磷脂酶sPLA2G2E缺失导致动脉粥样硬化新机制的研究

Secreted phospholipases A2 (sPLA2s) are enzymes that catalyze the hydrolysis of glycerophospholipids at the sn-2 position to release free fatty acids and lysophospholipids and also can bind to receptors, involving in many physiological functions including metabolism, immune response and inflammation. Previously, we resolved the crystal structure of sPLA2G1B (JBC，2009) which is associated with metabolic regulation. Subsequently, we discovered that sPLA2G12B knockout mouse had disorders in lipid metabolism and fatty liver, with a mechanism associated with HNF4α signaling (Hepatology, 2011). Recently, we generated the knockout mouse for sPLA2G2E, a new member in sPLA2 family with unknown function. We found that the deficiency of sPLA2G2E resulted in hypercholesterolemia and hyperglyceridemia. Most intriguingly, sPLA2G2E-null mice developed severe atherosclerosis after high fat diet. This phenotype is opposite to other members of sPLA2s that have been studied so far. What is the underlying mechanism of sPLA2G2E deficiency that caused such severe atherosclerosis? sPLA2G2E hydrolyzes glycerophospholipids and aslo can bind to M-type sPLA2 Receptor. Could sPLA2G2E be a new key switch related to regulating atherosclerosis development? Our preliminary investigation revealed that the deficiency of sPLA2G2E resulted in decrease of low density lipoprotein receptor (LDLR) expression, a well known molecule in regulating lipid metabolism and its gene knockout mouse is a well recognized mouse model for atherosclerosis. We hypothesize that sPLA2G2E may play an important role as an essential regulator in mediating lipid metabolism which acts upon the upstream of LDLR. We will carry out transcriptomic, proteomic, and metabolomic analysis, generating sPLA2G2E+LDLR double knockout mouse, using adenovirus to deliver genes of LDLR or G2E, as well as siRNA and chemical probes to further study the biological function of sPLAG22E. The objective is to elucidate the role and the mechanism of sPLA2G2E deficiency in inducing atherosclerosis. This work could potentially identify a new target for diagnosis, prevention and treatment of atherosclerosis.

分泌型磷脂酶A2(sPLA2)是一类水解磷脂的酶并参与脂质代谢与免疫调控，既有酶活性又可与受体结合。我们先前解析了其成员G1B的晶体结构(JBC，2009)，并在小鼠发现其成员G12B缺失导致脂肪肝且受HNF4α调控(Hepatology，2011)。最近，我们在小鼠中敲除了另一成员G2E，发现其血脂升高并发生严重的动脉粥样硬化(AS)，该表型与已报道的PLA2其他成员促进AS的功能相反。G2E缺失导致AS的机制是什么，G2E是否是拮抗AS的关键分子？研究发现，G2E缺失降低了低密度脂蛋白受体(LDLR，其敲除小鼠是公认的AS模型)表达。由此我们提出假设：G2E可能是一个新的调控脂质代谢的重要节点并位于LDLR上游。我们将采用基因和蛋白芯片、G2E/LDLR双敲除小鼠、腺病毒过表达、RNA干扰 及化学探针等分析相关信号通路，探索G2E拮抗AS的作用机制，为寻找防治AS的新靶点提供科学依据。

PLA2G2E是sPLA2家族成员之一，之前我们发现PLA2G2E基因敲除小鼠发生严重的动脉粥样硬化、血脂升高。在PLA2G2E基因敲除小鼠的巨噬细胞中，胆固醇大量积累，形成泡沫细胞。但是，PLA2G2E在巨噬细胞的脂质转运过程中发挥何种作用尚不清楚。进一步分析发现PLA2G2E的缺失降低了小鼠肝脏组织表皮生长因子(epidermal growth factor receptor, EGFR)的磷酸化，抑制了丝裂原活化蛋白激酶(Mitogen-activated protein kinases, MAPK)信号通路，进而导致低密度脂蛋白受体(low density lipoprotein receptor, LDLR)表达水平显著降低。另外，在PLA2G2E基因敲除小鼠的巨噬细胞中，胆固醇合成的限速酶HMGCR和HMGCS表达下降，胆固醇摄入相关的受体CD36和TLR4表达升高，而胆固醇流出的关键分子ABCA1和ABCG1表达也下降。在体外，我们用siRNA敲低了小鼠巨噬细胞系Raw264.7中PLA2G2E的表达，发现胆固醇合成的限速酶HMGCR和HMGCS及胆固醇摄入相关的受体CD36和TLR4表达没有变化，而胆固醇流出的关键分子ABCA1和ABCG1表达下降；并且ABCA1和ABCG1介导的巨噬细胞胆固醇流出也减少。这些结果说明，PLA2G2E主要参与调节了巨噬细胞胆固醇的流出。进一步研究发现，PLA2G2E调节巨噬细胞胆固醇流出作用依赖于其酶活性。另外，PLA2G2E敲除下调了L-PGDS表达和PGD2的生成，而PGD2可以促进ABCA1和ABCG1的表达。在PLA2G2E敲除的巨噬细胞中加入PGD2处理，可以增加细胞胆固醇的外流，从而减少细胞内脂滴的积累。本项目证实了PLA2G2E缺失导致动脉粥样硬化发生发展的分子机制，充实我们对sPLA2家族成员功能的多样性和复杂性的认知，为提出PLA2G2E可能是治疗动脉粥样硬化的新靶点提供科学依据。