APOPTOSIS,5 LIPOXYGENASE ACTIVATING PROTEIN AND BCL X

活性氧与细胞凋亡的几种效应物之间的关系已有报道。 尽管氧化剂诱导细胞凋亡的机制尚不清楚，但很可能会产生一些信号因子。 由于多不饱和脂肪酸非常容易氧化，因此已知可诱导细胞凋亡的氧化产物是合理的候选者。脂氧合酶是此类产品的可能来源，并且已使用各种抑制剂来使这些酶参与细胞凋亡。 然而，其中一些抑制剂，包括阻断 5-脂氧合酶激活蛋白 (FLAP) 的 MK886，即使在缺乏 LOX 的细胞中也会诱导细胞凋亡。 我们还表明，NM886 不依赖于 FLAP 诱导细胞凋亡。 然而，我们的发现提示了 FLAP 和 BCL 抗凋亡蛋白家族之间的联系，即过表达 bcl-xL 的 FL5.12 细胞的 FLAP 水平降低，并且在撤除 IL-3 后，此类细胞中的 FLAP 迅速丧失。 在用 mK886 处理的细胞中，bcl-xL 和 bcl-2 蛋白也快速丢失。 该项目的总体目标是通过确定 MK886 的细胞凋亡机制来增强我们对细胞凋亡相关机制的理解。 一个相关的目标是阐明 FLAP 和 bcl-XL 之间的关系以及细胞凋亡的诱导。 要测试的假设是：1) MK886 对过氧化物酶体增殖物激活受体 (PPAR) 的抑制在诱导细胞凋亡中发挥作用，2) MK886 阻断不饱和脂肪酸的结合，增加其细胞含量。 然后，这些脂肪酸直接或在转化为其他物种后激活细胞凋亡信号通路，3) 破坏脂肪酸信号传导调节 bcl-xL 的表达。 初步数据表明 MK886 是 PPARα 的有效抑制剂。 我们将更详细地研究这种独特的效应，并通过上调和下调 PPAR 并确定 MK886 的细胞凋亡效力来研究与细胞凋亡的任何联系。 第二个假设将通过确定用 MK886 处理后的脂肪酸氧化、比较 MK886 与外源脂肪酸（包括氧化物质）的效果以及检查谷胱甘肽是否可以影响 MK886 诱导的细胞凋亡来研究。 FLAP和bcl-XL之间的任何关系将通过测量bcl-XL过表达对其他细胞系中FLAP表达的影响、评估IL-3撤除或用MK886处理后bcl-XL过表达细胞中的FLAP和bcl-XL蛋白和mRNA水平以及评估各种蛋白水解途径在bcl-XL和FLAP损失中的作用来确定。 这些研究的结果将提高我们对与 FLAP 和 bcl 蛋白相关的脂肪酸信号传导和细胞凋亡的理解。

A relationship between reactive oxygen species and several effectors of apoptosis has been reported. Although the mechanism by which oxidants induce apoptosis is unknown, it is likely some signaling factor is generated. Since polyunsaturated fatty acids are highly susceptible to oxidation, oxidized products that are known to induce apoptosis are reasonable candidates. Lipoxygenase enzymes are a possible source of such products and various inhibitors have been used to implicate these enzymes in apoptosis. However, several of these inhibitors, including MK886 which blocks 5-lipoxygenase activating protein (FLAP), induce apoptosis even in cells lacking LOX. We have also shown that NM886 induces apoptosis independent of FLAP. Nevertheless, a link between FLAP and the BCL family of anti-apoptotic proteins is suggested by our findings that FL5.12 cells overexpressing bcl-xL have diminished levels of FLAP and there is a rapid loss of FLAP from such cells upon withdrawal of IL-3. There is also a rapid loss of bcl-xL and bcl-2 protein in cells treated with mK886. The overall goal of this project is to enhance our understanding of the mechanisms associated with apoptosis by determining the apoptotic mechanism of MK886. A related goal will be to clarify the relationship between FLAP and bcl-XL, and the induction of apoptosis. The hypotheses to be tested are that: 1) the inhibition of peroxisome proliferator activated receptors (PPAR) by MK886 plays a role in the induced apoptosis, 2) MK886 blocks the binding of unsaturated fatty acids increasing their cellular content. These fatty acids then activate apoptotic signaling pathways either directly or following conversion to other species, and 3) disrupting fatty acid signaling modulates the expression of bcl-xL. Preliminary data show that MK886 is a potent inhibitor of PPARalpha. This unique effect will be studied in more detail and any link to apoptosis investigated by up- and down-regulating PPAR and determining the apoptotic potency of MK886. The 2nd hypothesis will be studied by determining fatty acid oxidation after treatment with MK886, comparing the effects of MK886 to those of exogenous fatty acids including oxidized species, and examining whether glutathione can affect MK886-induced apoptosis. Any relationship between FLAP and bcl-XL will be determined by measuring the effect of bcl-XL overexpression on FLAP expression in other cell lines, assessing FLAP and bcl-XL protein and mRNA levels in bcl-XL overexpressing cells following withdrawal of IL-3 or treatment with MK886, and assessing the role of various proteolytic pathways in the loss of bcl-XL and FLAP. The results of these studies will improve our understanding of fatty acid signaling and apoptotic death in conjunction with FLAP and bcl proteins.