Downregulation Of Osteoprotegerin Research Articles

Downregulation of osteoprotegerin in colorectal cancer cells promotes liver metastasis via activating tumor-associated macrophage

Osteoprotegerin (OPG) is a secreted cytokine that functions as a decoy receptor for receptor activator of nuclear factor kappa-B (RANK) ligand (RANKL). Anti-RANKL treatment for bone metastasis has been widely accepted for solid tumors. However, the mechanism of OPG-RANKL-RANK signaling in systemic colorectal cancer (CRC) metastasis remains unclear. In this study, we investigated the relevance and function of OPG expression in CRC liver metastasis. First, we performed in silico analysis using The Cancer Genome Atlas public database and found that lower OPG expression in CRC was associated with poor overall survival. Immunohistochemistry analyses using resected specimen from patients with CRC in our institute confirmed the result. Patient-matched primary CRC and liver metastases showed a significant downregulation of OPG expression in metastatic lesions. In CRC cell lines, OPG expression did not suppress cell proliferation and migration. However, OPG expression inhibited macrophage migration by suppressing the RANKL-RANK pathway. Moreover, in vivo mouse liver metastasis models showed that OPG expression in CRC cells suppressed liver metastases. In addition, treatment with an anti-RANKL neutralizing antibody also suppressed liver metastases. These results showed that downregulation of OPG expression in CRC cells promotes liver metastasis by activating tumor-associated macrophage, which can become a candidate for targeted therapy with anti-RANKL neutralizing antibody for CRC liver metastasis.

Abaloparatide outperforms teriparatide in protecting against alveolar bone loss in experimental periodontitis.

The aim of this study was to compare the effects of two osteoanabolic drugs, abaloparatide (ABL) and teriparatide (TPTD), on protecting alveolar bone in experimental periodontitis. Twenty-four 9-week-old, male, Sprague-Dawley rats were placed with silk suture around the right maxillary second molar, and then were randomly divided into three groups, that is, the ABL, TPTD, and saline group, receiving intermittent subcutaneous injections of ABL (80μg/kg), TPTD (80μg/kg) or saline respectively every other day for 4weeks. Samples on both sides were assessed through micro-computerized tomography, histological, and immunohistochemical analysis. Mouse pre-osteoblast MC3T3 cell was cultured with lipopolysaccharide (LPS) and treated with ABL or TPTD, before assays of cell proliferation, alkaline phosphatase (ALP) activity and real-time polymerase chain reaction. On the ligature side, both ABL and TPTD significantly reduced alveolar bone loss, and ABL had significantly better effects with higher expression of runt-related transcription factor 2 (RUNX2) and Bglap (formerly called osteocalcin); meanwhile, the ligature induced osteoclastogenesis and down-regulation of osteoprotegerin (OPG) was affected by neither drug. On the non-ligature side, ABL also showed better osteoanabolic effects. In vitro studies revealed that, in the presence of LPS, ABL, and TPTD similarly promoted MC3T3 proliferation, whereas ABL induced higher ALP activity and osteoblastic gene expression compared to TPTD. Both ABL and TPTD protect and regenerate alveolar bone in experimental periodontitis, and ABL behaves even better than TPTD at the same dose, attributed to its stronger osteoanabolic effects in this context.

Radiation induces primary osteocyte senescencephenotype and affects osteoclastogenesis invitro.

Irradiation-induced bone remodeling imbalances arise as a consequence of the dysregulation of bone formation and resorption. Due to the abundance of osteocytes, their long life and their dual-regulatory effects on both osteoblast and osteoclast function, they serve as critical coordinators of bone remolding. In the present study, femur and tibia-derived primary osteocytes were cultured and irradiated to observe the functional changes and the cellular senescence phenotype in vitro. Irradiation directly reduced cell viability, affected the crucial dendritic morphology and altered the expression of functional proteins, including upregulation of receptor activator of nuclear factor-κB ligand and sclerostin, and downregulation of osteoprotegerin. Irradiated osteocytes were shown to exhibit notable DNA damage, which resulted in the initiation of a typical cellular senescence phenotype. Furthermore, it was found that irradiation-induced prematurely senescent osteocytes stimulate molecular secretion, referred to as senescence-associated secretory phenotype (SASP), which may be involved in modulation of the bone microenvironment, including the promotion of osteoclastogenesis. Taken together, the results showed that irradiation triggered osteocyte senescence and the acquisition of an associated secretory phenotype. This further resulted in an imbalance of bone remodeling through senescent influence on proliferation, morphology and marker protein production, but also indirectly via a paracrine pathway through SASP secretion. The results of the present study may highlight the potential of SASP-targeted interventions for the management of radiation-induced bone loss.

