COMPLEX SPHINGOLIPIDS: INDUCTION OF CYP1A1

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。本研究项目的总体目标是阐明复合鞘脂在3-甲基胆蒽（MC）诱导细胞色素P450 1A 1（CYP 1A 1）的信号转导途径中的作用。这对癌症研究很重要，因为CYP 1A 1在许多大的有机环境污染物转化为体内致癌物中起着关键作用。我们最近发现，ISP 1对复合鞘脂合成的抑制显著抑制了MC诱导CYP 1A 1的能力。此外，我们发现神经酰胺的短链类似物（N-乙酰鞘氨醇或C2-神经酰胺）不仅逆转这种抑制，而且在酶活性和蛋白质和mRNA水平上显著增强MC对CYP 1A 1的诱导。因此，本项目下一阶段的具体目标是：（1）确定复杂鞘脂对mRNA水平的调节是否是mRNA合成增加或mRNA稳定性增强的结果;（2）确定鞘脂是否影响其他多环芳烃诱导CYP 1A 1的能力;（3）测定MC、MC+ ISP 1、MC+ ISP 1 +C2-神经酰胺处理细胞后AHR和ARNT量的变化（如果有的话）;（4）确定鞘脂的其他途径相互作用，例如参与AHR-MC缔合、AHr-Hsp 90解离、AHR-MC穿过核膜，AHR-MC-ARNT在细胞核中的结合或AHR-MC-ARNT与CYP 1A 1基因上的XRE的结合;（5）确定神经酰胺对CYP 1A 1表达的作用的结构特异性，两者均相对于神经酰胺的性质（例如，长链碱基骨架的类型、酰胺连接的脂肪酸的长度和其它类型的化合物，包括结构类似物）;和（6）确定复合鞘脂是否调节P450的其它同种型和与AHR依赖性信号转导途径相关的酶的表达。这些研究将为CYP 1A 1和其他异生物质代谢酶的调节提供潜在的重要途径的基本信息。 这些研究还可能为鞘脂如何影响细胞行为提供新的见解，特别是在基因表达水平上，并可能确定鞘脂如何对癌症治疗至关重要。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this proposed research project is to elucidate the role(s) of complex sphingolipids in the signal transduction pathway of 3-methylcholanthrene (MC) induction of cytochrome P450 1A1 (CYP1A1). This is important to cancer research because of the key role CYP1A1 plays in the conversion of many large, organic environmental pollutants to carcinogens in vivo. We have recently discovered that inhibition of the synthesis of complex sphingolipids by ISP1 significantly suppresses the ability of MC to induce CYP1A1. In addition, we discovered that a short-chain analog of ceramide (N-acetyl-sphingosine or C2-ceramide) not only reverses this inhibition, but also significantly enhances CYP1A1 induction by MC at the levels of enzyme activity and protein and mRNA quanitites. Therefore, the specific objectives of the next phase of this project are: (1) to determine whether complex sphingolipid modulation of mRNA levels is the result of either increased mRNA synthesis or enhanced mRNA stability; (2) to determine whether sphingolipids effect the ability of other polycyclic aromatic hydrocarbons to induce CYP1A1; (3) to determine changes (if any) in the amount of AHR and ARNT after cells are treated with MC, MC+ISP1, MC+ISP1+C2-ceramide; (4) to determine other pathway interactions for sphingolipids, such as involvement in AHR-MC association, AHr-Hsp 90 dissociation, AHR-MC passage across the nuclear membrane, AHR-MC-ARNT association in the nucleus or AHR-MC-ARNT binding to an XRE on the CYP1A1 gene; (5) to determine the structural specificity of the effects of ceramide on the expression of CYP1A1, both with respect to the nature of the ceramide (e.g., the types of long-chain base backbones, the length of the amide-linked fatty acid and other types of compounds, including structural analogs); and (6) to determine whether complex sphingolipids modulate the expression of other isoforms of P450 and enzymes linked to the AHR-dependent signal transduction pathway. These studies will provide fundamental information about a potentially important pathway for the regulation of CYP1A1 and other xenobiotic metabolizing enzymes. These studies may also provide new insights into how sphingolipids affect cell behavior, particularly at the level of gene expression, and may determine how sphingolipids might be critical to cancer therapeutics.