The present study aimed to systematically examine the molecular mechanisms of papillary thyroid cancer (PTC), and identify potential biomarkers and drugs for the treatment of PTC. Two microarray data sets (GSE3467 and GSE3678), containing 16 PTC samples and 16 paired normal samples, were downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified using the Linear Models for Microarray Analysis package. Subsequently, the common DEGs were screened for functional and pathway enrichment analysis using the Database for Annotation Visualization and Integrated Discovery. The representative interaction subnetwork was further derived using Molecular Complex Detection software. In addition, the potential drugs for the hub DEGs in the subnetwork were screened from DrugBank and the potential drug‑like ligands, which interacted with genes, were selected using MTiOpenScreen. A total of 167 common DEGs, including 77 upregulated and 90 downregulated DEGs, were screened. The common DEGs were associated with the functions of plasma membrane, extracellular matrix, response to steroid hormone stimulus and cell adhesion, and the pathways of tyrosine metabolism and cell adhesion molecules were significantly enriched. A total of eight common DEGs (MET, SERPINA1, LGALS3, FN1, TNFRSF11B, LAMB3 and COL13A1) were involved in the subnetwork. The two drugs, lanoteplase and ocriplasmin, and four drugs, β‑mercaptoethanol, recombinant α 1‑antitrypsin, PPL‑100 and API, were found for FN1 and SERPINA1, respectively. The common DEGs identified may be potential biomarkers for PCT. FN1 and SERPINA1 may be involved in PTC by regulating epithelial‑to‑mesenchymal transition and responding to steroid hormone stimuli, respectively. Ocriplasmin, β‑mercaptoethanol and recombinant α 1‑antitrypsin may be potential drugs for the treatment of PTC.

OBJECTIVE

A main challenge in the therapy of osteoarthritis (OA) is the development of drugs that will modify the disease. Reliable test systems are necessary to enable an efficient screening of therapeutic substances. We therefore established a chondrocyte-based in vitro cell culture model in order to characterize different p38MAPK inhibitors.

METHODS

Interleukin-1beta (IL-1beta)-stimulated human OA chondrocytes were treated with the p38MAPK inhibitors Birb 796, pamapimod, SB203580 and the new substance CBS-3868. Birb 796- and SB203580-treated cells were analysed in a genome-wide microarray analysis. The efficacy of all inhibitors was characterized by quantitative gene expression analysis and the quantification of PGE(2) and NO release.

RESULTS

Microarray analysis revealed inhibitor-specific differences in gene expression. Whereas SB203580 had a broad effect on chondrocytes, Birb 796 counteracted the IL-1beta effect more specifically. All p38MAPK inhibitors significantly inhibited the IL-1beta-induced gene expression of COX-2, mPGES1, iNOS, matrix metalloproteinase 13 (MMP13) and TNFRSF11B, as well as PGE(2) release. Birb 796 and CBS-3868 showed a higher efficacy than SB203580 and pamapimod at inhibiting the expression of COX-2 and MMP13 genes, as well as PGE(2) release. In the case of mPGES1 and TNFRSF11B gene expression, CBS-3868 exceeded the efficacy of Birb 796.

CONCLUSIONS

Our test system could differentially characterize inhibitors of the same primary pharmaceutical target. It reflects processes relevant in OA and is based on chondrocytes that are mainly responsible for cartilage degradation. It therefore represents a valuable tool for drug screening in between functional in vitro testing and in vivo models.

BACKGROUND

The pathogenesis of osteoarthritis is related to abnormal mechanical stresses that alter cartilage metabolism and chondrocyte survival. Among the mechanical stresses, shear stress is held responsible for the development of arthritis.

METHODS

Monolayer cultures of human osteoarthritic chondrocytes were subjected to fluid-induced shear stress in vitro. A cDNA microarray technology was used to screen the differentially regulated genes and quantitative real-time poly-merase chain reaction (Q-RT-PCR) was used to confirm the results. The significance of the expression ratio for each gene was determined on the lowest associated false discovery rate calculated from the changes of gene expression in relation to the standard deviation of repeated measurements for that gene.

RESULTS

Exposure of human osteoarthritic chondrocytes to shear stress (0.82 Pa) for 2 hours differentially regulated 373 and 227 clones in two independent microarray analyses with at least a 1.7-fold change. By comparing the differentially regulated clones, 14 upregulated and 6 downregulated genes were identified. Many of the differentially expressed genes were related to cell proliferation/differentiation (TGF-beta, acidic FGF), cell survival/apoptosis (CYP1B1, BCL2L3, TNFRSF11B, chemokine ligands, ADM), and matrix homeostasis (DCN, SDC2, MGP, WISP2).

CONCLUSIONS

The gene expression patterns following shear stress show a high similarity to the gene expression in the reparative process of osteoarthritis chondrocytes. Using microarray analysis, this study suggests a close interaction between shear stress and the pathogenesis of osteoarthritis.