Osteoprotegerin Promotes Liver Steatosis by Targeting the ERK-PPAR-γ-CD36 Pathway.

Previous cross-sectional studies have established that circulating osteoprotegerin (OPG) levels are associated with nonalcoholic fatty liver disease (NAFLD). However, the role of OPG in metabolic diseases, such as diabetes and NAFLD, is still unclear. In the current study, we demonstrated that hepatic OPG expression was downregulated in NAFLD individuals and in obese mice. OPG deficiency decreased lipid accumulation and expression of CD36 and peroxisome proliferator-activated receptor-γ (PPAR-γ) in the livers of OPG-/- mice and cultured cells, respectively, whereas OPG overexpression elicited the opposite effects. The stimulatory role of OPG in lipid accumulation was blocked by CD36 inactivation in hepatocytes isolated from CD36-/- mice. The overexpression of OPG led to a decrease in extracellular signal-regulated kinase (ERK) phosphorylation in the livers of OPG-/- mice and in cultured cells, while OPG deficiency resulted in the opposite effect. The inhibition of PPAR-γ or the activation of ERK blocked the induction of CD36 expression by OPG in cultured cells. Mechanistically, OPG facilitated CD36 expression by acting on PPAR response element (PPRE) present on the CD36 promoter. Taken together, our study revealed that OPG signaling promotes liver steatosis through the ERK-PPAR-γ-CD36 pathway. The downregulation of OPG in NAFLD might be a compensatory response of the body to dampen excess hepatic fat accumulation in obesity.

Promoter Methylation Down-regulates Osteoprotegerin Expression in Ovarian Carcinoma.

Previous studies have documented that osteoprotegerin (OPG) is involved in the development and progression of several human malignancies. However, OPG has also been shown to act as a tumor suppressor. The aim of this study was to examine the expression status of OPG in ovarian carcinoma cells and investigate the underlying mechanism responsible for alterations in OPG expression. The expression levels of OPG mRNA and protein were assessed in human ovarian carcinoma cell lines. The methylation status of the OPG promoter region was determined using the bisulfite pyrosequencing technique. The effects of demethylation on OPG expression were also analyzed. The human ovarian carcinoma cell lines, SW 626, OVCAR-3, ES-2, TOV-112D, and TOV-21G, expressed significantly lower levels of OPG mRNA and protein than the normal human ovarian epithelial cell line, HS823.Tc. Moreover, three CpG sites in the OPG promoter region were highly methylated in the SW 626, OVCAR-3, ES-2, and TOV-112D ovarian carcinoma cell lines compared to normal control cells. Furthermore, in all the examined ovarian carcinoma cell lines, treatment with the demethylating agent, 5-aza-2-deoxycytidine, resulted in significantly increased expression levels of OPG mRNA and protein compared to the respective pre-treatment levels. OPG expression was down-regulated in the studied ovarian carcinoma cells compared to the normal control cells, while demethylation significantly restored OPG expression in the OPG-down-regulated cell lines. Our results suggest that OPG down-regulation in ovarian carcinoma occurs, at least partly, through epigenetic repression, suggesting its involvement in ovarian carcinogenesis.

