Soluble RANKL Induces High Bone Turnover and Decreases Bone Volume, Density, and Strength in Mice S. A. J. Lloyd Æ Y. Y. Yuan Æ P. J. Kostenuik Æ M. S. Ominsky Æ A. G. Lau Æ S. Morony Æ M. Stolina Æ F. J. Asuncion Æ Ted A. Bateman Received: 30 January 2008 / Accepted: 11 April 2008 / Published online: 9 May 2008 Ó Springer Science+Business Media, LLC 2008 Abstract Receptor activator for nuclear factor-j B ligand (RANKL) is an essential mediator of osteoclasto- genesis. We hypothesized that administration of soluble RANKL to mice would result in high turnover and dele- terious effects on both cortical and trabecular bone. For 10 days, 10-week-old C57BL/6J female mice (n = 12/ group) were given twice-daily subcutaneous injections of human recombinant RANKL (0.4 or 2 mg/kg/day) or inert vehicle (VEH). Bone turnover was greatly accelerated by RANKL, as evidenced by the 49–84% greater levels of serum TRAP-5b (bone resorption marker) and 300–400% greater levels of serum alkaline phosphatase (bone for- mation marker). RANKL resulted in significantly greater endocortical bone erosion surface (79–83%) and periosteal bone formation rate (64–87%) vs. VEH. Microcomputed tomographic (microCT) analysis of the proximal tibia indicated a reduction in trabecular volume fraction (–84%) for both doses of RANKL. Cortical bone geometry and strength were also negatively influenced by RANKL. MicroCT analysis of the femoral diaphysis indicated sig- nificantly lower cortical bone volume (-10% to –13%) and greater cortical porosity (8–9%) relative to VEH. Biomechanical testing of the femur diaphysis revealed significantly lower maximum bending load (-19% to –25%) vs. VEH. Bone strength remained correlated with bone mass, independent of RANKL stimulation of bone turnover. These findings are consistent with the hypothesis that soluble RANKL could be an important etiologic factor in pathologic bone loss. RANKL also has potential utility as a model for studying the consequences of high bone turnover on bone quality and strength in animals. Keywords RANKL Á Bone strength Á Turnover Á Microcomputed tomography Á Osteoporosis Bone strength is determined by bone mass and bone quality [1, 2]. Bone mass reflects the balance between bone forma- tion and bone resorption, which involves cellular regulation of osteoblast/osteoclast number and activity. Moreover, the initiation of bone formation and resorption is coupled: cells of the osteoblast lineage regulate the recruitment and activity of osteoclasts through expression of receptor activator for nuclear factor-j B ligand (RANKL) and osteoprotegerin (OPG) [3, 4]. Balanced RANKL/OPG levels may be critical for maintaining precise, homeostatic control over bone remodeling [3–6]. Bone quality is determined by a number of variables, including microarchitecture, microdamage, degree of mineralization, and bone turnover. Abnormally high turnover reduces bone mass and may also reduce bone quality by accelerating the normal resorption and formation phases of the remodeling cycle, leading to reduced matrix mineralization [2, 7]. RANKL is an essential mediator of osteoclast formation, activation, and survival [3]. RANKL binds to its receptor, RANK, on the surface of osteoclasts, leading to their activation and differentiation and the subsequent induction of bone resorption [3, 8–10]. OPG, a member of the tumor necrosis factor (TNF) receptor superfamily, is a soluble decoy receptor that inhibits the interaction of RANKL with its receptor, RANK [4, 11]. Transgenic mice overexpress- ing RANKL and OPG knockout mice (OPG –/– ) both develop severe osteoporosis, accompanied by high bone S. A. J. Lloyd Á Y. Y. Yuan Á A. G. Lau Á T. A. Bateman (&) Department of Bioengineering, Clemson University, 501 Rhodes Research Center, Clemson, SC 29634, USA e-mail: bateman@clemson.edu P. J. Kostenuik Á M. S. Ominsky Á S. Morony Á M. Stolina Á F. J. Asuncion Metabolic Disorders, Amgen, Inc., Thousand Oaks, CA, USA 123 Calcif Tissue Int (2008) 82:361–372 DOI 10.1007/s00223-008-9133-6