Decreased bone density, elevated serum osteoprotegerin, and beta-cross-laps in Wilson disease.

Osteopathia has been reported in Wilson disease (WD), but bone density has not been measured; therefore, we performed bone mineral density (BMD), bone mineral content (BMC), and quantitative bone ultrasound (QUS) assessments, as well as measured the serum levels of osteocalcin (OCN), beta-cross-laps (beta-CTx's), and the recently discovered osteoprotegerin (OPG) and its ligand RANKL to investigate the underlying mechanism of osseous disorders. Serum OCN, beta-CTx, OPG, and RANKL levels were measured by ELISA in 21 WD patients and in 20 age- and gender-matched healthy subjects. BMD, BMC, and QUS parameters were also determined. Osteoporosis was present in 9/21 (43%) WD patients. Abnormal QUS parameters were found in 7 (33%) of the patients. Although serum OCN levels were similar in patients and controls (29.93 +/- 24.65 mg/ml vs. 29.84 +/- 6.89 mg/ml), beta-CTx and OPG levels were significantly increased in WD compared with the healthy controls (625.4 +/- 312.3 pg/ml vs. 423.6 +/- 144.3 pg/ml and p = 0.022 and 7.2 +/- 3.4 pM vs. 3.5 +/- 1.0 pM and p < 0.001, respectively). No difference was observed in the RANKL level. There was a positive correlation between OCN and beta-CTx (r = 0.55; p = 0.01). We proved high occurrence of osteoporosis in WD. Negative bone remodeling balance is a consequence of increased bone resorption, which is indicated by elevated beta-CTx. The novel finding of elevated serum OPG may reflect a compensatory reaction to enhanced osteoclast activity, despite the normal OCN level.     

Increased expression of IL-6 and RANK mRNA in human trabecular bone from fragility fracture of the femoral neck

Previous studies have implicated pro-inflammatory cytokines in the bone loss of estrogen deficiency. The aim of this study was to investigate the expression of key regulatory molecules of bone remodeling in the trabecular bone microenvironment in osteoporosis. Bone samples were taken from the intertrochanteric region of the proximal femur of patients undergoing total hip arthroplasty for a subcapital fragility fracture of the femoral neck (#NOF). For comparison, samples were taken from age-matched control individuals at routine autopsy. Expression of RANKL, RANK, osteoprotegerin (OPG), IL-6, IL-11, osteocalcin (OCN), and calcitonin receptor (CTR) messenger RNA (mRNA) species were analyzed and the data were nonparametrically distributed. The median expression of the proresorptive genes, RANK and IL-6, were significantly elevated in the fracture group compared to an age-matched control group (2.2 [1.9-2.9; 25th-75th percentiles] > 1.0 [0.4-2.1], P < 0.03; 3.9 [1.8-6.2] > 0.8 [0.7-1.5], P < 0.002, respectively). In contrast, there were no significant differences in expression of RANKL, OPG, CTR, or OCN mRNA between the #NOF and control groups. The median RANKL/OPG mRNA ratio was significantly greater in hip fracture bone than in bone from controls (4.8 [3.8-7.6] > 3.2 [2.1-4.0], P < 0.05). IL-6 mRNA levels associated strongly with RANKL mRNA levels in the #NOF group (r = 0.77, P < 0.001), but not in the control group. A strong positive association was found between IL-11 mRNA levels and RANKL mRNA levels in the #NOF group (r = 0.81, P < 0.001), consistent with the apparent coordinated regulation of IL-6 and IL-11 in bone samples from the #NOF group (r = 0.93, P < 0.0001). These data suggest a relative increase in the expression of the molecular promoters of osteoclast formation and activity in #NOF bone, which may lead to the imbalance between bone formation and resorption associated with fragility fracture.     