Activation of Notch1 in cells of the osteoblastic lineage inhibits osteoblast differentiation/function and causes osteopenia, whereas its activation in osteocytes causes a distinct osteopetrotic phenotype. To explore mechanisms responsible, we established the contributions of canonical Notch signaling (Rbpjκ dependent) to osteocyte function. Transgenics expressing Cre recombinase under the control of the dentin matrix protein-1 (Dmp1) promoter were crossed with Rbpjκ conditional mice to generate Dmp1-Cre(+/-);Rbpjκ(Δ/Δ) mice. These mice did not have a skeletal phenotype, indicating that Rbpjκ is dispensable for osteocyte function. To study the Rbpjκ contribution to Notch activation, Rosa(Notch) mice, where a loxP-flanked STOP cassette is placed between the Rosa26 promoter and the NICD coding sequence, were crossed with Dmp1-Cre transgenic mice and studied in the context (Dmp1-Cre(+/-);Rosa(Notch);Rbpjκ(Δ/Δ)) or not (Dmp1-Cre(+/-);Rosa(Notch)) of Rbpjκ inactivation. Dmp1-Cre(+/-);Rosa(Notch) mice exhibited increased femoral trabecular bone volume and decreased osteoclasts and bone resorption. The phenotype was reversed in the context of the Rbpjκ inactivation, demonstrating that Notch canonical signaling was accountable for the phenotype. Notch activation downregulated Sost and Dkk1 and upregulated Axin2, Tnfrsf11b, and Tnfsf11 mRNA expression, and these effects were not observed in the context of the Rbpjκ inactivation. In conclusion, Notch activation in osteocytes suppresses bone resorption and increases bone volume by utilization of canonical signals that also result in the inhibition of Sost and Dkk1 and upregulation of Wnt signaling.

Osteoporosis is a common disease that is characterized by low bone mineral density (BMD), deterioration in bone microarchitecture, and increased fracture risk. Due to its important role in bone biology, the TNFRSF11B gene, coding for OPG, has been considered as a candidate gene for osteoporosis. In this study, single nucleotide polymorphisms (SNPs) A163G, T245G, and G1181C (rs3102735, rs3134069, and rs2073618, respectively) within the TNFRSF11B gene were studied for association with BMD and fracture incidence in a cohort of 327 postmenopausal Slovak women. Genomic DNA was extracted and purified from peripheral blood leukocytes by the commercial kit JetQuick (Genomed GmbH, Germany) using a standard protocol. Genotyping was performed using the Custom TaqMan® SNP Genotyping Assays. The lumbar L1-L4 spine BMD (g/cm(2)) and T-score in the subgroup of Slovak postmenopausal women with osteoporotic fractures were significantly lower than those in the subgroup of women without fracture (p = 0.0025; p = 0.0009). We identified the T245G (rs3134069) polymorphism in the TNFRSF11B gene associated with osteoporotic fractures (vertebral fractures: p = 0.0320; non-vertebral fractures: p = 0.0005; all fractures: 0.0000). The polymorphism T245G (rs3134069) in the TNFRSF11B gene could be used together with other genetic markers to identify individuals at high risk of osteoporotic fractures. The results from the present study provided more evidence to reveal the role of TNFRSF11B gene polymorphisms in BMD and the risk of osteoporotic fractures.

This one-year double blind randomized control trial assessed the effects of nightly melatonin, strontium (citrate), vitamin D3 and vitamin K2 (MK7; MSDK) on bone mineral density (BMD) and quality of life (QOL) in postmenopausal osteopenic women (ages 49-75). Compared to placebo, MSDK treatment increased BMD in lumbar spine (4.3%) and left femoral neck (2.2%), with an upward trend for total left hip (p=0.069). MSDK increased serum P1NP levels and reduced bone turnover (CTx:P1NP). Psychometric analyses indicated that mood and sleep quality improved for the MSDK group. MSDK-exposed human mesenchymal stem cells (hMSCs) and human peripheral blood monocytes (hPBMCs) plated in transwells or layered demonstrated increases in osteoblastogenesis, decreases in osteoclastogenesis, increases in OPG (TNFRSF11B) and decreases in RANKL (TNFSF11) levels. In transwell osteoblasts, MSDK increased pERK1/2 (MAPK1/MAPK3) and RUNX2 levels; decreased ERK5 (MAPK7); and did not affect the expression of NFκB (NFKB1) and β1integrin (ITGB1). In layered osteoblasts, MSDK also decreased expression of the metabolic proteins PPARγ (PPARG) and GLUT4 (SLC2A4). In adipose-derived human MSCs, MSDK induced osteoblastogenesis. These findings provide both clinical and mechanistic support for the use of MSDK for the prevention or treatment of osteopenia, osteoporosis or other bone-related diseases.

