Functional analysis of human alpha 1(I) procollagen gene promoter. Differential activity in collagen-producing and -nonproducing cells and response to transforming growth factor beta 1.
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 alpha 1(I) procollagen gene promoter. A series of deletion constructs containing portions of the alpha 1(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 alpha 1(I) procollagen gene (-2.3 or -5.3 kb) showed reduced CAT activity compared with the -804 bp construct. The activity of the alpha 1(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 approximately 2-fold in NIH/3T3 cells treated with transforming growth factor beta 1 (TGF beta 1). Electrophoretic mobility shift assays indicated that protein-DNA complex formation with a probe corresponding to the -170 to -80 bp fragment of the alpha 1(I) procollagen promoter was markedly enhanced in nuclear extracts prepared from TGF beta 1-treated fibroblasts as compared with untreated fibroblasts. The DNA binding activity stimulated by TGF beta 1 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 alpha 1(I) procollagen promoter, 2) these regulatory regions function differently in collagen-producing and -nonproducing cells, 3) the alpha 1(I) procollagen promoter contains TGF beta 1-responsive sequences located between -174 and -84 bp from the transcription start site, and 4) TGF beta 1 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 alpha 1(I) procollagen promoter.
Jimenez
S A
SA
Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Varga
J
J
Olsen
A
A
Li
L
L
Diaz
A
A
Herhal
J
J
Koch
J
J
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003094:Collagen / Q000096:biosynthesis*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011347:Procollagen / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D009687:Nuclear Proteins; D011347:Procollagen; D016329:Sp1 Transcription Factor; D016212:Transforming Growth Factor beta; D003094:Collagen; D015500:Chloramphenicol O-Acetyltransferase