Compression and tension variably alter Osteoprotegerin expression via miR-3198 in periodontal ligament cells

BackgroundOsteoclasts play a critical role in bone resorption due to orthodontic tooth movement (OTM). In OTM, a force is exerted on the tooth, creating compression of the periodontal ligament (PDL) on one side of the tooth, and tension on the other side. In response to these mechanical stresses, the balance of receptor activator of nuclear-factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) shifts to stimulate osteoclastogenesis. However, the mechanism of OPG expression in PDL cells under different mechanical stresses remains unclear. We hypothesized that compression and tension induce different microRNA (miRNA) expression profiles, which account for the difference in OPG expression in PDL cells.To study miRNA expression profiles resulting from OTM, compression force (2 g/cm2) or tension force (15% elongation) was applied to immortalized human PDL (HPL) cells for 24 h, and miRNA extracted. The miRNA expression in each sample was analyzed using a human miRNA microarray, and the changes of miRNA expression were confirmed by real-time RT-PCR. In addition, miR-3198 mimic and inhibitor were transfected into HPL cells, and OPG expression and production assessed.ResultsWe found that certain miRNAs were expressed differentially under compression and tension. For instance, we observed that miR-572, − 663, − 575, − 3679-5p, UL70-3p, and − 3198 were upregulated only by compression. Real-time RT-PCR confirmed that compression induced miR-3198 expression, but tension reduced it, in HPL cells. Consistent with previous reports, OPG expression was reduced by compression and induced by tension, though RANKL was induced by both compression and tension. OPG expression was upregulated by miR-3198 inhibitor, and was reduced by miR-3198 mimic, in HPL cells. We observed that miR-3198 inhibitor rescued the compression-mediated downregulation of OPG. On the other hand, miR-3198 mimic reduced OPG expression under tension. However, RANKL expression was not affected by miR-3198 inhibitor or mimic.ConclusionsWe conclude that miR-3198 is upregulated by compression and is downregulated by tension, suggesting that miR-3198 downregulates OPG expression in response to mechanical stress.

TLR2 Promotes Vascular Smooth Muscle Cell Chondrogenic Differentiation and Consequent Calcification via the Concerted Actions of Osteoprotegerin Suppression and IL-6-Mediated RANKL Induction.

Objective- Vascular smooth muscle cell (VSMC) transformation to an osteochondrogenic phenotype is an initial step toward arterial calcification, which is highly correlated with cardiovascular disease-related morbidity and mortality. TLR2 (Toll-like receptor 2) plays a pathogenic role in the development of vascular diseases, but its regulation in calcification of arteries and VSMCs remains unclear. We postulate that TLR2-mediated inflammation participates in mediating atherosclerotic arterial calcification and VSMC calcification. Approach and Results- We found that ApoE-/- Tlr2-/- genotype in mice suppressed high-fat diet-induced atherosclerotic plaques formation during initiation but progressively lost its preventative capacity, compared with ApoE-/- mice. However, TLR2 deficiency prohibited high-fat diet-induced advanced atherosclerotic calcification, chondrogenic metaplasia, and OPG (osteoprotegerin) downregulation in the calcified lesions. Incubation of VSMCs in a calcifying medium revealed that TLR2 agonists significantly increased VSMC calcification and chondrogenic differentiation. Furthermore, TLR2 deficiency suppressed TLR2 agonist-mediated VSMC chondrogenic differentiation and consequent calcification, which were triggered via the concerted actions of IL (interleukin)-6-mediated RANKL (receptor activator of nuclear factor κB ligand) induction and OPG suppression. Inhibition experiments with pharmacological inhibitors demonstrated that IL-6-mediated RANKL induction is signaled by p38 and ERK1/2 (extracellular signal-regulated kinase 1/2) pathways, whereas the OPG is suppressed via NF-κB (nuclear factor κB) dependent signaling mediated by ERK1/2. Conclusions- We concluded that on ligand binding, TLR2 activates p38 and ERK1/2 signaling to selectively modulate the upregulation of IL-6-mediated RANKL and downregulation of OPG. These signaling pathways act in concert to induce chondrogenic transdifferentiation of VSMCs, which in turn leads to vascular calcification during the pathogenesis of atherosclerosis.

