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.
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.
Yan
Chunhong
C
Department of Cancer Biology, M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Wang
Heng
H
Aggarwal
Bharat
B
Boyd
Douglas D
DD
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
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