Estradiol interacts with insulin through membrane receptors to induce an antimitogenic effect on lactotroph cells.

The signaling mechanisms of estrogens interact with those of growth factors to control the pituitary gland functions. The contribution of the membrane bound estrogen receptor in these actions is not fully understood. In this study, we focused on the regulatory action of estradiol in interaction with insulin on the secretory and proliferative lactotroph cell activities from primary pituitary cell cultures. Furthermore, we studied the involvement of ERK1/2, PKC epsilon and Pit-1 in these actions. In serum free conditions, estradiol and estradiol-BSA promoted a differential secretory activity on PRL cells but were unable to induce lactotroph cell proliferation. However, both free and conjugated estradiol were competent arresting the mitogenic activity promoted by insulin. Estradiol, estradiol-BSA and insulin stimuli increased the PKC epsilon, phosphorylated ERK 1/2 and Pit-1 expression, although combined treatments with estradiol/insulin or estradiol-BSA/insulin induced a significant reduction in these levels, in close correlation with the decrease of lactotroph cell proliferation. The pre-treatment with PKC inhibitor BIM significantly inhibited the ERK activation promoted by insulin without modifying the ERK expression levels induced by estradiol or estradiol-BSA. By immuno-electron-microscopy the alpha nuclear estrogen receptor was localized in the plasma membrane of lactotroph cells. These findings suggest that the membrane bound ER participates modulating lactotroph cells proliferation via PKC epsilon, ERK1/2 and Pit-1. The interactions between estradiol and growth factors, inducing both mitogenic and antimitogenic effects, could provide glandular plasticity preventing an over-proliferation induced by growth factors.

Ultrastructural immunolocalization of IGF-1 and insulin receptors in rat pituitary culture: evidence of a functional interaction between gonadotroph and lactotroph cells.

We have investigated the expression of receptors for insulin and insulin-like growth factor 1 (IGF-1) in rat pituitary cells in vitro and examined the morphological and proliferative changes induced in adenohypophyseal cells by insulin and IGF-1. The proliferation of lactotrophs was determined by double-immunostaining for bromodeoxyuridine and prolactin. Incubation with insulin (10, 100 or 1000 ng/ml) or IGF-1 (5, 30 or 100 ng/ml) for 48 or 72 h significantly increased the number of lactotrophs undergoing mitosis. Co-incubation of insulin or IGF-1 with genistein (25 microM), an inhibitor of the tyrosine kinase receptor, reduced the proliferation of lactotrophs elicited by the hormone and the growth factor. The receptors for insulin and IGF-1 were localized in intact pituitary cells by ultrastructural immunocytochemistry with the colloidal gold-protein A technique. Gonadotrophs expressed both receptors, specific labelling being restricted to this cell type. Electron-microscopical observations of pituitary cell cultures incubated with insulin or IGF-1 revealed gonadotroph cells exhibiting the fine-structural features of enhanced protein synthetic activity. These findings suggest that both insulin and IGF-1 are able to induce the proliferation of lactotrophs through an indirect mechanism mediated by a factor synthesized by gonadotroph cells, in addition to stimulating the biosynthetic activity of the gonadotroph in a direct manner.

Antagonic effects of oestradiol in interaction with IGF-1 on proliferation of lactotroph cells in vitro.

The effects of IGF-1, 17 beta oestradiol and its functional interaction on lactotrophs cell proliferation were evaluated. In addition we investigated the involvement of PKC alpha, epsilon and phosphorilated ERK, in the mitogenic process. Primary cell cultures of adenohypophysis from female Wistar rats were studied in serum free conditions. The proliferation of lactotrophs was determined by double immunostaining for BrdU and PRL. The incubation with IGF-1 5, 30 or 100 ng/ml during 48 or 72 h increased lactotrophs proliferation two-threefold depending on IGF-1 concentration. Co-incubation of IGF-1 (30 ng/ml) with genistein (25 microM) or BIM (0.5 or 2 microM), lowered of tyrosine kinase receptor or of PKC respectively, inhibited the induced IGF-1 lactotrophs proliferation. 17 beta oestradiol (1, 10 or 100 nM) had not mitogenic effect, whereas in the presence of serum PRL cells proliferation was stimulated. Co-incubation with 1 nM oestradiol and IGF-1 significantly decreased the lactotroph BrdU-labelling achieved with IGF-1. PKC alpha, epsilon and ERK1/2 levels measured by western blot augmented in the presence of IGF-1 and were inhibited with the addition of genistein, supporting a participation of these enzymes in the proliferate process. Co-incubation of IGF-1 with 1 nM oestradiol decreased both PKC isoforms and activated ERK1/2 levels, suggesting that oestradiol would exert its antiproliferative effect by acting on the signalling pathway of IGF-1. The results revealed antagonic effects of oestradiol on lactotroph proliferation depending on its concentration and the presence of IGF-1.