Therapeutic targeting of GPCR Gbetagamma-GRK2 in osteoarthritis

摘要 骨关节炎(OA)是一种关节衰弱的疾病，涉及进行性软骨退化，没有疾病 可用的修改疗法。软骨细胞是软骨的细胞成分，通过以下方式调节软骨的动态平衡 通过分泌蛋白多糖和胶原以及基质降解酶来调节基质周转。在办公自动化中， 关节软骨细胞在经历分化和肥大(CH)的过程中获得异常表型 基质降解酶的产生增加，导致软骨退化。G蛋白偶联受体 GPCRs是一个与细胞内G蛋白偶联的受体超家族。激动剂与GPCR结合可激活其G- 蛋白a亚单位(GA)的生理信号。一种平行的G蛋白BG亚单位(GBG)的激活和募集 激酶(尤其是GRK2)导致GPCR内化和信号终止。GRK2表达与GBG-GRK2 在几种疾病中，信号在病理上升高，导致GPCR脱敏和GA信号丢失。 重要的是，我们和其他人最近报道了GBG-GRK2抑制的治疗效果，这两种方法都是通过挽救GA 通过抑制直接的下游病理信号促进细胞生长和 差异化；这是通过使用新型小分子药物和FDA批准的药物实现的。软骨细胞富含 1型甲状旁腺激素受体(PTH1R，一种GRCR)，其中Gas和PTH1R是CH的关键调节因子，两者都低于 由GBG-GRK2严格监管。然而，GBG-GRK2信号在CH和OA中的作用仍不清楚。我们的 初步确定GBG-GRK2在骨性关节炎中的作用表明，GRK2的表达和活性是 骨性关节炎患者和创伤后骨性关节炎小鼠的关节软骨细胞均增加。有趣的是，在体外和体内 GBG-GRK2抑制减轻DMM小鼠的CH和软骨退变(手术治疗内侧失稳) 半月板以诱发骨性关节炎)。这些初步发现支持我们的总体假设，即：GBG-GRK2信号发挥作用 在骨性关节炎的CH中起中心作用，其抑制代表了一种治疗骨性关节炎的新策略。要验证我们的 假设我们将在体内使用软骨细胞条件基因敲除小鼠和小分子GBG和GRK2抑制剂 建立小鼠骨性关节炎模型，并利用不同的体外系统(小鼠和人)，以(1)确定GRK2的作用 (2)确定小分子GBG-GRK2抑制剂在骨关节炎中的治疗效果，以及 (3)探讨GBG-GRK2信号在PTH1R关节软骨保护/再生中的作用。这些研究 使我们能够发现一种新的治疗策略和治疗药物(包括新的和FDA批准的)在 从而为我们未来的临床研究建立一种新的有效的骨性关节炎治疗方法奠定了基础。

Abstract Osteoarthritis (OA) is a debilitating disease of the joints involving progressive cartilage degeneration, with no disease modifying therapy available. Chondrocytes, the cellular component of cartilage, regulate cartilage homeostasis by modulating matrix turnover via secretion of proteoglycans and collagen as well as matrix-degrading enzymes. In OA, articular chondrocytes acquire an aberrant phenotype where they undergo differentiation and hypertrophy (CH) with increased production of matrix degrading enzymes leading to cartilage degeneration. G protein-coupled receptors (GPCRs) is a superfamily of receptors coupled to intracellular G-proteins. Agonist binding to a GPCR activates its G- protein a subunit (Ga) physiologic signaling. A parallel G-protein bg subunit (Gbg) activation and recruitment of GPCR kinases (GRK2 particularly) leads to GPCR internalization and signal termination. GRK2 expression and Gbg-GRK2 signaling are pathologically elevated in several diseases, leading to GPCR desensitization and loss of Ga signaling. Importantly, we and others have recently reported the therapeutic efficacy of Gbg-GRK2 inhibition, both by rescuing Ga physiologic activity and by inhibiting direct downstream pathological signaling that promotes cell growth and differentiation; this was achieved using both novel small molecules and FDA-approved drugs. Chondrocytes are rich in type 1 parathyroid hormone receptor (PTH1R, a GRCR), where Gas and PTH1R are key regulators of CH, both are under tight regulation by Gbg-GRK2. However, the role of Gbg-GRK2 signaling in CH and OA remains unknown. Our preliminary work to establish the role of Gbg-GRK2 in CH during OA indicates that GRK2 expression and activity are increased in articular chondrocytes of both OA patients and mice with posttraumatic OA. Interestingly, in vitro and in vivo Gbg-GRK2 inhibition attenuated CH and cartilage degeneration in mice with DMM (surgical destabilization of the medial meniscus to induce OA). These preliminary findings support our overarching hypothesis that: Gbg-GRK2 signaling plays a central role in CH during OA, and its inhibition represents a novel therapeutic strategy for OA. To validate our hypothesis we will use chondrocyte conditional knockout mice and small molecule Gbg and GRK2 inhibitors in an in vivo OA mouse model and utilize different in vitro systems (both murine and human), to (1) determine the role of GRK2 signaling in articular CH in OA, (2) determine the therapeutic efficacy of small molecule Gbg-GRk2 inhibitors in OA, and (3) determine the role of Gbg-GRK2 signaling in PTH1R chondro-protective/regenerative effect in OA. These studies enable us to discover a novel therapeutic strategy and therapeutic agents for OA (both novel and FDA-approved) in a preclinical setting, thus laying the grounds for our future clinical study to establish a novel effective OA therapy.