The Role of PLEKHM1 in Osteoclast Function

描述（由申请人提供）：骨稳态需要破骨细胞的骨吸收和成骨细胞的骨形成之间的紧密平衡。在病理学背景下，增强的破骨细胞骨吸收导致骨量减少，如在骨质疏松症中所见，而破骨细胞分化或功能缺陷导致骨的异常积聚，如在骨硬化症中所观察到的。对石骨症患者的遗传学研究和啮齿动物石骨症模型中天然发生或靶向基因敲除揭示了骨钙素骨吸收的重要调控机制。基因如c-src、α 3整联蛋白亚基、组织蛋白酶K、TCIRG 1（编码液泡H+-ATP酶α 3亚基）、CLCN 7（编码clc-7氯离子通道）和OSTM 1（灰色致死）的破坏会破坏破骨细胞功能。尽管TCIRG 1和CLCN 7的突变在人类骨石化病患者中最丰富，但在约25%的病例中尚未确定相关基因。最近，PLEKHM 1被鉴定为在骨硬化症ia/ia（门齿缺失）大鼠和骨硬化症患者的子集中突变的基因。来自ia/ia大鼠的破骨细胞由于皱褶边缘形成和分泌受损而功能障碍。然而，PLEKHM 1调节破骨细胞功能的详细机制仍然未知。我们发现1）PLEKHM 1的表达在破骨细胞分化期间增加2）通过shRNA敲低PLEKHM 1抑制组织蛋白酶K分泌和破骨细胞功能，而不减少破骨细胞形成和3）plekhm 1结合成熟破骨细胞中的TRAF 6和rab 7，这两种蛋白质对细胞的骨吸收能力至关重要。因此，我们推测，plekhm 1调节破骨细胞的功能，至少部分，通过与TRAF 6和rab 7的相互作用。由于我们已经开发了一种逆转录病毒系统，通过该系统我们可以敲除内源性基因，同时用RNAi抗性突变体拯救它，因此我们能够通过进行结构/功能分析来探索plekhm 1在破骨细胞功能中的作用。因此，我们的具体目标是鉴定plekhm 1中对1）plekhm 11/TRAF 6相互作用和破骨细胞功能以及2）rab 7结合和破骨细胞功能至关重要的结构域或基序。破骨细胞是一种专门存在于骨中的细胞，负责溶解这一重要组织。由于破骨细胞的活动，老龄化的美国人口继续失去骨骼，导致目前每年花费超过150亿美元的重大公共卫生问题。我们在破骨细胞中发现了一种新的蛋白质，这种蛋白质有助于破骨细胞的骨吸收活性。我们计划确定这种新蛋白质如何调节破骨细胞，从而提高我们开发预防骨质流失新药的可能性。

DESCRIPTION (provided by applicant): A tight balance between bone resorption by osteoclasts and bone formation by osteoblasts is required for bone homeostasis. In a pathological context, enhanced osteoclastic bone resorption leads to a reduction of bone mass, as seen in osteoporosis, while defects in either osteoclast differentiation or function result in an abnormal accumulation of bone, as observed in osteopetrosis. Genetic studies of patients with osteopetrosis and naturally occurring or targeted gene knockout in rodent osteopetrotic models have unveiled important regulatory mechanisms of osteoclastic bone resorption. Disruption of genes such as c-src, ¿3 integrin subunit, cathepsin K, TCIRG1 (encoding a3 subunit of vacuolar H+-ATPase), CLCN7 (encoding clc-7 chloride channel) and OSTM1 (grey lethal) disrupts osteoclast function. Although mutations in TCIRG1 and CLCN7 are most abundant in human osteopetrosis patients, the involved genes have not been identified in about 25% of cases. Recently, PLEKHM1 was identified as a gene mutated in the osteopetrotic ia/ia (incisors absent) rat and a subset of patients with osteopetrosis. Osteoclasts from ia/ia rats are dysfunctional due to impaired ruffled border formation and secretion. However, the detailed mechanisms by which PLEKHM1 regulates osteoclast function remain unknown. We find that 1) expression of PLEKHM1 increases during osteoclast differentiation 2) knockdown of PLEKHM1 by shRNA inhibits cathepsin K secretion and osteoclast function, without a decrease in osteoclast formation and 3) plekhm1 binds to TRAF6 and rab7 in mature osteoclasts, two proteins critical for the cell's capacity to resorb bone. Thus, we hypothesize that plekhm1 regulates osteoclast function, at least in part, through interaction with TRAF6 and rab7. Since we have developed a retroviral system by which we can knockdown an endogenous gene and simultaneously rescue it with an RNAi-resistant mutant, we are in a position to explore the role of plekhm1 in osteoclast function by performing structure/function analysis. Therefore, our specific aims are to identify the domain or motif in plekhm1 essential for 1) plekhm11/TRAF6 interaction and osteoclast function and 2) rab7 binding and osteoclast function. The osteoclast, a cell found exclusively in bone, is responsible for dissolution of this important tissue. The aging American population continues to lose bone because of osteoclast activity, leading to major public health problems that currently cost in excess of $15 billion dollars annually. We have identified a novel protein in osteoclasts which contributes to their bone resorbing activity. We plan to determine how this new protein regulates the osteoclast, raising the possibility that we will be able to develop a new drug that prevents bone loss.