Summary The ratio of osteoprotegerin [OPG, tumour necrosis factor receptor superfamily, member 11b (TNFRSF11B)] to receptor activator of nuclear factor kappaB ligand [RANKL, tumour necrosis factor (ligand) superfamily, member 11 (TNFSF11)] in bone is critical for the regulation of bone remodelling. Myeloma cells can home to bone, triggering increased RANKL and decreased OPG expression by stromal cells, leading to osteolysis. Whether myeloma cells contribute directly to the pool of RANKL or OPG in bone has been contentious. Here we provide evidence of RANKL expression by reverse transcription polymerase chain reaction and in situ hybridization, demonstrating transcripts encoding both the membrane-bound and secreted forms of RANKL in five human multiple myeloma cell lines (LP-1, NCI-H929, OPM-2, RPMI8226, U266) and myeloma cells purified from bone marrow aspirates of myeloma patients. We demonstrated that RANKL encoding mRNAs are translated to protein by antibody detection of RANKL. In vitro assays showed that myeloma cells induced bone marrow derived mononuclear cells to differentiate into adherent tartrate-resistant acid phosphatase positive multinucleated cells, indicative of the formation of functional osteoclasts. This differentiation could also be achieved with passaged myeloma media alone, implicating secreted products. Finally, we provide evidence that the differentiation observed is at least in part the result of myeloma cell expression of RANKL. We therefore conclude that myeloma cells can directly contribute to the pool of RANKL in bone.

Identification of the RANKL/OPG/RANK/NF-kB (receptor activator of nuclear factor kappa-B ligand / osteoprotegerin) signaling pathway as the major regulatory system for osteoclastogenesis began with discovery of these ligands and receptors in the tumor necrosis factor (TNF) superfamily. Subsequently, genetically altered mice revealed physiologic roles for these proteins in bone biology. However, full appreciation of their significance for the human skeleton came from clinical characterization of several extremely rare, heritable disorders followed by discovery of their genetic bases. Familial expansile osteolysis (FEO) is an autosomal dominant disorder featuring constitutive activation of RANK due to an 18-bp tandem duplication in its gene (TNFRSF11A). A similar, 27-bp duplication causes what has been called a familial form of early-onset Paget's disease of bone (PDB2). Expansile skeletal hyperphosphatasia (ESH) is allelic to FEO and PDB2 and involves a 15-bp tandem duplication in TNFRSF11A. Autosomal recessive inheritance of deactivating mutations of the gene encoding OPG (TNFRSF11B) causes most cases of juvenile Paget disease. These disorders feature high bone turnover, deafness during early childhood, "idiopathic external lysis" of adult teeth, and sometimes focal lesions in appendicular bones that mimic active PDB. Biochemical markers indicate rapid skeletal remodeling. In FEO, osteolysis progresses to fat-filled bone rather than to osteosclerosis. Antiresorptive therapy with bisphosphonates can be effective for each disorder.

To identify the susceptibility genes for osteoporotic fracture in postmenopausal Chinese women, a two-stage case-control association study using joint analysis was conducted in 1046 patients with nontraumatic vertebra, hip, or distal radius fractures and 2303 healthy controls. First, 113 single-nucleotide polymorphisms (SNPs) in 16 potential osteoporosis candidate genes reported in recent genomewide association studies, meta-analyses studies, large-scale association studies, and functional studies were genotyped in a small-sample-size subgroup consisting of 541 patients with osteoporotic fractures and 554 healthy controls. Variants and haplotypes in SPTBN1, TNFRSF11B, CNR2, LRP4, and ESR1 that have been identified as being associated with osteoporotic fractures were further reanalyzed in the entire case-control group. We identified one SNP in TNFRSF11B (rs3102734), three SNPs in ESR1 (rs9397448, rs2234693, and rs1643821), two SNPs in LRP4 (rs17790156 and rs898604), and four SNPs in SPTBN1 (rs2971886, rs2941583, rs2941584, and rs12475342) were associated with all of the broadly defined osteoporotic fractures. The most significant polymorphism was rs3102734, with increased risk of osteoporotic fractures (odds ratio, 1.35; 95% confidence interval [CI], 1.17-1.55, Bonferroni p = 2.6 × 10(-4) ). Furthermore, rs3102734, rs2941584, rs12475342, rs9397448, rs2234693, and rs898604 exhibited significant allelic, genotypic, and/or haplotypic associations with vertebral fractures. SNPs rs12475342, rs9397448, and rs2234693 showed significant genotypic associations with hip fractures, whereas rs3102734, rs2073617, rs1643821, rs12475342, and rs2971886 exhibited significant genotypic and/or haplotypic associations with distal radius fractures. Accordingly, we suggest that in addition to the clinical risk factors, the variants in TNFRSF11B, SPTBN1, ESR1, and LRP4 are susceptibility genetic loci for osteoporotic fracture in postmenopausal Chinese women.

