Copy Number Variation and Osteoporosis.

PURPOSE OF REVIEW: The purpose of this review is to summarize recent findings on copy number variations and susceptibility to osteoporosis. RECENT FINDINGS: Osteoporosis is highly influenced by genetic factors, including copy number variations (CNVs). The development and accessibility of whole genome sequencing methods has accelerated the study of CNVs and osteoporosis. Recent findings include mutations in novel genes and validation of previously known pathogenic CNVs in monogenic skeletal diseases. Identification of CNVs in genes previously associated with osteoporosis (e.g. RUNX2, COL1A2, and PLS3) has confirmed their importance in bone remodelling. This process has been associated also with the ETV1-DGKB, AGBL2, ATM, and GPR68 genes, identified by comparative genomic hybridisation microarray studies. Importantly, studies in patients with bone pathologies have associated bone disease with the long non-coding RNA LINC01260 and enhancer sequences residing in the HDAC9 gene. Further functional investigation of genetic loci harbouring CNVs associated with skeletal phenotypes will reveal their role as molecular drivers of osteoporosis.

Anaphase-Promoting Complex Subunit 1 Associates with Bone Mineral Density in Human Osteoporotic Bone.

Genome-wide association studies (GWAS) are one of the most common approaches to identify genetic loci that are associated with bone mineral density (BMD). Such novel genetic loci represent new potential targets for the prevention and treatment of fragility fractures. GWAS have identified hundreds of associations with BMD; however, only a few have been functionally evaluated. A locus significantly associated with femoral neck BMD at the genome-wide level is intronic SNP rs17040773 located in the intronic region of the anaphase-promoting complex subunit 1 (ANAPC1) gene (p = 1.5 × 10-9). Here, we functionally evaluate the role of ANAPC1 in bone remodelling by examining the expression of ANAPC1 in human bone and muscle tissues and during the osteogenic differentiation of human primary mesenchymal stem cells (MSCs). The expression of ANAPC1 was significantly decreased 2.3-fold in bone tissues and 6.2-fold in muscle tissue from osteoporotic patients as compared to the osteoarthritic and control tissues. Next, we show that the expression of ANAPC1 changes during the osteogenic differentiation process of human MSCs. Moreover, the silencing of ANAPC1 in human osteosarcoma (HOS) cells reduced RUNX2 expression, suggesting that ANAPC1 affects osteogenic differentiation through RUNX2. Altogether, our results indicate that ANAPC1 plays a role in bone physiology and in the development of osteoporosis.

Zirconia-Toughened Alumina Ceramic Wear Particles Do Not Elicit Inflammatory Responses in Human Macrophages.

Ten percent of patients undergoing total hip arthroplasty (THA) require revision surgery. One of the reasons for THA are wear particles released from the implants that can activate the immune defense and cause osteolysis and failure of the joint implant. The discrepancies between reports on toxicity and immunogenicity of the implant materials led us to this study in which we compared toxicity and immunogenicity of well-defined nanoparticles from Al2O3, zirconia-toughened alumina (ZTA), and cobalt chrome (CoCr), a human THP-1 macrophage cell line, human PBMCs, and therefrom-derived primary macrophages. None of the tested materials decreased the viability of THP-1 macrophages nor human primary macrophages at the 24 h time point, indicating that at concentrations from 0.05 to 50 µm3/cell the tested materials are non-toxic. Forty-eight hours of treatment of THP-1 macrophages with 5 µm3/cell of CoCr and Al2O3 caused 8.3-fold and 4.6-fold increases in TNF-α excretion, respectively, which was not observed for ZTA. The comparison between THP-1 macrophages and human primary macrophages revealed that THP-1 macrophages show higher activation of cytokine expression in the presence of CoCr and Al2O3 particles than primary macrophages. Our results indicate that ZTA is a non-toxic implant material with no immunogenic effects in vitro.

Glucocorticoid Receptor Regulates TNFSF11 Transcription by Binding to Glucocorticoid Responsive Element in TNFSF11 Proximal Promoter Region.

