Osteoclast-derived IGF1 induces RANKL production in osteocytes and contributes to pagetic lesion formation.

We previously reported that measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) of patients with Paget disease (PD) or targeted to the OCL lineage in MVNP-transgenic mice (MVNP mice) increases IGF1 production in osteoclasts (OCL-IGF1) and leads to development of PD OCLs and pagetic bone lesions (PDLs). Conditional deletion of Igf1 in OCLs of MVNP mice fully blocked development of PDLs. In this study, we examined whether osteocytes (OCys), key regulators of normal bone remodeling, contribute to PD. OCys in PDLs of patients and of MVNP mice expressed less sclerostin, and had increased RANKL expression compared with OCys in bones from WT mice or normal patients. To test whether increased OCL-IGF1 is sufficient to induce PDLs and PD phenotypes, we generated TRAP-Igf1 (T-Igf1) transgenic mice to determine whether increased IGF1 expression in the absence of MVNP in OCLs is sufficient to induce PDLs and pagetic OCLs. We found that T-Igf1 mice at 16 months of age developed PD OCLs, PDLs, and OCys, with decreased sclerostin and increased RANKL, similar to MVNP mice. Thus, pagetic phenotypes could be induced by OCLs expressing increased IGF1. OCL-IGF1 in turn increased RANKL production in OCys to induce PD OCLs and PDLs.

Increased S1P expression in osteoclasts enhances bone formation in an animal model of Paget's disease.

Paget's disease (PD) is characterized by increased numbers of abnormal osteoclasts (OCLs) that drive exuberant bone formation, but the mechanisms responsible for the increased bone formation remain unclear. We previously reported that OCLs from 70% of PD patients express measles virus nucleocapsid protein (MVNP), and that transgenic mice with targeted expression of MVNP in OCLs (MVNP mice) develop bone lesions and abnormal OCLs characteristic of PD. In this report, we examined if OCL-derived sphingosine-1-phosphate (S1P) contributed to the abnormal bone formation in PD, since OCL-derived S1P can act as a coupling factor to increase normal bone formation via binding S1P-receptor-3 (S1PR3) on osteoblasts (OBs). We report that OCLs from MVNP mice and PD patients expressed high levels of sphingosine kinase-1 (SphK-1) compared with wild-type (WT) mouse and normal donor OCLs. SphK-1 production by MVNP-OCLs was interleukin-6 (IL-6)-dependent since OCLs from MVNP/IL-6-/- mice expressed lower levels of SphK-1. Immunohistochemistry of bone biopsies from a normal donor, a PD patient, WT and MVNP mice confirmed increased expression levels of SphK-1 in OCLs and S1PR3 in OBs of the PD patient and MVNP mice compared with normal donor and WT mice. Further, MVNP-OCLs cocultured with OBs from MVNP or WT mice increased OB-S1PR3 expression and enhanced expression of OB differentiation markers in MVNP-OBs precursors compared with WT-OBs, which was mediated by IL-6 and insulin-like growth factor 1 secreted by MVNP-OCLs. Finally, the addition of an S1PR3 antagonist (VPC23019) to WT or MVNP-OBs treated with WT and MVNP-OCL-conditioned media (CM) blocked enhanced OB differentiation of MVNP-OBs treated with MVNP-OCL-CM. In contrast, the addition of the SIPR3 agonist, VPC24191, to the cultures enhanced osterix and Col-1A expression in MVNP-OBs treated with MVNP-OCL-CM compared with WT-OBs treated with WT-OCL-CM. These results suggest that IL-6 produced by PD-OCLs increases S1P in OCLs and S1PR3 on OBs, to increase bone formation in PD.

Osteoclast-derived IGF1 is required for pagetic lesion formation in vivo.

We report that transgenic mice expressing measles virus nucleocapsid protein (MVNP) in osteoclasts (OCLs) (MVNP mice) are Paget's disease (PD) models and that OCLs from patients with PD and MVNP mice express high levels of OCL-derived IGF1 (OCL-IGF1). To determine OCL-IGF1's role in PD and normal bone remodeling, we generated WT and MVNP mice with targeted deletion of Igf1 in OCLs (Igf1-cKO) and MVNP/Igf1-cKO mice, and we assessed OCL-IGF1's effects on bone mass, bone formation rate, EphB2/EphB4 expression on OCLs and osteoblasts (OBs), and pagetic bone lesions (PDLs). A total of 40% of MVNP mice, but no MVNP/Igf1-cKO mice, had PDLs. Bone volume/tissue volume (BV/TV) was decreased by 60% in lumbar vertebrae and femurs of MVNP/Igf1-cKO versus MVNP mice with PDLs and by 45% versus all MVNP mice tested. Bone formation rates were decreased 50% in Igf1-cKO and MVNP/Igf1-cKO mice versus WT and MVNP mice. MVNP mice had increased EphB2 and EphB4 levels in OCLs/OBs versus WT and MVNP/Igf1-cKO, with none detectable in OCLs/OBs of Igf1-cKO mice. Mechanistically, IL-6 induced the increased OCL-IGF1 in MVNP mice. These results suggest that high OCL-IGF1 levels increase bone formation and PDLs in PD by enhancing EphB2/EphB4 expression in vivo and suggest OCL-IGF1 may contribute to normal bone remodeling.

