title: Early growth response gene-1 regulates the expression of the rat luteinizing hormone receptor gene.
abstract: LH receptor gene expression is primarily regulated via specific interactions of trans-acting proteins and cis-acting DNA sequences in the upstream region of the gene. In this study, we report, using luciferase assays, that the region between -171 and -137 base pairs (bp) is essential for basal expression of the rat LH receptor gene. To identify factors that interact with the region between -171 and -137 bp and regulate expression of the gene, a rat granulosa cell cDNA library was screened using a yeast one-hybrid system. A positive clone, isolated by the screening, encodes a transcription factor early growth response gene-1 (Egr-1). To determine the sequence to which Egr-1 protein binds, electrophoretic mobility shift assay (EMSA) was employed. The Egr-1 protein was produced by an in vitro transcription/translation system using a full-length rat Egr-1 cDNA. The upstream region between -171 and -137 bp contains 2 overlapping Egr-1 consensus sequences. The EMSA revealed that Egr-1 binds independently to both sites. The overexpression of Egr-1 in MA-10 cells caused an approximately 2-fold increase in reporter luciferase activity. However, no induction of the luciferase activity was observed when luciferase constructs that lacked or had mutations in either or both of the Egr-1 sites were used, indicating that Egr-1 positively regulates LH receptor gene expression. In differentiated granulosa cells that had been pretreated with FSH for 48 h, the levels of both mRNA and Egr-1 protein were induced by hCG or cAMP, reaching maximal levels approximately 1.5 h after treatment and then returning to basal levels 8 h thereafter. No Egr-1 mRNA or protein was detected in undifferentiated granulosa cells, even after stimulation with 8-bromoadenosine-cAMP. These results suggest that Egr-1 functions only in luteinized granulosa cells after stimulation with hCG or cAMP. In conclusion, the findings demonstrate that Egr-1 actually binds to the regulatory upstream region of the LH receptor gene and positively regulates receptor gene expression. In addition, Egr-1 expression was observed only in luteinized granulosa cells after stimulation with hCG or cAMP. The present study provides further support to the hypothesis that Egr-1 plays important roles in the pituitary-gonadal axis.
authors.affiliation: Department of Biochemistry, Fukui Medical University, Shimoaizuki, Matsuoka, Fukui 910-, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Yoshino
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Mizutani
authors.forename: Kazuya
authors.initials: K
authors.lastname: Yamada
authors.forename: Megumi
authors.initials: M
authors.lastname: Tsuchiya
authors.forename: Takashi
authors.initials: T
authors.lastname: Minegishi
authors.forename: Takashi
authors.initials: T
authors.lastname: Yazawa
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kawata
authors.forename: Toshio
authors.initials: T
authors.lastname: Sekiguchi
authors.forename: Takashi
authors.initials: T
authors.lastname: Kajitani
authors.forename: Kaoru
authors.initials: K
authors.lastname: Miyamoto
mesh_terms: D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D051766:Early Growth Response Protein 1; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D006107:Granulosa Cells / Q000737:chemistry / Q000378:metabolism; D017874:Immediate-Early Proteins; D007984:Leydig Cell Tumor; D016296:Mutagenesis; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D011974:Receptors, LH / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D006063:Chorionic Gonadotropin; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D011974:Receptors, LH; D014157:Transcription Factors; D005640:Follicle Stimulating Hormone; D004247:DNA