Glucocorticoid osteoporosis is a serious side effect of long term glucocorticoid uptake and it is caused by osteoblast apoptosis and imbalance in the major bone remodeling pathway RANK/RANKL/OPG. The impact of glucocorticoid on the maintenance of RANK/RANKL/OPG is well explored; dexamethasone was shown to disturb the ratio between OPG and RANKL level by decreasing the expression level of OPG and increasing level of RANKL. Here, were aimed to decipher whether glucocorticoid receptor directly influences RANKL promoter activity and its transcriptional regulation. We demonstrate that overexpression of glucocorticoid receptor (GR) NR3C1 increased RANKL promoter activity in human osteosarcoma, cervical cancer (2-fold) and adenocarcinoma cells (4.5-fold). Mutational analysis revealed that +352 site in the RANKL promoter is functional glucocorticoid responsive element (GRE) since the effect of GR on RANKL promoter activity was diminished by mutation at this site. Overexpression of NR3C1 upregulated RANKL mRNA expression 1.5-fold in human A549 and HOS cells. On the other hand silencing of NR3C1 caused slight decrease in RANKL mRNA level, suggesting that NR3C1 directly accounts for RANKL transcriptional regulation. Using electrophoretic mobility shift assay we demonstrate that NR3C1 binds to the proximal RANKL promoter region. Our study provides evidences that NR3C1 directly upregulates RANKL transcription in human cell lines and connects the missing link in the mechanism of RANK/RANKL/OPG imbalance of glucocorticoid induced osteoporosis.

Genetic effects on bone health.

PURPOSE OF REVIEW: In recent years, the lower costs of arrays and sequencing technologies, and the better availability of data from genome-wide association studies (GWASs) have led to more reports on genetic factors that are associated with bone health. However, there remains the need for a summary of the newly identified genetic targets that are associated with bone metabolism, and the status of their functional characterization. RECENT FINDINGS: GWASs revealed dozens of novel genetic loci that are associated with bone mineral density (BMD). Some of these targets have been functionally characterized, although the vast majority have not. Glypican 6, a membrane surface proteoglycan involved in cellular growth control and differentiation, was identified as a novel determinant of BMD and represents a possible drug target for treatment of osteoporosis. Pathway analysis also showed that cell-growth pathways and the SMAD proteins associated with low BMD. SUMMARY: Hits that were significantly associated with BMD in different studies represent likely candidates (e.g. SOST, WNT16, ESR1 and RANKL) for functional characterization and development of osteoporosis treatments. Indeed, currently available treatment for osteoporosis (antibody against RANKL) appeared a significant target in four recent GWAS studies indicating their applicability and importance for future treatment development.

The Intricate Interplay between APOBEC3 Proteins and DNA Tumour Viruses.

APOBEC3 proteins are cytidine deaminases that play a crucial role in the innate immune response against viruses, including DNA viruses. Their main mechanism for restricting viral replication is the deamination of cytosine to uracil in viral DNA during replication. This process leads to hypermutation of the viral genome, resulting in loss of viral fitness and, in many cases, inactivation of the virus. APOBEC3 proteins inhibit the replication of a number of DNA tumour viruses, including herpesviruses, papillomaviruses and hepadnaviruses. Different APOBEC3s restrict the replication of different virus families in different ways and this restriction is not limited to one APOBEC3. Infection with DNA viruses often leads to the development and progression of cancer. APOBEC3 mutational signatures have been detected in various cancers, indicating the importance of APOBEC3s in carcinogenesis. Inhibition of DNA viruses by APOBEC3 proteins appears to play a dual role in this process. On the one hand, it is an essential component of the innate immune response to viral infections, and, on the other hand, it contributes to the pathogenesis of persistent viral infections and the progression of cancer. The current review examines the complex interplay between APOBEC3 proteins and DNA viruses and sheds light on the mechanisms of action, viral countermeasures and the impact on carcinogenesis. Deciphering the current issues in the interaction of APOBEC/DNA viruses should enable the development of new targeted cancer therapies.

Differential inhibition of LINE1 and LINE2 retrotransposition by vertebrate AID/APOBEC proteins.

BACKGROUND: The role of AID/APOBEC proteins in the mammalian immune response against retroviruses and retrotransposons is well established. G to A hypermutations, the hallmark of their cytidine deaminase activity, are present in several mammalian retrotransposons. However, the role of AID/APOBEC proteins in non-mammalian retroelement restriction is not completely understood. RESULTS: Here we provide the first evidence of anti-retroelement activity of a reptilian APOBEC protein. The green anole lizard A1 protein displayed potent DNA mutator activity and inhibited ex vivo retrotransposition of LINE1 and LINE2 ORF1 protein encoding elements, displaying a mechanism of action similar to that of the human A1 protein. In contrast, the human A3 proteins did not require ORF1 protein to inhibit LINE retrotransposition, suggesting a differential mechanism of anti-LINE action of A1 proteins, which emerged in amniotes, and A3 proteins, exclusive to placental mammals. In accordance, genomic analyses demonstrate differential G to A DNA editing of LINE retrotransposons in the lizard genome, which is also the first evidence for G to A DNA editing in non-mammalian genomes. CONCLUSION: Our data suggest that vertebrate APOBEC proteins differentially inhibit the retrotransposition of LINE elements and that the anti-retroelement activity of APOBEC proteins predates mammals.

