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Find video protocols related to scientific articles indexed in Pubmed.
Blocking the expression of both bone sialoprotein (BSP) and osteopontin (OPN) impairs the anabolic action of PTH in mouse calvaria bone.
J. Cell. Physiol.
PUBLISHED: 08-27-2014
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Osteopontin (OPN) and bone sialoprotein (BSP) are coexpressed in osteoblasts and osteoclasts, and display overlapping properties. We used daily injection of parathyroid hormone 1-84 (iPTH) over the calvaria of BSP knockout (-/-) mice to investigate further their functional specificity and redundancy. iPTH stimulated bone formation in both +/+ and -/- mice, increasing to the same degree periosteum, osteoid and total bone thickness. Expression of OPN, osterix, osteocalcin (OCN) and DMP1 was also increased by iPTH in both genotypes. In contrast to +/+, calvaria cell cultures from -/- mice revealed few osteoblast colonies, no mineralization and little expression of OCN, MEPE or DMP1. In contrast, OPN levels were 5x higher in -/- vs +/+ cultures. iPTH increased alkaline phosphatase (ALP) activity in cell cultures of both genotypes, with higher OCN and the induction of mineralization in -/- cultures. siRNA blocking of OPN expression did not alter the anabolic action of the hormone in BSP+/+ calvaria, while it blunted iPTH effects in -/- mice, reduced to a modest increase in periosteum thickness. In -/- (not +/+) cell cultures, siOPN blocked the stimulation by iPTH of ALP activity and OCN expression, as well as the induction of mineralization. Thus, full expression of either OPN or BSP is necessary for the anabolic effect of PTH at least in the ectopic calvaria injection model. This suggests that OPN may compensate for the lack of BSP in the response to this hormonal challenge, and provides evidence of functional overlap between these cognate proteins. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
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The "connexin" between bone cells and skeletal functions.
J. Cell. Biochem.
PUBLISHED: 04-30-2014
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The processes of bone modeling and remodeling are crucial in the skeleton's functions as a supportive and protective structure, a mineral reservoir, and an endocrine organ. The coordination between bone cell activities (bone formation and bone resorption), necessary to maintain the integrity of the skeleton during these processes, is mediated at least in part by cell-cell and cell-environment interactions across gap junctions and hemichannels. The increasing number of genetically engineered Connexin 43 (Cx43) knockout and missense mouse models have provided insight into the complex and critical roles of Cx43-containing gap junctions and hemichannels in the development and turnover of the skeleton, in differentiation, activity and survival of the bone cell lineages, and in the cellular and molecular mechanisms by which Cx43 functions and assists in mediating cellular responses to stimuli in bone. Cx43 may be an important potential therapeutic target, making it crucial that we continue to gain understanding of the multiple and complex roles of Cx43 in bone.
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First mouse model for combined osteogenesis imperfecta and Ehlers-Danlos syndrome.
J. Bone Miner. Res.
PUBLISHED: 01-11-2014
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By using a genome-wide N-ethyl-N-nitrosourea (ENU)-induced dominant mutagenesis screen in mice, a founder with low bone mineral density (BMD) was identified. Mapping and sequencing revealed a T to C transition in a splice donor of the collagen alpha1 type I (Col1a1) gene, resulting in the skipping of exon 9 and a predicted 18-amino acid deletion within the N-terminal region of the triple helical domain of Col1a1. Col1a1(Jrt) /+ mice were smaller in size, had lower BMD associated with decreased bone volume/tissue volume (BV/TV) and reduced trabecular number, and furthermore exhibited mechanically weak, brittle, fracture-prone bones, a hallmark of osteogenesis imperfecta (OI). Several markers of osteoblast differentiation were upregulated in mutant bone, and histomorphometry showed that the proportion of trabecular bone surfaces covered by activated osteoblasts (Ob.S/BS and N.Ob/BS) was elevated, but bone surfaces undergoing resorption (Oc.S/BS and N.Oc/BS) were not. The number of bone marrow stromal osteoprogenitors (CFU-ALP) was unaffected, but mineralization was decreased in cultures from young Col1a1(Jrt) /+ versus +/+ mice. Total collagen and type I collagen content of matrices deposited by Col1a1(Jrt) /+ dermal fibroblasts in culture was ?40% and 30%, respectively, that of +/+ cells, suggesting that mutant collagen chains exerted a dominant negative effect on type I collagen biosynthesis. Mutant collagen fibrils were also markedly smaller in diameter than +/+ fibrils in bone, tendon, and extracellular matrices deposited by dermal fibroblasts in vitro. Col1a1(Jrt) /+ mice also exhibited traits associated with Ehlers-Danlos syndrome (EDS): Their skin had reduced tensile properties, tail tendon appeared more frayed, and a third of the young adult mice had noticeable curvature of the spine. Col1a1(Jrt) /+ is the first reported model of combined OI/EDS and will be useful for exploring aspects of OI and EDS pathophysiology and treatment.
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ERR? Is Not Required for Skeletal Development but Is a RUNX2-Dependent Negative Regulator of Postnatal Bone Formation in Male Mice.
