NADPH oxidase 4 limits bone mass by promoting osteoclastogenesis

ROS are implicated in bone diseases. NADPH oxidase 4 (NOX4), a constitutively active enzymatic source of ROS, may contribute to the development of such disorders. Therefore, we studied the role of NOX4 in bone homeostasis. Nox4^–/– mice displayed higher bone density and reduced numbers and markers of osteoclasts. Ex vivo, differentiation of monocytes into osteoclasts with RANKL and M-CSF induced Nox4 expression. Loss of NOX4 activity attenuated osteoclastogenesis, which was accompanied by impaired activation of RANKL-induced NFATc1 and c-JUN. In an in vivo model of murine ovariectomy–induced osteoporosis, pharmacological inhibition or acute genetic knockdown of Nox4 mitigated loss of trabecular bone. Human bone obtained from patients with increased osteoclast activity exhibited increased NOX4 expression. Moreover, a SNP of NOX4 was associated with elevated circulating markers of bone turnover and reduced bone density in women. Thus, NOX4 is involved in bone loss and represents a potential therapeutic target for the treatment of osteoporosis.     

热损伤对人皮肤成纤维细胞氧化应激的影响

目的探讨体外培养人皮肤成纤维细胞热损伤后氧化应激及前列腺素E2（PGE2）分泌的变化及其可能的机制。方法体外培养人皮肤成纤维细胞,制作热损伤模型后分为损伤组、DPI预处理组、NAC预处理组三组,以未受热损伤细胞作为对照组。CCK8比色法检测细胞增殖。荧光探针法及化学发光法观察细胞内活性氧（ROS）含量和NADPH氧化酶（Nox）活性变化,EIA检测上清液中PGE2的变化。半定量RT—PCR法检测人皮肤成纤维细胞表达的Nox亚型的mRNA水平。Westernblot法检测热损伤后Noxl蛋白水平的变化。结果热损伤明显抑制人皮肤成纤维细胞的增殖;热损伤后即刻细胞内ROS含量和Nox活性明显升高,在4h后二者均抵达峰值,而DPI预处理组ROS含量和Nox酶活性明显低于损伤组（P〈0．叭或P〈0．05）;热损伤后细胞分泌PGE2明显增高（P〈0．01）,而DPI预处理组和NAC预处理组均显著低于损伤组（P〈0．01）。RT—PCR结果提示正常人皮肤成纤维细胞表达Noxl、Nox3和Nox4三种亚型,三者表达量无明显差异（P〉0．05）。Westernblot结果显示热损伤后不同时间点Noxl的蛋白表达量逐渐升高。结论热损伤可明显抑制人皮肤成纤维细胞增殖并诱导Nox活性升高和Noxl蛋白表达增高,从而使细胞内ROS量升高,进一步引起PGE2分泌增多。     

三七总皂苷干预股骨头缺血性坏死兔成骨细胞护骨素基因的表达

背景:前期研究已明确三七总皂苷能抑制乙醇诱导的兔骨髓基质干细胞成脂分化。目的:进一步观察三七总皂苷对兔成骨细胞的增殖和分化以及骨保护素、细胞核因子κB受体活化因子配体mRNA表达的影响。方法:白酒灌胃4周制备新西兰兔股骨头缺血坏死模型,随机分为生理盐水组、复方骨肽组和三七总皂苷组。通过组织块培养法提取各组兔成骨细胞,检测细胞的分化,骨保护素、细胞核因子κB受体活化因子配体基因表达情况,检测各目的基因杂交信号的强弱。结果与结论:三七总皂苷组兔碱性磷酸酶活性、钙结节计数及骨保护素、细胞核因子κB受体活化因子配体的表达强度、骨保护素mRNA/细胞核因子κB受体活化因子配体mRNA均高于生理盐水组(P<0.01);与复方骨肽组效果接近。证实三七总皂苷能够促进兔成骨细胞的增殖和分化,并能提高成骨细胞的骨保护素mRNA的相对表达量而对其细胞核因子κB受体活化因子配体mRNA有抑制作用。     

Extracorporeal Shock Waves Stimulate Osteoblast Activities

The extracorporeal shock wave therapy (ESWT) is an extensively applied treatment for musculoskeletal disorders because it promotes bone repair. The aim of this study was to evaluate the direct effect of ESWT on murine osteoblasts to clarify the cellular mechanism that leads to the induction of osteogenesis. Osteoblasts in culture flasks were treated with ESWT pulses (500 impulses of 0.05 mJ/mm²) generated by an electromagnetic device. Using western blot analysis 3 h after ESWT, an increased expression of Bax was found, indicating a fast pro-apoptotic effect of treatment on some of the osteoblasts. Activation of the cyclin E2/CDK2 is the complex that regulates the G1-S transition and is essential for cell proliferation. It was evident 24 to 72 h after treatment, indicating a proliferative stimulus. A decreased expression of osteoprotegerin (OPG) and receptor activator NF kappa B ligand (RANKL) 24 and 48 h after ESW, followed by a later increase of OPG, paired with a much smaller increase of RANKL, was evident by real-time polymerase chain reaction (PCR). The decreased RANKL/OPG ratio suggests inhibition of osteoclastogenesis. We can conclude that ESWT induces bone repair through the proliferation and differentiation of osteoblasts and the reduction of their secretion of pro-osteoclastogenic factors. (E-mail: angelanotarnicola@yahoo.it)     

