Post-transcriptional Regulation of NO Synthase

内皮细胞一氧化氮合酶(ENOS)的表达增加是血管对层流切应力的一种重要适应。这不仅发生在培养的内皮细胞中，也发生在暴露在高水平血流中的血管中，例如在运动训练期间。我们实验室的研究表明，eNOS对剪切力的反应通过两条不同的途径发生，这两条途径都依赖于酪氨酸激酶CSRC。一条途径导致转录短暂增加一小时，依赖于经典的ERK1/2途径。随后，持续的切应力导致enos mRNA的长期稳定，这种稳定不依赖于ERK1/2或其上游信号。调节enos mRNA稳定性的机制仍不清楚。在初步研究中，我们发现层流切应力显著增加eNOS转录本的3‘多聚腺苷；eNOS 3’ 在基本条件下，75nT到160nT的聚(A)尾对剪切力的反应比25nT的聚(A)尾要好。具有较长PolyA尾巴的eNOS转录本比具有较短Poly(A)尾巴的eNOS转录本更稳定，在翻译上也更活跃。这种对RNA3‘加工的调节似乎代表了一种全新的机制，用于调节基因表达和翻译以响应机械刺激。在拟议的研究中，我们计划对这一现象有更多的了解。在第一个目标中，我们将确定eNOS聚腺化信号中的六核苷酸元件，并确定其在剪切诱导的聚腺化中的作用。我们将检测含有野生型序列元件的eNOS构建体的多聚腺苷化和切割效率，并将其与含有规范序列元件的eNOS构建体进行比较。这将使用体外分析和暴露在剪切力下的eNOS-/-细胞进行研究。在目标2中，我们将通过连续删除eNOS聚(A)信号上游的保守序列，并在体外和细胞中检测多聚腺苷酸化效率，来检测其对剪切的反应性。我们有初步数据表明，HMG CoA还原酶抑制剂也能增加eNOS 3‘聚(A)尾长。在目标3中，我们将研究导致这一现象的机制。为此，我们将确定小G蛋白Rho、其靶标Rho激酶和细胞骨架组织在eNOS mRNA 3‘聚(A)尾部加工中的作用。最后，在目标4中，我们将确定运动训练相关的剪切力是否在体内调节eNOS 3‘聚腺苷酸化。在这些实验中，我们将训练野生型小鼠和中国证监会杂合子小鼠(其中eNOS表达不因运动而增加)。总体而言，这些研究有望提供关于机械力和其他刺激如何通过调节mRNA3‘聚(A)尾长来调节基因表达的非常新颖的信息。

An important vascular adaptation to laminar shear stress is increased expression of the endothelial cell nitric oxide synthase (eNOS). This not only occurs in cultured endothelial cells, but it also occurs in vessels exposed to high levels of flow, such as during exercise training. Research from our laboratory has shown that eNOS upregulation in response to shear stress occurs via two divergent pathways, both of which are dependent on the tyrosine kinase cSrc. One pathway leads to a brief one hour increase in transcription that is dependent on a classical ERK1/2 pathway. Following this, continued shear stress leads to a prolonged stabilization of the eNOS mRNA that is independent of ERK1/2 or its upstream signals. The mechanisms regulating eNOS mRNA stability remain poorly defined. In preliminary studies, we have found that laminar shear stress dramatically increases the 3' polyadenylation of eNOS transcripts; eNOS 3' poly(A) tails of 75 nt to 160 nt are expressed in response to shear stress compared to poly(A) tails of <25 nt under basal conditions. ENOS transcripts with longer poly(A) tails are more stable and are translationally more active than those with short poly(A) tails. This modulation of RNA 3' processing seems to represent a completely novel mechanism for regulation of gene expression and translation in response to mechanical stimuli. In the proposed studies, we plan to gain additional insight into this phenomenon. In aim one, we will identify the hexanucleotide element in the eNOS polyadenylation signal and determine its role in shear-induced polyadenylation. We will examine the polyadenylation and cleavage efficiency of eNOS constructs containing wild-type sequence element and compare it to eNOS constructs with canonical sequence element. This will be studied using in vitro assays and in eNOS-/- cells exposed to shear. In aim 2, we will examine conserved sequences upstream to the eNOS poly(A) signal for their responsiveness to shear by making sequential deletions of this region and examining polyadenylation efficiency in vitro and in cells. We have preliminary data suggesting that HMG CoA reductase inhibitors also increase eNOS 3' poly(A) tail length. In aim 3, we will examine mechanisms responsible for this phenomenon. In this aim, we will determine the roles of the small G protein Rho, its target Rho kinase, and cytoskeleton organization in eNOS mRNA 3' poly(A) tail processing. Finally, in aim 4, we will determine if shear stress associated with exercise training modulates eNOS 3' polyadenylation in vivo. In these experiments, we will exercise train wild-type mice and mice heterozygotic for cSrc (in whom eNOS expression is not increased by exercise). Overall, these studies promise to provide very novel information regarding how mechanical forces and other stimuli modulate gene expression via regulation of mRNA 3' poly(A) tail length.