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

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.

p53 negatively regulates the expression of FAT10, a gene upregulated in various cancers

“…Regions upstream of the 5 0 UTR (exon 1) do not confer any promoter activity (Figure 1a, right panel). Basal promoter activity residing entirely in exon 1 was also reported in a few other genes including the SCA2 (Aguiar et al, 1999) and TIMP-1 (Clark et al, 1997) genes. Although the consensus sequence for a TATA box was identified in the promoter of FAT10 (Figure 1b), this box was found not to be essential for promoter activity but to play a role in enhancing its activity.…”

Farnesyltransferase inhibitor SCH‐66336 downregulates secretion of matrix proteinases and inhibits carcinoma cell migration

“…[45][46][47] Promoters of genes encoding TIMP-1 and TIMP-2 also contain consensus sequences for AP-1. 48,49 uPA expression is regulated through AP-1 binding sites in human prostate cancer. 50,51 NF-B signal blockade downregulates MMP-9 and uPA but upregulates TIMP-1, TIMP-2 and PAI-2.…”

Isoginkgetin inhibits tumor cell invasion by regulating phosphatidylinositol 3-kinase/Akt–dependent matrix metalloproteinase-9 expression

“…Regulation of TIMP-1 expression has not been well elucidated up to now. Although TIMP-1 promoter contains activator protein-1 binding site (31), inhibition of activator protein-1 does not always lead to TIMP-1 down-regulation. For example, in hepatic stellate cells, induction of c-Fos and c-Jun is unlikely to result in transactivation of the TIMP-1 promoter (32).…”