Hagihara Keisuke, Nishikawa Teppei, Isobe Tomoyasu, Song Jian, Sugamata Yasuhiro, Yoshizaki Kazuyuki
Department of Medical Science I, School of Health and Sport Sciences, Osaka University, Osaka, Japan.
Biochem Biophys Res Commun. 2004 Feb 6;314(2):363-9. doi: 10.1016/j.bbrc.2003.12.096.
Serum amyloid A (SAA) is known to be a precursor of amyloid A (AA) protein in AA (secondary) amyloidosis and SAA1 to be mainly involved in AA amyloidosis. We established an SAA isoform real-time quantitative RT-PCR assay and found that beta-2 microglobulin is more stable as an internal control than GAPDH and beta-actin for our system. Either IL-6 and IL-1beta or IL-6 and TNFalpha, but not IL-1beta and TNFalpha, induced the synergistic induction of SAA1 and SAA2 genes. Anti-IL-6 receptor monoclonal antibody completely inhibited the synergistic induction of SAA1 and SAA2 during triple stimulation with IL-6, IL-1beta, and TNFalpha, but, IL-1 receptor antagonist or anti-TNFalpha monoclonal antibody was only partially inhibited in HepG2, Hep3B, and PLC/PRF/5 cells. Although the SAA1 promoter has no STAT3 consensus sequence, the JAK2 inhibitor-AG490 reduced SAA1 gene expression to 30%, suggesting the involvement of STAT3. We were able to demonstrate that IL-6 plays a critical role in the synergistic induction of human SAA gene when stimulated with proinflammatory cytokines.
血清淀粉样蛋白A（SAA）是AA（继发性）淀粉样变性中淀粉样蛋白A（AA）的前体，已知SAA1主要参与AA淀粉样变性。我们建立了一种SAA亚型实时定量RT-PCR检测方法，发现对于我们的系统而言，β2微球蛋白作为内参比甘油醛-3-磷酸脱氢酶（GAPDH）和β-肌动蛋白更稳定。单独的白细胞介素-6（IL-6）和白细胞介素-1β（IL-1β）或IL-6和肿瘤坏死因子α（TNFα），而非IL-1β和TNFα，可诱导SAA1和SAA2基因的协同诱导。抗IL-6受体单克隆抗体完全抑制了在IL-6、IL-1β和TNFα三联刺激期间SAA1和SAA2的协同诱导，但在HepG2、Hep3B和PLC/PRF/5细胞中，IL-1受体拮抗剂或抗TNFα单克隆抗体仅部分抑制了这种协同诱导。尽管SAA1启动子没有信号转导和转录激活因子3（STAT3）共有序列，但JAK2抑制剂AG490将SAA1基因表达降低至30%，提示STAT3参与其中。我们能够证明，当受到促炎细胞因子刺激时，IL-6在人SAA基因的协同诱导中起关键作用。
