1 in the regulation of MMP-2 production. Anti-alphavbeta3 totally abolished the effects of hGH and hIGF-1 on metalloproteinase release, but had no influence on cathepsin release. The results suggest that cysteine-proteinase modulation is not mediated by alphavbeta3 integrin (strongly expressed on osteoclastic surface) whereas the resorption process and metalloproteinase modulation are clearly mediated by this integrin. Our finding about the collagen coating also suggests that hGH- and hIGF-1-stimulated MMP-2 activity are mediated, along with alphavbeta3 integrin, by another adhesion molecule.     

Involvement of PKC-beta in PTH, TNF-alpha, and IL-1 beta effects on IL-6 promoter in osteoblastic cells and on PTH-stimulated bone resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-beta, LY379196, was used to determine the role of the PKC-beta isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) induced translocation of PKC-alpha and -beta(I) to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-beta(I) translocation by PTH, TNF-alpha or IL-1 beta was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-alpha-, or IL-1 beta-stimulated translocation of PKC-alpha. PTH, TNF-alpha, and IL-1 beta increased luciferase expression in UMR-106 cells transiently transfected with a -224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-beta(I) is a component of the signaling pathway that mediates PTH-, TNF-alpha-, and IL-1 beta-stimulated IL-6 expression and PTH-stimulated bone resorption.     

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.     

Mechanisms underlying catabolic and anabolic functions of parathyroid hormone on bone by combination of culture systems of mouse cells

Since bone resorption and formation by continuous and intermittent parathyroid hormone (PTH) treatments involve various types of cells in bone, this study examined the underlying mechanism by combining culture systems using mouse primary calvarial osteoblasts and bone marrow cells. The PTH/PTHrP receptor (PTH1R) expression and the cAMP accumulation in response to PTH were increased in accordance with the differentiation of osteoblasts. Osteoclast formation was strongly induced by continuous PTH treatment in the monolayer co‐culture of osteoblasts and bone marrow cells, which was associated with RANKL expression in differentiated osteoblasts. Bone formation determined by ALP activity and the type I collagen mRNA expression was stimulated by intermittent PTH treatment in the monolayer co‐culture and in the bone marrow cell layer of the separated co‐culture in a double chamber dish, but not in the culture of bone marrow cells alone. The stimulation in the separated co‐culture, accompanied by IGF‐I production by osteoblasts, was abolished when bone marrow cells were derived from knockout mice of insulin‐receptor substrate‐1 (IRS‐1?/?) or when osteoblasts were from PTH1R?/? mice. We conclude that differentiated osteoblasts are most likely the direct target of both continuous and intermittent PTH, while bone marrow cells are likely the effector cells. The osteoblasts stimulated by continuous PTH express RANKL which causes osteoclastogenesis from the precursors in bone marrow via cell‐to‐cell contact, leading to bone resorption; while the osteoblasts stimulated by intermittent PTH secrete IGF‐I which activates IRS‐1 in osteoblast precursors in bone marrow via a paracrine mechanism, leading to bone formation. J. Cell. Biochem. 109: 755–763, 2010. © 2010 Wiley‐Liss, Inc.     

Cysteine protease production by human osteosarcoma cells (MG63, SAOS2) and its modulation by soluble factors

The production of cysteine protease by two human osteosarcoma cell lines (MG-63 and SaOS2) was analyzed, as well as their modulation by interleukin 1beta (hIL-1 beta), interleukin 6 (hIL-6), insulin growth factor-1 (hIGF-1), oncostatin M (hOSM), leukemia inhibitory factor (hLIF) and growth hormone (hGH). Cysteine protease activities were detected using a synthetic substrate. The protease activities (especially cathepsin L activity) of both cell lines were increased significantly in the presence of hIL-1 beta, hIL-6 and hOSM. In contrast, hIGF-1 and hGH decreased these activities, and no effect was detectable in the presence of hLIF. The addition of antibodies against the gp-130 chain of the hIL-6 and hOSM receptors totally inhibited the stimulating effect of these two cytokines on cysteine protease activities. In increasing collagen type I degradation, hIL-1beta, hIL-6 and hOSM could be involved in bone resorption, whereas the inhibitory action of hIGF-1 and hGH on collagen type I degradation suggest that this factor could play a role in bone formation.     

Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin

Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.     

Mouse osteoblasts synthesize collagenase in response to bone resorbing agents

Bone cells isolated from mouse calvariae by a sequential digestion procedure have many osteoblast characteristics: they respond to PTH and prostaglandin E2 by activation of adenylate cyclase but not to calcitonin, they stain for alkaline phosphatase and they make only type I collagen. In confluent monolayer culture, they do not secrete collagenase in appreciable quantities, unless stimulated with resorptive substances such as PTH, prostaglandin E2, 1,25(OH)2 vitamin D-3 and monocyte-conditioned medium. This suggests they play a direct role in bone resorption.