John G Logan

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites

Artemisinin‐ and artesunate‐resistant Plasmodium chabaudi mutants, AS‐ART and AS‐ATN, were previously selected from chloroquine‐resistant clones AS‐30CQ and AS‐15CQ respectively. Now, a genetic cross between AS‐ART and the artemisinin‐sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS‐30CQ and AS‐ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS‐ATN to artemisinin derivates, the other cannot account solely for the resistance of AS‐ART, relative to the responses of its sensitive progenitor AS‐30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug‐response phenotype was not genetically stable. No mutations in the UBP‐1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.

Identification of 153 new loci associated with heel bone mineral density and functional involvement of GPC6 in osteoporosis

Osteoporosis is a common disease diagnosed primarily by measurement of bone mineral density (BMD). We undertook a genome-wide association study (GWAS) in 142,487 individuals from the UK Biobank to identify loci associated with BMD as estimated by quantitative ultrasound of the heel. We identified 307 conditionally independent single-nucleotide polymorphisms (SNPs) that attained genome-wide significance at 203 loci, explaining approximately 12% of the phenotypic variance. These included 153 previously unreported loci, and several rare variants with large effect sizes. To investigate the underlying mechanisms, we undertook (1) bioinformatic, functional genomic annotation and human osteoblast expression studies; (2) gene-function prediction; (3) skeletal phenotyping of 120 knockout mice with deletions of genes adjacent to lead independent SNPs; and (4) analysis of gene expression in mouse osteoblasts, osteocytes and osteoclasts. The results implicate GPC6 as a novel determinant of BMD, and also identify abnormal skeletal phenotypes in knockout mice associated with a further 100 prioritized genes.

Hydrogen sulphide‐releasing diclofenac derivatives inhibit breast cancer‐induced osteoclastogenesis in vitro and prevent osteolysis ex vivo

BACKGROUND AND PURPOSE Hydrogen sulphide (H2S) and prostaglandins are both involved in inflammation, cancer and bone turnover, and non‐steroidal anti‐inflammatory drugs (NSAIDs) and H2S donors exhibit anti‐inflammatory and anti‐tumour properties. H2S‐releasing diclofenac (S‐DCF) derivatives are a novel class of NSAIDs combining the properties of a H2S donor with those of a conventional NSAID.

Advanced Bone Formation in Mice with a Dominant-negative Mutation in the Thyroid Hormone Receptor β Gene due to Activation of Wnt/β-Catenin Protein Signaling

Background: Thyroid hormone (T3) regulates skeletal development by unknown mechanisms. Results: Canonical Wnt signaling was inhibited by T3 in osteoblastic cells, but activated in ThrbPV/PV mutant mice that display advanced bone formation. Conclusion: The mutant thyroid hormone receptor (TRβPV) activates skeletal Wnt signaling in vivo. Significance: T3 and Wnt signaling pathways interact during bone development. Thyroid hormone (T3) acts in chondrocytes and bone-forming osteoblasts to control bone development and maintenance, but the signaling pathways mediating these effects are poorly understood. ThrbPV/PV mice have a severely impaired pituitary-thyroid axis and elevated thyroid hormone levels due to a dominant-negative mutant T3 receptor (TRβPV) that cannot bind T3 and interferes with the actions of wild-type TR. ThrbPV/PV mice have accelerated skeletal development due to unknown mechanisms. We performed microarray studies in primary osteoblasts from wild-type mice and ThrbPV/PV mice. Activation of the canonical Wnt signaling in ThrbPV/PV mice was confirmed by in situ hybridization analysis of Wnt target gene expression in bone during postnatal growth. By contrast, T3 treatment inhibited Wnt signaling in osteoblastic cells, suggesting that T3 inhibits the Wnt pathway by facilitating proteasomal degradation of β-catenin and preventing its accumulation in the nucleus. Activation of the Wnt pathway in ThrbPV/PV mice, however, results from a gain of function for TRβPV that stabilizes β-catenin despite the presence of increased thyroid hormone levels. These studies demonstrate novel interactions between T3 and Wnt signaling pathways in the regulation of skeletal development and bone formation.

Generation and culture of osteoclasts

Osteoclasts are highly specialized cells of haematopoietic lineage that are uniquely responsible for bone resorption. In the past, osteoclasts were isolated as mature cells from chicken long bones, or were generated using osteoblasts or stromal cells to induce osteoclast formation in total bone marrow from mice or rabbits. The Copernican revolution in osteoclast biology began with the identification of macrophage-colony stimulating factor (M-CSF) and receptor activator NFκB-ligand (RANKL ) as the key regulators of osteoclast formation, fusion and function. The availability of recombinant human and mouse M-CSF and RANKL has enabled researchers to reliably generate osteoclasts from primary monocyte/macrophage cells as well as from cell lines such as RAW 264.7. This article summarizes the most commonly used procedures for the isolation, generation and characterization of human, rodent and chicken osteoclasts in vitro. Lists of further reading and recommendations are included to facilitate a successful application by the reader.

