Publications by authors named "Antonia Sophocleous"

23 Publications

  • Page 1 of 1

JZL184, A Monoacylglycerol Lipase Inhibitor, Induces Bone Loss in a Multiple Myeloma Model of Immunocompetent Mice.

Calcif Tissue Int 2020 07 13;107(1):72-85. Epub 2020 Apr 13.

Department of Oncology and Metabolism, Medical School, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX, UK.

Multiple myeloma (MM) patients develop osteolysis characterised by excessive osteoclastic bone destruction and lack of osteoblast bone formation. Pharmacological manipulation of monoacylglycerol lipase (MAGL), an enzyme responsible for the degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG), reduced skeletal tumour burden and osteolysis associated with osteosarcoma and advanced breast and prostate cancers. MM and hematopoietic, immune and bone marrow cells express high levels of type 2 cannabinoid receptor and osteoblasts secrete 2-AG. However, the effects of MAGL manipulation on MM have not been investigated. Here, we report that treatment of pre-osteoclasts with non-cytotoxic concentrations of JZL184, a verified MAGL inhibitor, enhanced MM- and RANKL-induced osteoclast formation and size in vitro. Exposure of osteoblasts to JZL184 in the presence of MM cell-derived factors reduced osteoblast growth but had no effect on the ability of these cells to mature or form bone nodules. In vivo, administration of JZL184 induced a modest, yet significant, bone loss at both trabecular and cortical compartments of long bones of immunocompetent mice inoculated with the syngeneic 5TGM1-GFP MM cells. Notably, JZL184 failed to inhibit the in vitro growth of a panel of mouse and human MM cell lines, or reduce tumour burden in mice. Thus, MAGL inhibitors such as JZL184 can exacerbate MM-induced bone loss.
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http://dx.doi.org/10.1007/s00223-020-00689-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271071PMC
July 2020

Conditional deletion of E11/Podoplanin in bone protects against ovariectomy-induced increases in osteoclast formation and activity.

Biosci Rep 2020 01;40(1)

Roslin Institute and R(D)SVS, The University of Edinburgh, Easter Bush, Midlothian, U.K.

E11/Podoplanin (Pdpn) is implicated in early osteocytogenesis and the formation of osteocyte dendrites. This dendritic network is critical for bone modelling/remodelling, through the production of receptor activator of nuclear factor κ B (RANK)-ligand (RANKL). Despite this, the role of Pdpn in the control of bone remodelling is yet to be established in vivo. Here we utilised bone-specific Pdpn conditional knockout mice (cKO) to examine the role of Pdpn in the bone loss associated with ovariectomy (OVX). MicroCT revealed that Pdpn deletion had no significant effect on OVX-induced changes in trabecular microarchitecture. Significant differences between genotypes were observed in the trabecular pattern factor (P<0.01) and structure model index (P<0.01). Phalloidin staining of F-actin revealed OVX to induce alterations in osteocyte morphology in both wild-type (WT) and cKO mice. Histological analysis revealed an expected significant increase in osteoclast number in WT mice (P<0.01, compared with sham). However, cKO mice were protected against such increases in osteoclast number. Consistent with this, serum levels of the bone resorption marker Ctx were significantly increased in WT mice following OVX (P<0.05), but were unmodified by OVX in cKO mice. Gene expression of the bone remodelling markers Rank, Rankl, Opg and Sost were unaffected by Pdpn deletion. Together, our data suggest that an intact osteocyte dendritic network is required for sustaining osteoclast formation and activity in the oestrogen-depleted state, through mechanisms potentially independent of RANKL expression. This work will enable a greater understanding of the role of osteocytes in bone loss induced by oestrogen deprivation.
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http://dx.doi.org/10.1042/BSR20190329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954370PMC
January 2020

Liver-derived IGF-I is not required for protection against osteoarthritis in male mice.

Am J Physiol Endocrinol Metab 2019 12 22;317(6):E1150-E1157. Epub 2019 Oct 22.

Centre for Bone and Arthritis Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Sweden.

