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* Department of Medicine, University of Alberta, Edmonton, Alberta, Canada;
Northern Ontario School of Medicine, Lakehead University, Thunder Bay, Ontario, Canada;
University of Dublin Trinity College, Dublin, Ireland; and
Department of Laboratory Medicine and Pathology, University of Alberta Hospitals, Edmonton, Alberta, Canada
Matrix metalloproteinase-9 (MMP-9) is released by human lung epithelial cells (LEC) in conditions such as asthma and chronic obstructive pulmonary disease and expression of MMP-9 correlates with the severity of these disorders. MMP-9 production has been reported to be regulated by a NO/soluble guanylate cyclase-dependent pathway. Transcriptional regulation of this enzyme, however, is poorly understood. Using phylogenetic analysis, we observed a highly conserved sequence in the 5' flanking region of the MMP-9 gene containing binding sites for the transcription factor Wilms tumor 1 (WT1). We confirmed the presence of WT1 in human LEC and that treatment with TNF or a mixture containing LPS, PMA, and IFN-
resulted in translocation of WT1 from the nucleus to the cytosol. This translocation coincided with increased expression of MMP-9 and could be blocked by inhibitors of the NO/soluble guanylate cyclase pathway. WT1 knockdown using small-interfering RNA up-regulated MMP-9 expression in the presence of the NO synthase inhibitor 1400W. Using either WT1 pulldown with probes for the conserved region of the MMP-9 promoter or chromatin immunoprecipitation, we confirmed WT1 binding to the MMP-9 promoter. These findings indicate WT1 is a repressor of MMP-9, regulated by a NO-mediated pathway in human LEC. To our knowledge, this is the first report of WT1 regulating MMP-9 expression. Further study is needed to determine whether clinical conditions exhibiting tissue remodeling, such as asthma and/or chronic obstructive pulmonary disease, demonstrate reduced levels of WT1 or its repressor activity.
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1 Address correspondence and reprint requests to Dr. A. Dean Befus, Glaxo-Heritage Asthma Research Laboratories, Room 550A Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada. E-mail address: dean.befus{at}ualberta.ca
2 Abbreviations used in this paper: MMP, matrix metalloproteinase; COPD, chronic obstructive pulmonary disease; LEC, lung epithelial cell; sGC, soluble guanylate cyclase; NOS, NO synthase; iNOS, inducible NOS; WT1, Wilms tumor 1; DAF-FM, 4-amino-5-methylamino-2',7'-difluorofluorescein; PKA, protein kinase A; EC, epithelial cell; PBEC, primary normal human bronchial EC; L-NAME, N
-nitro-L-arginine methyl ester; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PKG, protein kinase G; ChIP, chromatin immunoprecipitation; 2-D, two dimensional; PMP, paramagnetic particle; pI, isoelectric point; siRNA, small-interfering RNA; DAPI, 4',6'-diamidino-2-phenylindole.
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