Sphingolipid Signaling and Pteridine Biosynthesis

鞘脂信号传导和蝶啶生物合成

• 批准号: 6979882
• 负责人: SHELDON MILSTIEN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF MENTAL HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6979882
• 关键词: G protein; biological signal transduction; biosynthesis; cell growth regulation; cell migration; ceramides; chemotaxis; enzyme activity; epidermal growth factor; immunocytochemistry; laboratory rat; mast cell; nerve growth factors; phenylalanine; phenylketonurias; phosphotransferases; pteridines; receptor coupling; sphingolipids; sphingosine; tetrahydrobiopterin; tissue /cell culture

Sphingosine-1-phosphate (S1P) is now recognized as a potent lipid mediator that regulates many vital biological processes, including cell growth, death, and differentiation. In a continuing and highly successful collaboration with Spiegel?s lab at Virginia Commonwealth University, we are elucidating the mechanisms by which S1P is produced, how its levels are regulated, and how it mediates its diverse actions. S1P is produced by two sphingosine kinase (SphK) isoforms after activation by diverse types of external stimuli. For example, we found that activation of mast cells by crosslinking of the high affinity IgE receptor not only increased cellular levels of S1P by activating SphK1, an enzyme originally cloned by us, degranulation and motility of RBL 2H3 and bone marrow derived mast cells were dependent on formation and release of S1P which subsequently transactivated specific mast cell S1P receptors. Our results suggest that this process plays a crucial role in mast cell functions in inflammatory responses. In another study with important implications for immunology, we discovered that SphK2 is the enzyme that phosphorylates the novel immunosuppressive agent, FTY720, converting it to a S1P mimetic that is highly effective in preventing organ transplant rejection. Phosphorylated FTY720 acting through S1P receptors prevents egress of activated lymphocytes from secondary lymphoid tissues. Unexpectedly, we found that the second sphingosine kinase isozyme that we cloned, SphK2, has completely the opposite effects on cells as SphK1 has. SphK2 is larger than SphK1, has a different tissue distribution, and contains a putative BH3-only motif, similar to that in a large sub-family of Bcl-2 proteins important for apoptosis. While SphK1 promotes cell growth and survival, SphK2 inhibits cell growth and induces cell death. How these two very closely related and similar enzymes that use the same substrate and produce the same product can have such opposite effects and their importance in regulating cell functions is now under investigation. While many important biological effects of S1P are now known to be mediated by activation of S1P receptors, we have previously suggested that S1P might also have direct intracellular actions. This concept has been challenged due to the scarcity of identified intracellular targets. We have now found that in contrast to treatment of cells with exogenous S1P, which increases proliferation by binding to S1P receptors on the cell surface, increasing intracellular S1P formation stimulated cell growth and promoted survival independently of these receptors. Thus, exogenous and intracellularly generated S1P can affect cell growth