An Activator Protein-1 (AP-1) Response Element on Pro α1(l) 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 proα1(l) collagen promoter activity. In this study we determined the sequences on proα1(l) 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(l) collagen promoter demonstrated that the inhibitory effect of T3is 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 typesαandβthyroid hormone receptors identified the lower molecular weight DNA-binding protein asβreceptor 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 typeβand 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 proα1(l) collagen promoter activity. These data identify molecular mechanisms involved in thyroid hormon-induced inhibition of collagen expression in the heart.