Insulin-like growth factor-I (IGF-I) is anabolic for cartilage and important for cartilage integrity, which might suggest a connection between IGF-I and osteoarthritis (OA) development. However, the results of studies performed so far are conflicting, and we aimed to clarify the role of endocrine IGF-I in rodent OA. Male mice with inducible inactivation of circulating, liver-derived IGF-I (LI-IGF-I mice, serum IGF-I reduced by ~80%) were used. Experimental OA was induced in young adult LI-IGF-I and control mice by destabilization of the medial meniscus (DMM); age-related OA was also evaluated in 1-yr-old mice. DMM-operated LI-IGF-I mice had thinner lateral subchondral bone plate in tibia compared with control mice, whereas osteophyte volume and articular cartilage damage were unaffected at the medial side of the DMM knee. However, the control mice but not the LI-IGF-I mice also developed mild OA on the lateral side of the DMM knee compared with the unoperated knee. One-year-old LI-IGF-I mice had lower mid-diaphyseal cortical bone area than the 1-yr-old control mice, whereas analyses of joint tissues displayed smaller osteophyte volume and thicker calcified cartilage than the control mice. There was no difference in OA severity in the articular cartilage between old LI-IGF-I and control mice. Our study is the first to investigate whether there is an association between circulating IGF-I and OA in mice. We conclude that, although there is an ~80% reduction of circulating IGF-I and a decrease in cortical bone in male LI-IGF-I mice, cartilage damage is clearly not intensified and may instead be slightly reduced.
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http://dx.doi.org/10.1152/ajpendo.00330.2019DOI Listing
December 2019

Paradoxical effects of JZL184, an inhibitor of monoacylglycerol lipase, on bone remodelling in healthy and cancer-bearing mice.

EBioMedicine 2019 Jun 29;44:452-466. Epub 2019 May 29.

Department of Oncology and Metabolism, University of Sheffield, Medical School, Beech Hill Road, Sheffield S10 2RX, UK; Bone and Cancer Group, Edinburgh Cancer Research Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, EH4 2XR, UK. Electronic address:

Background: Cancer-associated bone disease is a serious complication in bone sarcomas and metastatic carcinomas of breast and prostate origin. Monoacylglycerol lipase (MAGL) is an enzyme of the endocannabinoid system, and is responsible for the degradation of the most abundant endocannabinoid in bone, 2-arachidonoyl glycerol (2AG).

Methods: The effects of the verified MAGL inhibitor on bone remodelling were assessed in healthy mice and in mouse models of bone disease caused by prostate and breast cancers and osteosarcoma.

Findings: JZL184 reduced osteolytic bone metastasis in mouse models of breast and prostate cancers, and inhibited skeletal tumour growth, metastasis and the formation of ectopic bone in models of osteosarcoma. Additionally, JZL184 suppressed cachexia and prolonged survival in mice injected with metastatic osteosarcoma and osteotropic cancer cells. Functional and histological analysis revealed that the osteoprotective action of JZL184 in cancer models is predominately due to inhibition of tumour growth and metastasis. In the absence of cancer, however, exposure to JZL184 exerts a paradoxical reduction of bone volume via an effect that is mediated by both Cnr1 and Cnr2 cannabinoid receptors.

Interpretation: MAGL inhibitors such as JZL184, or its novel analogues, may be of value in the treatment of bone disease caused by primary bone cancer and bone metastasis, however, activation of the skeletal endocannabinoid system may limit their usefulness as osteoprotective agents.
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http://dx.doi.org/10.1016/j.ebiom.2019.05.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606522PMC
June 2019

Raman spectroscopy as a predictive tool for monitoring osteoporosis therapy in a rat model of postmenopausal osteoporosis.

J Mater Sci Mater Med 2019 Feb 12;30(2):25. Epub 2019 Feb 12.

Department of Oncology and Metabolism, Medical School, University of Sheffield, Beech Hill Road, Sheffield, UK.

Pharmacological therapy of osteoporosis reduces bone loss and risk of fracture in patients. Modulation of bone mineral density cannot explain all effects. Other aspects of bone quality affecting fragility and ways to monitor them need to be better understood. Keratinous tissue acts as surrogate marker for bone protein deterioration caused by oestrogen deficiency in rats. Ovariectomised rats were treated with alendronate (ALN), parathyroid hormone (PTH) or estrogen (E2). MicroCT assessed macro structural changes. Raman spectroscopy assessed biochemical changes. Micro CT confirmed that all treatments prevented ovariectomy-induced macro structural bone loss in rats. PTH induced macro structural changes unrelated to ovariectomy. Raman analysis revealed ALN and PTH partially protect against molecular level changes to bone collagen (80% protection) and mineral (50% protection) phases. E2 failed to prevent biochemical change. The treatments induced alterations unassociated with the ovariectomy; increased beta sheet with E2, globular alpha helices with PTH and fibrous alpha helices with both ALN and PTH. ALN is closest to maintaining physiological status of the animals, while PTH (comparable protective effect) induces side effects. E2 is unable to prevent molecular level changes associated with ovariectomy. Raman spectroscopy can act as predictive tool for monitoring pharmacological therapy of osteoporosis in rodents. Keratinous tissue is a useful surrogate marker for the protein related impact of these therapies.The results demonstrate utility of surrogates where a clear systemic causation connects the surrogate to the target tissue. It demonstrates the need to assess broader biomolecular impact of interventions to examine side effects.
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http://dx.doi.org/10.1007/s10856-019-6226-xDOI Listing
February 2019

Osteoarthritis Mouse Model of Destabilization of the Medial Meniscus.

