Enhanced prostacyclin formation and Wnt signaling in sclerostin deficient osteocytes and bone
Introduction
Excessive bone loss causes osteoporosis and enhances susceptibility to fractures, leading to significant morbidity, mortality and excess health care costs [1]. Understanding mechanisms by which bone mass can be enhanced or maintained could result in new strategies effective in increasing or preserving skeletal integrity and promoting fracture healing, thus reducing costs and co-morbidities associated with osteoporosis and fractures.
The balance between bone loss and deposition is important for normal bone growth and remodeling, and depends on a complex regulatory interplay among resident bone cells such as osteoclasts, osteoblasts, and osteocytes whose activities are altered by serum concentrations of hormones such as parathyroid hormone and 1α,25-dihydroxyvitamin D, and several regulatory proteins such as bone morphogenetic proteins, receptor activator of nuclear factor κ-B ligand (RANKL), and lipid mediators such as prostaglandins produced by such cells [2], [3], [4], [5], [6], [7], [8], [9]. Previous work demonstrated that the prostaglandin PGE2 can either increase bone formation and osteoblastic activity [10] or increase bone resorption [11] depending on experimental conditions. The prostaglandin, PGI2 or prostacyclin also alters bone formation and bone resorption depending on the experimental model [12], [13], [14], [15], [16], [17], [18], [19], [20]. Deletion of prostacyclin synthase, the enzyme responsible for prostacyclin synthesis, results in a decrease in bone mineral density in young mice and an increase in bone mineral density in older mice [14].
Human patients and mice with inactivating mutations of the sclerostin (SOST, Sost) gene have significantly increased bone density [7], [21], [22]. The Sost gene product, sclerostin, is a secreted glycoprotein that alters Wnt, bone morphogenetic protein and other signaling pathways [23], [24], [25], [26], [27]. We previously examined the mechanisms by which bone mass is increased in a novel sclerostin-deficient mouse model [21] which shows an exceptionally dense skeleton and rapid fracture healing [28]. We now show that the production of prostacyclin (PGI2), a cyclic prostanoid, is greatly increased in bone and osteocytes of sclerostin-deficient mice. We demonstrate that prostacyclin, and a prostacyclin analog, increase osteoblast differentiation and mineralization. In the absence of sclerostin production, β-catenin activity in osteocytes is increased, and PGI2 production is influenced by Wnt signaling pathways.
Section snippets
Animal studies
Animal research was conducted according to National Institutes of Health and the Institute of Laboratory Animal Resources, National Research Council guidelines. The Mayo Clinic Institutional Animal Care and Use Committee approved all animal studies.
Generation of Sost knock-out mice
Mice were generated as described earlier [21].
Isolation of osteocytes from mouse femurs
Osteocytes were isolated from 8-week-old Sost KO and WT mice as described by Stern et al. [29].
Immortalization of osteocytes
Osteocytes were immortalized using an SV40 T antigen viral construct as described earlier [30]. Clonal
Results
Arachidonic acid (AA), the precursor to prostaglandins, is converted via the cyclooxygenase pathway to prostaglandin PGG2 and subsequently to PGH2 [33]. The latter is converted to PGI2, PGD2, PGE2, PGF2 and thromboxane A2 by specific synthases; PGF2 is also directly produced from PGE2 [33]. In femoral bone extracts, higher concentrations of 6-keto PGF1α, the stable metabolite of PGI2, were detected in bone from Sost KO mice compared with those measured in WT mice (Fig. 1A). Concentrations of PGE
Discussion
Relative rates of bone formation and resorption determine the amount of mineralized bone present in the skeleton. These processes are under cellular control and the activities of osteoblasts and osteoclasts that form and resorb bone, respectively, are dependent on physiological needs and mechanical stimulation. Among the physiological regulators are endocrine factors, such as parathyroid hormone and 1α,25-dihydroxyvitamin D and the availability of calcium and phosphorus in the extra-cellular
Acknowledgments
The NIH (R01 DE020194, AR60869, T32 AR056950, F32 AR60140) a grant from the Dr. Ralph and Marion Falk Foundation.
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