Transcriptional activity of the human tissue inhibitor of metalloproteinases 1 (TIMP-1) gene in fibroblasts involves elements in the promoter, exon 1 and intron 1.
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.
Clark
I M
IM
Rheumatology Research Unit, Addenbrooke's Hospital, Cambridge, CB2 2QQ, U.K.
Rowan
A D
AD
Edwards
D R
DR
Bech-Hansen
T
T
Mann
D A
DA
Bahr
M J
MJ
Cawston
T E
TE
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D005091:Exons / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D015723:Gene Library; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D019714:Tissue Inhibitor of Metalloproteinases; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D018808:Transcription Factor AP-1; D014157:Transcription Factors; C085968:UBP1 protein, human