Exogenous PTH and Endogenous 1,25‐Dihydroxyvitamin D Are Complementary in Inducing an Anabolic Effect on Bone

PTH and 1,25(OH)₂D each exert dual anabolic and catabolic skeletal effects. We assessed the potential interaction of PTH and 1,25(OH)₂D in promoting skeletal anabolism by comparing the capacity of exogenous, intermittently injected PTH(1‐34) to produce bone accrual in mice homozygous for the 1α(OH)ase‐null allele [1α(OH)ase^?/? mice] and in wildtype mice. In initial studies, 3‐mo‐old wildtype mice were either injected once daily (40 μg/kg) or infused continuously (120 μg/kg/d) with PTH(1–34) for up to 1 mo. Infused PTH reduced BMD, increased the bone resorption marker TRACP‐5b, and raised serum calcium but did not increase serum 1,25(OH)₂D. Injected PTH increased serum 1,25(OH)₂D and BMD, raised the bone formation marker osteocalcin more than did infused PTH, and did not produce sustained hypercalcemia as did PTH infusion. In subsequent studies, 3‐mo‐old 1α(OH)ase^?/? mice, raised on a rescue diet, and wildtype littermates were injected with PTH(1–34) (40 μg/kg) either once daily or three times daily for 1 mo. In 1α(OH)ase^?/? mice, baseline bone volume (BV/TV) and bone formation (BFR/BS) were lower than in wildtype mice. PTH administered intermittently increased BV/TV and BFR/BS in a dose‐dependent manner, but the increases were always less than in wildtype mice. These studies show that exogenous PTH administered continuously resorbs bone without raising endogenous 1,25(OH)₂D. Intermittently administered PTH can increase bone accrual in the absence of 1,25(OH)₂D, but 1,25(OH)₂D complements this PTH action. An increase in endogenous 1,25(OH)₂D may therefore facilitate an optimal skeletal anabolic response to PTH and may be relevant to the development of improved therapeutics for enhancing skeletal anabolism.     

The role of estrogen receptor α in growth plate cartilage for longitudinal bone growth

Estrogens enhance skeletal growth during early sexual maturation, whereas high estradiol levels during late puberty result in growth plate fusion in humans. Although the growth plates do not fuse directly after sexual maturation in rodents, a reduction in growth plate height is seen by treatment with a high dose of estradiol. It is unknown whether the effects of estrogens on skeletal growth are mediated directly via estrogen receptors (ERs) in growth plate cartilage and/or indirectly via other mechanisms such as the growth hormone/insulin‐like growth factor 1 (GH/IGF‐1) axis. To determine the role of ERα in growth plate cartilage for skeletal growth, we developed a mouse model with cartilage‐specific inactivation of ERα. Although mice with total ERα inactivation displayed affected longitudinal bone growth associated with alterations in the GH/IGF‐1 axis, the skeletal growth was normal during sexual maturation in mice with cartilage‐specific ERα inactivation. High‐dose estradiol treatment of adult mice reduced the growth plate height as a consequence of attenuated proliferation of growth plate chondrocytes in control mice but not in cartilage‐specific ERα^?/? mice. Adult cartilage‐specific ERα^?/? mice continued to grow after 4 months of age, whereas growth was limited in control mice, resulting in increased femur length in 1‐year‐old cartilage‐specific ERα^?/? mice compared with control mice. We conclude that during early sexual maturation, ERα in growth plate cartilage is not important for skeletal growth. In contrast, it is essential for high‐dose estradiol to reduce the growth plate height in adult mice and for reduction of longitudinal bone growth in elderly mice. © 2010 American Society for Bone and Mineral Research.     

Intermittent PTH Administration Stimulates Pre‐Osteoblastic Proliferation Without Leading to Enhanced Bone Formation in Osteoclast‐Less c‐fos−/− Mice

This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH‐driven bone anabolism. After administering PTH intermittently to wildtype and c‐fos^?/? mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH‐treated wildtype mice, whereas in the osteoclast‐deficient c‐fos^?/? mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double‐positive for alkaline phosphatase and BrdU, suggesting active pre‐osteoblastic proliferation. Ultrastructural examinations showed two major pre‐osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c‐fos^?/? mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH‐administered wildtype mice. We concluded that the absence of osteoclasts in c‐fos^?/? mice might hinder PTH‐driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration.     