The TNF-family molecule receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL) (OPGL, TRANCE, ODF) and its receptor activator of NFkappaB (RANK) are key regulators of bone remodeling and regulate T cell/dendritic cell communications, and lymph node formation. Moreover, RANKL and RANK are expressed in mammary gland epithelial cells and control the development of a lactating mammary gland during pregnancy. Genetically, RANKL and RANK are essential for the development and activation of osteoclasts and bone loss in response to virtually all triggers tested. Inhibition of RANKL function via the natural decoy receptor osteoprotegerin (OPG, TNFRSF11B) prevents bone loss in postmenopausal osteoporosis and cancer metastases. Importantly, RANKL appears to be the pathogenetic principle that causes bone and cartilage destruction in arthritis, and OPG treatment prevents bone loss at inflamed joints and has partially beneficial effects on cartilage destruction in all arthritis models studied so far. Modulation of these systems provides a unique opportunity to design novel therapeutics to inhibit bone loss and crippling in arthritis.

OBJECTIVE

Paget's disease of bone (PDB), with a prevalence of 2 to 5% in Caucasians >55 years, is the second most frequent metabolic bone disease, after osteoporosis. PDB characteristics are bone lesions with an imbalanced bone remodeling, resulting in disorganized and nonfully fledged new bone. PDB etiology is not completely understood. In this review, current views on the etiology, clinical aspects, and PDB treatment are summarized and discussed.

METHODS

The PubMed database was searched using the keywords PDB, sequestosome1 (SQSTM1), valosin-containing protein (VCP), receptor activator of nuclear factor-κB (RANK), osteoprotegerin (OPG), RANK ligand (RANKL), mutation, genetic variants, virus, osteosarcoma, bisphosphonates, and denosumab.

RESULTS

Environmental evidence (e.g. viruses) and also genetic risk factors have been found for PDB. Until now, SQSTM1 was the only PDB-causing gene identified. However, PDB patients without SQSTM1 mutations seem to have susceptibility genetic polymorphisms in regions containing the CaSR, ESR1, TNFRSF11B (OPG), TNFRSF11A (RANK), CSF1 (M-CSF), OPTN, TM7SF4 (DC-STAMP), VCP, NUP205, RIN3, PML, and GOLGA6A genes, resulting in an increased risk of developing PDB. The nature of these genes indicates that the regulation of osteoclastogenesis is a key process in PDB pathogenesis. Furthermore, with the involvement of SQSTM1 and VCP in autophagy and in forming protein aggregates, this might also indicate that a disturbance of these processes might be a risk factor.

CONCLUSIONS

Unraveling the PDB genetic background is instrumental to understanding the PDB pathogenesis and the role of slow viruses. Furthermore, it might make early detection and subsequently treatment of risk individuals possible.

Genetic deficiency in granulocyte-macrophage colony-stimulating factor (CSF2, GM-CSF) results in altered placental structure in mice. To investigate the mechanism of action of CSF2 in placental morphogenesis, the placental gene expression and cell composition were examined in Csf2 null mutant and wild-type mice. Microarray and quantitative RT-PCR analyses on Embryonic Day (E) 13 placentae revealed that the Csf2 null mutation caused altered expression of 17 genes not previously known to be associated with placental development, including Mid1, Cd24a, Tnfrsf11b, and Wdfy1. Genes controlling trophoblast differentiation (Ascl2, Tcfeb, Itgav, and Socs3) were also differentially expressed. The CSF2 ligand and the CSF2 receptor alpha subunit were predominantly synthesized in the placental junctional zone. Altered placental structure in Csf2 null mice at E15 was characterized by an expanded junctional zone and by increased Cx31(+) glycogen cells and cyclin-dependent kinase inhibitor 1C (CDKN1C(+), P57(Kip2+)) giant cells, accompanied by elevated junctional zone transcription of genes controlling spongiotrophoblast and giant cell differentiation and secretory function (Ascl2, Hand1, Prl3d1, and Prl2c2). Granzyme genes implicated in tissue remodeling and potentially in trophoblast invasion (Gzmc, Gzme, and Gzmf) were downregulated in the junctional zone of Csf2 null mutant placentae. These data demonstrate aberrant placental gene expression in Csf2 null mutant mice that is associated with altered differentiation and/or functional maturation of junctional zone trophoblast lineages, glycogen cells, and giant cells. We conclude that CSF2 is a regulator of trophoblast differentiation and placental development, which potentially influences the functional capacity of the placenta to support optimal fetal growth in pregnancy.

