Open source software for semi-automated histomorphometry of bone resorption and formation parameters

Micro-CT analysis has become the standard method for assessing bone volume and architecture in small animals. However, micro-CT does not allow the assessment of bone turnover parameters such as bone formation rate and osteoclast (OC) number and surface. For these crucial variables histomorphometric analysis is still an essential technique. Histomorphometry however, is time consuming and, especially in mouse bones, OCs can be difficult to detect. The main purpose of this study was to develop and validate a relatively easy and rapid method to measure static and dynamic bone histomorphometry parameters. Here we present the adaptation of established staining protocols and three novel open source image analysis packages: TrapHisto, OsteoidHisto and CalceinHisto that allow rapid, semi-automated analysis of histomorphometric bone resorption, osteoid, and calcein double labelling parameters respectively. These three programs are based on ImageJ, but use a relatively simple user interface that hides the underlying complexity of the image analysis

Analysis of Bone Architecture in Rodents Using Micro-Computed Tomography.

This chapter describes the use of micro-computed tomography scanning for analyzing bone structure, focussing on rodent bone. It discusses sample preparation, the correct setup of the scanner, the impact of some of the important scanner settings and new applications

Analysis of Nkx3.1:Cre-driven Erk5 deletion reveals a profound spinal deformity which is linked to increased osteoclast activity

Extracellular signal-regulated protein kinase 5 (ERK5) has been implicated during development and carcinogenesis. Nkx3.1-mediated Cre expression is a useful strategy to genetically manipulate the mouse prostate. While grossly normal at birth, we observed an unexpected phenotype of spinal protrusion in Nkx3.1:Cre;Erk5fl/fl (Erk5fl/fl) mice by ~6–8 weeks of age. X-ray, histological and micro CT (µCT) analyses showed that 100% of male and female Erk5fl/fl mice had a severely deformed curved thoracic spine, with an associated loss of trabecular bone volume. Although sex-specific differences were observed, histomorphometry measurements revealed that both bone resorption and bone formation parameters were increased in male Erk5fl/fl mice compared to wild type (WT) littermates. Osteopenia occurs where the rate of bone resorption exceeds that of bone formation, so we investigated the role of the osteoclast compartment. We found that treatment of RANKL-stimulated primary bone marrow-derived macrophage (BMDM) cultures with small molecule ERK5 pathway inhibitors increased osteoclast numbers. Furthermore, osteoclast numbers and expression of osteoclast marker genes were increased in parallel with reduced Erk5 expression in cultures generated from Erk5fl/fl mice compared to WT mice. Collectively, these results reveal a novel role for Erk5 during bone maturation and homeostasis in vivo

Reduced expression of PML predisposes to Paget's disease of bone by increasing osteoclast differentiation and bone resorption

Paget's disease of bone (PDB) is characterized by focal increases in bone remodelling. Genome-wide association studies identified a susceptibility locus for PDB tagged by rs5742915, which is located within the PML gene. Here, we have assessed the candidacy of PML as the predisposing gene for PDB at this locus. We found that the PDB-risk allele of rs5742915 was associated with lower PML expression and that PML expression in blood cells from individuals with PDB was lower than in controls. The differentiation, survival and resorptive activity of osteoclasts prepared from Pml-/- mice was increased compared with wild type. Furthermore, the inhibitory effect of IFN-γ on osteoclast formation from Pml-/- was significantly blunted compared with wild type. Bone nodule formation was also increased in osteoblasts from Pml-/- mice when compared with wild type. Although microCT analysis of trabecular bone showed no differences between Pml-/- mice and wild type, bone histomorphometry showed that Pml-/- mice had high bone turnover with increased indices of bone resorption and increased mineral apposition rate. These data indicate that reduced expression of PML predisposes an individual to PDB and identify PML as a novel regulator of bone metabolism. This article has an associated First Person interview with the first author of the paper

Zoledronic acid prevents pagetic-like lesions and accelerated bone loss in the p62^P394L mouse model of Paget's disease

Paget's disease of bone (PDB) is an age-related metabolic bone disorder, characterised by focally increased and disorganised bone remodelling initiated by abnormal and hyperactive osteoclasts. The germline P392L mutation of SQSTM1 (encoding p62) is a strong genetic risk factor for PDB in humans, and the equivalent mutation in mice (P394L) causes a PDB-like disorder. However, it is unclear why pagetic lesions become more common with age. Here, we assessed the effect of the p62 P394L mutation on osteoclastogenesis and bone morphometry in relation to ageing, the natural history of lesion progression in p62P394L mice and the effect of zoledronic acid (ZA) on lesion development. p62P394L+/+ osteoclast precursors had increased sensitivity to RANKL (also known as TNFSF11) compared with wild-type (WT) cells, and the sensitivity further increased in both genotypes with ageing. Osteoclastogenesis from 12-month-old p62P394L+/+ mice was twofold greater than that from 3-month-old p62P394L+/+ mice (P<0.001) and three-fold greater than that from age-matched WT littermates. The p62P394L+/+ mice lost 33% more trabecular bone volume in the long bones by 12 months compared with WT mice (P<0.01), and developed pagetic-like lesions in the long bones which progressed with ageing. ZA prevented the development of pagetic-like lesions, and increased trabecular bone volume tenfold compared with vehicle by 12 months of age (P<0.01). This demonstrates that ageing has a pro-osteoclastogenic effect, which is further enhanced by the p62 P394L mutation, providing an explanation for the increased penetrance of bone lesions with age in this model. Lesions are prevented by ZA, providing a rationale for early intervention in humans

