v-Ha-RaS oncogene upregulates the 92-kDa type IV collagenase (MMP-9) gene by increasing cellular superoxide production and activating NF-kappaB.
Matrix metalloproteinase 9 (MMP-9) degrades basement membrane type IV collagen and is expressed during cellular migration and invasion. Here we show that v-Ha-Ras overexpression in rat kidney epithelial cells (REC) caused upregulation of MMP-9 gene expression in part by increasing cellular oxidant levels. v-Ha-Ras mediated the production of superoxide in Ras-transfected cells, which was associated with upregulated MMP-9 gene expression. Conversely, v-Ha-Ras expression decreased steady-state levels of mRNAs from tissue inhibitor of metalloproteinase 1 (TIMP-1), an inhibitor of MMP-9; plasminogen activator inhibitor 1 (PAI-1), which indirectly activates MMP-9 by increasing plasmin levels; and collagen IV, a substrate of MMP-9 and a major component of basement membrane. Gel mobility shift assays demonstrated that Ras overexpression enhanced NF-kappaB, but not AP-1 DNA binding to motifs in the MMP-9 gene promoter. The Ras-induced increase in NF-kappaB DNA binding could be inhibited by treatment with the antioxidants N-acetyl-L-cysteine and glutathione monoester, suggesting that intracellular oxidant levels can mediate MMP-9 transcription. Our findings identify an important role for Ras in the regulation of MMP-9 expression, and suggest that increased superoxide production can upregulate MMP-9 expression and thus contribute to malignant conversion.
Yang
J Q
JQ
Free Radical and Radiation Biology Program and Holden Comprehensive Cancer Center, Department of Radiology, The University of Iowa, Iowa City, IA 52242, USA.
Zhao
W
W
Duan
H
H
Robbins
M E
ME
Buettner
G R
GR
Oberley
L W
LW
Domann
F E
FE
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D003574:Cytochrome c Group / Q000037:antagonists & inhibitors / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004847:Epithelial Cells / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D011905:Genes, ras / Q000502:physiology*; D007668:Kidney / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D016328:NF-kappa B / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000502:physiology
D003574:Cytochrome c Group; D017931:DNA Primers; D005353:Fibronectins; D016328:NF-kappa B; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D018808:Transcription Factor AP-1; D013481:Superoxides; D013482:Superoxide Dismutase; D020780:Matrix Metalloproteinase 9