Paget's disease of bone is a common condition characterised by increased and disorganised bone turnover which can affect one or several bones throughout the skeleton. These abnormalities disrupt normal bone architecture and lead to various complications such as bone pain osteoarthritis, pathological fracture, bone deformity, deafness, and nerve compression syndromes. Genetic factors play an important role in PDB and mutations or polymorphisms have been identified in four genes that cause classical Paget's disease and related syndromes. These include TNFRSF11A, which encodes RANK, TNFRSF11B which encodes osteoprotegerin, VCP which encodes p97, and SQSTM1 which encodes p62. All of these genes play a role in the RANK-NFkappaB signalling pathway and it is likely that the mutations predispose to PDB by disrupting normal signalling, leading to osteoclast activation. Although Paget's has traditionally be considered a disease of the osteoclast there is evidence that stromal cell function and osteoblast function are also abnormal, which might account for the fact that the disease is associated with increased bone formation as well as resorption. Environmental factors also contribute to Paget's disease. Most research has focused on paramyxovirus infection as a possible environmental trigger but evidence in favour of the involvement of viruses in the disease remains conflicting. Other factors which have been implicated as possible disease triggers include mechanical loading, dietary calcium and environmental toxins. Further work will be required to identify additional genetic variants that predispose to Paget's disease and to determine how the causal mutations and predisposing polymorphisms interact with environmental factors to influence bone cell function and cause the focal bone lesions that are characteristic of the disease.

PDB (Paget's disease of bone) is a common condition characterized by focal increases in bone turnover affecting one or more sites throughout the skeleton. Genetic factors are important in the pathogenesis of PDB and many families have been described where PDB is inherited in an autosomal-dominant fashion. Several candidate loci for susceptibility to PDB and related syndromes have been identified by genome-wide scans and recent evidence suggests that mutations in genes that encode components of the RANK [receptor activator of NF-kappaB (nuclear factor-kappaB)]/NF-kappaB signalling pathway play an important role in the pathogenesis of this group of diseases. Insertion mutations in the TNFRSF11A gene encoding RANK have been identified as the cause of familial expansile osteolysis, some cases of early onset PDB and expansile skeletal hyperphosphatasia. Inactivating mutations in the TNFRSF11B gene that encodes OPG (osteoprotegerin) have been found to cause the syndrome of juvenile PDB. Polymorphisms in OPG also appear to increase the risk of developing PDB. The most important causal gene for classical PDB is Sequestosome 1 (SQSTM1), which is a scaffold protein in the NF-kappaB signalling pathway, and mutations affecting the UBA (ubiquitin-associated) domain of this protein occur in between 20-50% of familial and 10-20% of sporadic PDB cases. The rare syndrome of IBMPFD (inclusion body myopathy, PDB and fronto-temporal dementia) is due to mutations in the VCP gene and these also cluster in the domain of VCP that interacts with ubiquitin, suggesting a common disease mechanism with SQSTM1-mediated PDB.

BACKGROUND

In psoriasis, only limited overlap between sets of genes identified as differentially expressed (psoriatic lesional vs. psoriatic non-lesional) was found using statistical and fold-change cut-offs. To provide a framework for utilizing prior psoriasis data sets we sought to understand the consistency of those sets.

RESULTS

Microarray expression profiling and qRT-PCR were used to characterize gene expression in PP and PN skin from psoriasis patients. cDNA (three new data sets) and cRNA hybridization (four existing data sets) data were compared using a common analysis pipeline. Agreement between data sets was assessed using varying qualitative and quantitative cut-offs to generate a DEG list in a source data set and then using other data sets to validate the list. Concordance increased from 67% across all probe sets to over 99% across more than 10,000 probe sets when statistical filters were employed. The fold-change behavior of individual genes tended to be consistent across the multiple data sets. We found that genes with <2-fold change values were quantitatively reproducible between pairs of data-sets. In a subset of transcripts with a role in inflammation changes detected by microarray were confirmed by qRT-PCR with high concordance. For transcripts with both PN and PP levels within the microarray dynamic range, microarray and qRT-PCR were quantitatively reproducible, including minimal fold-changes in IL13, TNFSF11, and TNFRSF11B and genes with >10-fold changes in either direction such as CHRM3, IL12B and IFNG.

CONCLUSIONS

Gene expression changes in psoriatic lesions were consistent across different studies, despite differences in patient selection, sample handling, and microarray platforms but between-study comparisons showed stronger agreement within than between platforms. We could use cut-offs as low as log10(ratio) = 0.1 (fold-change = 1.26), generating larger gene lists that validate on independent data sets. The reproducibility of PP signatures across data sets suggests that different sample sets can be productively compared.

A number of polymorphisms in various genes have been identified and associated with bone mineral density (BMD) and with an increased risk of osteoporosis.

