PREGNANCY CHANGES IN UTERINE ARTERY SIGNALING PATHWAYS

在怀孕期间，激动剂诱导的NO以及子宫动脉产生的PGI2显著增加，从而导致血管阻力减少，支持发育的胎儿所需的血流量增加。比较七螺旋受体激动剂AII和ATP与生长因子bFGF和VEGF在P-UAEC中的作用的研究进一步表明，虽然不是所有这些激动剂都能在这些细胞中动员钙离子，但七螺旋受体和生长因子受体激活都会导致P-UAEC细胞外信号调节蛋白激酶1和2(ERK-1/2)的激活，并且对于每类激动剂来说，这与它们刺激NO产生的能力直接相关(见下文)。此外，在NP-UAEC中的平行研究表明，广泛的激动剂，包括ATP，AII和TPA，不能刺激NO的产生或有效地偶联ERK-1/2激活，否则在P-UAEC中会有效偶联。进一步识别和了解MAPK细胞信号通路的偶联和功能，以及这种信号通路在妊娠中的变化是本提案的目标。此外，目前尚不清楚的信号通路也可能介导生长因子而不是七螺旋受体效应在NP-UAEC中产生NO，因此妊娠引起的UAEC功能的变化可能涉及与未知的ERK-1/2独立通路的协同作用，这是由于与ERK-1/2激活的偶联增加。为此，我们提出：特定的目标1：确定在P-UAEC中，是否通过生长因子受体自身磷酸化和随后的磷酸化酪氨酸募集(Shc/Grb2/SOS)导致MEK的RAS/Raf激活，从而在NP-UAEC中解偶联MEK，从而确定在MEK介导的ERK-1/2激活中，bFGF或VEGF而不是AII或ATP的作用是否发生。具体目的2：确定AII和ATP是否通过与生长因子受体磷酸化和接头蛋白募集无关的替代途径，即通过P-UAEC中的Src/Raf/MEK级联，以及在NP-UAEC中这种反应在何种水平上解偶联来实现MEK的激活。具体目的3：进一步阐明细胞信号从非妊娠到妊娠过程中的变化，从而进一步阐明这些变化的机制基础。这将通过(3A)进一步比较P-UAEC和NP-UAEC中bFGF、VEGF、ALL、ATP和佛波酯对Shc/Grb2/SOS/RAF/Raf和Src/Raf/MEK通路激活的时间进程，以及(3B)进一步研究在NP-UAEC和P-UAEC中可能通过P13-Kinase/AKT不依赖MEK的信号转导途径。具体目的4：通过观察选择性抑制剂对Np-UAEC和P-UAEC信号中间产物活性的影响及其与NO产生和eNOS磷酸化的关系，确定AIMS 1-3中确定的这些信号中间产物在激动剂刺激的NP-UAEC和P-UAEC中产生NO的重要性。具体目标5：(5A)将这些观察到的变化与体内妊娠引起的变化联系起来。因此，如果在以前的特定目标1-4中已显示信号通路中间产物在功能水平上丢失或获得，或者在5B中编码信号中间产物的mRNA发生改变，我们将对UA内皮进行蛋白质分析，并对卵泡、卵泡期、黄体期和怀孕绵羊的UA切片进行免疫组织化学分析，或者对同一动物的UA-内皮细胞mRNA进行RT-PCR定量，以确定是否在体内发生了表达变化。(5B)利用最近发展的分子技术进一步研究P-UAEC和NP-UAEC在mRNA种类上的可能差异，从而确定其他信号分子以及其他因怀孕而诱导和/或丢失但目前可能尚未确定的因素。我们相信，这些研究将在了解UA内皮功能的正常控制和导致妊娠期间对多种血管收缩药物的耐受性增加的潜在机制方面取得重大进展，并可能提供一种新的内皮细胞功能模型，未来可能会在此基础上寻找导致子痫前期和IUGR的功能障碍。

During pregnancy, there is a dramatic enhancement of agonist induced NO as well as PGI2 production by the uterine artery, so leading to a reduction in vascular resistance and increase in blood flow necessary to support the growing fetus. Studies comparing the effects of heptahelical receptor agonists AII and ATP vs growth factors bFGF and VEGF in P- UAEC have further revealed that while not all these agonists can mobilize Ca2+ in these cells, both heptahelical receptors and growth factor receptor activation results in activation of Extracellular signal Regulated Protein Kinase 1 and 2 (ERK-1/2) in P-UAEC, and for each class of agonist this correlates directly to their ability to stimulate NO production in particular (see below). In addition, parallel studies in NP- UAEC have shown a broad range of agonists, including ATP, AII and TPA, fail to stimulate NO production or couple efficiently to ERK-1/2 activation that otherwise couple efficiently in P-UAEC. Further identification and understanding of coupling to and function of the MAPK cell signaling pathways, and how this changes in pregnancy is the goal of this proposal. Furthermore, as yet unidentified signaling pathways may also be mediating the growth factor but not heptahelical receptor effects in NP-UAEC to produce NO, and therefore in turn that pregnancy induced changes in UAEC function may involve a possible synergy with unidentified ERK-1/2 independent pathways due