The biological function of DMP‐1 in osteocyte maturation is mediated by its 57‐kDa c‐terminal fragment

Dentin matrix protein 1 (DMP‐1) is a key molecule in controlling osteocyte formation and phosphate homeostasis. Based on observations that full‐length DMP‐1 is not found in bone, but only cleaved fragments of 37 and 57 kDa are present, and in view of the finding that mutations in the 57‐kDa fragment result in disease, we hypothesized that the 57‐kDa C‐terminal fragment is the functional domain of DMP‐1. To test this hypothesis, a 3.6‐kb type I collagen promoter was used to express this 57‐kDa C‐terminal fragment for comparison with full‐length DMP‐1 in Dmp1 null osteoblasts/osteocytes. Not only did expression of the full‐length DMP‐1 in bone cells fully rescue the skeletal abnormalities of Dmp1 null mice, but the 57‐kDa fragment also had similar results. This included rescue of growth plate defects, osteomalacia, abnormal osteocyte maturation, and the abnormal osteocyte lacunocanalicular system. In addition, the abnormal fibroblast growth factor 23 (FGF‐23) expression in osteocytes, elevated circulating FGF‐23 levels, and hypophosphatemia were rescued. These results show that the 57‐kDa C‐terminal fragment is the functional domain of DMP‐1 that controls osteocyte maturation and phosphate metabolism. © 2011 American Society for Bone and Mineral Research.     

Conditional Deletion of Murine Fgf23: Interruption of the Normal Skeletal Responses to Phosphate Challenge and Rescue of Genetic Hypophosphatemia

The transgenic and knockout (KO) animals involving Fgf23 have been highly informative in defining novel aspects of mineral metabolism, but are limited by shortened lifespan, inability of spatial/temporal FGF23 control, and infertility of the global KO. To more finely test the role of systemic and genetic influences in FGF23 production, a mouse was developed that carried a floxed (“f”)‐Fgf23 allele (exon 2 floxed) which demonstrated in vivo recombination when bred to global‐Cre transgenic mice (eIIa‐cre). Mice homozygous for the recombined allele (“Δ”) had undetectable serum intact FGF23, elevated serum phosphate (p < 0.05), and increased kidney Cyp27b1 mRNA (p < 0.05), similar to global Fgf23‐KO mice. To isolate cellular FGF23 responses during phosphate challenge, Fgf23^Δ/f mice were mated with early osteoblast type Iα1 collagen 2.3‐kb promoter‐cre mice (Col2.3‐cre) and the late osteoblast/early osteocyte Dentin matrix protein‐1‐cre (Dmp1‐cre). Fgf23^Δ/f/Col2.3‐cre⁺ and Fgf23^Δ/f/Dmp1‐cre⁺ exhibited reduced baseline serum intact FGF23 versus controls. After challenge with high‐phosphate diet Cre^– mice had 2.1‐fold to 2.5‐fold increased serum FGF23 (p < 0.01), but Col2.3‐cre⁺ mice had no significant increase, and Dmp1‐cre⁺ mice had only a 37% increase (p < 0.01) despite prevailing hyperphosphatemia in both models. The Fgf23^Δ/f/Col2.3‐cre was bred onto the Hyp (murine X‐linked hypophosphatemia [XLH] model) genetic background to test the contribution of osteoblasts and osteocytes to elevated FGF23 and Hyp disease phenotypes. Whereas Hyp mice maintained inappropriately elevated FGF23 considering their marked hypophosphatemia, Hyp/Fgf23^Δ/f/Col2.3‐cre⁺ mice had serum FGF23 <4% of Hyp (p < 0.01), and this targeted restriction normalized serum phosphorus and ricketic bone disease. In summary, deleting FGF23 within early osteoblasts and osteocytes demonstrated that both cell types contribute to baseline circulating FGF23 concentrations, and that targeting osteoblasts/osteocytes for FGF23 production can modify systemic responses to changes in serum phosphate concentrations and rescue the Hyp genetic syndrome. © 2016 American Society for Bone and Mineral Research.     

