Oestradiol is a potent mitogen and modulator of GnRH signalling in alpha T3-1 cells: are these effects causally related?

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. Wie have shown that oe stradiol can stimulate proliferation and modulate GnRH-stimulated [H-3]inos itol phosphate ([H-3]IPx) accumulation (used as a measure of PLC activity) in a gonadotroph-derived cell line (alpha T3-1). Here we show that when alp ha T3-1 cells were incubated in medium with 2% foetal calf serum (FCS), [H- 3]thymidine incorporation was not stimulated by oestradiol but was reduced to <2% of control by the oestrogen antagonist, raloxifene. The inhibitory e ffect of 10 or 1000 nM raloxifene was reversed competitively by oestradiol. A similar pattern of effects was seen when effects of oestradiol and ralox ifene on the proportion of cells in the S-phase of the cell cycle (as measu red 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-PC R revealed expression of alpha and beta (but not beta 2) subtypes of oestro gen receptors. Thus, oestrogen is an essential mitogen for alpha T3-1 cells , its mitogenic effect is oestrogen receptor mediated and is associated wit h a marked alteration of cell cycle distribution, and the full extent of th ese effects are best revealed in the presence of raloxifene. Using this str ategy, we found that cells cultured for 4 days with 10 nM raloxifene expres sed GnRH receptors (K-d for I-125-buserelin 4.33 nM) and that their activat ion by GnRH caused a concentration-dependent increase in [H-3]IPx (in cells labelled with [H-3]inositol) and inositol 1,4,5 trisphophate (in unlabelle d cells). Addition of 10 nM oestradiol (to overcome receptor blockade by ra loxifene) reduced GnRH receptor number by 31% but increased maximal effects on [H-3]IPx and Ins(1,4,5)P-3 approximately 4-fold. Thr effects of oestrad iol on GnRH receptor number and signalling were not, however, mimicked by c ulture 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 tre atments which altered cell cycle transition (hydroxyurea, colcemid, methotr exate) also failed to alter GnRH receptor number or signalling and no corre lation was seen between GnRH receptor number or GnRH-stimulated [H-3]IPx ac cumulation 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, pr oliferation and cell cycle distribution in alpha T3-1 cells, but these trop hic effects do not underlie the modulation of GnRH signalling.