RANKL Induction Research Articles

Parathyroid hormone(1-34) regulates the expression of osteoclast inhibitory lectin via multiple signaling pathways in UMR106 osteoblast-like cells

Objective To investigate the regulation of parathyroid hormone(1-34) on mRNA expression of osteoclast inhibitory lectin (OCIL) gene in UMR106 osteoblastic-like cells and involved signaling pathway.Methods Rat UMR106 osteoblastic-like cells were cultured and treated with various concentration of PTH(1-34) and specific agonists or inhibitors of PKA,PKC,Ca2+/calmodulin-dependent protein kinase (CaMK) and mitogen-activated protein kinase (MAPK) signal pathways for indicated time intervals.Then the cells were gathered at indicated time points and total RNA were extracted.OCIL mRNA expression was analyzed using real-time PCR technique.Results PTH(1-34) stimulated OCIL mRNA expression in a time-and dose-dependentmanner.A dose of 10 nmol/L PTH(1-34) started to induce OCIL mRNA from 6 h,with a highest increase of about 2.8-fold vs.control group (without PTH treatment) at 24 h.The up-regulation of OCIL mRNA began and reached maximum later than RANKL induction and OPG suppression effected by PTH(1-34).Protein Kinase A (PKA) signaling activators forskolin(FSK) and dibutyryl cAMP (db-cAMP),as well as calcium ionophore A23187 all up-regulated OCIL mRNA with the maximal induction of about 4.2-fold,4.5-fold and 5.1-fold.Protein Kinase C (PKC) activator phorbol-12-myristate-13-acetate(PMA) reduced OCIL mRNA expression at the early stage(2-6 h),with the highest down-regulation of 50％ at 6 h.However,the inhibitory effect on OCIL mRNA turned into slightly stimulatory effect later (24 h).PKA inhibitor KT5720,calmodulin antagonist W-7,CaMK Ⅱ inhibitor KN-62 and mitogen-activated protein kinase (MAPK) inhibitor PD98059 all blocked PTH(1-34)-induced OCIL mRNA expression by the maximal reduction of 56％,61％,63％ and 50％ respectively.There also exist cross-talks between different signal pathways.MAPK inhibitor PD98059 blocked the expression of OCIL mRNA which was stimulated by PKA activators FSK or db-cAMP,with the reduction of 98％ and 63％ respectively,while the OCIL mRNA expression stimulated by A23187 remained unaffected.Conclusion PTH(1-34) increased OCIL mRNA expression in vitro through cAMP/PKA,Ca2+/CaMK and MAPK signaling pathways. Key words: Parathyroid hormone; Osteoclasts; Gene expression; Agglutinins

Progesterone receptor and Stat5 signaling cross talk through RANKL in mammary epithelial cells.

Progesterone (P4) stimulates proliferation of the mammary epithelium by a mechanism that involves paracrine signaling mediated from progesterone receptor (PR)-positive to neighboring PR-negative cells. Here we used a primary mouse mammary epithelial cell (MEC) culture system to define the molecular mechanism by which P4 regulates the expression of target gene effectors of proliferation including the paracrine factor receptor and activator of nuclear factor κB ligand (RANKL). MECs from adult virgin mice grown and embedded in three-dimensional basement-membrane medium resemble mammary ducts in vivo structurally and with respect to other properties including a heterogeneous pattern of PR expression, P4 induction of RANKL and other target genes in a PR-dependent manner, and a proliferative response to progestin. RANKL was demonstrated to have multiple functional P4-responsive enhancers that bind PR in a hormone-dependent manner as detected by chromatin immunoprecipitation assay. P4 also stimulated recruitment of signal transducer and activator of transcription (Stat)5a to RANKL enhancers through an apparent tethering with PR. Analysis of primary MECs from Stat5a knockout mice revealed that P4 induction of RANKL and a broad range of other PR target genes required Stat5a, as did P4-stimulated cell proliferation. In the absence of Stat5a, PR binding was lost at selective RANKL enhancers but was retained with others, suggesting that Stat5a acts to facilitate PR DNA binding at selective sites and to function as a coactivator with DNA-bound PR at others. These results show that RANKL is a direct PR target gene and that Stat5a has a novel role as a cofactor in PR-mediated transcriptional signaling in the mammary gland.

AB0116 HIF-2alpha dependent rankl induction and osteoclastogenesis is augmented by inflammatory cytokines

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation and consecutive local hypoxia, leading to cartilage and bone destruction. Hypoxia promotes osteoclasts formation in vitro but its...