and survival by divergent pathways and our study describes a receptor-independent, intracellular function of S1P, reminiscent of its action in yeast and plants that lack S1P receptors. In a study that may have importance for development of the nervous system, we found that differential transactivation of S1P receptors on neurons regulates NGF-induced neurite extension. The process of neurite extension after activation of the TrkA tyrosine kinase receptor by NGF involves complex signaling pathways. Stimulation of SphK1 is part of the functional TrkA signaling repertoire. We found that in PC12 cells and dorsal root ganglion neurons, NGF translocates SphK1 to the plasma membrane and differentially activates the S1P receptors S1P1 and S1P2 in a SphK1-dependent manner. NGF-induced neurite extension was suppressed by down-regulation of S1P1 expression with antisense RNA. Conversely, when overexpressed in PC12 cells, transactivation of S1P1 by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation of neurons down-regulated expression of S1P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S1P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension. In an effort to further understanding of the regulation of SphK1, we used a yeast two-hybrid screen to search for SphK1-interacting proteins that might regulate its activity and/or its cellular localization. One of these was identified as a C-terminal fragment of aminoacylase 1 (Acy1), a metalloenzyme that removes amide-linked acyl groups from amino acids and may play a role in regulating responses to oxidative stress. Acy1 co-immunoprecipitated with SphK1. Though both C-terminal and full-length Acy1 reduced SphK1 activity measured in vitro, the C-terminal fragment inhibited while full-length Acy1 potentiated the growth promoting and anti-apoptotic effects of SphK1. Interestingly, Acy1 expression induced redistribution of SphK1 determined by immunocytochemistry and subcellular fractionation. Our results suggest that Acy1 physically interacts with SphK1 and may influence its physiological functions. Because of the pivotal role of S1P in cells, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by SphKs and degradation by S1P lyase and a specific S1P phosphatase (SPP-1). Surprisingly, we found that down-regulation of SPP-1 enhanced migration of cells towards EGF and conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration towards EGF. We examined whether the inhibitory effect on EGF-induced migration was because of decreased S1P or increased ceramide as a consequence of acylation of increased sphingosine by ceramide synthase. Although the ceramide synthase inhibitor fumonisin B1 blocked ceramide production and increased sphingosine, it did not reverse the negative effect of SPP-1 expression on EGF- or S1P-induced chemotaxis. We then examined the possibility that intracellularly generated S1P might be involved in activating a G protein-coupled S1P receptor important for EGF-directed migration. Treatment with pertussis toxin to inactivate Galpha(i) suppressed EGF-induced migration. Collectively, our results suggest that metabolism of S1P by SPP-1 is important for EGF-directed cell migration. We previously found that S1P and ceramide also play important roles in regulating tetrahydrobiopterin synthesis as well