Abstract
The active forms of all of the matrix metalloproteinases (MMPs) are inhibited by a family of specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Inhibition represents a major level of control of MMP activity. A detailed knowledge of the mechanisms controlling TIMP gene expression is therefore important. We have isolated a genomic clone of the human TIMP-1 gene. A 3 kbp XbaI fragment has been sequenced; this fragment contains 1718 bp 5' flanking sequences, exon 1, a 929 bp intron 1 and part of exon 2. Computer analysis reveals 10 consensus sequences for Sp1, six for activating protein 1 (AP-1), six for polyoma enhancer A3 (PEA3), 12 for AP-2 and five CCAAT boxes. The region hybridizing with a murine TIMP-1 promoter fragment has been subcloned and analysed further. RNase protection identifies six transcription start points, making exon 1 up to 48 bp in length. Transient transfection of promoter-chloramphenicol O-acetyltransferase reporter constructs into primary human connective tissue fibroblasts shows that a 904 bp fragment that hybridizes to a murine TIMP-1 promoter fragment contains a functional promoter. Constructs of -738/+95 to -194/+21 are inducible with serum or phorbol ester to a similar extent to the endogenous TIMP-1 gene. These results and further mapping with 5' deletion mutants from the -738/+95 region have demonstrated that an AP-1 site at -92/-86 is essential for basal expression of the gene. Point mutations within this region have further confirmed the role of this site, along with a more minor role for a neighbouring PEA3 site, in basal expression. Deletions from the 3' end also implicate a region across the exon 1/intron 1 boundary and especially +21 to +58 in basal expression. The +21/+58 region contains a putative binding site for the transcription factor leader-binding protein 1 (LBP-1). Gel-shift analysis shows that protein binds specifically to this region, but competition studies suggest that it is unlikely to be LBP-1.
Original language | English |
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Pages (from-to) | 611-617 |
Number of pages | 7 |
Journal | Biochemical Journal |
Volume | 324 |
Issue number | 2 |
DOIs | |
Publication status | Published - 1 Jun 1997 |
Keywords
- Animals
- Base Sequence
- Binding Sites
- Cells, Cultured
- Cloning, Molecular
- Consensus Sequence
- DNA-Binding Proteins
- Exons
- Fibroblasts
- Gene Expression Regulation
- Gene Library
- Glycoproteins
- Humans
- Introns
- Mice
- Molecular Sequence Data
- Point Mutation
- Promoter Regions, Genetic
- Recombinant Fusion Proteins
- Tissue Inhibitor of Metalloproteinases
- Transcription Factor AP-1
- Transcription Factors
- Transcription, Genetic
- Transfection