DMP1 C‐terminal mutant mice recapture the human ARHR tooth phenotype

DMP1 mutations in autosomal recessive hypophosphatemic rickets (ARHR) patients and mice lacking Dmp1 display an overlapping pathophysiology, such as hypophosphatemia. However, subtle differences exist between the mouse model and human ARHR patients. These differences could be due to a species specificity of human versus mouse, or it may be that the mutant DMP1 in humans maintains partial function of DMP1. In this study we report a deformed tooth phenotype in a human DMP1 deletion mutation case. Unexpectedly, the deletion of nucleotides 1484 to 1490 (c.1484_1490delCTATCAC, delMut, resulting in replacement of the last 18 residues with 33 random amino acids) showed a severe dentin and enamel defect similar to a dentinogenesis imperfecta (DI) III–like phenotype. To address the molecular mechanism behind this phenotype, we generated delMut transgenic mice with the endogenous Dmp1 gene removed. These mutant mice did not recapture the abnormal phenotype observed in the human patient but displayed a mild rachitic tooth phenotype in comparison with that in the Dmp1‐null mice, suggesting that the DI III–like phenotype may be due to an as‐yet‐undetermined acquired gene modifier. The mechanism studies showed that the mutant fragment maintains partial function of DMP1 such as stimulating MAP kinase signaling in vitro. Last, the in vitro and in vivo data support a role of odontoblasts in the control of fibroblast growth factor 23 (FGF‐23) regulation during early postnatal development, although this regulation on P_i homeostasis is likely limited. © 2010 American Society for Bone and Mineral Research.     

Cell line IDG‐SW3 replicates osteoblast‐to‐late‐osteocyte differentiation in vitro and accelerates bone formation in vivo

Osteocytes are the most abundant cells in bone yet are the most challenging to study because they are embedded in a mineralized matrix. We generated a clonal cell line called IDG‐SW3 (for Immortomouse/Dmp1‐GFP‐SW3) from long‐bone chips from mice carrying a Dmp1 promoter driving GFP crossed with the Immortomouse, which expresses a thermolabile SV40 large T antigen regulated by interferon γ (IFN‐γ). Cells from these mice can be expanded at 33 °C in the presence of IFN‐γ and then allowed to resume their original phenotype at 37 °C in the absence of IFN‐γ. IDG‐SW3 cells are Dmp1‐GFP^? and T antigen⁺ under immortalizing conditions but Dmp1‐GFP⁺ and T antigen^? under osteogenic conditions. Like osteoblasts, they express alkaline phosphatase and produce and mineralize a type 1 collagen matrix containing calcospherulites. Like early osteocytes, they express E11/gp38, Dmp1, MEPE, and Phex. Like late osteocytes, they develop a dendritic morphology and express SOST/sclerostin and fibroblast growth factor 23 (FGF‐23), regulated by parathyroid hormone (PTH) and 1,25‐dihydroxyvitamin D₃. When cultured on 3D matrices, they express Dmp1‐GFP and sclerostin. When the 3D cultures are implanted in calvarial defects in vivo, they accelerate bone healing. This cell line should prove useful for studying osteoblast‐to‐osteocyte transition, mechanisms for biomineralization, osteocyte function, and regulation of SOST/sclerostin and FGF‐23. © 2011 American Society for Bone and Mineral Research     

Leptin stimulates fibroblast growth factor 23 expression in bone and suppresses renal 1α,25‐dihydroxyvitamin D3 synthesis in leptin‐deficient ob/ob Mice