PLoS ONE
PUBLISHED: 01-01-2014
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To assess the effects of the orphan nuclear Estrogen receptor-related receptor gamma (ERR?) deficiency on skeletal development and bone turnover, we utilized an ERR? global knockout mouse line. While we observed no gross morphological anomalies or difference in skeletal length in newborn mice, by 8 weeks of age ERR? +/- males but not females exhibited increased trabecular bone, which was further increased by 14 weeks. The increase in trabecular bone was due to an increase in active osteoblasts on the bone surface, without detectable alterations in osteoclast number or activity. Consistent with the histomorphometric results, we observed an increase in gene expression of the bone formation markers alkaline phosphatase (Alp) and bone sialoprotein (Bsp) in bone and increase in serum ALP, but no change in the osteoclast regulators receptor activator of NF-?B ligand (RANKL) and osteoprotegerin (OPG) or the resorption marker carboxy-terminal collagen crosslinks (CTX). More colony forming units-alkaline phosphatase and -osteoblast (CFU-ALP, CFU-O respectively) but not CFU-fibroblast (CFU-F) formed in ERR? +/- versus ERR? +/+ stromal cell cultures, suggesting that ERR? negatively regulates osteoblast differentiation and matrix mineralization but not mesenchymal precursor number. By co-immunoprecipitation experiments, we found that ERR? and RUNX2 interact in an ERR? DNA binding domain (DBD)-dependent manner. Treatment of post-confluent differentiating bone marrow stromal cell cultures with Runx2 antisense oligonucleotides resulted in a reduction of CFU-ALP/CFU-O in ERR? +/- but not ERR? +/+ mice compared to their corresponding sense controls. Our data indicate that ERR? is not required for skeletal development but is a sex-dependent negative regulator of postnatal bone formation, acting in a RUNX2- and apparently differentiation stage-dependent manner.
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Skeletal development of mice lacking bone sialoprotein (BSP)--impairment of long bone growth and progressive establishment of high trabecular bone mass.
PLoS ONE
PUBLISHED: 01-01-2014
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Adult Ibsp-knockout mice (BSP-/-) display shorter stature, lower bone turnover and higher trabecular bone mass than wild type, the latter resulting from impaired bone resorption. Unexpectedly, BSP knockout also affects reproductive behavior, as female mice do not construct a proper "nest" for their offsprings. Multiple crossing experiments nonetheless indicated that the shorter stature and lower weight of BSP-/- mice, since birth and throughout life, as well as their shorter femur and tibia bones are independent of the genotype of the mothers, and thus reflect genetic inheritance. In BSP-/- newborns, µCT analysis revealed a delay in membranous primary ossification, with wider cranial sutures, as well as thinner femoral cortical bone and lower tissue mineral density, reflected in lower expression of bone formation markers. However, trabecular bone volume and osteoclast parameters of long bones do not differ between genotypes. Three weeks after birth, osteoclast number and surface drop in the mutants, concomitant with trabecular bone accumulation. The growth plates present a thinner hypertrophic zone in newborns with lower whole bone expression of IGF-1 and higher IHH in 6 days old BSP-/- mice. At 3 weeks the proliferating zone is thinner and the hypertrophic zone thicker in BSP-/- than in BSP+/+ mice of either sex, maybe reflecting a combination of lower chondrocyte proliferation and impaired cartilage resorption. Six days old BSP-/- mice display lower osteoblast marker expression but higher MEPE and higher osteopontin(Opn)/Runx2 ratio. Serum Opn is higher in mutants at day 6 and in adults. Thus, lack of BSP alters long bone growth and membranous/cortical primary bone formation and mineralization. Endochondral development is however normal in mutant mice and the accumulation of trabecular bone observed in adults develops progressively in the weeks following birth. Compensatory high Opn may allow normal endochondral development in BSP-/- mice, while impairing primary mineralization.
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Osteogenesis and expression of the bone marrow niche in endothelial cell-depleted HipOPs.
J. Cell. Biochem.
PUBLISHED: 08-28-2013
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The identification and purification of murine multipotent mesenchymal stem cells (MSCs) have been difficult due to their low frequency, the presence of contaminating cell types and lack of unambiguous markers. Using a magnetic micro-beads negative selection technique to remove hematopoietic cells from mouse bone marrow stromal cells (BMSCs), our lab recently isolated a highly purified osteoprogenitor (HipOP) population that was also enriched for other mesenchymal precursors, including MSCs [Itoh and Aubin, 2009]. We now report that HipOPs are also highly enriched in vascular endothelial cells (VECs), which we hypothesized were an accessory cell type regulating osteogenesis. However, when VECs were immunodepleted from HipOPs with anti-CD31 antibodies, the resulting CD31(-) HipOP population had equal osteogenic capacity to the HipOPs in vitro and in vivo. Analysis of gene expression of Ncad, Pth1r, Ang1, Cxcl12, Jag1, Pdgfr-?, ?-sma, Desmin, and Ng2 suggested that both HipOPs and CD31(-) HipOPs are hemopoietic stem cell (HSC) niche populations. However, the data support the view that osteoblast differentiation and depletion of VECs modulate the HSC niche.
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The R740S mutation in the V-ATPase a3 subunit results in osteoclast apoptosis and defective early-stage autophagy.