Methods Mol Biol 2019 ;1914:281-293

Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh, Scotland, UK.

This chapter describes the surgical procedure for destabilization of medial meniscus in mice. Details on subsequent microCT and histological analysis are also provided, as well as details on osteoarthritis evaluation.
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http://dx.doi.org/10.1007/978-1-4939-8997-3_15DOI Listing
June 2019

Ovariectomy/Orchiectomy in Rodents.

Methods Mol Biol 2019 ;1914:261-267

Department of Oncology and Metabolism, University of Sheffield, Sheffield, UK.

This chapter describes the surgical procedures for ovariectomy and orchiectomy in mice and rats. In addition to providing technical details of the surgical techniques, details of anesthesia options and pre-, peri-, and postoperative care are also included.
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http://dx.doi.org/10.1007/978-1-4939-8997-3_13DOI Listing
June 2019

Analysis of Transcriptional Regulation in Bone Cells.

Methods Mol Biol 2019 ;1914:145-167

Rheumatic Diseases Unit, Centre for Genomics and Experimental Medicine, MRC IGMM, Western General Hospital, University of Edinburgh, Edinburgh, UK.

Transcription is a process by which the rate of RNA synthesis is regulated. Here we describe the techniques for carrying out promoter-reporter assays, electrophoretic mobility shift assays, chromosome conformation capture (3C) assays, chromatin immunoprecipitation assays, and CRISPR-Cas9 assay, five commonly used methods for studying and altering gene transcription.
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http://dx.doi.org/10.1007/978-1-4939-8997-3_8DOI Listing
June 2019

Regulation of breast cancer induced bone disease by cancer-specific IKKβ.

Oncotarget 2018 Mar 23;9(22):16134-16148. Epub 2018 Mar 23.

Department of Oncology and Metabolism, University of Sheffield, Sheffield, UK.

NFκB is implicated in breast cancer bone metastasis and skeletal remodelling. However, the role of IKKβ, a key component of the canonical NFκB pathway, in the regulation of breast cancer osteolytic metastasis has not been investigated. Here, we describe the cancer-specific contribution of IKKβ to bone metastasis, skeletal tumour growth and osteolysis associated with breast cancer. IKKβ is highly expressed in invasive breast tumours and its level of expression was higher in patients with bone metastasis. IKKβ overexpression in parental MDA-MD-231 breast cancer cells, promoted mammary tumour growth but failed to convey osteolytic potential to these cells in mice. In contrast, IKKβ overexpression in osteotropic sub-clones of MDA-MB-231 cells with differing osteolytic phenotypes increased incidence of bone metastasis, exacerbated osteolysis and enhanced skeletal tumour growth, whereas its knockdown was inhibitory. Functional and mechanistic studies revealed that IKKβ enhanced the ability of osteotropic MDA-MB-231 cells to migrate, increase osteoclastogenesis, and to inhibit osteoblast differentiation via a mechanism mediated, at least in part, by cytoplasmic sequestering of FoxO3a and VEGFA production. Thus, tumour-selective manipulation of IKKβ and its interaction with FoxO3a may represent a novel strategy to reduce the development of secondary breast cancer in the skeleton.
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http://dx.doi.org/10.18632/oncotarget.24743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882323PMC
March 2018

Raman spectroscopy predicts the link between claw keratin and bone collagen structure in a rodent model of oestrogen deficiency.

Biochim Biophys Acta Mol Basis Dis 2018 Feb 21;1864(2):398-406. Epub 2017 Oct 21.

Rheumatology and Bone Diseases Unit, Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, UK; Department of Oncology and Metabolism, Medical School, University of Sheffield, Beech Hill Road, Sheffield, UK. Electronic address:

Osteoporosis is a common disease characterised by reduced bone mass and an increased risk of fragility fractures. Low bone mineral density is known to significantly increase the risk of osteoporotic fractures, however, the majority of non-traumatic fractures occur in individuals with a bone mineral density too high to be classified as osteoporotic. Therefore, there is an urgent need to investigate aspects of bone health, other than bone mass, that can predict the risk of fracture. Here, we successfully predicted association between bone collagen and nail keratin in relation to bone loss due to oestrogen deficiency using Raman spectroscopy. Raman signal signature successfully discriminated between ovariectomised rats and their sham controls with a high degree of accuracy for the bone (sensitivity 89%, specificity 91%) and claw tissue (sensitivity 89%, specificity 82%). When tested in an independent set of claw samples the classifier gave 92% sensitivity and 85% specificity. Comparison of the spectral changes occurring in the bone tissue with the changes occurring in the keratin showed a number of common features that could be attributed to common changes in the structure of bone collagen and claw keratin. This study established that systemic oestrogen deficiency mediates parallel structural changes in both the claw (primarily keratin) and bone proteins (primarily collagen). This strengthens the hypothesis that nail keratin can act as a surrogate marker of bone protein status where systemic processes induce changes.
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http://dx.doi.org/10.1016/j.bbadis.2017.10.020DOI Listing
February 2018

The Reply.

