Molecular characterization of myocardial fibrosis during hypothyroidism: evidence for negative regulation of the pro-alpha 1(I) collagen gene expression by thyroid hormone receptor

The purpose of this study was to gain insights into the underlying mechanis m of myocardial fibrosis during hypothyroidism. Treatment of cardiac fibrob lasts with a medium lacking thyroid hormone led to a 47% increase in [H-3]t hymidine incorporation into the cell nuclei compared with that in untreated cells. Northern blot analysis of RNA from cardiac fibroblasts grown in a t hyroid 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 ty pe I collagen, as determined by immunoprecipitation, was increased either i ll 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, c ontains the 5'-flanking region of the rat pro-alpha 1(I) collagen gene (fro m bases - 3520 to + 115) fused to the chloramphenicol acetyltransferase (CA T) gene. The plasmid was cotransfected with thyroid hormone receptor (TR) e xpression plasmid into rat cardiac fibroblasts and COS-1 cells (monkey mesa ngial 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-de pendent repression was preserved with the deletion from 3520 bp of the flan king sequence to a 5' end point at position - 224, indicating that a thyroi d 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 + 11 5 in the 5'-flanking region of the rat pro-alpha 1(I) collagen gene. (C) 20 00 Elsevier Science Ireland Ltd. All rights reserved.