Stromal cells osteoblasts also secrete osteoprotegerin, a soluble glycoprotein of the tumor necrosis factor receptor super family. OPG fda approved acts as a decoy receptor for RANKL, competing against RANK and thereby inhibiting osteoclast differentiation. In addition, the Inhibitors,Modulators,Libraries macrophage colony stimulating factor is also produced by stromal cells and induces RANKL like effects in osteoclasts. It is essential for the survival and proliferation of macrophages and the regula tion of osteoclastogenesis. Furthermore, various cyto kines or hormones influence the complex osteoclast differentiation by regulating expression of RANKL, M CSF and OPG on stromal cells or osteoblasts. However, the function of RANK is not limited to cell differentiation.
As a member of the tumor necrosis fac tor receptor family RANK is expressed on the surface of osteoclast progenitor cells and plays an Inhibitors,Modulators,Libraries im portant role in bone Inhibitors,Modulators,Libraries homeostasis. RANK transduces intracellular Inhibitors,Modulators,Libraries signals upon ligand binding by recruiting various adaptor proteins through specific motifs in the cytoplasmic domain. Thus, the central role of RANKRANKL interaction in osteoclastogenesis makes RANK an attractive target for potential therapies. The aim of this study was to assess the effect of inhib ition of RANK expression in mouse bone marrow macro phages on osteoclast differentiation and bone resorption. We also studied the extent of osteoclast inhib ition by targeting RANK with retrovirus mediated shRNA. Methods Isolation and culture of bone marrow macrophages All experimental procedures involving animals were per formed in compliance with the regulations of the Guide for the Care and Use of Laboratory Animals.
Furthermore, the proce dures complied with the institutional ethical guidelines for animal experiments and approval was received Inhibitors,Modulators,Libraries from the institutional review board. The femora of 5 week old BALBc mice fresh cadavers were aseptically removed and dissected free of adhering tissues. The bone ends were cut off with scissors and the marrow cavity was flushed slowly by injecting Dulbeccos Modified Eagles Medium. The bone marrow cells were harvested from one end using a sterile needle. 5 106 of these cells were adhered to tissue culture dishes in 10 ml of DMEM containing 10% fetal bovine serum, 100 IUml penicillin G and 100 ug 10 minutes to eliminate non specific staining. The ex cess TBS was removed from the slides before incubation with primary antibody.
The immunohistochemical PD 0332991 procedure and prepar ation of controls were carried out according to the manufacturing companys standards and guidelines. The controls were used to assess the specificity of the reaction as follows pre incubation of the cells with the peptide in order to obtain the first antibody, incuba tion of the cells with an irrelevant primary antibody of the same isotype followed by the second antibody.