Large scale analysis of osteocyte lacunae in klotho hypomorphic mice using high-resolution micro-computed tomography.

Background: Osteocytes are the most abundant cell type in adult bone, and the morphological characteristics of osteocytes and their lacunae appear to influence bone mass and fragility. Although conventional computed tomography (CT) has contributed greatly to advances in bone morphometry, capturing details of the entire hierarchical assembly, e.g.

Developmental impairments of craniofacial bone and cartilage in transgenic mice expressing FGF10.

Mutations in a common extracellular domain of fibroblast growth factor receptor (FGFR)-2 isoforms (type IIIb and IIIc) cause craniosynostosis syndrome and chondrodysplasia syndrome. FGF10, a major ligand for FGFR2-IIIb and FGFR1-IIIb, is a key participant in the epithelial-mesenchymal interactions required for morphogenetic events. FGF10 also regulates preadipocyte differentiation and early chondrogenesis in vitro, suggesting that FGF10-FGFR signaling may be involved in craniofacial skeletogenesis in vivo.

Active sites of human MEPE-ASARM regulating bone matrix mineralization.

The proteolytic fragment ASARM (acidic serine- and aspartate-rich motif) of MEPE (matrix extracellular phosphoglycoprotein) (MEPE-ASARM) may act as an endogenous anti-mineralization factor involved in X-linked hypophosphatemic rickets/osteomalacia (XLH). We synthesized MEPE-ASARM peptides and relevant peptide fragments with or without phosphorylated Ser residues (pSer) to determine the active site(s) of MEPE-ASARM in a rat calvaria cell culture model. None of the synthetic peptides elicited changes in cell death, proliferation or differentiation, but the peptide (pASARM) with three pSer residues inhibited mineralization without causing changes in gene expression of osteoblast markers tested.

Emerging roles of microRNAs as extracellular vesicle cargo secreted from osteoblasts.

Background: Extracellular vesicles (EVs) have come into the spotlight as messengers, delivering cargo for cell-cell communication. Concomitantly, increasing attention has been focused on microRNAs (miRNAs) as EV cargo. Besides their well-known role in extracellular matrix mineralization, whether matrix vesicles (MVs) - which are in a broad sense a class of EV - also deliver miRNAs to regulate the function of recipient cells remains unclear.

The matrix vesicle cargo miR-125b accumulates in the bone matrix, inhibiting bone resorption in mice.

Communication between osteoblasts and osteoclasts plays a key role in bone metabolism. We describe here an unexpected role for matrix vesicles (MVs), which bud from bone-forming osteoblasts and have a well-established role in initiation of bone mineralization, in osteoclastogenesis. We show that the MV cargo miR-125b accumulates in the bone matrix, with increased accumulation in transgenic (Tg) mice overexpressing miR-125b in osteoblasts.

Soluble Klotho causes hypomineralization in Klotho-deficient mice.

The type I transmembrane protein αKlotho (Klotho) serves as a coreceptor for the phosphaturic hormone fibroblast growth factor 23 (FGF23) in kidney, while a truncated form of Klotho (soluble Klotho, sKL) is thought to exhibit multiple activities, including acting as a hormone, but whose mode(s) of action in different organ systems remains to be fully elucidated. FGF23 is expressed primarily in osteoblasts/osteocytes and aberrantly high levels in the circulation acting via signaling through an FGF receptor (FGFR)-Klotho coreceptor complex cause renal phosphate wasting and osteomalacia. We assessed the effects of exogenously added sKL on osteoblasts and bone using Klotho-deficient () mice and cell and organ cultures.

Imaging and mapping of mouse bone using MALDI-imaging mass spectrometry.

Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) is an advanced method used globally to analyze the distribution of biomolecules on tissue cryosections without any probes. In bones, however, hydroxyapatite crystals make it difficult to determine the distribution of biomolecules using MALDI-IMS. Additionally, there is limited information regarding the use of this method to analyze bone tissues.

Comparative proteome analysis of wild-type and klotho-knockout mouse kidneys using a combination of MALDI-IMS and LC-MS/MS.

Purpose: Mutation of the klotho gene in mice elicits a syndrome resembling accelerated human aging. However, there is limited evidence for the role of Klotho in the kidney. We conducted a comparative proteome analysis of wild-type (WT) and klotho-knockout (kl ) mouse kidneys to identify proteins involved in Klotho deficiency.

The dynamics of DNA methylation and hydroxymethylation during amelogenesis.

Amelogenesis is a multistep process that relies on specific temporal and spatial signaling networks between the dental epithelium and mesenchymal tissues. Epigenetic modifications of key developmental genes in this process may be closely linked to a network of molecular events. However, the role of epigenetic regulation in amelogenesis remains unclear.

Functional diversity of fibroblast growth factors in bone formation.

The functional significance of fibroblast growth factor (FGF) signaling in bone formation has been demonstrated through genetic loss-of-function and gain-of-function approaches. FGFs, comprising 22 family members, are classified into three subfamilies: canonical, hormone-like, and intracellular. The former two subfamilies activate their signaling pathways through FGF receptors (FGFRs).

The bioactive acidic serine- and aspartate-rich motif peptide.

