Stearns Mark E, Kim Greg, Garcia Fernando, Wang Min
Department of Pathology, College of Medicine, Drexel University, MS 435, 15th and Vine Streets, Philadelphia, PA 19085, USA.
Mol Cancer Res. 2004 Jul;2(7):403-16.
Aberrant expression of the 72-kDa type IV collagenase [matrix metalloproteinase (MMP)-2] is implicated in the invasion and angiogenesis process of malignant tumors. We investigated the effects of interleukin (IL)-10 on MMP-2 expression in CPTX-1532 human prostate tumor cells. Our results demonstrate that IL-10 significantly inhibited MMP-2 transcription and protein expression induced by a phorbol ester, phorbol 12-myristate 13-acetate. The inhibitory effects of IL-10 on MMP-2 expression correlated with the suppression of MMP-2 promoter activity. To determine the mechanism of IL-10 action, we examined IL-10-dependent promoter activity with luciferase constructs from a 2-kbp promoter region of the human MMP-2 gene. We functionally characterized the promoter fragments by transient transfection experiments with CPTX-1532 cells. The experiments revealed that a cAMP responsive element binding protein (CREB) consensus domain was identified upstream of the 5' transcriptional start site, which was highly responsive to IL-10-dependent down-regulation of promoter luciferase activity. Electrophoretic mobility shift assays combined with antibody "supershift assays" confirmed the data from the luciferase assays. Immunoblot assays of activating transcription factor (ATF) 3 immunoprecipitates with tyrosine specific antibodies revealed that IL-10 stimulated tyrosine phosphorylation of ATF3 to activate binding to the CREB domain and suppress MMP-2 expression. Studies with stable, IL-10 transfected CPTX-1532 subclones further showed that IL-10 failed to suppress MMP-2 expression in ATF3-deficient CPTX-1532 cells, where the ATF3 mRNA was destroyed with a DNAzyme oligonucleotide targeting the 5' region of the mRNA. Finally, reconstitution of ATF3 successfully restored the inhibitory effects of IL-10 on MMP-2 gene expression. Taken together, these data demonstrate the critical role of tyrosine phosphorylated ATF3 and the CREB consensus domain in IL-10 suppression of MMP-2 gene expression in primary human prostate tumor cells.
72-kDa IV型胶原酶[基质金属蛋白酶（MMP）-2]的异常表达与恶性肿瘤的侵袭和血管生成过程有关。我们研究了白细胞介素（IL）-10对CPTX-1532人前列腺肿瘤细胞中MMP-2表达的影响。我们的结果表明，IL-10显著抑制佛波酯（佛波醇12-肉豆蔻酸酯13-乙酸酯）诱导的MMP-2转录和蛋白表达。IL-10对MMP-2表达的抑制作用与MMP-2启动子活性的抑制相关。为了确定IL-10的作用机制，我们用人MMP-2基因2-kbp启动子区域的荧光素酶构建体检测了IL-10依赖性启动子活性。我们通过CPTX-1532细胞的瞬时转染实验对启动子片段进行了功能表征。实验显示，在5'转录起始位点上游鉴定出一个cAMP反应元件结合蛋白（CREB）共有结构域，其对IL-10依赖性的启动子荧光素酶活性下调高度敏感。电泳迁移率变动分析结合抗体“超迁移分析”证实了荧光素酶分析的数据。用酪氨酸特异性抗体对激活转录因子（ATF）3免疫沉淀进行免疫印迹分析显示，IL-10刺激ATF3的酪氨酸磷酸化，以激活其与CREB结构域的结合并抑制MMP-2表达。对稳定转染IL-10的CPTX-1532亚克隆的研究进一步表明，在ATF3缺陷的CPTX-1532细胞中，IL-10无法抑制MMP-2表达，在这些细胞中，ATF3 mRNA被靶向mRNA 5'区域的脱氧核酶寡核苷酸破坏。最后，ATF3的重建成功恢复了IL-10对MMP-2基因表达的抑制作用。综上所述，这些数据证明了酪氨酸磷酸化的ATF3和CREB共有结构域在IL-10抑制原发性人前列腺肿瘤细胞中MMP-2基因表达中的关键作用。