to increased coupling to ERK-1/2 activation. To this end we propose: Specific Aim 1: to establish if the action of bFGF or VEGF, but not AII or ATP on MEK mediated activation of ERK-1/2 occurs via growth factor receptor autophosphorylation and subsequent phosphotyrosine recruitment of adapter proteins (Shc/Grb2/SOS) leading to Ras/Raf activation of MEK in P-UAEC and at what level this is uncoupled in NP-UAEC. Specific Aim 2: to establish if AII and ATP mediated activation of MEK occurs through alternate pathways independent of growth factor receptor phosphorylation and adapter protein recruitment, namely via the Src/Raf/MEK cascade in P-UAEC and at what level this response is uncoupled in NP-UAEC. Specific Aim 3: to further delineate the changes which occur in cell signaling in progression from the nonpregnant to pregnant state and so further clarify the mechanistic basis for these changes. This will be investigated by: (3A) further comparing the time course of activation of the Shc/Grb2/SOS/Ras/Raf pathway and the Src/Raf/MEK pathway in response to bFGF, VEGF, All, ATP and phorbol ester (TPA -control) in P-UAEC vs NP-UAEC, and (3B) to further investigate possible MEK independent signaling via P13- kinase/AKT in NP-UAEC and P-UAEC. Specific Aim 4: To establish the importance of these signaling intermediates so identified in Aims 1-3 at the level of agonist-stimulated NO production in NP-UAEC and P-UAEC by observing the effects of selective inhibitors on activity of signaling intermediates and relating any such effects to changes in NO production and eNOS phosphorylation respectively. Specific Aim 5:(5A) to relate these observed changes back to the pregnancy induced changes in vivo. Thus where signaling pathway intermediates have been shown to be lost or gained at the level of function in previous specific aims 1-4, or where mRNA encoding a signaling intermediate is altered in 5B, we will perform western analysis on UA endothelium and immunohistochemistry on UA sections from ovex, follicular phase, luteal phase and pregnant ewes, or RT-PCR quantification of UA-endothelial cell mRNA from the same animals to determine if a change in expression occurs in vivo. (5B) to use recently developed molecular techniques to investigate further possible differences in mRNA species from P-UAEC vs NP-UAEC and so identify additional signaling molecules as well as other factors which are induced and/or lost by pregnancy but may not yet be identified as such at this time. We believe these studies will provide a significant advance in both the understanding of normal control of UA endothelial function and the mechanism underlying pregnancy-induction of increased refractoriness to a variety of vasoconstrictors in pregnancy, and may also provide a new model of endothelial cell function on which future studies searching for the dysfunction leading to preeclampsia and IUGR may be based.