An NF-Y binding site is important for basal, but not gonadotropin-releasing hormone-stimulated, expression of the luteinizing hormone beta subunit gene.

Regulated synthesis of luteinizing hormone (LH) requires coordinated transcriptional control of the alpha and LHbeta subunits in pituitary gonadotropes. Several cis-acting elements and trans-acting factors have been defined for control of the LHbeta promoter through heterologous cell culture models. In this report, we describe the identification of bipartite NF-Y (CBF/CP1) binding sites within the proximal bovine LHbeta promoter. When multimerized, one of these sites activates the heterologous, minimal HSV thymidine kinase promoter in the gonadotrope-derived cell line alphaT3-1. The functional role of the promoter-distal site in regulating the full-length bovine LHbeta promoter was assessed in vivo using transgenic mice harboring a mutant promoter linked to the chloramphenicol acetyltransferase reporter gene. While this element is important for conferring high level activity of the LHbeta promoter in pituitary, it does not appear to be essential for mediating gonadotropin-releasing hormone (GnRH) regulation. This is the first characterization of a cis-acting element within this GnRH-dependent promoter that is restricted to regulating basal expression and not GnRH-induced activity.

Chronic hypersecretion of luteinizing hormone in transgenic mice selectively alters responsiveness of the alpha-subunit gene to gonadotropin-releasing hormone and estrogens.

Steroid hormones can act either at the level of the hypothalamus or the pituitary to regulate gonadotropin subunit gene expression. However, their exact site of action remains controversial. Using the bovine gonadotropin alpha-subunit promoter linked to an expression cassette encoding the beta-subunit of LH, we have developed a transgenic mouse model where hypersecretion of LH occurs despite the presence of elevated ovarian steroids. We used this model to determine how hypersecretion of LH could occur when steroid levels are pathological. During transition from the neonatal period to adulthood, the endogenous LHbeta subunit gene becomes completely silent in these mice, whereas the alpha-directed transgene and endogenous alpha-subunit gene remain active. Interestingly, gonadectomy stimulates expression of the endogenous alpha and LHbeta subunit genes as well as the transgene; however, only the endogenous LHbeta gene retains responsiveness to 17beta-estradiol and GnRH. In contrast, LH levels remain responsive to negative regulation by androgen. Thus, alpha-subunit gene expression, as reflected by both the transgene and the endogenous gene, has become independent of GnRH regulation and, as a result, unresponsive to estradiol-negative feedback. This process is accompanied by a decrease in estrogen receptor alpha gene expression as well as an increase in the expression of transcription factors known to regulate the alpha-subunit promoter, such as cJun and P-LIM. These studies provide in vivo evidence that estrogen-negative feedback on alpha and LHbeta subunit gene expression requires GnRH input, reflecting an indirect mechanism of action of the steroid. In contrast, androgen suppresses alpha-subunit expression in both transgenic and nontransgenic mice. This suggests that androgens must regulate alpha-subunit promoter activity independently of GnRH. In addition to allowing the assessment of site of action of sex steroids on alpha-subunit gene expression, these studies also indicate that chronic exposure of the pituitary to LH-dependent ovarian hyperstimulation leads to a heretofore-undescribed pathological condition, whereby normal regulation of alpha, but not LHbeta, subunit gene expression becomes compromised.