Disruption of 12/15-lipoxygenase expression in peritoneal macrophages - Enhanced utilization of the 5-lipoxygenase pathway and diminished oxidation of low density lipoprotein
Disruption of 12/15-lipoxygenase expression in peritoneal macrophages - Enhanced utilization of the 5-lipoxygenase pathway and diminished oxidation of low density lipoprotein
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DOI：
10.1074/jbc.271.39.24055
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发表时间：
1996-09-27
影响因子：
4.8
通讯作者：
Funk, CD
中科院分区：
文献类型：
--
作者：
Sun, DX;Funk, CD
以前，我们从主要由白细胞(Chen，X.-S.，Kurre，U，Jenkins，NA，Copeland，N.G.和Funk，C.D.(1994)J.Biol)组成的腹膜诱导细胞的RNA中分离出小鼠‘’白细胞型‘’12-脂氧合酶(L-12LO)的cDNA.化学。269、13979-13987)。通过原位杂交，我们发现L-12LO基因在部分腹膜巨噬细胞中大量表达，而在诱导的白细胞、肺泡巨噬细胞和骨髓来源的巨噬细胞中不表达。L-12LO与人和兔的15-脂氧合酶高度相关，这些酶参与了红细胞的成熟过程，以及低密度脂蛋白的氧化修饰，参与了动脉粥样硬化的形成。因此，这些酶被称为12/15-脂氧合酶。我们利用胚胎干细胞中的同源重组技术，在小鼠体内灭活了L-12LO基因。在纯合缺陷小鼠，巨噬细胞L-12LO的表达被取消，12-羟基二十碳四烯酸(12-HETE)的形成也被取消。在酵母多糖刺激的细胞中，代谢明显转向5-脂氧合酶产物白三烯C-4和5-HETE，而在A23187处理的细胞中，代谢仅转向5-HETE。5-脂氧合酶代谢产物的增加不是由于5-脂氧合酶或5-脂氧合酶激活蛋白的代偿变化，而是一种明显的底物转移。L-12LO基因缺陷小鼠未见明显的网织红细胞或成熟红细胞异常，提示该途径对小鼠红细胞发育功能不重要。低密度脂蛋白氧化指标(硫代巴比妥酸反应物质或氧化应激标志物异前列腺素8-表前列腺素F-2α)在未刺激的野生型和L-12LO缺乏的巨噬细胞中是相同的，但酵母多糖诱导的野生型巨噬细胞的增加在L-12LO缺陷的细胞中被消除，因此，12/15脂氧合酶缺陷小鼠将有助于研究脂氧合酶途径之间的相互作用，并确定12/15脂氧合酶催化的低密度脂蛋白氧化在动脉粥样硬化形成中的体内作用。
Previously, we isolated the murine ''leukocyte-type'' 12-lipoxygenase (L-12LO) cDNA from RNA of peritoneal-elicited cells that consisted predominantly of leukocytes (Chen, X.-S., Kurre, U., Jenkins, N. A., Copeland, N. G., and Funk, C. D. (1994) J. Biol. Chem. 269, 13979-13987). By in situ hybridization we show that the L-12LO gene is expressed abundantly in a subset of peritoneal macrophages but not in elicited leukocytes, alveolar macrophages, or bone marrow-derived macrophages. L-12LO is highly related to human and rabbit 15-lipoxygenases, enzymes that have been implicated in the maturation process of red blood cells, and the oxidative modification of low density lipoproteins that is implicated in atherogenesis. Accordingly, these enzymes have been referred to as 12/15-lipoxygenases. We have inactivated the L-12LO gene in mice using homologous recombination in embryonic stem cells. Macrophage expression of L-12LO was abolished in homozygous deficient mice as was formation of 12-hydroxyeicosatetraenoic acid (12-HETE). In zymosan-stimulated cells, there was significant diversion of metabolism to the 5-lipoxygenase products leukotriene C-4 and 5-HETE and in A23187-treated cells to 5-HETE only. The enhanced formation of 5-lipoxygenase metabolites was not due to compensatory changes of 5-lipoxygenase or 5-lipoxygenase activating protein but rather an apparent substrate diversion. L-12LO-deficient mice have no obvious abnormalities in reticulocyte or mature red blood cells, which suggest that in mice this pathway is not functionally important for erythrocytic development. Indices for oxidation of low density lipoprotein (measured as either thiobarbituric acid-reactive substances or the oxidant stress marker isoprostane 8-epi-prostaglandin F-2 alpha) were identical in incubations with unstimulated wild type and L-12LO deficient macrophages, but the zymosan-induced increase observed with wild-type macrophages was abolished in L-12LO-deficient cells, Thus, 12/15-lipoxygenase-deficient mice will be useful for the study of interaction between lipoxygenase pathways and determination of the in vivo role of 12/15-lipoxygenase-catalyzed oxidation of LDL in atherogenesis.