Effects of medicinal herb salvia miltiorrhiza on osteoblastic cells in vitro

Sufficient osteoinduction is essential for the success and effectiveness of bone grafting. It was previously found that Salvia Miltiorrhiza (SM), a commonly used Chinese herb increased osteogenesis in vivo. The aim of this study is to investigate the effects of SM on bone cells in vitro, in an attempt to get a better understanding on how SM can promote bone remodeling. MC3T3‐E1, an osteoblastic cell line, was cultured with SM for different time intervals (24, 48, and 72 h), whereas the control group consisted of cells cultured without any intervention. The mRNA expression of alkaline phosphatase (ALP), osteocalcin (OCN), osteoprotegerin (OPG), and the receptor activator of nuclear factor kappa B ligand (RANKL) were examined by real‐time polymerase chain reaction (qPCR). The expression of ALP showed an early increase at 24 h by 50% (p < 0.001) and at 48 h by 13% (p < 0.001). OCN was decreased by 22% at 24 h (p < 0.001) but increased by 50% and 88% at 48 and 72 h, respectively (p < 0.001). RANKL showed an early increase at the first two time points of 24 and 48 h by 45% (p < 0.001) and 36% (p < 0.01), respectively, while OPG was up‐regulated at the latter two time points by 10% at 48 h (p < 0.01) and 68% at 72 h (p < 0.001). Thus, OPG/RANKL was down‐regulated first, and then up‐regulated. SM enhances bone remodeling by regulating the gene expression of ALP, OCN, OPG, and RANKL. It is a potential medicinal herb to be utilized in the application that requires stimulation in bone cell activities. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1059–1063, 2011     

二甲双胍对去卵巢大鼠骨质疏松症的影响及机制研究

目的 以雌二醇为对照,观察二甲双胍(metformin, MF)对去卵巢大鼠骨密度及骨矿含量的影响,并从细胞、分子水平探究MF可能的骨保护机制。方法 将60只雌性SD大鼠随机均分4组：假手术（SHAM）组、去卵巢（OVX）组、去卵巢+二甲双胍（OVX+MF）组和去卵巢+雌二醇(OVX+E2 )组。分组灌胃给药60 d后测量大鼠右侧胫骨骨密度和骨矿含量;分离培养各组大鼠骨髓间充质干细胞（BMSCs）并诱导其向成骨细胞分化,用MTT法测定细胞活性及增殖能力;测定各组碱性磷酸酶（ALP）活性、矿化结节数目、钙含量以及I型胶原（collagen type I）、骨钙素（OC）、骨保护素 (OPG)、NFκB受体的配体 (RANKL)、白细胞介素-6（IL-6）基因表达水平。结果 与OVX组相比,OVX+MF组和OVX+E2组成骨细胞的增殖能力与ALP活性明显增强,骨密度、骨矿含量以及钙沉积量显著增加(P均<0.05),且两组collagen type I、OC、OPG mRNA的表达水平显著升高,而RANKL、IL-6mRNA表达明显受到抑制;但OVX+MF组去卵巢大鼠成骨细胞的增殖能力、ALP活性、钙沉积量、collagen type I、OC、OPG mRNA表达水平低于OVX+E2组,RANKL、IL-6mRNA表达高于OVX+E2组（P均<0.05）;与SHAM组比较,OVX+MF组的collagen type I、OC、OPG mRNA的表达水平更高（P<0.05）。结论 二甲双胍可能通过OPG/RANKL/RANK信号通路促进BMSCs向成骨细胞分化,有效逆转去卵巢大鼠骨质疏松的状态,这种潜在的骨保护作用可能会改善糖尿病引起的骨质疏松。     

Imbalance of RANKL/RANK/OPG system in interface tissue in loosening of total hip replacement

In the differentiation of osteoclasts the differentiation factor (RANKL) interacts with the receptor activator of NF-kappaB (RANK) in a direct cell-to-cell contact between osteoblast and (pre)osteoclast. This is inhibited by soluble osteoprotegerin (OPG). The mRNA levels of both RANKL (p < 0.01) and RANK (p < 0.05) were high in peri-implant tissue and RANKL+ and RANK+ cells were found in such tissue. Double labelling also disclosed soluble RANKL bound to RANK+ cells. We were unable to stimulate fibroblasts to express RANKL in vitro, but monocyte activation with LPS gave a fivefold increase in RANK mRNA levels. In contrast to RANKL and RANK expression in peri-implant tissue, expression of OPG was restricted to vascular endothelium. Endothelial cell OPG mRNA levels were regulated by TNF-alpha and VEGF, but not by hypoxia. It is concluded that activated cells in the interface tissue overproduce both RANKL and RANK and they can interact without interference by OPG.     

