Conservation of mechanisms mediating gonadotrophin-releasing hormone 1 stimulation of human luteinizing hormone β subunit transcription

Abstract

Gonadotrophin-releasing hormone (GNRH1) regulates pituitary luteinizing hormone (LH). Previous studies have delineated a mechanism for GNRH1-induced LHb subunit gene (Lhb) transcription, the rate-limiting step in LH production. GNRH1 induces expression of early growth response 1 (EGR1), which interacts with steroidogenic factor 1 (SF1) and paired-like homeodomain transcription factor 1 (PITX1) to regulate Lhb promoter activity. Though the cis-elements for these factors are conserved across species, regulation of human LHB transcription has not been thoroughly investigated. We therefore characterized LHB transcriptional regulation by GNRH1 using promoter-reporter analyses in LbT2 cells. GNRH1 stimulated LHB transcription via an extracellular signal-regulated kinase 1/2 pathway. EGR1 bound to two binding sites on the LHB promoter and this binding was increased by GNRH1. Mutation of either site or knockdown of endogenous EGR1 decreased basal and/or GNRH1-regulated promoter activity. The human LHB promoter also contains low and high affinity SF1 binding sites. Mutation of these elements or depletion of endogenous SF1 impaired basal and ligand-induced transcription. Knockdown of PITX1 or PITX2 isoforms impaired GNRH1 induction, and endogenous PITX1 bound to the candidate PITX binding site on the LHB promoter. Thus, the mechanism described for GNRH1 regulation of Lhb in other species is largely conserved for human LHB. We also uncover a previously unappreciated role for PITX2 isoforms in this process.

Gene Expression Changes Underlying Idiopathic Central Hypogonadism in Cryptorchidism with Defective Mini-Puberty

“…A specif- ic function of PITX1 and PITX2 in the testis is yet unknown. However, all 4 genes, EGR4 , EGR2 , PITX1, and PITX2, are directly involved in the regulation and expression of the LHβ subunit in the pituitary gland [Kaiser et al, 1998[Kaiser et al, , 2000Wei et al, 2002;Fortin et al, 2009]. Noticeably, we identified a decreased expression of the genes MSX1 , DLX2 , DLX3 , NR4A1, and LHX3 in the HIR group ( table 2 ).…”

CREB Binding Protein (CBP) Activation Is Required for Luteinizing Hormone Beta Expression and Normal Fertility in Mice

“…In contrast, pituitary glandspecific SF-1 knockout mice have markedly decreased expression of LH and FSH, but they are able to produce LH in the pituitary gland in response to exogenous GNRH (55). In the L␤T2 gonadotroph cell line, mutation in the PITX binding site of the human Lhb promoter decreased basal, but not GNRH1-induced, transcriptional activity (13). These data indicate that while SF-1 and Pitx1 enhance the transcriptional response, Egr-1 is the major mediator of GNRH-stimulated Lhb gene expression.…”

“…In rat, Egr-1 expression increases in proestrous, suggesting that it may be an important signal for ovulation (39). Loss of Egr-1 in the gonadotroph, unlike loss of SF-1 or Pitx1, renders the cell unable to respond to GNRH, and Egr-1 knockout mice have a selective defect in Lhb expression that is not responsive to gonadectomy (13,29). In contrast, pituitary glandspecific SF-1 knockout mice have markedly decreased expression of LH and FSH, but they are able to produce LH in the pituitary gland in response to exogenous GNRH (55).…”

GnRH pulse frequency differentially regulates steroidogenic factor 1 (SF1), dosage-sensitive sex reversal-AHC critical region on the X chromosome gene 1 (DAX1), and serum response factor (SRF): potential mechanism for GnRH pulse frequency regulation of LH beta transcription in the rat

“…Egr1-and pituitary-specific SF1 knockout mice are either completely infertile or subfertile, due to a lack of LHb synthesis [10][11][12][13]. Two Egr1-binding sites have been identified in the proximal GnRH responsive region of the LHb promoter, which are highly conserved across species [11,[14][15][16][17][18], and mutations within these Egr1-binding sites abrogate the GnRH induction of LHb promoter reporter constructs in gonadotrope-derived cell lines [18][19][20][21]. However, recently we did not find that pituitary Egr1 (transcripts or protein) or the two Egr1 inhibitors (NAB1 and/or 2) were differentially regulated by GnRH pulse frequency in rats in vivo [22].…”

“…Adjacent to the two Egr1-binding sites on the LHb promoter are two SF1-binding sites [14,16,17,23]. Both SF1 sites are required for maximal induction of LHb by GnRH [12,13] and overexpression analyses indicate that Egr1 and SF1 act in tandem to stimulate LHb transcription [16][17][18]20].…”