Lipopolysaccharide-induced osteoclastogenesis in Src homology 2-domain phosphatase-1-deficient viable motheaten mice

Osteoclasts are hemopoietic cells that participate in bone resorption and remodeling. Receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are critical for development of osteoclasts. The Toll-like receptor (TLR) family shares some of the downstream signaling with RANK. The TLR4 ligand, lipopolysaccharide (LPS), is reported to accelerate bone lysis; however, signaling via TLRs has never been reported to induce osteoclastogenesis without RANKL. In this study we showed that significant numbers of mature osteoclasts were generated from protein tyrosine phosphatase Src homology 2-domain phosphatase-1-defective Hcph(me-v)/Hcph(me-v) (me(v)/me(v)) bone marrow cells in the presence of M-CSF and LPS without addition of RANKL in culture. This M-CSF plus LPS-induced osteoclastogenesis was not inhibited by an anti-TNFalpha antagonistic antibody or by osteoprotegerin, a decoy receptor for RANKL. The replacement of RANKL by TLR ligands only occurred with LPS. Other ligands, a peptidoglycan for TLR2 or an unmethylated CpG oligonucleotide for TLR9, did not support osteoclast generation. The osteoclast precursors as well as RANKL-responsive osteoclast precursors were present in the Kit-positive cell-enriched fraction of bone marrow cells. Although me(v)/me(v) bone marrow cells required a comparable concentration of RANKL or TNFalpha as wild-type cells for the initiation of osteoclastogenesis, the numbers of multinucleated osteoclasts in me(v)/me(v) bone marrow cultures were significantly increased by the equivalent dose of RANKL or TNFalpha in the presence of M-CSF. These results indicate that a defect of Src homology 2-domain phosphatase-1 function not only accelerates physiological osteoclast development by RANKL/RANK, but also acquires a novel pathway for osteoclastogenesis by LPS.     

重组人结缔组织生长因子对人成骨细胞骨保护素/RANKL表达的影响

目的 研究重组人结缔组织生长因子(CTGF)对体外培养的人成骨细胞骨保护素/RANKL(receptor activator of NF-κB ligand)表达的影响,并探讨重组人CTGF调节骨保护素/RANKL表达的信号转导机制.方法 用重组人CTGF干预体外培养的人成骨细胞,采用Western印迹法检测骨保护素/RANKL蛋白表达水平的变化,同时观察重组人CTGF对人成骨细胞FAK、MAPK磷酸化的影响.结果 重组人CTGF可呈时间-剂量依赖性抑制人成骨细胞RANKL的表达,200 ng/ml重组人CTGF作用24～48 h达最大抑制效果,而对人成骨细胞骨保护素的表达无明显影响.重组人CTGF可明显增强p38MAPK磷酸化,并减少FAK磷酸化,重组人CTGF干预对ERK、JNK磷酸化无明显影响;p38MAPK阻断剂SB23058可阻断重组人CTGF对RANKL表达的抑制效应.结论 重组人CTGF通过增强p38MAPK磷酸化,减少FAK磷酸化下调人成骨细胞RANKL的表达.     

脂联素调控人成骨细胞OPG/RANKL表达的信号转导机制

目的 研究脂联素调控人成骨细胞护骨素(OPG)和NF-кB受体活化凶子配体(RANKL)表达的作用机制.方法 人成骨细胞OPG和RANKL mRNA的表达以实时PCR检测,p38丝裂原活化蛋白激酶(p38 MAPK)、细胞外信号调节激酶(ERK1/2)、c-Jun氨基端激酶(JNK)的磷酸化水平用Western印迹法检测.廊用小分子RNA干扰技术(siRNA)阻断脂联素受体(AdR)表达,并以p38 MAPK抑制剂(SB203580)和JNK抑制剂(SP600125)干预,以观察脂联素对人成骨细胞OPG/RANKL作用的调节机制.结果 用siRNA沉默AdRl的表达可消除脂联素促进人成骨细胞RANKL表达和抑制OPG表达的作用;脂联素干预前予SB203580阻断p38 MAPK后,也可消除脂联素对成骨细胞RANKL和OPG的作用,而SP600125并无作用.结论 在人成骨细胞中,脂联素通过AdR1/p38 MARK途径促进RANKL表达和抑制OPG的表达.     

