Decoy Receptor Osteoprotegerin Research Articles

Serum Levels of Osteoprotegerin and Receptor Activator of Nuclear Factor-κB Ligand as Markers of Periprosthetic Osteolysis

Previous studies have suggested that the balance between receptor activator of nuclear factor-kappaB ligand (RANKL) and its decoy-receptor osteoprotegerin (OPG) in local tissue seems to play a crucial role in the loosening of the total hip replacement. The aim of this study was to evaluate whether the circulating levels of OPG and RANKL, as well as their ratio, could be different in patients with aseptic loosening compared with patients with stable implants. One hundred and twenty-eight subjects were recruited. They included thirty-nine patients with osteoarthritis who had not yet undergone total hip arthroplasty, thirty-three patients who had undergone total hip arthroplasty and had clinically and radiographically stable implants, thirty-six patients with aseptic loosening of total hip arthroplasty components, and twenty healthy volunteers. Serum levels of OPG and RANKL were measured with use of an immunoenzymatic method, and in each individual the OPG-to-RANKL ratio was calculated. In every group, a significant correlation was detected between OPG concentration and age (r = 0.58, p < 0.0001), especially in individuals older than fifty years, while gender and underlying disease were not found to influence serum levels of the tested parameters. In comparison with the levels in healthy donors and patients with a stable total hip replacement, the serum levels of OPG were increased in the patients who had not yet had an arthroplasty, those with aseptic loosening of a total hip replacement, and those with a cemented total hip replacement. Moreover, the OPG serum level provided good diagnostic accuracy in detecting the implant failure. A correlation was found between the sum of the osteolytic areas seen radiographically around the femoral stem and the RANKL level (r = 0.38, p = 0.02) and the OPG-to-RANKL ratio (r = -0.29, p = 0.04). An increase in OPG levels may reflect a protective mechanism of the skeleton to compensate for the osteolytic activity that occurs in severe osteoarthritis and in aseptic loosening. Prospective studies are needed to determine whether serum OPG levels could be used as markers for monitoring the stability of the implant, as well as for predicting aseptic loosening. Diagnostic study, Level III. See Instructions to Authors for a complete description of levels of evidence.

SERUM LEVELS OF OSTEOPROTEGERIN AND RECEPTOR ACTIVATOR OF NUCLEAR FACTOR-κB LIGAND AS MARKERS OF PERIPROSTHETIC OSTEOLYSIS

Background: Previous studies have suggested that the balance between receptor activator of nuclear factor-κB ligand (RANKL) and its decoy-receptor osteoprotegerin (OPG) in local tissue seems to play a crucial role in the loosening of the total hip replacement. The aim of this study was to evaluate whether the circulating levels of OPG and RANKL, as well as their ratio, could be different in patients with aseptic loosening compared with patients with stable implants. Methods: One hundred and twenty-eight subjects were recruited. They included thirty-nine patients with osteoarthritis who had not yet undergone total hip arthroplasty, thirty-three patients who had undergone total hip arthroplasty and had clinically and radiographically stable implants, thirty-six patients with aseptic loosening of total hip arthroplasty components, and twenty healthy volunteers. Serum levels of OPG and RANKL were measured with use of an immunoenzymatic method, and in each individual the OPG-to-RANKL ratio was calculated. Results: In every group, a significant correlation was detected between OPG concentration and age (r = 0.58, p < 0.0001), especially in individuals older than fifty years, while gender and underlying disease were not found to influence serum levels of the tested parameters. In comparison with the levels in healthy donors and patients with a stable total hip replacement, the serum levels of OPG were increased in the patients who had not yet had an arthroplasty, those with aseptic loosening of a total hip replacement, and those with a cemented total hip replacement. Moreover, the OPG serum level provided good diagnostic accuracy in detecting the implant failure. A correlation was found between the sum of the osteolytic areas seen radiographically around the femoral stem and the RANKL level (r = 0.38, p = 0.02) and the OPG-to-RANKL ratio (r = –0.29, p = 0.04). Conclusions: An increase in OPG levels may reflect a protective mechanism of the skeleton to compensate for the osteolytic activity that occurs in severe osteoarthritis and in aseptic loosening. Prospective studies are needed to determine whether serum OPG levels could be used as markers for monitoring the stability of the implant, as well as for predicting aseptic loosening. Level of Evidence: Diagnostic study, Level III. See Instructions to Authors for a complete description of levels of evidence.

