Identification of a retinoic acid response domain involved in the activation of the beta 1-adrenergic receptor gene by retinoic acid in F9 teratocarcinoma cells.

The density of beta 1-adrenergic receptors (beta 1-AR) is up-regulated upon differentiation of embryonic F9 teratocarcinoma cells by retinoic acid (RA) to the primitive endodermal phenotype. To identify the domains involved in RA-mediated activation of beta 1-AR gene transcription, three kb of 5'-flanking sequence of the beta 1-AR gene were ligated to a luciferase reporter gene and transiently transfected into F9 cells that were pre-exposed to 100 nM RA for 2 days. By generating deletions in the beta 1-AR promoter, a region between -125 and -100 was found to mediate a 3-fold induction in cells exposed to RA for an additional 2 days. Through site-directed mutagenesis of this region, it was determined that the RA responsive element (RARE) was organized as a direct repeat separated by 5 nucleotides in which the 5'-most AGGTCG half-site was between nucleotides -106 and -101 and the 3'-most AGGTCA half-site was between nucleotides -117 and -112. The RA receptor alpha (RAR alpha) isoform bound to the oligomer representing the sequences between -125 and -100 as a heterodimer complex with the retinoid X receptor alpha (RXR alpha). In a separate study, it was determined that the nucleotides between -125 and -100 are involved in thyroid hormone-mediated activation of the beta 1-AR gene in ventricular myocytes. Therefore, transcriptional activation of the beta 1-AR gene by thyroid hormone or RA involves a single binding site in the promoter.

Promoter analysis of the rat beta1-adrenergic receptor gene identifies sequences involved in basal expression.

The beta1-adrenergic receptor (beta1-AR) mediates several functions of catecholamines in the heart, including the stimulation of heart rate and contractility. The expression of the rat beta1-AR gene was assessed by transiently transfecting chimeric genes containing the beta1-AR promoter, driving the luciferase reporter gene into various cell lines. beta1-AR/luciferase vectors containing 3 kb of the 5'-flanking region and extending to -126 relative to the start site of translation were expressed at high levels in ventricular myocytes, SK-N-MC cells, and HepG2 cells. The addition of 26 nucleotides from -125 to -100 to the -3311 beta1-AR/luciferase chimeric gene reduced expression in myocytes and SK-N-MC cells while eliminating expression in HepG2 cells. This element is located 125 base-pairs 3' to the transcriptional start site. The mutation of four nucleotides between -121 and -118 diminished the inhibitory effect of this element. The inhibitory activity of the -125 to -100 sequence was completely dependent on promoter context and positioning. In addition to this 3' element, sequences between -3311 and -2740 in the 5'-flanking region of the beta1-AR gene were required for the full transcriptional suppression. Using DNase I footprinting and gel mobility assays, it was determined that within the 26-bp region, rat heart nuclear proteins bound to two sites between nucleotides -123 and -112 and -106 and -100. Therefore, appropriate basal expression of the beta1-AR gene involves widely separated sequences 3' and 5' to the transcriptional start site.

Expression of leptin and beta 3-adrenergic receptors in rat adipose tissue in altered thyroid states.

The level of leptin [the obese (ob) gene product] mRNA is markedly elevated in hypothyroid male rats. The administration of tri-iodothyronine (T3) to hypothyroid rats resulted in a 40% decrease in leptin mRNA at 8 h. This decrease in leptin mRNA was associated with a parallel decline in circulating leptin levels of about 50% at 24 h. Conversely, beta 3-adrenergic receptor mRNA levels were markedly decreased in epididymal adipose tissue from hypothyroid rats. T3 administration resulted in a 147% increase at 12 h in beta 3-adrenergic receptor mRNA. There was a corresponding increase due to T3 in the lipolytic response to the specific beta 3-adrenergic agonist CL 316,243 that paralleled the increase in beta 3-adrenergic receptor mRNA. T3-mediated changes in leptin and beta 3-adrenergic receptor mRNAs were blocked by cycloheximide, suggesting the involvement of short-lived proteins in these effects. The present results indicate that T3 has opposite effects to those of insulin on the white adipose tissue of rats with respect to leptin mRNA expression.

Thyroid hormone induces beta1-adrenergic receptor gene transcription through a direct repeat separated by five nucleotides.

Transcription of the rat gene for the beta1-adrenergic receptor (beta1-AR) is stimulated by thyroid hormone (T3) in ventricular myocytes. To identify the domains involved in the regulation of beta1-AR gene transcription by T3, three kb of 5'-flanking sequence of the rat beta1-AR gene were ligated to a luciferase reporter gene and transiently transfected into ventricular myocytes. By generating deletions in the rat beta1-AR promoter, a region between -125 and -100 was found to mediate a three-fold induction by T3. This element was able to confer T3 responsiveness to a neutral promoter driving the luciferase reporter gene. Through site directed mutagenesis of this region, it was determined that the T3 responsive element (TRE) was organized as a direct repeat separated by five nucleotides in which the 5'-most AGGTCG half-site was between nucleotides -105 to -102 and the 3'-most AGGTCA half-site between nucleotides -116 and -113. Both the thyroid hormone receptor isoforms alpha and beta bound to the oligomer representing the sequences between -125 and -100 most efficiently as heterodimers with the retinoid X receptor. This TRE is unusual in that it is a direct repeat separated by five nucleotides which is located 3' to the transcriptional start site.