Prostate-specific antigen stimulates osteoprotegerin production and inhibits receptor activator of nuclear factor-kappaB ligand expression by human osteoblasts

BACKGROUND: Prostate cancer cells produce a large amount of prostate-specific antigen (PSA), which is widely used as a marker for this cancer. Even though it is widely used in the diagnosis of prostate cancer, many aspects of the pathophysiologic role of PSA in bone metastasis remain obscure. The receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for the activation of osteoclasts, while osteoprotegerin (OPG) neutralizes the action of RANKL. Various substances that act on bone have been shown to modulate the production of RANKL and OPG by osteoblasts. METHODS: In this study, we investigated the effect of PSA on the expression of OPG and RANKL mRNA and on protein production in human osteoblast-like cells. RESULTS: After addition of PSA and culture for 72 h, OPG mRNA expression and protein secretion by MG-63 and SaOS-2 cells showed a concentration-dependent increase. When osteoblasts were incubated with PSA (100 ng/ml), OPG mRNA expression and protein secretion increased with the passage of time. alpha1 -antichymotrypsin (ACT), which inactivates the serine protease activity of PSA, inhibited the increase of OPG mRNA expression and protein production in response to PSA, and this effect of PSA was also inhibited by anti-transforming growth factor-beta antibody. CONCLUSIONS: Based on our findings, PSA acts on human osteoblast-like cells via its own serine protease activity and promotes osteoblast differentiation. In addition, PSA stimulates OPG production and inhibits RANKL expression of osteoblasts, and inhibits bone resorption by osteoclasts, suggesting that it contributes to the characteristic osteoblastic features of bone metastases of prostate cancer.     

Regulation of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin mRNA expression by parathyroid hormone is predominantly mediated by the protein kinase a pathway in murine bone marrow cultures

Parathyroid hormone (PTH) stimulates receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and inhibits osteoprotegerin (OPG) mRNA expression in murine bone marrow cultures. To understand the mechanisms influencing these responses, we investigated the role of the protein kinase A (PKA) and protein kinase C (PKC) pathways in the regulation of RANKL and OPG mRNA expression in murine bone marrow cultures. Murine bone marrow cells were stimulated with bovine PTH(1-34) and (1-34) amide, which activate both pathways; PTH(3-34), which more selectively activates the PKC and calcium pathways; and human PTH (1-31), which stimulates adenylyl cyclase, but not protein kinase C. We also examined agents that more directly activate either the PKA pathway (forskolin [FSK] and 8-bromo cAMP [8-Br-cAMP]) or the PKC pathway (phorbol 12-myristate 13-acetate [PMA]) in murine bone marrow cultures. After 1 h, RANKL mRNA expression was stimulated to a similar degree by agents that activate either or both the PKA and PKC pathways. However, this effect was sustained for 24 h only with agents that stimulated PKA. OPG mRNA expression was inhibited by all agents that stimulated PKA at 6 h. In contrast, PKC-specific stimulators [PMA and bPTH(3-34)] had no effect on OPG regulation in this culture system. To determine the involvement of the PKC signaling pathway in responses of RANKL, bone marrow cells were pretreated with PMA for 24 h and then treated with PTH(1-34) or FSK for 2 h. PMA pretreatment did not alter the ability of PTH or FSK to stimulate RANKL or inhibit OPG mRNA expression. Treatment of cells with H-89, a PKA inhibitor, significantly reduced the ability of PTH and FSK to induce RANKL and inhibit OPG mRNA expression. Calphostin C, a PKC inhibitor, significantly reduced PMA-stimulated RANKL mRNA expression without altering PTH- or FSK-mediated effects on RANKL or OPG mRNA. Cycloheximide, an inhibitor for protein synthesis, inhibited PTH-stimulated RANKL mRNA expression by 60% without altering the effect of PTH on OPG mRNA expression. To examine the involvement of prostaglandin in PMA-mediated responses, cells were treated with indomethacin, a nonspecific prostaglandin G/H synthase (PGHS) inhibitor, or NS-398, a selective inhibitor of PGHS-2. Neither PGHS inhibitor altered PMA-induced effects on RANKL and OPG mRNA expression. These results demonstrate that the PKA pathway is predominantly involved in the effects of PTH on RANKL mRNA expression in murine bone marrow cultures, but there is also a PKC-mediated response, which is not sustained. Inhibition of OPG by PTH appears to be a selective PKA response.     

Expression of bone resorption genes in osteoarthritis and in osteoporosis

Cathepsin K and MMP-9 are considered to be the most abundant proteases in osteoclasts. TRAP is a marker for osteoclasts, and there is increasing evidence of its proteolytic role in bone resorption. RANKL is a recently discovered regulator of osteoclast maturation and activity and induces expression of many genes. This study compared cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin gene expression in the proximal femur of patients with osteoarthritis with that of patients with femoral neck fracture. Fifty-six patients undergoing arthroplasty because of osteoarthritis or femoral neck fracture were included in the study. Total mRNA was extracted from the bone samples obtained from the intertrochanteric region of the proximal femur. Real-time RT-PCR was used to quantify CTSK (cathepsin K), MMP-9 (matrix metalloproteinase 9), ACP5 (TRAP), TNFSF11 (RANKL), TNFRSF11B (OPG), and BGLAP (osteocalcin) mRNAs. The levels of mRNAs coding for MMP-9 and osteocalcin indicated higher expression in the osteoarthritic group (P = 0.011, P = 0.001, respectively), whereas RANKL expression and the ratio RANKL/OPG were both significantly lower in the osteoarthritic group than in the fracture group. Expression of cathepsin K, MMP-9, and TRAP relative to RANKL was significantly higher in the osteoarthritic group. Ratios of all three proteolytic enzymes relative to formation marker osteocalcin were higher in the fracture group. Gene expression of cathepsin K, MMP-9, TRAP, RANKL, OPG, and osteocalcin and the association between their mRNA levels pointed to higher bone resorption and bone formation in osteoarthritis, differences in balance between them, and differences in regulation of bone resorption in osteoarthritic and osteoporotic bone.     

Effects of coumestrol on expression of bone markers during primary osteoblastic cells differentiation

目的 观察植物雌激素香豆雌酚对成骨细胞增殖分化的作用并探讨其作用机制.方法 从小鼠颅盖骨获得成骨细胞并用0,10-9～10-5 M香豆雌酚孵育48 h,以17β雌二醇为阳性对照,用酶消化法测定碱性磷酸酶及Ⅰ型胶原含量,放免法测定骨钙素含量,RT-PCR法测定OPG及RANKL mRNA表达情况,Western Blot测定OPG蛋白含量.结果 干预48 h,不同浓度香豆雌酚呈剂量依赖性增加碱性磷酸酶及Ⅰ型胶原含量,10-6 M时达到最大效应(P<0.05),但10-5 M效应有所降低,香豆雌酚轻度增加成骨细胞骨钙素含量,各组间无统计学差异.香豆雌酚呈剂量依赖性增加OPG基因及蛋白的表达(P<0.05),轻度降低RANKL基因的表达.结论 香豆雌酚能增加成骨细胞增殖及分化,可能其部分通过OPG/RANKL发挥作用.     

共轭亚油酸c9,t11-CLA及t10,c12-CLA对大鼠骨髓细胞骨代谢相关基因表达的影响

目的 观察c9,t11-CLA及t10,c12-CLA干预后,大鼠骨髓细胞过氧化物酶体增殖物激活受体2(PPARγ2)及核因子(NF)-κB活化受体配体(RANKL)、碱性磷酸酶(ALP)、骨保护素(OPG)、NF-κB活化受体(RANK)、抗酒石酸酸性磷酸酶(TRAP) mRNA表达的变化,探讨其对骨代谢的影响.方法 体外培养大鼠骨髓细胞,分别加入c9,t11-CLA及t10,c12-CLA干预24h,逆转录-聚合酶链反应(RT-PCR)检测PPARγ2、RANKL、ALP、OPG、RANK、TRAP mRNA表达水平,比较其对骨髓细胞上述基因表达的影响.结果 (1)c9,t11-CLA呈剂量依赖性下调RANK、TRAP mRNA表达,组间比较差异有统计学意义(P＜0.05,P＜0.01),而其对RANKL、ALP、OPG及PPARγ2 mRNA 表达的影响不明显.(2) t10,c12-CLA呈剂量依赖性上调RANKL、OPG mRNA的表达,同时下调 RANK、TRAP及PPARγ2 mRNA的表达,组间比较差异有统计学意义(P＜0.05,P＜0.01),但对ALP mRNA的表达无明显影响.结论 c9,t11-CLA可能通过抑制破骨细胞标记物基因表达阻断骨髓细胞向破骨细胞分化,而t10,c12-CLA在抑制破骨细胞标记物基因表达的同时也可促进成骨细胞标记物基因表达,两者均有利于骨形成.     

