title: A novel homologous recombination system to study 92 kDa type IV collagenase transcription demonstrates that the NF-kappaB motif drives the transition from a repressed to an activated state of gene expression.
abstract: The 92-kDa type IV collagenase (MMP-9) contributes to tissue remodeling in both physiology and pathology. Previous studies on the transcriptional regulation of this gene have used transiently transfected promoter-reporter constructs. However, this approach suffers from several limitations including (a) multiple copies of the plasmid and (b) the plasmid is not genomically integrated and consequently poorly chromatinized. We developed a novel system for studying MMP-9 transcription in which a single copy of a MMP-9 promoter-luciferase construct(s) is integrated at an identical genomic locus in HT1080 cells by homologous recombination. We report that the activity of a genomic-integrated 2.2 kb MMP-9 promoter sequence mirrors expression of the endogenous MMP-9 gene in response to both physiological and pharmacological (curcumin) cues. Further, when constrained into chromatin, the integrated NF-kappaB-mutated MMP-9 promoter is repressed by PMA, a situation not apparent using nonintegrated plasmids. Thus, we have developed a novel method for studying MMP-9 expression that overcomes some of the limitations associated with transient transfection approaches and which may be of utility in screening for agents that repress the expression of this gene.
authors.affiliation: Department of Cancer Biology, M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
authors.forename: Chunhong
authors.initials: C
authors.lastname: Yan
authors.forename: Heng
authors.initials: H
authors.lastname: Wang
authors.forename: Bharat
authors.initials: B
authors.lastname: Aggarwal
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Boyd
mesh_terms: D002460:Cell Line; D003474:Curcumin / Q000494:pharmacology; D004254:DNA Nucleotidyltransferases / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D020780:Matrix Metalloproteinase 9 / Q000235:genetics*; D016328:NF-kappa B / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011995:Recombination, Genetic; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D006877:Hydroxamic Acids; D016328:NF-kappa B; C012589:trichostatin A; D008156:Luciferases; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D020780:Matrix Metalloproteinase 9; D003474:Curcumin; D013755:Tetradecanoylphorbol Acetate