OBJECTIVE

In this study, three single nucleotide polymorphisms (SNPs) within the TNFRSF11B gene were studied for association with an increased risk of osteoporosis in postmenopausal Maltese women (n=126).

METHODS

Analysis was performed by PCR restriction fragment length polymorphism (RFLP) while BMD at the lumbar spine, femoral neck, Ward's triangle and trochanter was measured by DEXA.

RESULTS

No significant association was observed between genotypes and BMD for all polymorphisms studied within this gene. Homozygotes CC (T(950)-C) were observed to have the highest BMD at all anatomical sites although statistical significance was not reached when comparing the three genotypes. A statistical significant difference was observed in the distribution of genotype frequencies for this polymorphism between normal individuals and those that were either osteopenic or osteoporotic at one or both anatomical sites, with the TT genotype associated more frequently with low BMD. The T(950)-C and G(1181)-C polymorphisms were in strong linkage disequilibrium with each other but not with the A(163)-G polymorphism further upstream in the OPG promoter. Statistical significance was reached when constructing haplotypes, where the A-T-G haplotype was found to be more frequent in individuals with low BMD.

CONCLUSIONS

These results indicate the possible role of TNFRSF11B gene variants in postmenopausal bone loss in women in Malta.

BACKGROUND

The PI3K/AKT pathway plays a pivotal role in breast cancer development and maintenance. PIK3CA, encoding the PI3K catalytic subunit, is the oncogene exhibiting a high frequency of gain-of-function mutations leading to PI3K/AKT pathway activation in breast cancer. PIK3CA mutations have been observed in 30% to 40% of ERα-positive breast tumors. However the physiopathological role of PIK3CA mutations in breast tumorigenesis remains largely unclear.

RESULTS

To identify relevant downstream target genes and signaling activated by aberrant PI3K/AKT pathway in breast tumors, we first analyzed gene expression with a pangenomic oligonucleotide microarray in a series of 43 ERα-positive tumors with and without PIK3CA mutations. Genes of interest were then investigated in 249 ERα-positive breast tumors by real-time quantitative RT-PCR. A robust collection of 19 genes was found to be differently expressed in PIK3CA-mutated tumors. PIK3CA mutations were associated with over-expression of several genes involved in the Wnt signaling pathway (WNT5A, TCF7L2, MSX2, TNFRSF11B), regulation of gene transcription (SEC14L2, MSX2, TFAP2B, NRIP3) and metal ion binding (CYP4Z1, CYP4Z2P, SLC40A1, LTF, LIMCH1).

CONCLUSIONS

This new gene set should help to understand the behavior of PIK3CA-mutated cancers and detailed knowledge of Wnt signaling activation could lead to novel therapeutic strategies.

We profiled the global gene expression of a bone marrow-derived mesenchymal pluripotent cell line in response to Runx2 expression. Besides osteoblast differentiation, Runx2 promoted the osteoclastogenesis of co-cultured splenocytes. This was attributable to the upregulation of many novel osteoclastogenic genes and the downregulation of anti-osteoclastogenic genes.

BACKGROUND

In addition to being a master regulator for osteoblast differentiation, Runx2 controls osteoblast-driven osteoclastogenesis. Previous studies profiling gene expression during osteoblast differentiation had limited focus on Runx2 or paid little attention to its role in mediating osteoblast-driven osteoclastogenesis.

METHODS

ST2/Rx2(dox), a bone marrow-derived mesenchymal pluripotent cell line that expresses Runx2 in response to Doxycycline (Dox), was used to profile Runx2-induced gene expression changes. Runx2-induced osteoblast differentiation was assessed based on alkaline phosphatase staining and expression of classical marker genes. Osteoclastogenic potential was evaluated by TRAP staining of osteoclasts that differentiated from primary murine splenocytes co-cultured with the ST2/Rx2(dox) cells. The BeadChip™ platform (Illumina) was used to interrogate genome-wide expression changes in ST2/Rx2(dox) cultures after treatment with Dox or vehicle for 24 or 48 h. Expression of selected genes was also measured by RT-qPCR.

RESULTS

Dox-mediated Runx2 induction in ST2 cells stimulated their own differentiation along the osteoblast lineage and the differentiation of co-cultured splenocytes into osteoclasts. The latter was attributable to the stimulation of osteoclastogenic genes such as Sema7a, Ltc4s, Efnb1, Apcdd1, and Tnc as well as the inhibition of anti-osteoclastogenic genes such as Tnfrsf11b (OPG), Sema3a, Slco2b1, Ogn, Clec2d (Ocil), Il1rn, and Rspo2.

CONCLUSIONS

Direct control of osteoblast differentiation and concomitant indirect control of osteoclast differentiation, both through the activity of Runx2 in pre-osteoblasts, constitute a novel mechanism of coordination with a potential crucial role in coupling bone formation and resorption.

