Transcriptional activation of the gonadotropin-releasing hormone receptor gene by activin A.

Abstract

It has been reported that activin A stimulates the synthesis of the GnRH receptors (GnRHR) in rat pituitary cultures. However, the role of activin A in the regulation of the GnRHR gene at the molecular level is not known. In the present work, we have studied the regulation of the GnRHR gene by activin A in the gonadotrope cell line, alpha T3-1, where the GnRHR gene is highly expressed. First, we demonstrate that these cells express the mRNAs of three types of activin receptors: I, II, and IIB. Activin A increases GnRHR mRNA levels in a dose-and time-dependent manner, with maximal stimulation (2.5 +/- 0.5-fold) occurring with a dose of 20 ng/ml after 36 h of incubation. To ascertain whether this effect occurs at the transcriptional level, we performed nuclear run-off experiments in alpha T3-1 cells, which demonstrate a 1.6-fold increase in the levels of newly synthesized GnRHR mRNA in response to activin A. To investigate further the effect of activin A on the transcription of the GnRHR gene, alpha T3-1 cells were transiently transfected with a mouse GnRHR promoter/luciferase reporter gene (GnRHR-Luc) and challenged with activin A. Luciferase activity increases in response to activin A to the same extent (2.4 +/- 0.4-fold) and with similar dose-response and time-course profiles as the mRNA levels. Follistatin (100 ng/ml), a well known activin antagonist, completely abolishes the activin A effect on both mRNA levels and GnRHR-Luc activity. Follistatin also decreases the basal expression of the GnRHR gene by 33% as determined by GnRHR-Luc activity. This, together with our demonstration of the presence of the inhibin beta B-subunit mRNA in alpha T3-1 cells, suggests a potential paracrine/autocrine role of endogenous activin B on the regulation of the GnRHR gene in these cells. To provide evidence for biological significance of activin A stimulation of GnRHR gene expression, the response of a human gonadotropin alpha-subunit promoter/luciferase reporter gene (alpha Gon-Luc) to GnRH was assessed in alpha T3-1 cells pretreated with activin A. Activin enhances the stimulation of alpha Gon-Luc activity by GnRH by 1.6 +/- 0.4-fold. These data demonstrate that activin A can stimulate the expression of the GnRHR gene at the transcriptional level. Furthermore, transfection studies localize the activin responsive element to 1.2 kb of the 5'-flanking region of the GnRHR gene. Transcriptional activation of the GnRHR gene by activin A may serve as a mechanism for the modulation of gonadotrope responsiveness to GnRH.