GROUP VIA PHOSPHOLIPASE A2 (IPLA2BETA) IN TRANSCRIPTIONAL UPREGULATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 RGS2(G蛋白信号转导调节因子-2)基因缺陷的小鼠表现出严重的高血压，高血压患者中存在RGS2的遗传变异。血管紧张素II(Ang II)上调血管平滑肌细胞(VSMC)RGS2基因表达是血压稳态的一种潜在的负反馈机制，但其发生机制尚不清楚。在此，我们证明了通过磷脂酶A2的组(iPLA2β)在Ang II诱导的VSMC RGS2 mRNA上调中起关键作用的三种独立途径，包括用溴烯醇内酯自杀底物进行药物抑制，用反义寡核苷酸抑制iPLA2β的表达，以及在iPLA2β缺失的小鼠中进行基因缺失。这些途径中的每一种选择性抑制iPLA2β均可取消Ang II诱导的RGS2基因表达上调。此外，使用腺病毒介导的基因转移，我们证明了在iPLA2β缺失的VSMC中恢复iPLA2β表达重建了Ang II上调RGS2 mRNA表达的能力。相反，Ang II诱导的血管扩张剂刺激的磷蛋白磷酸化和Ang II受体的表达不受影响。此外，在野生型而不是iPLA2β缺失的VSMC中，Ang II显著刺激iPLA2的酶活性。IPLA2β作用的产物花生四烯酸和溶血磷脂酰胆碱均可诱导RGS2基因表达上调。抑制脂氧合酶，特别是15-脂氧合酶和环氧合酶，而不是细胞色素P450依赖的环氧合酶，可以抑制Ang II或AA诱导的RGS2 mRNA的表达。此外，血管紧张素转换酶II上调RGS2蛋白的表达，这种上调作用可被溴烯醇内酯减弱。阻断iPLA2β缺失细胞中的Ang II/iPLA2β/RGS2反馈通路，以时间依赖的方式增强Ang II诱导的血管扩张剂刺激的磷蛋白和Akt的磷酸化。综上所述，我们的结果表明iPLA2β参与了Ang II诱导的VSMC中RGS2的转录上调。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Rgs2 (regulator of G-protein signaling-2)-deficient mice exhibit severe hypertension, and genetic variations of RGS2 occur in hypertensive patients. RGS2 mRNA up-regulation by angiotensin II (Ang II) in vascular smooth muscle cells (VSMC) is a potentially important negative feedback mechanism in blood pressure homeostasis, but how it occurs is unknown. Here we demonstrate that group VIA phospholipase A2 (iPLA2beta) plays a pivotal role in Ang II-induced RGS2 mRNA up-regulation in VSMC by three independent approaches, including pharmacologic inhibition with a bromoenol lactone suicide substrate, suppression of iPLA2beta expression with antisense oligonucleotides, and genetic deletion in iPLA2beta-null mice. Selective inhibition of iPLA2beta by each of these approaches abolishes Ang II-induced RGS2 mRNA up-regulation. Furthermore, using adenovirus-mediated gene transfer, we demonstrate that restoration of iPLA2beta-expression in iPLA2beta-null VSMC reconstitutes the ability of Ang II to up-regulate RGS2 mRNA expression. In contrast, Ang II-induced vasodilator-stimulated phosphoprotein phosphorylation and Ang II receptor expression are unaffected. Moreover, in wild-type but not iPLA2beta-null VSMC, Ang II stimulates iPLA2 enzymatic activity significantly. Both arachidonic acid and lysophosphatidylcholine, products of iPLA2beta action, induce RGS2 mRNA up-regulation. Inhibition of lipoxygenases, particularly 15-lipoxygenase, and cyclooxygenases, but not cytochrome P450-dependent epoxygenases inhibits Ang II- or AA-induced RGS2 mRNA expression. Moreover, RGS2 protein expression is also up-regulated by Ang II, and this is attenuated by bromoenol lactone. Disruption of the Ang II/iPLA2beta/RGS2 feedback pathway in iPLA2beta-null cells potentiates Ang II-induced vasodilator-stimulated phosphoprotein and Akt phosphorylation in a time-dependent manner. Collectively, our results demonstrate that iPLA2beta participates in Ang II-induced transcriptional up-regulation of RGS2 in VSMC.