We report a 32-year-old man and his 59-year-old mother with a unique and extensive variant of Camurati-Engelmann disease (CED) featuring histopathological changes of osteomalacia and alterations within TGFβ1 and TNFSF11 encoding TGFβ1 and RANKL, respectively. He suffered leg pain and weakness since childhood and reportedly grew until his late 20s, reaching 7 feet in height. He had deafness, perforated nasal septum, torus palatinus, disproportionately long limbs with knock-knees, low muscle mass, and pseudoclubbing. Radiographs revealed generalized skeletal abnormalities, including wide bones and cortical and trabecular bone thickening in keeping with CED, except that long bone ends were also affected. Lumbar spine and hip BMD Z-scores were + 7.7 and + 4.4, respectively. Biochemical markers of bone turnover were elevated. Hypocalciuria accompanied low serum 25-hydroxyvitamin D (25[OH]D) levels. Pituitary hypogonadism and low serum insulin-like growth factor (IGF)-1 were present. Karyotype was normal. Despite vitamin D repletion, iliac crest histology revealed severe osteomalacia. Exon 1 of TNFRSF11A (RANK), exons 2, 3, and 4 of LRP5, and all coding exons and adjacent mRNA splice junctions of TNFRSF11B (OPG), SQSTM1 (sequestosome 1), and TNSALP (tissue nonspecific alkaline phosphatase) were intact. His asymptomatic and less dysmorphic 5'11″ mother, also with low serum 25(OH)D, had milder clinical, radiological, biochemical, and histopathological findings. Both individuals were heterozygous for a novel 12-bp duplication (c.27_38dup, p.L10_L13dup) in exon 1 of TGFβ1, predicting four additional leucine residues in the latency-associated-peptide segment of TGFβ1, consistent with CED. The son was also homozygous for a single base transversion in TNFSF11, predicting a nonconservative amino acid change (c.107C>> G, p.Pro36Arg) in the intracellular domain of RANKL that was heterozygous in his nonconsanguineous parents. This TNFSF11 variant was not found in the SNP Database, nor in published TNFSF11 association studies, but it occurred in four of the 134 TNFSF11 alleles (3.0%) we tested randomly among individuals without CED. Perhaps the unique phenotype of this CED family is conditioned by altered RANKL activity.

We studied the role of TNFRSF11B polymorphisms on the risk to develop Paget's disease of bone in a Belgian study population. We observed no association in men, but a highly significant association was found in women, and this was confirmed in a population from the United Kingdom.

BACKGROUND

Juvenile Paget's disease has been shown to be caused by mutations in TNFRSF11B encoding osteoprotegerin. Although mutations in this gene have never been found in patients with typical Paget's disease of bone (PDB), there are indications that polymorphisms in TNFRSF11B might contribute to the risk of developing PDB.

METHODS

We recruited a population of 131 Belgian patients with sporadic PDB and 171 Belgian controls. By means of the HapMap, we selected 17 SNPs that, in combination with four multimarker tests, contain most information on common genetic variation in TNFRSF11B. To replicate the findings observed in the Belgian study population, genotyping data of SNPs generated in a UK population were reanalyzed.

RESULTS

In our Belgian study population, associations were found for two SNPs (rs11573871, rs1485286) and for one multimarker test involving rs1032129. When subsequently analyzing men and women separately, these associations turned out to be driven by women (56 cases, 78 controls). In addition, three other tagSNPs turned out to be associated in women only. These were rs2073617 (C950T), rs6415470, and rs11573869. Reanalysis of genotyping data from a UK study population indicated that the associations found for C950T and C1181G were also exclusively driven by women (146 cases, 216 controls). Meta-analysis provided evidence for risk increasing effects of the T allele of C950T and the G allele of C1181G in the female population (p = 0.002 and 0.003, respectively). The haplotypes formed by the SNPs associated in the Belgian population were also distributed differentially between female cases and controls.

CONCLUSIONS

We showed for the first time that SNPs influencing the risk to develop PDB could be sex-specific. Further research is necessary to identify the causative variants in TNFRSF11B and to elucidate the molecular pathogenic mechanism.

To clarify the role of the TNFRSF11B gene encoding osteoprotegerin (OPG), in Paget's disease of bone (PDB) we studied TNFRSF11B polymorphisms in an association study of 690 UK subjects and in a worldwide familial study of 66 kindreds. We found that the G1181 allele of TNFRSF11B, encoding lysine at codon 3 of the OPG protein, predisposes to both sporadic and familial PDB.

BACKGROUND

Paget's disease of bone (PDB) is a common disorder characterized by focal abnormalities of bone turnover. Genetic factors are important in the pathogenesis of PDB, and studies have shown that inactivating mutations of the TNFRSF11B gene, encoding osteoprotegerin (OPG), cause the rare syndrome of juvenile Paget's disease. In this study, we sought to determine whether polymorphisms of the TNFRSF11B gene contribute to the pathogenesis of classical PDB.