J. Cell. Biochem.
PUBLISHED: 07-16-2013
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Vacuolar-type H(+)-ATPases (V-ATPases) are located in lysosomes and at the ruffled border in osteoclasts. We showed previously that the R740S mutation is dominant negative for V-ATPase activity, uncouples proton transport from ATP hydrolysis and causes osteopetrosis in heterozygous mice (+/R740S). Here we show mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis and die by postnatal day 14. Although R740S/R740S osteoclasts express wild-type levels of a3, it is mislocalized. Acridine orange staining of R740S/R740S osteoclasts grown on a Corning resorptive surface reveals no resorption and no acidification of intracellular compartments. Whereas osteoblast and osteocyte apoptosis is normal, R740S/R740S osteoclasts exhibit increased apoptosis compared with wild-type osteoclasts. Localization of the enzyme tartrate-resistant acid phosphatase (TRAP) is also aberrant. Transmission electron microscopy reveals that R740S/R740S osteoclasts do not polarize, lack ruffled borders, and contain fewer autophagosomes. Consistent with an early stage defect in autophagy, expression of LC3II is reduced and expression of p62 is increased in R740S/R740S compared to wild-type osteoclasts. These results indicate the importance of intracellular acidification for the early stages of autophagy as well as for osteoclast survival, maturation, and polarization with appropriate cytoplasmic distribution of key osteoclast enzymes such as TRAP.
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An energetic orphan in an endocrine tissue: a revised perspective of the function of estrogen receptor-related receptor alpha in bone and cartilage.
J. Bone Miner. Res.
PUBLISHED: 06-28-2013
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Estrogen receptor-related receptor alpha (ERR?) is an orphan nuclear receptor with sequence homology to the estrogen receptors, ER?/?, but it does not bind estrogen. ERR? not only plays a functional role in osteoblasts but also in osteoclasts and chondrocytes. In addition, the ERRs, including ERR?, can be activated by coactivators such as peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC1? and ?) and are implicated in adipogenesis, fatty acid oxidation, and oxidative stress defense, suggesting that ERR?-through its activity in bone resorption and adipogenesis--may regulate the insulin and leptin pathways and contribute to aging-related changes in bone and cartilage. In this review, we discuss data on ERR? and its cellular and molecular modes of action, which have broad implications for considering the potential role of this orphan receptor in cartilage and bone endocrine function, on whole-organism physiology, and in the bone aging process.
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EB1 levels are elevated in ascorbic Acid (AA)-stimulated osteoblasts and mediate cell-cell adhesion-induced osteoblast differentiation.
J. Biol. Chem.
PUBLISHED: 06-05-2013
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Osteoblasts are differentiated mesenchymal cells that function as the major bone-producing cells of the body. Differentiation cues including ascorbic acid (AA) stimulation provoke intracellular changes in osteoblasts leading to the synthesis of the organic portion of the bone, which includes collagen type I ?1, proteoglycans, and matrix proteins, such as osteocalcin. During our microarray analysis of AA-stimulated osteoblasts, we observed a significant up-regulation of the microtubule (MT) plus-end binding protein, EB1, compared with undifferentiated osteoblasts. EB1 knockdown significantly impaired AA-induced osteoblast differentiation, as detected by reduced expression of osteoblast differentiation marker genes. Intracellular examination of AA-stimulated osteoblasts treated with EB1 siRNA revealed a reduction in MT stability with a concomitant loss of ?-catenin distribution at the cell cortex and within the nucleus. Diminished ?-catenin levels in EB1 siRNA-treated osteoblasts paralleled an increase in phospho-?-catenin and active glycogen synthase kinase 3?, a kinase known to target ?-catenin to the proteasome. EB1 siRNA treatment also reduced the expression of the ?-catenin gene targets, cyclin D1 and Runx2. Live immunofluorescent imaging of differentiated osteoblasts revealed a cortical association of EB1-mcherry with ?-catenin-GFP. Immunoprecipitation analysis confirmed an interaction between EB1 and ?-catenin. We also determined that cell-cell contacts and cortically associated EB1/?-catenin interactions are necessary for osteoblast differentiation. Finally, using functional blocking antibodies, we identified E-cadherin as a major contributor to the cell-cell contact-induced osteoblast differentiation.
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The R740S mutation in the V-ATPase a3 subunit increases lysosomal pH, impairs NFATc1 translocation, and decreases in vitro osteoclastogenesis.
J. Bone Miner. Res.
PUBLISHED: 05-23-2013
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Vacuolar H(+) -ATPase (V-ATPase), a multisubunit enzyme located at the ruffled border and in lysosomes of osteoclasts, is necessary for bone resorption. We previously showed that heterozygous mice with an R740S mutation in the a3 subunit of V-ATPase (+/R740S) have mild osteopetrosis resulting from an ?90% reduction in proton translocation across osteoclast membranes. Here we show that lysosomal pH is also higher in +/R740S compared with wild-type (+/+) osteoclasts. Both osteoclast number and size were decreased in cultures of +/R740S compared with +/+ bone marrow cells, with concomitant decreased expression of key osteoclast markers (TRAP, cathepsin K, OSCAR, DC-STAMP, and NFATc1), suggesting that low lysosomal pH plays an important role in osteoclastogenesis. To elucidate the molecular mechanism of this inhibition, NFATc1 activation was assessed. NFATc1 nuclear translocation was significantly reduced in +/R740S compared with +/+ cells; however, this was not because of impaired enzymatic activity of calcineurin, the phosphatase responsible for NFATc1 dephosphorylation. Protein and RNA expression levels of regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of NFATc1 activation and a protein degraded in lysosomes, were not significantly different between +/R740S and +/+ osteoclasts, but the RCAN1/NFATc1 ratio was significantly higher in +/R740S versus +/+ cells. The lysosomal inhibitor chloroquine significantly increased RCAN1 accumulation in +/+ cells, consistent with the hypothesis that higher lysosomal pH impairs RCAN1 degradation, leading to a higher RCAN1/NFATc1 ratio and consequently NFATc1 inhibition. Our data indicate that increased lysosomal pH in osteoclasts leads to decreased NFATc1 signaling and nuclear translocation, resulting in a cell autonomous impairment of osteoclastogenesis in vitro.