OCT4B1 is a newly discovered spliced variant of OCT4 which is primarily expressed in pluripotent and tumor cells. Based on our previous studies, OCT4B1 is significantly overexpressed in tumors, where it endows an anti-apoptotic property to tumor cells. However, the mechanism by which OCT4B1 regulates the apoptotic pathway is not yet elucidated. Here, we investigated the effects of OCT4B1 suppression on the expression alteration of 84 genes involved in apoptotic pathway. The AGS (gastric adenocarcinoma), 5637 (bladder tumor), and U-87MG (brain tumor) cell lines were transfected with OCT4B1 or irrelevant siRNAs. The expression level of apoptotic genes was then quantified using a human apoptosis panel-PCR kit. Our data revealed an almost similar pattern of alteration in the expression profile of apoptotic genes in all three studied cell lines, following OCT4B1 suppression. In general, the expression of more than 54 apoptotic genes (64 % of arrayed genes) showed significant changes. Among these, some up-regulated (CIDEA, CIDEB, TNFRSF1A, TNFRSF21, TNFRSF11B, TNFRSF10B, and CASP7) and down-regulated (BCL2, BCL2L11, TP73, TP53, BAD, TRAF3, TRAF2, BRAF, BNIP3L, BFAR, and BAX) genes had on average more than tenfold gene expression alteration in all three examined cell lines. With some minor exceptions, suppression of OCT4B1 caused upregulation of pro-apoptotic and down-regulation of anti-apoptotic genes in transfected tumor cells. Uncovering OCT4B1 down-stream targets could further elucidate its part in tumorigenesis, and could lead to finding a new approach to combat cancer, based on targeting OCT4B1.

Several rare inherited osteolytic disorders have been described that show phenotypic overlap with Paget's disease of bone (PDB). Familial expansile osteolysis, early-onset familial PDB and expansile skeletal hyperphosphatasia are related disorders caused by mutations affecting the TNFRSF11A gene, which encodes the receptor activator of NFkappaB (RANK). The mutations result in failure of normal processing of RANK and osteoclast activation. Inactivating mutations in the TNFRSF11B gene, which encodes osteoprotegerin, cause idiopathic hyperphosphatasia which is a severe disorder that shares phenotypic similarities with PDB. The syndrome of hereditary inclusion body myopathy, PDB and frontotemporal dementia is caused by mutations in the gene encoding for valosin-containing protein which is involved in regulating degradation of ubiquitinated proteins. Anecdotal reports indicate that osteoclast inhibitors such as bisphosphonates are effective for suppressing bone turnover and improving symptoms in these disorders, although the long-term effects on clinical outcomes are unclear.

OBJECTIVE

Osteoprotegerin (OPG) is a secretory glycoprotein that belongs to the tumor necrosis factor receptor family and plays a role in atherosclerosis. OPG has been hypothesized to modulate vascular functions; however, its role in mediating atherosclerosis is controversial. Epidemiological data in patients with cardiovascular disease (CVD) indicate that OPG serum levels are associated with several inflammatory markers, myocardial infarction events, and calcium scores, suggesting that OPG may be causative for CVD.

METHODS

The present study aimed to evaluate whether the OPG gene (TNFRSF11B) polymorphisms are involved in the development of peripheral arterial occlusive disease (PAOD) and critical limb ischemia (CLI) in patients with type 2 diabetes. This genetic association study included 402 diabetic patients (139 males and 263 females) with peripheral arterial occlusive disease and 567 diabetic subjects without peripheral arterial occlusive disease (208 males and 359 females). The T245G, T950C, and G1181C polymorphisms of the OPG gene were analyzed by polymerase chain reaction and restriction fragment length polymorphism.

RESULTS

We found that the T245G, T950C, and G1181C gene polymorphisms of the OPG gene were significantly (27.9 vs. 12.2 %, P < 0.01; 33.6 vs. 10.4 %, P < 0.01 and 24.4 vs. 12.7 %, P < 0.01, respectively) and independently (adjusted OR 4.97 (3.12-6.91), OR 7.02 (4.96-11.67), and OR 2.85 (1.95-4.02), respectively) associated with PAOD. We also found that these three polymorphisms act synergistically in patients with PAOD and are associated with different levels of risk for PAOD and CLI, depending on the number of high-risk genotypes carried concomitantly by a given individual.

CONCLUSIONS

The TNFRSF11B gene polymorphisms under study are associated with PAOD, and synergistic effects between these genotypes might be potential markers for the presence and severity of atherosclerotic disorders.

