Sphingosine-1-phosphate Signaling and the Chronic Glucocorticoid Exposure Induced Glucose Homeostasis Disorder

总结 长期和/或过量糖皮质激素（GC）暴露，如长期应激和长期GC治疗， 导致代谢紊乱包括高血糖症。细胞内GC受体（GR）已被证明直接 刺激Pck 1和G6 pc等促癌基因的转录，促进肿瘤的发生。 然而，慢性GC暴露可诱导其他机制，以进一步增强肝硬化的发生。 我们的初步研究发现，慢性GC暴露升高肝鞘氨醇-1-磷酸（S1 P）水平。 S1 P被输出到细胞外表面，并与膜S1 P受体（S1 PRs）结合以发挥其作用。 我们发现，降低小鼠肝脏中S1 PR 2的表达， 异源发生激活肝癌细胞中的S1 PR 2增强GC诱导的Pck 1和G6 pc表达。 染色质免疫沉淀实验发现GC诱导GR募集到GC反应元件 Pck 1和G6 pc的GRES通过肝S1 PR 2敲低而降低。全基因表达分析 发现RAR相关孤儿受体C（Rorc）表达通过肝S1 PR 2敲低而降低。 Rorc拮抗剂减弱GC诱导的肝癌细胞凋亡相关基因表达及过表达 肝S1 PR 2基因敲除小鼠肝脏中的Rorc增强了GC诱导的GR向Pck 1和G6 pc的募集 GRES以及它们的表达。有趣的是，GC抑制Rorc的表达，这是拮抗 S1 PR 2信号。基于这些结果，我们假设慢性GC暴露激活了S1 PR 2信号传导 通过抑制GC对Rorc表达的抑制作用来增强GC诱导的胚胎发生， 可与GR共同作用，强烈增强致突变基因的转录。在目标1中，我们将测试是否改变 肝细胞内S1 P水平通过过度表达影响慢性GC处理诱导的高血糖 或者敲低Sphk 1和Sphk 2（两者都将鞘氨醇转化为S1 P）、Sgpl 1（水解S1 P）和Spns 2 （将S1 P输出到细胞外空间）。在目标2中，我们将在GC诱导的肝脏中建立Rorc's 通过降低肝脏Rorc表达或用Rorc拮抗剂治疗小鼠来诱导肝细胞生成。值得注意的是，4a- 羧基，4 b-甲基-酵母甾醇（4ACD 8），胆固醇生物合成的代谢产物，已被证明是Rorc 激动剂我们将研究减少Sc 4 mol（一种产生4ACD 8的酶）的表达， 减弱GC对肝硬化发生的作用。在目标3中，我们将识别由以下物质激活的信号分子： S1 PR 2参与GC对致瘤基因和Rorc表达的调控。我们还将分析 Rorc在GR调节的整体肝基因转录中的作用最后，GC和S1 PR 2信号如何调节 将研究Rorc表达。总体而言，拟议的研究将建立一个新的S1 PR 2-Rorc轴 在慢性GC暴露后特异性诱导，以增强GC激活的细胞生成。这不仅会 知识促进了我们对慢性GC暴露病理生理学理解，但它也将提供新的 用于针对类固醇诱导的高血糖症的治疗性干预的靶点。

SUMMARY Chronic and/or excess glucocorticoid (GC) exposure, such as prolonged stress and long-term GC therapy, causes metabolic disorders including hyperglycemia. Intracellular GC receptor (GR) has been shown to directly stimulate the transcription of gluconeogenic genes, such as Pck1 and G6pc, to promote gluconeogenesis. However, chronic GC exposure can induce additional mechanisms to further enhance hepatic gluconeogenesis. Our preliminary studies found that chronic GC exposure elevated hepatic sphingosine-1-phosphate (S1P) levels. S1P is exported to the extracellular surface and binds to membrane S1P receptors (S1PRs) to exert its actions. We found that reducing S1PR2 expression in mouse liver attenuated chronic GC exposure-promoted gluconeogenesis. Activating S1PR2 in hepatoma cells enhanced GC-induced Pck1 and G6pc expression. Chromatin immunoprecipitation assay found that GC-induced GR recruitment to the GC response elements (GREs) of Pck1 and G6pc was reduced by hepatic S1PR2 knockdown. Global gene expression analysis identified that RAR-related orphan receptor C (Rorc) expression was reduced by hepatic S1PR2 knockdown. Rorc antagonist attenuated GC-induced gluconeogenic gene expression in hepatoma cells and overexpressing Rorc in the liver of hepatic S1PR2 knockout mice enhanced GC-induced GR recruitment to the Pck1 and G6pc GREs as well as their expression. Intriguingly, GC suppressed Rorc expression, which was antagonized by S1PR2 signaling. Based on these results, we hypothesize that chronic GC exposure activates S1PR2 signaling to enhance GC-induced gluconeogenesis by inhibiting GC’s suppressive effect on the expression of Rorc, which can act with GR to strongly augment gluconeogenic gene transcription. In Aim 1, we will test if altering intracellular S1P levels in hepatocytes affects chronic GC treatment-induced hyperglycemia by overexpressing or knocking down Sphk1 and Sphk2 (both convert sphingosine to S1P), Sgpl1 (hydrolyzes S1P) and Spns2 (exports S1P to extracellular space) in mouse liver. In Aim 2, we will establish Rorc’s in GC-induced hepatic gluconeogenesis by reducing hepatic Rorc expression or treating mice with Rorc antagonist. Notably, 4a- carboxy, 4b-methyl-zymosterol (4ACD8), a metabolite of cholesterol biosynthesis, has been shown to be a Rorc agonist. We will investigate whether reducing the expression of Sc4mol, an enzyme generating 4ACD8, attenuates the GC effect on hepatic gluconeogenesis. In Aim 3, we will identify signaling molecules activated by S1PR2 that participate in GC regulation of gluconeogenic genes and Rorc expression. We will also analyze the role of Rorc in GR regulated global hepatic gene transcription. Finally, how GC and S1PR2 signaling regulate Rorc expression will be investigated. Overall, the proposed studies will establish a novel S1PR2-Rorc axis induced specifically upon chronic GC exposure to enhance GC activated gluconeogenesis. Not only will this knowledge advance our understand on pathophysiology of chronic GC exposure, but it also will provide novel targets for therapeutic intervention against steroid induced hyperglycemia.