Green tea extract suppresses osteoclastogenesis and opposes glucocorticoid-induced osteoporosis in rats

Background/aim Glucocorticoids-induced osteoporosis (GIO) through suppression of osteoblast activity, induction of receptor activator of nuclear factor-κB ligand (RANKL), and downregulation of osteoprotegerin (OPG). Green tea has antioxidant and anti-inflammatory properties. This study aims to evaluate the effects of green tea extract (GTE) supplementation on bone proteins, mineralization, and markers in GIO. Materials and methods The study was performed on 30 adult male Wistar rats, which were divided into three groups: control (C, n=10); GIO (n=10), which received dexamethasone 7 mg/kg body weight intramuscular once/week for 4 weeks; and GTE-supplemented osteoporotic (GT-GIO, n=10), which received dexamethasone, followed by 300 mg/kg per day of GTE by gavage for 4 weeks, with continuation of the dexamethasone. Serum calcium, phosphate, alkaline phosphatase activity, parathyroid hormone, deoxypyridinoline, osteocalcin, OPG, and RANKL were determined. Samples of left tibia were used for histopathological examination of bone. Results A significant decrease in serum Ca++, PO4, osteocalcin, and OPG, accompanied by a significant increase in serum bone-specific alkaline phosphatase, deoxypyridinoline, and RANKL was detected in the GIO group compared with the control group. GT-GIO group showed a significant increase in serum Ca++, PO4, osteocalcin, and OPG, with a significant decrease in serum bone-specific alkaline phosphatase, deoxypyridinoline, and RANKL compared with the GIO group. Bone examination of GIO rats revealed thinning, loss of architecture, erosions, and increased osteoclasts. Bone sections of the GT-GIO group revealed significant improvement, regain of normal architecture, and decreased osteoclast number. Conclusion GTE had an antiosteoporotic effect in GIO, and its effects are much more than its antioxidant characteristics.

Abstract 1836: Triple negative breast cancer: Metabolomics and flux analysis to identify targets for drug development

Abstract To date, no targeted therapies are clinically available to treat triple negative breast cancer (TNBC). This highly aggressive form of breast cancer (BCa) derives its name from the absence of estrogen receptor (ER+), progesterone receptor (PR+), and HER2/neu receptor (HER2+). These receptors are expressed in other subtypes of BCa (i.e. Luminal, HER2-enriched) and have been successfully targeted with therapeutics such as Tamoxifen and Herceptin. New therapeutics are needed to improve the prognosis for patients with TNBC. Metabolomics and metabolic flux analysis (MFA) were conducted to reveal differences in TNBC cell lines compared with hormone receptor-positive cell lines (ER+ and +/-HER2+) prior to and following treatment with chemotherapy drugs. Two triple-negative (MDA-MB-231 and MDA-MB-468) and two hormone-responsive (BT474 and MCF-7) cell lines were compared before and after treatment with Taxol®/Paclitaxel. The metabolic profiles were assessed using 1H NMR broad spectrum metabolomics, MFA using [U-13C]-glucose and [U-13C]-glutamine enriched media, and cytokine profiling of the media. Broad spectrum metabolomics analysis demonstrated differences in the metabolic profiles between the TNBC and hormone-responsive cell lines in the absence of treatment, with a higher level of amino acids (e.g. alanine, glutamate, glutamine, glutathione), short chain fatty acids (isobutyrate, β-hydroxybutyrate), and nucleotides and nucleotide sugars (ADP, ATP, UDP-glucose, UDP-glucuronate) in the TNBC cell lines. Further, MFA demonstrated that glucose utilization was greater in the TNBC cells (MDA-MB-468) compared to the hormone-responsive cells (MCF-7). In addition, 13C-labeled glycine and 13C-labeled glutathione were only observed in TNBC cell lines treated in the presence of [U-13C]-glucose. In response to Taxol treatment, more metabolites were altered in the hormone-responsive cell lines compared with the TNBC cells. Profiling of 80 inflammatory cytokines (RayBiotech arrays) also demonstrated different responses in secreted inflammatory signals following treatment. We observed significant upregulation in interleukins-4 and -16 in MDA-MB-468 cells, while there was a downregulation of osteoprotegerin in all four cell lines after treatment with Taxol. This approach holds promise for identifying biomarkers which may be leveraged for development of targeted treatments. Citation Format: Delisha A. Stewart, Jason Winnike, Susan McRitchie, Wimal Pathmasiri, Susan Sumner. Triple negative breast cancer: Metabolomics and flux analysis to identify targets for drug development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1836. doi:10.1158/1538-7445.AM2015-1836

Achyranthes bidentata extract exerts osteoprotective effects on steroid-induced osteonecrosis of the femoral head in rats by regulating RANKL/RANK/OPG signaling.

