Intermittent administration of parathyroid hormone (PTH) increases bone mass, at least in part, by increasing the number of osteoblasts. One possible source of osteoblasts might be conversion of inactive lining cells to osteoblasts, and indirect evidence is consistent with this hypothesis. To better understand the possible effect of PTH on lining cell activation, a lineage tracing study was conducted using an inducible gene system. Dmp1‐CreERt2 mice were crossed with ROSA26R reporter mice to render targeted mature osteoblasts and their descendents, lining cells and osteocytes, detectable by 5‐bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactopyranoside (X‐gal) staining. Dmp1‐CreERt2(+):ROSA26R mice were injected with 0.25 mg 4‐OH‐tamoxifen (4‐OHTam) on postnatal days 3, 5, 7, 14, and 21. The animals were euthanized on postnatal day 23, 33, or 43 (2, 12, or 22 days after the last 4‐OHTam injection). On day 43, mice were challenged with a subcutaneous injection of human PTH (1–34, 80 µg/kg) or vehicle once daily for 3 days. By 22 days after the last 4‐OHTam injection, most X‐gal (+) cells on the periosteal surfaces of the calvaria and the tibia were flat. Moreover, bone formation rate and collagen I(α1) mRNA expression were decreased at day 43 compared to day 23. After 3 days of PTH injections, the thickness of X‐gal (+) cells increased, as did their expression of osteocalcin and collagen I(α1) mRNA. Electron microscopy revealed X‐gal–associated chromogen particles in thin cells prior to PTH administration and in cuboidal cells following PTH administration. These data support the hypothesis that intermittent PTH treatment can increase osteoblast number by converting lining cells to mature osteoblasts in vivo. © 2012 American Society for Bone and Mineral Research.

Coupling is the process that links bone resorption to bone formation in a temporally and spatially coordinated manner within the remodeling cycle. Several lines of evidence point to the critical roles of osteoclast-derived coupling factors in the regulation of osteoblast performance. Here, we used a fractionated secretomic approach and identified the axon-guidance molecule SLIT3 as a clastokine that stimulated osteoblast migration and proliferation by activating β-catenin. SLIT3 also inhibited bone resorption by suppressing osteoclast differentiation in an autocrine manner. Mice deficient in Slit3 or its receptor, Robo1, exhibited osteopenic phenotypes due to a decrease in bone formation and increase in bone resorption. Mice lacking Slit3 specifically in osteoclasts had low bone mass, whereas mice with either neuron-specific Slit3 deletion or osteoblast-specific Slit3 deletion had normal bone mass, thereby indicating the importance of SLIT3 as a local determinant of bone metabolism. In postmenopausal women, higher circulating SLIT3 levels were associated with increased bone mass. Notably, injection of a truncated recombinant SLIT3 markedly rescued bone loss after an ovariectomy. Thus, these results indicate that SLIT3 plays an osteoprotective role by synchronously stimulating bone formation and inhibiting bone resorption, making it a potential therapeutic target for metabolic bone diseases.

Osterix (Osx) is essential for osteoblast differentiation and bone formation, because mice lacking Osx die within 1 h of birth with a complete absence of intramembranous and endochondral bone formation. Perinatal lethality caused by the disruption of the Osx gene prevents studies of the role of Osx in bones that are growing or already formed. Here, the function of Osx was examined in adult bones using the time‐ and site‐specific Cre/loxP system. Osx was inactivated in all osteoblasts by Col1a1‐Cre with the activity of Cre recombinase under the control of the 2.3‐kb collagen promoter. Even though no bone defects were observed in newborn mice, Osx inactivation with 2.3‐kb Col1a1‐Cre exhibited osteopenia phenotypes in growing mice. BMD and bone‐forming rate were decreased in lumbar vertebra, and the cortical bone of the long bones was thinner and more porous with reduced bone length. The trabecular bones were increased, but they were immature or premature. The expression of early marker genes for osteoblast differentiation such as Runx2, osteopontin, and alkaline phosphatase was markedly increased, but the late marker gene, osteocalcin, was decreased. However, no functional defects were found in osteoclasts. In summary, Osx inactivation in growing bones delayed osteoblast maturation, causing an accumulation of immature osteoblasts and reducing osteoblast function for bone formation, without apparent defects in bone resorption. These findings suggest a significant role of Osx in positively regulating osteoblast differentiation and bone formation in adult bone.

