Impact of aging on intracellular ceramide-mediated periodontal bone lesions

摘要 通过发现TLR信号在老年人中减弱，免疫老年学已经发生了范式转变， 提示另一种促炎机制可能参与了年龄相关性牙周炎。 构成脊椎动物细胞膜的神经酰胺作为一种信号媒介引起了人们的兴趣。 各种细胞事件，包括细胞凋亡和细胞衰老。我们最近报道了细胞内神经酰胺 通过作用于非肌肉肌球蛋白IIA(Myh9)促进破骨细胞的形成 破骨细胞形成中细胞融合事件的因子。另一方面，最近有证据表明 细胞内神经酰胺直接作用于组织蛋白酶B，激活其催化活性。已知组织蛋白酶B 通过时间受控的Myh9蛋白降解促进RANKL介导的破骨细胞形成 核聚变。作为对衰老的反应，细胞质中神经酰胺的数量随着 酸性神经酰胺酶(ACDase)水平降低，这是一种降解神经酰胺的脂质水解酶。我们的预赛 结果表明，高迁移率族蛋白B1(HMGB1)是一种与衰老相关的关键促炎因子 中介，下调Raw264.7细胞中aCDase的表达。基于这些证据，我们 假设在年龄相关性牙周炎的情况下，内毒素诱导的信号似乎是 减少，HMGB1通过作用于组织蛋白酶增加细胞内神经酰胺促进OC介导的骨丢失 B，破骨细胞生成的促进剂。在这个R15项目中，具体目标1将确定HMGB1在 通过下调aCDase活性使衰老OCPs细胞内神经酰胺积累。我们会 牙龈卟啉单胞菌和大肠埃希氏菌HMGB1和内毒素的作用比较 对青年OCPs中aCDase活性的下调和神经酰胺在胞浆中的积聚的影响 (两个月大)和老年(二十四个月大)野生型小鼠，体外破骨细胞生成试验和 活体小鼠牙周炎模型。在特定的目标2中，我们将研究细胞内神经酰胺的影响 组织蛋白酶B对破骨细胞生成的上调作用。我们将评估细胞内的影响 神经酰胺上调组织蛋白酶B在破骨细胞形成中诱导细胞融合事件的催化活性 经Myh9体外蛋白水解法。拟议的研究项目将首次调查可能的 细胞内神经酰胺在年龄依赖性升高的破骨细胞生成中的致病作用，并将提供 为老年人群牙周炎治疗新方法的开发奠定基础。最多的 重要的是，拟议的R15区域项目将为本科生和研究生提供一个独特的机会 学生参加最大的拉美裔学术机构之一的生物医学研究 诺瓦东南大学的。

ABSTRACT A paradigm-shift in immunogerontology has been occurring by finding that TLR signal is attenuated in elders, suggesting that an alternative pro-inflammatory mechanism may be engaged in the age-associated periodontitis. Ceramides that compose the cell membrane of vertebrates are attracting interests as signaling mediators in a variety of cell events, including, apoptosis and cell senescence. We recently reported that intracellular ceramides promote osteoclastogenesis by acting on a non-muscle myosin IIA (Myh9), an intracellular down-regulatory factor for cell fusion event in osteoclastogenesis. On the othernhad, it was recently demonstrated that intracellular ceramides directly act on cathepsin B to activate its catalytic activity. It is known that cathepsin B promotes RANKL-mediated osteoclastogenesis via temporally controlled proteolysis of Myh9 for initiation of cell fusion. In response to aging, the amount of ceramides increases in the cytoplasm of cells in conjunction with the diminished level of acid ceramidases (aCDase), a lipid hydrolase that degrades ceramides. Our preliminary results indicate that high mobility group protein B1 (HMGB1), a key senescence-associated proinflammatory mediator, downregulated aCDase expression in Raw264.7 cells. Based on these lines of evidence, we hypothesize that in the context of age associated periodontitis, where the LPS-induced signaling appears to be diminished, HMGB1 increases intracellular ceramides to advance OC-mediated bone loss by acting on cathepsin B, a promoter of osteoclastogenesis. In this R15 project, the Specific Aim 1 will establish the role of HMGB1 in accumulation of intracellular ceramide of senescence OCPs via downregulation of aCDase activity. We will compare the role of HMGB1 and LPS isolated from either Porphyromonas gingivalis or Escherichia coli relative to the downregulation of aCDase activity and resulting accumulation of ceramides in cytoplasm of OCPs of young (two month-old) and aged (twenty four month-old) wild type mice, both in vitro osteoclastogenesis assays and in in vivo mouse model of periodontitis. In Specific Aim 2, we will investigate the impact of intracellular ceramides on cathepsin B-dependent upregulation of osteoclastogenesis. We will evaluate the effects of intracellular ceramides on upregulation of cathepsin B’s catalytic activity that induce cells fusion event in osteoclastogenesis via Myh9 proteolysis in vitro. The proposed research project will, for the first time, investigate the possible pathogenic role of intracellular ceramide in age-dependently elevated osteoclastogenesis and will provide a foundation for the development of novel therapeutic approach for periodontitis in aging population. The most importantly, the proposed R15-AREA project will provide a unique opportunity for undergraduate and graduate students to participate in the biomedical researches at one of the largest academic Hispanic-serving institutions of Nova Southeastern University.