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首页> 外文期刊>International Journal of Environmental Research and Public Health >Novel Functional Association of Serine Palmitoyltransferase Subunit 1-A Peptide in Sphingolipid Metabolism with Cytochrome P4501A1 Transactivation and Proliferative Capacity of the Human Glioma LN18 Brain Tumor Cell Line
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Novel Functional Association of Serine Palmitoyltransferase Subunit 1-A Peptide in Sphingolipid Metabolism with Cytochrome P4501A1 Transactivation and Proliferative Capacity of the Human Glioma LN18 Brain Tumor Cell Line

机译：新型功能性丝氨酸棕榈酰转移酶亚基1-A肽在鞘脂代谢中的细胞色素P4501A1反式激活和人类神经胶质瘤LN18脑肿瘤细胞系的增殖能力。

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Some chemical modulators of cytochrome P4501A1, Cyp1A1, expression also perturb the activity of serine palmitoyltransferase, SPT, a heterodimeric protein responsible for catalyzing the first reaction in sphingolipid biosynthesis. The effect of altered SPT activity on Cyp1A1 expression has generally been attributed to changes in the composition of bioactive sphingolipids, generated downstream in the SPT metabolic pathway, but the precise mechanism remains poorly defined. A generally accepted model for chemical-induced transactivation of the Cyp1A1 gene involves intracellular signaling mediated by proteins including the arylhydrocarbon receptor, AhR, whose interaction with the 90 kilo Dalton heat shock protein, Hsp90, is essential for maintaining a high affinity ligandbinding receptor conformation. Because ligand-induced Cyp1A1 expression is important in the bioactivation of environmentally relevant compounds to genotoxic derivatives capable of perturbing cellular processes, binding to Hsp90 represents an important regulatory point in the cytotoxicity process. In the present study, based on evidence that indicates subunit 1 of serine palmitoyltransferase, SPT1, interacts with Hsp90, both ligand-induced Cyp1A1 transactivation and capacity for proliferation were evaluated using the wild type Glioma LN18 human brain cancer cell line and its recombinant counterparts expressing green fluorescent SPT1 fusion proteins. Exposure to the prototypical Cyp1A1 inducer, 3-methylcholanthrene, 3-MC, resulted in the translocation of SPT1 from a primarily cytoplasmic domain to sites of focal adhesion complexes. Immunolabel for Hsp90, which was dispersed throughout the cell, became primarily cytoplasmic, while the distribution of AhR remained unaffected. When compared to the wild type, cells transfected with recombinant SPT1-GFP vectors had significantly attenuated levels of 3-MC-induced Cyp1A1 mRNA, as determined by quantitative reverse transcription PCR. Although all the Glioma cell lines exhibited mitogenic proliferative response in dose response assay with the potent Cyp1A1 inducers 3-MC, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo [k] fluoranthene, BKF, only the recombinant cell line designated - 75SPT1-GFP, which was transfected with a mutant deletion of SPT1, retained its proliferative capacity at the highest PAH doses used in this study. The results suggest that overexpressing SPT1 as a green fluorescent fusion protein has a modulating effect on the transactivation of Cyp1A1. This is possibly due to SPT1 interacting with Hsp90 to modulate AhR-Hsp90 interaction, and altering downstream events such as in downregulating the transactivation and metabolic activity of Cyp1A1. This is supported by the fact that the -75SPT1-GFP recombinant cell line, with much lower capacity for Cyp1A1 induction, exhibited sustained mitogenic response to high doses of AhR ligands, but not the Cyp1A1 inducible wild type. Conceivably, the effect mediated by SPT1 on the AhR signaling pathway is an important underlying factor contributing to variability in Cyp1A1 gene expression and consequently, cytotoxic response to environmentally relevant compounds that pose risk to human health.
机译：细胞色素P4501A1，Cyp1A1，表达的某些化学调节剂也会干扰丝氨酸棕榈酰转移酶SPT的活性，SPT是负责鞘脂生物合成中第一个反应的异二聚体蛋白。 SPT活性改变对Cyp1A1表达的影响通常归因于SPT代谢途径下游产生的生物活性鞘脂组成的变化，但确切的机制仍然不清楚。 Cyp1A1基因化学诱导的反式激活的一种普遍接受的模型涉及由包括芳烃受体AhR在内的蛋白质介导的细胞内信号传导，其与90千道尔顿热激蛋白Hsp90的相互作用对于维持高亲和力的配体结合受体构象至关重要。由于配体诱导的Cyp1A1表达在环境相关化合物对能够扰乱细胞过程的遗传毒性衍生物的生物激活中很重要，因此与Hsp90的结合代表了细胞毒性过程中的重要调控点。在本研究中，基于表明丝氨酸棕榈酰转移酶SPT1的亚基1与Hsp90相互作用的证据，使用野生型Glioma LN18人脑癌细胞系及其重组对应物表达了配体诱导的Cyp1A1反式激活和增殖能力绿色荧光SPT1融合蛋白。暴露于典型的Cyp1A1诱导剂3-甲基胆碱3-MC，导致SPT1从主要的细胞质域转移到粘着斑复合物的位点。 Hsp90的免疫标签分散在整个细胞中，主要变为细胞质，而AhR的分布不受影响。与野生型相比，重组SPT1-GFP载体转染的细胞具有3MC诱导的Cyp1A1 mRNA显着降低的水平，这是通过定量逆转录PCR确定的。尽管所有的神经胶质瘤细胞系在有效的Cyp1A1诱导剂3-MC，2,3,7,8-四氯二苯并-p-二恶英（TCDD）和苯并[k]荧蒽，BKF的剂量反应分析中均显示出促有丝分裂增殖反应。重组细胞系-75SPT1-GFP，经SPT1突变缺失转染，在本研究中使用的最高PAH剂量下仍保持其增殖能力。结果表明，过表达SPT1为绿色荧光融合蛋白对Cyp1A1的反式激活具有调节作用。这可能是由于SPT1与Hsp90相互作用以调节AhR-Hsp90相互作用，并改变下游事件，例如下调Cyp1A1的反式激活和代谢活性。具有以下事实的事实支持了这一点：-75SPT1-GFP重组细胞系具有低得多的Cyp1A1诱导能力，对高剂量的AhR配体表现出持续的促有丝分裂反应，但对Cyp1A1诱导的野生型却没有。可以想象，由SPT1介导的对AhR信号通路的影响是一个重要的潜在因素，可导致Cyp1A1基因表达的变异，进而导致对环境相关化合物的细胞毒性反应，从而对人类健康构成威胁。

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