Leptin is the LEP (ob) gene product secreted by adipocytes. We previously reported that leptin decreases renal expression of the 25‐hydroxyvitamin D₃ 1α‐hydroxylase (CYP27B1) gene through the leptin receptor (ObRb) by indirectly acting on the proximal tubules. This study focused on bone‐derived fibroblast growth factor 23 (FGF‐23) as a mediator of the influence of leptin on renal 1α‐hydroxylase mRNA expression in leptin‐deficient ob/ob mice. Exposure to leptin (200 ng/mL) for 24 hours stimulated FGF‐23 expression by primary cultured rat osteoblasts. Administration of leptin (4 mg/kg i.p. at 12‐hour intervals for 2 days) to ob/ob mice markedly increased the serum FGF‐23 concentration while significantly reducing the serum levels of calcium, phosphate, and 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Administration of FGF‐23 (5 µg i.p. at 12‐hour intervals for 2 days) to ob/ob mice suppressed renal 1α‐hydroxylase mRNA expression. The main site of FGF‐23 mRNA expression was the bone, and leptin markedly increased the FGF‐23 mRNA level in ob/ob mice. In addition, leptin significantly reduced 1α‐hydroxylase and sodium‐phosphate cotransporters (NaP_i‐IIa and NaP_i‐IIc) mRNA levels but did not affect Klotho mRNA expression in the kidneys of ob/ob mice. Furthermore, the serum FGF‐23 level and renal expression of 1α‐hydroxylase mRNA were not influenced by administration of leptin to leptin receptor–deficient (db/db) mice. These results indicate that leptin directly stimulates FGF‐23 synthesis by bone cells in ob/ob mice, suggesting that inhibition of renal 1,25(OH)₂D₃ synthesis in these mice is at least partly due to elevated bone production of FGF‐23. © 2010 American Society for Bone and Mineral Research     

Pharmacological inhibition of fibroblast growth factor (FGF) receptor signaling ameliorates FGF23‐mediated hypophosphatemic rickets

Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25‐dihydroxyvitamin D₃ (1,25[OH]₂D₃). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)₂D₃ levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X‐linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23‐mediated hypophosphatemic disorders using NVP‐BGJ398, a novel selective, pan‐specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1‐null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long‐term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP‐BGJ398 treatment as a novel approach for the therapy of FGF23‐mediated hypophosphatemic diseases. © 2013 American Society for Bone and Mineral Research.     

Fibroblast growth factor 23 regulates renal 1,25‐dihydroxyvitamin D and phosphate metabolism via the MAP kinase signaling pathway in Hyp mice

In X‐linked hypophosphatemia (XLH) and in its murine homologue, the Hyp mouse, increased circulating concentrations of fibroblast growth factor 23 (FGF‐23) are critical to the pathogenesis of disordered metabolism of phosphate (P_i) and 1,25‐dihydroxyvitamin D [1,25(OH)₂D]. In this study, we hypothesized that in Hyp mice, FGF‐23‐mediated suppression of renal 1,25(OH)₂D production and P_i reabsorption depends on activation of mitogen‐activated protein kinase (MAPK) signaling. Wild‐type and Hyp mice were administered either vehicle or the MEK inhibitor PD0325901 (12.5 mg/kg) orally daily for 4 days. At baseline, the renal abundance of early growth response 1 (egr1) mRNA was approximately 2‐fold greater in Hyp mice than in wild‐type mice. Treatment with PD0325901 greatly suppressed egr1 mRNA abundance in both wild‐type and Hyp mice. In Hyp mice, PD0325901 induced an 8‐fold increase in renal 1α‐hydroxylase mRNA expression and a 4‐fold increase in serum 1,25(OH)₂D concentrations compared with vehicle‐treated Hyp mice. Serum P_i levels in Hyp mice increased significantly after treatment with PD0325901, and the increase was associated with increased renal Npt2a mRNA abundance and brush‐border membrane Npt2a protein expression. These findings provide evidence that in Hyp mice, MAPK signaling is constitutively activated in the kidney and support the hypothesis that the FGF‐23‐mediated suppression of renal 1,25(OH)₂D production and P_i reabsorption depends on activation of MAPK signaling via MEK/ERK1/2. These findings demonstrate the physiologic importance of MAPK signaling in the actions of FGF‐23 in regulating renal 1,25(OH)₂D and P_i metabolism. © 2011 American Society for Bone and Mineral Research     

VDR in Osteoblast‐Lineage Cells Primarily Mediates Vitamin D Treatment‐Induced Increase in Bone Mass by Suppressing Bone Resorption