Mendelian disorders of RANKL/OPG/RANK signaling feature the extremes of aberrant osteoclastogenesis and cause either osteopetrosis or rapid turnover skeletal disease. The patients with autosomal dominant accelerated bone remodeling have familial expansile osteolysis, early-onset Paget's disease of bone, expansile skeletal hyperphosphatasia, or panostotic expansile bone disease due to heterozygous 18-, 27-, 15-, and 12-bp insertional duplications, respectively, within exon 1 of TNFRSF11A that encodes the signal peptide of RANK. Juvenile Paget's disease (JPD), an autosomal recessive disorder, manifests extremely fast skeletal remodeling, and is usually caused by loss-of-function mutations within TNFRSF11B that encodes OPG. These disorders are ultra-rare. A 13-year-old Bolivian girl was referred at age 3years. One femur was congenitally short and curved. Then, both bowed. Deafness at age 2years involved missing ossicles and eroded cochleas. Teeth often had absorbed roots, broke, and were lost. Radiographs had revealed acquired tubular bone widening, cortical thickening, and coarse trabeculation. Biochemical markers indicated rapid skeletal turnover. Histopathology showed accelerated remodeling with abundant osteoclasts. JPD was diagnosed. Immobilization from a femur fracture caused severe hypercalcemia that responded rapidly to pamidronate treatment followed by bone turnover marker and radiographic improvement. No TNFRSF11B mutation was found. Instead, a unique heterozygous 15-bp insertional tandem duplication (87dup15) within exon 1 of TNFRSF11A predicted the same pentapeptide extension of RANK that causes expansile skeletal hyperphosphatasia (84dup15). Single nucleotide polymorphisms in TNFRSF11A and TNFRSF11B possibly impacted her phenotype. Our findings: i) reveal that JPD can be associated with an activating mutation within TNFRSF11A, ii) expand the range and overlap of phenotypes among the Mendelian disorders of RANK activation, and iii) call for mutation analysis to improve diagnosis, prognostication, recurrence risk assessment, and perhaps treatment selection among the monogenic disorders of RANKL/OPG/RANK activation.

The oldest person (60 yr) with juvenile Paget's disease is homozygous for the TNFRSF11B mutation 966_969delTGACinsCTT. Elevated circulating levels of immunoreactive OPG and soluble RANKL accompany this genetic defect that truncates the OPG monomer, preventing formation of OPG homodimers.

BACKGROUND

Juvenile Paget's disease (JPD), a rare autosomal recessive disorder, features skeletal pain, fracture, and deformity from extremely rapid bone turnover. Deafness and sometimes retinopathy also occur. Most patients have diminished osteoprotegerin (OPG) inhibition of osteoclastogenesis caused by homozygous loss-of-function defects in TNFRSF11B, the gene that encodes OPG. Circulating immunoreactive OPG (iOPG) is undetectable with complete deletion of TNFRSF11B but normal with a 3-bp in-frame deletion.

METHODS

We summarize the clinical course of a 60-yr-old Greek man who is the second reported, oldest JPD patient, including his response to two decades of bisphosphonate therapy. Mutation analysis involved sequencing all exons and adjacent mRNA splice sites of TNFRSF11B. Over the past 4 yr, we used ELISAs to quantitate his serum iOPG and soluble RANKL (sRANKL) levels.

RESULTS

Our patient suffered progressive deafness and became legally blind, although elevated markers of bone turnover have been normal for 6 yr. He carries the same homozygous mutation in TNFRSF11B (966_969delTGACinsCTT) reported in a seemingly unrelated Greek boy and Croatian man who also have relatively mild JPD. This frame-shift deletes 79 carboxyterminal amino acids from the OPG monomer, including a cysteine residue necessary for homodimerization. Nevertheless, serum iOPG and sRANKL levels are persistently elevated.

CONCLUSIONS

Homozygosity for the TNFRSF11B "Balkan" mutation (966_969delTGACinsCTT) causes JPD in the second reported, oldest patient. Elevated circulating iOPG and sRANKL levels complement evidence that this deletion/insertion omits a cysteine residue at the carboxyterminus needed for OPG homodimerization.

BACKGROUND

Arterial calcification and osteoporosis often coexist, especially in postmenopausal women. Osteoporosis associates with a substantially increased risk of stroke in elderly women, suggesting that impaired estrogen signaling may link stroke and osteoporosis. Osteoprotegerin (OPG, TNFRSF11B) and interleukin-6 (IL-6, IL6) are putative target genes for estrogen signaling and have been implicated in both cardiovascular diseases and osteoporosis. We hypothesized that specific polymorphisms in these genes may be associated with increased risk of ischemic stroke or intracerebral hemorrhage (ICH).

METHODS

We performed a population-based prospective nested case-control study, in which the relationships between polymorphisms (OPG-1181G/C, OPG-950T/C and IL6-174G/C) and ischemic stroke and ICH were examined. Definitive first-ever stroke events (n = 388), i.e. ischemic stroke (n = 320), ICH (n = 61) and unspecified stroke (n = 7) cases, and controls without cardiovascular disease (n = 773), matched for age, sex and geographical region were studied. Univariate and multivariate models using conditional logistic regression, which included traditional risk factors, were used to test for association.

