acid-phosphatase and echistatin

Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK) differentiate into osteoclasts following stimulation with the RANK ligand (RANKL). Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition) or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition). BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS) and tumor necrosis factor -αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6) in BMMs induced their differentiation into osteoclasts even under the non-adherent condition. These results suggest that cell adhesion signaling regulates RANK expression in osteoclast precursors.

Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Cell Adhesion; Cell Differentiation; Cells, Cultured; Genetic Vectors; Humans; Integrins; Intercellular Signaling Peptides and Proteins; Isoenzymes; Lipopolysaccharides; Macrophages; Male; Mice; NF-kappa B; Osteoclasts; Peptides; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Retroviridae; Signal Transduction; Stem Cells; Tartrate-Resistant Acid Phosphatase; TNF Receptor-Associated Factor 6; Tumor Necrosis Factor-alpha

The stimulators of bone resorption, prostaglandin E(2) (PGE(2)) and 1,25-dihydroxyvitamin D(3) (1,25D(3)), act through osteoblast-like cells to activate osteoclasts. One candidate for the intermediary produced by osteoblasts that subsequently stimulates the osteoclast is osteoprotegerin ligand (OPGL). OPGL has been shown to stimulate osteoclast differentiation and activation. The aim of the work reported here was to determine if soluble recombinant extracellular domain of human OPGL would bring about the change in osteoclast adhesion from the periosteum of mouse calvaria to the adjacent bone surface that occurs with the above-mentioned stimulators of resorption. This change in adherence or translocation of osteoclasts onto the bone surface required the expression and functioning of the integrin subunit, beta 3. We show that this soluble OPGL, like PGE(2) and 1,25D(3), stimulated the release of osteoclasts from the periosteum and their adherence to the bone surface accompanied by an increase in staining for immunolocalized integrin subunit beta 3. Recombinant human osteoprotegerin (OPG), which binds strongly to OPGL, inhibited this translocation of osteoclasts that occurred with PGE(2) and 1,25D(3), leaving integrin beta-3-negative osteoclasts on the periosteum. PGE(2) and 1,25D(3) increased the expression of messenger RNA for OPGL compared with indomethacin-treated controls after 6 h exposure. Evidence is presented that the change in the adhesion of osteoclasts from the periosteum to the bone surface, resulting in osteoclast activation, is mediated by OPGL.

Topics: Acid Phosphatase; Animals; Antigens, CD; Bone Resorption; Calcitriol; Carrier Proteins; Cell Adhesion; Cell Count; Cells, Cultured; Dinoprostone; Dose-Response Relationship, Drug; In Vitro Techniques; Indomethacin; Integrin beta3; Intercellular Signaling Peptides and Proteins; Isoenzymes; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Osteoclasts; Peptides; Periosteum; Platelet Membrane Glycoproteins; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skull; Tartrate-Resistant Acid Phosphatase

We have shown that, when mouse parietal bones were incubated in culture medium containing indomethacin, the number of tartrate-resistant acid phosphatase-positive osteoclasts (TRAP + OCs) on the bone surface was drastically reduced (down-regulation), and the number on the periosteal membrane adjacent to the resorbing surface was increased. Subsequent incubation of bones with prostaglandin E2 (PGE2) rapidly reversed these changes (up-regulation). In the work reported here, the osteoclast-associated integrin subunit beta3 was stained by immunohistochemistry. The beta3-positive osteoclast (beta3 + OC) population on freshly isolated bone was comprised of about 67% TRAP + OCs and 33% TRAP OCs. Like TRAP + OCs, beta3 + OCs were reduced in number on the surface of bones incubated with indomethacin, but, in contrast to the TRAP + OCs, beta3 + OCs were not seen on the periosteal membrane. Following up-regulation of TRAP + OCs with PGE2, large numbers of beta3 + OCs appeared on the bone surface and, again, were not seen on the periosteal membrane. Echistatin, a peptide that binds to the alphavbeta3 integrin on osteoclasts, was found to inhibit the up-regulation of TRAP + OCs in a dose-dependent manner but had no effect on the down-regulation of TRAP + OCs. Similarly, echistatin inhibited the upregulation of beta3 + OCs on the bone surface, and, under these conditions, beta3 + OCs were observed on the periosteal membrane. The addition of anti-beta3 antibody also inhibited the up-regulation of TRAP + OCs in response to PGE2. The association of beta3 protein expression with the up-regulated osteoclast and the inhibition of up-regulation by echistatin and by anti-beta3 antibody provide strong evidence that beta3 plays an essential role in the movement of osteoclasts from the membrane to the bone.

Topics: Acid Phosphatase; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibodies, Monoclonal; Antigens, CD; Cell Movement; Cells, Cultured; Dinoprostone; Indomethacin; Integrin beta3; Integrins; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred CBA; Osteoclasts; Oxytocics; Parietal Bone; Peptides; Periosteum; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins