A novel secreted glycoprotein that regulates bone resorption has been identified. The protein, termed Osteoprotegerin (OPG), is a novel member of the TNF receptor superfamily. In vivo, hepatic expression of OPG in transgenic mice results in a profound yet nonlethal osteopetrosis, coincident with a decrease in later stages of osteoclast differentiation. These same effects are observed upon administration of recombinant OPG into normal mice. In vitro, osteoclast differentiation from precursor cells is blocked in a dose-dependent manner by recombinant OPG. Furthermore, OPG blocks ovariectomy-associated bone loss in rats. These data show that OPG can act as a soluble factor in the regulation of bone mass and imply a utility for OPG in the treatment of osteoporosis associated with increased osteoclast activity.

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.

SumlnaryCD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14, rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14. R cent work has described several serum and cell surface proteins that are necessary for responses of leukocytes to low concentrations of bacterial LPS (endotoxin) (1). LPSbinding protein (LBP),I an acute phase reactant, binds LPS (2) and greatly enhances the sensitivity of cells to LPS (3). Normal serum and plasma also enhance responses to LPS, and a multicomponent factor termed septin has been proposed to serve this function (4). LBP (5) and septin (4) each bind to LPS-coated particles and promote the interaction of these particles with CD14 (6, 7), a glycosylphosphatidylinositol (GPI)-anchored protein of monocytes, macrophages, and PMN (8, 9, 10). CD14 is necessary for serum-or LBPmediated responses of cells to LPS, such as the production 1 Abbreviations used in this paper: CHO, Chinese hamster ovary; ELPS, sheep erythrocytes coated with LPS; ELPS-LBP, ELPS opsonized with LBP; GPI, glycosylphosphatidylinositol; HAP, Dulbecco's PBS with 0.5 U/ml aprotinin, 0.05% human serum albumin, 3 mM D-glucose; HUVEC, human umbilical vein endothelial cells; LBP, LPS-binding protein; NHP, normal human plasma; PD-EDTA, Dulbecco's PBS lacking Ca 2+ and Mg 2+ with 1 mM EDTA; Ra, strain R60; Re, strain R595; rLBP, recombinant LBP; rsCD14, recombinant soluble CD14. of TNF by monocytes (6) and an increase in the adhesive properties of 132-integrins on PMN (7).Cells that do not express CD14, such as endothelial cells, also respond to low concentrations of LPS in the presence of serum. We have shown that these resp...

Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309–319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165–176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects.

Angiopoietin-2 (Ang2) exhibits broad expression in the remodeling vasculature of human tumors but very limited expression in normal tissues, making it an attractive candidate target for antiangiogenic cancer therapy. To investigate the functional consequences of blocking Ang2 activity, we generated antibodies and peptide-Fc fusion proteins that potently and selectively neutralize the interaction between Ang2 and its receptor, Tie2. Systemic treatment of tumor-bearing mice with these Ang2-blocking agents resulted in tumor stasis, followed by elimination of all measurable tumor in a subset of animals. These effects were accompanied by reduced endothelial cell proliferation, consistent with an antiangiogenic therapeutic mechanism. Anti-Ang2 therapy also prevented VEGF-stimulated neovascularization in a rat corneal model of angiogenesis. These results imply that specific Ang2 inhibition may represent an effective antiangiogenic strategy for treating patients with solid tumors.