Am J Med 2017 10;130(10):e457

Rheumatology and Bone Diseases Unit, Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, UK.

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http://dx.doi.org/10.1016/j.amjmed.2017.05.033DOI Listing
October 2017

Combined deficiency of the Cnr1 and Cnr2 receptors protects against age-related bone loss by osteoclast inhibition.

Aging Cell 2017 10 28;16(5):1051-1061. Epub 2017 Jul 28.

Bone and Cancer Group, Edinburgh Cancer Research Centre, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XR, UK.

The endocannabinoid system plays a role in regulating bone mass and bone cell activity and inactivation of the type 1 (Cnr1) or type 2 (Cnr2) cannabinoid receptors influences peak bone mass and age-related bone loss. As the Cnr1 and Cnr2 receptors have limited homology and are activated by different ligands, we have evaluated the effects of combined deficiency of Cnr1 and 2 receptors (Cnr1/2 ) on bone development from birth to old age and studied ovariectomy induced bone loss in female mice. The Cnr1/2 mice had accelerated bone accrual at birth when compared with wild type littermates, and by 3 months of age, they had higher trabecular bone mass. They were also significantly protected against ovariectomy-induced bone loss due to a reduction in osteoclast number. The Cnr1/2 mice had reduced age-related bone loss when compared with wild-type due to a reduction in osteoclast number. Although bone formation was reduced and bone marrow adiposity increased in Cnr1/2 mice, the osteoclast defect outweighed the reduction in bone formation causing preservation of bone mass with aging. This contrasts with the situation previously reported in mice with inactivation of the Cnr1 or Cnr2 receptors individually where aged-related bone loss was greater than in wild-type. We conclude that the Cnr1 and Cnr2 receptors have overlapping but nonredundant roles in regulating osteoclast and osteoblast activities. These observations indicate that combined inhibition of Cnr1 and Cnr2 receptors may be beneficial in preventing age-related bone loss, whereas blockade of individual receptors may be detrimental.
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http://dx.doi.org/10.1111/acel.12638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595693PMC
October 2017

Heavy Cannabis Use Is Associated With Low Bone Mineral Density and an Increased Risk of Fractures.

Am J Med 2017 02 2;130(2):214-221. Epub 2016 Sep 2.

Rheumatology and Bone Diseases Unit, Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, UK. Electronic address:

Purpose: To investigate possible associations between recreational cannabis use and bone health in humans.

Methods: Cross-sectional study of individuals recruited from primary care in the UK between 2011 and 2013. Cases were regular smokers of cannabis divided into moderate (n = 56) and heavy user (n = 144) subgroups depending on whether they reported fewer or more than 5000 cannabis smoking episodes during their lifetime. Controls comprised 114 cigarette smokers.

Results: Heavy cannabis users had lower total hip bone mineral density (mean ± SD Z-score: -0.20 ± 0.9 vs +0.2 ± 0.9, P < .0005), lower spine bone mineral density (-0.5 ± 1.2 vs 0.0 ± 1.2, P < .0005), and lower body mass index (BMI; 26.5 ± 6.0 vs 29.0 ± 7.0, P = .01) than controls. Fracture rate was also increased in heavy users (rate ratio = 2.17; 95% confidence interval, 1.59-2.95; P < .001). When compared with controls, serum cross-linked C-telopeptide of type 1 collagen (CTX) concentrations were raised in heavy cannabis users (0.3 ± 0.1 vs 0.2 ± 0.1 pg/mL, P = .045), as were serum N-terminal propeptide of type 1 procollagen (P1NP) concentrations (47.1 ± 19.2 vs 41.2 ± 17.8 pg/mL, P = .01). Serum total 25-hydroxyvitamin D concentrations were reduced in heavy users compared with controls (25.3 ± 16.8 vs 36.9 ± 26.7 nmol/L, P = .002). Multiple regression analysis revealed that heavy cannabis use was an independent predictor of spine bone mineral density, accounting for 5.4% of the variance (P = .035), and total hip bone mineral density, accounting for 5.8% of the variance (P = .001), but mediation analysis suggested that the effect on spine bone mineral density was indirect and mediated through low body mass index.

