ABSTRACT: Estrogens are well known steroid hormones necessary to maintain bone health. In addition, mechanical loading, which estrogen signaling may intersect with the Wnt/β-catenin pathway, is also essential for bone health. As osteocytes are known as the major mechanosensory cells embedded in mineralized bone matrix, osteocyte ERα deletion mice (ERαÎOcy/ÎOcy) were generated by mating ERα floxed mice with Dmp1-Cre mice to determine functions of ERα in osteocytes. Trabecular bone mineral density of female, but not male ERαÎOcy/ÎOcy mice was significantly decreased. Bone formation parameters in ERαÎOcy/ÎOcy were significantly decreased while osteoclast parameters were unchanged. This suggests that ERα in osteocytes exerts osteoprotective function by positively controlling bone formation. To identify potential targets of ERα, gene array analysis of Dmp1-GFP osteocytes FACS sorted from ERαÎOcy/ÎOcy and control mice was performed. Expression of Mdk and Sostdc1, both known inhibitors of Wnt, were significantly increased without alteration of the mature osteocyte marker Sost or β-catenin. Hindlimb unloading exacerbated the trabecular bone loss, but surprisingly cortical bone was resistant. These studies show that ERα in osteocytes has osteoprotective effects in trabecular bone through regulating expression of Wnt antagonists, but conversely plays a negative role in cortical bone loss due to unloading. Wild type and osteocyte-specific Estrogen Receptor alpha knock-out mice were generated. The number of both genotypes of mice was three. Calvarial osteocytes of both genotypes harboring Dmp1-GFP were extracted by sequential enzymatic digestion, followed by FACS Aria sorting and total RNAs were purified for Affymetix GeneChip microarray analysis without pooling.
Project description:Estrogens are well known steroid hormones necessary to maintain bone health. In addition, mechanical loading, which estrogen signaling may intersect with the Wnt/β-catenin pathway, is also essential for bone health. As osteocytes are known as the major mechanosensory cells embedded in mineralized bone matrix, osteocyte ERα deletion mice (ERαΔOcy/ΔOcy) were generated by mating ERα floxed mice with Dmp1-Cre mice to determine functions of ERα in osteocytes. Trabecular bone mineral density of female, but not male ERαΔOcy/ΔOcy mice was significantly decreased. Bone formation parameters in ERαΔOcy/ΔOcy were significantly decreased while osteoclast parameters were unchanged. This suggests that ERα in osteocytes exerts osteoprotective function by positively controlling bone formation. To identify potential targets of ERα, gene array analysis of Dmp1-GFP osteocytes FACS sorted from ERαΔOcy/ΔOcy and control mice was performed. Expression of Mdk and Sostdc1, both known inhibitors of Wnt, were significantly increased without alteration of the mature osteocyte marker Sost or β-catenin. Hindlimb unloading exacerbated the trabecular bone loss, but surprisingly cortical bone was resistant. These studies show that ERα in osteocytes has osteoprotective effects in trabecular bone through regulating expression of Wnt antagonists, but conversely plays a negative role in cortical bone loss due to unloading.
Project description:Jason M. Graham, Bruce P. Ayati, Sarah A. Holstein & James A. Martin. The role of osteocytes in targeted bone remodeling: a mathematical model. PLoS ONE 8, 5 (2013).
Until recently many studies of bone remodeling at the cellular level have focused on the behavior of mature osteoblasts and osteoclasts, and their respective precursor cells, with the role of osteocytes and bone lining cells left largely unexplored. This is particularly true with respect to the mathematical modeling of bone remodeling. However, there is increasing evidence that osteocytes play important roles in the cycle of targeted bone remodeling, in serving as a significant source of RANKL to support osteoclastogenesis, and in secreting the bone formation inhibitor sclerostin. Moreover, there is also increasing interest in sclerostin, an osteocyte-secreted bone formation inhibitor, and its role in regulating local response to changes in the bone microenvironment. Here we develop a cell population model of bone remodeling that includes the role of osteocytes, sclerostin, and allows for the possibility of RANKL expression by osteocyte cell populations. We have aimed to give a simple, yet still tractable, model that remains faithful to the underlying system based on the known literature. This model extends and complements many of the existing mathematical models for bone remodeling, but can be used to explore aspects of the process of bone remodeling that were previously beyond the scope of prior modeling work. Through numerical simulations we demonstrate that our model can be used to explore theoretically many of the qualitative features of the role of osteocytes in bone biology as presented in recent literature.