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The G60S connexin 43 mutation activates the osteoblast lineage and results in a resorption-stimulating bone matrix and abrogation of old-age-related bone loss.
J. Bone Miner. Res.
PUBLISHED: 04-08-2013
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We previously isolated a low bone mass mouse, Gja1(Jrt) /?+?, with a mutation in the gap junction protein, alpha 1 gene (Gja1), encoding for a dominant negative G60S Connexin 43 (Cx43) mutant protein. Similar to other Cx43 mutant mouse models described, including a global Cx43 deletion, four skeletal cell conditional-deletion mutants, and a Cx43 missense mutant (G138R/?+), a reduction in Cx43 gap junction formation and/or function resulted in mice with early onset osteopenia. In contrast to other Cx43 mutants, however, we found that Gja1(Jrt) /+ mice have both higher bone marrow stromal osteoprogenitor numbers and increased appendicular skeleton osteoblast activity, leading to cell autonomous upregulation of both matrix bone sialoprotein (BSP) and membrane-bound receptor activator of nuclear factor-?B ligand (mbRANKL). In younger Gja1(Jrt) /+ mice, these contributed to increased osteoclast number and activity resulting in early onset osteopenia. In older animals, however, this effect was abrogated by increased osteoprotegerin (OPG) levels and serum alkaline phosphatase (ALP) so that differences in mutant and wild-type (WT) bone parameters and mechanical properties lessened or disappeared with age. Our study is the first to describe a Cx43 mutation in which osteopenia is caused by increased rather than decreased osteoblast function and where activation of osteoclasts occurs not only through increased mbRANKL but an increase in a matrix protein that affects bone resorption, which together abrogate age-related bone loss in older animals.
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Characterization of esophageal defects in the Crouzon mouse model.
Birth Defects Res. Part A Clin. Mol. Teratol.
PUBLISHED: 04-06-2013
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Mutations in Fibroblastic Growth Factor Receptors (FGFR) have been associated with human craniosynostotic birth defects like Crouzon syndrome. Several anecdotes and case reports have indicated higher incidence of gastrointestinal tract disorders in FGFR-associated craniosynostotic birth defects. Our objective was to characterize esophageal defects in a mouse model of human Crouzon syndrome, with a mutation in codon 290 of FGFR2.
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Both chondroinduction and proliferation account for growth of cartilage nodules in mouse limb bud cultures.
Stem Cell Rev
PUBLISHED: 03-01-2013
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High density micromass culture of limb bud mesenchymal stem cells isolated from mouse embryos represents a well-established model to study chondro- and osteogenesis. In spite of wide usage of the limb bud model, the mechanisms underlying cartilage nodule growth remain unclear. To determine whether cartilage nodules grow solely by induction of surrounding cells or proliferation of cells within the nodules, we performed BrdU/Collagen II (Col II) double-labelling and 3D reconstruction of growing cartilage nodules. We demonstrated that Col II-positive replicating chondrocytes are present throughout the nodules with the majority of replicating cells localized on the top (cell-medium interface) and periphery/sides of nodules. Kinetic analysis of cellular proliferation within the nodules demonstrated the time-dependent reduction in number of Col II-positive replicating cells. The sequential expression of Col I, Col II, Col X, parathyroid hormone related peptide receptor 1 (Pthr1), bone sialoprotein (Bsp) and osteocalcin (Ocn) mRNAs was similar to that characterizing chondrocyte differentiation and maturation in vivo. We conclude that the limb bud model recapitulates events seen during endochondral bone formation: cellular aggregation, proliferation, differentiation and maturation to hypertrophy. We also conclude that not only induction of peri-nodular mesenchymal cells but also proliferation of chondrocytes within cartilage nodules contribute to cartilage nodule growth.
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Cartilage-Specific Overexpression of ERR? Results in Chondrodysplasia and Reduced Chondrocyte Proliferation.
PLoS ONE
PUBLISHED: 01-01-2013
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While the role of estrogen receptor-related receptor alpha (ERR?) in chondrogenesis has been investigated, the involvement of ERR gamma (ERR?) has not been determined. To assess the effect of increased ERR? activity on cartilage development in vivo, we generated two transgenic (Tg) lines overexpressing ERR?2 via a chondrocyte-specific promoter; the two lines exhibited ?3 and ?5 fold increased ERR?2 protein expression respectively in E14.5 Tg versus wild type (WT) limbs. On postnatal day seven (P7), we observed a 4-10% reduction in the size of the craniofacial, axial and appendicular skeletons in Tg versus WT mice. The reduction in bone length was already present at birth and did not appear to involve bones that are derived via intramembranous bone formation as the bones of the calvaria, clavicle, and the mandible developed normally. Histological analysis of P7 growth plates revealed a reduction in the length of the Tg versus WT growth plate, the majority of which was attributable to a reduced proliferative zone. The reduced proliferative zone paralleled a decrease in the number of Ki67-positive proliferating cells, with no significant change in apoptosis, and was accompanied by large cell-free swaths of cartilage matrix, which extended through multiple zones of the growth plate. Using a bioinformatics approach, we identified known chondrogenesis-associated genes with at least one predicted ERR binding site in their proximal promoters, as well as cell cycle regulators known to be regulated by ERR?. Of the genes identified, Col2al, Agg, Pth1r, and Cdkn1b (p27) were significantly upregulated, suggesting that ERR?2 negatively regulates chondrocyte proliferation and positively regulates matrix synthesis to coordinate growth plate height and organization.