Long‐term treatment with active vitamin D [1α,25(OH)₂D₃] and its derivatives is effective for increasing bone mass in patients with primary and secondary osteoporosis. Derivatives of 1α,25(OH)₂D₃, including eldecalcitol (ELD), exert their actions through the vitamin D receptor (VDR). ELD is more resistant to metabolic degradation than 1α,25(OH)₂D₃. It is reported that ELD treatment causes a net increase in bone mass by suppressing bone resorption rather than by increasing bone formation in animals and humans. VDR in bone and extraskeletal tissues regulates bone mass and secretion of osteotropic hormones. Therefore, it is unclear what types of cells expressing VDR preferentially regulate the vitamin D–induced increase in bone mass. Here, we examined the effects of 4‐week treatment with ELD (50 ng/kg/day) on bone using osteoblast lineage‐specific VDR conditional knockout (Ob‐VDR‐cKO) and osteoclast‐specific VDR cKO (Ocl‐VDR‐cKO) male mice aged 10 weeks. Immunohistochemically, VDR in bone was detected preferentially in osteoblasts and osteocytes. Ob‐VDR‐cKO mice showed normal bone phenotypes, despite no appreciable immunostaining of VDR in bone. Ob‐VDR‐cKO mice failed to increase bone mass in response to ELD treatment. Ocl‐VDR‐cKO mice also exhibited normal bone phenotypes, but normally responded to ELD. ELD‐induced FGF23 production in bone was regulated by VDR in osteoblast‐lineage cells. These findings suggest that the vitamin D treatment‐induced increase in bone mass is mediated by suppressing bone resorption through VDR in osteoblast‐lineage cells. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

EphA4 Receptor Is a Novel Negative Regulator of Osteoclast Activity

Of the ephrin (Eph) receptors, mature osteoclasts express predominantly EphA4. This study sought to determine if EphA4 has a regulatory role in osteoclasts. Treatment of RAW/C4 cells with Epha4 small interfering RNAs (siRNAs) increased average size, Ctsk mRNA expression level, and bone resorption activity of the derived osteoclast‐like cells. Activation of the EphA4 signaling in osteoclast precursors with EfnA4‐fc chimeric protein reduced cell size and resorption activity of the derived osteoclasts. Homozygous Epha4 null mice had substantially less trabecular bone in femur and vertebra compared to wild‐type controls. The bone loss was due to a decrease in trabecular number and an increase in trabecular spacing, but not to an increase in osteoclast‐lined bone surface or an increase in the number of osteoclasts on bone surface. Dynamic histomorphometry and serum biomarker analyses indicate that bone formation in Epha4 null mice was reduced slightly but not significantly. Osteoclasts of Epha4 null mice were also larger, expressed higher levels of Mmp3 and Mmp9 mRNAs, and exhibited greater bone resorption activity than wild‐type osteoclasts in vitro. Deficient Epha4 expression had no effects on the total number of osteoclast formed in response to receptor activator of NF‐κB ligand nor on apoptosis of osteoclasts in vitro. It also did not affect the protein‐tyrosine phosphorylation status of its ligands, EfnB2, EfnA2, and EfnA4, in osteoclasts. Deficient Epha4 expression in Epha4 null osteoclasts activated the β₃‐integrin signaling through reduced phosphorylation of the tyr‐747 residue, which led to increased binding of the stimulatory talin and reduced binding of the inhibitory Dok1 to β₃‐integrin. This in turn activated Vav3 and the bone resorption activity of osteoclasts. In conclusion, we demonstrate for the first time that EphA4 is a potent negative regulator of osteoclastic activity, mediated in part through increased Dok1 binding to β₃‐integrin via an increase in EphA4‐dependent tyr‐747 phosphorylation. © 2014 American Society for Bone and Mineral Research.     

Targeted Disruption of NF1 in Osteocytes Increases FGF23 and Osteoid With Osteomalacia‐like Bone Phenotype

