Effects of estradiol and progesterone on gonadotropin LHß- and FSHß-subunit promoter activities in gonadotroph LßT2 cells.

OBJECTIVES: Sex steroid hormones play roles in the regulation of pituitary hormone synthesis and secretion. Here we investigated the role of estradiol (E2) and progesterone (P4) on pituitary gonadotropin luteinizing hormone (LH)ß- and follicle stimulating hormone (FSH)ß-transcriptional activity in a single colony of gonadotroph LßT2 cells. METHODS: Pituitary gonadotroph cell line, LßT2 cells were used in this study. Cells were transfected with LHß- or FSHß-subunit promoter region-linked luciferase vector, and stimulated with gonadotropin-releasing hormone (GnRH) in the presence or absence of sex steroids. Transcriptional activity for LHß- and FSHß-subunit were determined by luciferase assay. Effects of sex steroids on cell proliferation was also determined by measurement of 5-bromoe-2'-deoxyuridine (BrdU) incorporation. RESULTS: The basal promoter activity of the LHß subunit was not modulated by 10 nM E2, but gonadotropin releasing hormone (GnRH)-induced LHß promoter activity was significantly increased by the same concentration of E2. Similarly, although the basal FSHß promoter was not modulated by 10 nM E2, GnRH-induced FSHß promoters were significantly potentiated in the presence of E2. One micromole E2 modulated neither basal nor GnRH-induced LHß and FSHß promoters. On the other hand, basal LHß promoter activity was enhanced by 1 µM P4, but the stimulatory response of GnRH on LHß promoters was significantly inhibited in the presence of 1 µM P4. Similar to LHß promoters, the basal activity of the FSHß promoter was increased by 1 µM P4; however, the response to GnRH was not modulated in the presence of P4. Ten micromoles P4 modified neither basal nor GnRH-induced promoter activity for LHß and FSHß. E2 had no antagonistic effect on P4-induced basal promoter activities of LHß or FSHß. A cell proliferation assay showed that neither E2 nor P4 modulated the growth of LßT2 cells, even in the presence or absence of GnRH. CONCLUSION: These observations suggest that both E2 and P4 uniquely modulate basal and GnRH-stimulated gonadotropin promoters without affecting cell growth.

Role of Neurokinin B and Dynorphin A in pituitary gonadotroph and somatolactotroph cell lines.

The role of Neurokinin B (NKB) and Dynorphin A (Dyn) in the regulation of the hypothalamic pituitary axis is an important area of recent investigation. These peptides are critical for the rhythmic release of GnRH, which subsequently stimulates the secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The present study utilized the gonadotroph cell line LßT2 and the somatolactotroph GH3 cell line to examine the possible role of these peptides in pituitary hormone secretion. The NKB receptor (NK3R) and the Dyn receptor (the κ-opiate receptor (KOR)) were both detected in LßT2 cells and GH3 cells. NKB, by itself, failed to increase gonadotropin LHß and FSHß promoter activities and did not modulate the effects of GnRH on gonadotropin promoter activity. In GH3 cells, NKB significantly increased TRH-induced PRL promoter activity although NKB alone did not have an effect on basal PRL promoter activity. Dyn had no effect on gonadotropin promoters alone or in combination with GnRH stimulation. PRL promoters stimulated by TRH were not significantly changed by Dyn. TRH-induced PRL promoter activity was further increased in the presence of higher concentrations of NKB, whereas Dyn did not have a significant effect on the PRL promoter even at a high concentration. In addition, TRH-induced ERK (Extracelluar signal-regulated kinase) activation was enhanced in the presence of NKB. Our current study demonstrated that NKB had a stimulatory effect on PRL expression in a PRL-producing cell, but had no effect on gonadotropin secretion from a gonadotroph cell line.