RESUMO
We recently found that the membrane-bound receptor activator of NF-κB ligand (RANKL) on osteoblasts works as a receptor to stimulate osteoblast differentiation, however, the reason why the RANKL-binding molecules stimulate osteoblast differentiation has not been well clarified. Since the induction of cell-surface receptor clustering is known to lead to cell activation, we hypothesized that the induction of membrane-RANKL clustering on osteoblasts might stimulate osteoblast differentiation. Immunoblotting showed that the amount of RANKL on the membrane was increased by the RANKL-binding peptide OP3-4, but not by osteoprotegerin (OPG), the other RANKL-binding molecule, in Gfp-Rankl-transfected ST2 cells. Observation under a high-speed atomic force microscope (HS-AFM) revealed that RANKL molecules have the ability to form clusters. The induction of membrane-RANKL-OPG-Fc complex clustering by the addition of IgM in Gfp-Rankl-transfected ST2 cells could enhance the expression of early markers of osteoblast differentiation to the same extent as OP3-4, while OPG-Fc alone could not. These results suggest that the clustering-formation of membrane-RANKL on osteoblasts could stimulate early osteoblast differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Oligopeptídeos/farmacologia , Osteoblastos/efeitos dos fármacos , Peptidomiméticos/farmacologia , Ligante RANK/genética , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Camundongos , Microscopia de Força Atômica , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Ligação Proteica , Ligante RANK/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
We recently found that the receptor activator of NF-κB ligand (RANKL)-binding peptide, OP3-4 stimulated the differentiation of both chondrocytes and osteoblasts. OP3-4 is also shown to inhibit cartilage degeneration. To clarify whether the peptide can inhibit cartilage degeneration without stimulating bone formation, we first performed a proliferation assay using C3H10T1/2 (the murine mesenchymal stem cell line), which is the common origin of both chondrocytes and osteoblasts. The RANKL-binding peptides, OP3-4 and W9, promoted cellular proliferation at 24 and 48 h, respectively. Next, we injected both peptides into the intra-articular space of the knee joints of mice with monosodium-iodoacetate (MIA)-induced osteoarthritis to clarify the effects of the peptides on cartilage tissue. Twenty-five nine-week-old male C57BL/6J mice received injections of vehicle, or the same molar amount of W9, OP3-4, or a control peptide (which could not stimulate osteoblast differentiation) on days 7, 14, and 21 after the injection of MIA. The mice were sacrificed on day 28. The histomorphometric analyses revealed that both peptides inhibited the degeneration of cartilage without enhancing bone formation activity. Our data suggest that the stimulation of mesenchymal cell proliferation by the RANKL-binding peptides might lead to the inhibition of cartilage degeneration.