BackgroundSteroid-induced osteonecrosis of the femoral head (steroid-induced ONFH) presents great challenges due to the various effects of steroids on multi-system pathways involved into osteoblast differentiation, osteoblast and osteoclast apoptosis, lipid metabolism, calcium metabolism and coagulation. As one of the most frequently used herbs in Traditional Chinese Medicine formulas that are prescribed for the regulation of bone and mineral metabolism, the therapeutic effects of Achyranthes bidentata on steroid-induced ONFH remain unclear. Thus, the aim of the current study was to verify whether Achyranthes bidentata extract (ABE) can be used to prevent steroid-induced ONFH and to investigate its underlying pharmacological mechanisms.MethodsSteroid-induced ONFH rat models were established to evaluate the effects of ABE treatment on osteonecrotic changes and repair processes. Microfocal computed tomography (Micro-CT) was performed to assess the effects of ABE treatment on bone mass, microstructure, and vascularization. Then, the effects of ABE treatment on osteoclast differentiation and bone formation were also evaluated in vivo and in vitro. In addition, receptor activator of nuclear factor kappa B (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) expression in sera, femoral heads and bone marrow-derived mesenchymal stem cells (BMSCs) were detected at both protein and mRNA levels.ResultsThe ratio of empty lacuna, adipose tissue area, and adipocyte perimeter in the bone marrow were markedly lower in the ABE treatment groups than in the model group. Micro-CT evaluation indicated that ABE treatment could improve the microstructure of the trabecular bone, increase bone mineral density and promote vascularization in steroid-induced ONFH rats. Moreover, ABE treatment inhibited osteoclast differentiation and activated bone formation markers. Interestingly, OPG downregulation, RANK and RANKL upregulation, and an increased ratio of RANKL to OPG in sera and necrotic femoral head could be reversed by ABE treatment, which also effectively inhibited RANKL-induced osteoclast differentiation and regulated RANKL and OPG expression of in vitro.ConclusionABE may prevent steroid-induced ONFH and alleviate steroid-induced bone deterioration by regulating the RANKL/RANK/OPG signaling pathway.

Role of GSH/GSSG redox couple in osteogenic activity and osteoclastogenic markers of human osteoblast‐like SaOS‐2 cells

This study comprised a comprehensive analysis of the glutathione (GSH) redox system during osteogenic differentiation in human osteoblast-like SaOS-2 cells. For the first time, a clear relationship between expression of specific factors involved in bone remodeling and the changes in the GSH/oxidized GSH (GSSG) redox couple induced during the early phases of the differentiation and mineralization process is shown. The findings show that the time course of differentiation is characterized by a decrease in the GSH/GSSG ratio, and this behavior is also related to the expression of osteoclastogenic markers. Maintenance of a high GSH/GSSG ratio due to GSH exposure in the early phase of this process increases mRNA levels of osteogenic differentiation markers and mineralization. Conversely, these events are decreased by a low GSH/GSSG ratio in a reversible manner. Redox regulation of runt-related transcription factor-2 (RUNX-2) activation through phosphorylation is shown. An inverse relationship between RUNX-2 activation and extracellular signal-regulated kinases related to GSH redox potential is observed. The GSH/GSSG redox couple also affects osteoclastogenesis, mainly through osteoprotegerin down-regulation with an increase in the ratio of receptor activator of NF-κB ligand to osteoprotegerin and vice versa. No redox regulation of receptor activator of NF-κB ligand expression was found. These results indicate that the GSH/GSSG redox couple may have a pivotal role in bone remodeling and bone redox-dysregulated diseases. They suggest therapeutic use of compounds that are able to modulate not just the GSH level but the intracellular redox system through the GSH/GSSG redox couple.

