To gain a further understanding of the regulation of human type I collagen gene expression under physiologic and pathologic conditions, we characterized 5.3 kilobase pairs (kb) of the human α1(I) procollagen gene promoter. A series of deletion constructs containing portions of the α1(I) procollagen 5'-flanking region (with end points from -5.3 kb to -84 base pairs (bp)) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into NIH/3T3 cells. Maximal CAT activity was observed with constructs having 5' end points from -804 to -174 bp. A further 5' deletion to -84 bp caused a marked reduction in CAT activity. Cells transfected with plasmids containing longer 5'-flanking fragments of the α1(I) procollagen gene (-2.3 or -5.3 kb) showed reduced CAT activity compared with the -804 bp construct. The activity of the α1(I) procollagen promoter was much lower in cells that do not normally express type I collagen (HeLa cells) compared with collagen-producing NIH/3T3 cells. The CAT activity of deletion constructs containing longer 5' regions than -84 bp was increased by ≃2-fold in NIH/3T3 cells treated with transforming growth factor β1 (TGFβ1). Electrophoretic mobility shift assays indicated that protein-DNA complex formation with a probe corresponding to the -170 to -80 bp fragment of the α1(I) procollagen promoter was markedly enhanced in nuclear extracts prepared from TGFβ1-treated fibroblasts as compared with untreated fibroblasts. The DNA binding activity stimulated by TGFβ1 was specific for an Sp1-like sequence at positions -164 to -142 bp in the promoter. These results demonstrate that 1) there are both positive and negative cis-acting regulatory elements in the human α1(I) procollagen promoter, 2) these regulatory regions function differently in collagen-producing and - nonproducing cells, 3) the α1(I) procollagen promoter contains TGFβ1- responsive sequences located between -174 and -84 bp from the transcription start site, and 4) TGFβ1 caused marked stimulation of the DNA binding activity of a nuclear factor interacting with an Sp1-like binding site located within a region encompassing -164 to -142 bp of the α1(I) procollagen promoter.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology