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

成骨细胞分化所需的半胱氨酸天冬氨酸酶的激活。骨形态发生蛋白S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的凋亡蛋白酶，即半胱氨酸氨基转移酶的活性。BMP-4诱导成骨细胞系MC3T3-E1的生长停滞和分化，表现为成骨细胞的碱性磷酸酶(ALP)激活和甲状旁腺激素(PTH)依赖的cAMP产生。令人惊讶的是，BMP-4按如下顺序诱导caspase-8、caspase-2和caspase-3的瞬时和有效激活。Annexin-V/Propodium碘双重染色流式细胞仪未检测到BMP-4处理的细胞发生凋亡或坏死。多肽抑制半胱氨酸天冬氨酸氨基转移酶活性显著降低碱性磷酸酶激活和甲状旁腺素诱导的…产生cAMP更多经BMP-4处理的细胞。虽然FACS细胞周期分析显示BMP-4处理导致细胞周期G0/G1期停滞，但caspase抑制剂(caspase-8、caspase-2和caspase-3抑制剂)可以阻断MC3T3-E1细胞的G0/G1期停滞。综上所述，这些结果证实了caspase介导的信号级联在成骨细胞分化中的独特和未被预期的作用。RANKT和骨保护素在牙周炎患者龈沟液中的不同表达。核因子-kB受体激活剂(RANKL)在破骨细胞形成中起重要作用。然而，还没有人检测过人体体液中RANKL的水平。本通讯报告了重度、中度和轻度牙周病患者的牙周沟液中RANKL和RANKL诱骗受体护骨素(OPG)的体内浓度。牙周炎患者牙周液中RANKL浓度升高，OPG浓度降低(与对照组比较，P<0.05)。牙周病患者牙周液中RANKL/OPG浓度的比值显著高于健康对照组(P<0.01)。综上所述，这些数据提示RANKL和OPG在牙周病中参与破骨细胞性骨破坏。血清剥夺对成骨细胞中核因子-kB的激活具有双重作用：成骨细胞凋亡和细胞周期停滞。为了阐明成骨细胞死亡的机制，我们检测了血清剥夺是否会导致小鼠成骨细胞MC3T3-E1细胞凋亡信号级联激活和细胞周期停滞。从成骨细胞培养中提取血清导致细胞生长停滞和细胞周期停滞于G0/G1期，这一作用伴随着按顺序短暂而有效的激活NF-kB、caspase-8、caspase-2、caspase-3和caspase-9。Annexin-V/丙二醛双重染色流式细胞仪可检测到无血清培养细胞的凋亡，但不能检测到细胞坏死。血清剥夺还导致20S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)可抑制无血清细胞中蛋白酶体的激活，但不能抑制NF-kB抑制剂SN50的激活。虽然Caspase抑制剂不能阻止无血清细胞的G0/G1期停滞，但SN50和20S蛋白酶体抑制剂可以阻断它。由于SN50、20S蛋白酶体抑制剂和caspase抑制剂可以将细胞从血清剥夺诱导的细胞凋亡中拯救出来，所以NF-kB/caspase激活的途径不依赖于NF-kB/细胞周期通路，而NF-kB/caspase-9级联反应的下游事件导致了细胞凋亡。综上所述，我们的研究结果确定了核因子-kB在细胞周期和凋亡调控中的新作用，并强调了在去血清的成骨细胞中每个独立的信号级联的重要性。较少

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