Comparative evaluation of bone microstructure in alveolar cleft repair by cone beam CT: influence of different autologous donor sites and additional application of ß-tricalcium phosphate.

OBJECTIVES: This study used cone beam computed tomography (CBCT) images to comparatively evaluate the three-dimensional microstructural features of reconstructed bone bridge based on the bone harvesting site and the presence/absence of artificial bone material, as well as the features of regenerated bone tissue after bone harvesting from mandibular symphysis in secondary alveolar bone grafting (SABG) for patients with cleft lip, with or without cleft palate. MATERIALS AND METHODS: Thirty-one patients were divided into three groups in which SABG was performed by autologous bone harvesting from iliac crest (IC), mandibular symphysis (MS), or MS combined with ß-TCP granules (MS+TCP). The microstructural trabecular bone parameters (TBPs) and bone structure indexes (SIs) were analyzed using datasets of CBCT images taken before and after SABG. RESULTS: TBPs showed differences between IC and MS groups (P < 0.05), resulting in greater values of bone volume density (P < 0.05) and inferior value of TBPf (P = 0.070) in IC group compared with MS group. Using MS+TCP or filling ß-TCP granules into donor site significantly improved reconstructed or regenerated BV/TV and Tb.Th (P < 0.05) compared with group without ß-TCP. CONCLUSIONS: Microstructural characteristics of reconstructed bone bridge were dependent on the donor site of bone harvesting; using an absorbable bone conductive material improved bone quality and increased bone volume density. CLINICAL RELEVANCE: Application of ß-TCP granules as a partial alternative with autologous bone from mandibular symphysis could obtain comparable outcomes in the microstructure of bone bridge to SABG with autologous iliac crest.

Aplidin (plitidepsin) is a novel anti-myeloma agent with potent anti-resorptive activity mediated by direct effects on osteoclasts.

Despite recent progress in its treatment, Multiple Myeloma (MM) remains incurable and its associated bone disease persists even after complete remission. Thus, identification of new therapeutic agents that simultaneously suppress MM growth and protect bone is an unmet need. Herein, we examined the effects of Aplidin, a novel anti-cancer marine-derived compound, on MM and bone cells. In vitro, Aplidin potently inhibited MM cell growth and induced apoptosis, effects that were enhanced by dexamethasone (Dex) and bortezomib (Btz). Aplidin modestly reduced osteocyte/osteoblast viability and decreased osteoblast mineralization, effects that were enhanced by Dex and partially prevented by Btz. Further, Aplidin markedly decreased osteoclast precursor numbers and differentiation, and reduced mature osteoclast number and resorption activity. Moreover, Aplidin reduced Dex-induced osteoclast differentiation and further decreased osteoclast number when combined with Btz. Lastly, Aplidin alone, or suboptimal doses of Aplidin combined with Dex or Btz, decreased tumor growth and bone resorption in ex vivo bone organ cultures that reproduce the 3D-organization and the cellular diversity of the MM/bone marrow niche. These results demonstrate that Aplidin has potent anti-myeloma and anti-resorptive properties, and enhances proteasome inhibitors blockade of MM growth and bone destruction.

Extracellular Phosphate Induces the Expression of Dentin Matrix Protein 1 Through the FGF Receptor in Osteoblasts.

Dentin matrix protein 1 (Dmp1) is an extracellular matrix protein involved in phosphate metabolism and biomineralization, and its expression markedly increases during the maturation of osteoblasts into osteocytes. We previously reported that an increased level of inorganic phosphate (Pi) in media up-regulated the expression of Dmp1 in primary osteocytes isolated from mouse bones. In the present study, we found that elevated extracellular Pi strongly induced the expression of Dmp1 in osteoblasts and explored its underlying mechanism of action. In an osteoblastic cell line MC3T3-E1, increases in extracellular Pi induced the phosphorylation of ERK1/2 and up-regulated the expression of Dmp1, fibroblast growth factor 2 (Fgf2), and Fgf receptor 1 (Fgfr1). A co-treatment with the MEK inhibitor U0126 abolished the increase in the expression of Dmp1 and Fgfr1 by elevated Pi, suggesting the involvement of the MEK/ERK pathway in this up-regulation. Elevated extracellular Pi also resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), which was diminished by knockdown of Slc20a1 encoding Pit1 sodium-phosphate co-transporter. The co-treatment with an inhibitor against FGFR (SU5402) abolished the up-regulation of Dmp1 induced by elevated extracellular Pi. In primary osteoblasts, a treatment with 4 mM Pi transiently increased the expression of early growth response 1 (Egr1) before the up-regulation of Dmp1. These results indicate that FGFR mediates the direct effects of extracellular Pi on the expression of Dmp1 in osteoblasts and enhance the close relationship between the signaling evoked by elevated extracellular Pi and FGF/FGFR signaling. J. Cell. Biochem. 118: 1151-1163, 2017. © 2016 Wiley Periodicals, Inc.