RANKL Stimulates Inducible Nitric-oxide Synthase Expression and Nitric Oxide Production in Developing Osteoclasts: AN AUTOCRINE NEGATIVE FEEDBACK MECHANISM TRIGGERED BY RANKL-INDUCED INTERFERON-β VIA NF-κB THAT RESTRAINS OSTEOCLASTOGENESIS AND BONE RESORPTION

Nitric oxide (NO) is a multifunctional signaling molecule and a key vasculoprotective and potential osteoprotective factor. NO regulates normal bone remodeling and pathological bone loss in part through affecting the recruitment, formation, and activity of bone-resorbing osteoclasts. Using murine RAW 264.7 and primary bone marrow cells or osteoclasts formed from them by receptor activator of NF-kappaB ligand (RANKL) differentiation, we found that inducible nitric-oxide synthase (iNOS) expression and NO generation were stimulated by interferon (IFN)-gamma or lipopolysaccharide, but not by interleukin-1 or tumor necrosis factor-alpha. Surprisingly, iNOS expression and NO release were also triggered by RANKL. This response was time- and dose-dependent, required NF-kappaB activation and new protein synthesis, and was specifically blocked by the RANKL decoy receptor osteoprotegerin. Preventing RANKL-induced NO (via iNOS-selective inhibition or use of marrow cells from iNOS-/- mice) increased osteoclast formation and bone pit resorption, indicating that such NO normally restrains RANKL-mediated osteoclastogenesis. Additional studies suggested that RANKL-induced NO inhibition of osteoclast formation does not occur via NO activation of a cGMP pathway. Because IFN-beta is also a RANKL-induced autocrine negative feedback inhibitor that limits osteoclastogenesis, we investigated whether IFN-beta is involved in this novel RANKL/iNOS/NO autoregulatory pathway. IFN-beta was induced by RANKL and stimulated iNOS expression and NO release, and a neutralizing antibody to IFN-beta inhibited iNOS/NO elevation in response to RANKL, thereby enhancing osteoclast formation. Thus, RANKL-induced IFN-beta triggers iNOS/NO as an important negative feedback signal during osteoclastogenesis. Specifically targeting this novel autoregulatory pathway may provide new therapeutic approaches to combat various osteolytic bone diseases.

Plasma levels of receptor activator of nuclear factor‐κB ligand and osteoprotegerin in patients with neuroblastoma

Earlier reports showed that the balance between receptor activator of nuclear factor-kappaB ligand (RANKL) and its decoy-receptor osteoprotegerin (OPG) plays an important role in the pathogenesis of metastatic osteolysis induced by neuroblastoma cells. In this study, we investigated whether circulating levels of OPG, RANKL and their ratio were associated to the presence of osteolytic lesions in advanced neuroblastoma, as well as whether they provided additional information on the severity and prognosis of the disease. Plasma levels of RANKL and OPG were measured in 54 newly diagnosed neuroblastomas; 27 of them showed metastatic disease (stage IV), including 19 bone dissemination. Thirty-five children who were admitted to the pediatric department for minor surgical problems served as control group. OPG was significantly lower in all patients compared with controls, while RANKL levels were significantly increased in advanced neuroblastoma. OPG-to-RANKL ratio decreased in stage-IV patients, and particularly in those who had bone metastases. The diagnostic accuracy of the OPG-to-RANKL ratio in discriminating the presence of osteolytic lesions was not confirmed statistically. OPG correlated significantly with other prognostic factors, namely, ferritin and neurone-specific enolase. In addition, an inverse relationship was found between OPG and event-free survival, and it was more significant in patients who had bone metastasis. This pilot study confirms that the production of OPG and RANKL is disregulated in neuroblastoma. Although the OPG-to-RANKL ratio does not have a predictive value in detecting bone metastasis, the measurement of the previously mentioned markers could be useful in decisions regarding the use of adjuvant therapies.

