Keum Young-Sam, Yu Siwang, Chang Peter Pil-Jae, Yuan Xiaoling, Kim Jung-Hwan, Xu Changjiang, Han Jiahuai, Agarwal Anupam, Kong Ah-Ng Tony
Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
Cancer Res. 2006 Sep 1;66(17):8804-13. doi: 10.1158/0008-5472.CAN-05-3513.
Exposure of sulforaphane to HepG2 cells increased heme oxygenase-1 (HO-1) expression by activating antioxidant response element (ARE) through induction of Nrf2 and suppression of Kelch-like ECH-associated protein 1 (Keap1). Using human HO-1 promoter reporter plasmids and ChIP assay, we have identified that sulforaphane transcriptionally activated the upstream ARE-rich enhancer region, located at -9.0 kb upstream human HO-1 promoter. Induction of HO-1 by sulforaphane was attenuated by overexpression of mutant Nrf2 plasmid in HepG2 cells and totally abolished in Nrf2 knockout mouse embryonic keratinocytes and fibroblasts. Overexpression of individual p38 mitogen-activated protein (MAP) kinase (MAPK) isoforms also suppressed constitutive as well as sulforaphane- or Nrf2-induced ARE-dependent gene expression. Among the upstream kinases, although MKK3 was not involved in suppression of ARE by any of p38 MAPK isoforms, MKK6 selectively suppressed ARE by p38 gamma or p38 delta, but not by p38 alpha or p38 beta. Importantly, sulforaphane not only activated MAP/extracellular signal-regulated kinase (ERK) kinases 1/2 and ERK1/2, but also strongly suppressed anisomycin-induced activation of p38 MAPK isoforms by blocking phosphorylation of upstream kinases, MKK3/6. Finally, we found that stimulation of p38 MAPK isoforms phosphorylated purified Nrf2 protein and caused an increase in the interaction between Nrf2 and Keap1 in vitro and the suppression of Nrf2 translocation into the nucleus. Collectively, our results indicate that transcriptional activation of Nrf2/ARE is critical in sulforaphane-mediated induction of HO-1, which can be modulated in part by the blockade of p38 MAPK signaling pathway. In addition, our study shows that p38 MAPK can phosphorylate Nrf2 and promotes the association between Nrf2 and Keap1 proteins, thereby potentially inhibiting nuclear translocation of Nrf2.
萝卜硫素作用于HepG2细胞可通过诱导Nrf2和抑制类ECH相关蛋白1（Keap1）激活抗氧化反应元件（ARE），从而增加血红素加氧酶-1（HO-1）的表达。利用人HO-1启动子报告质粒和染色质免疫沉淀分析，我们确定萝卜硫素可转录激活位于人HO-1启动子上游-9.0 kb处富含ARE的增强子区域。在HepG2细胞中，突变型Nrf2质粒的过表达减弱了萝卜硫素对HO-1的诱导作用，而在Nrf2基因敲除的小鼠胚胎角质形成细胞和成纤维细胞中，这种诱导作用完全消失。单独过表达p38丝裂原活化蛋白（MAP）激酶（MAPK）亚型也会抑制组成型以及萝卜硫素或Nrf2诱导的ARE依赖性基因表达。在上游激酶中，虽然MKK3不参与任何p38 MAPK亚型对ARE的抑制作用，但MKK6可选择性地通过p38γ或p38δ抑制ARE，而不是通过p38α或p38β。重要的是，萝卜硫素不仅激活了MAP/细胞外信号调节激酶（ERK）激酶1/2和ERK1/2，还通过阻断上游激酶MKK3/6的磷酸化，强烈抑制茴香霉素诱导的p38 MAPK亚型的激活。最后，我们发现刺激p38 MAPK亚型可使纯化的Nrf2蛋白磷酸化，并导致体外Nrf2与Keap1之间的相互作用增加以及Nrf2向细胞核的转位受到抑制。总体而言，我们的结果表明，Nrf2/ARE的转录激活在萝卜硫素介导的HO-1诱导中起关键作用，这可部分通过阻断p38 MAPK信号通路来调节。此外，我们的研究表明p38 MAPK可使Nrf2磷酸化并促进Nrf2与Keap1蛋白之间的结合，从而可能抑制Nrf2的核转位。
