Identification of distinct gene expression profiles associated with treatment of LβT2 cells with gonadotropin-releasing hormone agonist using microarray analysis
Introduction
Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide that is synthesized by hypothalamic neurons and released into the portal circulation in a pulsatile fashion (Fink, 1988). GnRH plays a pivotal role in the reproductive process by regulating the synthesis and secretion of gonadotropins (FSH; follicle stimulating hormone and LH; luteinizing hormone). These actions of GnRH are achieved through its high-affinity receptors that are expressed on the cell membranes of gonadotropes. Interestingly, GnRH desensitizes the pituitary gonadotropes unless it is presented in a pulsatile fashion. Continuous exposure to long-acting GnRH analogs or exposure to supra-physiologic concentrations of GnRH causes down-regulation and desensitization of the GnRH receptors on the pituitary cells, leading to a profound decrease in the secretion of LH and FSH and thereby gonadal hormones (Belchetz et al., 1978). It is this effect that forms the basis for the clinical use of GnRH analogs in the treatment of prostate cancer, endometriosis, and precocious puberty.
It is well established that GnRH receptor mRNA is expressed in the anterior pituitary. More recently, its expression in various extra-pituitary tissues, including the placenta, ovary, myometrium, endometrium, breast, prostate, and blood mononuclear cells, and various hormone-dependent tumors and cell lines derived from such tumors, has been reported (for review, see Emons et al., 1997, Schally, 1999). This suggests the possibility that GnRH may act directly on extra-pituitary tissues. Suppression of tumor cell growth in vitro by GnRH analogs suggests a direct antitumor effect of GnRH. However, the molecular mechanisms by which GnRH achieves its functions remain unclear. Although a limited number of individual target genes in gonadotropes and tumor cells have been identified, a large-scale profile of the target genes regulated by GnRH had not been undertaken at the time of this study. Microarray hybridization, which enables simultaneous measurement and comparison of the levels of expression of thousands of genes, allows the analysis of GnRH effects on cellular mechanisms through analysis of gene regulation on a genomic scale. For these studies, we used a mouse gonadotrope tumor cell line, LβT2, which was developed by utilizing the LHβ promoter for targeted expression of the SV40 T-antigen in transgenic mice. These cells express both LHβ and FSHβ genes, both of which are regulated by GnRH in these cells, as is secretion of LH protein (Thomas et al., 1996, Turgeon et al., 1996). The cells are therefore reasonably representative of mature pituitary gonadotropes.
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Cell culture and sample preparation
LβT2 cells were obtained from Dr. Pamela Mellon (University of California, San Diego) and were maintained at 37 °C in 5% CO2 in humidified air in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (Hyclone). For experiments, 4–5×106 cells were seeded in 15-cm flasks. After 24 h, the media were replaced with fresh media containing 0.1% fetal bovine serum. One hour later, the cells were treated with 100 nM of GnRHA or vehicle for 1 h or 24 h. After incubation, the cells
LβT2 cells express high affinity and biologically active GnRH receptors
Binding of GnRH to its specific high affinity receptors initiates activation and allows coupling of receptor to the Gαq11 protein with generation of several second messengers, most notable diacylglycerol and inositol production (IP3). To determine the expression and biological activity of the GnRH receptors on the plasma membranes of LβT2 cells under our culture conditions, LβT2 cells were treated with GnRHA and measured the levels of inositol triphosphate. As shown in Fig. 1, treatment of the
Discussion
Our global microarray analysis has provided interesting insight into regulation of gonadotropes by GnRHA. It has led to the identification of a number of genes that were found to be regulated by GnRH but had not been implicated previously in the mediation of its effects. The regulated genes include genes that encode proteins involved in transcriptional regulation, fuel metabolism, signal transduction, the cell cycle, oncogenesis, cell proliferation, apoptosis, and the formation of ion channels
Acknowledgements
Supported by Grant CA6087 from the National Cancer Institute. We wish to thank Dr. Fiona Hunter for editorial assistance.
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