Figure 7

Ubiquitin conjugating enzyme variant (Uev)1A regulates Matrix metalloproteinase 1 ( MMP1 ) expression through an NF-κB target sequence in the MMP1 promoter. (A) A schematic illustration of the MMP1 promoter and the P _MMP1 -Luciferase (Luc) reporter construct. A putative NF-κB binding site in the MMP1 promoter [GenBank: AJ002550.1] is mapped to 1133–1125 nucleotides upstream of its start codon. The mutated sequence in the P _MMP1 -NF-κBm construct is also shown. (B) The P _MMP1 -Luc reporter was co-transfected to MDA-MB-231 cells with constructs that overexpressed wild-type UEV1A or UEV1A-F38E. The data were normalized to the activity of cells transfected with the empty vector (pGL4.2). (C) Only UEV1A, but not UEV1C or hMMS2, was able to activate the MMP1 promoter, and this ability was dependent on its interaction with Ubc13. The experimental conditions were as described in Figure 7B. (D) TNFα activates NF-κB as measured by the nuclear localization of the p65 subunit of NF-κB. HeLa cells were treated with or without TNFα for 30 minutes, nuclear proteins were purified and the relative p65 level in the nuclear fraction was determined by western blot and the nuclear protein Lamin B was used as a loading control. (E) Physical interaction between NF-κB and the putative NF-κB target sequence in the MMP1 promoter as determined by electrophoretic mobility shift assay. Lane 1, biotin-labeled NF-κB target sequence probe (NF-κB*). Lane 2, biotin-labeled mutated NF-κB binding site probe (NF-κBm*). Lane 3, Unlabeled double-strand DNA containing the P _MMP1 NF-κB target sequence (NF-κB). Lanes 4 to 7 contain either wild-type or mutated P _MMP1 NF-κB target sequence as indicated in the upper panel, plus TNFα-treated or untreated cell extract. Lane 7 also contains excessive unlabeled DNA of P _MMP1 NF-κB target sequence.