RESULTS

Carriers of the OPG-1181C/C genotype had a significantly (p = 0.018) increased risk of ICH (OR, 2.69; 95% CI, 1.19-6.12) in the univariate analysis. After adjustments (hypertension, diabetes, BMI and triglycerides), this genotype remained significantly (p = 0.005) associated with ICH (OR, 6.04; 95% CI, 1.71-21.29). By contrast, no correlations were found between this genotype and ischemic stroke, nor between the OPG-950T/C or IL6-174G/C polymorphisms and stroke subtypes.

CONCLUSIONS

In this population, the OPG-1181C/C genotype associates with first-ever ICH, implying that alterations in OPG-mediated signaling in the vasculature may be involved in the pathophysiology of this disease.

BACKGROUND

Diabetic foot is a severe diabetic complication, which may result in ulcerations that are unresponsive to treatment and in lower limb amputation. Osteoprotegerin is a protein that is involved in the pathogenesis of diabetic foot.

OBJECTIVE

The aim of the study was to evaluate the frequency of alleles in the TNFRSF11B gene rs2073617, rs2073618, and rs3134069 polymorphisms in patients with diabetic foot, diabetes, and healthy controls.

METHODS

The study comprised 877 patients, including 122 with diabetic foot, 293 with type 2 diabetes without diabetic foot, and 462 healthy controls.

RESULTS

In the rs2073618 polymorphism, the C allele was a risk factor for diabetic foot in patients with diabetes in the allelic variants [CC] vs. [CG + GG] (odds ratio [OR], 1.72; 95% confidence interval [CI], 1.03-2.86; P = 0.035), and in men in the following allelic variants: CC vs. GG (OR, 3.16; 95% CI, 1.27-7.87; P = 0.011), CC vs. CG (OR, 3.33; 95% CI, 1.47-7.54; P = 0.002), and [CC] vs. [CG + GG] (OR, 3.28; 95% CI, 1.48-7.24; P = 0.002). A similar association was observed between men with diabetic foot and those only with diabetes in the following allelic variants: CC vs. GG (OR, 2.30; 95% CI, 0.91-5.85; P = 0.076), CC vs. CG (OR, 2.69; 95% CI, 1.16-6.22; P = 0.018) and [CC] vs. [CG + GG] (OR, 2.56; 95% CI, 1.13-5.77; P = 0.02). For patients with neuropathic diabetic foot, the association was demonstrated in variant CC vs. CG (OR, 2.5; 95% CI, 1.00-6.23; P = 0.044) and only for men in the following allelic variants: [CC] vs. [CG + GG] (OR, 3.17; 95% CI, 1.07-9.38; P = 0.029) and CC vs. CG (OR, 3.49; 95% CI, 1.15-10.58; P = 0.02). The A allele of the rs2073617 polymorphism protected women in variant AA vs. AG against diabetic foot compared with controls (OR, 0.45; 95% CI, 1.00-4.92; P = 0.045). The rs3134069 polymorphism was not observed to be a risk factor for diabetic foot.

CONCLUSIONS

The analysis of the TNFRSF11B gene may be used to assess the risk of diabetic foot and neuropathic diabetic foot in patients with type 2 diabetes.

Paget's disease of bone (PDB) is a common condition, which is characterised by focal areas of increased and disorganized bone remodeling. Genetic factors play an important role in the disease. In some cases, Paget's disease is inherited in an autosomal dominant manner and the most common cause for this is a mutation in the SQSTM1 gene. Other familial cases have been linked to the OPTN locus on Chromosome 10p13 and still other variants have been identified by genome wide association studies that lie within or close to genes that play roles in osteoclast differentiation and function. Mutations in TNFRSF11A, TNFRSF11B and VCP have been identified in rare syndromes with PDB-like features. These advances have improved understanding of bone biology and the causes of PDB. The identification of genetic markers for PDB also raises the prospect that genetic profiling could identify patients at high risk of developing complications, permitting enhanced surveillance and early therapeutic intervention.

A recent genome-wide association study (GWAS) identified association with variants in X-linked CLDN2 and MORC4, and PRSS1-PRSS2 loci with chronic pancreatitis (CP) in North American patients of European ancestry. We selected 9 variants from the reported GWAS and replicated the association with CP in Indian patients by genotyping 1807 unrelated Indians of Indo-European ethnicity, including 519 patients with CP and 1288 controls. The etiology of CP was idiopathic in 83.62% and alcoholic in 16.38% of 519 patients. Our study confirmed a significant association of 2 variants in CLDN2 gene (rs4409525-OR 1.71, P = 1.38 x 10-09; rs12008279-OR 1.56, P = 1.53 x 10-04) and 2 variants in MORC4 gene (rs12688220-OR 1.72, P = 9.20 x 10-09; rs6622126-OR 1.75, P = 4.04x10-05) in Indian patients with CP. We also found significant association at PRSS1-PRSS2 locus (OR 0.60; P = 9.92 x 10-06) and SAMD12-TNFRSF11B (OR 0.49, 95% CI [0.31-0.78], P = 0.0027). A variant in the gene MORC4 (rs12688220) showed significant interaction with alcohol (OR for homozygous and heterozygous risk allele -14.62 and 1.51 respectively, P = 0.0068) suggesting gene-environment interaction. A combined analysis of the genes CLDN2 and MORC4 based on an effective risk allele score revealed a higher percentage of individuals homozygous for the risk allele in CP cases with 5.09 fold enhanced risk in individuals with 7 or more effective risk alleles compared with individuals with 3 or less risk alleles (P = 1.88 x 10-14). Genetic variants in CLDN2 and MORC4 genes were associated with CP in Indian patients.