Conclusions: Heavy cannabis use is associated with low bone mineral density, low BMI, high bone turnover, and an increased risk of fracture. Heavy cannabis use negatively impacts on bone health both directly and indirectly through an effect on BMI.
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http://dx.doi.org/10.1016/j.amjmed.2016.07.034DOI Listing
February 2017

Bone Cell-autonomous Contribution of Type 2 Cannabinoid Receptor to Breast Cancer-induced Osteolysis.

J Biol Chem 2015 Sep 20;290(36):22049-60. Epub 2015 Jul 20.

Bone and Cancer Group, Edinburgh Cancer Research Centre, Western General Hospital, University of Edinburgh, Edinburgh EH4 2XR, United Kingdom, From the Academic Unit of Bone Biology, Mellanby Centre for Bone Research, Department of Human Metabolism, Medical School, Beech Hill Road, Sheffield S10 2RX, United Kingdom,

The cannabinoid type 2 receptor (CB2) has previously been implicated as a regulator of tumor growth, bone remodeling, and bone pain. However, very little is known about the role of the skeletal CB2 receptor in the regulation of osteoblasts and osteoclasts changes associated with breast cancer. Here we found that the CB2-selective agonists HU308 and JWH133 reduced the viability of a variety of parental and bone-tropic human and mouse breast cancer cells at high micromolar concentrations. Under conditions in which these ligands are used at the nanomolar range, HU308 and JWH133 enhanced human and mouse breast cancer cell-induced osteoclastogenesis and exacerbated osteolysis, and these effects were attenuated in cultures obtained from CB2-deficient mice or in the presence of a CB2 receptor blocker. HU308 and JWH133 had no effects on osteoblast growth or differentiation in the presence of conditioned medium from breast cancer cells, but under these circumstances both agents enhanced parathyroid hormone-induced osteoblast differentiation and the ability to support osteoclast formation. Mechanistic studies in osteoclast precursors and osteoblasts showed that JWH133 and HU308 induced PI3K/AKT activity in a CB2-dependent manner, and these effects were enhanced in the presence of osteolytic and osteoblastic factors such as RANKL (receptor activator of NFκB ligand) and parathyroid hormone. When combined with published work, these findings suggest that breast cancer and bone cells exhibit differential responses to treatment with CB2 ligands depending upon cell type and concentration used. We, therefore, conclude that both CB2-selective activation and antagonism have potential efficacy in cancer-associated bone disease, but further studies are warranted and ongoing.
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http://dx.doi.org/10.1074/jbc.M115.649608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571957PMC
September 2015

Targeted sequencing of the Paget's disease associated 14q32 locus identifies several missense coding variants in RIN3 that predispose to Paget's disease of bone.

Hum Mol Genet 2015 Jun 20;24(11):3286-95. Epub 2015 Feb 20.

Rheumatology and Bone Disease Section, Centre for Genomic & Experimental Medicine

Paget's disease of bone (PDB) is a common disorder with a strong genetic component characterized by increased but disorganized bone remodelling. Previous genome-wide association studies identified a locus on chromosome 14q32 tagged by rs10498635 which was significantly associated with susceptibility to PDB in several European populations. Here we conducted fine-mapping and targeted sequencing of the candidate locus to identify possible functional variants. Imputation in 741 PDB patients and 2699 controls confirmed that the association was confined to a 60 kb region in the RIN3 gene and conditional analysis adjusting for rs10498635 identified no new independent signals. Sequencing of the RIN3 gene identified a common missense variant (p.R279C) that was strongly associated with the disease (OR = 0.64; P = 1.4 × 10(-9)), and was in strong linkage disequilibrium with rs10498635. A further 13 rare missense variants were identified, seven of which were novel and detected only in PDB cases. When combined, these rare variants were over-represented in cases compared with controls (OR = 3.72; P = 8.9 × 10(-10)). Most rare variants were located in a region that encodes a proline-rich, intrinsically disordered domain of the protein and many were predicted to be pathogenic. RIN3 was expressed in bone tissue and its expression level was ∼10-fold higher in osteoclasts compared with osteoblasts. We conclude that susceptibility to PDB at the 14q32 locus is mediated by a combination of common and rare coding variants in RIN3 and suggest that RIN3 may contribute to PDB susceptibility by affecting osteoclast function.
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http://dx.doi.org/10.1093/hmg/ddv068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424954PMC
June 2015

Quantitative analysis of bone and soft tissue by micro-computed tomography: applications to ex vivo and in vivo studies.

Bonekey Rep 2014 20;3:564. Epub 2014 Aug 20.