RANKL/RANK/MMP-1 Molecular Triad Contributes to the Metastatic Phenotype of Breast and Prostate Cancer Cells In Vitro

The osteolytic nature of bone metastasis results from a tumor-driven increased bone resorption. Bone remodeling is orchestrated by the molecular triad RANK-RANKL-OPG. This process is dysregulated in bone metastases, mostly via induction of RANKL by tumor-derived factors. These factors increase expression of RANKL, which induce osteoclast formation, function, and survival, thereby increasing bone resorption. RANK is unexpectedly expressed by cancer cells, and the activation of RANKL-RANK pathway correlates with an increased invasive phenotype. To investigate the interaction between RANK expression in human breast and prostate cancer cells and their pro-metastatic phenotype we analyzed the activation of RANKL-RANK pathway and its effects on cell migration, invasion, gene expression in vitro, and osteolysis-inducing ability in vivo. RANKL activates kinase signaling pathways, stimulates cell migration, increases cell invasion, and up-regulates MMP-1 expression. In vivo, MMP-1 knockdown resulted in smaller x-ray osteolytic lesions and osteoclastogenesis, and decreased tumor burden. Therefore, RANKL inhibition in bone metastatic disease may decrease the levels of the osteoclastogenesis inducer MMP-1, contributing to a better clinical outcome.

PP090: TGF-B in jaw tumor fluids induces RANKL expression in stromal fibroblasts

Purpose Odontogenic tumor and odontogenic cyst, arising in jaw bone, grow with resorption and destruction of jaw bones. However, the mechanisms of bone resorption by odontogenic tumor and cyst are still unclear. These odontogenic lesions consist of odontogenic epithelial cells and stromal fibroblasts. It has been demonstrated that odontogenic epithelial cells of developing tooth germ induce osteoclastogenesis to prevent tooth germ from invasion of developing bone. Thus we hypothesized that odontogenic epithelial cells in odontogenic tumor and cyst induce osteoclastogenesis, and then that would play potential roles in tumor outgrowth into the jaw bones. Purpose of this study is to examine osteoclastogenesis by cytokines, focusing on TGF-b, produced by odontogenic epithelial cells. Results Concentration of TGF-b in fluids of odontogenic tumor and cyst (ameloblastoma, keratocystic odontogenic tumor and follicular cyst) was measured by ELISA, and expression of TGF-b, were examined by immunohistochemistry. Expression of RANKL, OPG and COX-2 in stromal fibroblasts in culture was assessed by RT-PCR and/or western blotting, and PGE2 synthesis was measured by ELISA. Fluids of jaw tumor and cyst increased expression of RANKL in stromal fibroblasts isolated from odontogenic tumor, but did not change expression of OPG. Induction of RANKL expression was further enhanced by acidified fluids. The induction of RANKL by the fluids was inhibited by SB-505124, a selective inhibitor of TGF-b receptor kinase or anti TGF-b neutralizing antibody, suggesting the fluids contain TGF-b. Immunohistochemistry revealed that TGF-b was positive in epithelial cells of odontogenic tumor/cyst, and phosphorylated smad3 was localized in the nuclei of odontogenic epithelial cells and stromal fibroblasts. TGF-b induced RANKL expression in the presence of cycloheximide, a protein synthesis inhibitor. Conclusions These results suggest that TGF-b signals directly enhanced RANKL gene transcription in stromal fibroblast, and then induced-RANKL would be participate in bone resorption by the lesions.

Novel anti‐osteoclastogenic activity of fisetin antagonizing RANKL‐induced bone resorption in murine macrophages

Bone resorption and formation is imbalance resulting in more bone resorption than bone formation leading to osteoporosis. Osteoclasts were multi‐nucleated cells formed by exposure to RANKL and activating bone resorption. Fisetin is a flavonol found naturally in many fruits and vegetables including strawberries, blueberries and the skin of cucumbers. This study investigated that fisetin RANKL‐induced osteoclast differentiation and fusion in RAW 264.7 murine macrophages incubated with ≤10 μM fisetin for 5d. Fisetin inhibited the RANKL‐induced multi‐nucleated osteoclast formation by suppressing tartrate‐resistance acid phosphatase activity. Additionally, Fisetin treatment reduced cellular expression carbonic anhydrase II and integrin β3 elevated by RANKL. When fisetin was treated to RANKL‐exposed macrophages, the elevated actin‐ring formation was attenuated, indicating fisetin inhibition of osteoclast fusion and resorption. It was also found the fisetin diminished the RANKL induction of gelsolin, an actin‐binding protein that is a key regulator of actin filament assembly and disassermbly. Therefore, fisetin was a natural therapeutic agent retarding osteoclast differentiation and bone resorption

197 PREMETASTATIC NICHE INVOLVES RANKL-RANK SIGNALING RECRUITING BYSTANDER CANCER CELLS TO PARTICIPATE CANCER SKELETAL METASTASIS

