P27 Suppresses Arsenite-induced Hsp27/Hsp70 Expression through Inhibiting JNK2/c-Jun- and HSF-1-dependent Pathways

Jinyi Liu,Dongyun Zhang,Xiaoyi Mi,Qing Xia,Yonghui Yu,Zhenghong Zuo,Wei Guo,Xuewei Zhao,Jia Cao,Qing Yang,Angela Zhu,Wancai Yang,Xianglin Shi,Jingxia Li,Chuanshu Huang

doi:10.1074/jbc.m110.100271

Abstract

p27 is an atypical tumor suppressor that can regulate the activity of cyclin-dependent kinases and G(0)-to-S phase transitions. More recent studies reveal that p27 may also exhibit its tumor-suppressive function through regulating many other essential cellular events. However, the molecular mechanisms underlying these anticancer effects of p27 are largely unknown. In this study, we found that depletion of p27 expression by either gene knock-out or knockdown approaches resulted in up-regulation of both Hsp27 and Hsp70 expression at mRNA- and promoter-derived transcription as well as protein levels upon arsenite exposure, indicating that p27 provides a negative signal for regulating the expression of Hsp27 and Hsp70. Consistently, arsenite-induced activation of JNK2/c-Jun and HSF-1 pathways was also markedly elevated in p27 knock-out (p27(-/-)) and knockdown (p27 shRNA) cells. Moreover, interference with the expression or function of JNK2, c-Jun, and HSF-1, but not JNK1, led to dramatic inhibition of arsenite-induced Hsp27 and Hsp70 expression. Collectively, our results demonstrate that p27 suppresses Hsp27 and Hsp70 expression at the transcriptional level specifically through JNK2/c-Jun- and HSF-1-dependent pathways upon arsenite exposure, which provides additional important molecular mechanisms for the tumor-suppressive function of p27.

Highlights

Ible by stress such as heat, oxidative stress, or anticancer drugs [2]
We have demonstrated that p27 had an inhibitory effect on activation of the JNK/c-Jun and HSF-1 pathways, by which p27 exhibited suppression of hsp27 and hsp70 transcriptional expression and in turn resulted in the reduction of Hsp27 and Hsp70 protein expression in the cellular response to arsenite exposure
HSF-1 is reported to be responsible for Heat shock or stress proteins (Hsps) transcriptional regulation in many experimental systems

Summary

MATERIALS AND METHODS

Cell Culture—Immortalized wild-type p27 (p27ϩ/ϩ) and p27-deficient (p27Ϫ/Ϫ) mouse embryonic fibroblasts (MEFs) [12], HSF-1ϩ/ϩ and HSF-1Ϫ/Ϫ MEFs [13, 14], and their stable transfectants were maintained at 37 °C in a 5% CO2 incubator with DMEM supplemented with 10% FBS (Nova Tech Inc., Grand Island, NE), 2 mM L-glutamine, and 25 ␮g/ml gentamycin. The proteins were transferred to PVDF membranes (Bio-Rad), blocked, and probed with antibody against Hsp or HSF-1 (Stressgen, Ann Arbor, MI); Hsp, non-phosphorylated c-Jun, JNK1/2, p38, phospho-Ser63/Ser c-Jun, phospho-Thr183/ Tyr185 JNK, phospho-Ser257/Thr261 MKK4, phospho-Ser271/ Thr275 MKK7, phospho-Thr308/Ser473 AKT (Cell Signaling Technology, Beverly, MA); JNK1 (BioSource, Invitrogen); p27 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); or ␤-actin (Sigma). P27ϩ/ϩ and p27Ϫ/Ϫ cells were left untreated or treated with arsenite (20 ␮M) for 9 h, and genomic DNA and the proteins were cross-linked with 1% formaldehyde for 10 min at room temperature. The cross-linked cells were pelleted, resuspended in lysis buffer, and sonicated to generate 200 –500-bp chromatin DNA fragments using a Sonic Dismembrator 550 (Thermo Fisher Scientific) for 2 min at output level 3.

RESULTS

DISCUSSION