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Dual function of ERR? in breast cancer and bone metastasis formation: implication of VEGF and osteoprotegerin.
Cancer Res.
PUBLISHED: 07-06-2011
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Bone metastasis is a complication occurring in up to 70% of advanced breast cancer patients. The estrogen receptor-related receptor alpha (ERR?) has been implicated in breast cancer and bone development, prompting us to examine whether ERR? may function in promoting the osteolytic growth of breast cancer cells in bone. In a mouse xenograft model of metastatic human breast cancer, overexpression of wild-type ERR? reduced metastasis, whereas overexpression of a dominant negative mutant promoted metastasis. Osteoclasts were directly affected and ERR? upregulated the osteoclastogenesis inhibitor, osteoprotegerin (OPG), providing a direct mechanistic basis for understanding how ERR? reduced breast cancer cell growth in bone. In contrast, ERR? overexpression increased breast cancer cell growth in the mammary gland. ERR?-overexpressing primary tumors were highly vascularized, consistent with an observed upregulation of angiogenic growth factor, the VEGF. In support of these findings, we documented that elevated expression of ERR? mRNA in breast carcinomas was associated with high expression of OPG and VEGF and with disease progression. In conclusion, our results show that ERR? plays a dual role in breast cancer progression in promoting the local growth of tumor cells, but decreasing metastatic growth of osteolytic lesions in bone.
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Estrogen receptor-related receptor ? regulation by interleukin-1? in prostaglandin E(2)- and cAMP-dependent pathways in osteoarthritic chondrocytes.
Arthritis Rheum.
PUBLISHED: 04-21-2011
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We reported previously that the orphan nuclear receptor, estrogen receptor-related receptor ? (ERR?), is expressed in articular chondrocytes and is dysregulated in a mouse model of inflammatory arthritis. The aim of this study, therefore, was to determine whether ERR? is also dysregulated in patients with osteoarthritis (OA).
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The V-ATPase a3 subunit mutation R740S is dominant negative and results in osteopetrosis in mice.
J. Bone Miner. Res.
PUBLISHED: 02-10-2011
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A mouse founder with high bone mineral density and an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) screen. It was found to carry a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit of the vacuolar type H(+)-ATPase (V-ATPase), resulting in replacement of a highly conserved amino acid (R740S). The +/R740S mice have normal appearance, size, and weight but exhibit high bone density. Osteoblast parameters are unaffected in bones of +/R740S mice, whereas osteoclast number and marker expression are increased, concomitant with a decrease in the number of apoptotic osteoclasts. Consistent with reduced osteoclast apoptosis, expression of Rankl and Bcl2 is elevated, whereas Casp3 is reduced. Transmission electron microscopy revealed that unlike other known mutations in the a3 subunit of V-ATPase, polarization and ruffled border formation appear normal in +/R740S osteoclasts. However, V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport, whereas ATP hydrolysis is not significantly affected. We show for the first time that a point mutation within the a3 subunit, R740S, which is dominant negative for proton pumping and bone resorption, also uncouples proton pumping from ATP hydrolysis but has no effect on ruffled border formation or polarization of osteoclasts. These results suggest that the V(0) complex has proton-pumping-independent functions in mammalian cells.
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Impaired mesenchymal stem cell differentiation and osteoclastogenesis in mice deficient for Igf2-P2 transcripts.
Development
PUBLISHED: 12-09-2010
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During embryonic development, Igf2 gene transcription is highly regulated through the use of several promoters whose specific roles are not defined. Here, we show that loss-of-function of one of these promoters, Igf2-P2, results in growth defects that are temporally and quantitatively different from those seen in Igf2-null mutants. In particular, Igf2-P2 mutants exhibit skeletal abnormalities characterized by thin and short bones with reduced mineralization and medullar cavity and with altered bone remodeling. These abnormalities are associated with decreased numbers of embryonic mesenchymal chondroprogenitors, adult mesenchymal stem cells and osteoprogenitors. Differentiation of osteoprogenitors into osteoblasts is impaired in the Igf2-P2 mutant mice in a cell-autonomous manner, and osteopontin is a target of the IGF2 signaling pathway during this differentiation. Igf2-P2 mutant mice also display impaired formation of giant osteoclasts owing to a defective micro-environment. These results support a model wherein transcriptional activity of the Igf2-P2 promoter regulates the fate of mesenchymal progenitors during bone development and remodeling in the adult, and regulates osteogenesis in a cell-autonomous and non-autonomous manner.
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Mice lacking bone sialoprotein (BSP) lose bone after ovariectomy and display skeletal site-specific response to intermittent PTH treatment.