Rheumatic Diseases Unit, MRC Institute of Genetics and Molecular Medicine, Centre for Genomic and Experimental Medicine, Western General Hospital, University of Edinburgh , Edinburgh, UK.

Micro-computed tomography (micro-CT) is a high-resolution imaging modality that is capable of analysing bone structure with a voxel size on the order of 10 μm. With the development of in vivo micro-CT, where disease progression and treatment can be monitored in a living animal over a period of time, this modality has become a standard tool for preclinical assessment of bone architecture during disease progression and treatment. For meaningful comparison between micro-CT studies, it is essential that the same parameters for data acquisition and analysis methods be used. This protocol outlines the common procedures that are currently used for sample preparation, scanning, reconstruction and analysis in micro-CT studies. Scan and analysis methods for trabecular and cortical bone are covered for the femur, tibia, vertebra and the full neonate body of small rodents. The analysis procedures using the software provided by ScancoMedical and Bruker are discussed, and the routinely used bone architectural parameters are outlined. This protocol also provides a section dedicated to in vivo scanning and analysis, which covers the topics of anaesthesia, radiation dose and image registration. Because of the expanding research using micro-CT to study other skeletal sites, as well as soft tissues, we also provide a review of current techniques to examine the skull and mandible, adipose tissue, vasculature, tumour severity and cartilage. Lists of recommended further reading and literature references are included to provide the reader with more detail on the methods described.
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http://dx.doi.org/10.1038/bonekey.2014.59DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140449PMC
September 2014

Rodent models of osteoporosis.

Bonekey Rep 2014 10;3:614. Epub 2014 Dec 10.

Muscular-skeletal pharmacology, Academic Unit of Bone Biology, Mellanby Centre for Bone Research, Department of human metabolism, University of Sheffield , Sheffield, UK.

The aim of this protocol is to provide a detailed description of male and female rodent models of osteoporosis. In addition to indications on the methods of performing the surgical procedures, the choice of reliable and safe anaesthetics is also described. Post-operative care, including analgesia administration for pain management, is also discussed. Ovariectomy in rodents is a procedure where ovaries are surgically excised. Hormonal changes resulting from ovary removal lead to an oestrogen-deprived state, which enhances bone remodelling, causes bone loss and increases bone fracture risk. Therefore, ovariectomy has been considered as the most common preclinical model for understanding the pathophysiology of menopause-associated events and for developing new treatment strategies for tackling post-menopausal osteoporosis. This protocol also provides a detailed description of orchidectomy, a model for androgen-deficient osteoporosis in rodents. Endocrine changes following testes removal lead to hypogonadism, which results in accelerated bone loss, increasing osteoporosis risk. Orchidectomised rodent models have been proposed to mimic male osteoporosis and therefore remain a valuable tool for understanding androgen deficiency in aged men. Although it would have been particularly difficult to assemble an internationally acceptable description of surgical procedures, here we have attempted to provide a comprehensive guide for best practice in performing ovariectomy and orchidectomy in laboratory rodents. Research scientists are reminded that they should follow their own institution's interpretation of such guidelines. Ultimately, however, all animal procedures must be overseen by the local Animal Welfare and Ethical Review Body and conducted under licences approved by a regulatory ethics committee.
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http://dx.doi.org/10.1038/bonekey.2014.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4388108PMC
April 2015

Modulation of strain-specific differences in gene expression by cannabinoid type 2 receptor deficiency.

Calcif Tissue Int 2014 Apr 27;94(4):423-32. Epub 2013 Dec 27.

Rheumatology and Bone Research Group, Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK.

Previous studies have shown that the skeletal consequences of cannabinoid receptor deficiency differ in different strains of mice. In order to explore the mechanisms responsible, we analysed global gene expression in bone from wild-type CD1 mice and littermates with targeted inactivation of the type 2 cannabinoid receptor (Cnr2 (-/-)) and compared the results with those obtained from a similar analysis of wild-type and Cnr2 (-/-) C57BL/6 mice. Trabecular bone volume was increased in Cnr2 (-/-) CD1 mice compared with wild-type littermates but decreased in Cnr2 (-/-) C57BL/6 mice. Microarray analysis identified 354 genes in which substantial differences in gene expression (>1.5-fold) were observed that were specifically affected by Cnr2 deficiency. Bioinformatic analysis of data from wild-type mice of each strain revealed Cnr2-dependent differences in expression of genes clustering within the gene ontology (GO) terms immune response (p < 0.0001), positive regulation of response to stimulus (p < 0.0001), nucleotide binding (p = 0.002), and ribonucleotide binding (p = 0.003). Bioinformatic analysis of data from Cnr2 (-/-) mice of each strain revealed associations between GO terms corresponding to the extracellular region (p = 0.002), the cell surface (p = 0.02), antigen binding (p = 0.03), external side of plasma membrane (p = 0.04), and regulation of the force of heart contraction (p = 0.04). We conclude that Cnr2 deficiency affects expression of a large number of genes in different strains of mice, and that these differences are likely to be responsible in part for the differences in skeletal phenotype that we and others have observed in mice with defective cannabinoid receptor signalling in different genetic backgrounds.
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http://dx.doi.org/10.1007/s00223-013-9823-6DOI Listing
April 2014

