Kim Yong Kee, Bae Gyu-Un, Kang Jae Ku, Park Jong Woo, Lee Eun Kyung, Lee Hoi Young, Choi Wahn Soo, Lee Hyang Woo, Han Jeung-Whan
College of Medicine, Kwandong University, Gangneung 210-701, Republic of Korea.
Cell Signal. 2006 Feb;18(2):236-43. doi: 10.1016/j.cellsig.2005.04.008. Epub 2005 Jun 24.
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.
尽管已经证明p21WAF1/Cip1可以由转化生长因子-β1（TGF-β1）以Smad依赖的方式诱导，但Smad信号通路与其他信号通路的相互作用仍知之甚少。在本研究中，我们调查了过氧化氢（H2O2）-ERK通路在人角质形成细胞HaCaT细胞中TGF-β1诱导p21WAF1/Cip1过程中的可能作用。使用针对MAP激酶家族成员的特异性药理抑制剂，我们发现TGF-β1诱导p21WAF1/Cip1需要ERK，而不是JNK或p38。用N-乙酰-L-半胱氨酸或过氧化氢酶处理可显著减弱TGF-β1对ERK的激活，表明TGF-β1产生的活性氧（ROS），主要是H2O2，刺激ERK信号通路诱导p21WAF1/Cip1表达。支持这一点的是，TGF-β1刺激导致细胞内ROS水平升高，而过氧化氢酶预处理可完全消除这种升高。ERK激活似乎与Smad-3的核转位无关，因为ERK抑制不影响TGF-β1诱导的Smads核转位，单独的H2O2处理也不会导致Smad-3的核转位。另一方面，ERK抑制消除了TGF-β1对Sp1的磷酸化，这伴随着Smad-3与Sp1之间相互作用的破坏以及TGF-β1诱导的Sp1募集到p21WAF1/Cip1启动子的破坏，表明ERK信号通路可能是它们相互作用所必需的。综上所述，这些结果表明，H2O2介导的ERK信号通路的激活对于TGF-β1诱导p21WAF1/Cip1表达是必需的，并使我们提出了一个协同模型，即TGF-β1诱导的受体激活不仅刺激Smad通路，还刺激平行的H2O2介导的ERK通路，该通路是Smads与Sp1转录因子之间关联的关键决定因素。
