Role of PPARG the PPARG Target Gene RBP7 in the Endothelium

 描述(申请人提供)：PPARγ是一种配体激活的转录因子，因其在调节胰岛素敏感性和脂肪组织的发育和扩张中的作用而闻名。现在有令人信服的证据支持内皮PPARγ在调节内皮功能中发挥重要的有益作用。然而，对于PPARγ的转录靶点，PPARγ如何选择性地激活某些靶点，以及它们在内皮细胞中的作用机制，人们知之甚少。在内皮细胞中选择性表达PPARγ的显性负突变(E-V290M)对年轻转基因小鼠的主动脉、颈动脉或基底动脉的内皮功能没有影响，但在以下情况下会导致严重的功能障碍 这些小鼠受到高脂肪饮食Ang-II的挑战，或者是变老了。大脑循环对氧化应激特别敏感，以响应对PPARγ信号的干扰。相反，内皮特异性野生型PPARγ的表达增加(E-PPARγ-WT)减少了对Ang-II的血管收缩。这些数据有力地表明，在应激条件下，PPARγ对血管内皮细胞具有保护作用，而PPARγ损伤导致的功能障碍是由氧化应激引起的。我们发现视黄醇结合蛋白7(RbP7)是血管内皮细胞中的视黄醇结合蛋白受体γ靶基因。RBP7是一种细胞内视黄醇结合蛋白，属于细胞内脂质和脂肪酸结合蛋白家族。RBP7的表达具有内皮细胞特异性。Rbp7基因缺陷小鼠表现出与E-V290M小鼠相同的高脂饮食诱导和血管紧张素Ⅱ诱导的表型，后者选择性地在内皮细胞中表达PPARγ的显性负突变。这促使我们考虑了一个创新的概念，即PPARγ在内皮中的有益作用可能是由RBP7介导的。据报道，细胞内的脂质和脂肪酸结合蛋白可以结合配体，从而激活其他配体激活的转录因子。因此，从概念上讲，我们假设pparγ和rbp7可能在内皮细胞中形成转录调控环(或中枢)，这是支持抗 氧化状态，当受损时，会导致促氧化状态。我们将从两个具体目标来考察这一概念。具体目标1将检验以下假设：a)野生型PPARγ在内皮细胞过度表达的有利作用需要RBP7；b)RBP7的作用是通过其视黄醇结合和脂质转运活性介导的；以及c)RBP7是PPARγ介导的抗氧化反应所必需的，包括内皮脂联素的表达和功能。特异性目标2将通过检验以下假设来评估RBP7调节PPARγ转录活性的分子机制：RBP7具有内在的视黄醇结合和核转位活性是PPARγ转录活性所必需的。

 DESCRIPTION (provided by applicant): PPARγ is a ligand activated transcription factor best known for its role in regulating insulin sensitivity and adipose tissue development and expansion. There is now convincing evidence to support an important beneficial role for endothelial PPARγ in the regulation of endothelial function. However, little is known about the transcriptional targets for PPARγ, how PPARγ selectively activates some targets over others, and their mechanisms of action in the endothelium. Selective expression of a dominant negative mutant of PPARγ in the endothelium (E-V290M) has no effect on endothelial function in aorta, carotid or basilar arteries in young transgenic mice at baseline, but induces severe dysfunction if the mice are challenged by a high fat diet, Ang-II, or are old. The cerebral circulation is particularly sensitive to oxidative stress in response to the interference with PPARγ signaling. Conversely, increased expression of wildtype PPARγ specifically in the endothelium (E- PPARγ-WT) reduced vasoconstriction to Ang-II. These data strongly suggest that PPARγ plays a protective role in the endothelium under stressed conditions, and the dysfunction resulting from PPARγ impairment is caused by oxidative stress. We identified retinol-binding protein 7 (RBP7) as a PPARγ target gene in the endothelium. RBP7 is an intracellular retinol binding protein belonging to the family of intracellular lipid and fatty acid-binding proteins. Expression of RBP7 is endothelial cell-specific. RBP7-deficient mice exhibit the same high fat diet-induced and Ang-II-induced phenotype as E-V290M mice which selectively express a dominant negative mutant of PPARγ in the endothelium. This led us to consider the innovative concept that the beneficial effects of PPARγ in the endothelium may be mediated by RBP7. Intracellular lipid and fatty acid binding proteins have been reported to bind ligands which activate other ligand activated transcription factors. Thus conceptually, we hypothesize that PPARγ and RBP7 may form a transcriptional regulatory loop (or hub) in endothelial cells, which is required to support an anti oxidant state and when impaired induces a pro-oxidant state. We will examine this concept in two Specific Aims. Specific Aim 1 will test the hypotheses that a) the beneficial effects of wildtype PPARγ over-expression in the endothelium require RBP7, b) that the effects of RBP7 are mediated by its retinol-binding and lipid transport activity, and c) RBP7 is required for the PPARγ- mediated anti-oxidant response, including the expression and function of endothelial adiponectin. Specific Aim 2 will evaluate the molecular mechanisms by which RBP7 regulates PPARγ transcriptional activity by testing the hypothesis that RBP7 with its intrinsic retinol binding and nuclear translocation activity is required for transcriptional activity of PPARγ.