RANKL increases migration of human lung cancer cells through intercellular adhesion molecule-1 up-regulation

Invasion of distant tissues by tumor cells is the primary cause of therapeutic failure in the treatment of malignant lung cancer cells. Receptor activator of nuclear factor-κB ligand (RANKL) and its receptor, RANK, play a key role in osteoclastogenesis and tumor metastasis. Intercellular adhesion molecule-1 (ICAM-1, also called CD54), a member of the immunoglobulin supergene family, is an inducible surface glycoprotein that mediates adhesion-dependent cell-to-cell interactions. The effects of RANKL on cell migration and ICAM-1 expression in human lung cancer cells are largely unknown. We found that RANKL directed the migration and increased ICAM-1 expression in human lung cancer (A549) cells. Pretreatment of A549 cells with the MAPK kinase (MEK) inhibitor PD98059 or U0126 inhibited RANKL-mediated migration and ICAM-1 expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, an NF-κB inhibitor (PDTC) and IκB protease inhibitor (TPCK) also inhibited RANKL-mediated cell migration and ICAM-1 up-regulation. Taken together, these results suggest that the RANKL and RANK interaction acts through MEK/ERK, which in turn activates NF-κB, resulting in the activation of ICAM-1 and contributing to the migration of human lung cancer cells.     

Functions of RANKL/RANK/OPG in bone modeling and remodeling

The discovery of the RANKL/RANK/OPG system in the mid 1990s for the regulation of bone resorption has led to major advances in our understanding of how bone modeling and remodeling are regulated. It had been known for many years before this discovery that osteoblastic stromal cells regulated osteoclast formation, but it had not been anticipated that they would do this through expression of members of the TNF superfamily: receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), or that these cytokines and signaling through receptor activator of NF-κB (RANK) would have extensive functions beyond regulation of bone remodeling. RANKL/RANK signaling regulates osteoclast formation, activation and survival in normal bone modeling and remodeling and in a variety of pathologic conditions characterized by increased bone turnover. OPG protects bone from excessive resorption by binding to RANKL and preventing it from binding to RANK. Thus, the relative concentration of RANKL and OPG in bone is a major determinant of bone mass and strength. Here, we review our current understanding of the role of the RANKL/RANK/OPG system in bone modeling and remodeling.     

仙灵骨葆对骨质疏松大鼠OPG/RANK/RANKL 表达的影响

目的:观察仙灵骨葆治疗骨质疏松模型大鼠后,对大鼠体内OPG/RANKL/RANK表达的影响。方法:卵巢摘除法建立SD大鼠骨质疏松模型,设立假手术组、对照组(单纯去卵巢组)、雌激素组(给予17β-雌二醇)和治疗组(给予仙灵骨葆)。术后1周开始给药,给药12周后检测各组大鼠股骨骨密度,ELISA法检测血清中OPG/RANKL含量,RT-PCR检测骨组织中OPG/RANK/RAN-KL mRNA表达,免疫组化检测骨组织中RANK的表达。结果:对照组大鼠骨密度显著低于假手术组;治疗组和雌激素组大鼠O-PG表达显著高于对照组,RANK及RANKL的表达显著低于对照组。结论:采用卵巢摘除法成功建立大鼠骨质疏松模型;仙灵骨葆可促进骨质疏松大鼠OPG的表达,并抑制RANK及RANKL的表达,对骨质疏松模型大鼠有治疗作用。     

CTGF enhances migration and MMP‐13 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CTGF increased the migration and expression of matrix metalloproteinase (MMP)‐13 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody (mAb) and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the CTGF‐induced increase of the migration and MMP‐13 up‐regulation of chondrosarcoma cells. CTGF stimulation increased the phosphorylation of focal adhesion kinase (FAK) and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited CTGF‐induced cell migration and MMP‐13 up‐regulation. Stimulation of JJ012 cells with CTGF also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The CTGF‐mediated increases in κB‐luciferase activities were inhibited by RGD, PD98059, U0126 or FAK, and ERK2 mutant. Taken together, our results indicated that CTGF enhances the migration of chondrosarcoma cells by increasing MMP‐13 expression through the αvβ3 integrin, FAK, ERK, and NF‐κB signal transduction pathway. J. Cell. Biochem. 107: 345–356, 2009. © 2009 Wiley‐Liss, Inc.     

Assessment and clinical implications of RANK/RANKL/OPG pathway as markers of bone tumor progression in patients with NET harboring bone metastases

Abstract

Introduction: The impact on the survival of bone metastases (BM) in patients with neuroendocrine tumor (NET) is a matter of debate. BM have a key role in causing symptoms and in decreasing patients’ quality of life. Although the mechanisms of the development of BM are not completely clear, it is now well understood that the Receptor Activator of Nuclear factor Kappa-B-/Ligand (RANK/RANKL)/osteoprotegerin (OPG) pathway plays a relevant role.

Aim: To characterize the RANK/RANKL/OPG pathway in patients affected with NET.

Patients and methods: Two cohorts of 15 patients each were enrolled in the study; one cohort was affected with NET without BM and the second cohort was affected with NET with BM. The serum RANK/RANKL/OPG pathway was assessed in both the groups.

Results: Serum OPG levels and RANKL/OPG ratio were lower and higher, respectively, in NET patients harboring BM than in those without BM. During the ROC analysis, a cut-off value of 1071?pg/ml for OPG and 0.62 for RANKL/OPG ratio were able to significantly distinguish between the two groups.

Conclusions: This study indicates that RANK/RANKL/OPG pathway is imbalanced in patients with NET harboring BM. Specific alterations of this pathway could predict an early development of BM.     

OPG、RANK、RANKL的结构、作用机制和在骨疾病中的作用

破骨细胞和成骨细胞分别介导骨的吸收过程和合成过程,而OPG、RANK、RANKL在调节二者的比例中发挥非常重要的作用.RANKL与RANK结合后可能通过三种途径:JNK途径、NF-κB途径和蛋白激酶B途径参与破骨细胞的分化,促进骨质的吸收;RANKL与OPG结合后能阻断RANKL与RANK的结合,由于缺乏RANKL-RANK产生的转录活化信号,破骨细胞分化成熟发生障碍,骨质的吸收受到抑制.OPG、RANK、RANKL同时也是免疫分子,在淋巴细胞、淋巴器官的分化、发育中起重要的作用,骨疾病与免疫系统之间存在着一定的关系.RANMKL/RANK与RANKI/OPG在生物体内保持着一定的比率,如果比率失衡,就会引起各种骨疾病.本篇综述总结了近年来OPG、RANK、RANKL结构、作用的新进展以及它们在骨疾病中的作用.     

Disturbance of the OPG/RANK/RANKL pathway and systemic inflammation in COPD patients with emphysema and osteoporosis

Background

Osteoporosis is one of the systemic features of COPD. A correlation between the emphysema phenotype of COPD and reduced bone mineral density (BMD) is suggested by some studies, however, the mechanisms underlying this relationship are unclear. Experimental studies indicate that IL-1β, IL-6 and TNF-α may play important roles in the etiology of both osteoporosis and emphysema. The OPG/RANK/RANKL system is an important regulator of bone metabolism, and participates in the development of post-menopausal osteoporosis. Whether the OPG/RANK/RANKL pathway is involved in the pathogenesis of osteoporosis in COPD has not been studied.

Methods

Eighty male patients (current or former smokers) completed a chest CT scan, pulmonary function test, dual x-ray absorptiometry measurements and questionnaires. Among these subjects, thirty patients with normal BMD and thirty patients with low BMD were selected randomly for measurement of IL-1β, IL-6, TNF-α (flow cytometry) and OPG/RANK/RANKL (ELISA). Twenty age-matched healthy volunteers were recruited as controls.

Results

Among these eighty patients, thirty-six had normal BMD and forty-four had low BMD. Age, BMI and CAT score showed significant differences between these two COPD groups (p < 0.05). The low-attenuation area (LAA%) in the lungs of COPD patients was negatively correlated with lumbar vertebral BMD (r = 0.741; p < 0.0001). Forward logistic regression analysis showed that only LAA% (p = 0.005) and BMI (p = 0.009) were selected as explanatory variables. The level of IL-1β was significantly higher in the COPD patients as compared to the normal controls (p < 0.05), but the difference between the two COPD groups did not reach significance. The levels of IL-6 and TNF-α among the three groups were significantly different (p < 0.05). The level of RANKL and the RANKL/OPG ratio were significantly higher in COPD patients with low BMD compared to those with normal BMD and the normal controls (p < 0.05), and correlated negatively with lumbar vertebral BMD, but positively with LAA%.

Conclusions

Radiographic emphysema is correlated with low BMD in current and former smokers with COPD. IL-1β, IL-6, TNF-α, and the osteoporosis-related protein system OPG/RANK/RANKL may have some synergetic effects on emphysema and bone loss in COPD.     

Changes in RANKL/OPG/RANK gene expression in peripheral mononuclear cells following treatment with estrogen or raloxifene

The RANKL/OPG/RANK pathway is the key mediator of osteoclastogenesis. Mononuclear cells may be implicated in post-menopausal osteoporosis. The effect of estrogen or raloxifene on bone resorption and the expression of RANKL/OPG/RANK in peripheral blood mononuclear cells (PBMCs) was examined. Twenty-nine women with post-menopausal osteoporosis were treated with estrogen (HRT) or raloxifene for 12 months. Bone mineral density (BMD) was measured at baseline and at 12 months at the spine and hip. Serum C-terminal telopeptide (CTX) and OPG were measured at baseline and at 1, 3, 6 and 12 months. PBMCs were isolated from 17 women and changes in RANKL, OPG and RANK mRNA were determined. The effects of estrogen or raloxifene in PBMCs in vitro were also assessed. BMD increased following treatment (lumbar spine % change mean [S.E.M.]: 4.3% [0.9], p<0.001). Serum CTX decreased (6 months: -43.7% [6.0], p<0.0001). Serum OPG declined gradually (12 months: -26.4% [4.4], p<0.001). RANKL, OPG and RANK gene expression decreased (6 months: RANKL 50.0% [24.8] p<0.001, OPG: 21.7% [28] p<0.001, RANK: 76.6% [10.2] p=0.015). Changes in OPG mRNA correlated with changes in BMD (r=-0.53, p=0.027) and CTX (r=0.7, p=0.0044). Down-regulation in RANKL, OPG, RANK mRNA and reduction in bone resorption was also seen in vitro. These results suggest that the expression of RANKL/OPG/RANK in PBMCs are responsive to the slowing in bone turnover/remodeling associated with treatment with estrogen or raloxifene. Further confirmatory studies are needed.     

Imbalance of RANK,RANKL and OPG expression during tibial fracture repair in diabetic rats

To clarify the mechanisms of altered bone repair in the diabetic state, we investigated RANK, RANKL and OPG expression by immunohistochemistry and RT-PCR in the fracture sites of rats that were either healthy or made diabetic by alloxan. Histomorphometric analysis of the fracture site at 7 days after fracture revealed that diabetic rats (db) have significantly less hard tissue formation at the fracture site, compared to controls. The number of RANK, RANKL and OPG positive cells was decreased in the db group; however, the RANKL/OPG ratio was similar in controls and db at this time. At day 14, numbers of RANKL and OPG positive cells and the mRNA expression for these markers were higher in the control group than in db (P = 0.008). The RANKL/OPG ratio in the db group was greater than in controls. Our results demonstrate an imbalance of RANKL/OPG expression associated with diabetes that may contribute to the delay of fracture repair during the course of diabetes.     

OPG/RANK/RANKL系统与骨质疏松研究最新进展

骨质疏松是严重威胁中老年人健康的骨科常见病,OPG/RANK/RANKL是参与调节骨重建的最重要的分子系统之一,与骨疾病相关的骨质疏松有密切联系,并已成为药物设计的新靶点.因此,对该系统的深入研究将为骨生理、病理机制阐明及骨疾病防治带来积极影响.     

Involvement of the JNK activation in terbinafine‐induced p21 up‐regulation and DNA synthesis inhibition in human vascular endothelial cells

Previously, we demonstrated that the extracellular signal‐regulated kinase (ERK)‐mediated pathway contributes to the terbinafine (TB)‐induced increases of p21 and p53 protein level as well as decrease of DNA synthesis in human umbilical venous endothelial cells (HUVEC). The aim of this study is to examine the involvement of c‐Jun NH2‐terminal kinase (JNK) in the TB‐induced increase of p21 protein level and DNA synthesis inhibition. Western blot analysis and kinase assay demonstrated that TB treatment increased both the protein level and the kinase activity of JNK1/2 in HUVEC. Transfection of HUVEC with JNK1 dominant negative (DN‐JNK1) prevented the TB‐induced increases of p21 and p53 protein level and decrease of DNA synthesis, suggesting that JNK1/2 activation is involved in the TB‐induced cell cycle arrest in HUVEC. Moreover, over‐expression of mitogen‐activated protein kinase (MEK)‐1 prevented the TB‐induced increase of JNK1/2 protein levels, suggesting that MEK‐1 is an upstream inhibitor of JNK. Transfection of HUVEC with DN‐JNK1 prevented the TB‐induced inhibition of ERK phosphorylation, suggesting that JNK1/2 might serve as a negative regulator of ERK. Taken together, our results suggest that JNK activation is involved in the TB‐induced inhibition of ERK phosphorylation, p53 and p21 up‐regulation and DNA synthesis inhibition in HUVEC. J. Cell. Biochem. 108: 860–866, 2009. © 2009 Wiley‐Liss, Inc.