Osteoprotegerin produced by osteoblasts is an important regulator in osteoclast development and function

Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoclast differentiation factor, inhibits both differentiation and function of osteoclasts. We previously reported that OPG-deficient mice exhibited severe osteoporosis caused by enhanced osteoclastic bone resorption. In the present study, potential roles of OPG in osteoclast differentiation were examined using a mouse coculture system of calvarial osteoblasts and bone marrow cells prepared from OPG-deficient mice. In the absence of bone-resorbing factors, no osteoclasts were formed in cocultures of wild-type (+/+) or heterozygous (+/-) mouse-derived osteoblasts with bone marrow cells prepared from homozygous (-/-) mice. In contrast, homozygous (-/-) mouse-derived osteoblasts strongly supported osteoclast formation in the cocultures with homozygous (-/-) bone marrow cells, even in the absence of bone-resorbing factors. Addition of OPG to the cocultures with osteoblasts and bone marrow cells derived from homozygous (-/-) mice completely inhibited spontaneously occurring osteoclast formation. Adding 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] to these cocultures significantly enhanced osteoclast differentiation. In addition, bone-resorbing activity in organ cultures of fetal long bones derived from homozygous (-/-) mice was markedly increased, irrespective of the presence and absence of bone-resorbing factors, in comparison with that from wild-type (+/+) mice. Osteoblasts prepared from homozygous (-/-), heterozygous (+/-), and wild-type (+/+) mice constitutively expressed similar levels of RANKL messenger RNA, which were equally increased by the treatment with 1alpha,25(OH)2D3. When homozygous (-/-) mouse-derived osteoblasts and hemopoietic cells were cocultured, but direct contact between them was prevented, no osteoclasts were formed, even in the presence of 1alpha,25(OH)2D3 and macrophage colony-stimulating factor. These findings suggest that OPG produced by osteoblasts/stromal cells is a physiologically important regulator in osteoclast differentiation and function and that RANKL expressed by osteoblasts functions as a membrane-associated form.     

Bone morphogenetic protein 2 stimulates osteoclast differentiation and survival supported by receptor activator of nuclear factor-kappaB ligand.

Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.     

Preptin对成骨细胞结缔组织生长因子表达的影响及其机制研究

目的 探讨Preptin对成骨细胞结缔组织生长因子(CTGF)表达的影响及其机制.方法采用人重组preptin干预人原代成骨细胞,CTGF蛋白水平用Western印迹法检测.丝裂原活化蛋白激酶p38(p38MAPK)、细胞外信号调节激酶(ERK1/2)、c-Jun氨基端激酶(JNK)及其磷酸化水平用Western印迹法检测.在preptin干预前用细胞信号阻断剂(PD98059、SP600125或SB203580)预处理阻断人成骨细胞MAPK信号转导,以分析preptin诱导人成骨细胞CTGF表达的作用机制.结果 Preptin可呈时间和剂量依赖性地促进人成骨细胞CTGF的分泌,并且preptin可诱导人成骨细胞ERK的活化,对p38MAPK或JNK无激活作用;人成骨细胞用ERK抑制剂PD98059预处理可使preptin诱导的CTGF分泌降低.结论Preptin增加CTGF的表达,并通过ERK/MAPK信号途径来介导.     

Preptin对人成骨细胞增殖和分化的影响及机制研究

目的 探讨preptin对人成骨细胞增殖和分化的影响及其信号途径.方法 体外培养人成骨细胞,用10-10、10-9、10-8和10-7mol/L preptin干预24 h,以[3H]脱氧胸腺嘧啶苷掺入法分析细胞增殖,用分光光度计法测定细胞碱性磷酸酶(ALP)活性判断细胞分化程度.Western印迹法检测细胞外信号调节激酶(ERK)、p38丝裂原活化蛋白激酶(p38MAPK)和c-Jun氨基末端激酶(JNK)的磷酸化水平.并在preptin干预前以ERK抑制剂(PD98059)、p38 MAPK抑制剂(SB203580)和JNK抑制剂(SP600125)预处理,观察preptin诱导人成骨细胞增殖和分化的途径.结果 Preptin剂量依赖地增加人成骨细胞的增殖和ALP活性,10-9mol/L浓度时达最大效应(均P<0.01).Preptin刺激人成骨细胞ERK的磷酸化,对p38MAPK和JNK无作用.PD98059阻断preptin刺激的成骨细胞增殖及ALP活性增加(均P<0.05),而SP600125和SB203580无此效应.结论 Preptin通过ERK途径促进人成骨细胞的增殖和分化.     

p38 mitogen-activated protein (MAP) kinase but not p44/p42 MAP kinase is involved in prostaglandin E1-induced vascular endothelial growth factor synthesis in osteoblasts

