Cooperation of H₂O₂-mediated ERK activation with Smad pathway in TGF-β1 induction of p21^WAF1/Cip1

Although it has been demonstrated that p21^WAF1/Cip1 could be induced by transforming growth factor-β1 (TGF-β1) 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-β1 induction of p21^WAF1/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-β1 induction of p21 ^WAF1/Cip1. ERK activation by TGF-β1 was significantly attenuated by treatment with N-acetyl-l-cysteine or catalase, indicating that reactive oxygen species (ROS) generated by TGF-β1, mainly H₂O ₂, stimulates ERK signaling pathway to induce the p21 ^WAF1/Cip1 expression. In support of this, TGF-β1 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-β1, 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-β1, which was accompanied with the disruption of interaction between Smad-3 and Sp1 as well as of the recruitment of Sp1 to the p21^WAF1/Cip1 promoter induced by TGF-β1, 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 p21^WAF1/Cip1 expression by TGF-β1 and led us to propose a cooperative model whereby TGF-β1-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.