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Molecular Parmacological Research for TNF-α receptor family on the pathogenetic mechanism in Periodontitis-related disease

TNF-α受体家族在牙周炎相关疾病发病机制中的分子生物学研究

基本信息

批准号：
15591986
负责人：
MOGI Makio
金额：
$ 1.92万
依托单位：
Aichi Gakuin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591986/
关键词：
osteoblast BMP4 caspase RANKL OPG apoptosis アポトーシス細胞周期歯周病破骨細胞顎関節症 OPG Caspase Apoptosis 分化 BMP

项目摘要

Activation of caspases required for osteoblastic differentiation.Bone morphogenetic protein (BMP)s are important regulators of osteoblast differentiation. Because regulation of osteoblastic differentiation is poorly understood, we sought to determine if BMP-4-induced differentiation of osteoblastic cells depends on the activity of the key apoptotic proteases, i.e.,the caspases. BMP-4 induced the growth arrest and differentiation of osteoblastic cell line MC3T3-E1,as evidenced by the appearance of osteoblastic phenotypes such as alkaline phosphatase (ALP) activation and parathyroid hormone (PTH)-dependent production of cAMP. Surprisingly, BMP-4 induced transient and potent activation of caspase-8,caspase-2,and caspase-3,in this order. However, no apoptosis or necrosis in BMP-4-treated cells could be detected by FACS using Annexin-V/propodium iodine double staining. Peptide inhibition of caspase activity led to a dramatic reduction in ALP activation and PTH-induced production of cAMP in … More BMP-4-treated cells. Although BMP-4 treatment resulted in cell-cycle G0/G1 arrest as detected by FACS cell-cycle analysis, caspase inhibitors (caspase-8,caspase-2,and caspase-3 inhibitors) could block the G0/G1 arrest in MC3T3-E1 cells. Taken together, these results confirm a unique and unanticipated role for the caspase-mediated signal cascade in the differentiation of osteoblasts.Differential expression of RANKT, and osteoprotegerin in gingival crevicular fluid of patients with Periodontitis.The receptor activator for NF-kB ligand (RANKL) plays an important role in osteoclast formation. However, no one has examined the level of RANKL in the body fluid of human subjects. This communication reports on the in vivo concentrations of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in thegingival crevicular fluid (GCF) of periodontal subjects with severe, moderate, and mild forms of the disease. An increased concentration o RANKL and a decreased concentration of OPG were detected in GCF from patients with periodontitis (^*p<0.05 vs.control subjects). The ratio of the concentration of RANKL to that of OPG in the GCF was significantly higher for periodontal disease patients than for healthy subjects (^*p<0.01). Taken together, these data suggest that RANKL and OPG contribute to osteoclastic bone destruction in periodontal disease.Dual roles for NF-kB activation in osteoblastic cells by serum deprivation : Osteoblastic apoptosis and cell-cycle arrest.To clarify the mechanisms of osteoblastic cell death, we examined whether serum deprivation would cause activation of the apoptotic signal cascade and arrest of the cell cycle in mouse osteoblastic MC3T3-E1 cells. Serum withdrawal from osteoblastic cell cultures resulted in growth arrest and cell-cycle arrest at G0/G1, which actions were accompanied by transient and potent activation of NF-kB,caspase-8,caspase-2,caspase-3,and caspase-9 in this order. Apoptosis, but not necrosis, in serum-deprived cells could be detected by FACS using Annexin-V/propidium iodine double staining. Serum deprivation also resulted in transient activation of the 20S proteasome, which is an important component for regulation of the cell cycle by the ubiquitin-proteasome system. The 20S proteasome inhibitor (PSI) but not NF-kB inhibitor SN50 suppressed the activation of proteasomes in serum-deprived cells. Although caspase inhibitors could not prevent the G0/G1 arrest in the serum-deprived cells, SN50 and the 20S proteasome inhibitor could block it. Since SN50,20S proteasome inhibitor, and caspase inhibitor could rescue cells from serum deprivation-induced apoptosis, the pathway for NF-kB/caspase activation is independent of the NF-kB/cell-cycle pathway, and the events down-stream of the NF-kB/caspase-9 cascade lead to apoptosis. Taken together, our present results identify a novel role for NF-kB in cell-cycle and apoptosis regulation, and underscore the significance of each independent signal cascade in serum-deprived osteoblastic cells. Less