You have accessJournal of UrologyProstate Cancer: Basic Research (I)1 Apr 2013197 PREMETASTATIC NICHE INVOLVES RANKL-RANK SIGNALING RECRUITING BYSTANDER CANCER CELLS TO PARTICIPATE CANCER SKELETAL METASTASIS Gina C.Y. Chu, Haiyen E. Zhau, Ruoxiang Wang, Andre Rogatko, Xu Feng, Majd Zayzafoon, and Leland W.K. Chung Gina C.Y. ChuGina C.Y. Chu Los Angeles, CA More articles by this author , Haiyen E. ZhauHaiyen E. Zhau Los Angeles, CA More articles by this author , Ruoxiang WangRuoxiang Wang Los Angeles, CA More articles by this author , Andre RogatkoAndre Rogatko Los Angeles, CA More articles by this author , Xu FengXu Feng Birmingham, AL More articles by this author , Majd ZayzafoonMajd Zayzafoon Birmingham, AL More articles by this author , and Leland W.K. ChungLeland W.K. Chung Los Angeles, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1577AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Activation of RANK, a receptor activator of NF-κB, by its ligand RANKL is critical for osteoclast differentiation/bone remodeling. RANKL can be derived from osteoblasts, infiltrating inflammatory cells and stromal fibroblasts. We found malignant prostate cancer (PCa) cells expressed RANKL; its expression correlated with clinical PCa progression and induced bone metastasis (BM) of RANK+ PCa cells. Our objective is to dissect the mechanism by which PCa-derived RANKL acts on RANK+ PCa cells to promote PCa BM. METHODS Human PCa LNCaP cells were stably transfected with a RANKL expression construct. We identified the common transcription factors (TFs) required for the induction of RANKL and c-Met expression by site-directed mutagenesis and TFs deletion/interference assays. We assessed EMT progression, stem, and neuroendocrine (NE) cell properties by determining changes in their specific markers. Tumorigenic and metastatic potential in vivo was examined by intratibial and intracardiac co-inoculation of RANKL+ and RANKL- PCa cells to male athymic mice. RANKL expression was detected by quantum dot staining and cell-based signal quantification from 20 primary PCa specimens with documented cancer-specific survival data. RESULTS We demonstrated that: 1) RANKL amplifies downstream signaling by activating RANKL and c-Met expression and signaling through a common TF complex, c-Myc/Max. 2) A few RANKL+ PCa cells are sufficient to initiate the metastatic cascade by recruiting non-tumorigenic RANKL- PCa cells to participate in the metastatic process via downstream signaling amplification. This is supported by the observation that recombinant RANKL protein alone is sufficient to induce BM of non-metastatic PCa cells. 3) RANKL also promotes EMT and confers stem and NE cell properties on participating cancer cells.4) RANKL expression at the single cell level in primary PCa specimens predicts PCa patient survival. CONCLUSIONS Small numbers of RANKL+ PCa cells are sufficient to initiate cancer bone colonization by recruiting non-tumorigenic/bystander PCa cells to participate in BM. RANKL-RANK signaling in PCa cells establishes a premetastatic niche through a “vicious cycle”, inducing RANKL and c-Met expression via activation of c-Myc/Max. RANKL-RANK interaction promotes PCa cells to undergo EMT and assume stem and NE cell properties. RANKL expression at the single cell level in primary human cancer tissues predicts PCa patient survival. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e81-e82 Peer Review Report Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gina C.Y. Chu Los Angeles, CA More articles by this author Haiyen E. Zhau Los Angeles, CA More articles by this author Ruoxiang Wang Los Angeles, CA More articles by this author Andre Rogatko Los Angeles, CA More articles by this author Xu Feng Birmingham, AL More articles by this author Majd Zayzafoon Birmingham, AL More articles by this author Leland W.K. Chung Los Angeles, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Runx2 Transcription Factor Regulates Heparanase-Induced Bone Resorption in Multiple Myeloma