Project description:Purpose of arrays were to determine what the effect of deletion of Mbtps1 gene was on gene expression of osteocytes in bone in vivo. DMP1 cre driver was used to delete the Mbtps1 gene in osteocytes and osteoblasts in bone. We then isolated osteocyte enriched bone particles from 40 week old male mice to determine the effect of this deletion on gene expression. We have previously shown that Mbtps1 is needed for transcription of Phex, DMP1, and MEPE genes in osteoblasts in culture. Arrays showed these genes were reduced as expected in osteocytes in vivo. Controls represent osteocyte enriched bone from 40 week old littermates. Also, as expected, Mbtps1 expression was reduced in these knockout mice
Project description:Glucocorticoids (GC) and parathyroid hormone (PTH) are widely used therapeutic endocrine hormones where their effects on bone and joint arise from actions on multiple skeletal cell types. In osteocytes, GC and PTH exert opposing effects on perilacunar canalicular remodeling (PLR). Suppressed PLR can impair bone quality and joint homeostasis, including in GC-induced osteonecrosis. However, combined effects of GC and PTH on PLR are unknown. Focusing on subchondral bone and joint homeostasis, we hypothesize that PTH, a PLR agonist, could rescue GC-suppressed PLR. The skeletal effects of GC and PTH, alone or combined, were examined in male and female mice by micro-computed tomography, mechanical testing, histology, and gene expression analysis. For each outcome, females were more responsive to GC and PTH than males. GC and PTH exerted regional differences, with GC increasing trabecular bone volume but reducing cortical bone thickness, stiffness, and ultimate force. Despite PTH’s anabolic effects on trabecular bone, it did not rescue GC’s catabolic effects on cortical bone. Likewise, cartilage integrity and subchondral bone apoptosis, alkaline phosphatase activity, and osteocyte lacunocanalicular networks showed no evidence that PTH could offset GC-dependent effects. Rather, GC and PTH each increased cortical bone gene expression implicated in bone resorption by osteoclasts and osteocytes, including Acp5, Mmp13, Atp6v0d2, Ctsk, differences maintained when GC and PTH were combined. Since PTH is insufficient to rescue GC’s effects on young female mouse bone, future studies are needed to determine if osteocyte PLR suppression, due to GC, aging, or other factors, can be offset by a PLR agonist.
Project description:RNA-seq analysis of transcriptome of osteocytes isolated from femora cortical bone of 5 mo old C57BL6 mice with WT or PPARa KO genotype. Conclusion: In osteocytes, PPARα controls large number of transcripts coding for signaling proteins and osteocyte secretome which regulates development of bone marrow adipose tissue and peripheral fat metabolism.
Project description:Some cuboidal osteoblasts differentiate into bone-embedded, dendrite-bearing osteocytes through the poorly-understood process of osteocytogenesis. Here, we report that the transcription factor Sp7 plays an essential role in osteocytogenesis. Severe defects in bone integrity and osteocyte dendrite morphology are noted in mice lacking Sp7 at the stage of the osteoblast-to-osteocyte transition. In osteocytes, Sp7 controls expression of a neuronally-enriched gene network. Analysis of the osteocyte-specific Sp7 cistrome reveals distinct genomic binding motifs and target sites distinct from those in osteoblasts. Amongst osteocyte-specific Sp7 targets, the secreted peptide osteocrin rescues Sp7-deficient defects. Single-cell transcriptional profiling of cells undergoing osteocytogenesis identifies novel Sp7-dependent transitional cell types enriched in genes linked to human fracture risk. Finally, humans with an SP7 R316C mutation display osteocyte morphology defects similar to those observed in mouse models. These findings demonstrate that cuboidal osteoblasts use a neuronally-enriched Sp7/osteocrin gene expression program to differentiate into dendrite-bearing osteocytes.
Project description:Some cuboidal osteoblasts differentiate into bone-embedded, dendrite-bearing osteocytes through the poorly-understood process of osteocytogenesis. Here, we report that the transcription factor Sp7 plays an essential role in osteocytogenesis. Severe defects in bone integrity and osteocyte dendrite morphology are noted in mice lacking Sp7 at the stage of the osteoblast-to-osteocyte transition. In osteocytes, Sp7 controls expression of a neuronally-enriched gene network. Analysis of the osteocyte-specific Sp7 cistrome reveals distinct genomic binding motifs and target sites distinct from those in osteoblasts. Amongst osteocyte-specific Sp7 targets, the secreted peptide osteocrin rescues Sp7-deficient defects. Single-cell transcriptional profiling of cells undergoing osteocytogenesis identifies novel Sp7-dependent transitional cell types enriched in genes linked to human fracture risk. Finally, humans with an SP7 R316C mutation display osteocyte morphology defects similar to those observed in mouse models. These findings demonstrate that cuboidal osteoblasts use a neuronally-enriched Sp7/osteocrin gene expression program to differentiate into dendrite-bearing osteocytes.
