Janja Marc

Expression of bone resorption genes in osteoarthritis and in osteoporosis.

Cathepsin K and MMP-9 are considered to be the most abundant proteases in osteoclasts. TRAP is a marker for osteoclasts, and there is increasing evidence of its proteolytic role in bone resorption. RANKL is a recently discovered regulator of osteoclast maturation and activity and induces expression of many genes. This study compared cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin gene expression in the proximal femur of patients with osteoarthritis with that of patients with femoral neck fracture. Fifty-six patients undergoing arthroplasty because of osteoarthritis or femoral neck fracture were included in the study. Total mRNA was extracted from the bone samples obtained from the intertrochanteric region of the proximal femur. Real-time RT-PCR was used to quantify CTSK (cathepsin K), MMP-9 (matrix metalloproteinase 9), ACP5 (TRAP), TNFSF11 (RANKL), TNFRSF11B (OPG), and BGLAP (osteocalcin) mRNAs. The levels of mRNAs coding for MMP-9 and osteocalcin indicated higher expression in the osteoarthritic group (P = 0.011, P = 0.001, respectively), whereas RANKL expression and the ratio RANKL/OPG were both significantly lower in the osteoarthritic group than in the fracture group. Expression of cathepsin K, MMP-9, and TRAP relative to RANKL was significantly higher in the osteoarthritic group. Ratios of all three proteolytic enzymes relative to formation marker osteocalcin were higher in the fracture group. Gene expression of cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin and the association between their mRNA levels pointed to higher bone resorption and bone formation in osteoarthritis, differences in balance between them, and differences in regulation of bone resorption in osteoarthritic and osteoporotic bone.

Osteoimmunology and the influence of pro-inflammatory cytokines on osteoclasts

Bone and immune system are functionally interconnected. Immune and bone cells derive from same progenitors in the bone marrow, they share a common microenvironment and are being influenced by similar mediators. The evidence on increased bone resorption associated with inappropriate activation of T cells such as during inflammation, is well established. However, the molecular mechanisms beyond this clinical observation have begun to be intensively studied with the advancement of osteoimmunology. Now days, we have firm evidence on the influence of numerous proinflammatory cytokines on bone cells, with the majority of data focused on osteoclasts, the bone resorbing cells. It has been shown that some proinflammatory cytokines could possess osteoclastogenic and/or anti-osteoclastogenic properties and can target osteoclasts directly or via receptor activator of nuclear factor κB (RANK)/RANK ligand(RANKL)/osteoprotegerin (OPG) system. Several studies have reported opposing data regarding (anti)osteoclastogenic properties of these cytokines. Therefore, the first part of this review is summarizing current evidence on the influence of pro-inflammatory cytokines on osteoclasts and thus on bone resorption. In the second part, the evidence on the role of pro-inflammatory cytokines in osteoporosis and osteoarthritis is reviewed to show that unravelling the mechanisms beyond such complex bone diseases, is almost impossible without considering skeletal and immune systems as an indivisible integrated system.

The many faces of estrogen signaling

Estrogens have long been known as important regulators of the female reproductive functions; however, our understanding of the role estrogens play in the human body has changed significantly over the past years. It is now commonly accepted that estrogens and androgens have important functions in both female and male physiology and pathology. This is in part due to the local synthesis and action of estrogens that broadens the role of estrogen signaling beyond that of the endocrine system. Furthermore, there are several different mechanisms through which the three estrogen receptors (ERs), ERα, ERβ and G protein-coupled estrogen receptor 1 (GPER1) are able to regulate target gene transcription. ERα and ERβ are mostly associated with the direct and indirect genomic signaling pathways that result in target gene expression. Membrane-bound GPER1 is on the other hand responsible for the rapid non-genomic actions of estrogens that activate various protein-kinase cascades. Estrogen signaling is also tightly connected with another important regulatory entity, i.e. epigenetic mechanisms. Posttranslational histone modifications, microRNAs (miRNAs) and DNA methylation have been shown to influence gene expression of ERs as well as being regulated by estrogen signaling. Moreover, several coregulators of estrogen signaling also exhibit chromatin-modifying activities further underlining the importance of epigenetic mechanisms in estrogen signaling. This review wishes to highlight the newer aspects of estrogen signaling that exceed its classical endocrine regulatory role, especially emphasizing its tight intertwinement with epigenetic mechanisms.