Thyrostimulin Regulates Osteoblastic Bone Formation During Early Skeletal Development

The ancestral glycoprotein hormone thyrostimulin is a heterodimer of unique glycoprotein hormone subunit alpha (GPA)2 and glycoprotein hormone subunit beta (GPB)5 subunits with high affinity for the TSH receptor. Transgenic overexpression of GPB5 in mice results in cranial abnormalities, but the role of thyrostimulin in bone remains unknown. We hypothesized that thyrostimulin exerts paracrine actions in bone and determined: 1) GPA2 and GPB5 expression in osteoblasts and osteoclasts, 2) the skeletal consequences of thyrostimulin deficiency in GPB5 knockout (KO) mice, and 3) osteoblast and osteoclast responses to thyrostimulin treatment. Gpa2 and Gpb5 expression was identified in the newborn skeleton but declined rapidly thereafter. GPA2 and GPB5 mRNAs were also expressed in primary osteoblasts and osteoclasts at varying concentrations. Juvenile thyrostimulin-deficient mice had increased bone volume and mineralization as a result of increased osteoblastic bone formation. However, thyrostimulin failed to induce a canonical cAMP response or activate the noncanonical Akt, ERK, or mitogen-activated protein kinase (P38) signaling pathways in primary calvarial or bone marrow stromal cell-derived osteoblasts. Furthermore, thyrostimulin did not directly inhibit osteoblast proliferation, differentiation or mineralization in vitro. These studies identify thyrostimulin as a negative but indirect regulator of osteoblastic bone formation during skeletal development.

Mice Lacking the Calcineurin Inhibitor Rcan2 Have an Isolated Defect of Osteoblast Function

Calcineurin-nuclear factor of activated T cells signaling controls the differentiation and function of osteoclasts and osteoblasts, and regulator of calcineurin-2 (Rcan2) is a physiological inhibitor of this pathway. Rcan2 expression is regulated by T(3), which also has a central role in skeletal development and bone turnover. To investigate the role of Rcan2 in bone development and maintenance, we characterized Rcan2(-/-) mice and determined its skeletal expression in T(3) receptor (TR) knockout and thyroid-manipulated mice. Rcan2(-/-) mice had normal linear growth but displayed delayed intramembranous ossification, impaired cortical bone formation, and reduced bone mineral accrual during development as well as increased mineralization of adult bone. These abnormalities resulted from an isolated defect in osteoblast function and are similar to skeletal phenotypes of mice lacking the type 2 deiodinase thyroid hormone activating enzyme or with dominant-negative mutations of TRα, the predominant TR isoform in bone. Rcan2 mRNA was expressed in primary osteoclasts and osteoblasts, and its expression in bone was differentially regulated in TRα and TRβ knockout and thyroid-manipulated mice. However, in primary osteoblast cultures, T(3) treatment did not affect Rcan2 mRNA expression or nuclear factor of activated T cells c1 expression and phosphorylation. Overall, these studies establish that Rcan2 regulates osteoblast function and its expression in bone is regulated by thyroid status in vivo.

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.

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.

Bone cell-autonomous contribution of type 2 cannabinoid receptor to breast cancer induced osteolysis

Background: CB2 is implicated in bone remodeling and tumor growth. Results: CB2 activation enhances breast cancer-induced bone cell activity and osteolysis via the PI3K/AKT pathway. Conclusion: CB2-selective antagonism has potential efficacy in cancer-associated bone disease. Significance: CB2 activation by phytocannabinoids might be detrimental in breast cancer patients with advanced malignancy. 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.

Quantitative X-ray microradiography for high-throughput phenotyping of osteoarthritis in mice

Summary Objective To investigate and validate digital X-ray microradiography as a novel, high-throughput and cost-effective screening approach to identify abnormal joint phenotypes in mice. Method Digital X-ray microradiography was used to quantify the subchondral bone mineral content (BMC) in the medial tibial plateau. Accuracy and reproducibility of the method were determined in 22 samples from C57BL/6(B6Brd;B6Dnk;B6N-Tyrc-Brd) wild-type mice. The method was then validated in wild-type mice that had undergone surgical destabilisation of medial meniscus (DMM) and in a genetically modified mouse strain with an established increase in trabecular bone mass. Results The measurement of subchondral BMC by digital X-ray microradiography had a coefficient of variation of 3.6%. Digital X-ray microradiography was able to demonstrate significantly increased subchondral BMC in the medial tibial plateau of male mice 4 and 8 weeks after DMM surgery and in female mice 8 weeks after surgery. Furthermore, digital X-ray microradiography also detected the increase in subchondral BMC in a genetically modified mouse strain with high trabecular bone mass. Conclusion Quantitation of subchondral BMC by digital X-ray microradiography is a rapid, sensitive and cost-effective method to identify abnormal joint phenotypes in mice of both genders at several ages.