Nuclear factor-κB Ligand Signaling Research Articles

The inhibitory roles of Ihh downregulation on chondrocyte growth and differentiation.

The proliferative rate of chondrocytes affects bone elongation. Chondrocyte hypertrophy is required for endochondral bone formation as chondrocytes secrete factors required for osteoblast differentiation and maturation. Previous studies have demonstrated that the Indian hedgehog (Ihh) signaling pathway is a key regulator of skeletal development and homeostasis. The aim of the present study was to investigate the function of Ihh in chondrocyte proliferation and differentiation, as well as the underlying mechanisms. Ihh was knocked down in mouse chondrocyte cells using short hairpin RNA. Chondrocyte apoptosis and cell cycle arrest were assessed using flow cytometry and the results indicated that knockdown of Ihh significantly inhibited cell growth (P<0.05) and increased apoptosis (P<0.001) compared with negative control cells. Downregulation of Ihh also resulted in cell cycle arrest at G1 to S phase in chondrocytes. It was also observed that knockdown of Ihh decreased alkaline phosphatase activity and mineral deposition of chondrocytes. The inhibitory roles of Ihh downregulation on chondrocyte growth and differentiation may be associated with the transforming growth factor-β/mothers against decapentaplegic and osteoprotegerin/receptor activator of nuclear factor κB ligand signaling pathway. The results of the present study suggest that chondrocyte-derived Ihh is essential for maintaining bone growth plates and that manipulation of Ihh expression or its signaling components may be a novel therapeutic technique for the treatment of skeletal diseases, including achondroplasia.

Cross-Talk of Receptor Activator of Nuclear Factor-κB Ligand Signaling With Renin–Angiotensin System in Vascular Calcification

Vascular calcification is accelerated by hypertension and also contributes to hypertension; however, it is an enigma why hypertension and vascular calcification are a vicious spiral. The present study elucidates the cross-talk between renin-angiotensin II system and receptor activator of nuclear factor-κB ligand (RANKL) system in vascular calcification. Angiotensin (Ang) II (10(-7) mol/L) significantly increased calcium deposition as assessed by Alizarin Red staining, associated with a significant increase in the expression of RANKL, RANK, and bone-related genes, such as cbfa1 and msx2, in human aortic vascular smooth muscle cells. Infusion of Ang II (100 ng/kg per minute) in ovariectomized ApoE(-/-) mice under high-fat diet significantly increased the expression of RANKL system and calcification in vivo, whereas administration of Ang II receptor blocker (olmesartan, 3 mg/kg per day) decreased the calcification and bone markers' expression. In addition, male OPG(-/-) mice showed a significant increase in vascular calcification followed by Ang II infusion as compared with wild type. Conversely, RANKL significantly increased Ang II type 1 receptor and angiotensin II-converting enzyme expression in vascular smooth muscle cells via extracellular signal-regulated protein kinase phosphorylation. The present study demonstrated that Ang II significantly induced vascular calcification in vitro and in vivo through RANKL activation. In addition, RANKL activated renin-angiotensin II system, especially angiotensin II-converting enzyme and Ang II type 1 receptor. Cross-talk between renin-angiotensin II system and RANKL system might work as a vicious cycle to promote vascular calcification in atherosclerosis. Further studies to inhibit renin-angiotensin II system and RANKL may provide new therapeutic options to prevent and regress vascular calcification.

Absence of Herpes Virus Entry Mediator (HVEM) Increases Bone Mass by Attenuating Receptor Activator of Nuclear Factor-κB ligand (RANKL)-Induced Osteoclastogenesis

Herpes virus entry mediator (HVEM), which is constitutively expressed at a high level on myeloid lineage cells, is also expressed on bone marrow-derived macrophages, suggesting that it may play a role in bone metabolism by affecting osteoclasts (OC) derived from bone marrow-derived macrophages. To address this question, we evaluated bone mass by micro-computed tomography and the number and activity of OC by tartrate-resistant acid phosphatase (TRAP) and pit formation on dentine slices, comparing HVEM-knockout mice with wild-type mice. The absence of HVEM led to a higher bone mass and to decreased levels of serum collagen type I fragments and serum TRACP5b in vivo. In vitro HVEM deficiency resulted in a reduced number and activity of OC and an impaired receptor activator of nuclear factor-κB ligand signaling through reduced activation of nuclear factor-κB and of nuclear factor of activated T-cells cytoplasmic 1. Exogenous soluble HVEM decreased expression of TRAP, whereas soluble LIGHT (a ligand of HVEM) increased it, indicating the occurrence of a positive signaling through HVEM during osteoclastogenesis. Our findings indicate that HVEM regulates bone remodeling via action on OC. The higher bone mass in the femurs of HVEM-knockout mice could be, at least in part, due to attenuated osteoclastogenesis and bone resorption resulting from decreased receptor activator of nuclear factor-κB ligand signaling in the OC.

The Roles of Lipid Oxidation Products and Receptor Activator of Nuclear Factor-κB Signaling in Atherosclerotic Calcification

This review focuses on the roles of oxylipids and receptor activator of nuclear factor-κB ligand signaling in calcific cardiovascular disease. Both intimal and valvular calcifications are closely associated with atherosclerosis, leading investigators to study the role of atherogenic oxidatively modified lipids (oxylipids). Results have identified the molecular signaling through which oxylipids induce osteogenic differentiation and calcification in vascular cells. A surprising concomitant finding was that, in bona fide osteoblasts from skeletal bone, oxylipids have the opposite effect, ie, inhibiting osteoblastic maturation. This is the basis for the lipid hypothesis of osteoporosis. Oxylipids also induce resorptive osteoclastic cells within the bone environment, raising the question of whether resorptive osteoclasts can be harnessed in the vascular context for cell-based therapy to remove artery wall mineral deposits. The challenge is that vascular cells produce antiosteoclastogenic factors, including the soluble decoy receptor for receptor activator of nuclear factor-κB ligand, possibly accounting for the paucity of resorptive cells and the dominance of mineral in atherosclerotic plaque. These factors may have therapeutic use in osteoclastogenic removal of mineral deposits from arteries.

NHE10, a novel osteoclast-specific member of the Na +/H + exchanger family, regulates osteoclast differentiation and survival

Bone homeostasis is tightly regulated by the balanced actions of osteoblasts (OBs) and osteoclasts (OCs). We previously analyzed the gene expression profile of OC differentiation using a cDNA microarray, and identified a novel osteoclastogenic gene candidate, clone OCL-1-E7 [J. Rho, C.R. Altmann, N.D. Socci, L. Merkov, N. Kim, H. So, O. Lee, M. Takami, A.H. Brivanlou, Y. Choi, Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis, DNA Cell Biol. 21 (2002) 541–549]. In this study, we have isolated full-length cDNAs corresponding to this clone from mice and humans to determine the functional roles of this gene in osteoclastogenesis. The full-length cDNA of OCL-1-E7 encodes 12 membrane-spanning domains that are typical of isoforms of the Na +/H + exchangers (NHEs), indicating that this clone is a novel member of the NHE family (hereafter referred to as NHE10). Here, we show that NHE10 is highly expressed in OCs in response to receptor activator of nuclear factor-κB ligand signaling and is required for OC differentiation and survival.

Osteoclast-targeted therapy for metastatic prostate cancer

Bone metastases and skeletal complications are major causes of morbidity in men with prostate cancer. Despite the osteoblastic appearance of bone metastases on imaging studies, patients with bone metastases have elevated markers of bone resorption, indicative of high osteoclast activity. Indeed, increased osteoclast activity is independently associated with higher risk of subsequent skeletal complications. Therapies aimed at reducing osteoclast activity would theoretically diminish disease-related skeletal complications, bone metastases and treatment-related fractures. Phase III clinical trials to address the role of osteoclast-targeted therapy in metastatic prostate cancer are reviewed here. Zoledronic acid, a potent bisphosphonate, significantly decreased the risk of skeletal complications in men with androgen-independent prostate cancer and bone metastases. Ongoing studies will evaluate the use of bisphosphonates or denosumab, a monoclonal antibody that inhibits the receptor activator of nuclear factor-κB ligand signaling in the prevention of bone metastases or skeletal complications. Additional studies are needed to determine the optimal timing, schedule and duration of bisphosphonate treatment in men with bone metastases. The results of these trials in the near future may herald further evolution in the targeting of osteoclasts and optimal supportive care in men with prostate cancer.

Protective Effects of Estradiol on Ethanol-Induced Bone Loss Involve Inhibition of Reactive Oxygen Species Generation in Osteoblasts and Downstream Activation of the Extracellular Signal-Regulated Kinase/Signal Transducer and Activator of Transcription 3/Receptor Activator of Nuclear Factor-κB Ligand Signaling Cascade

Bone loss occurs following chronic ethanol (EtOH) consumption in males and cycling females in part as a result of increased bone resorption. We have demonstrated in vivo that estradiol treatment can reverse this effect. Using osteoclast precursors from bone marrow and osteoblast/preosteoclast coculture, we found that EtOH-induced receptor activator of nuclear factor-kappaB ligand (RANKL) expression in osteoblasts was able to promote osteoclastogenesis. These effects were blocked by pretreatment of cells with either 17beta-estradiol (E(2)) or the anti-oxidant N-acetyl cysteine (NAC). EtOH treatment of stromal osteoblasts increased the intracellular level of reactive oxygen species (ROS). This was associated with induction of NADPH oxidase (NOX) and a downstream signaling cascade involving sustained activation of extracellular signal-regulated kinase (ERK) and activation of signal transducer and activator of transcription 3, resulting in increased gene expression of RANKL. In the presence of EtOH, sustained nuclear ERK translocation >24 h was observed in calvarial osteoblasts and UMR-106 cells transfected with green fluorescent protein-ERK2 plasmid. This was abolished by pretreatment with either E(2) or NAC. NOX subtypes 1, 2, and 4, but not 3, were expressed in stromal osteoblasts. Chemical inhibition of NOX by diphenylene iodonium also reversed the ability of EtOH to phosphorylate ERK and induce RANKL mRNA expression. Down-regulation of EtOH-induced ROS generation in osteoblasts was also observed after treatment with E(2) or NAC. These data suggest that the molecular mechanisms whereby E(2) prevents EtOH-induced bone loss involve interference with ROS generation and cytoplasmic kinase activation.

Assessment of a Serum Assay for Quantification of Abdominal Aortic Calcification

Intimal vascular calcification is an important marker of atherosclerosis, and its severity is an independent risk factor for cardiovascular events. Measurement of coronary or aortic calcification requires CT-based imaging and elaborate data analysis to ensure accuracy. A blood assay which could quantify the severity of vascular calcification would be less expensive, avoid exposure to radiation, and be more accessible than present imaging based methods. Osteoprotegerin (OPG) and osteopontin (OPN) are present in human serum and have been implicated in vascular calcification. In this prospective study we assessed the value of a number of serum assays for OPG and OPN in determining abdominal aortic calcification. Firstly, we investigated the assay characteristics and reproducibility of 5 ELISAs using a subset of patients. Based on the findings of these assessments, we selected 3 assays for full evaluation in the entire cohort.