TGF-ß in jaw tumor fluids induces RANKL expression in stromal fibroblasts.

Odontogenic tumors and cysts, arising in the jawbones, grow by resorption and destruction of the jawbones. However, mechanisms underlying bone resorption by odontogenic tumors/cysts remain unclear. Odontogenic tumors/cysts comprise odontogenic epithelial cells and stromal fibroblasts, which originate from the developing tooth germ. It has been demonstrated that odontogenic epithelial cells of the developing tooth germ induce osteoclastogenesis to prevent the tooth germ from invading the developing bone to maintain its structure in developing bones. Thus, we hypothesized that odontogenic epithelial cells of odontogenic tumors/cysts induce osteoclast formation, which plays potential roles in tumor/cyst outgrowth into the jawbone. The purpose of this study was to examine osteoclastogenesis by cytokines, focusing on transforming growth factor-ß (TGF-ß), produced by odontogenic epithelial cells. We observed two pathways for receptor activator of NF-κB ligand (RANKL) induction by keratocystic odontogenic tumor fluid: the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway through interleukin-1α (IL-1α) signaling and non-COX-2/PGE2 pathway through TGF-ß receptor signaling. TGF-ß1 and IL-1α produced by odontogenic tumors/cysts induced osteoclastogenesis directly in the osteoclast precursor cells and indirectly via increased RANKL induction in the stroma.

Interleukin-1-induced acute bone resorption facilitates the secretion of fibroblast growth factor 23 into the circulation.

Fibroblast growth factor 23 (FGF23), a central regulator of phosphate and vitamin D metabolism, is mainly produced by osteocytes in bone and exerts its effects on distant organs. Despite its endocrine function, the mechanism controlling serum FGF23 levels is not fully understood. Here we tested the hypothesis that osteoclastic bone resorption may play a role in regulating circulating levels of FGF23, using a mouse model where injections of interleukin (IL)-1ß into the subcutaneous tissue over the calvaria induced rapid bone resorption. A significant amount of FGF23 was detected in the extracts from mouse bones, which supports the idea that FGF23 stays in bone for a while after its production. IL-1ß-induced bone resorption was associated with elevated serum FGF23 levels, an effect abolished by pre-treatment with pamidronate. Fgf23 expression was not increased in either the calvariae or tibiae of IL-1ß-injected mice, which suggests that IL-1ß facilitated the entry of FGF23 protein into circulation by accelerating bone resorption rather than increasing its gene expression. The direct effect of IL-1ß on bone was confirmed when it increased FGF23 levels in the conditioned media of mouse calvariae in organ culture. Repeated treatment of the cultured calvariae with IL-1ß led to a refractory phase, where FGF23 was not mobilized by IL-1ß anymore. Consistent with the in vivo results, treatment with IL-1ß failed to increase Fgf23 mRNA in isolated primary osteocytes and osteoblasts. These results suggest that FGF23 produced by osteocytes remains in bone, and that rapid bone resorption facilitates its entry into the bloodstream.

Dysregulated gene expression in the primary osteoblasts and osteocytes isolated from hypophosphatemic Hyp mice.

Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH), Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT) control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na+/Pi) co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10-8 M 1,25(OH)2D3 increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10-8 M 1,25(OH)2D3 in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.

Elevated fibroblast growth factor 23 exerts its effects on placenta and regulates vitamin D metabolism in pregnancy of Hyp mice.

