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PREGNANCY CHANGES IN UTERINE ARTERY SIGNALING PATHWAYS

妊娠期子宫动脉信号通路的变化

基本信息

批准号：
6700771
负责人：
IAN M. BIRD
金额：
$ 21.83万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-02-01 至 2005-01-31
项目状态：
已结题

项目摘要

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.

在妊娠期间，子宫动脉的激动剂诱导的NO以及PGI 2产生显著增强，从而导致血管阻力降低和支持生长中的胎儿所需的血流增加。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揭示，虽然不是所有这些激动剂都可以动员这些细胞中的Ca 2+，但七螺旋受体和生长因子受体活化都导致P-UAEC中细胞外信号调节蛋白激酶1和2（ERK-1/2）的活化，并且对于每一类激动剂，这与它们刺激NO产生的能力直接相关，特别是（见下文）。此外，在NP-UAEC中的平行研究已经显示了广泛的激动剂，包括ATP、AII和TPA，不能刺激NO产生或有效地偶联到ERK-1/2活化，否则在P-UAEC中有效偶联。进一步鉴定和理解MAPK细胞信号通路的偶联和功能，以及这种变化在妊娠中的作用是本提案的目标。此外，尚未鉴定的信号传导途径也可能介导生长因子而不是NP-UAEC中的七螺旋受体效应以产生NO，因此反过来，妊娠诱导的UAEC功能变化可能涉及与未鉴定的ERK-1/2独立途径的可能协同作用，这是由于与ERK-1/2活化的偶联增加。为此，我们建议：具体目标1：确定bFGF或VEGF，而不是AII或ATP对MEK介导的ERK-1/2活化的作用是否通过生长因子受体自磷酸化和随后的衔接蛋白（Shc/Grb 2/SOS）的磷酸酪氨酸募集导致P-UAEC中MEK的Ras/Raf活化而发生，以及在NP-UAEC中这是在什么水平解偶联的。具体目标二：确定AII和ATP介导的MEK活化是否通过独立于生长因子受体磷酸化和衔接蛋白募集的替代途径发生，即通过P-UAEC中的Src/Raf/MEK级联反应，以及在NP-UAEC中该反应在何种水平下解偶联。具体目标3：进一步描述从非妊娠状态到妊娠状态的细胞信号传导过程中发生的变化，从而进一步阐明这些变化的机制基础。这将由以下人员进行调查：（3A）进一步比较P-UAEC与NP-UAEC中响应于bFGF、VEGF、A11、ATP和佛波醇酯（TPA -对照）的Shc/Grb 2/SOS/Ras/Raf途径和Src/Raf/MEK途径的活化的时程，以及（3B）进一步研究NP-UAEC和P-UAEC中可能的经由P13-激酶/AKT的MEK非依赖性信号传导。具体目标4：通过观察选择性抑制剂对信号传导中间体活性的影响，并将任何此类影响分别与NO产生和eNOS磷酸化的变化相关联，在NP-UAEC和P-UAEC中激动剂刺激的NO产生水平上确定目标1-3中如此鉴定的这些信号传导中间体的重要性。具体目的5：（5A）将这些观察到的变化与妊娠诱导的体内变化联系起来。因此，在先前的具体目标1-4中，信号传导途径中间体已显示在功能水平上丢失或获得，或者在5 B中编码信号传导中间体的mRNA改变的情况下，我们将对UA内皮进行western分析，并对来自子宫内膜、卵泡期、黄体期和妊娠母羊的UA切片进行免疫组织化学，或RT-PCR定量来自相同动物的UA-内皮细胞mRNA，以确定体内是否发生表达变化。(5B)使用最近开发的分子技术来研究P-UAEC与NP-UAEC的mRNA种类的进一步可能的差异，从而鉴定额外的信号分子以及由妊娠诱导和/或丢失的其他因子，但此时可能尚未被鉴定。我们相信，这些研究将提供一个显着的进步，在正常控制UA内皮功能的理解和机制的基础上妊娠诱导增加的各种血管收缩剂在怀孕期间的顽固性，也可能提供一个新的模型内皮细胞功能，未来的研究寻找功能障碍导致先兆子痫和IUGR可能是基于。