Abstract 567 Background.Despite advances in treatment strategies, myeloma remains incurable, and bone destruction is a major cause of morbidity in myeloma patients. We have documented in earlier studies that heparanase enzyme is preferentially expressed in myeloma cells and induces severe bone destruction in myeloma. We also discovered that heparanase increases the production of two major bone resorbing factors named Receptor Activator of NF-κB Ligand (RANKL) and Matrix Metalloproteinase 9 (MMP-9) by myeloma cells. Runx2, a member of the runt-related gene family, is a bone-specific transcription factor. Runx2 regulates osteoblast differentiation and is essential for bone tissue development. Interestingly, Runx2 also controls expression of RANKL and MMP-9 genes in osteoblasts. Recent evidence indicates that ectopic induction and overexpression of Runx2 in breast, uterine and prostate cancer cells is associated with bone-metastasis, and osteolytic bone disease in these cancers. However, very little is known about the function of Runx2 in myeloma cells. Here we report for the first time that heparanase engages the Runx2 pathway to promote expression of RANKL and MMP-9 in myeloma cells. Methods.Molecular, biochemical, cellular and in vivo approaches were used to assess the role of Runx2 in heparanase-induced expression of RANKL and MMP-9. These included: (1) Real-time PCR and Western blot analysis to monitor Runx2 levels in CAG myeloma cells expressing high level of heparanase (HPSE-high cells) or with knockdown of endogenous heparanase (HPSE k/d cells), and the corresponding control cells. (2) Chromatin Immunoprecipitation (ChIP) assay to determine in vivo occupancy of the RANKL and MMP-9 gene promoters in myeloma cells by Runx2. (3) Zymography to determine MMP-9 activity in both human and murine myeloma cells. (4) Real-time PCR to determine changes in RANKL and MMP-9 gene expression in Runx2 knockdown MM1.S and 5TGM1 myeloma cells. (5) Assessment of tumor growth/burden and bone resorption in the 5TGM1 syngenic model of murine myeloma. 5TGM1 cells with specific knockdown of Runx2 or non-target shRNA were injected into C57BL/KaLwRij mice through the tail vein and levels of IgG2b and TRAP5b were monitored in the sera of the mice by ELISA. Results.We find Runx2 expression is significantly increased in CAG myeloma cells expressing a high level of heparanase and dramatically reduced in HPSE k/d cells. Increased Runx2 in HPSE-high myeloma cells results in an increase in expression of RANKL and MMP-9 mRNA as well as MMP-9 activity. In sharp contrast, knockdown of Runx2 in human myeloma MM1.S and murine myeloma 5TGM1 cells results in a significant reduction of RANKL, MMP-9 gene expression and MMP-9 activity. Thus heparanase promotes RANKL and MMP-9 expression via Runx2. Analysis of initial 1-kb sequences of RANKL and MMP-9 gene promoter in human and mice, revealed presence of multiple high affinity Runx2-binding sites. ChIP assay confirmed that Runx2 mediates induction of RANKL and MMP-9 gene transcription in the both human and murine myeloma cells by direct association with the proximal promoter of these genes. Together these results demonstrate that Runx2 is a positive regulator of RANKL and MMP-9 gene expression in myeloma cells. Finally, in the syngenic model, knockdown of Runx2 in 5TGM1 murine myeloma cells remarkably inhibits the growth of these cells in vivo and bone resorption. Conclusions.Runx2 is a central regulator of heparanase induced myeloma bone disease. Our discoveries provide new insight into the mechanism of myeloma-induced bone disease and identify Runx2 as a novel target to block myeloma bone disease. Disclosures:No relevant conflicts of interest to declare.

Kinsenoside prevents ovariectomy-induced bone loss and suppresses osteoclastogenesis by regulating classical NF-κB pathways

SummaryKinsenoside is able to improve bone turnover rate in ovariectomized (OVX) mice. In vitro analysis shows that kinsenoside antagonizes osteoclast development and bone resorption.IntroductionKinsenoside, the main active compound of the traditional Taiwanese herb Anoectochilus formosanus, has an antiinflammatory effect. This study investigates whether kinsenoside inhibits osteoporosis and osteoclastogenesis.MethodsOVX mice were used to examine the antiosteoporotic activity of kinsenoside. The trabecular bone microarchitecture was assessed by microcomputed tomography. In vitro experiments were performed to determine the mechanisms of the antiosteoporotic effects of kinsenoside.ResultsMicrocomputed tomography scanning showed that kinsenoside suppresses bone loss in OVX mice. Kinsenoside decreases plasma CTx concentration. Reverse transcription polymerase chain reaction (RT-PCR) analysis also showed that kinsenoside reduces the femoral mRNA expression of tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase-9 (MMP-9). Kinsenoside inhibits osteoclast formation in bone marrow cells (BMs) and RAW 264.7 cells. Western blot was used to analyze osteoclast-associated signaling pathways in RAW 264.7 cells. Results show that kinsenoside does not inhibit IKK phosphorylation but suppresses the phosphorylation of IκBα and p65. Kinsenoside significantly inhibits the RANKL induction of IKK activity. Kinsenoside inhibits the RANKL-triggered nuclear translocations of NF-κB and nuclear factor of activated T cells c1 (NFATc1). RT-PCR was used to analyze osteoclast precursor fusion and resorption-associated gene expression in BMs. Kinsenoside inhibits the expression of cathepsin K (CAK), dendritic cell-specific transmembrane protein, MMP-9, and TRAP.ConclusionsKinsenoside inhibits osteoclastogenesis from macrophages by attenuating RANKL-induced NF-κB and NFATc1 activities, which in turn, prevents bone loss from OVX mice.

