Protein Phosphatase Phlpp1 in Cartilage Development & Osteoarthritis Progression

描述(由申请人提供)：骨关节炎(OA)是最常见的关节炎形式，是美国老年人残疾的主要原因，也是我们社会日益增长的经济负担。以关节软骨退化、滑膜炎、软骨下骨增厚、骨赘和其他关节改变为特征，骨性关节炎极其痛苦和虚弱。由于美国人口老龄化，迫切需要提供新的解决方案来预防骨性关节炎和/或促进软骨愈合。该提案中概述的实验旨在验证PHLPP1作为治疗靶点的有效性，并可迅速转化为治疗OA的方法。PHLPP1(pleckstrin同源域富含亮氨酸重复蛋白磷酸酶，“FliP”)是一种细胞内磷酸酶，终止了包括Akt和PKC在内的许多信号通路，抑制蛋白多糖和II型胶原的合成，影响细胞的增殖、分化和生存。我们发现PHLPP1在骨性关节炎(OA)患者的关节软骨中高表达。我们推测，PHLPP1通过调节Akt和其他信号通路促进了骨性关节炎进展过程中软骨细胞的肥大。该项目的目标是利用人体组织和动物模型确定PHLPP1如何促进骨关节炎的进展和骨骼发育，并验证PHLPP1作为骨关节炎的治疗靶点。该项目的具体目标是：1)通过对骨骼成熟的PHLPP1-/-小鼠进行DMM手术，并通过功能测试、成像和组织学监测OA的进展，确定PHLPP1在OA进展中的作用。Phlpp抑制剂也将在DMM模型中进行测试。2)通过显微CT成像、组织形态计量学和原位杂交检测PHLPP1-/-动物的骨和软骨发育，确定PHLPP1-/-在软骨内骨和关节形成中的作用；在存在或不存在Phlpp抑制剂的情况下，也将对来自PHLPP1-/-和野生型小鼠的原代软骨细胞、成骨细胞和破骨细胞进行体外分化分析；以及3)通过检验PHLPP1在OA软骨中的表达受DNA去甲基化和/或炎症诱导的HDAC共表达产物释放的表观遗传调控的假设，确定PHLPP1在OA软骨中表达的表观遗传事件和可溶性因素。这项工作的意义在于，PHLPP1是一个新的可药物靶点，其活性和/或表达可以被控制，以重置软骨细胞信号通路，减缓OA的疾病进展。这项工作具有创新性，因为PHLPP1在软骨发育和疾病中的作用从未被探索过。我们的团队拥有必要的试剂和专业知识，因此处于独特的地位，可以有效地完成这一项目。我们的结果将产生影响，因为它们将验证PHLPP1作为治疗目标的有效性，并可能迅速转化为数百万患有骨性关节炎的美国人的临床选择。

DESCRIPTION (provided by applicant): Osteoarthritis (OA) is the most common form of arthritis, a leading cause of disability in older Americans, and a growing economic burden to our society. Characterized by degradation of articular cartilage, synovitis, subchondral bone thickening, osteophytes, and other joint changes, OA is extremely painful and debilitating. Due to the aging population in the USA, there is an urgent need to provide new solutions that prevent osteoarthritis and/or promote cartilage healing. The experiments outlined in this proposal are designed to validate Phlpp1 as therapeutic target and could be rapidly translated into a therapeutic approach for OA. PHLPP1 (pleckstrin homology domain leucine-rich repeat protein phosphatase, "flip") is an intracellular phosphatase that terminates numerous signaling pathways, including Akt and PKC, to repress proteoglycan and type II collagen synthesis, and affect proliferation, differentiation, and survival. We discovered that PHLPP1 is highly expressed in articular cartilage from osteoarthritis (OA) patients. We hypothesize that PHLPP1 promotes chondrocyte hypertrophy during OA progression by virtue of its ability to regulate Akt and other signaling pathways. The objectives of this project are to define how PHLPP1 contributes to OA progression and skeletal development using both human tissues and animal models and to validate Phlpp1 as a therapeutic target for OA. The specific aims of this project are to: 1) Define the role of Phlpp1 in OA progression by performing DMM surgery on skeletally mature Phlpp1-/- mice and monitoring OA progression through functional testing, imaging and histology. Phlpp inhibitors will also be tested in the DMM model. 2) Determine the role of Phlpp1 in endochondral bone and joint formation by examining bone and cartilage development in Phlpp1-/- animals with microCT imaging, histomorphometry, and in situ hybridization; in vitro differentiation assays with primary chondrocytes, osteoblasts, and osteoclasts from Phlpp1-/- and wildtype mice will also be performed in the presence or absence of Phlpp inhibitors; and 3) Define the epigenetic events and soluble factors controlling PHLPP1 expression in OA cartilage by testing the hypothesis that PHLPP1 expression is epigenetically controlled by DNA demethylation in OA cartilage and/or by inflammation-induced release of Hdac co-repressors. The significance of this work is that PHLPP1 is a new and druggable target whose activities and/or expression could be controlled to reset chondrocyte signaling pathways and slow OA disease progression. The work is innovative because the role of PHLPP1 in cartilage development and disease has never been explored. Our team possesses necessary reagents and expertise and thus is uniquely positioned to efficiently complete this project. Our results will have an impact because they will validate PHLPP1 as therapeutic target and could be rapidly translated into a clinical option for millions of Americans suffering from OA.