活化的PLC-γ及与Akt关联调控OA软骨基质代谢的机制研究

Our previous study indicated that activated Phospholipase C-γ (PLC-γ) is higher in OA chondrocytes and activated Akt is lower in OA chondrocytes, compared with normal articular chondrocyte. Moreover, the increase of Akt expression caused by extracellular factors can promote extracellular matrix formation. PLC-γ is tyrosine phosphorylated by various growth factor receptors after binding of their cognate ligands. Tyrosine phosphorylation can turn protein structure into an enzymatically active form and generate second messengers by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol-trisphosphate (IP3). DAG subsequently activates protein kinase C(PKC ) and evokes phosphorylation of various cellular proteins. However, it is not clear to understand the role of PLC-γ in OA chondrocytes. Combined with other author’s studies on PLC-γ and Akt in other cell lines, we proposed that phosphorylation of PLC-γ1 at Tyr 783 and PLC-γ2 at Tyr759/Tyr1217 site is also the most crucial event in activating PLC-γ in articular chondrocyte as well as other cell lines, and the phosphorylation of PLC-γ at PLC-γ1-Ser 1248 and PLC-γ2-Ser677 site plays a negative role in PLC-γ activation. Moreover, Akt might interact with PLC-γ and result in the inhibition of PLC-γ activation by phosphorylating PLC-γ at PLC-γ1-Ser 1248 and PLC-γ2-Ser677 site. The increase of PLC-γ activation expression by suppressed-Akt activation in OA chondrocytes might trigger IP3/Ca2+ /MEK/ERK or P70S6K/S6 and DAG/PKCδ/MEK/ERK signaling axis, leading to the degradation of extracellular matrix in OA chondrocytes. In the present study, we will examine the protein and mRNA expression and distribution of activated PLC-γ and the activation of PLC-γ in normal and OA chondrocytes by western blot, the assay kit, and Real-time PCR, and analyze the difference. Meanwhile, the relevant signaling axis triggered by activation of PLC-γ will be monitored. We will also test the possibility that Akt regulates the activation of PLC-γ by binding to its SH3 domains and phosphorylating PLC-γ1-Ser 1248 and PLC-γ2-Ser677 site in normal and OA chondrocytes. The role of the inhibition of PLC-γ activation will be observed and analyzed in rat OA model. Eventually, the data will support the proposal that the activation of PLC-γ and the interaction with Akt lead to the degradation of extracellular matrix in OA chondrocytes, and it is helpful in understanding the role of PLC-γ in OA pathological progression and the prevention and therapy of OA.

前期实验显示OA软骨细胞内提高低表达的Akt可促进软骨基质合成；同时观察到PLC-γ在OA软骨细胞内呈高表达。已知激活后PLC-γ可水解PIP3产生IP3与DAG参与调节细胞代谢，但PLC-γ对OA软骨细胞调控意义尚且不清。结合前人和我们的研究推测：①活化的PLC-γ可通过IP3/Ca2+与DAG/PKCδ启动MEK/ERK/Sp1及P70S6K/S6信号轴调控OA软骨基质代谢；② PLC-γ与Akt间具有密切关联的结构基础，并可能存在功能上的相互拮抗。本研究以体外培养的人正常/OA软骨细胞、HEK293T细胞及大鼠OA动物模型为研究对象，转染相关表达载体/药物处理，借助WB与RT-PCR等技术，检测PLC-γ调节基质代谢的信号调控网络；探讨Akt调控PLC-γ活性的机制；观察抑制PLC-γ活性对活体软骨损伤的影响，验证上述推测、阐明PLC-γ在OA病理进程中的意义以及预防和治疗OA的价值

骨性关节炎（OA）主要病理表现为软骨细胞死亡、细胞外基质合成和降解之间平衡失调，最终导致软骨破坏。前期结果显示磷脂酶 C(PLC)-γ在OA软骨细胞内高表达，推测 PLC-γ的活化作用可能参与了 OA病理进程，尤其是软骨基质的代谢过程；并且与Akt间存在关联。为了证实上述推测，主要从下面四部分入手：（1）探讨PLC-γ在OA软骨基质代谢中的表达意义。（2）探讨PLC-γ调控OA软骨细胞基质代谢可能机制。（3）探讨Akt是否参与PLC-γ对OA软骨基质代谢调控过程。（4）构建大鼠OA模型，关节腔注射抑制PLC-γ或Akt活性的表达载体或药物，检测大鼠OA模型中软骨损伤情况。获得的重要研究结果：（1）OA软骨细胞内较高表达的PLC-γ1通过抑制Col II与Aggrecan的表达，提高MMP13、ADAMTS5的活性而促进软骨基质降解。（2）活化的PLC-γ1激活的两条经典途径 PLC-γ1/IP3/Ca(2+)/CaMKII与PLC-γ1/DAG/PKCδ 中PLC-γ1/IP3/Ca(2+)/CaMKII信号轴在调节软骨基质中发挥较大作用，其通过激活mTOR/P70S6K/S6通路参与软骨基质代谢调控。（3）PLC-γ1通过复杂的信号网络调控软骨基质代谢，发现OA软骨细胞中PLC-γ1可同时激活mTOR与ERK，但PLC-γ1/mTOR信号轴抑制了PLC-γ1/ERK信号轴，进而减弱PLC-γ1/ERK信号轴的作用，最终OA软骨细胞内高表达的PLC-γ1表现为抑制OA软骨基质合成。（4）活化Akt促进软骨基质合成，中药单体及miRNA-634通过PI3K/Akt调控OA软骨基质代谢。（5）PLC-γ1与Akt 的磷酸化作用参与OA软骨基质代谢调节，并作用相反：抑制p-PLC-γ1/Y783促进OA软骨基质合成；抑制p-Akt S473促进OA软骨基质降解。（6）OA软骨细胞中，PLC-γ1与Akt抑制剂可分别提高Akt与PLC-γ1的磷酸化水平，两者间存在相互拮抗作用；同时，Co-IP检测两者间存在结合，两者的磷酸化水平影响两者间结合状态。（7）ACLT+MMx手术建立OA大鼠模型，关节腔注射PLC-γ1或Akt的抑制剂检测发现：抑制PLCγ1有效延缓大鼠OA软骨损伤；抑制Akt并没有加重大鼠OA软骨损伤。上述结果初步阐明PLC-γ1在OA软骨基质代谢中的作用及机制