Induction of cytokines, MMP9, TIMPs, RANKL and OPG during orthodontic tooth movement

This controlled longitudinal intervention study investigated changes in cytokines and biomarkers of bone and tissue metabolism within gingival crevicular fluid (GCF) from patients undergoing orthodontic treatment. GCF was collected on Periopaper™ strips (Oraflow Inc., USA) from 20 volunteers at baseline, before tooth extraction and appliance placement and then at intervals during orthodontic treatment. Samples were taken 10 weeks following first appliance placement: at four hours; 7 days; and 42 days after application of distalising forces to maxillary canine teeth. Cytokines (GM-CSF, interferon-gamma, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and TNFalpha), tissue biomarkers (MMP-9, TIMP-1 & 2) and bone metabolism indicators (RANKL and OPG) were measured in GCF using multiplex assays. Tension sites adjacent to canines showed significant increases in IL-1beta, IL-8, TNFalpha, MMP-9 and TIMPs 1 and 2 across all time points following force application, while compression sites exhibited increases in IL-1beta and IL-8 after 4 hours, MMP-9 after 7 and 42 days and RANKL after 42 days. These data demonstrate that high levels of pro-inflammatory cytokines and biomarkers of tissue and bone metabolism in GCF are associated with orthodontic force application. Elevated levels were evident at 4 hours but continued for periods of up to 6 weeks. The data suggest that GCF biomarker analysis may help optimise orthodontic forces for individual patients.

RANKL Enhances Macrophage Paracrine Pro-Calcific Activity in High Phosphate-Treated Smooth Muscle Cells: Dependence on IL-6 and TNF-α

Background: Vascular calcification is highly correlated with cardiovascular disease (CVD) morbidity and mortality, and it is associated with inflammation. Receptor activator of NF-ĸB ligand (RANKL) inhibition in vivo has been shown to reduce vascular calcification in a mouse model of atherosclerosis. Therefore, we tested the hypothesis that RANKL regulates smooth muscle cell (SMC) calcification by modulating macrophage production of pro-calcific cytokines. Methods: We used a bone marrow-derived macrophage (BMDM)/SMC co-culture system and examined the effects of RANKL on BMDM activation and SMC matrix calcification. Results: Treatment with RANKL alone did not stimulate SMC calcification induced by elevated phosphate. BMDMs differentiated with macrophage colony-stimulating factor and placed in co-culture with SMCs increased phosphate-induced SMC calcification. RANKL added to the BMDM/SMC co-cultures further enhanced SMC calcification. Treatment of BMDMs with RANKL resulted in increased expression of IL-6 and TNF-α. Thus, increased expression of these pro-calcific cytokines in macrophages may mediate RANKL-induced SMC calcification in a paracrine fashion. Addition of neutralizing IL-6 and TNF-α antibodies together with RANKL treatment significantly reduced the RANKL induction of SMC calcification. Conclusion: RANKL activation of pro-inflammatory and pro-calcific pathways in macrophages may contribute to vascular calcification and inflammation.

Osteocyte Network; a Negative Regulatory System for Bone Mass Augmented by the Induction of Rankl in Osteoblasts and Sost in Osteocytes at Unloading

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.

Interleukin‐22 promotes osteoclastogenesis in rheumatoid arthritis through induction of RANKL in human synovial fibroblasts

To examine the regulatory role of interleukin-22 (IL-22) in the expression of RANKL and induction of osteoclastogenesis in rheumatoid arthritis (RA). Concentrations of IL-22 and RANKL in the serum and synovial fluid of RA patients were measured using enzyme-linked immunosorbent assay. RA synovial fibroblasts were treated with recombinant human IL-22 (rhIL-22), and the expression of RANKL messenger RNA (mRNA) and protein was measured using real-time polymerase chain reaction, Western blotting, and intracellular immunostaining. Human monocytes were cocultured with IL-22-prestimulated RA synovial fibroblasts and macrophage colony-stimulating factor, and osteoclastogenesis was assessed by counting the multinucleated cells (those staining positive for tartrate-resistant acid phosphatase). The IL-22 concentration in the synovial fluid was higher in RA patients than in patients with osteoarthritis (OA). The serum IL-22 concentration was also higher in RA patients than in OA patients and healthy volunteers, and this correlated with serum titers of rheumatoid factor and anti-cyclic citrullinated peptide antibodies. In RA synovial fibroblasts treated with rhIL-22, the expression of RANKL mRNA and protein was increased in a dose-dependent manner. IL-22-induced RANKL expression was down-regulated significantly by the inhibition of p38 MAPK/NF-κB or JAK-2/STAT-3 signaling. In human monocytes cocultured with IL-22-prestimulated RA synovial fibroblasts in the absence of exogenous RANKL, the monocytes differentiated into osteoclasts, but this osteoclastogenesis decreased after p38 MAPK/NF-κB or JAK-2/STAT-3 signaling was inhibited. These results show that IL-22 up-regulates RANKL expression in RA synovial fibroblasts and induces osteoclastogenesis. These effects are mediated by the p38 MAPK/NF-κB and JAK-2/STAT-3 signaling pathways.

