Transcriptional Repression of PPARgamma-2 by Glucocorticoid-Induced Leucine Zippe

描述(申请人提供)：研究目标和意义：骨质疏松症和肥胖是美国主要的公共健康威胁。然而，潜在的机制，以及这些看似无关的疾病是否有共同的因素，都是未知的。在衰老过程中，骨量会减少。与此同时，脂肪在腹部和骨髓等非脂肪组织中积聚，这表明成骨和成脂分化途径之间可能会出现失衡。PPAR(2)，也被称为脂肪生成的主调节因子，正向调节脂肪生成，但对成骨有相反的作用。我们的长期目标是确定PPAR(2)控制成脂和成骨途径的分子机制。本研究旨在探讨糖皮质激素诱导的亮氨酸拉链蛋白(GILZ)如何抑制PPAR(2)的表达。由于过量的腹部脂肪增加了冠状动脉疾病的风险并导致胰岛素抵抗，这两者都是主要的公共健康威胁，了解PPAR(2)的表达如何被负向调控将不仅为治疗骨质疏松症提供解决方案，也将为肥胖和肥胖相关疾病提供解决方案。 原理和假设：我们和其他人已经证明，PPAR(2)的表达是通过CCAAT/增强子结合蛋白(C/EBPs)与PPAR(2)启动子的直接结合而激活的。相比之下，人们对PPAR(2)如何受到负面调控知之甚少。我们最近的研究表明，GILZ可以抑制PPAR(2)转录，抑制成脂分化，促进骨髓前体细胞的成骨分化，骨髓前体细胞是脂肪、成骨脂肪细胞和成骨细胞的共同前体。根据这些研究和我们最近的初步数据显示，GILZ与C/EBPs相互作用并抑制C/EBP介导的PPAR(2)表达，我们假设GILZ通过抑制C/EBPs与PPAR(2)启动子的结合或通过GILZ-C/EBP相互作用破坏C/EBP的反式激活功能来抑制PPAR(2)的转录。基于最近的证据，1)PPAR(2)抑制Runx2/Cbfa1的表达和成骨细胞分化；2)PPAR(激动剂)诱导骨丢失和骨髓肥大；以及3)PPAR(不足)促进成骨和增加骨形成，并保护小鼠免受饮食诱导的肥胖和胰岛素抵抗的影响，我们还假设，GILZ在体内的过表达可降低PPAR(2)的表达水平，从而降低小鼠对PPAR(激动剂引起的骨丢失和骨髓肥胖)的易感性，从而防止高脂饮食诱导的肥胖和胰岛素抵抗。 具体目标和方法：我们的具体目标是使用最先进的分子生物学方法测试上述两个假说，包括DNA-蛋白质和蛋白质-蛋白质相互作用、转录活性和基因敲除方法(Aim#1，体外研究)，并使用我们实验室最近培育的GILZ转基因小鼠(Aim#2，体内研究)来验证我们在Aim#1中开发的假说。与公共健康相关：骨质疏松症和肥胖是美国主要的公共健康威胁。然而，潜在的机制，以及这些看似无关的疾病是否有共同的因素，都是未知的。在衰老过程中，骨量减少，腹部和骨髓中脂肪积累，导致骨质疏松症和与体内脂肪过多相关的疾病，如胰岛素抵抗和冠状动脉血管疾病。过氧化物酶体增殖物激活受体-γ2(PPAR(2))也被称为脂肪生成的“主”调节因子，它正向调节脂肪细胞的分化，但对成骨细胞的分化有相反的作用。因此，了解PPAR(2)如何被负调控，这是本研究的重点，可能会导致治疗骨质疏松症和肥胖症的新疗法。

DESCRIPTION (provided by applicant): Research Goals and Significance: Osteoporosis and obesity are major public health threats in the US. However, the underlying mechanisms, and whether these seemingly unrelated diseases have a common element, are unknown. During aging, bone mass decreases. At the same time, fat builds up in non-adipose tissues such as belly and bone marrow, suggesting that an imbalance may develop between the osteogenic and adipogenic differentiation pathways. PPAR(2, also known as the "master" regulator of adipogenesis, positively regulates adipogenesis but has the opposite effect on osteogenesis. Our long-term goal is to determine the molecular mechanisms by which PPAR(2 controls both adipogenic and osteogenic pathways. The purpose of this research is to address the question of how PPAR(2 expression can be repressed by glucocorticoid-induced leucine zipper (GILZ), a recently identified leucine zipper protein. Since excess belly fat increases the risks of coronary disease and causes insulin resistance, both of which are major public health threats, understanding how PPAR(2 expression can be negatively regulated will provide solutions not only for treating osteoporosis, but obesity and obesity-related disorders as well. Rationale and Hypotheses: We and others have shown that the expression of PPAR(2 is activated by direct binding of the CCAAT/enhancer-binding proteins (C/EBPs) to the PPAR(2 promoter. In contrast, very little is known about how PPAR(2 can be negatively regulated. Our recent studies show that GILZ can suppress PPAR(2 transcription, inhibit adipogenic differentiation, and enhance osteogenic differentiation of bone marrow progenitor cells, the common precursors of fat- and bone-forming adipocytes and osteoblasts. Based on these studies and our recent preliminary data showing that GILZ interacts with the C/EBPs and inhibits C/EBP-mediated PPAR(2 expression, we hypothesize that GILZ represses PPAR(2 transcription by either inhibiting the binding of C/EBPs to the PPAR(2 promoter or by disrupting C/EBP transactivation functions via GILZ-C/EBP interactions. Based on recent evidence that 1) PPAR(2 inhibits Runx2/Cbfa1 expression and osteoblast differentiation; 2) PPAR( agonists induce bone loss and marrow adiposity; and 3) PPAR( insufficiency enhances osteogenesis and increases bone formation, and protects mice against diet- induced obesity and insulin resistance, we also hypothesize that GILZ overexpression in vivo will reduce the level of PPAR(2 expression, thus reducing the susceptibility of mice to PPAR( agonist-induced bone loss and marrow adiposity, and protecting against high-fat diet-induced obesity and insulin resistance. Specific Aims and Approaches: Our Specific Aims are to test the above two hypotheses using state-of-the- art molecular biology approaches, including DNA-protein and protein-protein interactions, transcriptional activities, and knockdown approaches (Aim #1, in vitro studies), and to validate our hypothesis developed in Aim #1 using GILZ transgenic mice that have been recently generated in our laboratory (Aim #2, in vivo studies). PUBLIC HEALTH RELEVANCE: Osteoporosis and obesity are major public health threats in the US. However, the underlying mechanisms, and whether these seemingly unrelated diseases have a common element, are unknown. During aging, bone mass decreases and fat builds up in belly and bone marrow, resulting in osteoporosis and the diseases that are associated with excess body fat, such as insulin resistance and coronary vascular diseases. Peroxisome proliferators-activated receptor-gamma2 (PPAR(2), also known as the "master" regulator of adipogenesis, positively regulates adipocyte differentiation but has the opposite effect on osteoblast differentiation. Thus, understanding how PPAR(2 can be negatively regulated, a focus of this study, may lead to new therapies for treating osteoporosis and obesity.