Endocrinology
PUBLISHED: 09-15-2010
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Bone sialoprotein (BSP) belongs to the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family, whose members play multiple and distinct roles in the development, turnover, and mineralization of bone and dentin. The functions of BSP in bone remodeling are not yet well established. We previously showed that BSP knockout (BSP(-/-)) mice exhibit a higher trabecular bone volume, concomitant with lower bone remodeling, than wild-type (BSP(+/+)) mice. To determine whether bone turnover can be stimulated in the absence of BSP, we subjected BSP(+/+) and BSP(-/-) mice to catabolic [ovariectomy (OVX)] or anabolic (intermittent PTH administration) hormonal challenges. BSP(-/-) mice progressively develop hypocalcemia and high serum PTH between 2 and 4 months of age. Fifteen and 30 d after OVX, microtomography analysis showed a significant decrease of trabecular bone volume in tibiae of both genotypes. Histomorphometric parameters of bone formation and resorption were significantly increased by OVX. PTH treatment resulted in an increase of trabecular thickness and both bone formation and resorption parameters at all skeletal sites in both genotypes and a decrease of trabecular bone volume in tibiae of BSP(+/+) but not BSP(-/-) mice. PTH increased cortical thickness and bone area in BSP(+/+) but not BSP(-/-) mice and stimulated the bone formation rate specifically in the endosteum of BSP(+/+) mice and the periosteum of BSP(-/-) mice. PTH enhanced the expression of RANKL, MEPE, and DMP1 in both genotypes but increased OPG and OPN expression only in BSP(-/-) mice. In conclusion, despite the low basal turnover, both catabolic and anabolic challenges increase bone formation and resorption in BSP(-/-) mice, suggesting that compensatory pathways are operative in the skeleton of BSP-deficient mice. Although up-regulation of one or several other SIBLINGs is a possible mechanism, further studies are needed to analyze the interplay and cross-regulation involved in compensating for the absence of BSP.
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Involvement of the orphan nuclear estrogen receptor-related receptor ? in osteoclast adhesion and transmigration.
J. Mol. Endocrinol.
PUBLISHED: 09-14-2010
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The orphan nuclear receptor, estrogen receptor-related receptor ? (ERR?) is expressed in osteoblasts and osteoclasts (OCs) and has been proposed to be a modulator of estrogen signaling. To determine the role of ERR? in OC biology, we knocked down ERR? activity by transient transfection of an siRNA directed against ERR? in the RAW264.7 monocyte-macrophage cell line that differentiates into OCs in the presence of receptor activator of nuclear factor ?B-ligands and macrophage colony-stimulating factor. In parallel, stable RAW cell lines expressing a dominant-negative form of ERR? and green fluorescent protein (RAW-GFP-ERR??AF2) were used. Expression of OC markers was assessed by real-time PCR, and adhesion and transmigration tests were performed. Actin cytoskeletal organization was visualized using confocal microscopy. We found that RAW264.7 cells expressing siRNA directed against ERR? and RAW-GFP-ERR??AF2 OCs displayed abnormal spreading, and decreased osteopontin and ?3 integrin subunit expression compared with the corresponding control cells. Decreased adhesion and the absence of podosome belts concomitant with abnormal localization of c-src were also observed in RAW-GFP-ERR??AF2-derived OCs. In addition, RAW-GFP-ERR??AF2-derived OCs failed to transmigrate through osteoblast cell layers. Our data show that the impairment of ERR? function does not alter OC precursor proliferation and differentiation but does alter the adhesion/spreading and migration capacities of mature OCs.
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Bone sialoprotein deficiency impairs osteoclastogenesis and mineral resorption in vitro.
J. Bone Miner. Res.
PUBLISHED: 09-03-2010
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Bone sialoprotein (BSP) and osteopontin (OPN) belong to the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, whose members interact with bone cells and bone mineral. Previously, we showed that BSP knockout (BSP(-/-) ) mice have a higher bone mass than wild type (BSP(+/+) ) littermates, with very low bone-formation activity and reduced osteoclast surfaces and numbers. Here we report that approximately twofold fewer tartrate-resistant acid phosphatase (TRACP)-positive cells and approximately fourfold fewer osteoclasts form in BSP(-/-) compared with BSP(+/+) spleen cell cultures. BSP(-/-) preosteoclast cultures display impaired proliferation and enhanced apoptosis. Addition of RGD-containing proteins restores osteoclast number in BSP(-/-) cultures to BSP(+/+) levels. The expression of osteoclast-associated genes is markedly altered in BSP(-/-) osteoclasts, with reduced expression of cell adhesion and migration genes (?V integrin chain and OPN) and increased expression of resorptive enzymes (TRACP and cathepsin K). The migration of preosteoclasts and mature osteoclasts is impaired in the absence of BSP, but resorption pit assays on dentine slices show no significant difference in pit numbers between BSP(+/+) and BSP(-/-) osteoclasts. However, resorption of mineral-coated slides by BSP(-/-) osteoclasts is markedly impaired but is fully restored by coating the mineral substrate with hrBSP and partly restored by hrOPN coating. In conclusion, lack of BSP affects both osteoclast formation and activity, which is in accordance with in vivo findings. Our results also suggest at least some functional redundancy between BSP and OPN that remains to be clarified.
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1alpha,25-dihydroxyvitamin D3 acts predominately in mature osteoblasts under conditions of high extracellular phosphate to increase fibroblast growth factor 23 production in vitro.
J. Endocrinol.
PUBLISHED: 06-08-2010
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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. Pi alone and more so in combination with 1,25(OH)(2)D(3) increased FGF23 production and VDR mRNA expression. Parathyroid hormone, stanniocalcin 1, prostaglandin E(2), FGF2, and foscarnet did not increase FGF23 mRNA expression. Thus, these results suggest that 1,25(OH)(2)D(3) may exert its largest effect on FGF23 expression/production when exposed to high levels of extracellular Pi in osteoblasts/osteocytes.
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A subset of osteoblasts expressing high endogenous levels of PPARgamma switches fate to adipocytes in the rat calvaria cell culture model.
PLoS ONE
PUBLISHED: 04-12-2010
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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.
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Bone sialoprotein, but not osteopontin, deficiency impairs the mineralization of regenerating bone during cortical defect healing.
