Ceramide-mediated pathology in periodontitis

摘要 宿主细胞胞质中的神经酰胺在各种细胞事件中作为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 脊椎动物，不含神经酰胺，这表明细菌产生神经酰胺的情况很少。尽管如此， 牙龈卟啉单胞菌产生的磷酸甘油二氢神经酰胺(PGDHC)，而不是内毒素 在人类牙周炎病变中含量丰富。与膜上不透性神经酰胺形成对比 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，并 通过作用于非肌肉肌球蛋白IIA(Myh9)促进破骨细胞形成所需的细胞融合。Myh9是一位 破骨细胞形成中细胞融合事件的细胞内下调因子。相比之下，它也是 证明了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， 组织蛋白酶B通过时间受控的Myh9蛋白降解促进RANKL介导的破骨细胞生成 OCP之间细胞融合的启动。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的生成，这表明组织蛋白酶B也是 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，在生理环境中受神经酰胺酶调节。功能失调 酸性神经酰胺酶参与了Farber病和其他医学并发症的发病机制。我们 发现并发表了酸性神经酰胺酶在牙周炎患者中的表达减弱。 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介导的破骨细胞形成的分子机制尚不清楚。因此，我们 假设牙周病变中aCDase活性降低保留了组织的致病特性- 穿透PGDHC，进而通过骨骼中新的组织蛋白酶B/Myh9轴促进破骨细胞的形成 牙周炎的吸收损害。目的1是为了测试酸性神经酰胺酶的保护作用。 PGDHC介导的牙周炎小鼠模型中的破骨细胞形成。我们将评估这些措施的效果 内源性酸性神经酰胺酶与外源性重组酸性神经酰胺酶的中和作用 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参与经Myh9蛋白降解的PGDHC介导的OCP融合拟议的研究 该项目将首次阐明病因性升高的分子机制。 牙周骨丢失靶向牙龈假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Myh9，并研究宿主衍生酸性神经酰胺酶的作用 这可能会中和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.