Interleukin-10 induced activating transcription factor 3 transcriptional suppression of matrix metalloproteinase-2 gene expression in human prostate CPTX-1532 Cells.
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.
Stearns
Mark E
ME
Department of Pathology, College of Medicine, Drexel University, MS 435, 15th and Vine Streets, Philadelphia, PA 19085, USA. stearnsm1@aol.com
Kim
Greg
G
Garcia
Fernando
F
Wang
Min
M
D051700:Activating Transcription Factor 3; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D021881:DNA, Catalytic / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D006801:Humans; D047468:Immunoprecipitation; D016753:Interleukin-10 / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000235:genetics*; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011471:Prostatic Neoplasms / Q000235:genetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014407:Tumor Cells, Cultured
D051700:Activating Transcription Factor 3; D017362:Cyclic AMP Response Element-Binding Protein; D021881:DNA, Catalytic; D012333:RNA, Messenger; D014157:Transcription Factors; D016753:Interleukin-10; D008156:Luciferases; D020778:Matrix Metalloproteinase 2