Glucocorticoid-regulated transcription networks in macrophage biology

描述（由申请人提供）：肥胖、胰岛素抵抗和2型糖尿病及其相关并发症的流行是公共卫生领域一个令人生畏的问题。代谢性疾病涉及强烈的炎症成分，其中脂肪组织巨噬细胞（MΦ）起关键的致病作用。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，然而，GCs本身是致糖尿病的，这阻碍了它们的治疗效用。在MΦ中，GCs广泛抑制促炎介质的产生；此外，GCs促进“选择性激活”M2 MΦ的极化，显示出抗炎表型，并且矛盾的是，增加胰岛素敏感性。解开GCs的系统性与m2特异性代谢作用之间的对立对于理解胰岛素抵抗的发病机制和创造具有改善治疗效果的抗炎药物至关重要。GC通过GC受体（GR）发出信号，GC受体是核受体（NR）超家族的一种配体调节的转录因子，可招募许多共调节因子来激活或抑制转录。其中，GR相互作用蛋白（GRIP） 1是一种已建立的NR共激活因子，我们最近发现它是一种关键的GR共抑制因子，可减弱MΦ中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选择性地参与拮抗生物学途径或显示相反的转录活性尚不清楚。最近，我们发现GRIP1在多个位点经历gc诱导的GR相互作用依赖性磷酸化，这是诱导一部分GR靶点所必需的。本应用程序的目的是解剖gr诱导的GRIP1磷酸化在MΦ中的作用和机制，作为GCs的抗炎和代谢作用的调节剂。我们的中心假设是配体GR通过激活GC-response element （GRE）特异性募集GRIP1激酶来修饰其自身的共调节因子，从而决定GRIP1在不同GR转录复合物中的功能，或者优先利用其激活与抑制特性。我们的具体目标是：1)利用我们的MΦ-specific条件GRIP1缺陷小鼠和GRIP1磷酸化特异性抗体来评估MΦ中GRIP1磷酸化亚型的全基因组分布，并确定功能相关的结合位点；2)分析推测的grp1激酶、细胞周期蛋白依赖性激酶-9和酪蛋白激酶-2作为GR转录复合物的GR特异性组分的功能；3)通过阻断内源性GRIP1磷酸化或将GRIP1磷酸化突变体整合到GRIP1缺失细胞中，鉴定GRIP1磷酸化在GR转录复合物组装和基因调控中的作用，以及在MΦ极化和MΦ：脂肪细胞相互作用中的作用。这项工作将提供关于gr介导的基因表达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 (MΦ) 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 MΦ, GCs broadly inhibit the production of proinflammatory mediators; moreover, GCs promote the polarization of 'alternatively activated' M2 MΦ 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 MΦ. 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 MΦ 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 MΦ-specific conditional GRIP1-deficient mice and GRIP1 phosphosite-specific antibodies to assess the genome-wide distribution of GRIP1 phospho-isoforms in MΦ 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 MΦ polarization and the MΦ: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.