Why Bone Is Such an Attractive Target

Many cancers, including breast cancer malignant melanoma, metastasize to the bone, and the local factors in the host tissues that contribute to metastatic colonization are beginning to be uncovered. Chemokines in the target tissue to which the cancer cells will metastasize are important, and Jones et al. show that the cytokine RANKL (receptor activator of nuclear factor κB ligand) is another factor that governs metastasis to bone for tumor cells that express the receptor RANK. RANK was detected in primary human tumor samples from breast and prostate cancer, as well as in several cancer cell lines. Cancer cell lines (human breast and prostate and mouse melanoma) responded to RANKL with actin reorganization, activation of several signaling pathways, and cell migration. The decoy receptor osteoprotegerin (OPG) blocked actin polymerization and cell migration induced by RANKL. Injection of the mouse melanoma cell line B16F10 into the hearts of mice led to metastasis of the cancer cells to multiple organs, including the bones, which produced paralysis and ultimately death. Treatment of the mice with OPG specifically reduced metastasis to the bones, eliminated paralysis over the course of the experiment, and increased the survival of the mice. Thus, RANKL appears to be a key factor contributing to bone metastasis and may be a clinical target for reducing morbidity and pain associated with cancers that metastasize to the bone. Interestingly, RANK was found on normal untransformed mouse mammary epithelial cells, and RANKL stimulated the migration of these cells, suggesting that RANKL plays a role in normal breast tissue. D. H. Jones, T. Nakashima, O. H. Sanchez, I. Kozieradzki, S. V. Komarova, I. Sarosi, S. Morony, E. Rubin, R. Sarao, C. V. Hojilla, V. Komnenovic, Y.-Y. Kong, M. Schreiber, S. J. Dixon, S. M. Sims, R. Khokha, T. Wada, J. M. Penninger, Regulation of cancer cell migration and bone metastasis by RANKL. Nature 440 , 692-696 (2006). [PubMed]

Elevated Serum Levels of the Decoy Receptor Osteoprotegerin in Children with Langerhans Cell Histiocytosis

Langerhans cell histiocytosis (LCH) is characterized by an accumulation of dendritic Langerhans cells in granulomatous lesions in the bone, skin, and other sites in the body. The pathogenesis of the disease remains unknown. We measured serum levels of the decoy receptor osteoprotegerin (OPG), an important regulator of bone metabolism as well as immune responses, in 18 children with single system (SS) or multi-system (MS) forms of LCH and 20 pediatric controls. OPG levels were significantly increased in LCH patients at diagnosis when compared with controls, and pretreatment levels of OPG were elevated in MS compared with SS patients. Moreover, OPG levels in LCH patients were elevated in patients with involvement of risk organs (liver, lungs, hematopoietic system, or spleen) when compared with patients without risk organ involvement, indicative of an association between OPG values and disease severity. We also observed a positive correlation between serum levels of OPG and tumor necrosis factor (TNF)-alpha, a pro-inflammatory cytokine, at the time of onset of disease. These findings show, for the first time, that serum OPG levels are elevated in LCH patients, and suggest that OPG may play a role in the pathogenesis of this enigmatic disease.

Bacterial-Responsive B Lymphocytes Induce Periodontal Bone Resorption

Host immune responses play a key role in periodontal diseases. We have found that B lymphocytes in human periodontal lesions bear abundant receptor activator of NF-kappaB ligand (RANKL), a major factor in the regulation of osteoclast differentiation. The purpose of this study was to evaluate Actinobacillus actinomycetemcomitans-responsive B lymphocytes in their level of RANKL expression and their effects on periodontal bone resorption. Congenitally athymic Rowett rats received injections of formalin-fixed A. actinomycetemcomitans into the gingival papillae, and donor B cells from normal rats immunized with A. actinomycetemcomitans were transferred via tail vein injection. We demonstrated that B cells from A. actinomycetemcomitans-immunized animals had greater levels of RANKL expression and induced a significantly higher level of osteoclast differentiation from RAW 264.7 cells than did nonimmune B cells that were not Ag specific. This activity was eliminated by incubation with the RANKL decoy receptor osteoprotegerin fusion protein. A. actinomycetemcomitans-binding B cell (ABB) and RANKL-expressing B cells were recovered from the gingival tissues of recipient rats transferred with ABB, but not from recipients of PBS nonimmune B cells or A. actinomycetemcomitans nonbinding B cells. Also, recipients of ABB exhibited increased osteoclast formation on the alveolar bone surface and significant periodontal bone resorption. This effect was antagonized by injection of osteoprotegerin fusion protein into the local gingival tissues. In summary, this study suggests that B lymphocytes can contribute to increased periodontal bone resorption in the absence of T lymphocytes. This effect is associated with the up-regulation of RANKL expression.

EBF2 Regulates Osteoblast-Dependent Differentiation of Osteoclasts

Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:beta-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts.

