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Osteoclast (OC) differentiation requires that precursors, such as macrophage colony-stimulating factor (M-CSF)-dependent bone marrow macrophages, receive signals transduced by receptor activator of nuclear factor kappaB (RANK) and c-Fms, receptors for RANK ligand (RANKL) and M-CSF, respectively. Activated c-Fms autophosphorylates cytoplasmic tail tyrosine residues, which, by recruiting adaptor molecules, initiate specific signaling pathways. To identify which tyrosine residues are involved in c-Fms signaling in primary cells, we retrovirally transduced M-CSF-dependent bone marrow macrophages with a chimera comprising the external domain of the erythropoietin (Epo) receptor linked to the transmembrane and cytoplasmic domains of c-Fms. Transduced cells differentiate into bone-resorbing osteoclasts when treated with RANKL and either M-CSF or Epo, confirming that both endogenous and chimeric receptors transmit osteoclastogenic signals. Cells expressing chimeric receptors with Y(697)F, Y(706)F, Y(721)F, and Y(921)F single point mutations generate normal numbers of bone-resorbing OCs, with normal bone-resorbing activity when treated with RANKL and Epo. In contrast, those expressing Y(559)F generate fewer OCs, whereas theY807F mutant is incapable of osteoclastogenesis. Finally, although mature OCs expressing Y(559)F exhibit impaired bone resorption, those bearing Y807F do not. Thus, we have identified specific tyrosine residues in the cytoplasmic tail of c-Fms that are critical for transmitting M-CSF-initiated signals individually required for OC formation or function, respectively.

Receptor activator of nuclear factor-kappa B ligand [RANK ligand (RANK-L)] stimulates mature osteoclasts to resorb bone, a process associated with NF-kappa B activation. RANK-L also prompts macrophages to develop the osteoclast phenotype. Although NF-kappa B is essential for osteoclast differentiation, it is not known whether RANK-L activates this transcription complex in osteoclast precursors. We report that RANK-L rapidly induces NF-kappa B activation in both authentic osteoclast precursors, namely bone marrow macrophages, and RAW 264.7 cells, a murine macrophage line also capable of RANK-L-mediated osteoclastogenesis. Supershift studies reveal the RANK-L-induced DNA binding moiety contains p50/p65, the most common NF-kappa B complex. Subcellular translocation of p50 and p65 subunits is confirmed by Western blots and immunofluorescence analysis. RANK-L activates NF-kappa B in both bone marrow macrophages and RAW 264.7 cells by serine phosphorylation of I kappa B alpha within 5 min, resulting in rapid I kappa B alpha degradation and resynthesis. Attesting to function, RANK-L treatment of RAW 264.7 cells transiently transfected with a plasmid containing NF-kappa B consensus elements linked to luciferase greatly enhances reporter activity. Our data suggest that activation of the NF-kappa B pathway is an integral component of RANK-L-induced osteoclast differentiation.

