Publications (16) View all
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Article: Studying gene expression in bone by in situ hybridization.
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ABSTRACT: Here, we described a method for carrying out nonradioactive in situ hybridization to detect mRNA transcripts in cryostat sections of mouse bone using the CryoJane(®) Tape-Transfer System and digoxigenin (DIG)-labeled riboprobes.Methods in molecular biology (Clifton, N.J.) 01/2012; 816:305-20. -
Article: Isolation of mouse osteocytes using cell fractionation for gene expression analysis.
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ABSTRACT: Osteocytes are the terminally differentiated cells of the osteoblastic lineage embedded within the mineralized bone matrix. T: hey have been identified as key players in mechanotransduction and in mineral and phosphate homeostasis. In addition, they appear to have a role in mediating bone formation, since they secrete the bone formation inhibitor sclerostin. In contrast to osteoblasts and osteoclasts, which reside on the bone surface, it has been difficult to isolate and analyze cellular and molecular properties of osteocytes due to their specific location inside the "hard" mineralized bone compartment. This chapter describes a method to isolate osteocytes from newborn mouse calvaria and adult mouse long bone, followed by immediate total RNA extraction allowing to selectively study osteocytic versus osteoblastic gene expression by quantitative real-time polymerase chain reaction (qPCR). The osteocyte-enriched cell fraction isolated by this method can further be purified by FACS and selectively expresses osteocytic marker genes, such as Dmp1 and Sost.Methods in molecular biology (Clifton, N.J.) 01/2012; 816:55-66. -
Article: Mef2c deletion in osteocytes results in increased bone mass.
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ABSTRACT: Myocyte enhancer factors 2 (MEF2) are required for expression of the osteocyte bone formation inhibitor Sost in vitro, implying these transcription factors in bone biology. Here, we analyzed the in vivo function of Mef2c in osteocytes in male and female mice during skeletal growth and aging. Dmp1-Cre-induced Mef2c deficiency led to progressive decreases in Sost expression by 40% and 70% in femoral cortical bone at 3.5 months and 5 to 6 months of age. From 2 to 3 months onward, bone mass was increased in the appendicular and axial skeleton of Mef2c mutant relative to control mice. Cortical thickness and long bone and vertebral trabecular density were elevated. To assess whether the increased bone mass was related to the decreased Sost expression, we characterized 4-month-old heterozygous Sost-deficient mice. Sost heterozygotes displayed similar increases in long bone mass and density as Mef2c mutants, but the relative increases in axial skeletal parameters were mostly smaller. At the cellular level, bone formation parameters were normal in 3.5-month-old Mef2c mutant mice, whereas bone resorption parameters were significantly decreased. Correspondingly, cortical expression of the anti-osteoclastogenic factor and Wnt/β-catenin target gene osteoprotegerin (OPG) was increased by 70% in Mef2c mutant males. Furthermore, cortical expression of the Wnt signaling modulators Sfrp2 and Sfrp3 was strongly deregulated in both sexes. In contrast, heterozygous Sost deficient males displayed mildly increased osteoblastic mineral apposition rate, but osteoclast surface and cortical expression of osteoclastogenic regulators including OPG were normal and Sfrp2 and Sfrp3 were not significantly changed. Together, our data demonstrate that Mef2c regulates cortical Sfrp2 and Sfrp3 expression and is required to maintain normal Sost expression in vivo. Yet, the increased bone mass phenotype of Mef2c mutants is not directly related to the reduced Sost expression. We identified a novel function for Mef2c in control of adult bone mass by regulation of osteoclastic bone resorption.Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 12/2011; 27(2):360-73. · 6.04 Impact Factor -
Article: Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function.
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ABSTRACT: Humans lacking sclerostin display progressive bone overgrowth due to increased bone formation. Although it is well established that sclerostin is an osteocyte-secreted bone formation inhibitor, the underlying molecular mechanisms are not fully elucidated. We identified in tandem affinity purification proteomics screens LRP4 (low density lipoprotein-related protein 4) as a sclerostin interaction partner. Biochemical assays with recombinant proteins confirmed that sclerostin LRP4 interaction is direct. Interestingly, in vitro overexpression and RNAi-mediated knockdown experiments revealed that LRP4 specifically facilitates the previously described inhibitory action of sclerostin on Wnt1/β-catenin signaling. We found the extracellular β-propeller structured domain of LRP4 to be required for this sclerostin facilitator activity. Immunohistochemistry demonstrated that LRP4 protein is present in human and rodent osteoblasts and osteocytes, both presumed target cells of sclerostin action. Silencing of LRP4 by lentivirus-mediated shRNA delivery blocked sclerostin inhibitory action on in vitro bone mineralization. Notably, we identified two mutations in LRP4 (R1170W and W1186S) in patients suffering from bone overgrowth. We found that these mutations impair LRP4 interaction with sclerostin and its concomitant sclerostin facilitator effect. Together these data indicate that the interaction of sclerostin with LRP4 is required to mediate the inhibitory function of sclerostin on bone formation, thus identifying a novel role for LRP4 in bone.Journal of Biological Chemistry 04/2011; 286(22):19489-500. · 4.77 Impact Factor -
Article: Osteocyte Wnt/beta-catenin signaling is required for normal bone homeostasis.
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ABSTRACT: Beta-Catenin-dependent canonical Wnt signaling plays an important role in bone metabolism by controlling differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. To investigate its function in osteocytes, the cell type constituting the majority of bone cells, we generated osteocyte-specific beta-catenin-deficient mice (Ctnnb1(loxP/loxP); Dmp1-Cre). Homozygous mutants were born at normal Mendelian frequency with no obvious morphological abnormalities or detectable differences in size or body weight, but bone mass accrual was strongly impaired due to early-onset, progressive bone loss in the appendicular and axial skeleton with mild growth retardation and premature lethality. Cancellous bone mass was almost completely absent, and cortical bone thickness was dramatically reduced. The low-bone-mass phenotype was associated with increased osteoclast number and activity, whereas osteoblast function and osteocyte density were normal. Cortical bone Wnt/beta-catenin target gene expression was reduced, and of the known regulators of osteoclast differentiation, osteoprotegerin (OPG) expression was significantly downregulated in osteocyte bone fractions of mutant mice. Moreover, the OPG levels expressed by osteocytes were higher than or comparable to the levels expressed by osteoblasts during skeletal growth and at maturity, suggesting that the reduction in osteocytic OPG and the concomitant increase in osteocytic RANKL/OPG ratio contribute to the increased number of osteoclasts and resorption in osteocyte-specific beta-catenin mutants. Together, these results reveal a crucial novel function for osteocyte beta-catenin signaling in controlling bone homeostasis.Molecular and cellular biology 06/2010; 30(12):3071-85. · 6.06 Impact Factor
