Elsevier

Steroids

Volume 78, Issue 7, July 2013, Pages 717-725
Steroids

Agonistic activity of ICI 182 780 on activation of GSK 3β/AKT pathway in the rat uterus during the estrous cycle

https://doi.org/10.1016/j.steroids.2013.03.003Get rights and content

Highlights

  • ICI treatment in proestrus day inhibited 15% luminal epithelial proliferation.

  • ICI treatment in estrous day had no effect on uterine epithelial proliferation.

  • ICI exerts agonistic and antagonistic effects in the uterus during the estrous cycle.

  • Different molecular mechanisms regulate proliferation during the estrous cycle.

Abstract

We examined the ability of ICI 182,780 (ICI) to block uterine cell proliferation via protein kinase b/AKT pathway in the uterus of the rat during the estrous cycle. Intact rats, with regular estrous cycles, received a subcutaneous (s.c.) injection of either vehicle or ICI at 08:00 h on the day of proestrus or at 00:00 h on the day of estrus and sacrificed at 13:00 h of metaestrus. Estradiol (E2) and progesterone (P4) plasma levels were measured by radioimmunoassay. Both ICI treatments, induced a significant decrease (p < 0.01) in uterine estrogen receptor alpha (ERα) content, had no effect on uterine progesterone receptor (PR) protein expression and caused marked nuclear localization of cyclin D1, in both luminal and glandular uterine epithelium, as compared to vehicle-treated animals. Furthermore, we detected that ICI treatment induced glycogen synthase kinase (Gsk3-β) Ser 9 phosphorylation, which correlates with cyclin D1 nuclear localization. However, some differences were observed between the two different time schedules of administration. We observed that the administration of ICI at 08:00 h on proestrus day produced a 15% inhibition of luminal epithelial cell proliferation, reduced uterine wet weight by 21% and caused reduction of Akt phosphorylation at Ser 473 as compared to vehicle-treated animals, whereas ICI treatment at 00:00 h on estrus day had no effect on these parameters. The overall results indicate that ICI may exert agonistic and antagonistic effects on uterine cell proliferation through differential activation of the Akt pathway depending on the administration period during the estrous cycle, and indicates that the mechanism of cell proliferation during the physiological conditions of the estrous cycle, is under a different and more complex regulation than in the ovariectomized + E2 animal model.

Introduction

Antiestrogenic drugs have been developed for their ability to inhibit the physiological response of reproductive tissues to estrogen [1], [2], [3]. In the uterus, however, some antiestrogenic treatments mimic the actions of estradiol (E2) [1], [4], [5], [6]. The relative antiestrogenic and estrogenic effects depend on the antiestrogen used, the dose, the animal species and the molecular-physiological model under investigation [7], [8], [9].

ICI 182,780 (ICI) is thought to be a pure steroidal estrogen antagonist that blocks estrogen action by competing with endogenous estrogen for estrogen receptors (ER) present in the nuclei of estrogen responsive tissues [10], [11], [12], [13]. A large number of studies demonstrated that ICI antagonizes E2 induction of uterine cell proliferation and estrogen-regulated protein expression in ovariectomized (ovx) animals [14], [15], [16], [17], [18], [19], [20], [21], [22]. However, several reports indicate some estrogenic agonist effects of ICI in both in vivo and in vitro models. For example, in vitro, ICI induces 5% of E2 up regulated genes in MCF-7 cells [23], causes an increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) in neonatal rat primary cerebellar neurons [24], and does not block the activation of ERK1/2 induced by E2 in basal forebrain cholinergic neurons [25]. Also, in vivo, ICI increases endometrial weight and GAPDH mRNA levels in the stroma and glandular epithelium of the sheep uterus [26], induces secretion of oxytocin and prostanglandin F2 alpha in intact ewes during estrous cycle [27], potentiates the effect of E2 on plasma calcium and up regulates ERα in fish liver [28]. Taken together, these studies demonstrate that ICI display agonistic and antagonist effects on several typical estrogen responses in different tissues and models. Here, we examined whether ICI acts as an agonist or antagonist of E2 effects on uterine cell proliferation and protein kinase b/AKT pathway protein expression, in adult rat uterus during the estrous cycle.

Section snippets

Animal treatments

All procedures with animals were carried out according with the Guidelines of the Mexican Federal Law for Animal Protection and with the approval of the Laboratory Animal Care and Use Committee from Facultad de Química, UNAM. Rats were obtained from the animal center UNAM-Harlan. Adult female Wistar rats (Rattus Norvegicus) (200–250 g) were maintained under a 12:12 h light/dark cycle, lights on from 06:00 to 18:00 h, with food and water available ad libitum. Two experimental models were used: ovx

ICI 182,780 did not block BrdU DNA incorporation in cycling animals

To assess the relative efficacy of anti-estrogen ICI, the compound was first tested in ovx rats. Using BrdU incorporation after i.p. injection 2 h before death as a measure of DNA synthesis, we observed that in vehicle treated ovx rats, there was a low basal rate of DNA synthesis in both luminal and glandular epithelia (1.99 ± 0.5% and 2.21 ± 0.9% BrdU labeled cells, respectively) (Fig. 1A). As expected, E2 treatment induced a significant increase in the number of BrdU-positive luminal (20.96 ± 0.7%)

Discussion

ICI is the most complete and best characterized ER antagonist in both in vivo and in vitro models. Importantly, it has been described as a “pure” antagonist, but it is likely that this concept is not completely accurate for different functions and tissues [23], [24], [25], [26], [27], [28]. Several reports indicate some estrogenic effects of ICI in vitro and in vivo models. These results prompted us to evaluate whether ICI acts as an agonist or antagonist on uterine cell proliferation and

Acknowledgements

This work was supported by the National Council for Science and Technology (CONACyT Grant 80338), Mexico City Science and Technology Institute (ICyT D.F. 234/2010) and by PAPIIT (IN-210412) and PAIP (6190-08) from the National Autonomous University of Mexico (UNAM).

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