Osteoprotegerin (OPG) inhibits the ability of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) to stimulate the differentiation, activity, and survival of bone-resorbing osteoclasts. Genetic studies in mice show that osteocytes are an important source of RANKL, but the cellular sources of OPG are unclear. We use conditional deletion of Tnfrsf11b, which encodes OPG, from different cell populations to identify functionally relevant sources of OPG in mice. Deletion from B lymphocytes and osteocytes, two cell types commonly thought to supply OPG, has little or no impact on bone mass. By contrast, deletion of Tnfrsf11b from osteoblasts increases bone resorption and reduces bone mass to an extent similar to germline deletion, demonstrating that osteoblasts are an essential source of OPG. These results suggest that, in addition to producing new bone matrix, osteoblasts also play an active role in terminating the resorption phase of the bone remodeling cycle by suppressing RANKL activity.

Keywords: bone remodeling; osteoblast; osteoclast; osteocyte; osteoprotegerin.

Several rare inherited disorders have been described that show phenotypic overlap with Paget's disease of bone (PDB) and in which PDB is a component of a multisystem disorder affecting muscle and the central nervous system. These conditions are the subject of this review article. Insertion mutations within exon 1 of the TNFRSF11A gene, encoding the receptor activator of nuclear factor kappa B (RANK), cause severe PDB-like disorders including familial expansile osteolysis, early-onset familial PDB and expansile skeletal hyperphosphatasia. The mutations interfere with normal processing of RANK and cause osteoclast activation through activation of nuclear factor kappa B (NFκB) independent of RANK ligand stimulation. Recessive, loss-of-function mutations in the TNFRSF11B gene, which encodes osteoprotegerin, cause juvenile PDB and here the bone disease is due to unopposed activation of RANK by RANKL. Multisystem proteinopathy is a disorder characterised by myopathy and neurodegeneration in which PDB is often an integral component. It may be caused by mutations in several genes including VCP, HNRNPA1, HNRNPA2B1, SQSTM1, MATR3, and TIA1, some of which are involved in classical PDB. The mechanisms of osteoclast activation in these conditions are less clear but may involve NFκB activation through sequestration of IκB. The evidence base for management of these disorders is somewhat limited due to the fact they are extremely rare. Bisphosphonates have been successfully used to gain control of elevated bone remodelling but as yet, no effective treatment exists for the treatment of the muscle and neurological manifestations of MSP syndromes.

Genome-wide association studies (GWASs) have revealed numerous loci for areal bone mineral density (aBMD). We completed the first GWAS meta-analysis (n = 15,275) of lumbar spine volumetric BMD (vBMD) measured by quantitative computed tomography (QCT), allowing for examination of the trabecular bone compartment. SNPs that were significantly associated with vBMD were also examined in two GWAS meta-analyses to determine associations with morphometric vertebral fracture (n = 21,701) and clinical vertebral fracture (n = 5893). Expression quantitative trait locus (eQTL) analyses of iliac crest biopsies were performed in 84 postmenopausal women, and murine osteoblast expression of genes implicated by eQTL or by proximity to vBMD-associated SNPs was examined. We identified significant vBMD associations with five loci, including: 1p36.12, containing WNT4 and ZBTB40; 8q24, containing TNFRSF11B; and 13q14, containing AKAP11 and TNFSF11. Two loci (5p13 and 1p36.12) also contained associations with radiographic and clinical vertebral fracture, respectively. In 5p13, rs2468531 (minor allele frequency [MAF] = 3%) was associated with higher vBMD (β = 0.22, p = 1.9 × 10-8 ) and decreased risk of radiographic vertebral fracture (odds ratio [OR] = 0.75; false discovery rate [FDR] p = 0.01). In 1p36.12, rs12742784 (MAF = 21%) was associated with higher vBMD (β = 0.09, p = 1.2 × 10-10 ) and decreased risk of clinical vertebral fracture (OR = 0.82; FDR p = 7.4 × 10-4 ). Both SNPs are noncoding and were associated with increased mRNA expression levels in human bone biopsies: rs2468531 with SLC1A3 (β = 0.28, FDR p = 0.01, involved in glutamate signaling and osteogenic response to mechanical loading) and rs12742784 with EPHB2 (β = 0.12, FDR p = 1.7 × 10-3 , functions in bone-related ephrin signaling). Both genes are expressed in murine osteoblasts. This is the first study to link SLC1A3 and EPHB2 to clinically relevant vertebral osteoporosis phenotypes. These results may help elucidate vertebral bone biology and novel approaches to reducing vertebral fracture incidence. © 2016 American Society for Bone and Mineral Research.

