Upstream tissue inhibitor of metalloproteinases-1 (TIMP-1) element-1, a novel and essential regulatory DNA motif in the human TIMP-1 gene promoter, directly interacts with a 30-kDa nuclear protein

Elevated expression of the tissue inhibitor of metalloproteinases-l (TIMP-1 ) protein and mRNA has been reported in human diseases including cancers an d tissue fibrosis. Regulation of TIMP-1 gene expression is mainly mediated at the level of gene transcription and involves the activation of several w ell known transcription factors including those belonging to the AP-1, STAT , and Pea3/Ets families. In the current study, we have used DNase-l footpri nting to identify a new regulatory element (5'-TGTGGTTTCCG-3') present in t he human TIMP-1 gene promoter. Mutagenesis and transfection studies in cult ure-activated rat hepatic stellate cells and the human Jurkat T cell line d emonstrated that the new element named upstream TIMP-1 element-1 (UTE-1) is essential for transcriptional activity of the human TIMP-1 promoter. Elect rophoretic mobility shift assay studies revealed that UTE-1 can form protei n-DNA complexes of distinct mobilities with nuclear extracts from a variety of mammalian cell types and showed that induction of a high mobility UTE-1 complex is associated with culture activation of freshly isolated rat hepa tic stellate cells. A combination of W-cross-linking and Southwestern blott ing techniques demonstrated that UTE I directly interacts with a 30-kDa nuc lear protein that appears to be present in all cell types tested. We conclu de that UTE-1 is a novel regulatory element that in combination with its ce llular binding proteins may be an important component of the mechanisms con trolling TIMP-1 expression in normal and pathological states.