GPCR Kinase-2 in TNFalpha Signaling in Macrophages

描述（申请人提供）：巨噬细胞是参与包括动脉粥样硬化在内的许多炎症性疾病发病的重要免疫细胞，是动脉粥样硬化病变的主要免疫细胞之一。巨噬细胞除了在血管壁上摄取修饰脂蛋白后成为泡沫细胞外，还通过产生细胞因子、趋化因子和生长因子介导与动脉粥样硬化斑块形成相关的炎症反应。这些药物进一步影响更多单核细胞和其他免疫细胞的化学吸引，并深刻影响附近平滑肌细胞和内皮细胞的功能。因此，巨噬细胞在动脉粥样硬化的各个阶段都起着至关重要的作用，从脂肪条纹到晚期斑块，最终到斑块破裂。因此，了解巨噬细胞生物学（包括炎症介质产生和巨噬细胞凋亡/存活）调控的生化机制是非常关键的。初步数据表明，巨噬细胞生物学受丝氨酸/苏氨酸激酶，即g蛋白偶联受体激酶-2 （GRK2）的调控。GRK2最初是因其在-肾上腺素能受体和其他gpcr的磷酸化中的作用而被发现的。最近的研究表明，GRK2还有其他细胞质靶点。然而，GRK2在巨噬细胞中的作用，特别是与非gpcr和细胞质靶标相关的作用尚不清楚。初步数据表明，肿瘤坏死因子- (TNF)诱导的NFB信号，以及随之而来的巨噬细胞炎症介质的表达和存活受到GRK2的关键调控。初步研究结果还表明，GRK2通过与NFB信号抑制剂IB的相互作用和潜在的磷酸化来调节其对NFB信号的影响。这些结果表明，该激酶在巨噬细胞生物学中具有新的重要作用，因此在动脉粥样硬化的发病机制中具有重要作用。本研究的目的是进一步扩展我们的初步研究结果，并检查GRK2调节巨噬细胞生物学的机制，重要的是，也测试GRK2的丢失是否影响动脉粥样硬化的病理生理。总的假设是GRK2与IB的相互作用和磷酸化调节tnf诱导的NFB信号、炎症介质的产生和巨噬细胞的存活/凋亡，因此在动脉粥样硬化的发病机制中起着至关重要的作用。为了验证这一假设，我们将研究以下具体目标：1。确定GRK-2调控巨噬细胞tnf诱导的NFB通路的机制。2. 研究巨噬细胞中GRK2在TNF信号传导中的功能相关性。3. 确定GRK2的缺失是否影响Apo-E敲除小鼠动脉粥样硬化的发病机制。综上所述，我们的研究应该为巨噬细胞中TNF信号传导的机制提供重要的见解，并确定慢性炎症性疾病治疗的潜在治疗靶点。公共卫生相关性：tnf - α在包括动脉粥样硬化在内的慢性炎症性疾病的发展中起重要作用。因此，了解调节tnf - α信号的各种机制将有助于我们找到治疗动脉粥样硬化的新的治疗靶点。

DESCRIPTION (provided by applicant): Macrophages are important immune cells involved in the pathogenesis of many inflammatory diseases, including atherosclerosis and are one of the major immune cells in the atherosclerotic lesions. In addition to becoming foam cells upon uptake of modified lipoproteins in the vessel walls, macrophages also mediate the inflammatory responses that are associated with atherosclerotic plaque formation by producing cytokines, chemokines and growth factors. These agents further influence chemoattraction of more monocytes and other immune cells as well as profoundly affect the functions of smooth muscle cells and endothelial cells that are in the vicinity. Therefore, macrophages play an essential role in all stages of atherogenesis including fatty streaks to advanced plaques and eventually to plaque rupture. Thus, understanding the biochemical mechanisms by which macrophage biology (including inflammatory mediator production, and macrophage apoptosis/survival) is regulated is highly critical. Preliminary data demonstrates that macrophage biology is regulated by a serine/threonine kinase, namely, G-protein coupled receptor kinase-2 (GRK2). GRK2 was originally discovered for its role in the phosphorylation of -adrenergic receptor and other GPCRs. Recent studies indicate that there are other cytosolic targets of GRK2. However, the role of GRK2 in macrophages, particularly as it relates to non-GPCRs and cytosolic targets is not well known. Preliminary data demonstrates that tumor necrosis factor- (TNF)-induced NFB signaling, and the consequent inflammatory mediator expression and survival of macrophages are critically regulated by GRK2. Preliminary findings also suggest that GRK2 mediates its effects on NFB signaling via interaction with, and potentially phosphorylation of the inhibitor of NFB signaling namely IB. These results suggest a novel and significant role for this kinase in macrophage biology and therefore in the pathogenesis of atherosclerosis. The objective of this proposal is to further expand on our preliminary findings and examine the mechanisms by which GRK2 regulates macrophage biology and importantly, also test if, loss of GRK2 affects the pathophysiology of atherosclerosis. The overall hypothesis is that GRK2 interaction with and phosphorylation of IB regulates TNF-induced NFB signaling, inflammatory mediator production, and macrophage survival/apoptosis and therefore, plays a crucial role in the pathogenesis of atherosclerosis. To test this hypothesis we will examine the following specific aims: 1. Determine the mechanisms by which GRK-2 regulates TNF-induced NFB pathway in macrophages. 2. Examine the functional relevance of GRK2 in TNF signaling in macrophages. 3. Determine whether loss of GRK2 affects the pathogenesis of atherosclerosis in Apo-E knockout mice. Taken together, our studies should provide important insight into the mechanisms of TNF signaling in macrophages as well as identify potential therapeutic targets in the treatment of chronic inflammatory diseases. PUBLIC HEALTH RELEVANCE: TNF-alpha plays a major role in the development of chronic inflammatory diseases including atherosclerosis. Therefore, understanding the various mechanisms by which TNF-alpha signaling is regulated will help us identify new and novel therapeutic targets to treat atherosclerosis.