Abstract
Mounting evidence suggests a role for matrix metalloproteinase (MMP)-2 in the malignant progression of breast cancer cells. We showed previously that H-Ras, but not N-Ras, induced invasion of MCF10A human breast epithelial cells through Rac-MKK3/6-p38 pathway resulted in MMP-2 up-regulation. Activation of p38 pathway by MKK6 caused a selective up-regulation of MMP-2. In this study, we aimed to elucidate the transcriptional regulation of MMP-2 by p38 pathway leading to the invasive phenotype of MCF10A cells. By using 5' deletion mutant constructs of MMP-2 promoter, we showed that deletion of the region containing activator protein-1 (AP-1) site caused the greatest reduction of MMP-2 promoter activity both in MKK6- and H-Ras-activated MCF10A cells, suggesting that the AP-1 binding site is critical for the MMP-2 promoter activation. DNA binding and transcriptional activities of AP-1 were increased by MKK6 or H-Ras as evidenced by electrophoretic mobility shift assay and luciferase assay using an AP-1-driven plasmid. By doing immunoinhibition assay and chromatin immunoprecipitation assay, we revealed the activating transcription factor (ATF) 2 as a transcription factor for MMP-2 gene expression through binding to the functional AP-1 site. Activation of ATF2, which depended on p38 activity, was crucial for MMP-2 promoter activity as well as induction of invasive and migrative phenotypes in MCF10A cells. This is the first report revealing ATF2 as an essential transcription factor linking MKK3/6-p38 signaling pathway to MMP-2 up-regulation, providing evidence for a direct role of ATF2 activation in malignant phenotypic changes of human breast epithelial cells.
Highlights
Tumor invasion and metastasis are often associated with enhanced synthesis of matrix metalloproteinases (MMPs), among which MMP-2 (72-kDa type IV collagenase) and MMP-9 (92-kDa type IV collagenase) are of central importance [1,2,3,4]
Increased activity of MMP-9 promoter was detected only in H-Ras MCF10A cells but not in MKK6-transfected cell lines. These results show that MKK6 induces up-regulation of MMP-2 by transcriptional activation, whereas it does not affect MMP-9 expression in MCF10A cells
We showed that the activation of MKK6/p38 pathway phosphorylated and activated ATF2, which bound to the activator protein-1 (AP-1) site critical for MMP-2 promoter activity, suggesting that ATF2 may be involved in the transcriptional regulation of the MMP-2 gene in MCF10A cells
Summary
Tumor invasion and metastasis are often associated with enhanced synthesis of matrix metalloproteinases (MMPs), among which MMP-2 (72-kDa type IV collagenase) and MMP-9 (92-kDa type IV collagenase) are of central importance [1,2,3,4]. Mounting evidence from laboratories, including ours, suggests a role for MMP-2 in the invasion of breast cancer cells and the risk for a relapse in breast cancer patients [5,6,7]. Activity of MMP-2 can be regulated by transcriptional gene activation, proenzyme activation, and inhibition of enzyme activity. Song: Korea Food and Drug Administration, Seoul 122-704, Korea. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1461
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