橙皮苷对去卵巢骨质疏松症大鼠骨质流失和机制的影响

目的研究橙皮苷对去卵巢骨质疏松大鼠骨质流失和骨保护素(osteoprotegerin,OPG)/核因子kappa B配体的受体激活物(receptor activator of nuclear factor kappa B ligand,RANKL)/RANK通路的影响。方法将48只雌性Sprague-Dawley(SD)大鼠随机分为假手术组、去卵巢骨质疏松组、橙皮苷低剂量组、橙皮苷中剂量、橙皮苷高剂量组和雌激素组。检测骨组织骨体积分数(bone volume fraction,BV/TV)、骨小梁厚度(trabecular thickness,Tb.Th)、骨密度(bone mineral density,BMD)、骨小梁数量(trabecular number,Tb.N)、骨小梁分离度(trabecular separation,Tb.Sp),通过HE染色观察骨组织病理损伤,采用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测人Ⅰ型胶原C端肽(C-terminal peptide collagen typeⅠ,CTX-Ⅰ)和血清骨钙素(bone glaprotein,BGP)的表达量。运用qRT-PCR和Western bolt分别检测骨组织OPG、RANKL、RANK的mRNA和蛋白表达。结果在橙皮苷治疗后,与去卵巢骨质疏松组相比,骨质疏松标志物BV/TV、BMD、Tb.Th和Tb.N水平升高,Tb.Sp水平降低;大鼠骨小梁数量明显增加,且骨小梁连接更为紧密;骨吸收标志物CTX-Ⅰ水平降低;骨形成标志物BGP水平升高。OPG mRNA相对表达量和蛋白的表达量升高,RANKL和RANK mRNA相对表达量和蛋白的表达量降低。结论橙皮苷能缓解去卵巢骨质疏松大鼠骨质流失并调节OPG/RANKL/RANK信号通路。     

黄瓜籽总皂苷对MC3T3-E1细胞分化及SPARC、OPG/RANKL表达的影响

目的观察黄瓜籽总皂苷提取物(cucumber seed saponins,CSS)对小鼠成骨细胞MC3T3-E1增殖、分化和矿化的影响,以及与骨质疏松相关的SPARC、OPG/RANKL/RANK信号通路的作用。方法通过MTT实验、碱性磷酸酶(alkaline phosphatase,ALP)活性检测、茜素红染色,考察不同浓度CSS对MC3T3-E1细胞增殖、分化及矿化的影响;采用RT-PCR方法检测SPARC、OPG/RANKL mRNA表达水平; Western blot检测SPARC、OPG/RANKL的蛋白表达量。结果与阳性对照组相比,CSS能明显促进MC3T3-E1细胞增殖(P0.05),CSS高、中剂量组能明显提高MC3T3-E1细胞ALP活性及钙化结节数量(P0.05);与空白组相比,CSS不同剂量组均明显上调SPARC、OPG/RANKL mRNA及蛋白表达水平(P0.05)。结论黄瓜籽总皂苷能够促进成骨细胞MC3T3-E1的增殖、分化及矿化能力,并通过上调SPARC、OPG/RANKL的表达水平提高MC3T3-E1细胞的成骨能力。     

Expression of RANKL and OPG correlates with age-related bone loss in male C57BL/6 mice.

