Glucocorticoid-regulated transcription networks in macrophage biology

描述(由申请人提供)：肥胖、胰岛素抵抗和2型糖尿病的流行及其相关的共病是公共卫生中的一个令人望而生畏的问题。代谢性疾病涉及一种强烈的炎症成分，其中脂肪组织巨噬细胞(M？)起着关键的致病作用。糖皮质激素(GCs)仍然是治疗炎症性疾病最常见和最具成本效益的一类药物，但GCs本身是糖尿病原性的，这阻碍了它们的治疗效果。在M？中，GCs广泛抑制促炎介质的产生；此外，GCs促进表现为抗炎表型的“交替激活”M2 M？的极化，并矛盾地增加胰岛素敏感性。解开GCs全身性和M2特异性代谢活动之间的二分法，对于理解胰岛素抵抗的发病机制和创造具有改进治疗方案的抗炎药物至关重要。GCS通过GC受体(GR)发出信号，GR是核受体(NR)超家族的一种配体调节的转录因子，它招募许多辅助调节因子来激活或抑制转录。其中，GR相互作用蛋白(GRIP)1是一种已被证实的NR辅活化子，我们最近发现它是一种关键的GR辅阻遏子，可以减弱M？的炎症基因表达程序。出乎意料的是，GRIP1还被发现与免疫系统中的非受体调节因子合作。是什么使GRIP1能够选择性地参与拮抗的生物学途径或显示相反的转录活动尚不清楚。最近，我们发现GRIP1经历了GC诱导的GR相互作用依赖的多个位点的磷酸化，这是诱导一组GR靶点所必需的。本应用的目的是分析GR诱导的GRIP1在M？中的磷酸化作为GCs抗炎和代谢效应的调节器的作用和机制。我们的中心假设是，连接的GR通过使GC反应元件(GRE)特异性地招募GRIP1激酶来修改自己的辅助调节因子，从而决定GRIP1在不同的GR转录复合体中的功能，或者优先利用其激活和抑制特性。我们的具体目标是：1)利用我们的M？特异性条件性GRIP1缺陷小鼠和GRIP1亚磷酸盐特异性抗体来评估GRIP1磷酸异构体在M？中的全基因组分布并确定功能相关的结合位点；2)剖析推测的GRIP1激酶、细胞周期蛋白依赖的激酶-9和酪蛋白激酶-2作为GRE特异性转录复合体的功能；3)(通过阻断内源性GRIP1的磷酸化或将GRIP1亚磷酸突变体整合到GRIP1缺失的细胞中)确定GRIP1磷酸化在组装和GR转录复合体调控基因中的作用，以及在M？极化和M：脂肪细胞相互作用中的作用。这项工作将提供有关GR介导的基因表达的特异性的新机制的详细信息，这将揭示代谢控制的一个重要方面，并为设计更安全的炎症性疾病治疗方法提供新的机会。

DESCRIPTION (provided by applicant): The epidemic of obesity, insulin resistance and type 2 diabetes with associated co-morbidities is a daunting problem in public health. Metabolic disease involves a strong inflammatory component in which adipose tissue macrophages (M¿) play a critical pathogenic role. Glucocorticoids (GCs) remain the most common and cost-effective class of medications for managing inflammatory diseases however, GCs are themselves diabetogenic, which hampers their therapeutic utility. In M¿, GCs broadly inhibit the production of proinflammatory mediators; moreover, GCs promote the polarization of 'alternatively activated' M2 M¿ that display an anti-inflammatory phenotype, and, paradoxically, increase insulin sensitivity. Untangling the dichotomy between the systemic vs. M2-specific metabolic actions of GCs is crucial for understanding the pathogenesis of insulin resistance and for creating anti-inflammatory drugs with improved therapeutic profiles. GCs signal through the GC receptor (GR) a ligand-regulated transcription factor of the nuclear receptor (NR) superfamily that recruits numerous coregulators to activate or repress transcription. Among those, the GR-interacting protein (GRIP) 1 is an established NR coactivator, that we have recently shown to serve as a key GR corepressor attenuating the inflammatory gene expression program in M¿. Unexpectedly, GRIP1 was also found to cooperate with non-receptor regulators in the immune system. What enables GRIP1 to selectively engage in antagonistic biological pathways or display opposite transcriptional activities is unknown. Recently, we found that GRIP1 undergoes GC-induced, GR interaction-dependent phosphorylation at multiple sites that was required for the induction of a subset of GR targets. The objective of this application is to dissect the role and mechanisms of GR-induced GRIP1 phosphorylation in M¿ as a modulator of anti-inflammatory and metabolic effects of GCs. Our central hypothesis is that liganded GR modifies its own coregulator by enabling GC-response element (GRE)-specific recruitment of GRIP1 kinases, thus dictating GRIP1 function in distinct GR transcription complexes, or preferential utilization of its activating vs. repressing properties. Our Specific Aims are to: 1) utilize our M¿-specific conditional GRIP1-deficient mice and GRIP1 phosphosite-specific antibodies to assess the genome-wide distribution of GRIP1 phospho-isoforms in M¿ and determine the functionally relevant binding sites; 2) dissect the function of putative GRIP1 kinases, cyclin-dependent kinase-9 and casein kinase-2, as GRE-specific components of GR transcription complexes; 3) identify (by blocking phosphorylation of endogenous GRIP1 or integrating GRIP1 phosphosite mutants into GRIP1-null cells) the role of GRIP1 phosphorylation in the assembly and gene regulation by GR transcription complexes, as well as in M¿ polarization and the M¿:adipocyte interactions. This work will yield detailed information on a novel mechanism contributing specificity to GR-mediated gene expression, which should reveal an important facet of metabolic control and new opportunities for the design of safer therapies for inflammatory diseases.