Kang Li-Jung, Nguyen Khoa V A, Eom Sanung, Choi Yeo-Jin, Nguyen Cam Ngoc, Lee Jaeeun, Kim Chaelin, Lee Shinhui, Lee Seong-Gene, Lee Jun-Ho
Department of Biotechnology, Chonnam National University, Gwangju, Republic of Korea.
J Cell Biochem. 2019 Oct;120(10):18193-18208. doi: 10.1002/jcb.29125. Epub 2019 Jun 6.
DDX3 is a host viral factor that can inhibit the hepatitis B virus-induced innate immune responses. In this study, the 20 bioactive compounds have screened the effects on DDX3 and we found that 5-HT upregulated DDX3 promoter activity via the 5-HT receptor on liver hepatocellular cells (HepG2 cells) by using a luciferase assay, reverse transcription-polymerase chain reaction analysis, and Western blot analysis. Furthermore, we are trying to elucidate the pathways involved in the stimulating effect of 5-HT on DDX3 expression to induce innate immune responses against hepatitis B virus infection. A knockdown of the 5-HT receptor by transfection si-5-HT receptors or si-control into HepG2 cells treated by 5-HT (or 5-HT plus agonist) confirmed the role of the 5-HT receptor in DDX3 expression. The IFN-β-Luc expression and level of hepatitis B virus surface Antigen (HBsAg) showed that DDX3 mediated by the 5-HT agonist (AS-19) increased IFN-β expression and inhibited HBV replication. Luciferase assays showed the involvement of 5-HT receptors in DDX3 expression via cAMP/AC/PKA pathways by using protein kinase A (PKA) and adenylyl cyclase inhibitor (MDL 12330A). AS-19 mediated DDX3 promoter activated PKA extracellular signal-regulated kinase ERK signaling the p53 phosphorylation (-1080/-1070) resulted in upregulation of DDX3 promoter transactivation via the 5-HT receptors agonist. Overall, 5-HT was found to be a new potential target to inhibit hepatitis B infection by activating AC/PKA/ERK pathways by phosphorylating p53 via the 5-HT agonist response by mediating DDX3 expression.
DDX3是一种宿主病毒因子，可抑制乙型肝炎病毒诱导的先天免疫反应。在本研究中，我们筛选了20种生物活性化合物对DDX3的影响，通过荧光素酶测定、逆转录-聚合酶链反应分析和蛋白质印迹分析发现，5-羟色胺（5-HT）通过肝细胞（HepG2细胞）上的5-HT受体上调DDX3启动子活性。此外，我们试图阐明5-HT刺激DDX3表达以诱导针对乙型肝炎病毒感染的先天免疫反应所涉及的途径。通过将si-5-HT受体或si对照转染到经5-HT（或5-HT加激动剂）处理的HepG2细胞中敲低5-HT受体，证实了5-HT受体在DDX3表达中的作用。干扰素-β-荧光素酶（IFN-β-Luc）表达和乙型肝炎病毒表面抗原（HBsAg）水平表明，由5-HT激动剂（AS-19）介导的DDX3增加了IFN-β表达并抑制了HBV复制。荧光素酶测定表明，通过使用蛋白激酶A（PKA）和腺苷酸环化酶抑制剂（MDL 12330A），5-HT受体通过环磷酸腺苷/腺苷酸环化酶/蛋白激酶A（cAMP/AC/PKA）途径参与DDX3表达。AS-19介导DDX3启动子激活PKA细胞外信号调节激酶ERK信号，p53磷酸化（-1080/-1070）导致通过5-HT受体激动剂上调DDX3启动子反式激活。总体而言，发现5-HT是通过5-HT激动剂反应介导DDX3表达，通过磷酸化p53激活AC/PKA/ERK途径来抑制乙型肝炎感染的新潜在靶点。