METHODS

We screened for polymorphisms of the TNFRSF11B gene by DNA sequencing of the proximal promoter, coding exons, and intron-exon boundaries in 20 PDB patients and 10 controls. Informative single nucleotide polymorphisms (SNPs), including a G1181C SNP, which predicts a lysine-asparagine substitution at codon 3 of the OPG signal peptide and haplotypes, were related to the presence of PDB in 312 cases compared with 378 controls and to transmission of PDB in 140 affected offspring from 66 kindreds with familial PDB.

CONCLUSIONS

The G1181 allele was significantly over-represented in PDB patients (chi(2) = 5.7, df = 1, p = 0.017, adjusted alpha = 0.024), equivalent to an odds ratio for PDB of 1.55 (95% CI: 1.11-2.16). The distribution of TNFRSF11B haplotypes significantly differed in sporadic PDB cases and controls (chi(2) = 30.2, df = 9, p < 0.001) because of over-representation of haplotypes containing the G1181 allele in cases. The family study showed that the most common haplotype containing the G1181 allele was transmitted more frequently than expected to 140 individuals with familial PDB (chi(2) = 7.35, df = 1, p < 0.01), and the transmission disequilibrium was even more pronounced in a subgroup of 78 familial PDB patients who did not carry mutations of the SQSTM1 gene (chi(2) = 8.44, df = 1, p < 0.005). We conclude that the G1181 allele of TNFRSF11B, encoding lysine at codon 3 of the OPG protein, predisposes to the development of sporadic PDB and familial PDB that is not caused by SQSTM1 mutations.

OBJECTIVE

Osteoprotegerin (OPG) is a secretory glycoprotein which belongs to the tumor necrosis factor receptor family. Various mechanisms have been suggested by which calcification might alter atherosclerotic plaque stability, but the significance of this intimal calcification is controversial. High concentrations of OPG have been associated with the presence of vascular and cardiovascular diseases. This study was designed to assess the association between gene polymorphisms of the OPG gene (TNFRSF11B), the serum OPG level, and plaque stability in patients with carotid atherosclerosis.

METHODS

We studied 177 patients with internal carotid artery stenosis who underwent carotid endarterectomy and also 303 controls. Carotid endarterectomy samples removed from patients were assessed by immunohistochemistry. Concentrations of OPG were measured and gene polymorphisms were examined by polymerase chain reaction and restriction enzyme analysis and were compared, initially between patients with carotid atherosclerosis and controls, and subsequently between stable and unstable carotid plaques.

RESULTS

We found that the GG genotype of the T245G polymorphism, the CC genotype of the T950C polymorphism, and the CC genotype of the G1181C polymorphism were significantly higher in patients with carotid plaque than in controls (21.5% versus 10.9% , P<0.01; 15.8% versus 7.6%, P<0.01; and 20.3% versus 10.9%, P<0.01, respectively) and that these polymorphisms were associated with high serum OPG levels (4.02 [3.07] versus 2.94 [1.81] pmol/L; P<0.01), which were significantly higher in patients with unstable atherosclerotic plaques (5.86 [4.02] versus 3.53 [1.87] pmol/L; P<0.01).

CONCLUSIONS

The TNFRSF11B gene polymorphisms studied are associated with high serum OPG levels and might be potential markers for plaque instability.

Figure 7 summarizes the heritable disorders identified to date that directly involve the RANKL/OPG/RANK signaling pathway in humans. Activating mutations in TNFRSF11A encoding RANK and deactivating mutations in TNFRSF11B encoding OPG cause systemic bone disease (FEO, PDB2, ESH and JPD) featuring accelerated bone turnover, low bone mass, deafness early in life, and loss of dentition by enhancing signaling. No human disease has been identified involving defects in the TNFSF11 gene encoding RANKL. Despite genetic bases for these autosomal dominant and recessive conditions involving bone cell receptors, focal expansile osteolytic lesions are common and can occur perhaps from further local activation of osteoclast-mediated bone resorption following trauma. These disorders resemble PDB which can be inherited as an autosomal dominant trait with focal osteolytic disease, sometimes with deafness and tooth loss, and increasingly associated with mutations, but in other genes.