Sex and age modify biochemical and skeletal manifestations of chronic hyperparathyroidism by altering target organ responses to Ca2+ and parathyroid hormone in mice

We studied mice with or without heterozygous deletion of the Casr in the parathyroid gland (PTG) [^PTGCaSR(+/–)] to delineate effects of age and sex on manifestations of hyperparathyroidism (HPT). In control mice, aging induced a left‐shift in the Ca²⁺/parathyroid hormone (PTH) set point accompanied by increased PTG CaSR expression along with lowered serum Ca²⁺ and mildly increased PTH levels, suggesting adaptive responses of PTGs to aging‐induced changes in mineral homeostasis. The aging effects on Ca²⁺/PTH set point and CaSR expression were significantly blunted in ^PTGCaSR(+/–) mice, who showed instead progressively elevated PTH levels with age, especially in 12‐month‐old females. These 12‐month‐old knockout mice demonstrated resistance to their high PTH levels in that serum 1,25‐dihydroxyvitamin D (1,25‐D) levels and RNA expression of renal Cyp27b1 and expression of genes involved in Ca²⁺ transport in kidney and intestine were unresponsive to the rising PTH levels. Such changes may promote negative Ca²⁺ balance, which further exacerbate the HPT. Skeletal responses to HPT were age‐, sex‐, and site‐dependent. In control mice of either sex, trabecular bone in the distal femur decreased whereas cortical bone in the tibiofibular junction increased with age. In male ^PTGCaSR(+/–) mice, anabolic actions of the elevated PTH levels seemed to protect against trabecular bone loss at ≥3 months of age at the expense of cortical bone loss. In contrast, HPT produced catabolic effects on trabecular bone and anabolic effects on cortical bone in 3‐month‐old females; but these effects reversed by 12 months, preserving trabecular bone in aging mice. We demonstrate that the CaSR plays a central role in the adaptive responses of parathyroid function to age‐induced changes in mineral metabolism and in target organ responses to calciotropic hormones. Restraining the ability of the PTG to upregulate CaSRs by heterozygous gene deletion contributes to biochemical and skeletal manifestations of HPT, especially in aging females. © 2013 American Society for Bone and Mineral Research.     

Calcium-Sensing Receptors in Chondrocytes and Osteoblasts Are Required for Callus Maturation and Fracture Healing in Mice

Calcium and its putative receptor (CaSR) control skeletal development by pacing chondrocyte differentiation and mediating osteoblast (OB) function during endochondral bone formation—an essential process recapitulated during fracture repair. Here, we delineated the role of the CaSR in mediating transition of callus chondrocytes into the OB lineage and subsequent bone formation at fracture sites and explored targeting CaSRs pharmacologically to enhance fracture repair. In chondrocytes cultured from soft calluses at a closed, unfixed fracture site, extracellular [Ca²⁺] and the allosteric CaSR agonist (NPS-R568) promoted terminal differentiation of resident cells and the attainment of an osteoblastic phenotype. Knockout (KO) of the Casr gene in chondrocytes lengthened the chondrogenic phase of fracture repair by increasing cell proliferation in soft calluses but retarded subsequent osteogenic activity in hard calluses. Tracing growth plate (GP) and callus chondrocytes that express Rosa26-tdTomato showed reduced chondrocyte transition into OBs (by >80%) in the spongiosa of the metaphysis and in hard calluses. In addition, KO of the Casr gene specifically in mature OBs suppressed osteogenic activity and mineralizing function in bony calluses. Importantly, in experiments using PTH (1-34) to enhance fracture healing, co-injection of NPS-R568 not only normalized the hypercalcemic side effects of intermittent PTH (1-34) treatment in mice but also produced synergistic osteoanabolic effects in calluses. These data indicate a functional role of CaSR in mediating chondrogenesis and osteogenesis in the fracture callus and the potential of CaSR agonism to facilitate fracture repair. © 2019 American Society for Bone and Mineral Research.     

Calcilytic Ameliorates Abnormalities of Mutant Calcium‐Sensing Receptor (CaSR) Knock‐In Mice Mimicking Autosomal Dominant Hypocalcemia (ADH)