TNF-related apoptosis-inducing ligand and its decoy receptor osteoprotegerin in nonischemic dilated cardiomyopathy

Apoptosis has been attributed an essential role in dilated cardiomyopathy (DCM) recently. We assessed expression of TNF-related apoptosis-inducing ligand (TRAIL) and its decoy receptor osteoprotegerin (OPG) in men with nonischemic DCM, who underwent coronary angiography and endomyocardial biopsy (EMB) after exclusion of coronary artery disease compared to control patients. TRAIL plasma concentrations were elevated in DCM ( p = 0.02 vs. controls), and were positively correlated with left ventricular enddiastolic diameter ( r = 0.15, p = 0.04), whereas OPG plasma levels did not differ between both groups ( p = 0.96). In EMB of DCM patients, TRAIL and OPG protein were detected by immunohistochemistry but not in controls. Furthermore, gene expression in EMB or peripheral blood leukocytes (PBL) of DCM patients assessed by real-time PCR showed an increase of TRAIL mRNA in PBL ( p = 0.01 vs. controls), whereas OPG mRNA was upregulated in endomyocardial specimens ( p < 0.001 vs. controls). In conclusion, myocardial overexpression of antiapoptotic OPG in DCM patients may represent a compensatory mechanism to limit systemic activation of TRAIL in patients with congestive heart disease.

RANK ligand and TNF-α mediate acid-induced bone calcium efflux in vitro

Chronic metabolic acidosis stimulates net calcium efflux from bone due to increased osteoclastic bone resorption and decreased osteoblastic collagen synthesis. Previously, we determined that incubation of neonatal mouse calvariae in medium simulating physiological metabolic acidosis leads to a significant, cyclooxygenase-dependent, increase in RNA for bone cell receptor activator of NF-kappaB ligand (RANKL) compared with incubation in neutral pH medium. In this study, we tested the hypothesis that the acid-mediated increase in RANKL expression is a primary mechanism for the stimulated osteoclastic resorption. Acid medium increased the medium concentration of sRANKL without altering the concentration of the decoy receptor osteoprotegerin (OPG). Inhibition of the RANKL pathway with concentrations of OPG up to 25 ng/ml, far greater than physiological, did not significantly decrease the robust acid-induced Ca efflux from bone nor did incubation of the calvariae with a different inhibitor, RANK/Fc (up to 50 ng/ml). Thus acid-induced net Ca efflux appears to involve mechanisms in addition to the RANK/RANKL pathway. Osteoblasts also produce TNF-alpha, another cytokine that stimulates the maturation and activity of osteoclasts. Incubation of calvariae in acid medium caused a significant increase in TNF-alpha levels. Incubation of calvariae with anti-TNF (up to 250 ng/ml) did not significantly decrease acid-induced net Ca efflux. However, the combination of RANK/Fc plus anti-TNF caused a significant but subtotal reduction in acid-induced Ca efflux, whereas the combination of RANK/Fc plus an isotype-matched control for the anti-TNF had no effect on Ca release. Thus simultaneous inhibition of RANKL and TNF-alpha is necessary to reduce acid-induced, cell-mediated net Ca efflux from bone; however, additional osteoblast-produced factors must also be involved in acid-induced, cell-mediated bone resorption.