VDR Genotype and Response to Etidronate Therapy in Late Postmenopausal Women

Abstract: Twenty-four late postmenopausal women with osteoporosis were studied. The patients were separated in three subgroups according to the BsmI polymorphism of the vitamin D receptor (VDR) gene: BB (n= 8), Bb (n = 10) and bb (n = 6). They did not differ in age (mean ages were 66.0 years, 65.9 years and 63.9 years, respectively), years after menopause (18.7 years, 18.1 years and 18.4 years) or body weight (64.9 kg, 65.3 kg and 63.8 kg), the variables known to be associated with bone mineral density (BMD). The results show that the response to antiresorptive bisphosphonate therapy in combination with calcium supplementation is modified by VDR genotype. The lumbar spine BMD increased significantly faster in the BB and Bb groups (7.3% and 7.0%, respectively) compared with the bb group (2.5%) during 1 year of cyclic etidronate therapy (400 mg/day) and calcium supplementation (1000 mg/day). The biochemical marker of bone resorption (urinary hydroxyproline excretion) as well as the bone formation marker (serum levels of osteocalcin) decreased during the treatment. With respect to VDR genotype, a significantly higher decrease in osteocalcin level was observed in bb as compared with BB subjects. We conclude that the VDR genotype is involved in an individual’s response to cyclic etidronate therapy with calcium supplementation.

A microarray based identification of osteoporosis-related genes in primary culture of human osteoblasts

Genetic factors influencing the pathogenesis of osteoporosis are still largely unknown. We employed genome-wide gene expression approach in order to discover novel genes involved in the pathogenesis of osteoporosis. To this end, primary cultures of osteoblasts isolated from osteoporotic and non-osteoporotic human bone tissue samples were prepared. One thousand six hundred six genes were found to be differentially expressed, indicating increased demand for protein synthesis and decreased cell proliferation rate in osteoblasts from osteoporotic tissue as compared to osteoblasts from non-osteoporotic tissue. At first, top four genes, based on the microarray data and potential role in bone metabolism, were further studied in bone tissue samples of 55 patients. PTN and COL15A1 were both downregulated in osteoporotic bone tissue (6.2- and 3.4-fold, respectively, both p<0.05), while IBSP and CXCL2 were both upregulated (5.7-fold, p<0.05, and 2.1-fold, p>0.05). Further biostatistical analysis of the microarray data by gene set enrichment analysis suggested oxidative stress may have an important part in the pathogenesis of osteoporosis. Thus, secondly, we tested it by an in vitro assay on human osteosarcoma cell line cells treated with hydrogen peroxide. After 72 h of treatment with 500 microM hydrogen peroxide, the upregulation of the same genes involved in the response to oxidative stress as on the microarrays was observed: MT1G (metallothionein 1G, 22.1-fold, p<0.05), TXNRD1 (thioredoxin reductase 1, 3.7-fold, p<0.05), AOX1 (aldehyde oxidase 1, 24.5-fold, p<0.05) and GSR (glutathione reductase, 4.7-fold, p<0.05). Our results present a novel list of genes and metabolic pathways that may be associated with the pathogenesis of osteoporosis. PTN, CXCL2, COL15A1, IBSP, AOX1, MT1G, GSR and TXNRD1 are candidate genes for further studies in the assessment of the genetic susceptibility to osteoporosis. In addition, differences in protein synthesis, cell proliferation rate and response to oxidative stress may also be involved in the pathogenesis of osteoporosis.