Early osteoclast precursors, in the form of murine bone marrow macrophages (BMMs), while expressing no detectable alpha(v)beta3 integrin, contain abundant alpha(v)beta5 and attach to matrix in an alpha(v) integrin-dependent manner. Furthermore, alpha(v)beta5 expression by osteoclast precursors progressively falls as they assume the resorptive phenotype. We find the osteoclastogenic agent, tumor necrosis factor-alpha, (TNF) down-regulates alpha(v)beta5 expression by BMMS via attenuation of beta5 messenger RNA (mRNA) t1/2. Using BMMs from TNF receptor knockout mice we establish the p55 receptor transmits the beta5 suppressive effect. The functional implications of TNF-mediated alpha(v)beta5 down-regulation are underscored by the capacity of an alpha(v) inhibitory peptide mimetic to prevent spreading by BMMs expressing abundant alpha(v)beta5 while failing to impact those in which the integrin has been diminished by TNF. Finally, beta5 mRNA in BMMs of wild-type mice administered lipopolysaccharide (LPS) progressively falls with time of in vivo treatment. Alternatively, beta5 mRNA does not decline in BMMs of LPS-treated mice lacking both TNF receptors, documenting down-regulation of the beta5 integrin subunit, in vivo, is mediated by TNF. Thus, matrix attachment of osteoclast precursors and mature osteoclasts are governed by distinct alpha(v) integrins which are differentially regulated by specific cytokines.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and macrophage colony-stimulating factor (M-CSF) both accelerate differentiation of marrow macrophages from which osteoclasts are derived. Previously, we showed that the steroid's effect on early macrophage precursors may be mediated through M-CSF, as the steroid enhances cytokine receptor expression. In contrast, 1,25-(OH)2D3 blunts M-CSF receptor expression on more mature, yet still pluripotential, hematopoietic precursors. Extending these observations to marrow cells committed to macrophage differentiation, we found that 1,25-(OH)2D3 causes a marked decrease in cellular proliferation despite a 2- to 3-fold increase in [125I]M-CSF binding in a similar dose-dependent metabolite-specific manner. Scatchard analysis demonstrated that increased binding reflects increased receptor capacity without an alteration in affinity. Steroid-induced M-CSF receptor enhancement reflects acceleration of protein appearance rather than overexpression, as treated and untreated cells ultimately exhibit equivalent binding. Increased M-CSF receptor expression is mirrored by increased c-fms messenger RNA levels, and actinomycin D or cycloheximide experiments indicate that new receptor synthesis, rather than mobilization of intracellular pools, is required. Thus, 1,25-(OH)2D3 differentially impacts on M-CSF receptor expression throughout the spectrum of bone marrow macrophage differentiation.

Osteoclasts are polykaryons and the principal, if not exclusive, resorptive cell of bone. They are members of the monocyte/macrophage family whose precursors differentiate under the influence of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone resorption is dependent on osteoclast-bone attachment, and we have shown that the integrin alpha v beta 3 is critical to the resorptive process. Thus, we asked whether 1,25-(OH)2D3 enhances the expression of alpha v beta 3 on the surface of osteoclast precursors and if the steroid modulates expression of the beta 3-integrin subunit. We found that 1,25-(OH)2D3 promotes the plasma membrane appearance of alpha v beta 3 on avian bone marrow-derived osteoclast precursors and does so at physiological concentrations (10(-11) M) of the steroid. The effect is time dependent, appearing within 1 day of treatment. A full-length avian cDNA was cloned to explore the molecular mechanisms of beta 3 expression. The deduced amino acid sequence of the cDNA is 81% identical and 89% similar to that of human beta 3. Northern analysis demonstrates that beta 3 mRNA levels in vitamin D-treated osteoclast precursors mirror protein expression. Nuclear run-on experiments document the transcriptional nature of the event.

Several lines of indirect evidence suggest that a monocyte family precursor gives rise to the osteoclast, although this hypothesis is controversial. Starting with a uniform population of nonspecific esterase positive, tartrate-sensitive, acid phosphatase-producing, mannose receptor-bearing mononuclear cells, prepared from dispersed marrow of calcium-deprived laying hens by cell density separation and selective cellular adherence, we generated multinucleated cells in vitro. When cultured with devitalized bone, these cells show, by electron microscopy, the characteristic osteoclast morphology in that they are mitochondria-rich, multinucleated, and, most importantly, develop characteristic ruffled membranes at the matrix attachment site. Moreover, as documented by scanning electron microscopy, these cells pit bone slices in a manner identical to freshly isolated osteoclasts. In addition, isoenzymes of acid phosphatase from generated osteoclasts, separated by 7.5% polyacrylamide gel electrophoresis at pH 4, are identical to those of mature osteoclasts in migration pattern and tartrate resistance, although the precursor cells from which the osteoclasts are generated produce an entirely different isoenzyme, which is tartrate-sensitive and migrates less rapidly at pH 4. The fused cells also exhibit a cAMP response to prostaglandin E2. Therefore, osteoclast-like cells can be derived by in vitro culture of a marrow-derived monocyte cell population.