Activating mutations of calcium‐sensing receptor (CaSR) cause autosomal dominant hypocalcemia (ADH). ADH patients develop hypocalcemia, hyperphosphatemia, and hypercalciuria, similar to the clinical features of hypoparathyroidism. The current treatment of ADH is similar to the other forms of hypoparathyroidism, using active vitamin D₃ or parathyroid hormone (PTH). However, these treatments aggravate hypercalciuria and renal calcification. Thus, new therapeutic strategies for ADH are needed. Calcilytics are allosteric antagonists of CaSR, and may be effective for the treatment of ADH caused by activating mutations of CaSR. In order to examine the effect of calcilytic JTT‐305/MK‐5442 on CaSR harboring activating mutations in the extracellular and transmembrane domains in vitro, we first transfected a mutated CaSR gene into HEK cells. JTT‐305/MK‐5442 suppressed the hypersensitivity to extracellular Ca²⁺ of HEK cells transfected with the CaSR gene with activating mutations in the extracellular and transmembrane domains. We then selected two activating mutations locating in the extracellular (C129S) and transmembrane (A843E) domains, and generated two strains of CaSR knock‐in mice to build an ADH mouse model. Both mutant mice mimicked almost all the clinical features of human ADH. JTT‐305/MK‐5442 treatment in vivo increased urinary cAMP excretion, improved serum and urinary calcium and phosphate levels by stimulating endogenous PTH secretion, and prevented renal calcification. In contrast, PTH(1‐34) treatment normalized serum calcium and phosphate but could not reduce hypercalciuria or renal calcification. CaSR knock‐in mice exhibited low bone turnover due to the deficiency of PTH, and JTT‐305/MK‐5442 as well as PTH(1‐34) increased bone turnover and bone mineral density (BMD) in these mice. These results demonstrate that calcilytics can reverse almost all the phenotypes of ADH including hypercalciuria and renal calcification, and suggest that calcilytics can become a novel therapeutic agent for ADH. © 2015 American Society for Bone and Mineral Research.     

β‐Arrestin2 Regulates RANKL and Ephrins Gene Expression in Response to Bone Remodeling in Mice

PTH‐stimulated intracellular signaling is regulated by the cytoplasmic adaptor molecule β‐arrestin. We reported that the response of cancellous bone to intermittent PTH is reduced in β‐arrestin2^?/? mice and suggested that β‐arrestins could influence the bone mineral balance by controlling RANKL and osteoprotegerin (OPG) gene expression. Here, we study the role of β‐arrestin2 on the in vitro development and activity of bone marrow (BM) osteoclasts (OCs) and Ephrins ligand (Efn), and receptor (Eph) mRNA levels in bone in response to PTH and the changes of bone microarchitecture in wildtype (WT) and β‐arrestin2^?/? mice in models of bone remodeling: a low calcium diet (LoCa) and ovariectomy (OVX). The number of PTH‐stimulated OCs was higher in BM cultures from β‐arrestin2^?/? compared with WT, because of a higher RANKL/OPG mRNA and protein ratio, without directly influencing osteoclast activity. In vivo, high PTH levels induced by LoCa led to greater changes in TRACP5b levels in β‐arrestin2^?/? compared with WT. LoCa caused a loss of BMD and bone microarchitecture, which was most prominent in β‐arrestin2^?/?. PTH downregulated Efn and Eph genes in β‐arrestin2^?/?, but not WT. After OVX, vertebral trabecular bone volume fraction and trabecular number were lower in β‐arrestin2^?/? compared with WT. Histomorphometry showed that OC number was higher in OVX‐β‐arrestin2^?/? compared with WT. These results indicate that β‐arrestin2 inhibits osteoclastogenesis in vitro, which resulted in decreased bone resorption in vivo by regulating RANKL/OPG production and ephrins mRNAs. As such, β‐arrestins should be considered an important mechanism for the control of bone remodeling in response to PTH and estrogen deprivation.     

Estrogen receptor‐α is required for the osteogenic response to mechanical loading in a ligand‐independent manner involving its activation function 1 but not 2

Estrogen receptor‐α (ERα) is crucial for the adaptive response of bone to loading but the role of endogenous estradiol (E2) for this response is unclear. To determine in vivo the ligand dependency and relative roles of different ERα domains for the osteogenic response to mechanical loading, gene‐targeted mouse models with (1) a complete ERα inactivation (ERα^?/?), (2) specific inactivation of activation function 1 (AF‐1) in ERα (ERαAF‐1⁰), or (3) specific inactivation of ERαAF‐2 (ERαAF‐2⁰) were subjected to axial loading of tibia, in the presence or absence (ovariectomy [ovx]) of endogenous E2. Loading increased the cortical bone area in the tibia mainly as a result of an increased periosteal bone formation rate (BFR) and this osteogenic response was similar in gonadal intact and ovx mice, demonstrating that E2 (ligand) is not required for this response. Female ERα^?/? mice displayed a severely reduced osteogenic response to loading with changes in cortical area (?78% ± 15%, p < 0.01) and periosteal BFR (?81% ± 9%, p < 0.01) being significantly lower than in wild‐type (WT) mice. ERαAF‐1⁰ mice also displayed a reduced response to mechanical loading compared with WT mice (cortical area ?40% ± 11%, p < 0.05 and periosteal BFR ?41% ± 8%, p < 0.01), whereas the periosteal osteogenic response to loading was unaffected in ERαAF‐2⁰ mice. Mechanical loading of transgenic estrogen response element (ERE)‐luciferase reporter mice did not increase luciferase expression in cortical bone, suggesting that the loading response does not involve classical genomic ERE‐mediated pathways. In conclusion, ERα is required for the osteogenic response to mechanical loading in a ligand‐independent manner involving AF‐1 but not AF‐2. © 2013 American Society for Bone and Mineral Research     

