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Estrogen Enhances Gonadotropin-Releasing Hormone-Stimulated Transcription of the Luteinizing Hormone Subunit Promoters via Altered Expression of Stimulatory and Suppressive Transcription Factors

Division of Endocrinology (T.K., M.A.S.), Department of Medicine, University of Virginia School of Medicine, Neuroscience Graduate Program (H.E.W.), University of Virginia, Charlottesville, Virginia 22908; and Center for Oral Health and Systemic Disease (D.S.D.), Periodontics, Endodontics and Dental Hygiene, University of Louisville School of Dentistry, Louisville, Kentucky 40292

Address all correspondence and requests for reprints to: Margaret A. Shupnik, Ph.D., Box 800578 HSC, University of Virginia, Charlottesville, Virginia 22908. E-mail: mas3x{at}virginia.edu.

Transcription of the LH subunit genes is stimulated by GnRH and may be modulated physiologically by steroids such as 17ß-estradiol (E). We found that E treatment amplified GnRH stimulation of the rat LHß and -subunit promoters, and expression of the endogenous mRNA, in LßT2 gonadotrope cells 2- to 5-fold above GnRH alone. We examined gene expression in LßT2 cells after E and/or GnRH treatment, and found that E suppressed expression of transcription factor Zfhx1a, and enhanced GnRH stimulation of Egr-1 mRNA and protein. E effects were abolished in the presence of antiestrogen. Egr-1 is critical for LHß expression; however, the role of Zfhx1a, which binds to E-box sequences, was untested. We found E-box motifs in both the rat LHß (–381, –182, and –15 bp) and -subunit (–292, –64, –58 bp) promoters. Zfhx1a overexpression suppressed basal and GnRH-stimulated activity of both promoters. Mutation of the -subunit promoter E boxes at either –64 or –58 bp eliminated Zfhx1a suppression, whereas mutation of the –292 bp E box had no effect. Gel shift assays demonstrated that Zfhx1a bound to the –64 and –58, but not –292, bp E-box DNA. Similarly, mutation of LHß promoter E boxes at either –381 or –182, but not –15, bp reduced Zfhx1a suppression, correlating with binding of Zfhx1a. The –381 bp LHß E box overlaps with an Sp1 binding site in the distal GnRH-stimulatory region, and increased Sp1 expression overcame Zfhx1a suppression. Thus, one mechanism by which E may enhance GnRH-stimulated LH subunit promoter activity is through regulation of both activators and suppressors of transcription.