The organic component of the bone matrix comprises 40% dry weight of bone. The organic component is mostly composed of type I collagen and small amounts of non-collagenous proteins (NCPs) (10-15% of the total bone protein content). The small integrin-binding ligand N-linked glycoprotein (SIBLING) family, a NCP, is considered to play a key role in bone mineralization.

The EP4-ERK-dependent pathway stimulates osteo-adipogenic progenitor proliferation resulting in increased adipogenesis in fetal rat calvaria cell cultures.

We previously reported that fetal rat calvaria (RC) cells are osteo-adipogenic bipotential and that PGE(2) receptors EP2 and EP4 are involved in bone nodule formation via both common and distinct MAPK pathways in RC cell cultures. Because PGE(2) participates in multiple biological processes including adipogenesis, it is of interest to determine the additional role(s) of PGE(2) in RC cells. PGE(2) increased the number of adipocyte colonies when RC cells were treated during proliferation but not other development stages.

Incisor enamel formation is impaired in transgenic rats overexpressing the type III NaPi transporter Slc20a1.

Inorganic phosphate (Pi) is required in many biological processes, including signaling cascades, skeletal development, tooth mineralization, and nucleic acid synthesis. Recently, we showed that Pi transport in osteoblasts, mediated by Slc20a1, a member of the type III sodium-dependent phosphate transporter family, is indispensable for osteoid mineralization in rapidly growing rat bone. In addition, we found that bone mineral density decreased slightly with dysfunction of Pi homeostasis in aged transgenic rats overexpressing mouse Slc20a1 (Slc20a1-Tg).

A subset of osteoblasts expressing high endogenous levels of PPARgamma switches fate to adipocytes in the rat calvaria cell culture model.

Background: Understanding fate choice and fate switching between the osteoblast lineage (ObL) and adipocyte lineage (AdL) is important to understand both the developmental inter-relationships between osteoblasts and adipocytes and the impact of changes in fate allocation between the two lineages in normal aging and certain diseases. The goal of this study was to determine when during lineage progression ObL cells are susceptible to an AdL fate switch by activation of endogenous peroxisome proliferator-activated receptor (PPAR)gamma.

Methodology/principal Findings: Multiple rat calvaria cells within the ObL developmental hierarchy were isolated by either fractionation on the basis of expression of alkaline phosphatase or retrospective identification of single cell-derived colonies, and treated with BRL-49653 (BRL), a synthetic ligand for PPARgamma.

1alpha,25-dihydroxyvitamin D3 acts predominately in mature osteoblasts under conditions of high extracellular phosphate to increase fibroblast growth factor 23 production in vitro.

Osteoblasts/osteocytes are the principle sources of fibroblast growth factor 23 (FGF23), a phosphaturic hormone, but the regulation of FGF23 expression during osteoblast development remains uncertain. Because 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and inorganic phosphate (Pi) may act as potent activators of FGF23 expression, we estimated how these molecules regulate FGF23 expression during rat osteoblast development in vitro. 1,25(OH)(2)D(3)-dependent FGF23 production was restricted largely to mature cells in correlation with increased vitamin D receptor (VDR) mRNA levels, in particular, when Pi was present.

EP2 and EP4 receptors differentially mediate MAPK pathways underlying anabolic actions of prostaglandin E2 on bone formation in rat calvaria cell cultures.

Of the four prostaglandin (PG) E receptor subtypes (EP1-EP4), EP2 and EP4 have been proposed to mediate the anabolic action of PGE(2) on bone formation but comparative evaluation studies of EPs on bone formation do not necessarily share a common mechanism, implying that their additional features including downstream MAPK pathways may be beneficial to resolve this issue. We systematically assessed the roles of EPs in the rat calvaria (RC) cell culture model by using four selective EP agonists (EPAs). Consistent with relative expression levels of the respective receptors, multiple phenotypic traits of bone formation in vitro, including proliferation of nodule-associated cells, osteoblast marker expression and mineralized nodule formation were upregulated not only by PGE(2) but equally by EP2A and EP4A, but not by EP1A and EP3A.

Overexpression of fibroblast growth factor 23 suppresses osteoblast differentiation and matrix mineralization in vitro.

Introduction: Fibroblast growth factor (FGF)23 is produced primarily in bone and acts on kidney as a systemic phosphaturic factor; high levels result in rickets and osteomalacia. However, it remains unclear whether FGF23 acts locally and directly on bone formation.

Materials And Methods: We overexpressed human FGF23 in a stage-specific manner during osteoblast development in fetal rat calvaria (RC) cell cultures by using the adenoviral overexpression system and analyzed its effects on osteoprogenitor proliferation, osteoid nodule formation, and mineralization.

Mineralized tissue cells are a principal source of FGF23.

While fibroblast growth factor (FGF) 23 is known as a phosphaturic factor in inherited and/or acquired hypophosphatemic disorders, it also serves an endocrine role in normal phosphate homeostasis. FGF23 acts negatively on the NaPi2a cotransporter and 25-hydroxy D(3)-1 alpha-hydroxylase with a resultant decrease in renal phosphate (Pi) reabsorption, while osteoblasts appear to be a primary source of FGF23 whose expression is counter-upregulated by 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Here we have shown the distribution of FGF23 in normal rat bone and tooth, and its expression profile in fetal rat calvaria (RC) cell cultures.