Ceramide-mediated pathology in periodontitis

摘要 宿主细胞胞质中的神经酰胺作为多种细胞事件中的第二信使引起了人们的兴趣， 尤其是促炎反应。然而，大多数细菌的膜结构，不同于 在脊椎动物中，不含神经酰胺，这表明细菌很少产生神经酰胺。尽管如此， 发现牙龈卟啉单胞菌产生磷酸甘油二氢神经酰胺（PGDHC），而不是LPS 在人牙周炎病变中大量表达。与衍生自的膜不可渗透的神经酰胺相比， 在脊椎动物中，我们报道了PGDHC穿透宿主破骨细胞前体（OCP）的细胞膜， 通过作用于非肌肉肌球蛋白IIA（Myh 9）促进破骨细胞生成所需的细胞融合。MyH 9是一个 破骨细胞发生中细胞融合事件细胞内下调因子相比之下， 证明宿主神经酰胺直接作用于组织蛋白酶B以激活其催化活性。此外，委员会认为， 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， OCP之间细胞融合的起始。我们的初步数据表明，PGDHC介导的上调 组织蛋白酶B的化学抑制剂减弱了破骨细胞生成的作用，表明组织蛋白酶B也 参与PGDHC介导的破骨细胞生成。重要的是，神经酰胺的细胞内水平， 来源于宿主或细菌，在生理环境中由神经酰胺酶调节。功能失调 酸性神经酰胺酶的活性与法伯病和其他医学并发症的发病机制有关。我们 发现并发表了牙周炎患者中酸性神经酰胺酶的表达减少。 尽管如此，我们对酸性神经酰胺酶调节PGDHC水平的能力以及 PGDHC介导的破骨细胞生成的分子机制还不清楚。因此我们 假设牙周病变中aCD酶活性的降低保留了组织的致病特性- 穿透PGDHC，进而通过骨中的新型组织蛋白酶B/Myh 9轴提高破骨细胞生成 牙周炎的吸收性损害。Aim 1旨在测试酸性神经酰胺酶的保护作用， 小鼠牙周炎模型中PGDHC介导的破骨细胞生成我们将评估 内源性酸性神经酰胺酶以及外源性给予的重组酸性神经酰胺酶对中和 PGDHC的致病活性。在目标2中，使用功能损失和功能增益方法，我们将评估 组织蛋白酶B通过Myh 9蛋白水解参与PGDHC介导的OCP融合。拟议研究 该项目将首次阐明致病性升高的分子机制。 牙周骨丢失靶向独特的牙龈卟啉单胞菌神经酰胺，促进破骨细胞生成， 调节细胞融合调节因子Myh 9，并研究宿主来源的酸性神经酰胺酶的作用 其可以潜在地抵消PGDHC的这种致病作用。

ABSTRACT Ceramides in the cytoplasm of host cells are attracting interests as second messengers in a variety of cell events, especially pro-inflammatory response. However, membrane structures of most bacteria, as distinct from that of vertebrate, do not contain ceramide, indicating that production of ceramides by bacteria is rare. Nonetheless, phosphoglycerol dihydroceramide (PGDHC), but not LPS, produced by Porphyromonas gingivalis is found abundantly in the human periodontitis lesion. Contrast to the membrane impermeable ceramides derived from vertebrates, we reported that PGDHC penetrates the cellular membrane of host osteoclast precursor (OCP) and promotes cell fusion required for osteoclastogenesis by acting on a non-muscle myosin IIA (Myh9). Myh9 is an intracellular down-regulatory factor for cell fusion event in osteoclastogenesis. In contrast, it was also demonstrated that host ceramides directly act on cathepsin B to activate its catalytic activity. Furthermore, cathepsin B promotes RANKL-mediated osteoclastogenesis via temporally controlled proteolysis of Myh9 for initiation of cell fusion between OCPs. Our preliminary data demonstrated that PGDHC-mediated upregulation of osteoclastogenesis is attenuated by the chemical inhibitor for cathepsin B, indicating that cathepsin B is also engaged in the PGDHC-mediated osteoclastogenesis. Importantly, the intracellular levels of ceramides, either derived from host or bacteria, are regulated by ceramidases in the physiological context. Dysregulated function of acid ceramidase is implicated in the pathogenesis of Farber Disease and other medical complications. We discovered and published that expression of acid ceramidase in patients with periodontitis is diminished. Nonetheless, our knowledges about the ability of acid ceramidase to regulate the level of PGDHC as well as molecular mechanisms underlying PGDHC-mediated osteoclastogenesis are not yet understood well. Thus, we hypothesized that diminished aCDase activity in periodontal lesions retains the pathogenic property of tissue- penetrating PGDHC which, in turn, elevates the osteoclastogenesis via novel Cathepsin B/Myh9 axis in the bone resorption lesion of periodontitis. The Aim 1 is designed to test the protective role of acid ceramidase against PGDHC-mediated osteoclastognesis in a mouse model of periodontitis. We will evaluate the effect of endogenous acid ceramidase as well as exogenously administered recombinant acid ceramidase on neutralizing the PGDHC’s pathogenic activity. In Aim 2, using loss- and gain-of-function approaches, we will evaluate the engagement of cathepsin B in the PGDHC-mediated OCP fusion via Myh9 proteolysis. The proposed research project will, for the first time, elucidate the molecular mechanism underlying the pathogenically elevated periodontal bone loss targeting the unique P. gingivalis ceramides that promote osteoclastogenesis by modulating the cell fusion regulatory factor, Myh9, and investigate the effects of host-derived acid ceramidase that can potentially counteract such a pathogenic action of PGDHC.