Epigenetic mechanisms in bone

Abstract Epigenetics refers to the study of mechanisms able to influence gene expression in a stable and potentially heritable manner without altering the DNA sequence. These mechanisms include posttranslational histone modifications, miRNA-mediated post-transcriptional regulation and DNA methylation. The accumulation of molecular errors over time resulting, at least partly, in the alteration of normal epigenetic patterns is being widely associated with aging. Epigenetic processes are also considered important mechanisms through which environmental and stochastic stressors promote numerous pathologies in humans. It is, therefore, reasonable to expect that several complex multi-factorial late-onset disorders, like osteoporosis and osteoarthritis, could have a strong epigenetic component. The focal point of all skeletal pathologies is the deregulation of bone remodeling, mediated by bone-forming osteoblasts and bone-resorbing osteoclasts. In order to keep both processes in balance, the activity, differentiation and apoptosis of both cell types have to be tightly regulated. In particular, the differentiation of osteoblasts and osteoclasts is accompanied by profound changes in gene expression. It has been shown that histone deacetylation and DNA methylation negatively regulate the expression of several genes associated with different stages of osteoblast differentiation; however, several miRNAs promote osteoblastogenesis. Furthermore, inactivating mutations in the miRNA coding regions could be associated with the pathogenesis of osteoporosis. The aim of this review is to highlight the role of epigenetic mechanisms in bone remodeling and bone homeostasis, so as to implicate their diagnostic and therapeutic potential in skeletal diseases.

New insights into adipose tissue dysfunction in insulin resistance

Abstract In a state of caloric excess, adipose tissue plays an essential role by storing lipids. Its expandability determines the onset of metabolic syndrome (central obesity, dyslipidemia, glucose intolerance and hypertension). When the adipocyte endoplasmic reticulum is no longer capable of processing the excess nutrients, the so-called “endoplasmic reticulum stress” develops. This triggers efflux of free fatty acids from adipocytes into the circulation and causes triglyceride overload in skeletal muscle, liver and pancreas. Adipose tissue hypoxia then develops, due to the failure of vasculature to expand with adipocyte hypertrophy. Increased catabolism in mitochondria leads there to oxidative stress. Both phenomena cause deranged adipokine secretion and low-grade inflammation. Inflammatory cytokines, reactive oxygen species and ectopic lipid deposition are the main mediators of insulin resistance and vascular impairment, which both lead finally to diabetes type 2 and cardiovascular disease. Recently, fibrosis of adipose tissue was also demonstrated in obesity, contributing to the interplay of deleterious factors forcing inflammation. The present paper reviews recent evidence for adipose tissue dysfunction, trying to define causes and consequences. In conclusion, insulin resistance and associated complications originate from excess lipids, which cannot be stored without limit in adipose tissue, thus affecting its integrity and adipokine secretion.

The combinations of polymorphisms in vitamin D receptor, osteoprotegerin and tumour necrosis factor superfamily member 11 genes are associated with bone mineral density.

1alpha,25-dihydroxyvitamin D(3) upregulates tumour necrosis factor superfamily member 11 (TNFSF11) that codes for the receptor activator of nuclear factor kappaB ligand (RANKL), and downregulates osteoprotegerin (OPG) expression. We have analyzed the individual effects of polymorphisms in the vitamin D receptor gene (VDR), OPG and TNFSF11, and searched for interactions between them. Six hundred and forty one subjects were evaluated: 239 osteoporotic and 228 non-osteoporotic post-menopausal, 57 pre-menopausal women and 117 elderly men. The subjects were genotyped for BsmI, FokI and Cdx2 in VDR, K3N in OPG and -290C>T, -643C>T and -693G>C in TNFSF11 gene. Bone mineral density (BMD) and biochemical markers were measured. In the osteoporotic women, femoral neck BMD (BMD-fn) showed associations with BsmI(VDR) and Cdx2(VDR) (P=0.015 and 0.047 respectively), and lumbar spine BMD (BMD-ls) with K3N(OPG) and -290C>T(TNFSF11) (P=0.021 and 0.017). No association with BMD was found in the non-osteoporotic women. In the pre-menopausal women, the Cdx2(VDR) polymorphism was associated with BMD-fn and total hip BMD (P=0.011 and 0.011). In elderly men, FokI(VDR) was associated with BMD-fn and BMD-ls (P=0.040 and 0.036). Interestingly, the -290C>T(TNFSF11)-K3N(OPG) combination was associated with BMD-th (P=0.041) in the osteoporotic women. In the non-osteoporotic women, the combination K3N(OPG)-Cdx2(VDR) was associated with BMD-ls, BMD-th and BMD-fn (P=0.032, 0.049 and 0.022), and the combination -290C>T(TNFSF11)-K3N(OPG) with BMD-fn (P=0.029). For the first time, the presence of gene-gene interactions between VDR, OPG and TNFSF11 genes was studied. Our results strongly suggest further confirmation of their combined influence on larger cohorts.

