Genomic deletion of a long-range bone enhancer misregulates sclerostin in Van Buchem disease

Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Genome Research (Impact Factor: 14.63). 07/2005; 15(7):928-35. DOI: 10.1101/gr.3437105
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Mutations in distant regulatory elements can have a negative impact on human development and health, yet because of the difficulty of detecting these critical sequences, we predominantly focus on coding sequences for diagnostic purposes. We have undertaken a comparative sequence-based approach to characterize a large noncoding region deleted in patients affected by Van Buchem (VB) disease, a severe sclerosing bone dysplasia. Using BAC recombination and transgenesis, we characterized the expression of human sclerostin (SOST) from normal (SOST(wt)) or Van Buchem (SOST(vbDelta) alleles. Only the SOST(wt) allele faithfully expressed high levels of human SOST in the adult bone and had an impact on bone metabolism, consistent with the model that the VB noncoding deletion removes a SOST-specific regulatory element. By exploiting cross-species sequence comparisons with in vitro and in vivo enhancer assays, we were able to identify a candidate enhancer element that drives human SOST expression in osteoblast-like cell lines in vitro and in the skeletal anlage of the embryonic day 14.5 (E14.5) mouse embryo, and discovered a novel function for sclerostin during limb development. Our approach represents a framework for characterizing distant regulatory elements associated with abnormal human phenotypes.

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Available from: Hansjoerg Keller, Oct 01, 2015
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    • "Genetic changes in the gene encoding sclerostin (SOST), with consequences in downregulation or loss of gene function, induce phenotypes with high bone mass. This is the case of sclerosteosis or van Buchem's disease in humans [14] [15] [16]. Conversely, transgenic overexpression of the SOST gene results in pronounced osteopoenia and reduced bone formation [17]. "
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    ABSTRACT: Introduction: The biological mechanisms associated with an inadequate response to treatment with bisphosphonates are not well known. This study investigates the association between circulating levels of sclerostin and estradiol with an inadequate clinical outcome to bisphosphonate therapy in women with postmenopausal osteoporosis. Methods: This case-control study is based on 120 Spanish women with postmenopausal osteoporosis being treated with oral bisphosphonates. Patients were classified as adequate responders (ARs, n=66, mean age 68.2±8 years) without incident fractures during 5 years of treatment, or inadequate responders (IRs, n=54, mean age 67±9 years), with incident fractures between 1 and 5 years of treatment. Bone mineral density (DXA), structural analysis of the proximal femur and structural/fractal analysis of the distal radius were assessed. Sclerostin concentrations were measured by ELISA and 17β-estradiol levels by radioimmunoassay based on ultrasensitive methods. Results: In the ARs group, sclerostin serum levels were significantly lower (p=0.02) and estradiol concentrations significantly higher (p=0.023) than in the IRs group. A logistic regression analysis was performed, including as independent variables in the original model femoral fracture load, 25 hydroxyvitamin D, previus history of fragility fracture, sclerostin and estradiol. Only previous history of fragility fracture (OR 14.04, 95% CI 2.38-82.79, p=0.004) and sclerostin levels (OR 1.11, 95% CI 1.02-1.20, p=0.011), both adjusted by estradiol levels remained associated with IRs. Also, sclerostin concentrations were associated with the index of resistance to compression (IRC) in the fractal analysis of the distal radius, a parameter on bone microstructure. Conclusions: Sclerostin and estradiol levels are associated with the response to bisphosphonate therapy in women with postmenopausal osteoporosis.
    Maturitas 09/2015; DOI:10.1016/j.maturitas.2015.08.007 · 2.94 Impact Factor
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    • "). As a result of this mutation, SOST no longer exerts its inhibitory effect on bone formation, which leads to hyperplasia, a typical manifestation of sclerosteosis and van Buchem disease (Balemans et al. 2001, 2005, Brunkow et al. 2001, Loots et al. 2005, Kim et al. 2008). Understanding the molecular background of these two conditions provided novel therapeutic options for patients with excessive bone formation and individuals with osteoporosis (McClung et al. 2014). "
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    ABSTRACT: Bone is a dynamic tissue that undergoes constant remodeling. Appropriate course of this process determines development and regeneration of the skeleton. Tight molecular control of bone remodeling is vital for the maintenance of appropriate physiology and microarchitecture of the bone, providing homeostasis, also at the systemic level. The process of remodeling is regulated by a rich innervation of the skeleton, being the source of various growth factors, neurotransmitters and hormones regulating function of the bone. Although the course of bone remodeling at the cellular level is mainly associated with the activity of osteoclasts and osteoblasts, recently also osteocytes gain growing interest as the principal regulators of bone turnover. Osteocytes play a significant role in the regulation of osteogenesis, releasing sclerostin, an inhibitor of bone formation. The process of bone turnover, especially osteogenesis, is also modulated by extra-skeletal molecules. Proliferation and differentiation of osteoblasts are promoted by the brain-derived serotonin and hypothetically inhibited by its intestinal equivalent. The activity of sclerostin and serotonin is either directly or indirectly associated with the canonical Wnt/β-catenin signaling pathway, the main regulatory pathway of osteoblasts function. The impairment of bone remodeling may lead to many skeletal diseases, such as high bone mass syndrome or osteoporosis. In this paper, we review the most recent data on the cellular and molecular mechanisms of bone remodeling control, with the particular emphasis on the role of osteocytes and the nervous system in this process.
    Journal of Molecular Endocrinology 08/2015; DOI:10.1530/JME-15-0067 · 3.08 Impact Factor
    • "These cells are known to respond to loading by sending signals to osteoblasts and osteoclasts thereby affecting their number and function (Bonewald, 2011). Osteocytes secrete sclerostin, a major inhibitor of Wnt/b-catenin pathway, which blocks osteoblast differentiation (Balemans et al., 2001; Loots et al., 2005). Moreover, osteocytes regulate osteoclast function through the expression of the receptor activator of nuclear factor kappa B ligand (RANKL) (Xiong et al., 2012). "
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    ABSTRACT: Mechanical loading plays a key role in bone formation and maintenance. While unloading induces osteocyte apoptosis and bone loss in vivo, mechanical stimuli prevents osteocyte death through a mechanism involving β-catenin accumulation and ERK nuclear translocation. Vascular endothelial growth factor (VEGF) has a crucial role in bone formation, but its interaction with osteocytes is not completely understood. Of interest, VEGF receptor 2 (VEGFR2) has recently been shown to mediate the mechanical response of endothelial cells. The present study aimed to evaluate the putative role of the VEGF system in osteocyte mechanosensing. We show that either short (10 min) mechanical stimulus by pulsatile fluid flow (FF) (10 dyn/cm2, 8 Hz) or exogenous VEGF165 (6 ng/ml) similarly stimulated cell viability, ERK phosphorylation, and β-catenin membrane translocation. A VEGFR2 antagonist (SU5416) or transfection with specific VEGFR2 siRNAs (siVEGFR2) decreased these events. FF for 10 min increased VEGFR2 phosphorylation at both Tyr-1059 and Tyr-1175; an effect that was mimicked by VEGF165 but was unaffected by a VEGF neutralizing antibody. Subsequently (at 6 h), this mechanical stimulus induced VEGF gene overexpression, which was prevented by siVEGFR2 transfection. Depletion of the structural protein caveolin-1 by using siRNA technology impaired FF-induced VEGFR2 phosphorylation. In conclusion, these in vitro findings point to caveolin-1-dependent VEGFR2 activation as an important mechanism whereby mechanical stimuli promote osteocyte viability. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 02/2015; 230(2). DOI:10.1002/jcp.24734 · 3.84 Impact Factor
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