Immunomodulatory drugs lenalidomide and pomalidomide inhibit multiple myeloma-induced osteoclast formation and the RANKL/OPG ratio in the myeloma microenvironment targeting the expression of adhesion molecules

Multiple myeloma (MM)-induced osteoclast (OC) formation is mainly due to an imbalance of the receptor activator NF-κB ligand (RANKL)-osteoprotegerin (OPG) ratio in favor of RANKL in the bone microenvironment and to the CCL3 production by MM cells. The purpose of the study was to investigate the effect of the immunomodulatory drugs on RANKL/OPG ratio, the production of pro-osteoclastogenic cytokines, and MM-induced OC formation. We found that invivo concentrations of both lenalidomide (LEN) and pomalidomide (POM) significantly blunted RANKL upregulation normalizing the RANKL/OPG ratio in human osteoprogenitor cells (PreOBs) when co-cultured with MM cells and also inhibited CCL3 production by MM cells. A reduction in CD49d expression, a molecule critically involved in RANKL upregulation in the MM microenvironment, accompanied this effect. Consistently, the pro-osteoclastogenic property of MM cells co-cultured with PreOBs was reduced by both LEN and POM. We further investigated the effect of these drugs on the transcriptional profile of both MM cells and PreOBs by microarray analysis, which showed that adhesion molecules, such as ITGA8 and ICAM2, are significantly downregulated in MM cells. Our data suggest that LEN and POM inhibit MM-induced OC formation through normalization of the RANKL/OPG ratio targeting the expression of adhesion molecules by MM cells.

Understanding the role of Fas-Fas ligand system in bone

Fas ligand (CD 178) and its receptor Fas (CD 95) are members of the TNF superfamily of ligands and receptors involved in the activation of apoptosis. Our research group demonstrated that Fas and Fas ligand were expressed during osteoblast and osteoclast differentiation, and their expression may be modified by various cytokines. The lack of functional Fas signaling in murine models leads to altered endochondral ossification, increase of the bone mass in adult mice, and resistance to ovariectomy-induced bone loss. We also showed that mice with a Fas gene knockout lose less bone during antigen-induced arthritis. These changes seem to be, at least in part, mediated by increased expression of osteoprotegerin (OPG), another member of the TNF superfamily, which acts as a decoy receptor for receptor activator for nuclear factor κB (RANK) ligand (RANKL). The bone phenotype of mice lacking Fas signaling may be related to the immunological disturbance rather than intrinsic bone disorder. To address this question at molecular level, we performed a set of parabiotic experiments in mice with non-functional Fas ligand mutation (gld mice). Mice were kept in parabiosis for 1 to 4 weeks, and for 2 weeks after separation from 4-week parabiosis. We also analyzed OPG levels in the peripheral blood of patients with autoimmune lymphoproliferative syndrome (ALPS). Joined circulation between gld and wild-type mice led to increased expression of bone protective OPG in the wild-type animal, both at the gene and protein level at 4 weeks of parabiosis. This effect was sustained even after the separation of parabiotic mice. At the same time, double-negative T lymphocytes transferred from gld into wild-type member of a parabiotic pair rapidly vanished from the periphery of both gld and control mice in parabiosis. Patients with ALPS had increased OPG mRNA level in peripheral blood mononuclear cells, as assessed by real-time PCR, in comparison to age- and sex-matched controls. These findings show that bone and immune changes are uncoupled during Fas ligand deficiency. Under the assumption that OPG also acts as a molecular brake in the immune system, downregulation of OPG in gld mice during parabiosis with wild-type mice could be considered as a molecular marker of remission. Increased expression of OPG in children with ALPS leads to the hypothesis that a similar mechanism might be at play in humans.

Trail down‐regulates the release of osteoprotegerin (OPG) by primary stromal cells

The soluble member of the TNF-R superfamily osteoprotegerin (OPG) is abundantly released under basal conditions by both mesenchymal stem cells (MSC) and fibroblasts and by endothelial cells upon stimulation with inflammatory cytokines. Since MSC, fibroblasts and endothelial cells represent key elements of the normal and tumor microenvironment and express detectable levels of surface TRAIL receptors, we investigated the effect of TRAIL on OPG release. Unexpectedly, recombinant TRAIL decreased the spontaneous OPG release in all cell types examined. Moreover, TRAIL decreased OPG release also in stromal cells co-cultured with lymphoma cells and counteracted the OPG induction by TN-alpha in HUVEC and MSC. Such down-regulation was not due to a masking effect in the ELISA quantification of the OPG released in the culture supernatants due to binding of OPG to its ligands (TRAIL and RANKL), as demonstrated by competition experiments with recombinant TRAIL and by the lack of RANKL release/induction. In addition, OPG down-regulation was not due to induction of cytotoxic effects by TRAIL, since the degree of apoptosis in response to TRAIL was negligible in all primary cell types. With regards to the possible molecular mechanism accounting for the down-regulation of OPG release by TRAIL, we found that treatment of MSC with TRAIL significantly decreased the phosphorylation levels of p38/MAPK. There is a suggestion that this pathway is involved in the stabilization of OPG mRNA. In this respect, the ability of TRAIL to decrease the release of OPG, in the absence of cell cytotoxicity, was mimicked by the p38/MAPK inhibitor SB203580.