Neurofibromatosis type 1 (NF1, OMIM 162200), caused by NF1 gene mutations, exhibits multi‐system abnormalities, including skeletal deformities in humans. Osteocytes play critical roles in controlling bone modeling and remodeling. However, the role of neurofibromin, the protein product of the NF1 gene, in osteocytes is largely unknown. This study investigated the role of neurofibromin in osteocytes by disrupting Nf1 under the Dmp1‐promoter. The conditional knockout (Nf1 cKO) mice displayed serum profile of a metabolic bone disorder with an osteomalacia‐like bone phenotype. Serum FGF23 levels were 4 times increased in cKO mice compared with age‐matched controls. In addition, calcium‐phosphorus metabolism was significantly altered (calcium reduced; phosphorus reduced; parathyroid hormone [PTH] increased; 1,25(OH)₂D decreased). Bone histomorphometry showed dramatically increased osteoid parameters, including osteoid volume, surface, and thickness. Dynamic bone histomorphometry revealed reduced bone formation rate and mineral apposition rate in the cKO mice. TRAP staining showed a reduced osteoclast number. Micro‐CT demonstrated thinner and porous cortical bones in the cKO mice, in which osteocyte dendrites were disorganized as assessed by electron microscopy. Interestingly, the cKO mice exhibited spontaneous fractures in long bones, as found in NF1 patients. Mechanical testing of femora revealed significantly reduced maximum force and stiffness. Immunohistochemistry showed significantly increased FGF23 protein in the cKO bones. Moreover, primary osteocytes from cKO femora showed about eightfold increase in FGF23 mRNA levels compared with control cells. The upregulation of FGF23 was specifically and significantly inhibited by PI3K inhibitor Ly294002, indicating upregulation of FGF23 through PI3K in Nf1‐deficient osteocytes. Taken together, these results indicate that Nf1 deficiency in osteocytes dramatically increases FGF23 production and causes a mineralization defect (ie, hyperosteoidosis) via the alteration of calcium‐phosphorus metabolism. This study demonstrates critical roles of neurofibromin in osteocytes for osteoid mineralization. © 2017 American Society for Bone and Mineral Research.     

The calcium‐sensing receptor and 25‐hydroxyvitamin D–1α‐hydroxylase interact to modulate skeletal growth and bone turnover

We examined parathyroid and skeletal function in 3‐month‐old mice expressing the null mutation for 25‐hydroxyvitamin D–1α‐hydroxylase [1α(OH)ase^?/?] and in mice expressing the null mutation for both the 1α(OH)ase and the calcium‐sensing receptor [Casr^?/?1α(OH)ase^?/?] genes. On a normal diet, all mice were hypocalcemic, with markedly increased parathyroid hormone (PTH), increased trabecular bone volume, increased osteoblast activity, poorly mineralized bone, enlarged and distorted cartilaginous growth plates, and marked growth retardation, especially in the compound mutants. Osteoclast numbers were reduced in the Casr^?/?1α(OH)ase^?/? mice. On a high‐lactose, high‐calcium, high‐phosphorus “rescue” diet, serum calcium and PTH were normal in the 1α(OH)ase^?/? mice but increased in the Casr^?/?1α(OH)ase^?/? mice with reduced serum phosphorus. Growth plate architecture and mineralization were improved in both mutants, but linear growth of the double mutants remained abnormal. Mineralization of bone improved in all mice, but osteoblast activity and trabecular bone volume remained elevated in the Casr^?/?1α(OH)ase^?/? mice. These studies support a role for calcium‐stimulated maturation of the cartilaginous growth plate and mineralization of the growth plate and bone and calcium‐stimulated CaSR‐mediated effects on bone resorption. PTH‐mediated bone resorption may require calcium‐stimulated CaSR‐mediated enhancement of osteoclastic activity. © 2010 American Society for Bone and Mineral Research. © 2010 American Society for Bone and Mineral Research     

An essential role for the association of CD47 to SHPS‐1 in skeletal remodeling

Integrin‐associated protein (IAP/CD47) has been implicated in macrophage‐macrophage fusion. To understand the actions of CD47 on skeletal remodeling, we compared Cd47^?/? mice with Cd47^+/+ controls. Cd47^?/? mice weighed less and had decreased areal bone mineral density compared with controls. Cd47^?/? femurs were shorter in length with thinner cortices and exhibited lower trabecular bone volume owing to decreased trabecular number and thickness. Histomorphometry revealed reduced bone‐formation and mineral apposition rates, accompanied by decreased osteoblast numbers. No differences in osteoclast number were observed despite a nonsignificant but 40% decrease in eroded surface/bone surface in Cd47^?/? mice. In vitro, the number of functional osteoclasts formed by differentiating Cd47^?/? bone marrow cells was significantly decreased compared with wild‐type cultures and was associated with a decrease in bone‐resorption capacity. Furthermore, by disrupting the CD47–SHPS‐1 association, we found that osteoclastogenesis was markedly impaired. Assays for markers of osteoclast maturation suggested that the defect was at the point of fusion and not differentiation and was associated with a lack of SHPS‐1 phosphorylation, SHP‐1 phosphatase recruitment, and subsequent dephosphorylation of non–muscle cell myosin IIA. We also demonstrated a significant decrease in osteoblastogenesis in bone marrow stromal cells derived from Cd47^?/? mice. Our finding of cell‐autonomous defects in Cd47^?/? osteoblast and osteoclast differentiation coupled with the pronounced skeletal phenotype of Cd47^?/? mice support the conclusion that CD47 plays an important role in regulating skeletal acquisition and maintenance through its actions on both bone formation and bone resorption. © 2011 American Society for Bone and Mineral Research     