OBJECTIVE

Osteoporosis is a complex disease characterized principally by low bone mineral density (BMD), which is determined by an interaction of genetic, metabolic, and environmental factors. The aim of this study was to analyze the possible association among one polymorphism of LRP5 and three polymorphisms of TNFRSF11B as well as their haplotypes with BMD variations in Maya-Mestizo postmenopausal women.

METHODS

We studied 583 postmenopausal women of Maya-Mestizo ethnic origin. A structured questionnaire for risk factors was applied and BMD was measured in lumbar spine (LS), total hip (TH), and femoral neck (FN) by dual-energy X-ray absorptiometry. DNA was obtained from blood leukocytes. One single-nucleotide polymorphism of LRP5 (rs3736228, p.A1330V) and three of TNFRSF11B (rs4355801, rs2073618, and rs6993813) were studied using real-time PCR allelic discrimination for genotyping. Differences between the means of the BMDs according to the genotype were analyzed with covariance. Deviations from Hardy-Weinberg equilibrium were tested. Pairwise linkage disequilibrium between single nucleotide polymorphisms was calculated by direct correlation r(2), and haplotype analysis of TNFRSF11B was conducted.

RESULTS

The Val genotype of the rs3736228 (p.A1330V) of LRP5 was significantly associated with BMD variations at the LS, TH, and FN. None of the three polymorphisms of TNFRSF11B was associated with BMD variations.

CONCLUSIONS

Our results show that p.A1330V was significantly associated with BMD variations at all three skeletal sites analyzed; the Val allele and the Val/Val genotype were those most frequently found in our population.

OBJECTIVE

This paper aims to investigate the influence of single-nucleotide polymorphisms (SNPs) in the receptor of activator of nuclear factor kappaB ligand (RANKL) gene (TNFSF11) and osteoprotegerin (OPG) gene (TNFRSF11B) on bone and joint injury in patients with rheumatoid arthritis (RA).

METHODS

Two hundred RA patients and 201 matched controls were analysed by case-control design, and their samples were genotyped. Bone mineral density (BMD) and serum OPG and RANKL levels were measured. Clinical and laboratory parameters were recorded, and the radiographic changes in both hands of RA were evaluated by Sharp's method.

RESULTS

Our results showed no significant differences in the distribution frequency of the alleles and genotypes of TNFRSF11B (rs2073618 and rs3102735) and TNFSF11 (rs2277438) between the RA group and controls (p>0.05). Compared to patients with TNFSF11 (rs2277438) AA or GG genotype, RA with TNFSF11 (rs2277438) AG genotype had significantly decreased BMD values at lumbar spine 3, lumbar spine 4, lumbar spine 2-4 (p<0.05-0.01), and apparently elevated Sharp scores (p<0.05), respectively. The RA group showed significantly higher serum levels of RANKL, RANKL/OPG ratio and a lower serum level of OPG than that of the controls (p<0.05-0.0001). RA patients with RANKL-rs2277438 heterozygotic genotype (AG) had significantly increased serum levels of RANKL (p<0.05), compared to homozygotic genotype (AA or GG).

CONCLUSIONS

These results indicate that SNP of TNFRSF11B (rs2073618 and rs3102735) and TNFSF11 (rs2277438) may not be susceptibility factors for RA in Chinese Han population. SNP of TNFSF11 (rs2277438) may have an important influence on bone and joint injury in RA.

OBJECTIVE

In addition to genome-wide association studies (GWAS), height-associated genes may be uncovered by studying individuals with extreme short or tall stature.

METHODS

Genome-wide analysis for copy number variants (CNVs), using single nucleotide polymorphism (SNP) arrays, was performed in 49 index cases born small for gestational age with persistent short stature. Segregation analysis was performed, and genes in CNVs were compared with information from GWAS, gene expression in rodents' growth plates, and published information.

RESULTS

CNVs were detected in 13 cases. In 5 children a known cause of short stature was found: UPD7, UPD14, a duplication of the SHOX enhancer region, an IGF1R deletion, and a 22q11.21 deletion. In the remaining 8 cases, potential pathogenic CNVs were detected, either de novo (n = 1), segregating (n = 2), or not segregating with short stature (n = 5). Bioinformatic analysis of the de novo and segregating CNVs suggested that HOXD4, AGPS, PDE11A, OSBPL6, PRKRA and PLEKHA3, and possibly DGKB and TNFRSF11B are potential candidate genes. A SERPINA7 or NRK defect may be associated with an X-linked form of short stature.