as survival of neurons and glial cells. In collaborative studies with Dr. Cory Harding, we examined the role of tetrahydrobiopterin in the efficacy of heterologous muscle-directed gene therapy of PKU with phenylalanine hydroxylase. It appears that the effectiveness of this type of therapy will be limited by the ability to supply sufficient tetrahydrobiopterin to tissues. We also found in collaboration with Dr. R.J. Groszmann that bacterial translocation leads to endotoxemia, stimulation of GTP cyclohydrolase, the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, and enhancement of nitric oxide production which aggravates vasodilation. These results suggest that targeting tetrahydrobiopterin biosynthesis might be beneficial in treatment of septic shock.

现在，鞘氨醇1-磷酸盐（S1P）被认为是一种有效的脂质介质，可调节许多重要的生物学过程，包括细胞生长，死亡和分化。在与弗吉尼亚联邦大学（Virginia Commonwealth University）的Spiegel实验室的持续且非常成功的合作中，我们正在阐明生产S1P的机制，如何调节其水平以及如何介导其多样化的行动。 S1P是由两种类型的外部刺激激活后通过两种鞘氨醇激酶（SPHK）同工型产生的。 For example, we found that activation of mast cells by crosslinking of the high affinity IgE receptor not only increased cellular levels of S1P by activating SphK1, an enzyme originally cloned by us, degranulation and motility of RBL 2H3 and bone marrow derived mast cells were dependent on formation and release of S1P which subsequently transactivated specific mast cell S1P receptors.我们的结果表明，该过程在炎症反应中的肥大细胞功能中起着至关重要的作用。在另一项对免疫学意义的研究中，我们发现SPHK2是磷酸化的酶，它磷酸化了新型免疫抑制剂FTY720，将其转化为在防止器官移植抑制非常有效的S1P模拟物中。通过S1P受体作用的磷酸化FTY720可防止从继发性淋巴组织中流出活化的淋巴细胞。 出乎意料的是，我们发现我们克隆的第二个鞘氨醇激酶同工酶SPHK2完全对细胞的影响如SPHK1一样。 SPHK2大于SPHK1，具有不同的组织分布，并且包含推定的仅BH3基序，类似于在大型的Bcl-2蛋白中对凋亡重要的Bcl-2蛋白中的基序。 SPHK1促进细胞生长和存活，而SPHK2抑制细胞生长并诱导细胞死亡。这两个使用相同底物并产生相同产品的非常紧密相关和相似的酶如何具有相反的影响，并且它们在调节细胞功能方面的重要性现在正在研究中。 尽管现在已知S1P的许多重要生物学作用是通过S1P受体激活介导的，但我们以前已经提出S1P也可能具有直接的细胞内作用。由于鉴定出的细胞内靶标的稀缺性，该概念受到了挑战。现在，我们发现与外源S1P的细胞进行对比，这通过与细胞表面上的S1P受体结合而增加增殖，从而增加了细胞内S1P的形成刺激了细胞的生长，并独立于这些受体促进了生存。因此，外源性和细胞内产生的S1P可以通过不同的途径影响细胞的生长和存活，我们的研究描述了S1P的受体独立的，细胞内功能，使人联想到其在酵母中的作用以及缺乏S1P受体的植物。 在一项可能对神经系统发育具有重要意义的研究中，我们发现神经元上S1P受体的差异反式激活调节NGF诱导的神经突扩展。 NGF激活TRKA酪氨酸激酶受体后神经突延伸的过程涉及复杂的信号通路。 SPHK1的刺激是功能性TRKA信号传导库的一部分。我们发现，在PC12细胞和背根神经神经元中，NGF将SPHK1转移到质膜上，并以SPHK1依赖性方式差异地激活S1P受体S1P1和S1P2。 NGF诱导的神经突延伸通过用反义RNA下调S1P1表达来抑制。相反，当在PC12细胞中过表达时，NGF对S1P1的反式激活显着增强了神经突的延伸和小型GTPase RAC的刺激，对于神经突扩展所需的细胞骨架变化很重要。同时，神经元的分化下调S1P2的表达，其激活会刺激Rho并抑制神经突延伸。因此，NGF对S1P受体的差异反式激活调节调节神经突扩展的拮抗信号通路。 为了进一步了解SPHK1的调节，我们使用了酵母两杂交屏幕来搜索可能调节其活性和/或其细胞定位的SPHK1相互作用蛋白。其中之一被确定为氨基酰基酶1（ACY1）的C末端片段，这是一种去除氨基酸的酰胺连接酰基基团的金属酶，并且可能在调节对氧化应激的反应中起作用。 ACY1与SPHK1共免疫沉淀。尽管C末端和全长ACY1都降低了体外测量的SPHK1活性，但C末端片段抑制了全长ACY1，而全长ACY1则增强了SPHK1的生长促进和抗凋亡作用。有趣的是，ACY1表达诱导通过免疫细胞化学和亚细胞分馏确定的SPHK1的重新分布。我们的结果表明，ACY1与SPHK1物理相互作用，并可能影响其生理功能。 由于S1P在细胞中的关键作用，其水平较低，并通过其由SPHK催化的合成和S1P裂解酶和特定的S1P磷酸酶（SPP-1）降解，以时空的方式受到较低的调节。令人惊讶的是，我们发现SPP-1的下调细胞向EGF增强了迁移，相反，SPP-1的过表达位于内质网中，使迁移降低了向EGF的迁移。我们检查了对EGF诱导的迁移的抑制作用是由于神经酰胺合酶增加了S1P或神经酰胺增加的神经酰胺。尽管神经酰胺合酶抑制剂Fumonisin b1阻断了神经酰胺的产生并增加了鞘氨酸，但它并没有逆转SPP-1表达对EGF-或S1P诱导的趋化性的负面影响。然后，我们研究了细胞内产生的S1P可能与激活G蛋白偶联的S1P受体有关EGF指导迁移很重要的可能性。用百日咳毒素治疗使galpha（i）抑制了EGF诱导的迁移。总体而言，我们的结果表明，SPP-1对S1P的代谢对于EGF指导的细胞迁移很重要。 我们先前发现，S1P和神经酰胺在调节四氢无菌合成以及神经元和神经胶质细胞的存活中也起着重要作用。在与Cory Harding博士的合作研究中，我们研究了四氢无菌蛋白酶在PKU与苯丙氨酸羟化酶异源肌肉定向基因治疗功效中的作用。看来，这种类型的疗法的有效性将受到向组织提供足够的四氢无生物蛋白酶的能力的限制。我们还与R.J.博士合作发现格罗斯曼（Groszmann），细菌易位会导致内毒素血症，刺激GTP环丙基多酶，四氢无菌生物合成的速率限制酶以及一氧化氧化物产生的增强，从而使血管舒张加重。这些结果表明，靶向四氢无菌生物合成可能有益于治疗败血性休克。