Is Serum IL-17A a Useful Systemic Biomarker in Patients With Langerhans Cell Histiocytosis?

To the editor: In a recent article in Molecular Therapy, Hogarty1 elucidated the still unresolved issue regarding the role of interleukin-17A (IL-17A) in the pathogenesis of Langerhans cell histiocytosis (LCH) and whether serum IL-17A could be used as a systemic biomarker of the disease. In the same article, it was stated that the “IL-17A issue,” beyond its academic interest, has definite therapeutic implications, given that IL-17A-targeted treatments are now in preclinical development for LCH. Therefore, clear conclusions are urgently required that can be obtained only through collective work. LCH is a rare disease of obscure etiology, involving single or multiple systems of the human body. Although all lesions share common histopathological features, LCH has an unpredictable clinical course ranging from spontaneous resolution to disseminated disease, compromising vital functions with occasionally fatal consequences. It is characterized by the clonal accumulation and/or proliferation of specific dendritic cells that resemble the normal epidermal Langerhans cell; however, there is ongoing debate over whether the origin of the disease is immunological or neoplastic. IL-17, also known as IL-17A and cytotoxic T lymphocyte–associated antigen 8 (CTLA-8), is a T cell–specific proinflammatory cytokine involved in chronic inflammatory states presenting with granuloma formation and also in bone resorption through RANKL induction in osteoblasts.2 Histological features of LCH resembling granulomas, as well as osteolytic bone lesions of the disease, provide indications of a possible central role of IL-17A in LCH pathogenesis. Recently, Coury et al. implicated IL-17A, favoring the immune etiology of LCH.3 These authors demonstrated increased serum IL-17A concentrations in 13 subjects (2 adults) at various stages of LCH (12 patients with multiple-system disease; all patients had received several chemoimmunotherapy treatments).3 They also suggested that IL-17A was originating from the dendritic cells rather than the T cells, and, although total serum IL-17A concentrations did not correlate with the activity of the disease, serum IL-17A-dependent fusion activity did correlate with the activity of LCH.3 On the basis of these findings, a potential IL-17A-targeted therapy was proposed as a future therapeutic approach in LCH, whereas IL-17A appeared as a possible biomarker to monitor disease activity. Therefore, and according to these findings, IL-17A-targeted treatments, which are currently in phase I and phase II trials for other inflammatory conditions, are also considered in LCH.1 However, another research group was unable to identify any IL-17A messenger RNA expression in 12 lesions from 10 pediatric patients with LCH.4 Peters et al. studied plasma samples from 26 LCH patients with active disease and 46 pediatric control subjects and reported IL-17A at background levels in LCH patients at concentrations consistent with controls in two different standard enzyme-linked immunosorbent assay (ELISA) kits. In addition, IL-17A concentrations of both patients and controls were highly variable according to the development ELISA kit utilized in the study by Coury et al.3 Moreover, the principal antibody employed by Coury et al. cross-reacted with protein lysate from LCH lesions. Therefore, it was suggested that the lack of identifiable IL-17A transcripts or protein expression in the lesions precludes the possibility of lesional IL-17A production and subsequent autocrine and/or paracrine action.1 LCH is rarer in adults than in children, with an estimated annual incidence of 1 case per 560,000 inhabitants, and it is therefore regarded as an “orphan disease.”5 In our recent study of 25 adults with LCH at various disease stages who had previously received a variety of treatments, we found decreased bone mineral density as well as decreased bone turnover in those treated with chemotherapy or glucocorticoids or both.6 In the same patients, we have now measured serum IL-17A concentrations in duplicates (standard deviation and coefficient of variation values of 5.9 and 4.7%, respectively) using one of the three commercial kits employed in the study by Peters et al. (Human IL-17 Quantikine ELISA kit, D1700; RD Mann-Whitney test, P = 0.144), as well as no association between LCH activity and serum levels of IL-17A. These differences remained nonsignificant after exclusion of the two apparently healthy controls with high IL-17 values (outliers, Figure 1) (patients 154 ± 19 pg/ml vs. controls, 115 ± 10 pg/ml; Mann-Whitney test, P = 0.072). Figure 1 Interleukin-17A levels in patients and controls. From our findings, it seems that serum IL-17A is not ubiquitously increased among LCH patients with active disease. Multicenter studies with large numbers of patients are needed to define whether several parameters such as age (adults vs. children), disease extension and involvement, and treatment administered can influence IL-17A. Our findings are in agreement with those from the previous groups that failed to demonstrate a correlation between IL-17A concentrations and disease activity, and they suggest that serum IL-17A cannot serve as a biomarker to differentiate patients with LCH and active disease or indicate those who might benefit from an IL-17A-targeted therapeutic approach.