Farnesoid X receptor (FXR) is a nuclear receptor that functions as a bile acid sensor controlling bile acid homeostasis. We investigated the role of FXR in regulating bone metabolism. We identified the expression of FXR in calvaria and bone marrow cells, which gradually increased during osteoblastic differentiation in vitro. In male mice, deletion of FXR (FXR^−/−) in vivo resulted in a significant reduction in bone mineral density by 4.3% to 6.6% in mice 8 to 20 weeks of age compared with FXR^+/+ mice. Histological analysis of the lumbar spine showed that FXR deficiency reduced the bone formation rate as well as the trabecular bone volume and thickness. Moreover, tartrate-resistant acid phosphatase (TRACP) staining of the femurs revealed that both the osteoclast number and osteoclast surface were significantly increased in FXR^−/− mice compared with FXR^+/+ mice. At the cellular level, induction of alkaline phosphatase (ALP) activities was blunted in primary calvarial cells in FXR^−/− mice compared with FXR^+/+ mice in concert with a significant reduction in type I collagen a1(Col1a1), ALP, and runt-related transcription factor 2 (Runx2) gene expressions. Cultures of bone marrow–derived macrophages from FXR^−/− mice exhibited an increased number of osteoclast formations and protein expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). In female FXR^−/− mice, although bone mineral density (BMD) was not significantly different from that in FXR^+/+ mice, bone loss was accelerated after an ovariectomy compared with FXR^+/+ mice. In vitro, activation of FXR by bile acids (chenodeoxycholic acid [CDCA] or 6-ECDCA) or FXR agonists (GW4064 or Fexaramine) significantly enhanced osteoblastic differentiation through the upregulation of Runx2 and enhanced extracellular signal-regulated kinase (ERK) and β-catenin signaling. FXR agonists also suppressed osteoclast differentiation from bone marrow macrophages. Finally, administration of a farnesol (FOH 1%) diet marginally prevented ovariectomy (OVX)-induced bone loss and enhanced bone mass gain in growing C57BL/6J mice. Taken together, these results suggest that FXR positively regulates bone metabolism through both arms of the bone remodeling pathways; ie, bone formation and resorption. © 2013 American Society for Bone and Mineral Research.

Free fatty acid receptor 4 (FFA4) has been reported to be a receptor for n-3 fatty acids (FAs). Although n-3 FAs are beneficial for bone health, a role of FFA4 in bone metabolism has been rarely investigated. We noted that FFA4 was more abundantly expressed in both mature osteoclasts and osteoblasts than their respective precursors and that it was activated by docosahexaenoic acid. FFA4 knockout (Ffar4−/−) and wild-type mice exhibited similar bone masses when fed a normal diet. Because fat-1 transgenic (fat-1Tg+) mice endogenously converting n-6 to n-3 FAs contain high n-3 FA levels, we crossed Ffar4−/− and fat-1Tg+ mice over two generations to generate four genotypes of mice littermates: Ffar4+/+;fat-1Tg−, Ffar4+/+;fat-1Tg+, Ffar4−/−;fat-1Tg−, and Ffar4−/−;fat-1Tg+. Female and male littermates were included in ovariectomy- and high-fat diet-induced bone loss models, respectively. Female fat-1Tg+ mice decreased bone loss after ovariectomy both by promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption than their wild-type littermates, only when they had the Ffar4+/+ background, but not the Ffar4−/− background. In a high-fat diet-fed model, male fat-1Tg+ mice had higher bone mass resulting from stimulated bone formation and reduced bone resorption than their wild-type littermates, only when they had the Ffar4+/+ background, but not the Ffar4−/− background. In vitro studies supported the role of FFA4 as n-3 FA receptor in bone metabolism. In conclusion, FFA4 is a dual-acting factor that increases osteoblastic bone formation and decreases osteoclastic bone resorption, suggesting that it may be an ideal target for modulating metabolic bone diseases.