RESUMO
OBJECTIVE: Cathepsin K plays essential roles in bone resorption and is intensely investigated as a therapeutic target for the treatment of osteoporosis. Hence an assessment of the active form of cathepsin K may provide important biological information in metabolic bone diseases, such as osteoporosis or ankylosing spondylitis. METHODS: Presently there are no robust assays for the assessment of active cathepsin K in serum, and therefore an ELISA specifically detecting the N-terminal of the active form of cathepsin K was developed. RESULTS: The assay was technically robust, with a lowest limit of detection (LOD) of 0.085 ng/mL. The average intra- and inter-assay CV% were 6.60% and 8.56% respectively. The dilution recovery and spike recovery tests in human serum were within 100±20% within the range of the assay. A comparison of latent and active cathepsin K confirmed specificity towards the active form. Quantification of the levels of active cathepsin K in supernatants of purified human osteoclasts compared to corresponding macrophages showed a 30-fold induction (p<0.001). In contrast, in serum samples from osteoporotic women on estrogen or bisphosphonate therapy and from ankylosing spondylitis patients no clinically relevant differences were observed. CONCLUSION: In summary, we have developed a robust and sensitive assay specifically detecting the active form of cathepsin K; however, while it monitors osteoclasts with high specificity in vitro, it appears that circulating levels of active cathepsin K do not reflect bone changes under these circumstances.
Assuntos
Catepsina K/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Osteoclastos/enzimologia , Osteoporose/sangue , Espondilite Anquilosante/sangue , Animais , Anticorpos Monoclonais/química , Conservadores da Densidade Óssea/uso terapêutico , Osso e Ossos/enzimologia , Osso e Ossos/patologia , Difosfonatos/uso terapêutico , Ativação Enzimática , Feminino , Humanos , Macrófagos/citologia , Macrófagos/enzimologia , Camundongos , Pessoa de Meia-Idade , Osteoclastos/citologia , Osteoporose/tratamento farmacológico , Osteoporose/enzimologia , Osteoporose/patologia , Sensibilidade e Especificidade , Espondilite Anquilosante/enzimologia , Espondilite Anquilosante/patologiaRESUMO
Osteopetrosis is the result of mutations affecting osteoclast function. Careful analyses of osteopetrosis have provided instrumental information on bone remodeling, including the coupling of bone formation to bone resorption. Based on a range of novel genetic mutations and the resulting osteoclast phenotypes, we discuss how osteopetrosis models have clarified the function of the coupling of bone formation to bone resorption, and the pivotal role of the osteoclast and their function in this phenomenon. We highlight the distinct possibility that osteoclast activities can be divided into two separate avenues: bone resorption and control of bone formation.
Assuntos
Mutação , Osteoclastos/fisiologia , Osteopetrose/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Relacionadas à Autofagia , Remodelação Óssea/genética , Remodelação Óssea/fisiologia , Reabsorção Óssea/genética , Reabsorção Óssea/fisiopatologia , Anidrase Carbônica II/deficiência , Anidrase Carbônica II/genética , Catepsina K , Catepsinas/genética , Canais de Cloreto/genética , Modelos Animais de Doenças , Humanos , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Modelos Biológicos , Osteoblastos/patologia , Osteoblastos/fisiologia , Osteoclastos/patologia , Osteopetrose/etiologia , Osteopetrose/patologia , Osteopetrose/fisiopatologia , Ligante RANK/deficiência , Ligante RANK/genética , Receptor Ativador de Fator Nuclear kappa-B/deficiência , Receptor Ativador de Fator Nuclear kappa-B/genética , Ubiquitina-Proteína Ligases/genética , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Estrogen deficiency arising with the menopause promotes marked acceleration of bone resorption, which can be restored by hormone replacement therapy. The inhibitory effects of estrogen seem to involve indirect cytokine- mediated effects via supporting bone marrow cells, but direct estrogen-receptor mediated effects on the bone-resorbing osteoclasts have also been proposed. Little information is available on whether estrogens modulate human osteoclastogenesis or merely inhibit the functional activity of osteoclasts. To clarify whether estrogens directly modulate osteoclastic activities human CD14+ monocytes were cultured in the presence of M-CSF and RANKL to induce osteoclast differentiation. Addition of 0.1-10 nM 17beta-estradiol to differentiating osteoclasts resulted in a dose-dependent reduction in tartrate resistant acid phosphatase (TRACP) activity reaching 60% at 0.1 nM. In addition, 17beta-estradiol inhibited bone resorption, as measured by the release of the C-terminal crosslinked telopeptide (CTX), by 60% at 0.1 nM, but had no effect on the overall cell viability. In contrast to the results obtained with differentiating osteoclasts, addition of 17beta-estradiol (0.001-10 nM) to mature osteoclasts did not affect bone resorption or TRACP activity. We investigated expression of the estrogen receptors, using immunocytochemistry and Western blotting. We found that ER-alpha is expressed in osteoclast precursors, whereas ER- beta is expressed at all stages, indicating that the inhibitory effect of estrogen on osteoclastogenesis is mediated by ER-alpha for the major part. In conclusion, these results suggest that the in vivo effects of estrogen are mediated by reduction of osteoclastogenesis rather than direct inhibition of the resorptive activity of mature osteoclasts.