Role of DNA methylation in the regulation of the RANKL-OPG system in human bone

Osteoblasts are specialized cells that form new bone and also indirectly influence bone resorption by producing factors that modulate osteoclast differentiation. Although the methylation of CpG islands plays an important role in the regulation of gene expression, there is still scanty information about its role in human bone. The aim of this study was to investigate the influence of CpG methylation on the transcriptional levels of two osteoblast-derived critical factors in the regulation of osteoclastogenesis: the receptor activator of nuclear factor NF-κB ligand (RANKL) and its soluble decoy receptor osteoprotegerin (OPG). Quantitative methylation specific PCR (qMSP) and pyrosequencing analysis in various cell types showed that the methylation of regulatory regions of these genes, in the vicinity of the transcription start sites, repressed gene transcription, whereas an active transcription was associated with low levels of methylation. In addition, treatment with the DNA demethylating agent 5-azadeoxycitidine promoted a 170-fold induction of RANKL and a 20-fold induction of OPG mRNA expression in HEK-293 cells, which showed hypermethylation of the CpG islands and barely expressed RANKL and OPG transcripts at baseline. Transcriptional levels of both genes were also explored in bone tissue samples from patients with hip fractures and hip osteoarthritis. Although RANKL transcript abundance and the RANKL:OPG transcript ratio were significantly higher in patients with fractures than in those with osteoarthritis (RANKL: 0.76 ± 0.23 vs. 0.24 ± 0.08, p = 0.012; RANKL/OPG: 7.66 ± 2.49 vs. 0.92 ± 0.21, p = 0.002), there was no evidence for differential methylation across patient groups. In conclusion, the association between DNA methylation and the repression of RANKL and OPG expression strongly suggests that methylation-dependent mechanisms influence the transcription of these genes, which play a critical role in osteoclastogenesis. However, other mechanisms appear to be involved in the increased RANKL/OPG ratio of patients with osteoporotic fractures.

Sustained RANKL response to parathyroid hormone in oncostatin M receptor-deficient osteoblasts converts anabolic treatment to a catabolic effect in vivo

Parathyroid hormone (PTH) is the only approved anabolic agent for osteoporosis treatment. It acts via osteoblasts to stimulate both osteoclast formation and bone formation, with the balance between these two activities determined by the mode of administration. Oncostatin M (OSM), a gp130-dependent cytokine expressed by osteoblast lineage cells, has similar effects and similar gene targets in the osteoblast lineage. In this study, we investigated whether OSM might participate in anabolic effects of PTH. Microarray analysis and quantitative real-time polymerase chain reaction (qPCR) of PTH-treated murine stromal cells and primary calvarial osteoblasts identified significant regulation of gp130 and gp130-dependent coreceptors and ligands, including a significant increase in OSM receptor (OSMR) expression. To determine whether OSMR signaling is required for PTH anabolic action, 6-week-old male Osmr(-/-) mice and wild-type (WT) littermates were treated with hPTH(1-34) for 3 weeks. In WT mice, PTH increased trabecular bone volume and trabecular thickness. In contrast, the same treatment had a catabolic effect in Osmr(-/-) mice, reducing both trabecular bone volume and trabecular number. This was not explained by any alteration in the increased osteoblast formation and mineral apposition rate in response to PTH in Osmr(-/-) compared with WT mice. Rather, PTH treatment doubled osteoclast surface in Osmr(-/-) mice, an effect not observed in WT mice. Consistent with this finding, when osteoclast precursors were cultured in the presence of osteoblasts, more osteoclasts were formed in response to PTH when Osmr(-/-) osteoblasts were used. Neither PTH1R mRNA levels nor cAMP response to PTH were modified in Osmr(-/-) osteoblasts. However, RANKL induction in PTH-treated Osmr(-/-) osteoblasts was sustained at least until 24 hours after PTH exposure, an effect not observed in WT osteoblasts. These data indicate that the transient RANKL induction by intermittent PTH administration, which is associated with its anabolic action, is changed to a prolonged induction in OSMR-deficient osteoblasts, resulting in bone destruction.

