Role of 12/15-lipoxygenase pathway in implantation

描述(申请人提供)：本提案的目的是研究12/15-脂氧合酶(12/15-LOX)酶产生的脂质介质在着床期子宫功能控制中的功能作用。12/15-LOX催化20碳多不饱和脂肪酸、花生四烯酸和亚油酸的立体特定氧化反应，生成一系列复杂的衍生物HETE和HODES。最近利用高密度寡核苷酸芯片的研究表明，孕酮在着床时能显著诱导妊娠子宫表面上皮中编码12/15-LOX家族成员白细胞-12/15-LOX和表皮-12/15-LOX的mRNAs的合成。在小鼠延迟着床过程中，给予LOX特异性抑制剂显著抑制类固醇激素调节的血管通透性，表明这些酶在创建接受性子宫方面发挥了重要作用。这一建议的主要目的是：1.确定在植入过程中这些脂质代谢产物在子宫中的生理作用的基因网络。12/15-LOX调控的基因通路将通过DNA微阵列进行鉴定。将分析这些基因在植入期间子宫中的时空表达，并将通过新开发的反义寡核苷酸策略来确定它们的功能。2.探讨着床期PPARGamma在12/15-LOX信号通路中的作用。在基于细胞的分析中，花生四烯酸或亚油酸的12/15-LOX代谢物通过过氧化物酶体增殖物激活受体伽马(PPARGamma)激活基因转录。PPARGamma的表达也在着床期的子宫中被诱导。我们将测试PPARGamma是否确实是植入围植入期子宫中12/15-LOX衍生代谢物的内源性受体，并确定其在该组织中的靶基因。PPARGamma的作用将通过在子宫中产生其基因的条件敲除以及通过分析该受体在植入过程中功能丧失突变的功能后果来进一步研究。总的来说，拟议的实验将检验一种新的信号通路，涉及12/15-LOX衍生的脂质介质PPARGamma及其下游靶基因，调节着床过程中的关键事件。

DESCRIPTION (provided by applicant): The objective of this proposal is to investigate the functional role of the lipid mediators generated by the 12/15-lipoxygenase (12/15-LOX) enzymes in the control of uterine function during implantation. The 12/15-LOX catalyzes the stereo-specific oxygenation of the 20-carbon polyunsaturated fatty acids, arachidonic acid and linoleic acid, into a complex series of derivatives, HETEs and HODES. Recent studies, using high-density oligonucleotide microarrays, revealed that progesterone markedly induces the synthesis of mRNAs encoding the 12/15-LOX family members, leukocyte-12/15-LOX and epidermal-12/15-LOX, in the surface epithelium of pregnant uterus precisely at the time of implantation. Administration of a LOX-specific inhibitor markedly inhibited steroid hormone-regulated vascular permeability during delayed implantation in mice, indicating an important role of these enzymes in creating a receptive uterus. The major goals of this proposal are to: 1. Identify the gene networks that mediate the physiological effects of these lipid metabolites in the uterus during implantation. The 12/15-LOX-regulated gene pathways will be identified by DNA microarray. The spatio-temporal expression of these genes in the peri-implantation uterus will be analyzed and their function will be determined by a newly developed antisense oligonucleotide strategy. 2. Investigate the role of PPARgamma in the 12/15-LOX signaling pathway during implantation. The 12/15-LOX-derived metabolites of arachidonic or linoleic acid activate gene transcription by the peroxisome proliferator-activated receptor gamma (PPARgamma) in cell-based assays. PPARgamma expression is also induced in the uterus in the peri-implantation period. We will test whether PPARgamma is indeed the endogenous receptor for the 12/15-LOX-derived metabolites in the peri-implantation uterus and identify its target genes in this tissue. The role of PPARgamma will be further investigated by generating a conditional knockout of its gene in the uterus and by analyzing the functional consequences of the loss-of-function mutation of this receptor during implantation. Collectively, the proposed experiments will test the hypothesis that a novel signaling pathway, involving 12/15-LOX-derived lipid mediators, PPARgamma, and its downstream target genes, regulates critical events during implantation.