Taheri Faramarz, Bazan Haydee E P
Department of Biochemistry, Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112-2223, USA.
Invest Ophthalmol Vis Sci. 2007 May;48(5):1931-41. doi: 10.1167/iovs.06-1008.
Matrix metalloproteinase (MMP)-9 is induced in corneal epithelial cells stimulated with platelet-activating factor (PAF), and interferes with the normal reepithelialization of wounded cornea. Here the transcriptional regulation of MMP-9 gene expression by PAF was investigated in human corneal epithelial cells (HCECs).
DNA-binding activity of NFkappaB, Sp1, and AP-1 was determined in quiescent and PAF-stimulated HCECs by electrophoretic mobility shift assay (EMSA). A series of 5' deleted human MMP-9 promoter-luciferase reporter constructs was transiently transfected into HCECs, and luciferase activity was examined after stimulation with PAF. Mutagenesis and specific deletions of some elements in the MMP-9 promoter were also introduced and analyzed. Phosphorylation of Sp1 and MEK/ERK pathway proteins was examined by Western blot analysis. Activation of Sp1 and MMP-9 was also determined by ELISA and zymography, respectively, in the absence or presence of the MEK inhibitor PD98059.
DNA-binding activity of NFkappaB, Sp1, and AP-1 was upregulated by PAF with a peak at 1 hour after stimulation. A region spanning -670 to -460 relative to the transcription start point was required for the induction of the MMP-9 promoter by PAF. Mutation of the -79AP-1 or -600NFkappaB motif reduced the activity of MMP-9 promoter and the induction of gene expression by PAF. In untreated HCECs, mutation of the -558Sp1 motif upregulated gene expression, but it caused a significant decrease in the promoter activity induced by PAF. Inhibition of MEK activity eliminated the PAF-induced phosphorylation and activation of Sp1 and abolished the upregulation of MMP-9 expression and activity.
These findings demonstrate that collaboration between several regulatory elements is required for the induction of MMP-9 promoter activity by PAF and that PAF overturns the repressor effect of Sp1 through activation of the MEK/ERK signaling cascade.
基质金属蛋白酶（MMP）-9 在血小板活化因子（PAF）刺激的角膜上皮细胞中被诱导产生，并干扰受伤角膜的正常再上皮化过程。在此，我们研究了 PAF 对人角膜上皮细胞（HCECs）中 MMP-9 基因表达的转录调控作用。
通过电泳迁移率变动分析（EMSA）测定静止和 PAF 刺激的 HCECs 中 NFκB、Sp1 和 AP-1 的 DNA 结合活性。将一系列 5'端缺失的人 MMP-9 启动子-荧光素酶报告基因构建体瞬时转染至 HCECs 中，并用 PAF 刺激后检测荧光素酶活性。还对 MMP-9 启动子中的某些元件进行了诱变和特异性缺失，并进行分析。通过蛋白质印迹分析检测 Sp1 和 MEK/ERK 信号通路蛋白的磷酸化情况。在存在或不存在 MEK 抑制剂 PD98059 的情况下，分别通过 ELISA 和酶谱法测定 Sp1 和 MMP-9 的激活情况。
PAF 可上调 NFκB、Sp1 和 AP-1 的 DNA 结合活性，刺激后 １ 小时达到峰值。PAF 诱导 MMP-9 启动子需要一个相对于转录起始点跨度为 -670 至 -460 的区域。-79AP-1 或 -600NFκB 基序的突变降低了 MMP-9 启动子的活性以及 PAF 对基因表达的诱导作用。在未处理的 HCECs 中，-558Sp1 基序的突变上调了基因表达，但导致 PAF 诱导的启动子活性显著降低。抑制 MEK 活性可消除 PAF 诱导的 Sp1 磷酸化和激活，并消除 MMP-9 表达和活性的上调。
这些发现表明，PAF 诱导 MMP-9 启动子活性需要几种调控元件之间的协同作用，并且 PAF 通过激活 MEK/ERK 信号级联反应来消除 Sp1 的抑制作用。
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