Impact of aging on intracellular ceramide-mediated periodontal bone lesions

摘要 老年免疫学的范式转变已经发生，发现TLR信号在老年人中减弱， 提示在年龄相关性牙周炎中可能存在另一种促炎机制。 构成脊椎动物细胞膜的神经酰胺作为一种信号传导介质引起了人们的兴趣， 多种细胞事件，包括细胞凋亡和细胞衰老。我们最近报道细胞内神经酰胺 通过作用于非肌肉肌球蛋白IIA（Myh 9）促进破骨细胞生成， 破骨细胞生成中细胞融合事件的因子。另一方面，最近的研究表明， 细胞内神经酰胺直接作用于组织蛋白酶B以激活其催化活性。已知组织蛋白酶B 通过Myh 9的时间控制蛋白水解促进RANKL介导的破骨细胞生成，以启动细胞 核聚变作为对衰老的响应，神经酰胺的量在细胞的细胞质中增加， 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。我们的初步 结果表明，高迁移率族蛋白B1（HMGB 1），一种关键的衰老相关的促炎因子， 介质，下调Raw 264.7细胞中的aCD酶表达。根据这些证据，我们 假设在与年龄相关的牙周炎的背景下，脂多糖诱导的信号传导似乎是 减少，HMGB 1通过作用于组织蛋白酶增加细胞内神经酰胺以促进OC介导的骨丢失 B，破骨细胞生成的启动子。在这个R15项目中，具体目标1将确定HMGB 1在以下方面的作用： 衰老OCP的细胞内神经酰胺的积累通过下调α CD酶活性。我们将 比较牙龈卟啉单胞菌或大肠杆菌相关株中HMGB 1和LPS的作用 aCD酶活性下调，导致神经酰胺在年轻OCP细胞质中积累， (two月龄）和老龄（24月龄）野生型小鼠，体外破骨细胞生成测定和 牙周炎体内小鼠模型。在具体目标2中，我们将研究细胞内神经酰胺的影响， 破骨细胞生成的组织蛋白酶B依赖性上调。我们将评估细胞内 神经酰胺类药物对破骨细胞形成过程中组织蛋白酶B催化活性的影响 通过Myh 9蛋白水解在体外。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破骨细胞生成增加中的致病作用， 为老年人牙周炎的治疗提供了新的治疗方法。最 重要的是，拟议的R15-AREA项目将为本科生和研究生提供一个独特的机会， 学生参加在最大的学术西班牙裔服务机构之一的生物医学研究 诺瓦东南大学

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.