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Increased osteopontin contributes to inhibition of bone mineralization in FGF23-deficient mice.

ABSTRACT: 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.

SUBMITTER: Yuan Q

PROVIDER: S-EPMC3937302 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Increased osteopontin contributes to inhibition of bone mineralization in FGF23-deficient mice.

Yuan Quan Q Jiang Yan Y Zhao Xuefeng X Sato Tadatoshi T Densmore Michael M Schüler Christiane C Erben Reinhold G RG McKee Marc D MD Lanske Beate B

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 20140301 3

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 ...[more]

PMID: 24038141

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Project description:X-linked hypophosphatemia (XLH) is the most frequent form of inherited rickets in humans caused by mutations in the phosphate-regulating gene with homologies to endopeptidases on the X-chromosome (PHEX). Hyp mice, a murine homologue of XLH, are characterized by hypophosphatemia, inappropriately low serum vitamin D levels, increased serum fibroblast growth factor-23 (Fgf23), and osteomalacia. Although Fgf23 is known to be responsible for hypophosphatemia and reduced vitamin D hormone levels in Hyp mice, its putative role as an auto-/paracrine osteomalacia-causing factor has not been explored. We recently reported that Fgf23 is a suppressor of tissue nonspecific alkaline phosphatase (Tnap) transcription via FGF receptor-3 (FGFR3) signaling, leading to inhibition of mineralization through accumulation of the TNAP substrate pyrophosphate. Here, we report that the pyrophosphate concentration is increased in Hyp bones, and that Tnap expression is decreased in Hyp-derived osteocyte-like cells but not in Hyp-derived osteoblasts ex vivo and in vitro. In situ mRNA expression profiling in bone cryosections revealed a ~70-fold up-regulation of Fgfr3 mRNA in osteocytes versus osteoblasts of Hyp mice. In addition, we show that blocking of increased Fgf23-FGFR3 signaling with anti-Fgf23 antibodies or an FGFR3 inhibitor partially restored the suppression of Tnap expression, phosphate production, and mineralization, and decreased pyrophosphate concentration in Hyp-derived osteocyte-like cells in vitro. In vivo, bone-specific deletion of Fgf23 in Hyp mice rescued the suppressed TNAP activity in osteocytes of Hyp mice. Moreover, treatment of wild-type osteoblasts or mice with recombinant FGF23 suppressed Tnap mRNA expression and increased pyrophosphate concentrations in the culture medium and in bone, respectively. In conclusion, we found that the cell autonomous increase in Fgf23 secretion in Hyp osteocytes drives the accumulation of pyrophosphate through auto-/paracrine suppression of TNAP. Hence, we have identified a novel mechanism contributing to the mineralization defect in Hyp mice.

Project description:We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts regulating Fgf23 production and mineralization in Hyp mice. We discovered that activation of Fgf receptor- and Wnt-pathways contribute to increased Ffg23 gene transcription in Hyp bone. We found evidence in Hyp bone for increased expression of Fgf1, Fgf7, and Egr2 in the Fgf-signaling pathway and decrements in Sost and Cpz and increments in Sfrp1 and 4 in the Wnt-signaling pathway. Moreover, activation of Fgf and Wnt-signaling stimulated, whereas Tgf β inhibited Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the Fgf23 regulatory extracellular matrix protein Dmp1. These findings suggest that elevation of Fgf23 expression in osteocytes is regulated by interactions between cell surface expression of Phex, extracellular matrix proteins and paracrine effects of Fgf and Wnt. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced post-translational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regards to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone. We isolated total RNAs from long bones of both WT and Hyp mice at 12 days of age. Since the RNA yields from the long bones are very low, we combined 2 bone samples with same genotype (WT or Hyp) for one RNA extraction. We will compare the difference of the gene expressions between Hyp and WT. We will use 4 samples in each animal condition.

Project description:The periodontal complex is essential for tooth attachment and function and includes the mineralized tissues, cementum and alveolar bone, separated by the unmineralized periodontal ligament (PDL). To gain insights into factors regulating cementum-PDL and bone-PDL borders and protecting against ectopic calcification within the PDL, we employed a proteomic approach to analyze PDL tissue from progressive ankylosis knock-out (Ank-/-) mice, featuring reduced PPi, rapid cementogenesis, and excessive acellular cementum. Using this approach, we identified the matrix protein osteopontin (Spp1/OPN) as an elevated factor of interest in Ank-/- mouse molar PDL. We studied the role of OPN in dental and periodontal development and function. During tooth development in wild-type (WT) mice, Spp1 mRNA was transiently expressed by cementoblasts and strongly by alveolar bone osteoblasts. Developmental analysis from 14 to 240days postnatal (dpn) indicated normal histological structures in Spp1-/- comparable to WT control mice. Microcomputed tomography (micro-CT) analysis at 30 and 90dpn revealed significantly increased volumes and tissue mineral densities of Spp1-/- mouse dentin and alveolar bone, while pulp and PDL volumes were decreased and tissue densities were increased. However, acellular cementum growth was unaltered in Spp1-/- mice. Quantitative PCR of periodontal-derived mRNA failed to identify potential local compensators influencing cementum in Spp1-/- vs. WT mice at 26dpn. We genetically deleted Spp1 on the Ank-/- mouse background to determine whether increased Spp1/OPN was regulating periodontal tissues when the PDL space is challenged by hypercementosis in Ank-/- mice. Ank-/-; Spp1-/- double deficient mice did not exhibit greater hypercementosis than that in Ank-/- mice. Based on these data, we conclude that OPN has a non-redundant role regulating formation and mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. These findings may inform therapies targeted at controlling soft tissue calcification.

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Increased osteopontin contributes to inhibition of bone mineralization in FGF23-deficient mice.

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Increased osteopontin contributes to inhibition of bone mineralization in FGF23-deficient mice.

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