Stimulation of Luteinizing Hormone Gene Promoter Activity by the Orphan Nuclear Receptor, Steroidogenic Factor-1

doi:10.1074/jbc.271.12.6645

Stimulation of Luteinizing Hormone Gene Promoter Activity by the Orphan Nuclear Receptor, Steroidogenic Factor-1 (*)

From the (1)Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115 and the
(2)Department of Obstetrics and Gynecology, Tufts University School of Medicine, Boston, Massachusetts 02111

^¶ To whom correspondence should be addressed:
G. W. Thorn Research Building, Rm. 913, Brigham and Women's Hospital, 20 Shattuck St., Boston, MA 02115
. Tel.: 617-732-5856; Fax: 617-732-5123.

Abstract

The orphan nuclear receptor, steroidogenic factor-1 (SF-1), is expressed in the pituitary and in the gonadotrope precursor cell line, αT3-1, where it is believed to enhance expression of the common gonadotropin α-subunit gene through transactivation of the gonadotrope-specific element (GSE). Sequence analysis of the rat luteinizing hormone β-subunit (LHβ) gene promoter revealed the presence of a consensus GSE at −127 to −119 (TGACCTTGT). We have demonstrated the ability of SF-1 to bind specifically to this putative GSE sequence by electrophoretic mobility shift assay, utilizing both αT3-1 nuclear extracts and in vitro translated SF-1. In addition, mutation of the putative LHβ-GSE (TGAAATTGT) eliminated specific DNA binding. To examine the ability of SF-1 to enhance LHβ promoter activity, CV-1 cells, which lack endogenous SF-1, were cotransfected with an SF-1-containing expression vector and an LHβ-luciferase reporter construct. When cotransfected with −209/+5 of the LHβ promoter, SF-1 increased luciferase activity by 56-fold. SF-1 responsiveness was markedly diminished with loss of the putative GSE region in deletion constructs and in the presence of a two base pair mutation, analogous to the mutation which eliminated DNA binding. Finally, the LHβ-GSE was able to confer SF-1 responsiveness on a heterologous minimal growth hormone promoter, GH50 (57-fold). We conclude that SF-1 both binds to and transactivates the rat LHβ promoter. These data suggest that SF-1 may participate in the expression of the LHβ gene by the gonadotrope.

Footnotes

↵^§ Contributed equally to this work and both should be considered first authors.
↵* This work was supported in part by National Institutes of Health Grant HD000849 and the American Gynecological and Obstetrical Society/American Association of Obstetricians and Gynecologists Foundation through the Reproductive Scientist Development Program (to L. M. H.), the Medical Research Council of Canada Clinician-Scientist Award (to U. B. K.), and by National Institutes of Health Grant HD19938 (to W. W. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GnRH

gonadotropin-releasing hormone

CRE

cAMP response element

GSE

gonadotrope-specific element

SF-1

steroidogenic factor-1

LHβ

luteinizing hormone β-subunit

FSHβ

follicle-stimulating hormone β-subunit

EMSA

electrophoretic mobility shift assay

RSV

Rous sarcoma virus.
- Received September 6, 1995.
- Revision received December 13, 1995.