Myeloma cells suppress osteoblasts through sclerostin secretion

Wingless-type (Wnt) signaling through the secretion of Wnt inhibitors Dickkopf1, soluble frizzled-related protein-2 and -3 has a key role in the decreased osteoblast (OB) activity associated with multiple myeloma (MM) bone disease. We provide evidence that another Wnt antagonist, sclerostin, an osteocyte-expressed negative regulator of bone formation, is expressed by myeloma cells, that is, human myeloma cell lines (HMCLs) and plasma cells (CD138+ cells) obtained from the bone marrow (BM) of a large number of MM patients with bone disease. We demonstrated that BM stromal cells (BMSCs), differentiated into OBs and co-cultured with HMCLs showed, compared with BMSCs alone, reduced expression of major osteoblastic-specific proteins, decreased mineralized nodule formation and attenuated the expression of members of the activator protein 1 transcription factor family (Fra-1, Fra-2 and Jun-D). Moreover, in the same co-culture system, the addition of neutralizing anti-sclerostin antibodies restored OB functions by inducing nuclear accumulation of β-catenin. We further demonstrated that the upregulation of receptor activator of nuclear factor κ-B ligand and the downregulation of osteoprotegerin in OBs were also sclerostin mediated. Our data indicated that sclerostin secretion by myeloma cells contribute to the suppression of bone formation in the osteolytic bone disease associated to MM.

Wogonin inhibits osteoclast formation induced by lipopolysaccharide

To evaluate the inhibitory activity of wogonin against lipopolysaccharide (LPS)-induced bone resorption, we investigated the effect of wogonin on osteoclastogenesis induced by LPS. Wogonin inhibited LPS-induced osteoclastogenesis in co-cultures of mouse calvaria-derived osteoblasts and bone marrow-derived pre-osteoclasts. Wogonin also suppressed osteoclastogenesis in LPS-injected mouse calvaria. In osteoblasts, the upregulation of receptor activator of nuclear factor-kappaB (RANKL) expression and the downregulation of osteoprotegerin (OPG) expression by LPS were inhibited by wogonin. Wogonin and NS-398, a COX-2 inhibitor, suppressed LPS-stimulated PGE(2) production in osteoblasts. NS-398 inhibited the effect of LPS on RANKL and OPG expression in osteoblasts. These results suggest that wogonin acts as an inhibitor of LPS-induced osteoclastogenesis through downregulation of RANKL and upregulation of OPG expression via blockage of PGE(2) production. Based on these results, wogonin has potential for use as a therapeutic agent in bacteria-induced bone resorption.