We investigated the mechanism underlying vascular endothelial growth factor (VEGF) synthesis stimulated by prostaglandin E1 (PGE1) in osteoblast-like MC3T3-E1 cells. PGE1 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. SB203580, a specific inhibitor of p38 MAP kinase, inhibited the PGE1-stimulated VEGF synthesis as well as PGE1-induced phosphorylation of p38 MAP kinase. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, which reduced the PGE1-induced phosphorylation of p44/p42 MAP kinase, had little effect on the VEGF synthesis stimulated by PGE1. AH-6809, an antagonist of the subtypes of the PGE receptor, EP1 and EP2, or SC-19220, an antagonist of EP1 receptor, did not inhibit the PGE1-induced VEGF synthesis. H-89, an inhibitor of cAMP-dependent protein kinase, and SQ22536, an inhibitor of adenylate cyclase, reduced the VEGF synthesis induced by PGE1. Cholera toxin, an activator of G(s), and forskolin, an activator of adenylate cyclase, induced VEGF synthesis. SB203580 and PD169316, another specific inhibitor of p38 MAP kinase, reduced the cholera toxin-, forskolin- or 8bromo-cAMP-stimulated VEGF synthesis. However, PD98059 failed to affect the VEGF synthesis stimulated by cholera toxin, forskolin or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP). SB203580 reduced the phosphorylation of p38 MAP kinase induced by forskolin or 8bromo-cAMP. These results strongly suggest that p44/p42 MAP kinase activation is not involved in the PGE1-stimulated VEGF synthesis in osteoblasts but that p38 MAP kinase activation is involved.     

Cytochrome P4502E1 sensitizes to tumor necrosis factor alpha-induced liver injury through activation of mitogen-activated protein kinases in mice

The goal of this study was to evaluate the role of mitogen-activated protein kinase (MAPK) in cytochrome P4502E1 (CYP2E1) potentiation of lipopolysaccharide or tumor necrosis factor alpha (TNF-alpha)-induced liver injury. Treatment of C57/BL/6 mice with pyrazole (PY) plus lipopolysaccharide (LPS) induced liver injury compared with mice treated with PY or LPS alone. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 or p38 MAPK inhibitor SB203580 prevented this liver injury. PY plus LPS treatment activated p38 MAPK and JNK but not extracellular signal-regulated kinase (ERK). PY plus LPS treatment triggered oxidative stress in the liver with increases in lipid peroxidation, decrease of glutathione (GSH) levels, and increased production of 3-nitrotyrosine adducts and protein carbonyl formation. This oxidative stress was blocked by SP600125 or SB203580. PY plus LPS treatment elevated TNF-alpha production, and this was blocked by SP600125 or SB203580. Neither SP600125 nor SB203580 affected CYP2E1 activity or protein levels. Treating C57/BL/6 mice with PY plus TNF-alpha also induced liver injury and increased lipid peroxidation and decreased GSH levels. Prolonged activation of JNK and p38 MAPK was observed. All of these effects were blocked by SP600125 or SB203580. In contrast to wild-type SV 129 mice, treating CYP2E1 knockout mice with PY plus TNF-alpha did not induce liver injury, thus validating the role of CYP21E1 in this potentiated liver injury. Liver mitochondria from PY plus LPS or PY plus TNF-alpha treated mice underwent calcium-dependent, cyclosporine A-sensitive swelling, which was prevented by SB203580 or SP600125. CONCLUSION: These results show that CYP2E1 sensitizes liver hepatocytes to LPS or TNF-alpha and that the CYP2E1-enhanced LPS or TNF-alpha injury, oxidant stress, and mitochondrial injury is JNK or p38 MAPK dependent.     

A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation

Signaling by receptor activator of NF-kappaB (nuclear factor-kappaB) ligand (RANKL) is essential for differentiation of bone marrow monocyte-macrophage lineage (BMM) cells into osteoclasts. Here, we show RANKL stimulation of BMM cells transiently increased the intracellular level of reactive oxygen species (ROS) through a signaling cascade involving TNF (tumor necrosis factor) receptor-associated factor (TRAF) 6, Rac1, and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase (Nox) 1. A deficiency in TRAF6 or expression of a dominant-interfering mutant of TRAF6 blocks RANKL-mediated ROS production. Application of N-acetylcysteine (NAC) or blocking the activity of Nox, a protein leading to the formation of ROS, with diphenylene iodonium (DPI) inhibits the responses of BMM cells to RANKL, including ROS production, activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and extracellular signal-regulated kinase (ERK), and osteoclast differentiation. Moreover, both RANKL-mediated ROS production and osteoclast differentiation were completely blocked in precursors depleted of Nox1 activity by RNA interference or by expressing a dominant-negative mutant of Rac1. Together, these results indicate that ROSs act as an intracellular signal mediator for osteoclast differentiation.     

