Elsevier

Gene

Volume 308, 10 April 2003, Pages 67-77
Gene

Identification of distinct gene expression profiles associated with treatment of LβT2 cells with gonadotropin-releasing hormone agonist using microarray analysis

https://doi.org/10.1016/S0378-1119(03)00446-3Get rights and content

Abstract

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a pivotal role in reproductive processes. In recent years, it has become clear that it is also an anti-proliferative agent. GnRH analogs are now used clinically in the treatment of prostate cancer as well as endometriosis and precocious puberty. The target cells of GnRH include the gonadotropes of the anterior pituitary gland and the cells of various hormone-dependent tumors. Only a few target genes have been identified in these cells, however, and little is known concerning their regulation by GnRH. Therefore, we used a quantitative microarray assay to identify the genes that are regulated by GnRH in a murine gonadotrope tumor cell line (LβT2). Treatment of LβT2 cells with GnRH agonist des-gly10,[d-Ala6]GnRH (GnRHA) for 1 h resulted in alterations in the levels of expression of genes that ranged in magnitude from 1.3- to 159-fold, with a total of 232 genes exhibiting a twofold or greater alteration in expression compared to vehicle treated cells. Of these 232 genes, 149 were up-regulated and, surprisingly, 83 were down-regulated by GnRHA treatment. After 24 h of treatment, the expression of most of the genes that had exhibited altered expression after 1 h of treatment had returned to baseline levels. Moreover, a different profile was observed after 24 h of treatment with 208 genes exhibiting a twofold or greater alteration. Of these, 95 were up-regulated and 113 down-regulated. Most of the affected genes were not known to be responsive to GnRH prior to this study. Treatment with GnRHA was found to affect the expression of a diverse range of genes, including oncogenes and those that encode transcription factors, ion channel proteins, and cytoskeletal proteins as well as other proteins that are involved in signal transduction, the cell cycle, cell proliferation and apoptosis. The altered expression of six of the genes that were found by microarray analysis to be regulated by GnRHA was confirmed by semiquantitative reverse transcriptase–polymerase chain reaction. This is first application of the microarray technique in the study of the global profile of genes regulated by GnRH, and should prove to be a powerful tool for future analysis of the mechanisms by which GnRH regulates the expression of gonadotropins and the growth of tumor cells.

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.

Section snippets

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.

References (29)

  • E. Wurmbach et al.

    Gonadotropin-releasing hormone receptor-coupled gene network organization

    J. Biol. Chem.

    (2001)
  • M.M. Abbas et al.

    Regulation of C-fos protein in gonadotrope cells by oxytocin and gonadotropin-releasing hormone

    Neuroendocrinology

    (2000)
  • P.E. Belchetz et al.

    Hypophysial responses to continuous and intermittent delivery of hypopthalamic gonadotropin-releasing hormone

    Science

    (1978)
  • B.A. Christy et al.

    A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with ‘zinc finger’ sequences

    Proc. Natl. Acad. Sci. USA

    (1988)
  • Cited by (67)

    • Gonadotropin gene transcription is activated by menin-mediated effects on the chromatin

      2015, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms
    • Translational control of gene expression in the gonadotrope

      2014, Molecular and Cellular Endocrinology
      Citation Excerpt :

      ATF4 target genes are involved in amino acid transport and synthesis, metabolism, and the antioxidant response (Shan et al., 2009; Han et al., 2013). Microarray studies have shown that GNRH treatment of gonadotrope cells increases mRNA levels of Atf3 (Kakar et al., 2003; Zhang et al., 2006; Lawson et al., 2007), a transcription factor targeted by the EIF2AK3/ATF4 arm of the UPR (Jiang et al., 2004). EIF2AK3 phosphorylation is induced by GNRH in LβT2 cells, but is unique in that the overall level of activation is moderate in comparison to the typical pharmacological insult used in most studies (Do et al., 2009).

    • Polyribosome and ribonucleoprotein complex redistribution of mRNA induced by GnRH involves both EIF2AK3 and MAPK signaling

      2014, Molecular and Cellular Endocrinology
      Citation Excerpt :

      Measurements in vivo show only moderate increases in Lhb mRNA levels (Burger et al., 2002), although primary transcript levels and LH hormone output are increased substantially (Burger et al., 2004). Microarray based approaches of gene expression analysis have also shown only modest increases in mRNA levels in response to various condition of GnRH stimulation (Kakar et al., 2003; Lawson et al., 2007; Wurmbach et al., 2001). Increased LH protein synthesis can occur in the absence of transcriptional activity (Nguyen et al., 2004) and increased DUSP1 protein levels precedes increases in mRNA (Lawson et al., 2007; Nguyen et al., 2010), providing evidence in gonadotropes of an independent and complementary translational regulatory regime.

    • Optimized amplification and single-cell analysis identify GnRH-mediated activation of Rap1b in primary rat gonadotropes

      2012, Molecular and Cellular Endocrinology
      Citation Excerpt :

      In order to validate the microarray data, we conducted a real-time PCR assay on a subset of up-regulated and mechanistically interesting genes regulated in the primary gonadotropes. With the exception of rap1b and nr4a3, the genes selected for real-time PCR analysis were previously found to be activated in LβT2 cells exposed to GnRH for one hour (Kakar et al., 2003; Wurmbach et al., 2001; Yuen et al., 2002; Dorn et al., 1999 and Ebersole et al., unpublished data). Our quantitative assay confirmed the up-regulation of c-fos, fosB, egr2, and rap1b, while others that did not meet our criteria on the microarray (below 1.5-fold change, see Table 1) were found to be up-regulated, e.g., c-jun, egr1, egr3, and nr4a3 (Fig. 3).

    View all citing articles on Scopus
    View full text