Evaluation and prognostic value of serum osteoprotegerin in multiple myeloma

An essential cytokine system for osteoclast biology consists of the receptor activator of nuclear factor (NF)-B ligand (RANKL), its receptor RANK, and the soluble decoy receptor osteoprotegerin (OPG). Myeloma plasma cells have been shown to increase RANKL expression and to decrease OPG availability in the bone marrow microenvironment. These effects result in an increased RANKL:OPG ratio that favours the activation of osteoclasts. Conflicting reports exist regarding serum OPG levels in multiple myeloma (MM) and their correlation with lytic bone disease and survival (Seidel et al, 2001; Lipton et al, 2002; Kraj et al, 2003; Terpos et al, 2003; Corso et al, 2004; Kyrtsonis et al, 2004). We evaluated the serum levels of OPG in 140 newly diagnosed MM patients and assessed its prognostic value in the 101 symptomatic patients requiring treatment. The mean (± standard deviation) OPG levels in symptomatic MM, asymptomatic MM and controls represented by 41 healthy age- and sex-matched individuals were 3.4 ± 2.5, 2.9 ± 1.1 and 2.0 ± 0.7 ng/ml respectively. The median OPG concentration in symptomatic MM, asymptomatic MM and controls were 3.0 ng/ml (range, 0.8–24), 2.6 (1.6–7.2) and 1.6 ng/ml (1.2–3), respectively. OPG levels were higher in asymptomatic and symptomatic MM compared with controls (P < 0.001 in each case). In contrast, there was no statistically significant difference between asymptomatic and symptomatic MM patients (P = 0.28). Bone lesions were graded in three groups according to a standard radiographic evaluation: no osteolytic lesions, 1–6 lesions, >6 lesions. We found no significant differences of OPG values between the three groups. Significant correlations were found with β2-microglobulin (r = 0.23; P = 0.02) and creatinine (r = 0.22; P = 0.03) but not with haemoglobin, bone marrow plasmacytosis, M-component, albumin, lactate dehydrogenase (LDH) or C-reactive protein (CRP) levels. We assessed the prognostic value of OPG on overall survival (OS) and progression-free survival (PFS) in symptomatic patients. Median follow-up of the 101 patients was 68.4 months [95% confidence interval (CI): 60.2–76.6]. The median PFS and OS were 18.8 months (95% CI: 14.5–23.1) and 39.8 months (95% CI: 29.6–50.0) respectively. High serum levels of OPG were associated with a poorer OS and PFS in univariate analysis (P = 0.025 and P = 0.029 respectively) (Fig 1A,B). The other adverse prognostic factors were age >60 years (P = 0.019), albumin <35 g/l (P = 0.015), β2-microglobulin >2.5 mg/l (P = 0.025), bone marrow plasmacytosis >15% (P = 0.048), chromosome 13 deletion (P = 0.002), CRP > 16 mg/l (P = 0.037), IgA isotype (P = 0.012), LDH > 350 IU/l (P = 0.016), λ light chain (P = 0.021) for OS and β2-microglobulin >2.5 mg/l (P = 0.01), chromosome 13 deletion (P = 0.002), and IgA isotype (P = 0.012) for PFS. Interestingly, multivariate analysis showed that OPG ≥2.4 ng/ml remained an adverse independent prognostic factor for OS [relative risk (RR) 2.3, 95% CI: 1.2–4.5, P = 0.018] together with albumin <35 g/l (RR 2.4, 95% CI: 1.3–4.5, P = 0.005) and CRP >16 mg/l (RR 2.3, 95% CI 1.2–4.2, P = 0.008). Overall survival (A) and progression-free survival (PFS) (B) of the 101 symptomatic MM patients according to serum OPG level at diagnosis. The biological role of OPG in MM is possibly more complex than expected. There is no clear evidence that serum OPG reflects the availability of OPG in the bone marrow environment and we cannot exclude that OPG may have different functions on bone biology and tumor progression. OPG is a decoy receptor for tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), which inhibits TRAIL-induced apoptosis. In relation to this activity, it has been recently suggested that OPG may be a survival factor for human prostate cancer cells (Holen et al, 2002) and also function as a paracrine survival factor for human myeloma cells (Shipman & Croucher, 2003). These results, if they translate in vivo, raise the possibility that OPG might have the capacity to function as a deleterious factor on MM progression in some patients.

Estrogen specifically stimulates expression and production of osteoprotegerin from rheumatoid synovial fibroblasts

We studied the effects of estrogen on human fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS) focusing on receptor activator of NF-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), the osteoclast formation and function regulators that have a substantial role in bone erosion of RA. Estrogen influences osteoporosis and the onset of RA clinically. The cellular responses of RA-FLS to estrogen are initiated via two high-affinity estrogen receptors (ERs). Culture of RA-FLS in the presence of 10(-6) M 17beta-estradiol (E2) increased expression of estrogen receptor (ER)-alpha, but not ER-beta. OPG mRNA expression was significantly increased, whereas RANKL mRNA was unaffected. E2 treatment also significantly increased the amount of OPG released in the culture supernatant. The increase of OPG and ER-alpha was specifically antagonized by the pure estrogen antagonist ICI 182780. Tamoxifen, a selective ER moderator, did not increase OPG. The results indicate that estrogen stimulates secretion of OPG from RA-FLS by acting on ER-alpha, which likely prevents bone erosion in RA.

OPG/RANKL system imbalance in a case of hepatitis C-associated osteosclerosis: the pathogenetic key?