Investigating the role of optineurin in bone biology and Paget's disease of bone

Paget’s disease of bone (PDB) is a common disease with a strong genetic component. Approaches such as linkage analysis and candidate gene studies have shown that mutations in Sequestosome 1 (SQSTM1) explain up to 40% of familial cases and 10% of sporadic cases, however the majority of PDB patients have no mutations in this gene. Genome-wide association studies (GWAS) have recently identified new susceptibility loci for PDB including variants at CSF1, TNFRSF11A, OPTN, TM7SF4, PML, NUP205 and RIN3 loci. These loci were confirmed to be associated with PDB in various European populations. OPTN encodes optineurin, a widely expressed protein involved in many cellular processes but its role in bone metabolism is yet unknown. The aim of this PhD thesis was to investigate the role of OPTN in bone metabolism and PDB using in vitro and in vivo studies. In chapter 3, the OPTN rs1561570 identified by previous GWAS was examined for its association with the severity and clinical outcome of PDB in patients without SQSTM1 mutations. The results showed that rs1561570 was significantly associated with total disease severity score so that carriers of the risk allele “T” had higher severity score compared to non-carriers (P < 0.05). A trend for reduced quality of life physical scores (SF36) was also associated with the rs1561570 risk allele, but the relationship was not statistically significant. In order to identify functional variants within OPTN, the coding regions as well as the exon-intron boundaries were sequenced in 24 familial PDB cases and 19 controls. No mutation was found that could be predicted as pathogenic suggesting that disease susceptibility could be mediated by regulatory polymorphisms that influence gene expression. In chapter 4, the role of OPTN was investigated in osteoclast development using in vitro knockdown experiments. Optn was expressed in mouse bone marrow derived macrophages (BMDMs) as well as all stages of osteoclast development and it was significantly increased three days post RANKL treatment. Optn expression was knocked down in BMDMs and cells were induced to form osteoclast in the presence of RANKL and M-CSF. Compared to non-targeted cells, Optn depleted cells formed significantly more and larger osteoclasts (P< 0.05). Optn knockdown was also found to enhance osteoclast survival as well as RANKL-induced NFκB activation. In chapter 5, the role of OPTN was investigated in vitro from cells obtained from knock in mice with a loss-of-function mutation in Optn (OptnD477N/D477N). In agreement with the in vitro knockdown experiments, osteoclasts were significantly higher and larger in mutant mice compared to WT and the NF-B activity measured by luciferase reporter assay was significantly higher in cells from OptnD477N/D477N compared to WT during most stages of osteoclast development. OPTN from mutant and WT mice was co-precipitated with its CYLD binding-partner, which acts as a negative regulator to RANK signalling by inhibiting the TRAF6 downstream signalling. The data from this immunoprecipitation (IP) experiment revealed that defective OPTN interacted less with CYLD from mutant mice compared to WT. This study also showed that OPTN was expressed in osteoblasts and the expression rate did not change during osteoblast development. The data obtained from the mineralization assay revealed no significant difference between OptnD477N/D477N and WT. In chapter 6, I investigated the effect of the D477N loss of function mutation in Optn on bone metabolism. Bone Histomorphometrical analysis of OptnD477N/D477N mice showed higher bone resorption parameters (Oc.N/BS and Oc.S/BS) compared to wild type (WT). Osteoid analysis showed evidence of increased bone formation parameters (OS/BS and OV/BV) in mutant mice compared to WT. Calcein labelling showed a significant difference in mineral apposition rate (MAR) from mutant mice compared to WT. Analysis of serum biomarkers of bone turnover showed evidence of enhanced bone turnover in mutant mice compared to WT. Micro computed tomography (μCT) analysis of 4 and 14 months old mice showed no significant differences in bone morphology between WT and OptnD477N/D477N mice of both sexes. In conclusion, this study has shown for the first time that OPTN plays a role in regulating bone turnover by acting as a negative regulator of osteoclast differentiation. The data obtained from this study strongly suggest the crucial role of OPTN in RANK signalling. The effect of OPTN on osteoblast activity may be direct or indirect compensation for increased osteoclast activity. Further detailed studies will be required to explore the underlying mechanism of OPTN including downstream RANK signalling and a complete knockout model to corroborate these findings

Targeted Inactivation of Rin3 Increases Trabecular Bone Mass by Reducing Bone Resorption and Favouring Bone Formation