No major effect of estrogen receptor beta gene RsaI polymorphism on bone mineral density and response to alendronate therapy in postmenopausal osteoporosis

Genetic factors play an important role in the pathogenesis of osteoporosis. The genes involved are, however, still largely unknown. In the present study, we have investigated whether sequence variations in the estrogen receptor beta (ERbeta) gene are associated with bone mineral density (BMD) and biochemical markers of bone turnover in 79 Slovenian postmenopausal women with osteoporosis. We also assessed the response by BMD and bone markers to antiresorptive therapy with bisphosphonate alendronate. All eight exons of ERbeta gene were amplified by polymerase chain reaction and screened for mutations by single-strand conformation polymorphism analysis. Potentially mutated samples were found only in exon 5 and sequence analysis identified the presence of a silent mutation in codon 328 with a nucleotide substitution GTG to GTA. For easier detection of this silent mutation, the RsaI restriction fragment length polymorphism analysis was developed. The frequencies of genotypes were as follows: Rr 5.1% and RR 94.9%. Between both genotypes, no significant differences in baseline lumbar spine and femoral neck BMD or in bone markers osteocalcin and deoxypyridinoline were observed. Similarly, no significant difference between RR and Rr genotypes in BMD or bone markers after 1 year of therapy was found. The increase in lumbar spine BMD after therapy was the only parameter that approached statistical significance (P=0.099). Patients with genotype Rr showed a smaller increase compared to those with RR. Our results suggest that RsaI polymorphism of ERbeta gene is probably not an important genetic determinant of BMD and does not significantly influence the responsiveness to alendronate therapy.

Increased levels of osteoprotegerin in hemodialysis patients

Abstract Recently identified soluble circulating osteoprotegerin (OPG), a member of tumor necrosis factor receptor family, is the osteoclastogenesis inhibitory factor (OCIF). It acts as a “decoy” receptor for receptor activator of NF-κB ligand (RANKL) and antagonises RANKL/RANK activity. This way OPG exerts the protective effect on bone, which is also important in hyperparathyroidism. The studies measuring OPG levels in secondary hyperparathyroidism have shown contradictory results and inconsistent conclusions. The aim of our work was to evaluate OPG levels in hemodialysis patients and their correlation with the intensity of bone turnover, bone formation and bone resorption. Serum OPG levels, bone alkaline phosphatase activity (bALP) and β-CrossLaps (CTx) were measured in a control group (n = 20, age 30±6.7 years) and in two groups of dialysis patients: the first group with serum intact parathyroid hormone (iPTH) concentration below 200 pg/ml (n = 28, age 62.6±14.8 years) and the second group with iPTH concentration above 200 pg/ml (n = 16, age 63.7±14.8 years). Compared to controls, serum OPG levels were 6.4-fold higher in dialysis patients. OPG levels in patients with high PTH were approximately 1.2-fold higher than in the low-PTH group. OPG correlated weakly with bALP (r = 0.277, p = 0.153), as well as with CTx (r = 0.018, p = 0.929) in the low-PTH group, and there was an insignificant negative correlation in the high-PTH group (r = −0.145, p = 0.593 and r = −0.219, p = 0.416, respectively). In conclusion, 6.4-fold increase in OPG might protect bone against intensive bone loss in hemodialysis patients, but this increase is probably not mediated by the increased bone formation; rather, it seems to be the consequence of the imbalance of bone kinetics in renal disease. The precise role of OPG in the pathogenesis of renal osteodystrophy remains unknown and establishing it requires further studies.