The role of activation functions 1 and 2 of estrogen receptor‐α for the effects of estradiol and selective estrogen receptor modulators in male mice

Estradiol (E2) is important for male skeletal health and the effect of E2 is mediated via estrogen receptor (ER)‐α. This was demonstrated by the findings that men with an inactivating mutation in aromatase or a nonfunctional ERα had osteopenia and continued longitudinal growth after sexual maturation. The aim of the present study was to evaluate the role of different domains of ERα for the effects of E2 and selective estrogen receptor modulators (SERMs) on bone mass in males. Three mouse models lacking either ERαAF‐1 (ERαAF‐1⁰), ERαAF‐2 (ERαAF‐2⁰), or the total ERα (ERα^?/?) were orchidectomized (orx) and treated with E2 or placebo. E2 treatment increased the trabecular and cortical bone mass and bone strength, whereas it reduced the thymus weight and bone marrow cellularity in orx wild type (WT) mice. These parameters did not respond to E2 treatment in orx ERα^?/? or ERαAF‐2⁰ mirx ERαAF‐1⁰ mice were tissue‐dependent, with a clear response in cortical bone parameters and bone marrow cellularity, but no response in trabecular bone. To determine the role of ERαAF‐1 for the effects of SERMs, we treated orx WT and ERαAF‐1⁰ mice with raloxifene (Ral), lasofoxifene (Las), bazedoxifene (Bza), or vehicle. These SERMs increased total body areal bone mineral density (BMD) and trabecular volumetric BMD to a similar extent in orx WT mice. Furthermore, only Las increased cortical thickness significantly and only Bza increased bone strength significantly. However, all SERMs showed a tendency toward increased cortical bone parameters. Importantly, all SERM effects were absent in the orx ERαAF‐1⁰ mice. In conclusion, ERαAF‐2 is required for the estrogenic effects on all evaluated parameters, whereas the role of ERαAF‐1 is tissue‐specific. All evaluated effects of Ral, Las and Bza are dependent on a functional ERαAF‐1. Our findings might contribute to the development of bone‐specific SERMs in males. © 2013 American Society for Bone and Mineral Research.     

A G‐protein Subunit‐α11 Loss‐of‐Function Mutation,Thr54Met,Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2)

Familial hypocalciuric hypercalcemia (FHH) is a genetically heterogeneous disorder with three variants, FHH1 to FHH3. FHH1 is caused by loss‐of‐function mutations of the calcium‐sensing receptor (CaSR), a G‐protein coupled receptor that predominantly signals via G‐protein subunit alpha‐11 (Gα₁₁) to regulate calcium homeostasis. FHH2 is the result of loss‐of‐function mutations in Gα₁₁, encoded by GNA11, and to date only two FHH2‐associated Gα₁₁ missense mutations (Leu135Gln and Ile200del) have been reported. FHH3 is the result of loss‐of‐function mutations of the adaptor protein‐2 σ‐subunit (AP2σ), which plays a pivotal role in clathrin‐mediated endocytosis. We describe a 65‐year‐old woman who had hypercalcemia with normal circulating parathyroid hormone concentrations and hypocalciuria, features consistent with FHH, but she did not have CaSR and AP2σ mutations. Mutational analysis of the GNA11 gene was therefore undertaken, using leucocyte DNA, and this identified a novel heterozygous GNA11 mutation (c.161C>T; p.Thr54Met). The effect of the Gα₁₁ variant was assessed by homology modeling of the related Gα_q protein and by measuring the CaSR‐mediated intracellular calcium (Ca²⁺_i) responses of HEK293 cells, stably expressing CaSR, to alterations in extracellular calcium (Ca²⁺_o) using flow cytometry. Three‐dimensional modeling revealed the Thr54Met mutation to be located at the interface between the Gα₁₁ helical and GTPase domains, and to likely impair GDP binding and interdomain interactions. Expression of wild‐type and the mutant Gα₁₁ in HEK293 cells stably expressing CaSR demonstrate that the Ca²⁺_i responses after stimulation with Ca²⁺_o of the mutant Met54 Gα₁₁ led to a rightward shift of the concentration‐response curve with a significantly (p < 0.01) increased mean half‐maximal concentration (EC₅₀) value of 3.88 mM (95% confidence interval [CI] 3.76–4.01 mM), when compared with the wild‐type EC₅₀ of 2.94 mM (95% CI 2.81–3.07 mM) consistent with a loss‐of‐function. Thus, our studies have identified a third Gα₁₁ mutation (Thr54Met) causing FHH2 and reveal a critical role for the Gα₁₁ interdomain interface in CaSR signaling and Ca²⁺_o homeostasis. © 2016 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle–mediated mineralization,but lack of collagen X affects hematopoiesis and the Th1/Th2 response

