Identification of a hydrogen peroxide-induced PP1-JNK1-Sp1 signaling pathway for gene regulation

1 McGuire Veterans Affairs Medical Center, Richmond; and Departments of 2 Physiology and 3 Medicine, Virginia Commonwealth University, Richmond, Virginia Submitted 27 October 2005 ; accepted in final form 26 June 2006 Oxidative stress often results in changes in gene expression through the regulation of transcription factors. In this study, we examine how Sp1 phosphorylation is regulated by H 2 O 2 in a human alveolar epithelial cell line (HAE). Treatment of HAE cells with H 2 O 2 increases phosphorylation of Sp1 and activates JNK. To establish a relationship between JNK and Sp1, we show that JNK activator anisomycin increases Sp1 phosphorylation, and JNK inhibitors as well as dominant-negative JNK1 attenuate H 2 O 2 -induced Sp1 phosphorylation. Additionally, JNK1 directly phosphorylates Sp1 in vitro, reducing Sp1 binding to DNA. These results demonstrate the role of JNK in H 2 O 2 -induced Sp1 phosphorylation. Because H 2 O 2 inhibits Ser/Thr protein phosphatase-1 (PP1), we examined the role of PP1 in the regulation of JNK. Similar to H 2 O 2 , inhibition of PP1 induces phosphorylation of Sp1 and activation of JNK in HAE cells. Inhibition of JNK activity using either inhibitors or dominant-negative mutant JNK1 suppresses PP1 inhibition-induced Sp1 phosphorylation. Furthermore, PP1 directly inactivates JNK1 in vitro. These data suggest that 1 ) H 2 O 2 increases the phosphorylation level of Sp1, 2 ) Sp1 is a target of the JNK pathway, 3 ) PP1 regulates JNK activation, and 4 ) the "PP1-JNK" pathway plays a role in H 2 O 2 -induced Sp1 phosphorylation in lung epithelial cells. oxidative stress; reactive oxygen species; transcription factor; kinase; phosphatase; protein phosphatase-1 Address for reprint requests and other correspondence: S. Chu, McGuire VA Medical Center, 1201 Broad Rock Blvd., Richmond, VA 23249 (e-mail: schu{at}hsc.vcu.edu )