Bone
PUBLISHED: 08-07-2009
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Bone healing is a complex multi-step process, which depends on the position and size of the lesion, and on the mechanical stability of the wounded area. To address more specifically the mechanisms involved in cortical bone healing, we created drill-hole defects in the cortex of mouse femur, a lesion that triggers intramembranous repair, and compared the roles of bone sialoprotein (BSP) and osteopontin (OPN), two proteins of the extracellular matrix, in the repair process. Bone regeneration was analyzed by ex vivo microcomputerized X-ray tomography and histomorphometry of bones of BSP-deficient, OPN-deficient and wild-type mice. In all mouse strains, the cortical gap was bridged with woven bone within 2 weeks and no mineralized tissue was observed in the marrow. Within 3 weeks, lamellar cortical bone filled the gap. The amount and degree of mineralization of the woven bone was not affected by OPN deficiency, but cortical bone healing was delayed in BSP-deficient mice due to delayed mineralization. Gene expression studies showed a higher amount of BSP transcripts in the repair bone of OPN-deficient mice, suggesting a possible compensation of OPN function by BSP in OPN-null mice. Our data suggest that BSP, but not OPN, plays a role in primary bone formation and mineralization of newly formed bone during the process of cortical bone healing.
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A novel purification method for multipotential skeletal stem cells.
J. Cell. Biochem.
PUBLISHED: 07-11-2009
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At least some cells within bone marrow stromal populations are multipotential (i.e., differentiate in vitro into osteoblasts, chondrocytes, and adipocytes) and thus designated skeletal stem cells (SSCs) or mesenchymal stem cells (MSCs) amongst other names. Recently, a subpopulation of stromal cells, notably osteoblasts or their progenitors, has been identified as a definitive regulatory component of the hematopoietic stem cell (HSC) niche. Thus, the development of methods for purifying not only SSCs but cells comprising the HSC niche is of interest. Here, we report a method for purifying a novel bone marrow-derived population with a high frequency of osteoprogenitors and high expression levels of osteoblast differentiation markers (highly purified osteoprogenitors (HipOPs)) as well as markers of the bone niche for HSCs. In vivo transplantation experiments demonstrated that donor HipOPs differentiated into not only osteoblasts, osteocytes and cells around sinusoids but also hematopoietic cells. Thus, HipOPs represent a novel population for simultaneous reconstruction of bone and bone marrow microenvironments.
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Absence of bone sialoprotein (BSP) impairs cortical defect repair in mouse long bone.
Bone
PUBLISHED: 05-13-2009
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Matrix proteins of the SIBLING family interact with bone cells and with bone mineral and are thus in a key position to regulate bone development, remodeling and repair. Within this family, bone sialoprotein (BSP) is highly expressed by osteoblasts, hypertrophic chondrocytes and osteoclasts. We recently reported that mice lacking BSP (BSP-/-) have very low trabecular bone turnover. In the present study, we set up an experimental model of bone repair by drilling a 1 mm diameter hole in the cortical bone of femurs in both BSP-/- and +/+ mice. A non-invasive MRI imaging and bone quantification procedure was designed to follow bone regeneration, and these data were extended by microCT imaging and histomorphometry on undecalcified sections for analysis at cellular level. These combined approaches revealed that the repair process as reflected in defect-refilling in the cortical area was significantly delayed in BSP-/- mice compared to +/+ mice. Concomitantly, histomorphometry showed that formation, mineralization and remodeling of repair (primary) bone in the medulla were delayed in BSP-/- mice, with lower osteoid and osteoclast surfaces at day 15. In conclusion, the absence of BSP delays bone repair at least in part by impairing both new bone formation and osteoclast activity.
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EP2 and EP4 receptors differentially mediate MAPK pathways underlying anabolic actions of prostaglandin E2 on bone formation in rat calvaria cell cultures.
Bone
PUBLISHED: 02-08-2009
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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. EP2A and EP4A were effective when cells were treated chronically or pulse-treated during nascent nodule formation. EP2A and EP4A equally stimulated the endogenous PGE(2) production, while EP2A caused a greater increase in cAMP production and c-Fos gene expression compared to EP4A. EP2A and EP4A activated predominantly p38 MAPK and ERK respectively, while c-Jun N-terminal kinase (JNK) was equally activated by both agonists. SB203580 (p38 MAPK inhibitor) blocked the PGE(2) effect on mineralized nodule formation, while U0126 (ERK inhibitor) and dicumarol (JNK inhibitor) were less effective. PGE(2)-dependent phosphorylation of the MAPKs was affected not only by protein kinase (PK)A and PKC inhibitors but also by adenylate cyclase and PKC activators. Co-treatment of RC cells with EP2A or EP4A and bone morphogenetic protein (BMP)2, whose effects on bone nodule formation is known to be, in part, mediated through the PKA and p38 MAPK pathways, resulted in an additive effect on mineralized nodule formation. Further, PGE(2), EP2A and EP4A did not increase BMP2/4 mRNA levels in RC cells, and EP2-induced phosphorylation of p38 MAPK was not eliminated by Noggin. These results suggest that, in the RC cell model, the anabolic actions of PGE(2) on mineralized nodule formation are mediated at least in part by activation of the EP2 and EP4 receptor subtype-specific MAPK pathways, independently of BMP signaling, in cells associated with nascent bone nodules.
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Conservation of core gene expression in vertebrate tissues.
J. Biol.
PUBLISHED: 01-23-2009
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Vertebrates share the same general body plan and organs, possess related sets of genes, and rely on similar physiological mechanisms, yet show great diversity in morphology, habitat and behavior. Alteration of gene regulation is thought to be a major mechanism in phenotypic variation and evolution, but relatively little is known about the broad patterns of conservation in gene expression in non-mammalian vertebrates.
