Post-transcriptional Regulation of NO Synthase

一个重要的血管适应层流剪切应力的内皮细胞一氧化氮合酶（eNOS）的表达增加。这不仅发生在培养的内皮细胞中，而且也发生在暴露于高水平血流的血管中，例如在运动训练期间。我们实验室的研究表明，eNOS上调响应剪切应力发生通过两个不同的途径，这两个都是依赖于酪氨酸激酶cSrc。一种途径导致依赖于经典ERK 1/2途径的转录短暂增加1小时。在此之后，持续的剪切应力导致eNOS mRNA的长期稳定化，其独立于ERK 1/2或其上游信号。调节eNOS mRNA稳定性的机制仍然不清楚。在初步研究中，我们发现层流剪切应力显著增加eNOS转录物的3'多聚腺苷酸化; eNOS 3'多聚腺苷酸化是eNOS转录物的一个重要特征。 与基础条件下<25 nt的聚（A）尾相比，75 nt至160 nt的聚（A）尾响应于剪切应力而表达。具有较长poly（A）尾的ENOS转录物比具有短poly（A）尾的那些转录物更稳定并且在功能上更有活性。RNA 3'加工的这种调节似乎代表了一种响应机械刺激的基因表达和翻译调节的全新机制。在拟议的研究中，我们计划进一步深入了解这一现象。在目标一，我们将确定eNOS多聚腺苷酸化信号中的六核苷酸元件，并确定其在剪切诱导的多聚腺苷酸化中的作用。我们将研究含有野生型序列元件的eNOS构建体的多聚腺苷酸化和切割效率，并将其与具有典型序列元件的eNOS构建体进行比较。这将使用体外测定和在暴露于剪切的eNOS-/-细胞中进行研究。在目标2中，我们将检查保守序列上游的eNOS聚（A）信号的剪切反应，使该区域的顺序删除，并检查在体外和细胞中的聚腺苷酸化效率。我们有初步的数据表明HMG CoA还原酶抑制剂也增加eNOS 3' poly（A）尾长。在目标3中，我们将审查造成这一现象的机制。在这个目标中，我们将确定小G蛋白Rho，其目标Rho激酶，和细胞骨架组织eNOS mRNA 3' poly（A）尾加工的作用。最后，在目标4中，我们将确定与运动训练相关的剪切应力是否在体内调节eNOS 3'聚腺苷酸化。在这些实验中，我们将运动训练野生型小鼠和cSrc杂合子小鼠（其中eNOS表达不因运动而增加）。总之，这些研究有望提供关于机械力和其他刺激如何通过调节mRNA 3' poly（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.