三七总皂苷干预股骨头缺血性坏死兔成骨细胞护骨素基因的表达

背景：前期研究已明确三七总皂苷能抑制乙醇诱导的兔骨髓基质干细胞成脂分化。目的：进一步观察三七总皂苷对兔成骨细胞的增殖和分化以及骨保护素、细胞核因子κB受体活化因子配体mRNA表达的影响。方法：白酒灌胃4周制备新西兰兔股骨头缺血坏死模型,随机分为生理盐水组、复方骨肽组和三七总皂苷组。通过组织块培养法提取各组兔成骨细胞,检测细胞的分化,骨保护素、细胞核因子κB受体活化因子配体基因表达情况,检测各目的基因杂交信号的强弱。结果与结论：三七总皂苷组兔碱性磷酸酶活性、钙结节计数及骨保护素、细胞核因子κB受体活化因子配体的表达强度、骨保护素mRNA/细胞核因子κB受体活化因子配体mRNA均高于生理盐水组（P〈0.01）;与复方骨肽组效果接近。证实三七总皂苷能够促进兔成骨细胞的增殖和分化,并能提高成骨细胞的骨保护素mRNA的相对表达量而对其细胞核因子κB受体活化因子配体mRNA有抑制作用。     

Involvement of adenosine 5'-triphosphate in ultrasound-induced fracture repair

Ultrasound (US) accelerates fracture healing; however, the mechanism of this effect remains unclear. Adenosine 5'-triphosphate (ATP) stimulates bone remodeling and is released constitutively from intact osteoblasts; this is a process that is enhanced after mechanical stimulation. We hypothesized that ATP release from osteoblasts is increased after US stimulation and that this leads to accelerated fracture healing. US was applied to SaOS-2 human osteoblasts and the concentration of ATP in the cell culture medium was determined. Cell proliferation and gene expression were subsequently investigated. Increased concentrations of ATP were detected in the culture medium of US-treated cells and both ATP and US stimulation caused increased receptor activator of nuclear factor-kappa B ligand (RANKL), decreased osteoprotegerin expression and increased cell proliferation by SaOS-2 cells. These findings indicate that US causes ATP release by osteoblasts in vitro and that this may contribute to accelerated fracture healing by enhancing osteoblast proliferation and increasing RANKL expression and decreasing osteoprotegerin expression by osteoblasts to promote osteoclastogenesis.     

Activation of protein kinase A is necessary but not sufficient for ethanol-induced desensitization of cyclic AMP production.

Acute addition of EtOH to PC 12 pheochromocytoma cells increases cyclic AMP production, whereas chronic exposure to EtOH results in a decrease in the stimulation of cyclic AMP production in response to 2-chloroadenosine and forskolin. This EtOH-induced desensitization was not observed after chronic EtOH treatment of A126-1B2-1 cells which are a protein kinase A-deficient mutant cell line derived from PC 12 cells. Furthermore, in the parental PC 12 cell line the cell-permeable protein kinase A inhibitor, Rp-isomer of adenosine 3',5'-monophosphorothioate, blocked the development of EtOH-induced desensitization. Thus, activation of protein kinase A is apparently necessary for EtOH-induced desensitization of cyclic AMP production. Chronic treatment of PC 12 cells with forskolin qualitatively mimicked the desensitization observed with chronic EtOH exposure. However, the degree of desensitization induced by forskolin was significantly less than that caused by EtOH even though the acute addition of forskolin caused a greater increase in cyclic AMP production. Furthermore, the acute addition of EtOH inhibited forskolin-stimulated cyclic AMP production, yet inclusion of EtOH during the chronic forskolin treatment of PC 12 cells resulted in a greater degree of desensitization. These findings indicate an obligatory role of protein kinase A in EtOH-induced desensitization of cyclic AMP production in PC 12 cells. However, because protein kinase A activation alone is not sufficient to account for the degree of desensitization, EtOH probably also acts through a mechanism in addition to activation of protein kinase A.     

