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Regular Article |
-Subunit Gene Transcription in LßT2 Gonadotropes by Protein Kinase C and Extracellular Signal-Regulated Kinase 1/21
a Department of Endocrinology, St. Bartholomew's and the Royal London School of Medicine and Dentistry, West Smithfield, London EC1A 7BE, United Kingdom
Transcriptional activation of the human glycoprotein hormone 
-subunit (GSU) promoter in response to GnRH and phorbol-12-myristate-13-acetate (PMA) has been well characterized in T3-1 gonadotropes but not investigated in the more differentiated LßT2 clonal gonadotrope. We have evaluated GSU transcription in the more mature LßT2 cell line, using deletion and heterologous constructs of the GSU promoter linked to a luciferase reporter gene. Basal GSU-promoter activity was significantly less in LßT2 cells than in T3-1 cells, but stimulation of transfected cells with GnRH and PMA resulted in similar increases in GSU-promoter activity. Deletional analysis of the human GSU promoter in LßT2 cells indicated that sequences between -398 and -244 and between -244 and -195 base pairs (bp) were involved in regulating basal GSU-promoter transcription, whereas the region between -244 and -195 bp regulated PMA-stimulated promoter activity. Deletion of this promoter region containing a steroidogenic factor-1 (SF-1) binding site abolished basal and PMA-stimulated transcription. Site-directed mutagenesis of the SF-1 binding site resulted in a significant attenuation of basal and PMA-stimulated GSU transcription. Pretreatment of LßT2 cells with a mitogen-activated protein kinase kinase-specific inhibitor, U0126, abolished the PMA-stimulated increase in MAPK activity and significantly reduced basal and PMA-stimulated promoter activity. Electrophoretic mobility shift assays for SF-1 and GATA revealed that PMA failed to affect SF-1 binding but enhanced GATA binding to a consensus GATA oligonucleotide, an effect that was blocked with U0126 pretreatment, suggesting that GATA may mediate ERK activation of GSU transcription. Our data suggests that, in the mature LßT2 gonadotrope cell line, two regions of the human GSU promoter regulate basal transcription and that SF-1 is involved in mediating basal and PMA-stimulated promoter activity. Furthermore, PKC-stimulated transcription partially relies on ERK acting on elements downstream of -244 bp of the human GSU promoter.
1 Supported by Wellcome Trust grant RG8E8 to J.M.B.
2 Correspondence: Rob Fowkes, Molecular Endocrinology Lab, 1.4, 1st Floor Dominion House, 59 Bartholomew Close, West Smithfield, London EC1A 7BE, U.K. FAX: 44 0 207 601 8468; r.c.fowkes{at}qmul.ac.uk
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