Oestradiol is a potent mitogen and modulator of GnRH signalling in alphaT3-1 cells: are these effects causally related?

Abstract

GnRH acts via phospholipase C (PLC) activating G-protein coupled receptors to stimulate secretion of gonadotrophins from gonadotrophs. These cells are also regulated by gonadal steroids, which act centrally to influence GnRH secretion, and peripherally to modulate GnRH action. We have shown that oestradiol can stimulate proliferation and modulate GnRH-stimulated [ H]thymidine incorporation was not stimulated by oestradiol but was reduced to <2% of control by the oestrogen antagonist, raloxifene. The inhibitory effect of 10 or 1000 nM raloxifene was reversed competitively by oestradiol. A similar pattern of effects was seen when effects of oestradiol and raloxifene on the proportion of cells in the S-phase of the cell cycle (as measured by flow cytometry of propidium iodide-labelled cells) and on oestrogen receptor activity (as measured by transactivation of the oestrogen-response elements in the vitellogenin promoter) were quantified. In addition, RT-PCR revealed expression of and (but not 2) subtypes of oestrogen receptors. Thus, oestrogen is an essential mitogen for T3-1 cells, its mitogenic effect is oestrogen receptor mediated and is associated with a marked alteration of cell cycle distribution, and the full extent of these effects are best revealed in the presence of raloxifene. Using this strategy, we found that cells cultured for 4 days with 10 nM raloxifene expressed GnRH receptors (K d for 125 I-buserelin 4·33 nM) and that their activation by GnRH caused a concentration-dependent increase in 3 H]IP x and Ins(1,4,5)P 3 approximately 4-fold. The effects of oestradiol on GnRH receptor number and signalling were not, however, mimicked by culture for 2 days in medium with 10% FCS and the S-phase blocker, thymidine (15 mM). This treatment increased the proportion of cells in the S-phase 2-to 3-fold but did not alter GnRH receptor number or signalling. Other treatments which altered cell cycle transition (hydroxyurea, colcemid, methotrexate) also failed to alter GnRH receptor number or signalling and no correlation was seen between GnRH receptor number or GnRHstimulated [ 3 H]IP x accumulation and the proportion of cells in the S-phase or G2/M-phases of the cell cycle. Thus, oestradiol has pronounced effects on GnRH signalling, proliferation and cell cycle distribution in T3-1 cells, but these trophic effects do not underlie the modulation of GnRH signalling.

Signaling and Antiproliferative Effects of Type I and II Gonadotropin-Releasing Hormone Receptors in Breast Cancer Cells

“…Incorporation of [ 3 H]thymidine into newly synthesized DNA was used as a index of cell proliferation (23,37). Cells were seeded in 96-well plates in 250 l DMEM with 10% FCS at a density of 10,000 cells/ml.…”

Signalling and anti-proliferative effects mediated by gonadotrophin-releasing hormone receptors after expression in prostate cancer cells using recombinant adenovirus

“…3 H]thymidine into newly synthesized DNA was used as a measure of cell proliferation (Williams et al 2000, Everest et al 2001. Cells were seeded into 96-well plates at 2500 cells/well and 24 h later the media were replaced with medium containing 1% FCS and the required amount of Ad GnRH-R. Twenty-four hours later the cells were stimulated with the indicated peptides and 3 days later 10 µl [ 3 H]thymidine were added to each well.…”

Effect of treatment with the selective oestrogen receptor modulator LY117018-HCl on pituitary sensitivity to GnRH and subsequent ovulation

“…In both mice and rats, ER␤ is the predominant subtype in ovarian granulosa cells (Couse et al, 1997;Fitzpatrick et al, 1999;O'Brien et al, 1999). In addition, ER␤ seems to be the only ER expressed in the majority of GnRH neurones of female rats (Hrabovszky et al, 2000 a,b;Herbison et al, 2001). In contrast, ER␣ expression is high in both the uterus and the anterior pituitary of the rat (Kuiper et al, 1997;Pelletier et al, 2000;Schreihofer et al, 2000;Wang et al, 2000).…”

“…It has been shown that raloxifene diminished the stimulatory effect of GnRH on LH release in incubated anterior pituitaries (Simard and Labrie, 1987;González et al, 2000). Mechanistically, oestradiol increases the efficiency of GnRH receptors to stimulate phospholipase C and this effect is blocked by raloxifene in ␣T3-1 cells (Williams et al, 2000). In addition, it has been shown that oestradiol increases the number of GnRH receptors in cultured bovine pituitary cells and that this effect is blocked by treatment with tamoxifen (Gregg et al, 1990).…”

“…Similar effects of oestradiol on GnRH receptor mRNA have been described in granulosa cells and, as in the case of the pituitary receptor counterpart, oestradioldependent induction is blocked by tamoxifen (Nathwani et al, 2000). Therefore, treatment with raloxifene or its derivatives, that is, LY, in intact cyclic female rats could result in attenuation of oestradiol-induced sensitization of GnRH receptors by reducing efficiency of the signal transduction cascade (Williams et al, 2000), reducing the number of GnRH receptors (Gregg et al, 1990;Nathwani et al, 2000) or both mechanisms combined.…”