Osteoblasts regulate the recruitment and activity of osteoclasts through expression of RANKL and osteoprotegerin (OPG). To determine whether expression of RANKL and OPG change with age and how these changes relate to the bone loss of aging, we measured bone mass and cancellous volume, and expression of RANKL, OPG, alkaline phosphatase (AP), osteocalcin (OC), and alpha I collagen (COLL) in whole bone and osteoblast-like cells in culture using 6-week- (young), 6-month- (adult), and 24-month-old (old) mice. Cancellous volume decreased by 20% from young to adult and by 52% from adult to old. RANKL mRNA levels in whole bone were 2.1-fold and 4.4-fold higher in adult and old mice, respectively, compared with young mice, whereas OPG mRNA levels decreased with age slightly. RANKL expression was negatively (r = -0.99) and OPG was positively (r = 0.92) correlated with cancellous bone volume. Expression of RANKL was higher and OPG lower in cells from older animals early in culture (day 7). With cell maturation, RANKL mRNA levels in cells from young and adult mice increased, whereas levels in cells from old animals decreased. By 21 and 28 days of culture, no differences were found in RANKL mRNA in osteoblast-like cells among different age groups. We conclude that expression of RANKL and OPG change with age in whole bone and in cultured osteoblast-like cells. These changes favor increased osteoclast over osteoblast activity, and may explain, in part, the imbalance in bone formation and resorption associated with aging.     

Smad4促进成骨分化的机制研究

目的 探讨Smad4基因促进成骨分化的作用机制。方法 采用条件性基因敲除技术Cre/loxp,制备骨细胞特异性敲除Smad4小鼠(Smad4otcko),小鼠胚胎骨骼透明染色分析胚胎期小鼠长骨生长状况;待小鼠成长至1月龄,X-ray检测突变小鼠与对照组小鼠的骨密度差异;静态骨组织形态学分析检测突变鼠及对照鼠的骨量变化、成骨细胞数量变化等差异;实时荧光定量PCR检测Smad4突变鼠股骨成骨细胞相关因子Runx2、ALP、OSX及OCN;破骨细胞TRAP染色分析Smad4突变鼠破骨细胞形态及数量变化;qPCR检测突变鼠股骨破骨吸收标志基因RANKL、OPG,并计算RANKL/OPG比率。结果 Smad4基因敲除小鼠在胚胎期未出现长骨生长异常。X线结果显示,1月龄时,与对照组小鼠相比,Smad4突变鼠的骨密度降低（P＜0.05）,静态骨组织形态学分析表明突变鼠松质骨减少,皮质骨变薄,骨小梁数量减少（P＜0.05）;Smad4突变鼠成骨细胞标志基因表达量显著降低,成骨细胞的数量明显减少（P＜0.05）;RANKL作为破骨吸收标志物表达上调、作为其拮抗剂的OPG表达量下调,RANKL/OPG比率增高（P＜0.05）。结论 Smad4基因通过促进成骨分化,降低破骨吸收从而来维持骨稳态。     

Osteoprotegerin inhibits osteoclast formation and bone resorbing activity in giant cell tumors of bone

Osteolysis is a common complication of tumors that arise in, or metastasize to, bone. The recent discovery of key regulators of osteoclast formation and activity, including receptor activator of nuclear factor of kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG), may facilitate new treatment regimes for certain tumors associated with excessive bone loss. We recently showed that the stromal cells of osteolytic giant cell tumors (GCT) of bone express high levels of mRNA encoding RANKL, relative to mRNA for the RANKL antagonist, OPG, compared with the expression patterns of other lytic and nonlytic bone tumors. In this study, we found that expression of RANKL and OPG mRNA continued by the stromal element of these tumors in a constitutive manner for at least 9 days in the absence of giant cells. Immunostaining of unfractionated GCT cultured in vitro revealed punctate cytoplasmic/membranous staining for RANKL and both cytoplasmic and extracellular matrix staining for OPG in stromal cells. Giant cells (osteoclasts) were negative for RANKL staining, but stained brightly for cytoplasmic OPG protein. We also investigated the functional relevance of these molecules for GCT osteolysis by adding recombinant OPG and RANKL to cultured GCT cells. We found that OPG treatment potently and dose-dependently inhibited resorption of bone slices by GCT, and could also inhibit the formation of multinucleated osteoclasts from precursors within the GCT. These effects of OPG were reversed by stoichiometric concentrations of exogenous RANKL. These data indicate that both the processes of osteoclast formation and activation in GCT are promoted by RANKL. Therefore, GCT represent a paradigm for the direct stimulation of osteoclast formation and activity by tumor stromal cells, in contrast to the mechanisms described for osteolytic breast tumors and multiple myeloma. The demonstration of these relationships is important in developing approaches to limit tumor-induced osteolysis.     

Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing.