Fibroblast growth factor 23 (FGF23) functions in an endocrine fashion and requires α-Klotho to exert its effects on the target organs. We have recently demonstrated that the human placenta also expresses α-Klotho, which led us to hypothesize that FGF23 may exert effects on the placenta. Immunohistochemical analysis demonstrated the expression of FGF receptor 1 (FGFR1) as well as that of α-Klotho in the feto-maternal interface of both mouse and human normal-term placentas, which suggested that these areas might be receptive to FGF23. Therefore, we next investigated whether FGF23 has some roles in the placenta using Hyp mice with high levels of circulating FGF23. Hyp and wild-type (WT) females were mated with WT males, and the mothers and their male fetuses were analyzed. FGF23 levels in Hyp mothers were elevated. FGF23 levels were about 20-fold higher in Hyp fetuses than in Hyp mothers, whereas WT fetuses from Hyp mothers exhibited low levels of FGF23, as did fetuses from WT mothers. We analyzed the placental gene expression and found that the expression of Cyp24a1 encoding 25OHD-24-hydroxylase, a target gene for FGF23 in the kidney, was increased in the placentas of fetuses from Hyp mothers compared with fetuses from WT mothers. In an organ culture of WT placentas, treatment with plasma from Hyp mothers markedly increased the expression of Cyp24a1, which was abolished by the simultaneous addition of anti-FGF23 neutralizing antibody. The direct injection of recombinant FGF23 into WT placentas induced the expression of Cyp24a1. The increase in the placental expression of Cyp24a1 in fetuses from Hyp mothers resulted in decreased plasma 25-hydroxyvitamin D levels. These results suggest that increased levels of circulating FGF23 in pathological conditions such as Hyp mice exerts direct effects on the placenta and affects fetal vitamin D metabolism via the regulation of Cyp24a1 expression.

FGF23 suppresses chondrocyte proliferation in the presence of soluble α-Klotho both in vitro and in vivo.

Fibroblast growth factor-23 (FGF23) is well established to play crucial roles in the regulation of phosphate homeostasis. X-linked hypophosphatemic rickets (XLH) is characterized by impaired mineralization and growth retardation associated with elevated circulating FGF23 levels. Administration of phosphate and calcitriol is effective in improving growth retardation, but is not sufficient to fully reverse impaired growth, suggesting the existence of a disease-specific mechanism in the development of growth retardation in addition to dysregulated phosphate metabolism. However, the precise mechanisms of growth retardation in XLH remain elusive. Here, we postulated that FGF23 suppressed chondrocyte proliferation in the presence of soluble α-Klotho (sKL). In vitro and ex vivo studies revealed that FGF23 formed a protein complex with sKL through KL1 internal repeat and suppressed the linear growth of metatarsals in the presence of sKL, which was antagonized by co-incubation with neutralizing antibodies against FGF23 or by knocking-down FGFR3 expression. Additionally, FGF23 binding to FGFR3 was enhanced in the presence of sKL. Histologically, the length of the proliferating zone was diminished and was associated with decreased chondrocyte proliferation. FGF23/sKL suppressed Indian hedgehog (Ihh) expression and administration of Ihh protein partially rescued the suppressive effect of FGF23/sKL on metatarsal growth. Intraperitoneal administration of sKL in Hyp mice, a murine model for XLH, caused a decrease in the length of the proliferating zone associated with decreased chondrocyte proliferation without altering circulating phosphate levels. These findings suggest that suppression of chondrocyte proliferation by FGF23 could have a causative role in the development of growth retardation in XLH.

A novel underuse model shows that inactivity but not ovariectomy determines the deteriorated material properties and geometry of cortical bone in the tibia of adult rats.

Our goal in this study was to determine to what extent the physiologic consequences of ovariectomy (OVX) in bones are exacerbated by a lack of daily activity such as walking. We forced 14-week-old female rats to be inactive for 15 weeks with a unique experimental system that prevents standing and walking while allowing other movements. Tibiae, femora, and 4th lumbar vertebrae were analyzed by peripheral quantitative computed tomography (pQCT), microfocused X-ray computed tomography (micro-CT), histology, histomorphometry, Raman spectroscopy, and the three-point bending test. Contrary to our expectation, the exacerbation was very much limited to the cancellous bone parameters. Parameters of femur and tibia cortical bone were affected by the forced inactivity but not by OVX: (1) cross-sectional moment of inertia was significantly smaller in Sham-Inactive rat bones than that of their walking counterparts; (2) the number of sclerostin-positive osteocytes per unit cross-sectional area was larger in Sham-Inactive rat bones than in Sham-Walking rat bones; and (3) material properties such as ultimate stress of inactive rat tibia was lower than that of their walking counterparts. Of note, the additive effect of inactivity and OVX was seen only in a few parameters, such as the cancellous bone mineral density of the lumbar vertebrae and the structural parameters of cancellous bone in the lumbar vertebrae/tibiae. It is concluded that the lack of daily activity is detrimental to the strength and quality of cortical bone in the femur and tibia of rats, while lack of estrogen is not. Our inactive rat model, with the older rats, will aid the study of postmenopausal osteoporosis, the etiology of which may be both hormonal and mechanical.