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控因子。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取决于关键凋亡蛋白酶（即半胱天蛋白酶）的活性。BMP-4诱导成骨细胞系MC3T3-E1的生长停滞和分化，如碱性磷酸酶（ALP）激活和甲状旁腺激素（PTH）依赖性cAMP的产生等成骨表型的出现。令人惊讶的是，BMP-4依次诱导caspase-8、caspase-2和caspase-3的瞬时有效激活。然而，采用Annexin-V/propodium碘双染色，FACS未检测到bmp -4处理的细胞凋亡或坏死。在bmp -4处理的细胞中，多肽抑制caspase活性导致ALP激活和pth诱导的cAMP产生显著降低。尽管FACS细胞周期分析检测到BMP-4处理导致细胞周期G0/G1阻滞，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阻滞。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意想不到的作用。牙周炎患者龈沟液中RANKT和骨保护素的差异表达。NF-kB配体受体激活因子（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。本文报道了重度、中度和轻度牙周病患者牙龈沟液（GCF）中RANKL和RANKL诱饵受体骨保护素（OPG）的体内浓度。牙周炎患者GCF中检测到RANKL浓度升高，OPG浓度降低（^*p<0.05）。牙周病患者GCF中RANKL浓度与OPG浓度之比显著高于健康人（^*p<0.01）。综上所述，这些数据表明RANKL和OPG有助于牙周病的破骨细胞骨破坏。血清剥夺对成骨细胞NF-kB激活的双重作用：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。从成骨细胞培养中提取血清导致生长停滞和细胞周期停滞在G0/G1，这些作用伴随着NF-kB、caspase-8、caspase-2、caspase-3和caspase-9的短暂和有效激活。Annexin-V/丙碘双染色法可检测到血清剥夺细胞的凋亡，但未检测到坏死。血清剥夺还导致20S蛋白酶体的短暂激活，20S蛋白酶体是泛素-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）而非NF-kB抑制剂SN50抑制血清剥夺细胞中蛋白酶体的活化。虽然caspase抑制剂不能阻止血清剥夺细胞的G0/G1阻滞，但SN50和20S蛋白酶体抑制剂可以阻断这种阻滞。由于SN50、20S蛋白酶体抑制剂和caspase抑制剂可以拯救血清剥夺诱导的细胞凋亡，NF-kB/caspase激活途径独立于NF-kB/细胞周期途径，NF-kB/caspase-9级联下游的事件导致细胞凋亡。综上所述，我们目前的研究结果确定了NF-kB在细胞周期和凋亡调节中的新作用，并强调了血清缺失成骨细胞中每个独立信号级联的重要性。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