Insulin receptor substrate-1 in osteoblast is indispensable for maintaining bone turnover

Insulin receptor substrates (IRS-1 and -2) are essential for intracellular signaling by insulin and IGF-I, anabolic regulators of bone metabolism. Mice lacking the IRS-1 gene IRS-1(-/-) showed severe osteopenia with low bone turnover. IRS-1 was expressed in osteoblasts, but not in osteoclasts, of wild-type (WT) mice. IRS-1(-/-) osteoblasts treated with insulin or IGF-I failed to induce tyrosine phosphorylation of cellular proteins, and they showed reduced proliferation and differentiation. Osteoclastogenesis in the coculture of hemopoietic cells and osteoblasts depended on IRS-1 expression in osteoblasts and could not be rescued by IRS-1 expression in hemopoietic cells in the presence of not only IGF-I but also 1,25(OH)(2)D(3). In addition, osteoclast differentiation factor (RANKL/ODF) was not induced by these factors in IRS-1(-/-) osteoblasts. We conclude that IRS-1 deficiency in osteoblasts impairs osteoblast proliferation, differentiation, and support of osteoclastogenesis, resulting in low-turnover osteopenia. Osteoblastic IRS-1 is essential for maintaining bone turnover, because it mediates signaling by IGF-I and insulin and, we propose, also by other factors, such as 1,25(OH)(2)D(3).     

Anti-IL-20 monoclonal antibody inhibits the differentiation of osteoclasts and protects against osteoporotic bone loss

IL-20 is a proinflammatory cytokine of the IL-10 family that is involved in psoriasis, rheumatoid arthritis, atherosclerosis, and stroke. However, little is known about the role of IL-20 in bone destruction. We explored the function of IL-20 in osteoclastogenesis and the therapeutic potential of anti-IL-20 monoclonal antibody 7E for treating osteoporosis. Higher serum IL-20 levels were detected in patients with osteopenia and osteoporosis and in ovariectomized (OVX) mice. IL-20 mediates osteoclastogenesis by up-regulating the receptor activator of NF-κB (RANK) expression in osteoclast precursor cells and RANK ligand (RANKL) in osteoblasts. 7E treatment completely inhibited osteoclast differentiation induced by macrophage colony-stimulating factor (M-CSF) and RANKL in vitro and protected mice from OVX-induced bone loss in vivo. Furthermore, IL-20R1-deficient mice had significantly higher bone mineral density (BMD) than did wild-type controls. IL-20R1 deficiency also abolished IL-20-induced osteoclastogenesis and increased BMD in OVX mice. We have identified a pivotal role of IL-20 in osteoclast differentiation, and we conclude that anti-IL-20 monoclonal antibody is a potential therapeutic for protecting against osteoporotic bone loss.     

小分子肽对成骨前体细胞MC3T3-E1骨保护素和核转录因子κB受体活化因子配体表达的影响

背景：体内实验显示,小分子肽能明显增加去卵巢大鼠的骨钙含量,使其骨密度增加,能很好地预防骨质疏松。同时体外实验显示,小分子肽能促进小鼠成骨细胞和成骨前体细胞MC3T3-E1增殖、分化、矿化,并且可能是通过抑制核转录因子p50和p65的表达来起作用。而小分子肽对骨保护素/核转录因子κB受体活化因子配体的影响尚不明确。目的：观察小分子肽对MC3T3-E1在增殖、分化、矿化过程中骨保护素和RANKL表达的影响。方法：以体积分数10%胎牛血清的DMEM培养液为空白对照组,50,100mg/L质量浓度小分子肽作用小鼠成骨前体细胞MC3T3-E1,分别于作用3,6,12,18,24,30d后,收集细胞提取蛋白,Western Blot检测骨保护素和核转录因子κB受体活化因子配体蛋白的表达。结果与结论：50,100mg/L小分子肽作用MC3T3-E1后能明显促进作用骨保护素的表达（P〈0.01）,而对核转录因子κB受体活化因子配体无明显影响。小分子肽作用后MC3T3-E1中骨保护素/核转录因子κB受体活化因子配体的比值要明显高于空白对照组（P〈0.01）。因此,认为小分子肽可以通过增加骨保护素的表达来影响骨保护素/核转录因子κB受体活化因子配体系统,间接地抑制破骨细胞的数量和功能。     

IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis

IL-17 is a newly discovered T cell-derived cytokine whose role in osteoclast development has not been fully elucidated. Treatment of cocultures of mouse hemopoietic cells and primary osteoblasts with recombinant human IL-17 induced the formation of multinucleated cells, which satisfied major criteria of osteoclasts, including tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation on dentine slices. Direct interaction between osteoclast progenitors and osteoblasts was required for IL-17-induced osteoclastogenesis, which was completely inhibited by adding indomethacin or NS398, a selective inhibitor of cyclooxgenase-2 (COX-2). Adding IL-17 increased prostaglandin E2 (PGE2) synthesis in cocultures of bone marrow cells and osteoblasts and in single cultures of osteoblasts, but not in single cultures of bone marrow cells. In addition, IL-17 dose-dependently induced expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts. ODF is a membrane-associated protein that transduces an essential signal(s) to osteoclast progenitors for differentiation into osteoclasts. Osteoclastogenesis inhibitory factor (OCIF), a decoy receptor of ODF, completely inhibited IL-17-induced osteoclast differentiation in the cocultures. Levels of IL-17 in synovial fluids were significantly higher in rheumatoid arthritis (RA) patients than osteoarthritis (OA) patients. Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues. These findings suggest that IL-17 first acts on osteoblasts, which stimulates both COX-2-dependent PGE2 synthesis and ODF gene expression, which in turn induce differentiation of osteoclast progenitors into mature osteoclasts, and that IL-17 is a crucial cytokine for osteoclastic bone resorption in RA patients.     