Suppression of p38 mitogen-activated protein kinase inhibits hepatitis B virus replication in human hepatoma cell: the antiviral role of nitric oxide

Summary.  The role of the p38 mitogen-activated protein kinase (MAPK) pathway in hepatitis B virus (HBV) replication was investigated in this study. After transient transfection with HBV plasmid, p38 MAPK, but not JNK or ERK1/2, was significantly phosphorylated in human hepatoma cell Huh7. Interestingly, HBV proteins and RNA synthesis were significantly inhibited by a specific inhibitor of p38 MAPK, SB203580, in a dose-dependent manner. Intracellular core-associated DNA, extracellular virion-associated DNA and covalently closed circular DNA were also significantly inhibited by SB203580. Further results showed the antiviral role of nitric oxide (NO) on the suppression of HBV replication and downregulation of p38 MAPK phosphorylation. In conclusion, these results suggested that suppression of phosphorylation of p38 MAPK by inhibitor or NO could inhibit intracellular HBV replication.     

Cytokine-activated endothelium recruits osteoclast precursors

Osteoclast precursors reach sites of osteoclast formation and remodelling via the vasculature and are therefore destined to encounter endothelium before migrating to the bone surface. Here we investigated the hypothesis that endothelium may be involved in the regulation of osteoclast precursor recruitment to sites of bone resorption. Osteoclast precursors in human peripheral blood were identified by their ability to form mature osteoclasts in 21-day cultures supplemented with RANKLigand, M-CSF, 1,25(OH)(2)-vitamin D(3), dexamethasone and prostaglandin E(2). Under control conditions few osteoclast precursors adhered to endothelial cells (the human bone marrow-derived endothelial cell line BMEC-1). However, BMEC-1 cells treated with the resorption stimulating cytokines IL-1beta and TNFalpha depleted the PBMC population of all osteoclast precursors. These results provide the first evidence that osteoclast precursors can adhere to endothelium and suggest that endothelium could play an important role in the recruitment of osteoclast precursors to sites of bone resorption.     

辛伐他汀抑制人外周血单核巨噬细胞脂蛋白相关磷脂酶A2表达

目的 观察辛伐他汀对人外周血单核巨噬细胞脂蛋白相关磷脂酶A2(Lp-PLA2)表达的影响,并探讨其调控机制.方法 分离培养人外周血单核巨噬细胞,实验分为脂多糖(LPS)组、辛伐他汀组和丝裂原活化蛋白激酶(MAPK)干预组.LPS组:分别用不同浓度(0、1、10、102、103和104ng/ml)的LPS与细胞共同孵育6 h,观察不同浓度的辛伐他汀对LPS诱导的Lp-PLA2 mRNA和蛋白表达的影响;并用1μg/ml的LPS与细胞孵育不同时间(0、6、12、24和48 h),观察辛伐他汀作用不同时间对LPS诱导的Lp-PLA2 mRNA和蛋白表达的影响.辛伐他汀组:1 μg/ml的LPS+不同浓度的辛伐他汀(10-2～10-7mmol/L)与单核巨噬细胞共同孵育24 h,1 μg/ml LPS+10-3mmol/L的辛伐他汀与单核巨噬细胞孵育不同时间(0、6、24、24和48 h),观察辛伐他汀对LPS诱导的Lp-PLA2mRNA和蛋白表达及酶活性的影响.MAPK组:分别用10 μmol/L的p38抑制剂SB203580、20 μmol/L的ERK抑制剂U0126和20 μmol/L的JNK抑制剂SP600125预处理30 min后,将单核巨噬细胞与1μg/ml的LPS共同孵育24 h,观察MAPK信号通路在LPS介导的Lp-PLA2表达中的作用.逆转录-多聚酶链反应(RT-PCR)方法 检测Lp-PLA2 mRNA表达,比色法测定酶活性,Western blot方法 检测Lp-PLA2蛋白表达以及p38-MAPK蛋白及磷酸化水平.结果 (1)0.1μg/ml的LPS刺激6 h即可显著增加单核巨噬细胞Lp-PLA2 mRNA和蛋白的表达及其酶活性,并且随LPS浓度的增加和刺激时间的延长,该作用增强.(2)辛伐他汀可以明显抑制LPS诱导的Lp-PLA2的表达增加,并且降低酶活性,该作用呈浓度及时间依赖性.(3)辛伐他汀抑制LPS诱导的p38MAPK蛋白活化,p38MAPK的抑制剂SB203580可以完全阻断LPS介导的Lp-PLA2蛋白表达增加,与辛伐他汀作用相似.而MEK1/2的抑制剂U0126和JNK的抑制剂SP600125对LPS介导的Lp-PLA2蛋白表达的增加没有影响.结论 在培养的人外周血单核巨噬细胞中,辛伐他汀可以明显抑制LPS诱导的Lp-PLA2 mRNA和蛋白表达,降低Lp-PLA2酶活性,该作用至少部分由抑制p38MAPK信号转导通路介导.