Cooperation of H2O2-mediated ERK activation with Smad pathway in TGF-beta1 induction of p21WAF1/Cip1.
Although it has been demonstrated that p21WAF1/Cip1 could be induced by transforming growth factor-beta1 (TGF-beta1) in a Smad-dependent manner, the cross-talk of Smad signaling pathway with other signaling pathways still remains poorly understood. In this study, we investigated a possible role of hydrogen peroxide (H2O2)-ERK pathway in TGF-beta1 induction of p21WAF1/Cip1 in human keratinocytes HaCaT cells. Using pharmacological inhibitors specific for MAP kinase family members, we found that ERK, but not JNK or p38, is required for TGF-beta1 induction of p21WAF1/Cip1. ERK activation by TGF-beta1 was significantly attenuated by treatment with N-acetyl-l-cysteine or catalase, indicating that reactive oxygen species (ROS) generated by TGF-beta1, mainly H2O2, stimulates ERK signaling pathway to induce the p21WAF1/Cip1 expression. In support of this, TGF-beta1 stimulation caused an increase in intracellular ROS level, which was completely abolished by pretreatment with catalase. ERK activation does not appear to be associated with nuclear translocation of Smad-3, because ERK inhibition did not affect nuclear translocation of Smads by TGF-beta1, and H2O2 treatment alone did not cause nuclear translocation of Smad-3. On the other hand, ERK inhibition ablated the phosphorylation of Sp1 by TGF-beta1, which was accompanied with the disruption of interaction between Smad-3 and Sp1 as well as of the recruitment of Sp1 to the p21WAF1/Cip1 promoter induced by TGF-beta1, indicating that ERK signaling pathway might be necessary for their interaction. Taken together, these results suggest that activation of H2O2-mediated ERK signaling pathway is required for p21WAF1/Cip1 expression by TGF-beta1 and led us to propose a cooperative model whereby TGF-beta1-induced receptor activation stimulates not only a Smad pathway but also a parallel H2O2-mediated ERK pathway that acts as a key determinant for association between Smads and Sp1 transcription factor.
College of Medicine, Kwandong University, Gangneung 210-701, Republic of Korea. yksnbk@kwandong.ac.kr
Yong Kee
YK
Kim
Gyu-Un
GU
Bae
Jae Ku
JK
Kang
Jong Woo
JW
Park
Eun Kyung
EK
Lee
Hoi Young
HY
Lee
Wahn Soo
WS
Choi
Hyang Woo
HW
Lee
Jeung-Whan
JW
Han
D021581:Active Transport, Cell Nucleus; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis* / Q000235:genetics; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017382:Reactive Oxygen Species; D051785:Smad Proteins; D016329:Sp1 Transcription Factor; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D006861:Hydrogen Peroxide; D048049:Extracellular Signal-Regulated MAP Kinases