Numerous biochemical studies have pointed to an essential role of annexin A5 (AnxA5), annexin A6 (AnxA6), and collagen X in matrix vesicle–mediated biomineralization during endochondral ossification and in osteoarthritis. By binding to the extracellular matrix protein collagen X and matrix vesicles, annexins were proposed to anchor matrix vesicles in the extracellular space of hypertrophic chondrocytes to initiate the calcification of cartilage. However, mineralization appears to be normal in mice lacking AnxA5 and AnxA6, whereas collagen X–deficient mice show only subtle alterations in the growth plate organization. We hypothesized that the simultaneous lack of AnxA5, AnxA6, and collagen X in vivo induces more pronounced changes in the growth plate development and the initiation of mineralization. In this study, we generated and analyzed mice deficient for AnxA5, AnxA6, and collagen X. Surprisingly, mice were viable, fertile, and showed no obvious abnormalities. Assessment of growth plate development indicated that the hypertrophic zone was expanded in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? newborns, whereas endochondral ossification and mineralization were not affected in 13‐day‐ and 1‐month‐old mutants. In peripheral quantitative computed tomography, no changes in the degree of biomineralization were found in femora of 1‐month‐ and 1‐year‐old mutants even though the diaphyseal circumference was reduced in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? mice. The percentage of naive immature IgM⁺/IgM⁺ B cells and peripheral T‐helper cells were increased in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? mutants, and activated splenic T cells isolated from Col10a1^?/? mice secreted elevated levels of IL‐4 and GM‐CSF. Hence, collagen X is needed for hematopoiesis during endochondral ossification and for the immune response, but the interaction of annexin A5, annexin A6, and collagen X is not essential for physiological calcification of growth plate cartilage. Therefore, annexins and collagen X may rather fulfill functions in growth plate cartilage not directly linked to the mineralization process. © 2012 American Society for Bone and Mineral Research.     

Reversing LRP5‐Dependent Osteoporosis and SOST Deficiency–Induced Sclerosing Bone Disorders by Altering WNT Signaling Activity

The bone formation inhibitor sclerostin encoded by SOST binds in vitro to low‐density lipoprotein receptor‐related protein (LRP) 5/6 Wnt co‐receptors, thereby inhibiting Wnt/β‐catenin signaling, a central pathway of skeletal homeostasis. Lrp5/LRP5 deficiency results in osteoporosis‐pseudoglioma (OPPG), whereas Sost/SOST deficiency induces lifelong bone gain in mice and humans. Here, we analyzed the bone phenotype of mice lacking Sost (Sost^?/?), Lrp5 (Lrp5^?/?), or both (Sost^?/?;Lrp5^?/?) to elucidate the mechanism of action of Sost in vivo. Sost deficiency–induced bone gain was significantly blunted in Sost^?/?;Lrp5^?/? mice. Yet the Lrp5 OPPG phenotype was fully rescued in Sost^?/?;Lrp5^?/? mice and most bone parameters were elevated relative to wild‐type. To test whether the remaining bone increases in Sost^?/?;Lrp5^?/? animals depend on Lrp6, we treated wild‐type, Sost^?/?, and Sost^?/?;Lrp5^?/? mice with distinct Lrp6 function blocking antibodies. Selective blockage of Wnt1 class–mediated Lrp6 signaling reduced cancellous bone mass and density in wild‐type mice. Surprisingly, it reversed the abnormal bone gain in Sost^?/? and Sost^?/?;Lrp5^?/? mice to wild‐type levels irrespective of enhancement or blockage of Wnt3a class‐mediated Lrp6 activity. Thus, whereas Sost deficiency–induced bone anabolism partially requires Lrp5, it fully depends on Wnt1 class–induced Lrp6 activity. These findings indicate: first, that OPPG syndrome patients suffering from LRP5 loss‐of‐function should benefit from principles antagonizing SOST/sclerostin action; and second, that therapeutic WNT signaling inhibitors may stop the debilitating bone overgrowth in sclerosing disorders related to SOST deficiency, such as sclerosteosis, van Buchem disease, and autosomal dominant craniodiaphyseal dysplasia, which are rare disorders without viable treatment options. © 2014 American Society for Bone and Mineral Research.     

