Li Liuzhe, Zhang Jianliang, Jin Bilian, Block Edward R, Patel Jawaharlal M
Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32608-1197, USA.
Mol Cell Biochem. 2007 Nov;305(1-2):71-7. doi: 10.1007/s11010-007-9529-z. Epub 2007 Jun 13.
We recently reported that nitric oxide (NO) modulates expression of multiple genes associated with apoptotic pathways, including expression of caspase-8. The objective of the present study is to determine whether the NO-induced expression of the caspase-8 gene is regulated via signal transducers and activators of transcription-1 (STAT-1) signaling. The confluent monolayers of pulmonary artery endothelial cells (PAEC) were incubated with or without (control) 1 mM NOC-18, a NO donor, at 37 degrees C for 0-24 h. In some experiments PAEC were pretreated with a Janus kinase (JAK-2) inhibitor, AG490 (20 microM). Exposure of PAEC to NO-increased relative levels of caspase-8 mRNA as determined using quantitative real time PCR. Relative levels of phosphorylated STAT-1 at Serine (Ser)-727, but not total STAT-1 expression in NO-exposed cells, were upregulated significantly compared to control cells. AG490 attenuated NO-induced phosphorylation of STAT-1 at Ser 727 and expression of caspase-8 mRNA, suggesting JAK2 plays a role in the induction of caspase-8 mRNA. The promoter of caspase-8 has four gamma-activated sequence (GAS) and two interferon-stimulated response element (ISRE) transcription factor-binding sites. NO enhanced the STAT-1 binding activity to GAS/ISRE. Suppression of STAT-1 expression attenuated NO-induced elevation of caspase-8 mRNA. These studies demonstrate that a NO-dependent increase in caspase-8 mRNA levels is associated with phosphorylation of STAT-1 at Ser-727 and STAT1 binding to the caspase-8 promoter in cultured PAEC.
我们最近报道，一氧化氮（NO）可调节与凋亡途径相关的多个基因的表达，包括半胱天冬酶-8（caspase-8）的表达。本研究的目的是确定NO诱导的caspase-8基因表达是否通过信号转导和转录激活因子1（STAT-1）信号传导进行调控。将汇合的肺动脉内皮细胞（PAEC）单层在37℃下用或不用（对照）1 mM NOC-18（一种NO供体）孵育0至24小时。在一些实验中，PAEC用Janus激酶（JAK-2）抑制剂AG490（20 μM）进行预处理。使用定量实时PCR测定，PAEC暴露于NO后caspase-8 mRNA的相对水平增加。与对照细胞相比，暴露于NO的细胞中丝氨酸（Ser）-727处磷酸化STAT-1的相对水平显著上调，但总STAT-1表达未上调。AG490减弱了NO诱导的Ser 727处STAT-1的磷酸化以及caspase-8 mRNA的表达，表明JAK2在caspase-8 mRNA的诱导中起作用。caspase-8的启动子有四个γ-激活序列（GAS）和两个干扰素刺激反应元件（ISRE）转录因子结合位点。NO增强了STAT-1与GAS/ISRE的结合活性。抑制STAT-1表达减弱了NO诱导的caspase-8 mRNA升高。这些研究表明，在培养的PAEC中，NO依赖性的caspase-8 mRNA水平增加与Ser-727处STAT-1的磷酸化以及STAT1与caspase-8启动子的结合有关。