The pathophysiology of early‐stage chronic kidney disease–mineral bone disorder (CKD‐MBD) and response to phosphate binders in the rat

Chronic kidney disease–mineral bone disorder (CKD‐MBD) is a systemic disorder that describes the complex bone and mineral abnormalities that occur in CKD. To understand the pathophysiology of CKD‐MBD and determine whether the early use of phosphate binders would alter this physiology, we used a naturally occurring, slowly progressive model of CKD‐MBD, the Cy/+ rat. Male Cy/+ rats were compared with their normal littermates at 20 weeks of age after 1 week of no phosphate binder, calcium carbonate, or sevelamer carbonate. The Cy/+ rat had renal function that was 50% of that of normal littermates, elevated parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23), decreased 1,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] levels, but normal calcium and phosphorus levels. There was a significant positive correlation of blood FGF23 and phosphorus levels and blood FGF23 and urine phosphorus levels. There was an inverse correlation between FGF23 and calcium levels. mRNA from the kidney demonstrated 50% reduction in klotho and Npt2a expression but no difference in CYP27B1. In the intestine, CKD animals had reduced active phosphate absorption in the jejunum using modified Ussing chambers and a reduction in Npt2b expression throughout the small intestine compared with normal littermates. In bone, mRNA expression of FGF23 was reduced (driven by lowering with phosphate binders), and TRAP expression was increased in CKD. By histology, there was increased osteoclast activity and number, and there were reductions in some measures of femoral neck mechanical strength. One week of phosphate binders reduced intestinal phosphate flux, serum phosphorus levels, and urinary phosphate excretion. These results demonstrate marked abnormalities in kidney, intestine, and bone in early CKD‐MBD. While phosphate binders were effective in lowering urine phosphorus, they had little effect on end organs after 1 week of administration. © 2011 American Society for Bone and Mineral Research     

A Novel Osteogenic Cell Line That Differentiates Into GFP-Tagged Osteocytes and Forms Mineral With a Bone-Like Lacunocanalicular Structure

Osteocytes, the most abundant cells in bone, were once thought to be inactive, but are now known to have multifunctional roles in bone, including in mechanotransduction, regulation of osteoblast and osteoclast function and phosphate homeostasis. Because osteocytes are embedded in a mineralized matrix and are challenging to study, there is a need for new tools and cell models to understand their biology. We have generated two clonal osteogenic cell lines, OmGFP66 and OmGFP10, by immortalization of primary bone cells from mice expressing a membrane-targeted GFP driven by the Dmp1-promoter. One of these clones, OmGFP66, has unique properties compared with previous osteogenic and osteocyte cell models and forms 3-dimensional mineralized bone-like structures, containing highly dendritic GFP-positive osteocytes, embedded in clearly defined lacunae. Confocal and electron microscopy showed that structurally and morphologically, these bone-like structures resemble bone in vivo, even mimicking the lacunocanalicular ultrastructure and 3D spacing of in vivo osteocytes. In osteogenic conditions, OmGFP66 cells express alkaline phosphatase (ALP), produce a mineralized type I collagen matrix, and constitutively express the early osteocyte marker, E11/gp38. With differentiation they express osteocyte markers, Dmp1, Phex, Mepe, Fgf23, and the mature osteocyte marker, Sost. They also express RankL, Opg, and Hif1α, and show expected osteocyte responses to PTH, including downregulation of Sost, Dmp1, and Opg and upregulation of RankL and E11/gp38. Live cell imaging revealed the dynamic process by which OmGFP66 bone-like structures form, the motile properties of embedding osteocytes and the integration of osteocyte differentiation with mineralization. The OmGFP10 clone showed an osteocyte gene expression profile similar to OmGFP66, but formed less organized bone nodule-like mineral, similar to other osteogenic cell models. Not only do these cell lines provide useful new tools for mechanistic and dynamic studies of osteocyte differentiation, function, and biomineralization, but OmGFP66 cells have the unique property of modeling osteocytes in their natural bone microenvironment. © 2019 American Society for Bone and Mineral Research     

Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte‐Derived Matrix Vesicles in Phospho1–/– and Phospho1/Pit1 Double‐Knockout Mice

We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue‐nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis, but that their chondrocyte‐derived and osteoblast‐derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1‐mediated intravesicular generation of inorganic phosphate (P_i) and phosphate transporter‐mediated influx of P_i. To test this hypothesis, we generated mice with col2a1‐driven Cre‐mediated ablation of Slc20a1, hereafter referred to as P_it1, alone or in combination with a Phospho1 gene deletion. P_it1^col2/col2 mice did not show any major phenotypic abnormalities, whereas severe skeletal deformities were observed in the [Phospho1^–/–; P_it1^col2/col2] double knockout mice that were more pronounced than those observed in the Phospho1^–/– mice. Histological analysis of [Phospho1^–/–; P_it1^col2/col2] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1^–/–; P_it1^col2/col2] skeleton displayed significant decreases in BV/TV%, trabecular number, and bone mineral density, as well as decreased stiffness, decreased strength, and increased postyield deflection compared to Phospho1^–/– mice. Using atomic force microscopy we found that ～80% of [Phospho1^–/–; P_it1^col2/col2] MVs were devoid of mineral in comparison to ～50% for the Phospho1^–/– MVs and ～25% for the WT and P_it1^col2/col2 MVs. We also found a significant decrease in the number of MVs produced by both Phospho1^–/– and [Phospho1^–/–; P_it1^col2/col2] chondrocytes. These data support the involvement of phosphate transporter 1, hereafter referred to as P_iT‐1, in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis. © 2016 American Society for Bone and Mineral Research.     

Blockade of receptor‐activated Gi signaling in osteoblasts in vivo leads to site‐specific increases in cortical and cancellous bone formation

Osteoblasts play a critical role in the maintenance of bone mass through bone formation and regulation of bone resorption. Targeted expression of a constitutively active engineered G_i‐coupled G protein–coupled receptor (GPCR) to osteoblasts in vivo leads to severe osteopenia. However, little is known about the role of endogenous receptor‐mediated G_i signaling in regulating osteoblast function. In this study, we investigated the skeletal effects of blocking G_i‐coupled signaling in osteoblasts in vivo. This was accomplished by transgenic expression of the catalytic subunit of pertussis toxin (PTX) under control of the collagen Iα 2.3‐kb promoter. These mice, designated Col1(2.3)⁺/PTX⁺, showed increased cortical thickness at the femoral midshaft at 12 weeks of age. This correlated with increased periosteal bone formation associated with expanded mineralizing surface observed in 8‐week‐old mice of both genders. The cancellous bone phenotype of the Col1(2.3)⁺/PTX⁺ mice was sexually dimorphic, with increases in fractional bone volume at the distal femur seen only in females. Similarly, while cancellous bone‐formation rates were unchanged in males, they could not be quantified for female Col1(2.3)⁺/PTX⁺ mice owing to the disorganized nature of the labeling pattern, which was consistent with rapid formation of woven bone. Alterations in osteoclast activity did not appear to participate in the phenotype. These data demonstrate that G_i‐coupled signaling by GPCRs endogenous to osteoblasts plays a complex role in the regulation of bone formation in a manner that is dependent on both gender and the anatomic site within bone. © 2011 American Society for Bone and Mineral Research.     

