Abstract 325 Developmental Biology Platform, Sunday, 5/2

Classical models of hormonal regulation of gene expression have been defined whereby liganded receptors bind to unique sequence elements in the 5′ flanking region of genes and activate or repress gene transcription. However, this does not explain many complexities in hormonal regulation. How do hormone receptors bind to common DNA elements yet exert selective effects? How does this model explain changes in responsiveness at different developmental stages? We have identified a developmental switch in thyroid hormone regulation of expression of the ovine β1 adrenergic receptor (oβ1AR). o β1AR gene expression is not increased by thyroid hormones during fetal life but becomes thyroid responsive during postnatal life. Similar changes are seen in rodents during postnatal maturation of their endocrine axes. We hypothesize this represents a unique mode of developmental regulation of gene transcription. Our goal is to determine the underlying mechanism for this switch in transcriptional regulation. The o β1AR promoter, previously cloned and sequenced in our laboratory, was placed in a luciferase reporter vector (o β1ARLuc) and transfected into various cell lines. Rat C6 glioma cells and SK-N-SH cells (a human neuroblastoma cell line) are standard cell models in the study of adrenergic receptors. Because we are interested in the pathobiological role of β1AR expression in myocardium, we also used a newly described human embryonic myocardial cell line, W1 cells and primary neonatal rat cardiomyocytes. Cells were transiently transfected with the o β1ARLuc construct and treated with T3 alone, a thyroid receptor expression vector (TRα or TRβ) alone or T3 +TR. Luciferase activity (corrected for transfection efficiency) was measured. Treatment with either TRα alone or TRα + T3 induced a 3-5 fold increase in transcription in W1 but not C6 cells. Interestingly, in SK-N-SH cells, treatment with TRβ alone resulted in a 2 fold increase in gene expression while treatment with TRβ + T3 markedly decreased gene transcription. This "unliganded" TRβ activation of gene transcription was also seen in the neonatal rat cardiomyocytes. Liganded and unliganded thyroid receptor has been shown to influence transcription of certain genes in a cell and element specific manner. The results in W1 cells, SK-N-SH cells and neonatal rat cardiomyocytes suggest our promoter construct contains a functional thyroid response element. Work is in progress to map the specific sequences and thyroid receptor subtype conferring this activity and to determine the mechanism for this effect. We speculate that unique transcription factors and/or novel interactions with hormone receptors may explain differential thyroid responsiveness in fetal and postnatal life.