An activator protein-1 (AP-1) response element on pro α₁(I) collagen gene is necessary for thyroid hormone-induced inhibition of promoter activity in cardiac fibroblasts

Thyroid hormone-induced ventricular hypertrophy is characterized by the absence of fibrosis. Previously, we demonstrated that thyroid hormone inhibits collagen type I gene expression in the myocardium and in cardiac fibroblasts. We also demonstrated that thyroid hormones act as inhibitor of pro α₁(I) collagen promoter activity. In this study we determined the sequences on pro α₁(I) collagen gene and transcription factors in cardiac fibroblasts involved in the inhibitory effect of 3,3',5-triiodothyronine (T₃). Transient transfection of cells with chloramphenicol acetyl transferase (CAT)-linked deletion mutants of pro α₁(I)) collagen promoter demonstrated that the inhibitory effect of T₃ is transmitted via proximal sequences (-225/+115). Gel shift analysis using [³²P]-labeled -225/+115 gene fragment and nuclear proteins of cardiac fibroblasts showed T₃-induced DNA binding by two proteins. Analysis of non-overlapping restriction sub-fragments by gel shift along with supershift analysis with antibodies to types α and β thyroid hormone receptors identified the lower molecular weight DNA-binding protein as β receptor and confirmed that the T₃-induced protein-DNA binding sites are located at -15/+115. Selective base mutation (C in place of G at +93 and G in place of C at +97) in the activator protein-1 (AP-1) core binding motif (+92/+97) abolished the higher molecular weight T₃-induced DNA-protein complex obtained with [³²P]-labeled wild type sequences (-225/+115). Additional gel shift analyses using an oligonucleotide containing the AP-1 core binding motif, as an unlabeled competitor and as [³²P]-labeled probe. confirmed the T₃-induced protein binding to an AP-1 site. Transient transfection with CAT-linked -225/+115 sequences in which the AP-1 site was mutated abolished the T₃-induced inhibition of CAT activity. Together, these findings identify sequences necessary for T₃-induced inhibition of collagen type I promoter to which thyroid hormone receptor type β and protein(s) with affinity for AP-1 element bind. They also demonstrate that the AP-1 response element located on these sequences is necessary for T₃-induced inhibition of pro α₁(I) collagen promoter activity. These data identify molecular mechanisms involved in thyroid hormon-induced inhibition of collagen expression in the heart.