The cAMP signaling system regulates LHβ gene expression: Roles of early growth response protein-1, SP1 and steroidogenic factor-1

Abstract

Expression of the gonadotropin genes has been shown to be modulated by pharmacological or physiological activators of both the protein kinase C (PKC) and the cAMP second messenger signaling pathways. Over the past few years, a substantial amount of progress has been made in the identification and characterization of the transcription factors and cognate cis-elements which mediate the PKC response in the LH β-subunit (LHβ) gene. In contrast, little is known regarding the molecular mechanisms which mediate cAMP-mediated regulation of this gene. Using pituitary cell lines, we now demonstrate that rat LHβ gene promoter activity is stimulated following activation of the cAMP system by the adenylate cyclase activating agent, forskolin, or by the peptide, pituitary adenylate cyclase-activating peptide. The forskolin response was eliminated with mutation of a previously identified 3′ cis-acting element for the early growth response protein-1 (Egr-1) when evaluated in the context of region -207/+5 of the LHβ gene. Activation of the cAMP system increased Egr-1 gene promoter activity, Egr-1 protein levels and Egr-1 binding to the LHβ gene promoter, supporting the role of this transcription factor in mediating the cAMP response. Analysis of a longer LHβ promoter construct (-797/+5) revealed additional contribution by upstream Sp1 DNA-regulatory regions. Of interest, forskolin-induced stimulation of LHβ gene promoter activity was observed to increase synergistically with introduction of the transcription factor, steroidogenic factor-1 (SF-1). Although SF-1 is a critical mediator of the cAMP response in other genes, mutation of the SF-1 DNA-binding sites in the rat LHβ gene did not alter the forskolin response nor did forskolin increase SF-1 protein levels in a gonadotrope cell line. In a further set of experiments, it was determined that forskolin-responsiveness was maintained following mutation of the previously defined homeobox-binding element at position -100. We conclude that both Egr-1 and Sp1 contribute to cAMP-dependent transcription of the rat LHβ gene promoter. While SF-1 does not act independently to mediate the cAMP/PKA response, SF-1 is important for magnification of this response. © 2004 Society for Endocrinology.