Rankl-induced osteoclastogenesis leads to loss of mineralization in a medaka osteoporosis model

Osteoclasts are macrophage-related bone resorbing cells of hematopoietic origin. Factors that regulate osteoclastogenesis are of great interest for investigating the pathology and treatment of bone diseases such as osteoporosis. In mammals, receptor activator of NF-κB ligand (Rankl) is a regulator of osteoclast formation and activation: its misexpression causes osteoclast stimulation and osteoporotic bone loss. Here, we report an osteoporotic phenotype that is induced by overexpression of Rankl in the medaka model. We generated transgenic medaka lines that express GFP under control of the cathepsin K promoter in osteoclasts starting at 12 days post-fertilization (dpf), or Rankl together with CFP under control of a bi-directional heat-shock promoter. Using long-term confocal time-lapse imaging of double and triple transgenic larvae, we monitored in vivo formation and activation of osteoclasts, as well as their interaction with osteoblasts. Upon Rankl induction, GFP-positive osteoclasts are first observed in the intervertebral regions and then quickly migrate to the surface of mineralized neural and haemal arches, as well as to the centra of the vertebral bodies. These osteoclasts are TRAP (tartrate-resistant acid phosphatase) and cathepsin K positive, mononuclear and highly mobile with dynamically extending protrusions. They are exclusively found in tight contact with mineralized matrix. Rankl-induced osteoclast formation resulted in severe degradation of the mineralized matrix in vertebral bodies and arches. In conclusion, our in vivo imaging approach confirms a conserved role of Rankl in osteoclastogenesis in teleost fish and provides new insight into the cellular interactions during bone resorption in an animal model that is useful for genetic and chemical screening.

Interleukin‐4 inhibits RANKL‐induced NFATc1 expression via STAT6: A novel mechanism mediating its blockade of osteoclastogenesis

Interleukin-4 (IL-4) is an important immune regulatory protein that possesses potent anti-osteoclastogenic properties, and does so via the transcription factor STAT6. Previous studies have shown that IL-4 selectively blocks RANKL-induced activation of NF-κB and mitogen-activated protein kinase (MAPK) pathway molecules, suggesting that the cytokine arrests osteoclastogenesis by blockade of these signaling cascades. However, the fact that the inhibitory effect on these pathways requires prolonged IL-4 pretreatment, and that the cytokine fails to exert an anti-osteoclastogenic effect after short-term pre-exposure of RANKL to osteoclast precursors, suggests that an additional, more immediate mechanism may also be involved. In this study, we found that simultaneous exposure of IL-4 did not alter RANKL-dependent activation of NF-κB or MAPKs, whereas the cytokine did block RANKL-induced nuclear factor activated T cells c1 (NFATc1), a master osteoclastogenic transcription factor. This inhibitory effect of IL-4 required STAT6, consistent with its functional role in osteoclastogenesis. In addition, the cytokine also partially impaired RANKL-stimulated bone resorption. Furthermore, IL-4 suppressed expression of RANKL-induced osteoclast specific genes in a STAT6-dependent manner, but failed to do so when osteoclast precursors were pre-exposed to RANKL. Thus, we provide the first evidence that IL-4 inhibits osteoclast formation by inhibiting RANKL induction of NFATc1 via STAT6 as an early event, in addition to its suppression of other signaling pathways. The inhibitory effect is ultimately regulated at the gene expression transcriptional level.

The Roles of Interleukin-6 in the Pathogenesis of Rheumatoid Arthritis

Several clinical studies have demonstrated that the humanized anti-interleukin-6 (IL-6) receptor antibody tocilizumab (TCZ) improves clinical symptoms and prevents progression of joint destruction in rheumatoid arthritis (RA). However, the precise mechanism by which IL-6 blockade leads to the improvement of RA is not well understood. IL-6 promotes synovitis by inducing neovascularization, infiltration of inflammatory cells, and synovial hyperplasia. IL-6 causes bone resorption by inducing osteoclast formation via the induction of RANKL in synovial cells, and cartilage degeneration by producing matrix metalloproteinases (MMPs) in synovial cells and chondrocytes. Moreover, IL-6 is involved in autoimmunity by altering the balance between Th17 cells and Treg. IL-6 also acts on changing lipid concentrations in blood and on inducing the production of hepcidin which causes iron-deficient anemia. In conclusion, IL-6 is a major player in the pathogenesis of RA, and current evidence indicates that the blockade of IL-6 is a beneficial therapy for RA patients.

Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer

Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. The Women's Health Initiative and the Million Women Study have shown that hormone replacement therapy is associated with an increased risk of incident and fatal breast cancer. In particular, synthetic progesterone derivatives (progestins) such as medroxyprogesterone acetate (MPA), used in millions of women for hormone replacement therapy and contraceptives, markedly increase the risk of developing breast cancer. Here we show that the in vivo administration of MPA triggers massive induction of the key osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) in mammary-gland epithelial cells. Genetic inactivation of the RANKL receptor RANK in mammary-gland epithelial cells prevents MPA-induced epithelial proliferation, impairs expansion of the CD49f(hi) stem-cell-enriched population, and sensitizes these cells to DNA-damage-induced cell death. Deletion of RANK from the mammary epithelium results in a markedly decreased incidence and delayed onset of MPA-driven mammary cancer. These data show that the RANKL/RANK system controls the incidence and onset of progestin-driven breast cancer.