Abstract
Hormone regulation of anterior pituitary expression of the common glycoprotein hormone alpha-subunit (alphaGSU) is mediated by multiple response elements residing in the first -435 bp of the human promoter. In rat pituitary cells and mouse alphaT3-1 precursor gonadotrophs, the human alphaGSU promoter is strongly responsive to activators of the adenylyl cyclase/cAMP pathway, such as the hypothalamic releasing hormone, pituitary adenylate cyclase-activating polypeptide (PACAP) and forskolin (an adenylyl cyclase activator). However, the role of PACAP and cAMP in regulating alphaGSU transcription in the more differentiated LbetaT2 gonadotroph is unclear. Here, we investigate the regulation of the human alphaGSU promoter by PACAP and forskolin in LbetaT2 and alphaT3-1 gonadotrophs. PACAP failed to stimulate alphaGSU promoter activity or cAMP production in LbetaT2 cells, in marked contrast to alphaT3-1 cells. LbetaT2 gonadotrophs expressed extremely low levels of any PACAP type 1 receptors (PAC(1)-R) isoform by RT-PCR and lacked PAC(1)-R by radioligand binding. Forskolin stimulated the alphaGSU promoter in LbetaT2 cells, but by less than 30% of the response seen in alphaT3-1 gonadotrophs. This blunted cAMP transcriptional effect was not due to different levels of cAMP generation, or altered expression of the cAMP target proteins CREB, Akt, CBP or ICER. However, only LbetaT2 cells showed detectable expression of the protein kinase A type IIalpha regulatory subunit. Binding of activating transcription factor-2 and phosphorylated CREB to the consensus CRE was observed in both LbetaT2 and alphaT3-1 gonadotrophs, yet forskolin failed to stimulate either CRE- or CREB-mediated transcription in LbetaT2 cells. Collectively, these data demonstrate the lack of functional PACAP receptors in LbetaT2 gonadotrophs, and a pronounced attenuation in the responsiveness of this differentiated gonadotroph cell line to cAMP stimulus.
Highlights
Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are heterodimeric glycoproteins, which comprise a common -subunit ( GSU) and distinct -subunits that confer specificity (Gharib et al 1990)
We show that pituitary adenylate cyclase-activating polypeptide (PACAP) fails to stimulate GSU promoter activity or cAMP generation in L T2 cells, and that forskolinstimulated GSU transcription is significantly attenuated compared with similar observations in
Our previous studies comparing the activity of the human GSU promoter in L T2 and T3-1 cells had revealed that basal promoter activity was significantly reduced in L T2 cells compared with
Summary
Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are heterodimeric glycoproteins, which comprise a common -subunit ( GSU) and distinct -subunits that confer specificity (Gharib et al 1990). 264 R C FOWKES and others · Disrupted cAMP-mediated GSU transcription in L T2 cells et al 1996), whereas multiple elements contribute to gonadotroph GSU expression, including the pituitary glycoprotein hormone basal element, the CREs, a consensus GATA site and a steroidogenic factor-1 (SF-1) site (the gonadotroph-specific element) (Barnhart & Mellon 1994, Steger et al 1994, Heckert et al 1996). These sites have been implicated in basal and hormone-stimulated GSU promoter activity
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