Genetic background modifies the effects of type 2 cannabinoid receptor deficiency on bone mass and bone turnover.

Calcif Tissue Int 2014 Mar 14;94(3):259-68. Epub 2013 Sep 14.

Rheumatic Diseases Unit, Centre for Molecular Medicine, MRC Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, EH4 2XU, UK.

Cannabinoid receptors and their ligands play significant roles in regulating bone metabolism. Previous studies of type 1 cannabinoid receptor-deficient mice have shown that genetic background influences the skeletal phenotype. Here, we investigated the effects of genetic background on the skeletal phenotype of mice with type 2 cannabinoid receptor deficiency (Cnr2 (-/-)). We studied Cnr2 (-/-) mice on a CD1 background and compared the findings with those previously reported in Cnr2 (-/-) C57BL/6 mice. Young female Cnr2 (-/-) CD1 mice had low bone turnover and high trabecular bone mass compared with wild-type (WT), contrasting with the situation in Cnr2 (-/-) C57BL/6 mice where trabecular bone mass has been reported to be similar to WT. The Cnr2 (-/-) CD1 mice lost more trabecular bone at the tibia with age than WT due to reduced bone formation, and at 12 months there was no difference in trabecular bone volume between genotypes. This differs from the phenotype previously reported in C57BL/6 Cnr2 (-/-) mice, where bone turnover is increased and bone mass reduced with age. There were no substantial differences in skeletal phenotype between Cnr2 (-/-) and WT in male mice. Cortical bone phenotype was similar in Cnr2 (-/-) and WT mice of both genders. Deficiency of Cnr2 has site- and gender-specific effects on the skeleton, mainly affecting trabecular bone, which are influenced by genetic differences between mouse strains. Further evaluation of the pathways responsible might yield new insights into the mechanisms by which cannabinoid receptors regulate bone metabolism.
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http://dx.doi.org/10.1007/s00223-013-9793-8DOI Listing
March 2014

Selective tyrosine kinase inhibition of insulin-like growth factor-1 receptor inhibits human and mouse breast cancer-induced bone cell activity, bone remodeling, and osteolysis.

J Bone Miner Res 2013 May;28(5):1229-42

Bone and Cancer Group, Edinburgh Cancer Research Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, UK.

Insulin-like growth factor 1 (IGF-1) plays an important role in both bone metabolism and breast cancer. In this study, we investigated the effects of the novel IGF-1 receptor tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo[1,5-a]pyrazin-8-ylamine (PQIP) on osteolytic bone disease associated with breast cancer. Human MDA-MB-231 and mouse 4T1 breast cancer cells enhanced osteoclast formation in receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) stimulated bone marrow cultures, and these effects were significantly inhibited by PQIP. Functional studies in osteoclasts showed that PQIP inhibited both IGF-1 and conditioned medium-induced osteoclast formation by preventing phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) activation without interfering with RANKL or M-CSF signaling. Treatment of osteoblasts with PQIP significantly inhibited the increase in RANKL/osteoprotegerin (OPG) ratio by IGF-1 and conditioned medium and totally prevented conditioned medium-induced osteoclast formation in osteoblast-bone marrow (BM) cell cocultures, thereby suggesting an inhibitory effect on osteoblast-osteoclast coupling. PQIP also inhibited IGF-1-induced osteoblast differentiation, spreading, migration, and bone nodule formation. Treatment with PQIP significantly reduced MDA-MB-231 conditioned medium-induced osteolytic bone loss in a mouse calvarial organ culture system ex vivo and in adult mice in vivo. Moreover, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following 4T1 intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in bone resorption and formation indices, indicative of a reduced rate of cancer-associated bone turnover. We conclude that inhibition of IGF-1 receptor tyrosine kinase activity by PQIP suppresses breast cancer-induced bone turnover and osteolysis. Therefore, PQIP, and its novel derivatives that are currently in advanced clinical development for the treatment of a number of solid tumors, may be of value in the treatment of osteolytic bone disease associated with breast cancer.
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http://dx.doi.org/10.1002/jbmr.1847DOI Listing
May 2013

The type 2 cannabinoid receptor regulates bone mass and ovariectomy-induced bone loss by affecting osteoblast differentiation and bone formation.

