Li Changlin, Yu Yongshen, Wang Yun, Liu Lei, Zhang Min, Sugano Sumio, Wang Zhengguo, Chang Zongliang
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China.
Biol Cell. 2008 Jun;100(6):365-75. doi: 10.1042/BC20070140.
MD-2 is associated with the extracellular domain of TLR4 (Toll-like receptor 4) and augments TLR4-dependent LPS (lipopolysaccharide) responses in vitro. Our previous investigation found that PMA-induced HL-60 cell differentiation to macrophages is associated largely with TLR2 and CD14 and, to a much lesser extent, with TLR4.
We studied the MD-2 expression during differentiation of HL-60 cells induced by PMA. The results showed that PMA, but not VitD(3) (1alpha,25-dihydroxy-vitamin D(3)), strongly induces MD-2 gene expression by HL-60 cells in a time- and dose-dependent manner. Treatment with an MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] inhibitor (PD98059) and a JNK (c-Jun N-terminal kinase) inhibitor (SP600125) suppresses PMA-induced MD-2 gene expression, whereas impairment of p38 function by treatment with the inhibitor SB203580 has no effect on MD-2 mRNA. In order to reveal the possible molecular mechanism for such a regulation of MD-2 gene expression, we cloned and analysed the putative MD-2 gene promoter. Transient transfection of different deletion mutants demonstrated that the region -185/-171 (5'-TCCTTTACAGGAAGT-3') of the MD-2 gene promoter is closely related to gene transcription in response to PMA. Additionally, the transcription factor Elk-1 has been found to bind this specific motif.
These results suggest that ERK and JNK pathways are involved in PMA-mediated MD-2 gene expression during HL-60 cell differentiation, and the activation of the MEK/possible ERK/Elk signal pathway is the mechanism responsible for PMA-induced MD-2 gene expression in differentiated HL-60 cells.
MD-2与Toll样受体4（TLR4）的胞外结构域相关联，并在体外增强TLR4依赖性脂多糖（LPS）反应。我们之前的研究发现，佛波酯（PMA）诱导HL-60细胞分化为巨噬细胞主要与TLR2和CD14相关，而与TLR4的相关性较小。
我们研究了PMA诱导HL-60细胞分化过程中MD-2的表达情况。结果显示，PMA而非1α,25-二羟基维生素D3（VitD(3)）能以时间和剂量依赖性方式强烈诱导HL-60细胞表达MD-2基因。用MEK[丝裂原活化蛋白激酶（MAPK）/细胞外信号调节激酶（ERK）激酶]抑制剂（PD98059）和JNK（c-Jun氨基末端激酶）抑制剂（SP600125）处理可抑制PMA诱导的MD-2基因表达，而用抑制剂SB203580处理损害p38功能对MD-2 mRNA无影响。为揭示MD-2基因表达这种调控的可能分子机制，我们克隆并分析了假定的MD-2基因启动子。不同缺失突变体的瞬时转染表明，MD-2基因启动子的-185/-171区域（5'-TCCTTTACAGGAAGT-3'）与PMA刺激下的基因转录密切相关。此外，还发现转录因子Elk-1能结合这一特定基序。
这些结果表明，ERK和JNK信号通路参与了HL-60细胞分化过程中PMA介导的MD-2基因表达，MEK/可能的ERK/Elk信号通路的激活是PMA诱导分化的HL-60细胞中MD-2基因表达的机制。