APOBEC3 inhibits mouse mammary tumour virus replication in vivo.

Genomes of all mammals encode apobec3 genes, which are thought to have a function in intrinsic cellular immunity to several viruses including human immunodeficiency virus type 1 (HIV-1). APOBEC3 (A3) proteins are packaged into virions and inhibit retroviral replication in newly infected cells, at least in part by deaminating cytidines on the negative strand DNA intermediates. However, the role of A3 in innate resistance to mouse retroviruses is not understood. Here we show that A3 functions during retroviral infection in vivo and provides partial protection to mice against infection with mouse mammary tumour virus (MMTV). Both mouse A3 and human A3G proteins interacted with the MMTV nucleocapsid in an RNA-dependent fashion and were packaged into virions. In addition, mouse A3-containing and human A3G-containing virions showed a marked decrease in titre. Last, A3(-/-) mice were more susceptible to MMTV infection, because virus spread was more rapid and extensive than in their wild-type littermates.

Perspective of the GEMSTONE Consortium on Current and Future Approaches to Functional Validation for Skeletal Genetic Disease Using Cellular, Molecular and Animal-Modeling Techniques.

The availability of large human datasets for genome-wide association studies (GWAS) and the advancement of sequencing technologies have boosted the identification of genetic variants in complex and rare diseases in the skeletal field. Yet, interpreting results from human association studies remains a challenge. To bridge the gap between genetic association and causality, a systematic functional investigation is necessary. Multiple unknowns exist for putative causal genes, including cellular localization of the molecular function. Intermediate traits ("endophenotypes"), e.g. molecular quantitative trait loci (molQTLs), are needed to identify mechanisms of underlying associations. Furthermore, index variants often reside in non-coding regions of the genome, therefore challenging for interpretation. Knowledge of non-coding variance (e.g. ncRNAs), repetitive sequences, and regulatory interactions between enhancers and their target genes is central for understanding causal genes in skeletal conditions. Animal models with deep skeletal phenotyping and cell culture models have already facilitated fine mapping of some association signals, elucidated gene mechanisms, and revealed disease-relevant biology. However, to accelerate research towards bridging the current gap between association and causality in skeletal diseases, alternative in vivo platforms need to be used and developed in parallel with the current -omics and traditional in vivo resources. Therefore, we argue that as a field we need to establish resource-sharing standards to collectively address complex research questions. These standards will promote data integration from various -omics technologies and functional dissection of human complex traits. In this mission statement, we review the current available resources and as a group propose a consensus to facilitate resource sharing using existing and future resources. Such coordination efforts will maximize the acquisition of knowledge from different approaches and thus reduce redundancy and duplication of resources. These measures will help to understand the pathogenesis of osteoporosis and other skeletal diseases towards defining new and more efficient therapeutic targets.

Sex-determining region Y (SRY) attributes to gender differences in RANKL expression and incidence of osteoporosis.

Receptor activator of nuclear factor κB ligand (RANKL) plays a crucial role in bone metabolism. RANKL gene misregulation has been implicated in several bone and cancer diseases. Here, we aimed to identify novel transcription regulators of RANKL expression. We discovered that transcription factors, sex-determining region Y (SRY) and c-Myb, regulate RANKL expression. We demonstrated that c-Myb increases and male-specific SRY decreases RANKL expression through direct binding to its 5'-proximal promoter. These results are corroborated by the gene expression in human bone samples. In osteoporotic men, expression of RANKL is 17-fold higher, which correlates with the drastically reduced expression (200-fold) of Sry, suggesting that in osteoporotic men, the upregulation of RANKL is caused by a decrease of Sry. In healthy men, the expression of RANKL is 20% higher than that in healthy women. Our data suggest that gender differences in RANKL expression and bone quality could be due to the sex-specific transcription factor SRY.

APOBEC3 proteins inhibit LINE-1 retrotransposition in the absence of ORF1p binding.