成骨细胞分化所需的半胱天冬酶的激活骨形态发生蛋白（BMP）是成骨细胞分化的重要调节因子。由于对成骨细胞分化的调控知之甚少，我们试图确定BMP-4诱导的成骨细胞分化是否依赖于关键凋亡蛋白酶的活性，即，半胱天冬酶BMP-4诱导成骨细胞系MC3T3-E1的生长停滞和分化，如成骨细胞表型如碱性磷酸酶（ALP）活化和甲状旁腺激素（PTH）依赖性cAMP产生的出现所证明的。令人惊讶的是，BMP-4以这种顺序诱导半胱天冬酶-8、半胱天冬酶-2和半胱天冬酶-3的瞬时和有效活化。然而，在BMP-4处理的细胞中没有凋亡或坏死可以通过使用Annexin-V/碘丙啶双重染色的FACS检测。半胱天冬酶活性的肽抑制导致ALP激活和PTH诱导的cAMP产生的显着减少， ...更多信息 BMP-4处理的细胞。虽然BMP-4处理导致细胞周期G0/G1阻滞，如通过FACS细胞周期分析所检测到的，caspase抑制剂（caspase-8、caspase-2和caspase-3抑制剂）可以阻断MC3T3-E1细胞中的G0/G1阻滞。总之，这些结果证实了半胱天冬酶介导的信号级联在成骨细胞分化中的独特和未预料到的作用。RANKT和骨保护素在牙周炎患者龈沟液中的差异表达。然而，还没有人检测人类受试者体液中的RANKL水平。本通讯报告了RANKL和RANKL诱饵受体骨保护素（OPG）在重度、中度和轻度牙周病受试者龈沟液（GCF）中的体内浓度。在牙周炎患者的GCF中检测到RANKL浓度升高和OPG浓度降低（与对照受试者相比，^* p <0.05）。牙周病患者龈沟液中RANKL与OPG浓度的比值显著高于健康受试者（^* p <0.01）。综上所述，这些数据表明RANKL和OPG有助于牙周病中的成骨细胞骨破坏。成骨细胞凋亡和细胞周期阻滞：为了阐明成骨细胞死亡的机制，我们研究了血清剥夺是否会导致小鼠成骨细胞MC3T3-E1细胞凋亡信号级联的激活和细胞周期的阻滞。从成骨细胞培养物中撤出血清导致生长停滞和细胞周期停滞在G0/G1，其作用伴随着NF-kB，caspase-8，caspase-2，caspase-3和caspase-9的瞬时和有效激活。流式细胞仪Annexin-V/碘化丙啶双染法检测血清剥夺细胞凋亡而非坏死。血清剥夺也导致20 S蛋白酶体的瞬时激活，这是泛素-蛋白酶体系统调节细胞周期的重要组成部分。20S蛋白酶体抑制剂（PSI），而不是NF-kB抑制剂SN50抑制蛋白酶体在血清剥夺细胞的激活。虽然caspase抑制剂不能阻止血清剥夺细胞的G0/G1期阻滞，但SN50和20S蛋白酶体抑制剂可以阻断该阻滞，由于SN50、20S蛋白酶体抑制剂和caspase抑制剂可以从血清剥夺诱导的细胞凋亡中拯救细胞，NF-kB/caspase激活途径独立于NF-kB/细胞周期途径，NF-κ B/caspase-9级联反应下游的事件导致细胞凋亡。总之，我们目前的研究结果确定了一个新的作用，NF-κ B在细胞周期和凋亡的调节，并强调了每个独立的信号级联在血清剥夺成骨细胞的意义。少