AbstractCommon genetic variants at the RIN3 locus on chromosome 14q32 predispose to Paget’s disease of bone (PDB) but the mechanisms by which they do so are unknown. Here, we analysed the skeletal phenotype of female mice with targeted inactivation of the mouse Rin3 gene (Rin3−/−) as compared with wild-type littermates. The Rin3−/− mice had higher trabecular bone volume (BV/TV%) compared with wild type. Mean ± standard deviation values at the distal femur at 8 weeks were 9.0 ± 2.5 vs. 7.0 ± 1.5 (p = 0.002) and at 52 weeks were 15.8 ± 9.5 vs. 8.5 ± 4.2 (p = 0.002). No differences were observed in femoral cortical bone parameters with the exception of marrow diameter which was significantly smaller in 52-week-old Rin3−/− mice compared to wild type: (0.43 mm ± 0.1 vs. 0.57 mm ± 0.2 (p = 0.001). Bone histomorphometry showed a lower osteoclast surface / bone surface (Oc.S/BS%) at 8 weeks in Rin3−/− mice compared to wild type (24.1 ± 4.7 vs. 29.7 ± 6.6; p = 0.025) but there were no significant differences in markers of bone formation at this time. At 52 weeks, Oc.S/BS did not differ between genotypes but single labelled perimeter (SL.Pm/B.Pm (%)) was significantly higher in Rin3−/− mice (24.4 ± 6.4 vs. 16.5 ± 3.8, p = 0.003). We conclude that Rin3 negatively regulates trabecular bone mass in mice by inhibiting osteoclastic bone resorption and favouring bone formation. Our observations also suggest that the variants that predispose to PDB in humans probably do so by causing a gain-in-function of RIN3.</jats:p

A comprehensive candidate gene approach identifies genetic variation associated with osteosarcoma

<p>Abstract</p> <p>Background</p> <p>Osteosarcoma (OS) is a bone malignancy which occurs primarily in adolescents. Since it occurs during a period of rapid growth, genes important in bone formation and growth are plausible modifiers of risk. Genes involved in DNA repair and ribosomal function may contribute to OS pathogenesis, because they maintain the integrity of critical cellular processes. We evaluated these hypotheses in an OS association study of genes from growth/hormone, bone formation, DNA repair, and ribosomal pathways.</p> <p>Methods</p> <p>We evaluated 4836 tag-SNPs across 255 candidate genes in 96 OS cases and 1426 controls. Logistic regression models were used to estimate the odds ratios (OR) and 95% confidence intervals (CI).</p> <p>Results</p> <p>Twelve SNPs in growth or DNA repair genes were significantly associated with OS after Bonferroni correction. Four SNPs in the DNA repair gene <it>FANCM </it>(ORs 1.9-2.0, <it>P </it>= 0.003-0.004) and 2 SNPs downstream of the growth hormone gene <it>GH1 </it>(OR 1.6, <it>P </it>= 0.002; OR 0.5, <it>P </it>= 0.0009) were significantly associated with OS. One SNP in the region of each of the following genes was significant: <it>MDM2</it>, <it>MPG</it>, <it>FGF2</it>, <it>FGFR3</it>, <it>GNRH2</it>, and <it>IGF1</it>.</p> <p>Conclusions</p> <p>Our results suggest that several SNPs in biologically plausible pathways are associated with OS. Larger studies are required to confirm our findings.</p

Identification of a novel locus on chromosome 2q13, which predisposes to clinical vertebral fractures independently of bone density.

OBJECTIVES: To identify genetic determinants of susceptibility to clinical vertebral fractures, which is an important complication of osteoporosis. METHODS: Here we conduct a genome-wide association study in 1553 postmenopausal women with clinical vertebral fractures and 4340 controls, with a two-stage replication involving 1028 cases and 3762 controls. Potentially causal variants were identified using expression quantitative trait loci (eQTL) data from transiliac bone biopsies and bioinformatic studies. RESULTS: A locus tagged by rs10190845 was identified on chromosome 2q13, which was significantly associated with clinical vertebral fracture (P=1.04×10-9) with a large effect size (OR 1.74, 95% CI 1.06 to 2.6). Bioinformatic analysis of this locus identified several potentially functional SNPs that are associated with expression of the positional candidate genes TTL (tubulin tyrosine ligase) and SLC20A1 (solute carrier family 20 member 1). Three other suggestive loci were identified on chromosomes 1p31, 11q12 and 15q11. All these loci were novel and had not previously been associated with bone mineral density or clinical fractures. CONCLUSION: We have identified a novel genetic variant that is associated with clinical vertebral fractures by mechanisms that are independent of BMD. Further studies are now in progress to validate this association and evaluate the underlying mechanism

Toward the library generation of natural product-like polycyclic derivatives by stereocontrolled diversity-oriented synthesis

Due to the growing interest in small molecules that could help in understanding protein-protein interactions based on signal transduction, the demand for the generation of small-molecule libraries that are inspired by bioactive natural products has grown significantly. Many of these pathways are highly complex and present tremendous challenges with the use of classical tools. A rapid access to natural product-like small molecules having structural complexity and diversity is crucial for systematically dissecting the functions of complex protein networking and understanding cell signaling pathways. The complex nature, the three-dimensional architecture, and the number of protein binding functional groups presented in three-dimensional arrays are some of the attractive features to incorporate in smallmolecule chemical probes to be used as modulators of protein function.NRC publication: Ye