CONCLUSIONS

SNP arrays detected 5 known causes of short stature with prenatal onset and suggested several potential candidate genes.

OBJECTIVE

Cisplatin is an anti-neoplastic agent treatment with which causes many side effects including ototoxicity. The aim of this study was to investigate whether acetyl-L-carnitine would have protective effects on cisplatin-induced ototoxicity in vitro, and if present, to reveal roles of apoptotic gene expressions and pro-inflammatory cytokines.

METHODS

House Ear Institute-Organ of Corti 1 cell line was used for this study. Apoptotic genes were evaluated with an apoptosis PCR array and pro-inflammatory cytokine levels were measured using ELISA.

RESULTS

Apoptotic cell death reduced by around 22% with acetyl-L-carnitine-cisplatin treatment compared to cisplatin alone. Genes displaying increase in expression of apoptosis, related to cisplatin treatment, were Casp8, Bcl10, Bcl2, Bcl2l1, Bcl2l2, Bid, Naip1, Bnip3l, Card6, Pak7, Cd40, Trp 53inp1, Cideb and Cd70. The acetyl-L-carnitine-cisplatin combination caused reduced expression of genes Casp8, Fas, Casp1, Tnfrsf11b, Tnfrsf10b induced by cisplatin. Acetyl-L-carnitine-cisplatin also caused reduced levels of IL-6, IL-1β and TNF-α, pro-inflammatory cytokines, induced by cisplatin.

CONCLUSIONS

Protective mechanisms of aceytl-L-carnitine against cisplatin induced apoptosis, mainly due to activation of anti-apoptotic Bcl family members' genes, and in an Akt-related gene expression dependent manner. This is the first study to indicate that acetyl-L-carnitine can be an effective agent against cisplatin ototoxicity in auditory cells, with induction of anti-apoptotic gene expression and attenuating levels of pro-inflammatory cytokines.

BACKGROUND

Osteoprotegerin (OPG), an immunoregulatory member of the TNF receptor superfamily, is expressed in inflamed intestinal mucosa. We investigated whether OPG is produced by intestinal epithelial cells and tested the hypothesis that single-nucleotide polymorphisms (SNPs) in the gene encoding OPG (TNFRSF11B) are associated with traveler's diarrhea (TD) among North American travelers to Mexico.

METHODS

OPG concentration was measured in the supernatants of T84 cells infected with various diarrheagenic Escherichia coli pathotypes. Genotyping was performed for 4 SNPs in the OPG gene for 968 North American travelers with or without TD. Stool samples from travelers with TD were evaluated for the presence of enteric pathogens.

RESULTS

T84 cells produced higher OPG levels in response to infection with various diarrheagenic E. coli pathotypes than with E. coli controls (P<.05). A SNP in the exon 1 region of the OPG gene (OPG+1181G>C) was associated with TD in white travelers who stayed in Mexico for >1 week during the summer (P=.009) and for TD due to nonsecretory pathogens (P=.001).

CONCLUSIONS

Our study suggests that OPG is secreted by intestinal epithelial cells in response to enteropathogens and that a polymorphism in the OPG gene is associated with an increased susceptibility to TD.

An effective countermeasure against radiation damage to normal tissues is urgently needed. The major goal of the present study was to determine if minocycline could modify the immunomodulatory effects of radiation on the brain. C57BL/6 mice were treated with minocycline intraperitoneally for 5 days beginning immediately before total-body exposure to 0, 1, 2 and 3 Gray (Gy) (60)Co γ-rays. Brains were collected on days 4 and 32 post-irradiation for cytokine and gene analyses. Minocycline treatment significantly increased the levels of interleukin (IL)-10, IL-15 and vascular endothelial growth factor (VEGF) in the brain on day 4 in one or more irradiated groups compared to radiation-alone (p<0.05). IL-10 is anti-inflammatory, IL-15 can prevent apoptosis and VEGF is nuroprotective. On day 32, the drug decreased IL-1β in the 2- Gy group (p<0.05 vs. 2-Gy alone); this cytokine is implicated in immune-related central nervous system pathologies. Microarray analysis of brains on day 32 showed that while radiation increased expression of inflammatory genes such as Il1f10, Il17, Tnfrsf11b, Tnfsf12, Il12b and Il1f8, these were no longer up-regulated in the minocycline-treated groups. Similarly, the pro-apoptotic gene Bik and nitric oxide synthase producer (Nostrin) were no longer up-regulated in the drug-treated groups. Pathway analysis based on gene data suggested that catenin-β1 and tumor suppressor-related transcription regulation were significantly activated by radiation and/or minocycline (activation z-score >2.0). Overall, the data warrant further testing of minocycline as a potential neuroprotectant against radiation-induced damage.