Hepatitis C-associated osteosclerosis (HCAO) is an impressive example of acquired diffuse osteosclerosis in adults, recently described in ten patients infected with hepatitis C virus (HCV). Its hallmark is a painful and generalized increase of bone mass. Bone biopsies show enhanced accretion rate, usually without histological abnormalities. The HCAO pathogenesis is hitherto unknown. HCV might induce a slow bone cell infection and the production of bone growth factors, such as insulin-like growth factors. Recently, receptor activator of nuclear factor-kappaB (RANK), its ligand (RANKL), and soluble decoy receptor osteoprotegerin (OPG) have been identified as a pivotal cytokine system in the bone remodeling control. We describe the 11th case of HCAO. Notably, the patient's bone biopsy showed the presence of a high number of OPG-positive osteoblasts, a slight increase of RANKL-positive stromal cells, and a dramatic reduction of the osteoclasts. Moreover, OPG serum levels were increased. These findings reported here for the first time are consistent with a pathogenetic role of the OPG/RANKL system imbalance in HCAO.

Increased RANKL/OPG mRNA Ratio in Iliac Bone Biopsies From Women with Hip Fractures

RANKL (receptor activator of NF-kappaB) is a potent physiological inducer of osteoclastogenesis. Its actions are blocked by the decoy receptor osteoprotegerin (OPG), and treatment with OPG blocks bone resorption in postmenopausal women. Both positive and negative associations between serum OPG and bone mineral density (BMD) have been reported in the literature. We hypothesized that decreased OPG production relative to RANKL within bone itself could lead to increased risk of osteoporotic fracture. We included ten women with hip fracture (age 76.3 +/- 8.0 years, N.S, : hip BMD 0.686 +/- 1.3 g/cm2, P < 0.05) and 24 women with osteoarthrosis of the hip (age 72.8 +/- 7.2 years, hip BMD 0.832 +/- 1.1 g/cm(2)). Transiliac biopsies were obtained at the time of surgery. Total RNA was extracted from biopsies and reverse-transcribed. Real-time quantification of mRNA was performed with a SYBR Green I real time PCR assay, calculating relative gene expression with normalization of results for beta actin mRNA. Actin normalized mRNA levels for OPG and interleukin (IL)-6 were significantly lower in fracture patients, with a significantly higher RANKL/OPG ratio in patients with fractures. There was no significant difference in tumor necrosis factor (TNF), IL-1, IL-1ra, or IL-7 expression. IL-6 mRNA levels were lower in fracture patients (P < 0.05). The effect of increased RANKL/OPG ratio (Z = 2.08, P < 0.05) on fracture risk was additive to that of hip BMD T score (Z = -1.95, P < 0.05) when assessed using logistic regression. Elderly women with hip fractures exhibit an increased RANKL/OPG mRNA content of iliac bone. This is associated with increased fracture susceptibility, which is not in itself explained by low BMD.

Increased expression of receptor activator of NF-κB ligand (RANKL), its receptor RANK and its decoy receptor osteoprotegerin in the colon of Crohn's disease patients

Crohn's disease (CD) is associated with low bone mass due to chronic inflammation and other factors. Receptor activator of NF-kappaB ligand (RANKL), its receptor RANK and its decoy receptor osteoprotegerin (OPG) are potentially involved in this process as they regulate osteoclastogenesis and are influenced by pro-inflammatory cytokines. The aim of this study was to determine the levels of soluble RANKL (sRANKL), RANK and OPG expression both in the serum and in the colon of CD patients. Levels of sRANKL and OPG were assessed in the serum and the supernatants of cultured colonic biopsies in patients with CD and controls by ELISA. RANK expression was explored by immunostaining and immunofluorescence of fixed colonic samples. OPG and sRANKL levels were higher in the serum of CD patients as compared to age- and sex-matched controls. Levels of sRANKL and OPG were significantly enhanced in cultured colonic biopsies from CD, and OPG levels correlated with histological inflammation, and pro- and anti-inflammatory cytokine levels. No significant correlation was found for sRANKL. RANK+ cells were increased in the colon of CD, particularly in inflamed areas. These cells were positive for CD68 or S100 protein. We conclude that serum and local levels of sRANKL and OPG are increased in CD. Moreover, RANK is expressed in the colonic mucosa by subpopulations of activated macrophages or dendritic cells at higher levels in CD compared to normal colon.

