Molecular characterization of myocardial fibrosis during hypothyroidism:: evidence for negative regulation of the pro-α1(I) collagen gene expression by thyroid hormone receptor

The purpose of this study was to gain insights into the underlying mechanism of myocardial fibrosis during hypothyroidism. Treatment of cardiac fibroblasts with a medium lacking thyroid hormone led to a 47% increase in [H-3]thymidine incorporation into the cell nuclei compared with that in untreated cells. Northern blot analysis of RNA from cardiac fibroblasts grown in a thyroid hormone depleted medium resulted in a 38% increase in the abundance of mRNA for pro-alpha 1(I) collagen. At the protein level. the amount of type I collagen, as determined by immunoprecipitation, was increased either ill the cell lysate (46%) of cardiac fibroblasts grown in a thyroid hormone depleted medium or in the medium (44%). The chimeric plasmid, ColCAT 3.6, contains the 5'-flanking region of the rat pro-alpha 1(I) collagen gene (from bases - 3520 to + 115) fused to the chloramphenicol acetyltransferase (CAT) gene. The plasmid was cotransfected with thyroid hormone receptor (TR) expression plasmid into rat cardiac fibroblasts and COS-1 cells (monkey mesangial cells). Cells transfected with the ColCAT plasmid in the presence of thyroid hormone (100 nM T-3) had a significant decrease (39% in fibroblasts, P < 0.01; 52% in COS-I cells: P < 0.001) in CAT activity when compared to cells not exposed to thyroid hormone. Transient co-transfection of TR with various pro-alpha 1(I) collagen/CAT deletion constructs showed that T-3-dependent repression was preserved with the deletion from 3520 bp of the flanking sequence to a 5' end point at position - 224, indicating that a thyroid hormone-response element (TRE) was localized at the region - 224 to + 115. The TR-DNA binding assays demonstrated binding of the human TR beta 1 to a fragment containing a proposed TRE located between position - 35 and + 115 in the 5'-flanking region of the rat pro-alpha 1(I) collagen gene. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.