Osteolytic bone disease (OBD) in multiple myeloma (MM) is caused by interactions between MM cells and the bone marrow microenvironment and is characterized by increased osteoclastic bone resorption and decreased osteoblastic bone formation. Recently, the role of osteoblast inhibition has come into focus, especially the possible role of overexpression of DKK1, an inhibitor of the Wnt signalling pathway. Further, CKS2, PSME2 and DHFR have also been reported as candidate genes for OBD. We studied the gene expression by quantitative reverse transcription polymerase chain reaction of TNFSF11 (RANKL), TNFSF11A (RANK), TNFRSF11B (OPG), CCL3 (MIP1A), CCL4 (MIP1B), PTHR1 (PTHrp), DKK1, CKS2, PSME2 and DHFR in purified, immunophenotypic FACS-sorted plasma cells from 171 newly diagnosed MM patients, 20 patients with monoclonal gammopathy of undetermined significance and 12 controls. The gene expressions of the analysed genes were correlated with radiographically assessed OBD. Only overexpression of DKK1 was correlated to the degree of OBD. Myeloma cells did not express TNFSF11A, TNFSF11, or TNFRSF11B, and very rarely expressed CCL3 and PTHR11. CCL4, CKS2, PSME2 and DHFR were variably expressed, but the expression of these genes showed no correlation with OBD. In contrast, loss of PSME2 expression in MM plasma cells was significantly correlated with OBD.

Studying consanguineous families with Ghosal hematodiaphyseal dysplasia syndrome (GHDD), a disorder of increased bone density, we identified mutations in TBXAS1, which encodes thromboxane synthase (TXAS). TXAS, an enzyme of the arachidonic acid cascade, produces thromboxane A(2) (TXA(2)). Platelets from subjects with GHDD showed a specific deficit in arachidonic acid-produced aggregation. We also found that TXAS and TXA(2) modulated expression of TNFSF11 and TNFRSF11B (encoding RANKL and osteoprotegerin (OPG), respectively) in primary cultured osteoblasts.

Paget's disease of bone (PDB) is a common condition with a strong genetic component that is characterized by focal increases in bone turnover, leading to bone deformity, pathological fractures, and various other complications. Several rare disorders have also been described that show phenotypic overlap with PDB. Genome-wide searches have identified several susceptibility loci for PDB and PDB-like disorders, and mutations that cause these disorders have now been identified in four genes, all of which are involved in the RANK-NF-kappaB signaling pathway. Mutations in SQSTM1, which encodes an important scaffold protein in this pathway, have been found to be a common cause of classical PDB. Thus far, all disease-causing mutations in SQSTM1 affect the ubiquitin-associated (UBA) domain of the gene product and cause loss of ubiquitin binding. The rare PDB-like disorders of familial expansile osteolysis, early-onset familial PDB, and expansile skeletal hyperphosphatasia are caused by duplication mutations in exon 1 of the TNFRSF11A gene, which encodes the RANK receptor. This gene does not seem to be involved in the pathogenesis of classical PDB. Inactivating mutations in the TNFRSF11B gene, which encodes osteoprotegerin, cause juvenile PDB, and TNFRSF11B polymorphisms seem to increase the risk of classical PDB. The rare syndrome of hereditary inclusion body myopathy, PDB, and frontotemporal dementia (IBMPFD) is caused by mutations in the VCP gene, which is involved in regulating I-kappaB degradation by the proteasome. The disease-causing mutations in VCP cluster in and around a domain involved in ubiquitin binding. Whereas SQSTM1 has emerged as an important gene for classical PDB, most kindreds with familial PDB do not carry SQSTM1 mutations, indicating that additional genes for PDB remain to be discovered. In light of the molecular defects that have been identified thus far, it seems likely that these genes will also be involved in the RANK-NF-kappaB signaling pathway or its interactions with the ubiquitin-proteasome system.

OBJECTIVE

Osteoprotegerin (OPG) inhibits osteoclast function by acting as a decoy receptor for receptor activator of nuclear factor-κB ligand (RANKL), thus being an important candidate gene for osteoporosis. Three recent genome-wide association studies also identified the TNFRSF11B gene, coding for OPG, as playing a key role in bone mineral density (BMD) regulation. As variations in the TNFRSF11B gene could alter the susceptibility to osteoporosis, the aim of study was to investigate association of two TNFRSF11B gene polymorphisms with BMD and serum OPG concentration in postmenopausal women.

METHODS

478 postmenopausal women were genotyped for the presence of TNFRSF11B gene polymorphisms 245T>> G (rs3134069) and 1181G>> C (rs2073618). BMDs and serum OPG concentrations were measured.

RESULTS

Two common haplotypes GT and CT occurred in 41.2% and 52.4% of subjects. In osteoporotic postmenopausal women, lumbar spine BMD was associated with polymorphisms 245T>> G and 1181G>> C, as well as with CT haplotype (p values 0.013, 0.006 and 0.006, respectively). Additionally, femoral neck BMD showed the association with 245T>> G (p = 0.047). No other statistically significant associations with BMD were found for the studied SNPs and haplotypes. No association with serum OPG concentration was shown in any of the studied groups.