Runx-2 is not essential for the vitamin D-regulated expression of RANKL and osteoprotegerin in osteoblastic cells

The differentiation and activity of osteoclasts are positively and negatively controlled by receptor activator of nuclear factor-κB ligand (RANKL), which is expressed on the surface of osteoblasts and stromal cells, and its decoy receptor osteoprotegerin (OPG), which is secreted by osteoblasts and stromal cells, respectively. The expression of the genes for RANKL and OPG is regulated by 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. Runt-related gene-2 (Runx-2) is essential for osteoblast differentiation and there are several reports that Runx-2 is involved in osteoclast formation. Therefore, to clarify the role of Runx-2 in osteoclastogenesis, we designed a series of experiments using C2 cells and C6 cells, which are derived from calvariae of runx2-deficient mice. Treatment of C2 cells and C6 cells with 1α,25(OH)2D3 for 2–4 days increased and decreased the levels of expression of the mRNAs for RANKL and OPG, respectively, and the effects were dose-dependent. However, by day 8, the level of RANKL mRNA had fallen and that of OPG mRNA had risen. Furthermore, C6 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells (osteoclast-like cells) in the presence of 10−7M 1α,25(OH)2D3. Such formation of osteoclast-like cells was inhibited by exogenous OPG in a dose-dependent manner. Thus, our findings indicate that Runx-2 is not essential for the expression of RANKL and OPG, and the formation of osteoclast-like cells.

312 Pilot study of the use of infliximab for fatigue in advanced cancer

Bone metastases cause severe skeletal complications and are associated with osteoclast-mediated bone destruction. RANKL is essential for osteoclast formation, function, and survival, and is the primary effector of tumor-induced osteoclastogenesis and osteolysis. RANKL inhibition by its soluble decoy receptor osteoprotegerin (OPG) prevents tumor-induced osteolysis and decreases skeletal tumor burden. Because osteoclast-mediated bone resorption releases growth factors from the bone matrix, the host bone microenvironment induces a vicious cycle of bone destruction and tumor proliferation and survival. A prediction of this vicious cycle hypothesis is that targeting the host bone microenvironment by osteoclast inhibition would reduce tumor growth and survival and may enhance the anti-tumor effects of targeted therapies. The epidermal growth factor receptor (EGFR) pathway regulates critical processes such as cell growth and survival, and anti-EGFR therapies can cause tumor cell arrest and apoptosis. We evaluated whether reduction of osteolysis by RANKL inhibition could enhance the anti-tumor effects of an anti-EGFR antibody (panitumumab) in a novel murine model of human A431 epidermoid carcinoma bone metastasis. Skeletal tumor progression was assessed longitudinally by bioluminescence imaging. RANKL inhibition by OPG-Fc treatment resulted in a reduction in tumor progression in bony sites. OPG-Fc treatment also caused a dose-dependent reduction in tumor-induced osteolysis, supporting the essential role of RANKL in this process. In combination, RANKL inhibition increased the anti-tumor efficacy of an anti-EGFR antibody, and completely blocked tumor-induced bone breakdown, demonstrating that addition of the indirect anti-tumor effect of RANKL inhibition increases the anti-tumor efficacy of panitumumab, a targeted anti-EGFR antibody.

Clinical implications of the osteoprotegerin/RANKL/RANK system for bone and vascular diseases.

Bone resorption by osteoclasts is coupled with bone formation by osteoblasts, and this balanced process continuously remodels and adapts the skeleton. The receptor activator of nuclear factor kappaB ligand (RANKL) has been identified as an essential cytokine for the formation and activation of osteoclasts. The effects of RANKL are physiologically counterbalanced by the decoy receptor osteoprotegerin (OPG). Estrogen deficiency, glucocorticoid exposure, T-cell activation (eg, rheumatoid arthritis), and skeletal malignancies (eg, myeloma, metastases) enhance the ratio of RANKL to OPG and, thus, promote osteoclastogenesis, accelerate bone resorption, and induce bone loss. Moreover, alterations of the OPG/RANKL/RANK system have been implicated in vascular diseases. RANKL blockade (using OPG or RANK fusion proteins or RANKL antibodies) has prevented bone loss caused by osteoporosis, chronic inflammatory disorders, and malignant tumors in animal models and may emerge as a therapy in humans based on studies in postmenopausal osteoporosis, myeloma bone disease, and osteolytic metastases. This review summarizes the clinical implications of the OPG/RANKL/RANK system for bone and vascular diseases.

In vitro blockade of receptor activator of nuclear factor-kappaB ligand prevents osteoclastogenesis induced by neuroblastoma cells.

