Regulation of Hepatic SphK2 by Bile Acids: Effects on Lipid Metabolism

描述(由申请人提供)：胆汁酸对肝脏中的脂质平衡至关重要。在过去的十年里，研究人员发现胆汁酸不仅是洗涤剂，也是重要的信号分子。它们通过激活肝脏和胃肠道细胞中的特定核受体、G蛋白偶联受体(GPCR，TGR5)和多条信号通路，调节参与胆汁酸、葡萄糖、脂肪酸和脂蛋白代谢/合成以及能量代谢的许多酶/蛋白编码基因的表达。我们先前已经报道，结合胆汁酸通过G？I蛋白偶联受体激活AKT和ERK1/2通路(S)。我们最新的研究表明，胆汁酸上调鞘氨醇激酶2(SphK2)的表达(在mRNA和蛋白质水平)和酶活性，并增加血清鞘氨醇-1-磷酸(S1P)水平。被ERK1/2激活的SphK2增加了核S1P的水平，S1P被证明是特异组蛋白脱乙酰基酶(HDAC1和2)的抑制物。组蛋白乙酰化的增加通常与生理上的转录活性的增加有关。 相互关联的基因。我们的初步数据表明，结合胆汁酸和S1P一起激活了S1P受体2(S1P2)，进而激活了肝细胞中下游的ERK1/2和AKT信号通路。我们最近发现，在SphK2-/-小鼠中，涉及肝脏脂肪代谢的关键转录因子和关键酶的mRNA水平显着下调。与野生型对照小鼠相比，SphK2-/-小鼠的血清甘油三酯也增加了~18倍，高密度脂蛋白和低密度脂蛋白都降低了2到5倍。此外，牛磺酸熊去氧胆酸盐(TUDCA)对S1P2-/-和SphK2-/-小鼠原代肝细胞中脂质堆积的抑制作用被逆转。然而，胆汁酸、S1P、S1P受体之间的生理联系以及它们在调节肝脏类固醇/脂代谢中的作用尚未被揭示。根据最近的研究和我们的初步结果，我们假设胆汁酸介导的S1P2的激活通过核内SphK2的磷酸化和激活在调节肝脏脂质代谢中发挥关键作用。为了检验我们的中心假设，我们提出了以下三个具体目标。目的1：研究不同胆汁酸对S1P2的激活作用，确定S1P2与胆汁酸的结构与功能关系；目的2：利用S1P2-/-和SphK2-/-小鼠模型，阐明S1P2和SphK2在胆汁酸介导的肝脏类固醇/脂代谢调节中的作用；目的3：利用胆汁酸耗竭大鼠模型，研究不同胆汁酸(TUDCA、甘胆酸和脱氧胆酸盐)和S1P2拮抗剂对肝脏SphK2表达和活性以及HDAC1和HDAC2活性的影响。这项研究的完成不仅将建立胆汁酸生物学的新理论，而且还将 为血脂异常和包括非酒精性脂肪性肝病(NAFLD)在内的其他代谢性疾病的病理生理学提供新的机械见解。此外，这项研究对有效治疗NAFLD和其他相关代谢性疾病的新治疗靶点的确定和开发也具有潜在的影响。

DESCRIPTION (provided by applicant): Bile acids are crucial for lipid homeostasis in the liver. During the last decade, researchers have discovered that bile acids not only act as detergents, but also as important signaling molecules. They have been shown to regulate the expression of numerous genes encoding enzymes/proteins involved in the metabolism/synthesis of bile acids, glucose, fatty acids, and lipoproteins as well as energy metabolism by activating specific nuclear receptors, a G protein coupled receptor (GPCR, TGR5), and multiple signaling pathways in cells of the liver and gastrointestinal tract. We have previously reported that conjugated bile acids activate the AKT and ERK1/2 pathways via G¿i protein coupled receptor(s). Our most recent studies show that bile acids upregulate sphingosine kinase 2 (SphK2) expression (at both mRNA and protein levels) and enzyme activity and increase serum sphingosine-1-phosphate (S1P) levels. Activation of SphK2 by ERK1/2 increases nuclear S1P levels, which has been shown to be an inhibitor of specific histone deacetylases (HDAC 1 and 2). Increased histone acetylation is often associated with an increase in the transcriptional activity of physiologically linked genes. Our preliminary data strongly indicates that conjugated bile acids along with S1P activate the S1P receptor 2 (S1P2), which further activates the downstream ERK1/2 and AKT signaling pathways in hepatocytes. We recently discovered that in SphK2-/- mice the mRNA levels of key transcriptional factors and key enzymes involved in hepatic lipid metabolism were significantly down-regulated. The SphK2-/- mouse was also found to have an ~18-fold increase in serum triglycerides and a 2 to 5-fold decrease in both HDL and LDL as compared to wild-type control mice. Moreover, tauroursodeoxycholate (TUDCA)-mediated inhibition of lipid accumulation was reversed in the primary hepatocytes of S1P2-/- and SphK2-/- mice. However, the physiological link between bile acids, S1P, S1P receptors and their roles in regulating hepatic sterol/lipid metabolism have not been revealed. Based on the recent studies and our preliminary results, we hypothesize that bile acid-mediated activation of S1P2 plays a critical role in regulating hepatic lipid metabolism by phosphorylation and activation of SphK2 in the nucleus. The following three specific aims are proposed to test our central hypothesis. Aim 1: Characterize the activation of S1P2 by different bile acids and determine the structural-functional relationship of S1P2 and bile acids; Aim 2: Elucidate the role of S1P2 and SphK2 in bile acid-mediated regulation of hepatic sterol/lipid metabolism using S1P2-/- and SphK2-/- mice models; Aim 3: Determine the effects of administration of different bile acids (TUDCA, glycocholate and deoxycholate) and S1P2 antagonist on SphK2 expression and activity as well as activities of HDAC1 and HDAC2 in the liver using an in vivo rat model of bile acid depletion. Completion of this study will not only establish a novel theory in bile acid biology, but will also provide new mechanistic insights into the pathophysiology of dyslipidemia and other metabolic diseases including non-alcoholic fatty liver disease (NAFLD). Also, this study has potential impact on the identification and development of novel therapeutic targets for effective treatment of NAFLD and other related metabolic diseases.