Bisphosphonates in Patients with Glucocorticoids: Time for Implementation

Use of glucocorticoids (GC) may have devastating effects on bone quality1. From earlier histomorphometric data, we know that bone formation is inhibited by direct suppression of osteoblast function during the use of GC, while bone resorption is unchanged or even elevated, at least partly related to decreased intestinal calcium absorption, elevated urinary calcium excretion, and a tendency to secondary hyperparathyroidism2,3. However, exciting new data provide additional insights into the pathogenesis of Glucocorticoid Induced OsteoPorosis (GIOP)2,3: (1) upregulation of the Wnt inhibitor Dickkopf-1, leading to inhibition of bone formation; (2) increased apoptosis of osteoblasts and osteocytes; (3) differentiation of mesenchymal stem cells into the direction of adipocytes, instead of into osteoblasts; (4) elevated lifespan of osteoclasts, due to decreased apoptosis; and (5) upregulation of expression of RANKL in osteoblasts (and downregulation of osteoprotegerin). Nevertheless, it is important to realize that the (negative) direct and indirect effects of GC on bone are counteracted by the immunosuppressive effects of GC on the underlying disease2,3. Another complicating factor is that use of GC and the underlying disease may both be associated with muscular weakness and, subsequently, increased risk of falling. Use of GC is associated with bone loss, particularly in the early phase of treatment, when the dosage of GC is usually high and the underlying disease for which the GC are prescribed is active. In contrast, in the chronic phase, bone loss may be mild in GC-treated patients, related to the use of lower dosages of GC and to low or preferably absent disease activity. In addition, use of GC is also associated with elevated risk of fracture: In a retrospective cohort study with 244,235 GC-treated patients and the same number of age and sex matched controls, the fracture … Address correspondence to Prof. Lems. E-mail: wf.lems{at}vumc.nl

Bone morphogenetic proteins regulate osteoprotegerin and its ligands in human vascular smooth muscle cells

The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-beta1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-kappabeta ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-beta1 but were increased by both BMPs. Members of the TGF-superfamily, i.e. TGF-beta1, BMP-2 and BMP-7 exert effects on OPG and its ligands, indicating that these peptides may be involved in the development of vascular calcifications. The downregulation of OPG by these peptides does, however, not suggest that these factors are directly involved in OPG accumulation in diabetes.

Neuropeptides modulate RANKL and OPG expression in human periodontal ligament cells

Neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), may be associated with bone remodeling in response to mechanical stress during orthodontic tooth movement. To investigate this hypothesis, we examined the effects of neuropeptides on the expression of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) in human periodontal ligament (PDL) cells under compression in vitro. PDL cells were subjected to compressive force (2.0 g/cm 2) continuously in the presence or absence of SP or CGRP for 2–4 days. The expression of the SP receptor, neurokinin 1-receptor (NK 1-R), in PDL cells was confirmed by RT–PCR and immunofluorescent staining. The effects of neuropeptides (SP and CGRP) on the expression of RANKL and OPG mRNA were determined using RT–PCR. PDL cells constitutively expressed NK 1-R on both the mRNA and protein levels. Compressive force decreased OPG mRNA expression and increased RANKL mRNA expression. In the presence of neuropeptides, the OPG level decreased synergistically with compression. Neuropeptides stimulated RANKL expression without compression, whereas they decreased RANKL mRNA expression with compression. These results indicate that PDL cell compression induces the up-regulation of RANKL and down-regulation of OPG, whereas neuropeptides suppress the RANKL expression induced by compression. Therefore, the neuropeptides SP and CGRP may modulate bone remodeling by PDL cells during orthodontic tooth movement.

Soluble glucocorticoid-induced tumor necrosis factor receptor stimulates osteoclastogenesis by down-regulation of osteoprotegerin in bone marrow stromal cells

Soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) is a potent stimulator of osteoclastogenesis. The mechanism by which it induces osteoclastogenesis was studied by culturing bone-marrow-derived macrophages (BMM) with conditioned medium from mouse bone marrow stromal cells. GITR and GITR ligand (GITRL) were expressed on the surface of bone marrow stromal cells, and sGITR-induced osteoclastogenesis was inhibited by anti-GITRL Ab, indicating that stimulatory effect of osteoclastogenesis by sGITR involved signaling via GITRL. Bone marrow stromal cells up-regulated cyclooxygenase-2 (COX-2) and produced prostaglandin E 2 (PGE 2) early in their response to sGITR, and the stimulation of osteoclastogenesis was markedly inhibited by NS398, a COX-2 inhibitor. Later, sGITR markedly reduced the steady-state level of osteoprotegerin (OPG) mRNA and increased receptor activator of nuclear factor-κB ligand (RANKL) mRNA. NS398 blocked the sGITR-induced reduction of OPG mRNA but did not significantly affect the sGITR-induced rise in RANKL mRNA. This suggests that down-regulation of OPG by PGE 2 is involved in sGITR-induced osteoclast (OC) formation in the presence of conditioned medium from mouse bone marrow stromal cells.