Protein Palmitoylation Regulates Osteoblast Differentiation through BMP-Induced Osterix Expression

Osteoporosis is one of the most common diseases and can be treated by either anti-resorption drugs, anabolic drugs, or both. To search for anabolic drug targets for osteoporosis therapy, it is crucial to understand the biology of bone forming cells, osteoblasts, in terms of their proliferation, differentiation, and function. Here we found that protein palmitoylation participates in signaling pathways that control osterix expression and osteoblast differentiation. Mouse calvarial osteoblasts express most of the 24 palmitoyl transferases, with some being up-regulated during differentiation. Inhibition of protein palmitoylation, with a substrate-analog inhibitor, diminished osteoblast differentiation and mineralization, but not proliferation or survival. The decrease in differentiation capacity is associated with a reduction in osterix, but not Runx2 or Atf4. Inhibition of palmitoyl transferases had little effect in p53^−/− osteoblasts that show accelerated differentiation due to overexpression of osterix, suggesting that osterix, at least partially, mediated the effect of inhibition of palmitoyl transferases on osteoblast differentiation. BMPs are the major driving force of osteoblast differentiation in the differentiation assays. We found that inhibition of palmitoyl transferases also compromised BMP2-induced osteoblast differentiation through down-regulating osterix induction. However, palmitoyl transferases inhibitor did not inhibit Smad1/5/8 activation. Instead, it compromised the activation of p38 MAPK, which are known positive regulators of osterix expression and differentiation. These results indicate that protein palmitoylation plays an important role in BMP-induced MAPK activation, osterix expression, and osteoblast differentiation.     

低氧条件下成骨细胞的增殖与分化

背景：低氧可通过多种途径作用于成骨细胞影响骨代谢,对骨生成、骨愈合等产生负面影响。目的：观察低氧对体外培养大鼠成骨细胞增殖、分化的影响,并探讨其分子机制。方法：分离培养新生Wistar大鼠颅盖骨成骨细胞,取第2代细胞分别在常氧（体积分数20%O2）与低氧（体积分数3%O2）条件下培养。结果与结论：低氧组成骨细胞增殖、碱性磷酸酶活性、骨钙素水平及茜素红结节形成数量均明显低于常氧组（P〈0.05或P〈0.01）,说明缺氧条件对成骨细胞的增殖、分化及功能有抑制作用;低氧组骨形成发生蛋白2及Runx2表达低于常氧组（P〈0.05或P〈0.01）,说明低氧条件下大鼠成骨细胞Runx2、骨形成发生蛋白2的表达受抑制。结果表明低氧可通过抑制大鼠成骨细胞的Runx2、骨形成发生蛋白2的表达进一步抑制成骨细胞的增殖与分化。     

Anabolic effects of a G protein-coupled receptor kinase inhibitor expressed in osteoblasts

G protein-coupled receptors (GPCRs) play a key role in regulating bone remodeling. Whether GPCRs exert anabolic or catabolic osseous effects may be determined by the rate of receptor desensitization in osteoblasts. Receptor desensitization is largely mediated by direct phosphorylation of GPCR proteins by a family of enzymes termed GPCR kinases (GRKs). We have selectively manipulated GRK activity in osteoblasts in vitro and in vivo by overexpressing a GRK inhibitor. We found that expression of a GRK inhibitor enhanced parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor-stimulated cAMP generation and inhibited agonist-induced phosphorylation of this receptor in cell culture systems, consistent with attenuation of receptor desensitization. To determine the effect of GRK inhibition on bone formation in vivo, we targeted the expression of a GRK inhibitor to mature osteoblasts using the mouse osteocalcin gene 2 (OG2) promoter. Transgenic mice demonstrated enhanced bone remodeling as well as enhanced urinary excretion of the osteoclastic activity marker dexoypyridinoline. Both osteoprotegrin and OPG ligand mRNA levels were altered in calvaria of transgenic mice in a pattern that would promote osteoclast activation. The predominant effect of the transgene, however, was anabolic, as evidenced by an increase in bone density and trabecular bone volume in the transgenic mice compared with nontransgenic littermate controls.