Lee Yoon-Jin, Cho Hae-Nyun, Jeoung Doo-Il, Soh Ja-Won, Cho Chul Koo, Bae Sangwoo, Chung Hee-Yong, Lee Su-Jae, Lee Yun-Sil
Division of Molecular Life Science, College of Natural Science, Ewha Woman's University, Seoul, South Korea.
Free Radic Biol Med. 2004 Feb 15;36(4):429-44. doi: 10.1016/j.freeradbiomed.2003.11.009.
HSP25 has been shown to induce resistance to radiation and oxidative stress; however, its exact mechanisms remain unclear. In the present study, a high concentration of H2O2 was found to induce DNA fragmentation in L929 mouse fibroblast cells, and HSP25 overexpression attenuated this phenomenon. To elucidate the mechanisms of H2O2-mediated cell death, ERK1/2, p38 MAPK, and JNK1/2 phosphorylation in the cells after treatment with H2O2 were examined. ERK1/2 and JNK1/2 were activated by H2O2; ERK1/2 activation was inhibited in HSP25-overexpressed cells, while JNK1/2 was indifferent. Inhibition of ERK1/2 activation by treatment of the cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced cell death; similarly treated HSP25-overexpressed cells were not at all affected. Moreover, inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 transfection did not affect H2O2-mediated cell death in control cells. Dominant-negative Ras or Raf transfection inhibited H2O2-mediated ERK1/2 activation and cell death in control cells. On the contrary, HSP25-overexpressed cells did not show any differences. Upstream pathways of H2O2-mediated ERK1/2 activation and cell death involved both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta, while in HSP25-overexpressed cells these kinases did not respond to H2O2 treatment. Since HSP25 overexpression reduced reactive oxygen species (ROS), increased manganese superoxide dismutase (MnSOD) gene expression, and increased enzyme activity, involvement of MnSOD in HSP25-mediated attenuation of H2O2-mediated ERK1/2 activation and cell death was examined. Blockage of MnSOD with antisense oligonucleotides prevented DNA fragmentation and returned the ERK1/2 activation to the control level. Indeed, when MnSOD was overexpressed in L929 cells, similar to in HSP25-overexpressed cells, DNA fragmentation and ERK1/2 activation were reduced. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated downregulation of ERK1/2.
热休克蛋白25（HSP25）已被证明可诱导对辐射和氧化应激的抗性；然而，其确切机制仍不清楚。在本研究中，发现高浓度的过氧化氢（H2O2）可诱导L929小鼠成纤维细胞中的DNA片段化，而HSP25的过表达减弱了这种现象。为了阐明H2O2介导的细胞死亡机制，检测了用H2O2处理后细胞中细胞外信号调节激酶1/2（ERK1/2）、p38丝裂原活化蛋白激酶（p38 MAPK）和应激活化蛋白激酶1/2（JNK1/2）的磷酸化情况。H2O2激活了ERK1/2和JNK1/2；在HSP25过表达的细胞中，ERK1/2的激活受到抑制，而JNK1/2则不受影响。用PD98059处理细胞或转染显性负性ERK2抑制ERK1/2的激活可阻断H2O2诱导的细胞死亡；同样处理的HSP25过表达细胞则完全不受影响。此外，用显性负性JNK1或JNK2转染抑制JNK1/2并不影响对照细胞中H2O2介导的细胞死亡。显性负性Ras或Raf转染抑制了对照细胞中H2O2介导的ERK1/2激活和细胞死亡。相反，HSP25过表达的细胞没有表现出任何差异。H2O2介导的ERK1/2激活和细胞死亡的上游途径涉及酪氨酸激酶（血小板衍生生长因子β受体和Src）和蛋白激酶Cδ（PKCδ），而在HSP25过表达的细胞中，这些激酶对H2O2处理没有反应。由于HSP25的过表达减少了活性氧（ROS），增加了锰超氧化物歧化酶（MnSOD）基因的表达并提高了酶活性，因此研究了MnSOD在HSP25介导的减弱H2O2介导的ERK1/2激活和细胞死亡中的作用。用反义寡核苷酸阻断MnSOD可防止DNA片段化，并使ERK1/2的激活恢复到对照水平。实际上，当MnSOD在L929细胞中过表达时，与HSP25过表达的细胞类似，DNA片段化和ERK1/2的激活减少。根据上述结果，我们首次提出HSP25减少氧化损伤是由于MnSOD介导的ERK1/2下调。