Role of Bcl2 in Osteoclastogenesis and PTH Anabolic Actions in Bone

Introduction : B‐cell leukemia/lymphoma 2 (Bcl2) is a proto‐oncogene best known for its ability to suppress cell death. However, the role of Bcl2 in the skeletal system is unknown. Bcl2 has been hypothesized to play an important anti‐apoptotic role in osteoblasts during anabolic actions of PTH. Although rational, this has not been validated in vivo; hence, the impact of Bcl2 in bone remains unknown. Materials and Methods : The bone phenotype of Bcl2 homozygous mutant (Bcl2^?/?) mice was analyzed with histomorphometry and μCT. Calvarial osteoblasts were isolated and evaluated for their cellular activity. Osteoclastogenesis was induced from bone marrow cells using RANKL and macrophage‐colony stimulating factor (M‐CSF), and their differentiation was analyzed. PTH(1–3;34) (50 μg/kg) or vehicle was administered daily to Bcl2^+/+ and Bcl2^?/? mice (4 days old) for 9 days to clarify the influence of Bcl2 ablation on PTH anabolic actions. Western blotting and real‐time PCR were performed to detect Bcl2 expression in calvarial osteoblasts in response to PTH ex vivo. Results : There were reduced numbers of osteoclasts in Bcl2^?/? mice, with a resultant increase in bone mass. Bcl2^?/? bone marrow–derived osteoclasts ex vivo were significantly larger in size and short‐lived compared with wildtype, suggesting a pro‐apoptotic nature of Bcl2^?/? osteoclasts. In contrast, osteoblasts were entirely normal in their proliferation, differentiation, and mineralization. Intermittent administration of PTH increased bone mass similarly in Bcl2^+/+ and Bcl2^?/? mice. Finally, Western blotting and real‐time PCR showed that Bcl2 levels were not induced in response to PTH in calvarial osteoblasts. Conclusions : Bcl2 is critical in osteoclasts but not osteoblasts. Osteoclast suppression is at least in part responsible for increased bone mass of Bcl2^?/? mice, and Bcl2 is dispensable in PTH anabolic actions during bone growth.     

Increased Osteopontin Contributes to Inhibition of Bone Mineralization in FGF23‐Deficient Mice

Excessive FGF23 has been identified as a pivotal phosphaturic factor leading to renal phosphate‐wasting and the subsequent development of rickets and osteomalacia. In contrast, loss of FGF23 in mice (Fgf23^?/?) leads to high serum phosphate, calcium, and 1,25‐vitamin D levels, resulting in early lethality attributable to severe ectopic soft‐tissue calcifications and organ failure. Paradoxically, Fgf23^?/? mice exhibit a severe defect in skeletal mineralization despite high levels of systemic mineral ions and abundant ectopic mineralization, an abnormality that remains largely unexplained. Through use of in situ hybridization, immunohistochemistry, and immunogold labeling coupled with electron microscopy of bone samples, we discovered that expression and accumulation of osteopontin (Opn/OPN) was markedly increased in Fgf23^?/? mice. These results were confirmed by qPCR analyses of Fgf23^?/? bones and ELISA measurements of serum OPN. To investigate whether elevated OPN levels were contributing to the bone mineralization defect in Fgf23^?/? mice, we generated Fgf23^?/?/Opn^?/? double‐knockout mice (DKO). Biochemical analyses showed that the hypercalcemia and hyperphosphatemia observed in Fgf23^?/? mice remained unchanged in DKO mice; however, micro‐computed tomography (µCT) and histomorphometric analyses showed a significant improvement in total mineralized bone volume. The severe osteoidosis was markedly reduced and a normal mineral apposition rate was present in DKO mice, indicating that increased OPN levels in Fgf23^?/? mice are at least in part responsible for the osteomalacia. Moreover, the increased OPN levels were significantly decreased upon lowering serum phosphate by feeding a low‐phosphate diet or after deletion of NaPi2a, indicating that phosphate levels contribute in part to the high OPN levels in Fgf23^?/? mice. In summary, our results suggest that increased OPN is an important pathogenic factor mediating the mineralization defect and the alterations in bone metabolism observed in Fgf23^?/? bones. © 2014 American Society for Bone and Mineral Research.     

