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

ABSTRACT: 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.

SUBMITTER: Miyagawa K

PROVIDER: S-EPMC3977859 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Miyagawa Kazuaki K Yamazaki Miwa M Kawai Masanobu M Nishino Jin J Koshimizu Takao T Ohata Yasuhisa Y Tachikawa Kanako K Mikuni-Takagaki Yuko Y Kogo Mikihiko M Ozono Keiichi K Michigami Toshimi T

PloS one 20140407 4

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- ...[more]

PMID: 24710520

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Project description:Inactivating mutations of the "phosphate regulating gene with homologies to endopeptidases on the X chromosome" (PHEX/Phex) underlie disease in patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a murine homologue of the human disorder. Although increased serum fibroblast growth factor 23 (FGF-23) underlies the HYP phenotype, the mechanism(s) by which PHEX mutations inhibit FGF-23 degradation and/or enhance production remains unknown. Here we show that treatment of wild-type mice with the proprotein convertase (PC) inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (Dec), increases serum FGF-23 and produces the HYP phenotype. Because PC2 is uniquely colocalized with PHEX in osteoblasts/bone, we examined if PC2 regulates PHEX-dependent FGF-23 cleavage and production. Transfection of murine osteoblasts with PC2 and its chaperone protein 7B2 cleaved FGF-23, whereas Signe1 (7B2) RNA interference (RNAi) transfection, which limited 7B2 protein production, decreased FGF-23 degradation and increased Fgf-23 mRNA and protein. The mechanism by which decreased 7B2•PC2 activity influences Fgf-23 mRNA was linked to reduced conversion of the precursor to bone morphogenetic protein 1 (proBMP1) to active BMP1, which resulted in limited cleavage of dentin matrix acidic phosphoprotein 1 (DMP1), and consequent increased Fgf-23 mRNA. The significance of decreased 7B2•PC2 activity in XLH was confirmed by studies of hyp-mouse bone, which revealed significantly decreased Sgne1 (7B2) mRNA and 7B2 protein, and limited cleavage of proPC2 to active PC2. The expected downstream effects of these changes included decreased FGF-23 cleavage and increased FGF-23 synthesis, secondary to decreased BMP1-mediated degradation of DMP1. Subsequent Hexa-D-Arginine treatment of hyp-mice enhanced bone 7B2•PC2 activity, normalized FGF-23 degradation and production, and rescued the HYP phenotype. These data suggest that decreased PHEX-dependent 7B2•PC2 activity is central to the pathogenesis of XLH.

Project description:Osteocytes represent the most abundant cellular component of mammalian bones with important functions in bone mass maintenance and remodeling. To elucidate the differential gene expression between osteoblasts and osteocytes we completed a comprehensive analysis of their gene profiles. Selective identification of these two mature populations was achieved by utilization of visual markers of bone lineage cells. We have utilized dual GFP reporter mice in which osteocytes are expressing GFP (topaz) directed by the DMP1 promoter, while osteoblasts are identified by expression of GFP (cyan) driven by 2.3 kb of the Col1a1 promoter. Histological analysis of 7-day-old neonatal calvaria confirmed the expression pattern of DMP1GFP in osteocytes and Col2.3 in osteoblasts and osteocytes. To isolate distinct populations of cells we utilized fluorescent activated cell sorting (FACS). Cell suspensions were subjected to RNA extraction, in vitro transcription and labeling of cDNA and gene expression was analyzed using the Illumina WG-6v1 BeadChip. Following normalization of raw data from four biological replicates, 3444 genes were called present in all three sorted cell populations: GFP negative, Col2.3cyan(+) (osteoblasts), and DMP1topaz(+) (preosteocytes and osteocytes). We present the genes that showed in excess of a 2-fold change for gene expression between DMP1topaz(+) and Col2.3cyan(+) cells. The selected genes were classified and grouped according to their associated gene ontology terms. Genes clustered to osteogenesis and skeletal development such as Bmp4, Bmp8a, Dmp1, Enpp1, Phex and Ank were highly expressed in DMP1topaz(+)cells. Most of the genes encoding extracellular matrix components and secreted proteins had lower expression in DMP1topaz(+) cells, while most of the genes encoding plasma membrane proteins were increased. Interestingly a large number of genes associated with muscle development and function and with neuronal phenotype were increased in DMP1topaz(+) cells, indicating some new aspects of osteocyte biology. Although a large number of genes differentially expressed in DMP1topaz(+) and Col2.3cyan(+) cells in our study have already been assigned to bone development and physiology, for most of them we still lack any substantial data. Therefore, isolation of osteocyte and osteoblast cell populations and their subsequent microarray analysis allowed us to identify a number or genes and pathways with potential roles in regulation of bone mass.

Dataset Information

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

Publications

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

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