Osteoblast‐Derived Extracellular Vesicles Are Biological Tools for the Delivery of Active Molecules to Bone

Extracellular vesicles (EVs) are newly appreciated regulators of tissue homeostasis and a means of intercellular communication. Reports have investigated the role of EVs and their cargoes in cellular regulation and have tried to fine‐tune their biotechnological use, but to date very little is known on their function in bone biology. To investigate the relevance of EV‐mediated communication between bone cells, we isolated EVs from primary mouse osteoblasts and assessed membrane integrity, size, and structure by transmission electron microscopy (TEM) and fluorescence‐activated cell sorting (FACS). EVs actively shuttled loaded fluorochromes to osteoblasts, monocytes, and endothelial cells. Moreover, osteoblast EVs contained mRNAs shared with donor cells. Osteoblasts are known to regulate osteoclastogenesis, osteoclast survival, and osteoclast function by the pro‐osteoclastic cytokine, receptor activator of nuclear factor κ‐B ligand (Rankl). Osteoblast EVs were enriched in Rankl, which increased after PTH treatment. These EVs were biologically active, supporting osteoclast survival. EVs isolated from rankl^–/– osteoblasts lost this pro‐osteoclastic function, indicating its Rankl‐dependence. They integrated ex vivo into murine calvariae, and EV‐shuttled fluorochromes were quickly taken up by the bone upon in vivo EV systemic administration. Rankl^–/– mice lack the osteoclast lineage and are negative for its specific marker tartrate‐resistant acid phosphatase (TRAcP). Treatment of rankl^–/– mice with wild‐type osteoblast EVs induced the appearance of TRAcP‐positive cells in an EV density‐dependent manner. Finally, osteoblast EVs internalized and shuttled anti‐osteoclast drugs (zoledronate and dasatinib), inhibiting osteoclast activity in vitro and in vivo. We conclude that osteoblast EVs are involved in intercellular communication between bone cells, contribute to the Rankl pro‐osteoclastic effect, and shuttle anti‐osteoclast drugs, representing a potential means of targeted therapeutic delivery. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

Positive Regulation of Adult Bone Formation by Osteoblast‐Specific Transcription Factor Osterix

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.     

Genetic Ablation of Osteopontin in Osteomalacic Hyp Mice Partially Rescues the Deficient Mineralization Without Correcting Hypophosphatemia

PHEX is predominantly expressed by bone and tooth-forming cells, and its inactivating mutations in X-linked hypophosphatemia (XLH) lead to renal phosphate wasting and severe hypomineralization of bones and teeth. Also present in XLH are hallmark hypomineralized periosteocytic lesions (POLs, halos) that persist despite stable correction of serum phosphate (P_i) that improves bulk bone mineralization. In XLH, mineralization-inhibiting osteopontin (OPN, a substrate for PHEX) accumulates in the extracellular matrix of bone. To investigate how OPN functions in Hyp mice (a model for XLH), double-null (Hyp;Opn^−/−) mice were generated. Undecalcified histomorphometry performed on lumbar vertebrae revealed that Hyp;Opn^−/− mice had significantly reduced osteoid area/bone area (OV/BV) and osteoid thickness of trabecular bone as compared to Hyp mice, despite being as hypophosphatemic as Hyp littermate controls. However, tibias examined by synchrotron radiation micro-CT showed that mineral lacunar volumes remained abnormally enlarged in these double-null mice. When Hyp;Opn^−/− mice were fed a high-P_i diet, serum P_i concentration increased, and OV/BV and osteoid thickness normalized, yet mineral lacunar area remained abnormally enlarged. Enpp1 and Ankh gene expression were increased in double-null mice fed a high-P_i diet, potentially indicating a role for elevated inhibitory pyrophosphate (PP_i) in the absence of OPN. To further investigate the persistence of POLs in Hyp mice despite stable correction of serum P_i, immunohistochemistry for OPN on Hyp mice fed a high-P_i diet showed elevated OPN in the osteocyte pericellular lacunar matrix as compared to Hyp mice fed a control diet. This suggests that POLs persisting in Hyp mice despite correction of serum P_i may be attributable to the well-known upregulation of mineralization-inhibiting OPN by P_i, and its accumulation in the osteocyte pericellular matrix. This study shows that OPN contributes to osteomalacia in Hyp mice, and that genetic ablation of OPN in Hyp mice improves the mineralization phenotype independent of systemic P_i-regulating factors. © 2020 American Society for Bone and Mineral Research.