Proliferation and differentiation of osteoclasts are regulated by a cytokine system that includes RANKL, which binds 2 receptors: RANK, which activates osteoclast differentiation, and osteoprotegerin (OPG), a decoy receptor that limits RANKL action. We investigated the role of the OPG/RANKL/RANK network in the pathogenesis of skeletal metastasis in neuroblastoma. Four different neuroblastoma cell lines (NB100, CHP212, SH-SY5Y, SJ-NK-P) showed a large amount of OPG and RANKL transcripts. Soluble RANKL was detectable in all cell lines, but poor release of OPG was observed. SH-SY5Y showed the lowest OPG-to-RANKL ratio and promoted osteoclastic differentiation of FLG29.1 and peripheral mononuclear cells, inducing expression of the osteoclast markers RANK, c-src, c-fos, cathepsin-K and TRAP. SJ-N-KP, which released both OPG and RANKL, did not show the same capability. OPG, neutralizing anti-RANKL antibody and antisense oligonucleotides were evaluated for their ability to inhibit RANKL activity. The neutralizing antibody hampered osteoclastic differentiation by blocking both the juxtacrine and the paracrine activity of RANKL. Our findings confirm that neuroblastoma cells induce osteoclastogenesis via RANKL and suggest that the RANKL expression associated with lack of the decoy receptor OPG could be a peculiarity of some tumors that makes them able to induce metastatic osteolysis. Moreover, our results suggest that RANKL could be a relevant target in the adjuvant therapy of bone metastatic neuroblastoma as proper neutralization revokes completely osteoclastic differentiation.

PTH differentially regulates expression of RANKL and OPG.

RANKL and OPG gene expressions were measured with and without PTH at different stages of osteoblast development. Mouse stromal cells were cultured in osteoblast differentiating conditions, and RANKL, OPG, COLI, ALP, OC, and PTHRec genes were measured using qRT-PCR. OPG:RANKL ratios indicate that PTH may induce a possible switch in the regulatory mechanism of osteoclastogenesis where OPG is inhibited early and RANKL is increased at late stages of osteoblast differentiation. RANKL is essential for osteoclastogenesis, and its decoy receptor osteoprotegerin (OPG) negatively regulates this process. Both genes are expressed in cells of the osteoblast lineage, but the precise relationship between the state of osteoblast differentiation and RANKL and OPG expression is not clearly defined. The goal of this project was to quantify changes in RANKL and OPG gene expression in response to parathyroid hormone (PTH) at different stages of osteoblast differentiation. In this study, mouse primary bone marrow stromal cells (BMSCs) were cultured for up to 28 days. At specific time-points of cell culture, cells were stimulated with bovine PTH peptide [bPTH (1-34)] for 2 h. Levels of RANKL, OPG, alpha-1 (type I) collagen (COL1), alkaline phosphatase (ALP), osteocalcin (OC), and PTH receptor (PTHRec) mRNA were assayed using quantitative real-time reverse-transcriptase-polymerase chain reaction (qRT-PCR). In control cells, there was a gradual increase of RANKL gene expression with murine osteoblastic stromal cell maturation to a 3-fold level at day 28. In contrast, OPG mRNA levels were maximal at day 14 of cell culture and decreased through the latter stages of osteoblast differentiation. Exposing the cells to 100 ng/ml of bPTH (1-34) induced minimal increases in RANKL mRNA levels from days 7 to 14 but elevated expression significantly at days 21 (2-fold) and 28 (3-fold). PTH inhibited OPG gene expression maximally at day 14, but continued to have inhibitory effects on cultured cells at days 21 and 28. Alterations of RANKL and OPG mRNA levels by PTH in day 14 osteoblasts were sufficient to sustain a 5.6-fold increase in the number of TRACP+ cells when cocultured with osteoclast precursor cells. Cells in culture after 28 days showed a 1.9-fold increase in TRACP+ cells after PTH treatment. We conclude that (1) PTH significantly upregulates RANKL mRNA in primary bone marrow stromal osteoblasts with maximal sensitivity occurring late in osteoblast differentiation; (2) PTH inhibits OPG gene expression at all stages of osteoblast differentiation; and (3) changes in RANKL and OPG mRNA levels after exposure to PTH are associated with increased osteoclastogenesis as demonstrated by increased numbers of TRACP+ cells in cocultures. The results further suggest that the osteoclastogenic activity of PTH occurs primarily by suppression of OPG gene expression in early osteoblasts and elevation of RANKL gene expression in mature osteoblasts.