Project description:Some cuboidal osteoblasts differentiate into bone-embedded, dendrite-bearing osteocytes through the poorly-understood process of osteocytogenesis. Here, we report that the transcription factor Sp7 plays an essential role in osteocytogenesis. Severe defects in bone integrity and osteocyte dendrite morphology are noted in mice lacking Sp7 at the stage of the osteoblast-to-osteocyte transition. In osteocytes, Sp7 controls expression of a neuronally-enriched gene network. Analysis of the osteocyte-specific Sp7 cistrome reveals distinct genomic binding motifs and target sites distinct from those in osteoblasts. Amongst osteocyte-specific Sp7 targets, the secreted peptide osteocrin rescues Sp7-deficient defects. Single-cell transcriptional profiling of cells undergoing osteocytogenesis identifies novel Sp7-dependent transitional cell types enriched in genes linked to human fracture risk. Finally, humans with an SP7 R316C mutation display osteocyte morphology defects similar to those observed in mouse models. These findings demonstrate that cuboidal osteoblasts use a neuronally-enriched Sp7/osteocrin gene expression program to differentiate into dendrite-bearing osteocytes.
Project description:Estrogen clearly prevents osteoporotic bone loss by attenuating bone resorption. The molecular basis of how this is accomplished, however, remains elusive. Here we report a critical role of osteoclastic ERa in mediating estrogen action on bone in females. We selectively ablated ERa in differentiated osteoclasts (ERa dOc/dOc). ERa dOc/dOc females, but not males, exhibited clear trabecular bone loss, similar to the osteoporotic bone phenotype in post-menopausal women. Recovery of bone loss by estrogen treatment of the ovariectomized ERa dOc/dOc females was ineffective in the trabecular areas of the long bones and lumbar vertebral bodies. Osteoclastic apoptosis, induced by estrogen, occurred simultaneously with up-regulation of Fas ligand (FasL) expression in intact trabecular bones of ERa +/+mice, but not in ERa dOc/dOc mice. ERa was also required for similar effects of estrogen and tamoxifen in cultured osteoclasts. These findings suggest that the osteoprotective actions of estrogen and SERMS are mediated at least in part through osteoclastic ERa in trabecular bone; and the life span of mature osteoclasts is regulated through activation of the Fas/FasL system. Experiment Overall Design: Wild type and osteoclast-specific Estrogen Receptor alpha knock-out mice were ovariectomized. The number of both genotypes of mice was eight. The mice of each genotypes were divided to vehicle control and estrogen treated group. Four hours after chemical treatment, the distal 5 mm of the left femurs were harvested after sacrificing by cervical dislocation and total RNAs were purified for Affymetix GeneChip microarray analysis without pooling. Therefore, this experiment consists of four groups with four replicates per group.
Project description:Osteogenesis imperfecta (OI) is a group of diseases caused by defects in type I collagen processing which result in skeletal fragility. While these disorders have traditionally been regarded as defects in osteoblast function, the role of matrix-embedded osteocytes, descendants of osteoblasts, in OI pathogenesis remains unknown. The homozygous human SP7 (c.946C > T, R316C) mutation results in a recessive form of osteogenesis imperfecta characterized by short stature, fragility fractures, low bone mineral density, and osteocyte dendrite defects. To better understand how the osteogenesis imperfecta-causingSP7 R316Cmutation affects the function of this transcription factor in different osteoblast lineage cells in bone, we generatedSp7 R342Cknock-in mice. Homozygous mutantSp7 R342C/R342Cmice demonstrate increased cortical porosity and reduced cortical bone mineral density, findings consistent with phenotypes observed in patients with this mutation. Sp7 R342Cmice show osteocyte dendrite defects, increased osteocyte apoptosis, and intracortical bone remodeling characterized by ectopic intracortical osteoclasts and elevated Tnfsf11 expression by osteocytes. Remarkably, these overt defects in osteocyte function contrast to preserved osteoblast function, suggesting that this Sp7 point mutation selectively interferes with the function of this transcription factor in osteocytes but not osteoblasts. Osteocyte morphology changes in Sp7 R342C/R342Cmice were not restored by inhibiting osteoclast formation, indicating that dendrite defects lie upstream of high intra-cortical osteoclast activity in this model. Moreover, transcriptomic profiling reveals that the expression of a core set osteocyte-enriched genes is highly dysregulated by the R342C mutation. Thus, this model supports a model in which osteocyte dysfunction can drive osteogenesis imperfecta pathogenesis, and provides a valuable resource to test novel therapeutic approaches and to understand the osteocyte-specific role of SP7 in bone homeostasis and remodeling.