血管内皮细胞中G蛋白偶联受体4与血管内皮生长因子受体2之间crosstalk的发生机制研究

Receptor Crosstalks occur among different cell membrane receptors. These receptors crosstalks allow cells to integrate different cellular signals and pathways in order to carry out the biological regulations. Our previous study firstly found that, there exists crosstalk between SPC receptor, G protein coupled receptor 4(GPR4), and receptor 2 of vascular endothelial growth factor(VEGF). When SPC bound to GPR4, the proliferation and tube formation of vascular endothelial cell were stimulated. ERK and AKT were also activated. But to our surprise, when VEGF R2 inhibitors including VEGFR2 specific inhibitor and tyrosine kinase inhibitor were applied, the SPC effect was significantly blocked by VEGFR2 inhibitors. In another word, the effect of SPC/GPR4 on vascular endothelial cells is dependent on VEGF R2. That means, there exist crosstalk between GPR4 and VEGF R2. We speculate there are three possible mechanisms of this crosstalk. The first possible mechanism is the sequential model. The production and secretion of VEGF may be induced after SPC binds to GPR4. Extracellular VEGF then activates VEGF R2 and its downstream signaling pathways; The second possible mechanism is the integration model. The complex of GPR4 and VEGF R2 may form after SPC binds to GPR4. VEGF R2 is consequently activated; The third possible mechanism is the transactivation model. SPC may directly phosphorylate VEGF R2. This study aims to investigate these possible mechanisms. Understanding of this crosstalk mechanisms is helpful in understanding the development of tumor and cardiovascular diseases. Up to now, the similar study report has not been found.

细胞膜上不同受体间可发生Crosstalk，从而使细胞整合不同的信号和通路。我们前期首次发现血管内皮细胞上鞘氨醇磷酸胆碱SPC的受体GPR4与血管内皮生长因子受体2间存在crosstalk。SPC作用于GPR4后促进内皮细胞的增殖、血管生成、并激活ERK和AKT，而应用VEGFR2抑制剂：VEGFR2特异性抑制剂和酪氨酸激酶抑制剂均可阻断SPC/GPR4的作用，即GPR4作用依赖于VEGFR2，说明GPR4与VEGFR2间存在Crosstalk。我们推测有三种发生机制：SPC作用于GPR4后①刺激VEGF的产生，分泌致胞外的VEGF激活VEGFR2，再激活下游信号；②GPR4与VEGFR2间形成复体complex，使VEGFR2活化。③SPC/GPR4直接作用于VEGFR2磷酸化位点使其活化。本项目拟在血管内皮细胞中研究其机制，对了解心血管疾病和肿瘤的发展有重要意义。目前尚未见到相关报道。

一.GPR4与VEGFR2 crosstalk的作用机制研究：.1. 机制一：结合模式.①在酵母细胞中用LacZ方法进一步验证GPR4与VEGFR2间的关系：分别构建：含有pGADT7-VEGFR2 基因和LacZ 的N-端基因的质粒、含有pGBKT7-GPR4基因和LacZ 的C-端基因的质粒，将两种质粒同时转染酵母细胞内,转染后酵母能在SD/-Ade/-His/-Leu/-Trp培养基上生长，证实了VEGF R2 与GPR4 间确实形成了复体。.②将pcDNA-GPR4-Flag, pcDNA-VEGFR2-Myc质粒共同转入HMEC-1，经SPC刺激后， 用Co-IP发现，GPR4和VEGFR2抗体共沉淀下来，说明GPR4和VEGFR2在哺乳动物人血管内皮细胞HMEC-1中形成了复体。.2. 机制二：反式激活模式.通过SPC/GPR4 直接作用于VEGF R2 的酪氨酸磷酸化位点P951、P1212、P1175的磷酸化检测，发现GPR4与VEGFR2结合后能使VEGFR2的P951位点发生磷酸化。SPC/GPR4直接激活VEGF R2 的PY951酪氨酸磷酸化位点，使其磷酸化。.3. 机制三：序贯模式.当SPC作用于GPR4 后，发现①SPC能激活VEGF启动子；②SPC处理HMEC-1细胞后，ELISA和Western证实VEGF的表达增高。这证明SPC能刺激VEGF 的产生分泌，VEGF作用于细胞膜上的VEGF R2, 从而激活其下游的信号通路。当敲除GPR4后SPC刺激VEGF产生的作用消失。.二．MMP-2、MMP-9、MMP-13介导了SPC/GPR4对HMEC-1细胞迁移的促进作用.Western blot检测发现MMP-2，MMP-9以及MMP-13在SPC的刺激下均有不同程度的表达升高，进一步证实了细胞迁移的主要原因是MMP-2、MMP-9、MMP-13蛋白的促迁移作用。.三．下游机制：EST2.SPC激活PI3K/AKT，p38MAPK信号通路。我们筛选出了与GPR4密切相关的分子PPP1CC，ETS2，EIF4EBP2，PCDH20等分子。发现ETS2可能与SPC/GPR4密切相关。SPC处理HMEC-1细胞后，ETS2由细胞质定位转移到细胞核定位，ETS2表达显著升高，但VEGF抑制剂及干扰RNA可抑制其表达。