Nitric oxide signaling via nuclearized endothelial nitric-oxide synthase modulates expression of the immediate early genes iNOS and mPGES-1
Nitric oxide signaling via nuclearized endothelial nitric-oxide synthase modulates expression of the immediate early genes iNOS and mPGES-1
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DOI：
10.1074/jbc.m602219200
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发表时间：
2006-06-09
影响因子：
4.8
通讯作者：
Chemtob, Sylvain
中科院分区：
文献类型：
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作者：
Gobeil, Fernand, Jr.;Zhu, Tang;Chemtob, Sylvain
用溶血磷脂酸（LPA）刺激新鲜分离的大鼠肝细胞，导致LPA（1）受体介导的和一氧化氮依赖性的即刻早期基因iNOS（诱导型一氧化氮合酶（NOS））和mPGES-1（微粒体前列腺素E合酶-1）上调。由于LPA是细胞表面和细胞内受体位点的配体，并且是一种有效的内皮NOS（eNOS）激活剂，因此我们假设来自激活的核化NOS的NO可能参与基因调控。在此，我们通过对表达天然LPA（1）-受体和eNOS的猪脑内皮细胞以及用重组人LPA（1）-受体和融合eNOS-GFP cDNA共转染的HTC 4大鼠肝癌细胞进行共聚焦显微镜检查，发现在LPA刺激下，eNOS从外周区动态移位到核区。核定位eNOS及其下游效应，可溶性鸟苷酸环化酶，证明原位在大鼠肝脏标本中的免疫金标记使用特异性抗体。LPA和NO供体硝普钠刺激这一核组分分别导致亚硝酸盐（和eNOS磷酸化）和cGMP的产生增加; NOS抑制剂L-NAME和可溶性鸟苷酸环化酶抑制剂ODQ也相应地阻断了这些单独的反应。此外，硝普钠诱发的顺序增加核Ca 2+瞬变，激活p42 MAPK，NF-κ B结合DNA的共识序列，和依赖的iNOS RNA。这项研究描述了一个迄今未被认识的分子机制，即核eNOS通过随后的NO调节核钙稳态参与基因转录相关的事件。此外，我们的研究结果强烈支持核作为自主信号舱的概念。
Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA(1) receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearizede NOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA(1)-receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA(1)-receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor L-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca2+ transients, activation of p42 MAPK, NF-kappa B binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.