Endogenous parathyroid hormone–related protein compensates for the absence of parathyroid hormone in promoting bone accrual in vivo in a model of bone marrow ablation

To assess the effect of hypoparathyroidism on osteogenesis and bone turnover in vivo, bone marrow ablation (BMXs) were performed in tibias of 8‐week‐old wild‐type and parathyroid hormone–null (PTH^?/?) mice and newly formed bone tissue was analyzed from 5 days to 3 weeks after BMX. At 1 week after BMX, trabecular bone volume, osteoblast numbers, alkaline phosphatase‐positive areas, type I collagen‐positive areas, PTH receptor–positive areas, calcium sensing receptor–positive areas, and expression of bone formation–related genes were all decreased significantly in the diaphyseal regions of bones of PTH^?/? mice compared to wild‐type mice. In contrast, by 2 weeks after BMX, all parameters related to osteoblastic bone accrual were increased significantly in PTH^?/? mice. At 5 days after BMX, active tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts had appeared in wild‐type mice but were undetectable in PTH^?/? mice, Both the ratio of mRNA levels of receptor activator of NF‐κB ligand (RANKL)/osteoprotegerin (OPG) and TRAP‐positive osteoclast surface were still reduced in PTH^?/? mice at 1 week but were increased by 2 weeks after BMX. The expression levels of parathyroid hormone–related protein (PTHrP) at both mRNA and protein levels were upregulated significantly at 1 week and more dramatically at 2 weeks after BMX in PTH^?/? mice. To determine whether the increased newly formed bones in PTH^?/? mice at 2 weeks after BMX resulted from the compensatory action of PTHrP, PTH^?/?PTHrP^+/? mice were generated and newly formed bone tissue was compared in these mice with PTH^?/? and wild‐type mice at 2 weeks after BMX. All parameters related to osteoblastic bone formation and osteoclastic bone resorption were reduced significantly in PTH^?/?PTHrP^+/? mice compared to PTH^?/? mice. These results demonstrate that PTH deficiency itself impairs osteogenesis, osteoclastogenesis, and osteoclastic bone resorption, whereas subsequent upregulation of PTHrP in osteogenic cells compensates by increasing bone accrual. © 2013 American Society for Bone and Mineral Research     

SDF‐1/CXCR4 Axis in Tie2‐Lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing

CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal‐derived‐factor 1 (SDF‐1). SDF‐1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF‐1/CXCR4 pathway in Tie2‐lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2‐Cre^ER CXCR4 knockout mice (CXCR4^?/?) and wild‐type mice (WT). We report here that in vitro, EPCs derived from of CXCR4^?/? mouse bone marrow demonstrated severe reduction of migration activity and EPC colony‐forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4^?/? group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4^?/? group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4^?/? group. Especially, CXCR4^?/?group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real‐time RT‐PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE‐cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4^?/? group. In the gain‐of‐function study, the fracture in the SDF‐1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF‐1 injected CXCR4^?/? group. We demonstrated that an EPC SDF‐1/CXCR4 axis plays an important role in bone fracture healing using Tie2‐Cre^ER CXCR4 conditional knockout mice. © 2014 American Society for Bone and Mineral Research.     

Sustained RANKL response to parathyroid hormone in oncostatin M receptor-deficient osteoblasts converts anabolic treatment to a catabolic effect in vivo

