Triiodothyronine decreases the activity of the proximal promoter (PII) of the aromatase gene in the mouse sertoli cell line, TM4

Estrogens and thyroid hormones play a significant role in regulating functions and development of the testis. The synthesis of estrogens from androgens is catalyzed by the enzyme complex termed aromatase, which in the testis displays an age-related cellular compartmentalization, primarily in Sertoli cells in immature animals, whereas in adults it is expressed in Leydig and germ cells. T, induces a precocious terminal differentiation of prepubertal Sertoli cells together with a dramatic decrease of their aromatase activity. In the present work, we have examined the mechanism by which T, exerts this inhibitory action on aromatase expression. As an experimental model, we used the mouse Sertoli cell line TM4, which conserves a large spectrum of functional features present in immature Sertoli cells. For instance, after revealing the presence of aromatase by immunocytochemistry and measuring its enzymatic activity, we confirmed in this cell line the functional events previously characterized in primary cultures of immature rat Sertoli cells: 1) a strong stimulation of aromatase activity by dibutyryl-cAMP [(Bu)(2)cAMP] (simulating FSH action); and 2) the inhibition of aromatase activity by incubation with T-3 under basal condition and after (Bu)(2)cAMP stimulation. After identifying promoter 11 as the regulatory region located immediately upstream of the tran-scriptional initiation site in the TM4 cell line by rapid amplification of cDNA ends analysis, we conducted experiments to examine the molecular mechanism by which thyroid hormones modulate aromatase gene expression in this cell line. TM4 cells were transfected with plasmids containing different segments of the rat promoter 11 sequence ligated to a luciferase reporter gene. Analysis of the activities of these promoter fusions demonstrated that T, inhibits basal and (Bu)(2)cAMP-stimulated activity of the aromatase promoter. This effect was not revealed in T-3-treated cells transfected with construct in which the steroidogenic factor-1 (SF-1) response element was mutated. These results indicate that the inhibitory effect of T-3 requires the integrity of the SF-1 response element and are further supported in the EMSA. The EMSA experiments demonstrated that thyroid hormone/thyroid receptor alpha1 complex (TH/TRalpha1) is able to compete with SF-1 in binding to oligonu-cleotides containing an SF-1 motif, an element essential for the activity of the PII aromatase promoter. The findings suggest that the binding of the thyroid hormone/thyroid receptor alpha1 complex to the SF-1 motif is the molecular mechanism by which T, exerts an inhibitory effect on aromatase gene expression in the TM4 cell line.