CONCLUSIONS

Our results suggest that, in postmenopausal osteoporosis, polymorphisms 245T>> G and 1181G>> C, as well as haplotype CT in TNFRSF11B gene influence BMD.

Polymorphisms within the TNFRSF11B gene have been studied and associated with osteoporosis and fracture risk. Osteoprotegerin (OPG), the product of this gene, is a key negative regulator of osteoclastogenesis and is secreted by osteoblasts/stromal cells. A previous study in Maltese postmenopausal women showed positive association of low bone mineral density (BMD) with a polymorphism found within the promoter region of this gene (C950T). In this study, direct DNA sequencing revealed 12 variants with polymorphisms C950T, G1181C and rs4876869 observed to be in strong linkage disequilibrium. The constructed haplotype T-G-T was found to increase the risk for a low BMD, while C-G-T and C-C-C have a protective role; thus, we investigated the functional role of both C950T and rs4876869 in vitro. The promoter region, including the C950T alleles, was amplified by PCR, cloned into pGL3 enhancer vector and transfected into HeLa, COS-7 and RAW264.7 cell lines. After incubation, luciferase activity was measured. The T/C (rs4876869) change was tested for its possible effect on pre-mRNA splicing, using an exon-trapping vector. A statistical significant difference in gene expression was observed between the alleles for T950C, with the T allele showing a lower luciferase expression in all cell lines (P<0.01). For rs4876869, exon skipping was observed for the C allele, while only one transcript harbouring the whole exon was observed for the T allele. Our findings suggest that the T-G-T haplotype might be increasing the risk for osteoporosis due to lower quantities of the full OPG transcript being expressed resulting in a higher bone resorption.

In the thymus, medullary thymic epithelial cells (mTEC) regulate T cell tolerance via negative selection and Foxp3(+) regulatory T cell (Treg) development, and alterations in the mTEC compartment can lead to tolerance breakdown and autoimmunity. Both the receptor activator for NF-κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) axis and expression of the transcriptional regulator Aire are involved in the regulation of thymus medullary microenvironments. However, their impact on the mechanisms controlling mTEC homeostasis is poorly understood, as are the processes that enable the thymus medulla to support the balanced production of mTEC-dependent Foxp3(+) Treg. In this study, we have investigated the control of mTEC homeostasis and examined how this process impacts the efficacy of Foxp3(+) Treg development. Using newly generated RANK Venus reporter mice, we identify distinct RANK(+) subsets that reside within both the mTEC(hi) and mTEC(lo) compartments and that represent direct targets of OPG-mediated control. Moreover, by mapping OPG expression to a subset of Aire(+) mTEC, our data show how cis- and trans-acting mechanisms are able to control the thymus medulla by operating on multiple mTEC targets. Finally, we show that whereas the increase in mTEC availability in OPG-deficient (Tnfrsf11b(-/-)) mice impacts the intrathymic Foxp3(+) Treg pool by enhancing peripheral Treg recirculation back to the thymus, it does not alter the number of de novo Rag2pGFP(+)Foxp3(+) Treg that are generated. Collectively, our study defines patterns of RANK expression within the thymus medulla, and it shows that mTEC homeostasis is not a rate-limiting step in intrathymic Foxp3(+) Treg production.

OBJECTIVE

Transcorneal electrical stimulation (TES) has been beneficial in several neurodegenerative ocular diseases, but the exact mechanisms remain to be elucidated. This study was conducted to investigate the effects of TES on the retinas of wild-type Brown Norway (BN) rats by gene expression profiling and to assess its effects on retinal function and morphology.

METHODS

TES was applied to BN wild-type rat retinas in vivo for 1 hour (1-ms biphasic pulses at 20 Hz; 200 μA). RNA was isolated and processed for microarray-based profiling 4 hours after TES; differentially expressed genes from TES compared with those from sham-treated animals were validated by quantitative real-time polymerase chain reaction. Furthermore, the effect of TES was assessed at the structural and functional levels using electroretinography, confocal scanning laser ophthalmoscopy, optical coherence tomography, and immunohistochemistry.

RESULTS

Transcriptome changes associated with TES versus sham-stimulated BN wild-type retina were identified. Four hundred ninety genes were differentially expressed in TES and included potentially neuroprotective genes such as Bax or members of the tumor necrosis factor family (Tnfrsf11b, Tnrsf12a, Tnfsf13b, Tnfsf13). ERG recordings showed physiological retinal function after TES, and structural in vivo and ex vivo studies revealed intact retinal anatomy.

CONCLUSIONS

These results demonstrate that TES applied to the retina of the wild-type BN rats induces distinct transcriptome level changes and may help in the understanding of the mechanisms underlying TES. In addition, TES treatment indicates no negative effect on structure and function of the wild-type BN retina up to 35 hours after application.