Abstract
Elevated expression of the tissue inhibitor of metalloproteinases-1 (TIMP-1) protein and mRNA has been reported in human diseases including cancers and tissue fibrosis. Regulation of TIMP-1 gene expression is mainly mediated at the level of gene transcription and involves the activation of several well known transcription factors including those belonging to the AP-1, STAT, and Pea3/Ets families. In the current study, we have used DNase-1 footprinting to identify a new regulatory element (5'-TGTGGTTTCCG-3') present in the human TIMP-1 gene promoter. Mutagenesis and transfection studies in culture-activated rat hepatic stellate cells and the human Jurkat T cell line demonstrated that the new element named upstream TIMP-1 element-1 (UTE-1) is essential for transcriptional activity of the human TIMP-1 promoter. Electrophoretic mobility shift assay studies revealed that UTE-1 can form protein-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 hepatic stellate cells. A combination of UV-cross-linking and Southwestern blotting techniques demonstrated that UTE-1 directly interacts with a 30-kDa nuclear protein that appears to be present in all cell types tested. We conclude that UTE-1 is a novel regulatory element that in combination with its cellular binding proteins may be an important component of the mechanisms controlling TIMP-1 expression in normal and pathological states.
Highlights
The complex interactions between cells and components of their surrounding extracellular matrix (ECM)1 are critical for many cellular properties including division, migration, differentiation, and death [1]
We have previously reported a minimal active promoter for the human tissue inhibitors of metalloproteinases (TIMPs)-1 gene that was sufficient to support a high level of transcription in culture-activated rat hepatic stellate cells (HSCs) but was inert in freshly isolated or quiescent HSCs [31]
We have reasoned that the minimal tissue inhibitor of metalloproteinases-1 (TIMP-1) gene promoter may be used to identify transcription factors with activities that are induced during HSC activation
Summary
HSC Isolation and Culture—HSCs were isolated from the livers of normal Harlan Sprague Dawley rats (400 Ϯ 50 g) by sequential perfusion with pronase and collagenase as described previously [36]. Nuclear extract (10 g) from HSCs was incubated with 1 g of poly(dI-dC) nonspecific DNA competitor (Sigma) for 10 min, and the radiolabeled TIMP-1 minimal promoter (100 ng) was added and incubated for a further 20 min. For competition assays excess unlabeled oligonucleotide (2–20 ng; Table I) was added to the reaction mixture with the nuclear extract and poly(dI-dC) for the initial incubation, prior to the addition the probe. Cell extracts were prepared by repeated freeze-thaw cycles and normalized for protein content using the Bio-Rad DC protein assay, and CAT activities were determined using [14C]chloramphenicol (Amersham Pharmacia Biotech) and acetyl-CoA (Sigma). Nuclear extracts (30 g) from rat activated HSCs, Jurkat T cells, and Daudi B cells were separated on a 12.5% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane. The UTE-1 DNA probe (15 ng) was prepared as described for EMSA studies and hybridized to the membrane at room temperature for 30 min
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