Parathyroid hormone (PTH) is the only approved anabolic agent for osteoporosis treatment. It acts via osteoblasts to stimulate both osteoclast formation and bone formation, with the balance between these two activities determined by the mode of administration. Oncostatin M (OSM), a gp130‐dependent cytokine expressed by osteoblast lineage cells, has similar effects and similar gene targets in the osteoblast lineage. In this study, we investigated whether OSM might participate in anabolic effects of PTH. Microarray analysis and quantitative real‐time polymerase chain reaction (qPCR) of PTH‐treated murine stromal cells and primary calvarial osteoblasts identified significant regulation of gp130 and gp130‐dependent coreceptors and ligands, including a significant increase in OSM receptor (OSMR) expression. To determine whether OSMR signaling is required for PTH anabolic action, 6‐week‐old male Osmr^?/? mice and wild‐type (WT) littermates were treated with hPTH(1–34) for 3 weeks. In WT mice, PTH increased trabecular bone volume and trabecular thickness. In contrast, the same treatment had a catabolic effect in Osmr^?/? mice, reducing both trabecular bone volume and trabecular number. This was not explained by any alteration in the increased osteoblast formation and mineral apposition rate in response to PTH in Osmr^?/? compared with WT mice. Rather, PTH treatment doubled osteoclast surface in Osmr^?/? mice, an effect not observed in WT mice. Consistent with this finding, when osteoclast precursors were cultured in the presence of osteoblasts, more osteoclasts were formed in response to PTH when Osmr^?/? osteoblasts were used. Neither PTH1R mRNA levels nor cAMP response to PTH were modified in Osmr^?/? osteoblasts. However, RANKL induction in PTH‐treated Osmr^?/? osteoblasts was sustained at least until 24 hours after PTH exposure, an effect not observed in WT osteoblasts. These data indicate that the transient RANKL induction by intermittent PTH administration, which is associated with its anabolic action, is changed to a prolonged induction in OSMR‐deficient osteoblasts, resulting in bone destruction. © 2012 American Society for Bone and Mineral Research.     

Elevated serum IGF‐1 levels synergize PTH action on the skeleton only when the tissue IGF‐1 axis is intact

There is growing evidence that insulin‐like growth factor 1 (IGF‐1) and parathyroid hormone (PTH) have synergistic actions on bone and that part of the anabolic effects of PTH is mediated by local production of IGF‐1. In this study we analyzed the skeletal response to PTH in mouse models with manipulated endocrine or autocrine/paracrine IGF‐1. We used mice carrying a hepatic IGF‐1 transgene (HIT), which results in a threefold increase in serum IGF‐1 levels and normal tissue IGF‐1 expression, and Igf1 null mice with blunted IGF‐1 expression in tissues but threefold increases in serum IGF‐1 levels (KO‐HIT). Evaluation of skeletal growth showed that elevations in serum IGF‐1 in mice with Igf1 gene ablation in all tissues except the liver (KO‐HIT) resulted in a restoration of skeletal morphology and mechanical properties by adulthood. Intermittent PTH treatment of adult HIT mice resulted in increases in serum osteocalcin levels, femoral total cross‐sectional area, cortical bone area and cortical bone thickness, as well as bone mechanical properties. We found that the skeletal response of HIT mice to PTH was significantly higher than that of control mice, suggesting synergy between IGF‐1 and PTH on bone. In sharp contrast, although PTH‐treated KO‐HIT mice demonstrated an anabolic response in cortical and trabecular bone compartments compared with vehicle‐treated KO‐HIT mice, their response was identical to that of PTH‐treated control mice. We conclude that (1) in the presence of elevated serum IGF‐1 levels, PTH can exert an anabolic response in bone even in the total absence of tissue IGF‐1, and (2) elevations in serum IGF‐1 levels synergize PTH action on bone only if the tissue IGF‐1 axis is intact. Thus enhancement of PTH anabolic actions depends on tissue IGF‐1. © 2010 American Society for Bone and Mineral Research.     

Interferon‐γ plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice

Interferon γ (IFN‐γ) is a cytokine produced locally in the bone microenvironment by cells of immune origin as well as mesenchymal stem cells. However, its role in normal bone remodeling is still poorly understood. In this study we first examined the consequences of IFN‐γ ablation in vivo in C57BL/6 mice expressing the IFN‐γ receptor knockout phenotype (IFNγR1^?/?). Compared with their wild‐type littermates (IFNγR1^+/+), IFNγR1^?/? mice exhibit a reduction in bone volume associated with significant changes in cortical and trabecular structural parameters characteristic of an osteoporotic phenotype. Bone histomorphometry of IFNγR1^?/? mice showed a low‐bone‐turnover pattern with a decrease in bone formation, a significant reduction in osteoblast and osteoclast numbers, and a reduction in circulating levels of bone‐formation and bone‐resorption markers. Furthermore, administration of IFN‐γ (2000 and 10,000 units) to wild‐type C57BL/6 sham‐operated (SHAM) and ovariectomized (OVX) female mice significantly improved bone mass and microarchitecture, mechanical properties of bone, and the ratio between bone formation and bone resorption in SHAM mice and rescued osteoporosis in OVX mice. These data therefore support an important physiologic role for IFN‐γ signaling as a potential new anabolic therapeutic target for osteoporosis. © 2011 American Society for Bone and Mineral Research.