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Bone Marrow-Derived HipOP Cell Population Is Markedly Enriched in Osteoprogenitors.
Int J Mol Sci
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We recently succeeded in purifying a novel multipotential progenitor or stem cell population from bone marrow stromal cells (BMSCs). This population exhibited a very high frequency of colony forming units-osteoblast (CFU-O; 100 times higher than in BMSCs) and high expression levels of osteoblast differentiation markers. Furthermore, large masses of mineralized tissue were observed in in vivo transplants with this new population, designated highly purified osteoprogenitors (HipOPs). We now report the detailed presence and localization of HipOPs and recipient cells in transplants, and demonstrate that there is a strong relationship between the mineralized tissue volume formed and the transplanted number of HipOPs.
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Absence of bone sialoprotein (BSP) impairs primary bone formation and resorption: the marrow ablation model under PTH challenge.
Bone
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Bone sialoprotein (BSP) is highly expressed in early bone deposition and may play a part in primary bone mineralization. We previously showed that while BSP?/? mice have a mild secondary bone phenotype and are responsive to mechanical (unloading) and hormonal (ovariectomy, parathyroid hormone (PTH)) challenges, repair of a cortical bone defect, which involves primary bone deposition is significantly delayed in these mice. In the present study, we investigated the role of BSP in a pure model of primary bone modeling. Bone marrow was ablated by trans-epiphysis aspiration in the femora of BSP+/+ and BSP?/? mice, and 7 days post surgery ?CT analysis showed vigorous new bone formation in the shaft of BSP+/+ animals but much less in BSP?/? mice. After 14 days, the volume of medullary bone was significantly decreased as expected in BSP+/+ mice, while it remained stable in the BSP?/?. Osteoid thickness and surface were higher in BSP?/? at day 7, suggesting delayed mineralization, while osteoclast surface and number were significantly lower at day 14, a stage of high medullary bone resorption. At day 7, mRNA expression of early osteoblast marker genes (RUNX2, osterix, alkaline phosphatase, osteopontin) did not differ between the two genotypes, while markers of terminal differentiation (MEPE, DMP1, osteocalcin) as well as receptor activator of NF-kappaB ligand (RANKL) and tartrate-resistant acid phosphatase (TRAP) were significantly lower in BSP?/? than in BSP+/+ mice. PTH treatment maintained the volume of medullary bone up to 12 days after ablation in BSP+/+ mice, but failed to do so in BSP?/? mice. PTH significantly increased bone formation rate in both genotype, while it reduced osteoclast number and surface in BSP+/+, but not in BSP?/? medullary bone. In summary, medullary bone formation after marrow ablation is blunted in BSP?/? mice, with delayed resorption and impaired response to PTH. These findings confirm the hypothesis of a crucial role for BSP in primary ossification, which has long been suspected for mineralization, but here extends to bone deposition and turnover.
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A novel Phex mutation in a new mouse model of hypophosphatemic rickets.
J. Cell. Biochem.
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X-linked hypophosphatemic rickets (XLH) is a dominantly inherited disease characterized by renal phosphate wasting, aberrant vitamin D metabolism, and defective bone mineralization. It is known that XLH in humans and in certain mouse models is caused by inactivating mutations in PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). By a genome-wide N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a dominant mouse mutation that exhibits the classic clinical manifestations of XLH, including growth retardation, skeletal abnormalities (rickets/osteomalacia), hypophosphatemia, and increased serum alkaline phosphatase (ALP) levels. Mapping and sequencing revealed that these mice carry a point mutation in exon 14 of the Phex gene that introduces a stop codon at amino acid 496 of the coding sequence (Phex(Jrt) also published as Phex(K496X) [Ichikawa et al., 2012]). Fgf23 mRNA expression as well as that of osteocalcin, bone sialoprotein, and matrix extracellular phosphoglycoprotein was upregulated in male mutant long bone, but that of sclerostin was unaffected. Although Phex mRNA is expressed in bone from mutant hemizygous male mice (Phex(Jrt)/Y mice), no Phex protein was detected in immunoblots of femoral bone protein. Stromal cultures from mutant bone marrow were indistinguishable from those of wild-type mice with respect to differentiation and mineralization. The ability of Phex(Jrt)/Y osteoblasts to mineralize and the altered expression levels of matrix proteins compared with the well-studied Hyp mice makes it a unique model with which to further explore the clinical manifestations of XLH and its link to FGF23 as well as to evaluate potential new therapeutic strategies.
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The EP4-ERK-dependent pathway stimulates osteo-adipogenic progenitor proliferation resulting in increased adipogenesis in fetal rat calvaria cell cultures.
Prostaglandins Other Lipid Mediat.
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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. Of four EP agonists tested, the EP4 agonist ONO-AE1-437 (EP4A) was the most effective in promoting adipogenesis. Concomitantly, EP4A increased the number of cells with BrdU labeling and gene expression of CCAAT/enhancer binding protein (C/EBP)? and c-fos but not peroxisome proliferator-activated receptor ?2 and C/EBP?. Amongst MAPK inhibitors, U0126, an inhibitor of MEK1/2, abrogated the EP4A-dependent effects. Our results suggest that the PGE(2)-EP4-ERK pathway increases the number of osteo-adipogenic bipotential progenitor cells, with a resultant increase in adipogenesis in RC cell cultures.
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