Fracture healing is a unique biological process regulated by a complex array of signaling molecules and proinflammatory cytokines. Recent evidence for the role of tumor necrosis family members in the coupling of cellular functions during skeletal homeostasis suggests that they also may be involved in the regulation of skeletal repair. The expression of a number of cytokines and receptors that are of functional importance to bone remodeling (osteoprotegerin [OPG], macrophage colony-stimulating factor [M-CSF], and osteoprotegerin ligand [receptor activator of NF-kappaB ligand (RANKL)]), as well as inflammation (tumor necrosis factor alpha [TNF-alpha] and its receptors, and interleukin-1alpha [IL-1alpha] and -beta and their receptors) were analyzed over a 28-day period after the generation of simple transverse fractures in mouse tibias. OPG was expressed constitutively in unfractured bones and elevated levels of expression were detected throughout the repair process. It showed two distinct peaks of expression: the first occurring within 24 h after fracture and the second at the time of peak cartilage formation on day 7. In contrast, the expression of RANKL was nearly undetectable in unfractured bones but strongly induced throughout the period of fracture healing. The peak in expression of RANKL did not correlate with that of OPG, because maximal levels of expression were seen on day 3 and day 14, when OPG levels were decreasing. M-CSF expression followed the temporal profile of RANKL but was expressed at relatively high basal levels in unfractured bones. TNF-alpha, lymphotoxin-beta (LT-beta), IL-1alpha, and IL-1beta showed peaks in expression within the first 24 h after fracture, depressed levels during the period of cartilage formation, and increased levels of expression on day 21 and day 28 when bone remodeling was initiated. Both TNF-alpha receptors (p55 and p75) and the IL-1RII receptor showed identical patterns of expression to their ligands, while the IL-1R1 was expressed only during the initial period of inflammation on day 1 and day 3 postfracture. Both TNF-alpha and IL-1alpha expression were localized primarily in macrophages and inflammatory cells during the early periods of inflammation and seen in mesenchymal and osteoblastic cells later during healing. TNF-alpha expression also was detected at very high levels in hypertrophic chondrocytes. These data imply that the expression profiles for OPG, RANKL, and M-CSF are tightly coupled during fracture healing and involved in the regulation of both endochondral resorption and bone remodeling. TNF-alpha and IL-1 are expressed at both very early and late phases in the repair process, which suggests that these cytokines are important in the initiation of the repair process and play important functional roles in intramembraneous bone formation and trabecular bone remodeling.     

The effects of estrogen on osteoprotegerin,RANKL, and estrogen receptor expression in human osteoblasts

Estrogen is essential for bone growth and development and for the maintenance of bone health in adulthood. The cellular responses of osteoblasts and osteoclasts to estrogen are initiated via two high-affinity receptors (ERs). Osteoblasts synthesize RANKL (receptor activator of NF-kappaB ligand), necessary for osteoclast formation and function, and osteoprotegerin (OPG), its decoy receptor. To investigate the effects of estrogen on the expression of OPG, RANKL, and ERs in human osteoblasts, cells were cultured with physiological (10(-10) M) and high-dose (10(-7) M) 17beta-estradiol for 24 and 48 h. Proteins and corresponding mRNA levels were quantitatively determined by immunocytochemistry and RT-PCR. OPG expression was significantly increased three- and sevenfold at 24 h with 10(-10) M (P < 0.05) and 10(-7) M (P < 0.01) estradiol, respectively, compared to untreated cells. Similar but smaller increases were seen at 48 h (P < 0.05). Osteoblasts treated with estradiol demonstrated increased RANKL protein expression at 24 h (P < 0.05), but this was not maintained at 48 h. ERalpha expression was significantly increased by high-dose estradiol (P < 0.01) at 24 h and dose-dependently increased at 48 h (P < 0.01), while ERbeta was only increased at 24 h (P < 0.01). The estrogen-induced protein expression of ER, OPG, and RANKL was abrogated when cells were cultured in the presence of the estrogen antagonist ICI 182780. mRNA levels at 24 h demonstrated a significant suppression of RANKL with the low-dose but not the high dose. ERalpha mRNA but not ERbeta expression was up-regulated by estrogen. Our results suggest that estrogen may exert its anti-resorptive effects on bone, at least in part, by stimulating ER and OPG expression in osteoblasts.