Mechanisms underlying the tissue-specific and regulated activity of the Gnrhr promoter in mammals

Abstract

The GnRH receptor (GnRHR) plays a central role in the development and maintenance of reproductive function in mammals. Following stimulation by GnRH originating from the hypothalamus, GnRHR triggers multiple signaling events that ultimately stimulate the synthesis and the periodic release of the gonadotropins, luteinizing-stimulating hormone (LH) and follicle-stimulating hormones (FSH) which, in turn, regulate gonadal functions including steroidogenesis and gametogenesis. The concentration of GnRHR at the cell surface is essential for the amplitude and the specificity of gonadotrope responsiveness. The number of GnRHR is submitted to strong regulatory control during pituitary development, estrous cycle, pregnancy, lactation, or after gonadectomy. These modulations take place, at least in part, at the transcriptional level. To analyze this facet of the reproductive function, the 5' regulatory sequences of the gene encoding the GnRHR have been isolated and characterized through in vitro and in vivo approaches. This review summarizes results obtained with the mouse, rat, human, and ovine promoters either by transient transfection assays or by means of transgenic mice.

Keywords: GnRH receptor; gonadotrope cell lines; homeodomain proteins; promoter regions; steroidogenic factor 1; transcription; transgenic mice.

FIGURE 1

Structure of the GnRH receptor gene. The GnRH receptor gene is 15–31 kb in size depending on the species. This is primarily due to differences in intron sizes which are indicated at the top of the figure for mouse, rat, and human species. The coding sequence is boxed with the trans-membrane domains specified in black. The 5′ and 3′ untranslated regions (UTR) are designated by thick black lines whereas the two introns are specified by thick gray lines. The mature mRNA resulting from usual intron splicing is illustrated below the gene. Sizes of translated sequences included in the human mature transcript are indicated (987 bp corresponding to 328 codons excluding the stop codon). Rat and mouse translated sequences are 327 amino acids long, the exon II being three bases shorter (+523/+738).

FIGURE 2

Response elements identified within the mouse and rat promoters. The schema summarizes the functional elements characterized on mouse (A) and rat (B) promoters. These elements are involved in tissue-specific expression in gonadotrope cell lines, some of them being also involved in activin-, GnRH-, and PACAP-dependent regulation. Note that response elements directing gonadotrope-specific expression of the rat promoter are located within 1000 bp 5′ upstream of the ATG codon whereas those of the mouse promoter are confined within the most proximal 400 bp. The numbering is relative to the translation start codon, the adenine being considered at position +1. This numbering may therefore be different from that used in the original publications.

FIGURE 3

Sequence comparison of Gnrhr promoters in different mammalian species. (A) Dot matrix representation of pairwise alignment of rat versus mouse (left panel) or human (right panel) sequences. A strong sequence conservation between mouse and rat 5′ upstream flanking regions extends over 2 kb whereas that between rat (or mouse) and human sequences is limited to a proximal 0.5 kb region upstream of the ATG codon. Relaxed sequence identity between rat and human sequences is further observed between 0.5 and 1.2 kb upstream of the ATG codon. (B) Multiple sequence alignment of mammalian Gnrhr proximal promoters. Blocks of full identity are highlighted in gray. Some motifs of interest are boxed in the sequences of the species where they have been characterized (e.g., SURG1 in the mouse, AP1 in rat and mouse). Note that the SF1 response element characterized in the rat and mouse promoter sequences (−243/−235) is particularly well conserved among species contrasting with the human SF1 response element (−134/−142). The latter appears to be shared by bovine, porcine, ovine and canis species but not by rat and mouse.

FIGURE 4

Signaling pathways in the homologous up-regulation of the mouse GnRHR promoter by GnRH in gonadotrope and non-gonadotrope cells. In αT3−1 cells, GnRH-induced activation of the PKC-dependent pathway stimulates promoter activity through the MAP kinase signaling pathway, involving ERK1 and ERK2. Receptor-independent activation of the PKA-dependent pathway inhibits GnRH-stimulated promoter activity. In GGH3 cells, GnRH induces the simultaneous activation of the PKC and PKA-dependent pathways for stimulating promoter activity. Constitutive activation of the MAP kinase signaling pathway, involving ERK1 and ERK2, inhibits basal and GnRH-stimulated promoter activity. The three elements, SURG−1, SURG−2, and CRE therefore participate in GnRH up-regulation in both αT3−1 and GGH3 cells. In αT3−1 cells, the positive involvement of SURG−1 and SURG−2 have been demonstrated whereas that of the CRE element is only suspected to be inhibitory. In GGH3 cells, the CRE element is required for promoter stimulation and appears as the ultimate target of the PKA-dependent pathway. AP1, also referred to as SURG−2 is only potentially involved essentially because GnRH also activates the PKC-dependent signaling pathway in these cells.

FIGURE 5

Structure of the human GnRHR promoter. The elements identified in the proximal (A) and distal (B) promoter of the human gene and involved either in the tissue-specific or regulated activity are indicated. To facilitate comparison with the rodent promoters, the first nucleotide of the ATG codon was considered as position +1. The numbering may therefore be different from that used in the original publications.