Tomiyoshi Go, Nakamura Rika, Shinmen Natsuko, Yoshida Yoichi, Mine Seiichiro, Machida Toshio, Iwase Katsuro, Iwadate Yasuo, Hiwasa Takaki, Kuroda Hideyuki
Medical Project Division, Research Development Center, Fujikura Kasei Co., Saitama, Japan.
Department of Biochemistry and Genetics, Graduate School of Medicine, Chiba University, Chiba, Japan.
Front Med (Lausanne). 2023 May 9;10:1128921. doi: 10.3389/fmed.2023.1128921. eCollection 2023.
We previously identified growth arrest and DNA-damage-inducible gene 34 (GADD34) as a marker of ischemic stroke. In the present study, serum levels of anti-GADD34 antibodies were found to be significantly higher in patients with acute ischemic stroke or chronic kidney disease compared to healthy donors. We then examined the biological function of GADD34 by transfection into U2OS human osteosarcoma and U87 human glioblastoma cells. Knockdown of GADD34 by siRNA resulted in enhanced cell proliferation, which was reversed by co-knockdown of MDM2. Luciferase reporter assays revealed that the transactivation ability of p53 enhanced by genotoxic anticancer drugs such as camptothecin and etoposide was further potentiated by enforced expression of GADD34 but attenuated by co-transfection with p53 shRNA expression plasmids. Western blotting demonstrated increased p53 protein levels after treatment with camptothecin, which was also potentiated by GADD34 but suppressed by GADD34 siRNA, ATM siRNA, and ATM inhibitor wortmannin. GADD34 levels also increased in response to treatment with camptothecin or adriamycin, and this increase was attenuated by MDM2 siRNA. Immunoprecipitation with anti-GADD34 antibody followed by Western blotting with anti-MDM2 antibodies indicated ubiquitination of GADD34 is mediated by MDM2. Accordingly, GADD34 may function as a ubiquitination decoy to reduce p53 ubiquitination and increase p53 protein levels. Increased neuronal cell death due to activation of p53 by GADD34 may account for the elevated serum levels of anti-GADD34 antibodies observed in patients with acute ischemic stroke.
我们之前将生长停滞和DNA损伤诱导基因34（GADD34）鉴定为缺血性中风的一个标志物。在本研究中，发现急性缺血性中风患者或慢性肾病患者血清中抗GADD34抗体水平显著高于健康供者。然后，我们通过转染U2OS人骨肉瘤细胞和U87人胶质母细胞瘤细胞来检测GADD34的生物学功能。用小干扰RNA（siRNA）敲低GADD34导致细胞增殖增强，而MDM2的共敲低可逆转这种增强。荧光素酶报告基因检测显示，喜树碱和依托泊苷等基因毒性抗癌药物增强的p53反式激活能力，在强制表达GADD34时进一步增强，但在用p53短发夹RNA表达质粒共转染时减弱。蛋白质印迹法表明，喜树碱处理后p53蛋白水平升高，GADD34也可增强这种升高，但GADD34 siRNA、ATM siRNA和ATM抑制剂渥曼青霉素可抑制这种升高。喜树碱或阿霉素处理后GADD34水平也升高，而MDM2 siRNA可减弱这种升高。用抗GADD34抗体进行免疫沉淀，随后用抗MDM2抗体进行蛋白质印迹分析表明，GADD34的泛素化由MDM2介导。因此，GADD34可能作为一种泛素化诱饵，减少p53泛素化并增加p53蛋白水平。GADD34激活p53导致神经元细胞死亡增加，这可能是急性缺血性中风患者血清中抗GADD34抗体水平升高的原因。
