Regulation of aromatase promoter activity in human breast tissue by nuclear receptors

Abstract

Using the yeast one-hybrid approach to screen a human breast tissue hybrid cDNA expression library, we have found that four orphan/nuclear receptors, ERRα-1, EAR-2, COUP-TFI (EAR-3), and RARγ, bind to the silencer (S1) region of the human aromatase gene. S1 down regulates promoters I.3 and II of the human aromatase gene. In this study, the interaction of EAR-2, COUP-TFI, and RARγ with S1 was confirmed by DNA mobility shift analysis. In contrast to the findings that ERRα-1 behaves as a positive regulatory factor, these three nuclear receptors were found, by mammalian cell transfection experiments, to act as negative regulatory factors by binding to S1. Furthermore, the negative action of these three nuclear receptors could override the positive effect of ERRα-1. RT–PCR analysis of 11 cell lines and 55 human breast tumor specimens has shown that these nuclear receptors are expressed in human breast tissue. Since EAR-2, COUP-TFI, and RARγ are expressed at high levels, it is likely that S1 is a negative regulatory element that suppresses aromatase promoters I.3 and II in normal breast tissue. In cancer tissue, S1 may function as a positive element since ERRα-1 is expressed, but EAR-2 and RARγ are only present in a small number of tumor specimens. This hypothesis is sustained by the finding that there is a weak inverse correlation between the expression of COUP-TFI and that of aromatase in breast tumor tissue. Our studies have revealed that estrogen receptor alpha (ERα) can also bind to S1, in a ligand-dependent manner. By binding to S1, ERα down-regulates the aromatase promoter activity. These results demonstrate that nuclear receptors play important roles in modulating aromatase expression in human breast tissue.