Members of the apolipoprotein B mRNA editing complex polypeptide 1-like (APOBEC) family of enzymes exhibit inhibitory activity against a variety of exogenous and endogenous retroviruses including retrotransposons, such as long interspersed element 1 (LINE-1). Indeed, human APOBEC3A, APOBEC3B, and APOBEC3F inhibit retrotransposition of human LINE-1, mouse IAP and MusD retrotransposons. In our study, we examined whether the inhibitory effect of APOBEC3 proteins correlates with APOBEC3 ability to bind the LINE-1 ORF1 protein. We examined the interactions between the LINE-1 ORF1 protein and the most potent LINE-1 retrotransposon inhibitors, human APOBEC3A and APOBEC3B, by immunofluorescence and immunoprecipitation. Although human APOBEC3A shows the highest inhibitory potency against LINE-1 retrotransposon, no direct interactions were identified either by immunofluorescence or by co-immunoprecipitation. APOBEC3B binds to LINE-1 ORF1 protein, yet no co-localization was detected. We concluded that APOBEC3 proteins interfere indirectly with the LINE-1 retrotransposition pathway, probably through interference with RNA targeting.

Enhanced replication and pathogenesis of Moloney murine leukemia virus in mice defective in the murine APOBEC3 gene.

Human APOBEC3G (hA3G), a member of the AID/APOBEC family of deaminases, is a restriction factor for human immunodeficiency virus (HIV). In the absence of the viral Vif protein hA3G is packaged into virions and during reverse transcription in a recipient cell it deaminates cytosines, leading to G-->A hypermutation and inactivation of the viral DNA. Unlike humans, who carry seven APOBEC3 genes, mice only carry one, mA3. Thus the role of mA3 in restriction of retroviral infection could be studied in mA3 -/- knockout mice, where the gene is inactivated. M-MuLV-infected mA3 -/- mice showed substantially higher levels of infection at very early times compared to wild-type mice (ca. 2 logs at 0-10 days), particularly in the bone marrow and spleen. Restriction of M-MuLV infection was studied ex vivo in primary bone marrow-derived dendritic cells (BMDCs) that express or lack mA3, using an M-MuLV-based retroviral vector expressing enhanced jellyfish green fluorescent protein (EGFP). The results indicated that mA3 within the virions as well as mA3 in the recipient cell contribute to resistance to infection in BMDCs. Finally, M-MuLV-infected mA3 +/+ mice developed leukemia more slowly compared to animals lacking one or both copies of mA3 although the resulting disease was similar (T-lymphoma). These studies indicate that mA3 restricts replication and pathogenesis of M-MuLV in vivo.

Vpr.A3A chimera inhibits HIV replication.

Several APOBEC3 proteins (A3F and A3G), that are cytidine deaminases restrict human immunodeficiency virus (HIV) replication in the absence of the viral infectivity factor (Vif) protein. However, Vif leads to their degradation and counteracts their effects. Another member, A3A, restricts some retrotransposons and another virus but not HIV. We reasoned that this failure was due to the lack of appropriate targeting. Thus, we fused A3A to another viral protein, Vpr, which binds p6 in Gag and is incorporated into viral cores. Indeed, the Vpr.A3A chimera but not A3A was found abundantly in the viral core. It also restricted potently the replication of HIV and simian immunodeficiency virus in the presence and absence of Vif. Because we identified a high frequency of G to A mutations in viral cDNAs, this antiviral activity was mediated by DNA editing. Interestingly, our fusion protein did not restrict murine leukemia virus, which does not incorporate Vpr. Thus, by targeting appropriately a potent single domain cytidine deaminase, we rendered HIV and simian immunodeficiency virus restriction resistant to Vif.

Phylogenomic analysis of the L1 retrotransposons in Deuterostomia.

L1 retrotransposons constitute the largest single component of mammalian genomes. In contrast to the single remaining lineage of L1 retrotransposons in mammalian genomes, some teleost fishes contain a highly diverse L1 retrotransposon repertoire. Major evolutionary changes in L1 retrotransposon repertoires have therefore taken place in the land vertebrates (Tetrapoda). The lack of sequence data for L1 retrotransposons in the basal living Tetrapoda lineages prompted an investigation of their distribution and evolution in the genomes of the key tetrapod lineages, amphibians and reptiles, and in lungfishes. In this study, we combined genome database searches with PCR analysis to demonstrate that L1 retrotransposons are present in the genomes of lungfishes, amphibians, and lepidosaurs. Phylogenomic analysis shows that the genomes of Deuterostomia possess three highly divergent groups of L1 retrotransposons, with distinct distribution patterns. The analysis of L1 diversity shows the presence of a very large number of diverse L1 families, each with very low copy numbers, at the time of the origin of tetrapods. During the evolution of synapsids, all but one L1 lineage have been lost. This study establishes that the loss of L1 diversity and explosion in copy numbers occurred in the synapsid ancestors of mammals, and was most probably caused by severe population bottlenecks.