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

Abstract

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 [ 3H]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-α1(I) collagen. At the protein level, the amount of type I collagen, as determined by immunoprecipitation, was increased either in 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-αl(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-l 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-1 cells, P<0.001) in CAT activity when compared to cells not exposed to thyroid hormone. Transient co-transfection of TR with various pro-αl(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 TRβ1 to a fragment containing a proposed TRE located between position −35 and +115 in the 5′-flanking region of the rat pro-αl(I) collagen gene.