Osteoclasts resorb the mineralized matrices formed by chondrocytes or osteoblasts. The cytokine receptor activator of NFκB ligand (RANKL) is essential for osteoclast formation and thought to be supplied by osteoblasts or their precursors. However, RANKL is expressed by a variety of cell types and it is unclear which of them are essential sources for osteoclast formation. Here we have used a conditional mouse RANKL allele and a series of Cre-deleter strains to demonstrate that hypertrophic chondrocytes and osteocytes, both of which are embedded in matrix, are essential sources of the RANKL that controls mineralized cartilage resorption and bone remodeling, respectively. Moreover, osteocyte RANKL is responsible for the bone loss associated with unloading. Contrary to the current paradigm, RANKL produced by osteoblasts or their progenitors does not contribute to bone remodeling. These results suggest that the rate-limiting step of matrix resorption is controlled by cells embedded within the matrix itself.

The detection of estrogen receptor-α (ERα) in osteoblasts and osteoclasts over 20 years ago suggested that direct effects of estrogens on both of these cell types are responsible for their beneficial effects on the skeleton, but the role of ERα in osteoblast lineage cells has remained elusive. In addition, estrogen activation of ERα in osteoclasts can only account for the protective effect of estrogens on the cancellous, but not the cortical, bone compartment that represents 80% of the entire skeleton. Here, we deleted ERα at different stages of differentiation in murine osteoblast lineage cells. We found that ERα in osteoblast progenitors expressing Osterix1 (Osx1) potentiates Wnt/β-catenin signaling, thereby increasing proliferation and differentiation of periosteal cells. Further, this signaling pathway was required for optimal cortical bone accrual at the periosteum in mice. Notably, this function did not require estrogens. The osteoblast progenitor ERα mediated a protective effect of estrogens against endocortical, but not cancellous, bone resorption. ERα in mature osteoblasts or osteocytes did not influence cancellous or cortical bone mass. Hence, the ERα in both osteoblast progenitors and osteoclasts functions to optimize bone mass but at distinct bone compartments and in response to different cues. IntroductionThe volume of bone mass is determined by the balance between two opposing processes, bone removal (resorption) by osteoclasts and bone formation by osteoblasts. Under physiologic circumstances, formation compensates for the effect of resorption by adding bone either in the same anatomical site from which it was previously resorbed, in a process called remodeling, or in a different site, as in the case of the sculpting of bones during growth, in a process called modeling (1).After birth, long bones in both sexes increase in length. In parallel with linear growth, females and males experience an enlargement and thickening of the bone cortex, because bone apposition at the periosteal (outer) envelope exceeds the widening of the medullar cavity by endocortical resorption. Importantly, with the onset of puberty, estrogens inhibit, while androgens stimulate, periosteal bone formation, thereby contributing to sexual dimorphism and the greater bone mass in the male (2). At the same time, endocortical resorption is attenuated by either estrogens or androgens, producing increased cortical thickness at the end of puberty in both sexes. In line with the inhibitory effect of estrogens on periosteal bone formation, estrogen deficiency in female rodents and postmenopausal women increases periosteal apposition in an attempt to compensate for the endocortical loss of bone. At the end of puberty, estrogens are essential for the closure of the epiphyses and the cessation of linear growth (3).Estrogens also slow the rate of bone remodeling and help to maintain a balance between bone formation and resorption by attenuating the birth rate of osteoclast and osteoblast progenitors in the bone marrow and exerting a proapop...

The cytokine receptor activator of nuclear factor kappa B ligand (RANKL), encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.

Background:The contribution of B lymphocytes to the bone loss caused by estrogen deficiency is unclear. Results: Deletion of the cytokine receptor activator of NFB ligand from B lymphocytes, but not T lymphocytes, blunted bone loss in ovariectomized mice. Conclusion: Cytokine production by B lymphocytes contributes to ovariectomy-induced bone loss. Significance: This mechanism may be relevant to the mechanisms responsible for postmenopausal osteoporosis.