Endocrinology 2011 Jun 29;152(6):2141-9. Epub 2011 Mar 29.

Rheumatic Diseases Unit, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK.

The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass and bone turnover but the mechanisms responsible are incompletely understood. In this study we investigated the role that the CB2 pathway plays in bone metabolism using a combination of genetic and pharmacological approaches. Bone mass and turnover were normal in young mice with targeted inactivation of CB2 receptor (CB2(-/-)), but by 12 months of age, they had developed high-turnover osteoporosis with relative uncoupling of bone resorption from bone formation. Primary osteoblasts from CB2(-/-) mice had a reduced capacity to form bone nodules in vitro when compared with cells from wild-type littermates and also had impaired PTH-induced alkaline phosphatase (ALP) activity. The CB2-selective agonist HU308 stimulated bone nodule formation in wild-type osteoblasts but had no effect in CB2(-/-) osteoblasts. Further studies in MC3T3-E1 osteoblast like cells showed that HU308 promoted cell migration and activated ERK phosphorylation, and these effects were blocked by the CB2 selective inverse agonist AM630. Finally, HU308 partially protected against ovariectomy induced bone loss in wild-type mice in vivo, primarily by stimulating bone formation, whereas no protective effects were observed in ovariectomized CB2(-/-) mice. These studies indicate that the CB2 regulates osteoblast differentiation in vitro and bone formation in vivo.
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http://dx.doi.org/10.1210/en.2010-0930DOI Listing
June 2011

Cannabinoid receptor type 1 protects against age-related osteoporosis by regulating osteoblast and adipocyte differentiation in marrow stromal cells.

Cell Metab 2009 Aug;10(2):139-47

Rheumatic Diseases Unit, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK.

Age-related osteoporosis is characterized by reduced bone formation and accumulation of fat in the bone marrow compartment. Here, we report that the type 1 cannabinoid receptor (CB1) regulates this process. Mice with CB1 deficiency (CB1(-/-)) had increased peak bone mass due to reduced bone resorption, but developed age-related osteoporosis with reduced bone formation and accumulation of adipocytes in the bone marrow space. Marrow stromal cells from CB1(-/-) mice had an enhanced capacity for adipocyte differentiation, a reduced capacity for osteoblast differentiation, and increased expression of phosphorylated CREB (pCREB) and PPARgamma. Pharmacological blockade of CB1 receptors stimulated adipocyte differentiation, inhibited osteoblast differentiation, and increased cAMP and pCREB in osteoblast and adipocyte precursors. The CB1 receptor is therefore unique in that it regulates peak bone mass through an effect on osteoclast activity, but protects against age-related bone loss by regulating adipocyte and osteoblast differentiation of bone marrow stromal cells.
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http://dx.doi.org/10.1016/j.cmet.2009.07.006DOI Listing
August 2009

Regulation of bone mass, osteoclast function, and ovariectomy-induced bone loss by the type 2 cannabinoid receptor.

Endocrinology 2008 Nov 17;149(11):5619-26. Epub 2008 Jul 17.

Rheumatic Diseases Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom.

The endocannabinoid system has recently been shown to play a role in the regulation of bone metabolism. The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass, but conflicting results have been reported with regard to its effects on bone resorption and osteoclast function. Here we investigated the role that CB2 plays in regulating bone mass and osteoclast function using a combination of pharmacological and genetic approaches. The CB2-selective antagonist/inverse agonist AM630 inhibited osteoclast formation and activity in vitro, whereas the CB2-selective agonists JWH133 and HU308 stimulated osteoclast formation. Osteoclasts generated from CB2 knockout mice (CB2-/-) were resistant to the inhibitory effects of AM630 in vitro, consistent with a CB2-mediated effect. There was no significant difference in peak bone mass between CB2-/- mice and wild-type littermates, but after ovariectomy, bone was lost to a greater extent in wild-type compared with CB2-/- mice. Furthermore, AM630 protected against bone loss in wild-type mice, but the effect was blunted in CB2-/- mice. We conclude that CB2 regulates osteoclast formation and bone resorption in vitro and that under conditions of increased bone turnover, such as after ovariectomy, CB2 regulates bone loss. These observations indicate that CB2 regulates osteoclast formation and contributes to ovariectomy-induced bone loss and demonstrate that cannabinoid receptor antagonists/inverse agonists may be of value in the treatment of bone diseases characterized by increased osteoclast activity.
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http://dx.doi.org/10.1210/en.2008-0150DOI Listing
November 2008