Phlpp protein phosphatases in cartilage development and disease

摘要 软骨是一种软组织，参与骨骼延长、骨折修复和无痛关节运动。损坏 骨骼软骨会阻碍骨骼发育，并导致骨关节炎等疼痛的关节疾病， 在美国和世界各地，这是导致残疾和就医的主要原因。这个项目将 探索酶PHLPP1和Phlpp2如何调节骨骼的形成、维持和再生 软骨。PHLPP1和Phlpp2是细胞内的蛋白磷酸酶，通过以下途径控制软骨细胞的合成代谢 翻译后修饰蛋白质底物(如AKT、PKCα)。骨性关节炎患者关节软骨细胞的研究 表达异常高水平的PHLPP1和Phlpp2。PHLPP1基因整体缺失增加软骨细胞 增殖和基质合成，并在刺激软骨表达的同时减少损伤诱导的骨关节炎 生长因子和受体(Fgf18、转化生长因子β和Ptr1)。PHLPP1或Phlpp2耗尽也会诱导表达 一种小蛋白多糖(Prg4、DCN、Spp1)，可改善软骨组织完整性和关节润滑性。在一个 创伤后骨性关节炎小分子单次关节腔内注射的临床前动物模型 抑制剂可暂时减轻半月板损伤所致的软骨丢失和疼痛，且无不良反应 五个星期。这些Phlpp抑制剂还促进了软骨细胞的增殖和基质基因的产生。 因此，Phlpp抑制是一种新的治疗OA的策略，但对PHLPP1和Phlpp2如何 调节信号通路和促进软骨细胞合成代谢是最大限度地治疗软骨疾病所必需的 Phlpp抑制剂的潜力。这个项目的总体目标是阐明新的分子和细胞 PHLPP1和Phlpp2在软骨内发育和创伤后骨性关节炎中的作用机制 明确Phlpp抑制剂在关节损伤中的特异性。具体目标是 目的：1)明确PHLPP1和Phlpp2在2型胶原阳性细胞增殖和成熟中的作用 骨骼发育；2)成体aggrecan表达的PHLPP1和Phlpp2分子功能的测定 3)检测Phlpp抑制剂对PHLPP1和Phlpp2的特异性，并对其进行改进 创伤后骨性关节炎模型的有效性；以及4)阐明GIRK通道对 PHLPP1和Phlpp2与合成代谢信号事件的整合。

ABSTRACT Cartilage is a soft tissue involved in bone lengthening, fracture repair and painless joint motion. Damage to skeletal cartilages can hinder bone development and cause painful joint diseases like osteoarthritis (OA), which is a leading cause of disability and hospital visits in the USA and around the world. This project will explore how the enzymes Phlpp1 and Phlpp2 regulate the formation, maintenance and regeneration of skeletal cartilage. Phlpp1 and Phlpp2 are intracellular protein phosphatases that control chondrocyte anabolism by post-translationally modifying protein substrates (e.g., AKT, PKCα). Articular chondrocytes from OA patients express abnormally high levels of Phlpp1 and Phlpp2. Global deletion of Phlpp1 increases chondrocyte proliferation and matrix synthesis, and reduces injury-induced OA while stimulating the expression of cartilage growth factors and receptors (Fgf18, Tgfβ and Pthr1). Phlpp1 or Phlpp2 depletion also induces the expression of small proteoglycans (Prg4, Dcn, Spp1) that improve cartilage tissue integrity and joint lubrication. In a preclinical animal model of post-traumatic OA, a single intra-articular injection of a small molecule Phlpp inhibitor temporarily reduced cartilage loss and pain associated with a meniscal injury without adverse events for five weeks. These Phlpp inhibitors also promoted chondrocyte proliferation and production of matrix genes. Thus Phlpp inhibition is a new therapeutic strategy for OA, but additional knowledge of how Phlpp1 and Phlpp2 regulate signaling pathways and promote chondrocyte anabolism is necessary to maximize the therapeutic potential of Phlpp inhibitors. The overall goals of this project are to elucidate novel molecular and cellular mechanisms of Phlpp1 and Phlpp2 action during endochondral development and post-traumatic OA pathogenesis, and to define the specificity of Phlpp inhibitors in injured articulating joints. The specific aims are to: 1) define the roles of Phlpp1 and Phlpp2 in proliferation and maturation of collagen 2-positive cells during skeletal development; 2) determine the molecular functions of Phlpp1 and Phlpp2 in adult aggrecan-expressing cells following a joint injury; 3) test the specificity of Phlpp inhibitors to Phlpp1 and Phlpp2 and improve their effectiveness in post-traumatic OA models; and 4) elucidate the contribution of GIRK channels to the integration of Phlpp1 and Phlpp2 with anabolic signaling events.