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Oestrogens are the key factor in the sexual differentiation of the mammalian brain and play an important role in the activity of selected areas of the mature brain. To pursue the study of oestrogen action on neural cells at the molecular level, we developed a human neuroblastoma cell line (SK-ER3) expressing the oestrogen receptor (ER). Treatment of these cells with 17beta-oestradiol causes growth arrest and morphological and biochemical differentiation. The aim of the present study was to investigate whether oestrogen-differentiated SK-ER3 neuroblastoma cells acquire the ability to synthesize a specific neurotransmitter and whether the growth arrest previously reported can be ascribed to the blockage of the cells at a specific stage of the cell cycle. The results presented here indicate that oestrogens induce accumulation of SK-ER3 cells in the G0 phase of the cell cycle, underscoring the acquisition of a mature neural phenotype upon hormonal treatment. Most importantly, we show that in the differentiated cells the content of tyrosine hydroxylase and Na+-dependent dopamine uptake is significantly augmented, proving that the oestrogen-differentiated SK-ER3 cells can synthesize and store a specific neurotransmitter. In addition, we prove that the dopamine accumulated in differentiated SK-ER3 cells can be released. These studies therefore suggest that oestrogen treatment results in the acquisition of a fully functional dopaminergic phenotype of SK-ER3 cells. Ample evidence shows a link between dopaminergic neurons and oestrogen activity in hypothalamic and non-hypothalamic areas of the mammalian brain. Our study indicates that oestrogens might play a primary role in committing undifferentiated neuroblasts towards the dopaminergic phenotype.

Insulin is a well known mitotic agent for neuroblastoma cells. Human SK-N-BE neuroblastoma cells stably transfected with the estrogen receptor, however, undergo growth arrest and differentiation when treated with insulin. These effects were shown to be due to an insulin-dependent activation of the unliganded estrogen receptor. Here, we demonstrate that this activation involves the AF-2 COOH-terminal domain of the estrogen receptor and that the communication between estrogen and insulin receptor systems occurs via selected and specific transduction signals. In fact, by the use of dominant negative and dominant positive mutants we demonstrate that p21ras is essential for insulin and estrogen receptor coupling. With pharmacological tools, we prove that PI 3'kinase does not contribute to this cross-talk and that protein kinase C triggers transduction signals that act in synergism with p21ras. These results prove the intricacy of all these intracellular paths of communication. The finding that, in neuroblastoma cells, selected signal transduction systems are involved in the insulin-dependent activation of estrogen receptor is of particular interest considering that estrogen receptor might restrict the role played by insulin during the differentiation of neural cells and interfere with its proliferative potential while allowing its regulation of other functions related to cell survival.

Estrogens are known to modulate the growth rate and differentiation state of a number of cells. In uterine, as well as in mammary tumor cells, estrogen-dependent proliferation and differentiation are correlated to a series of biochemical responses, including increased expression of proto-oncogenes such as: c-fos, c-jun and c-myc. Since estrogens were shown to regulate the proliferation and the differentiation state of cells of nervous origin, the aim of the present study was to investigate whether these effects were associated to changes in the expression of early genes. In the model system utilized, the human cell line SK-ER3, an increase in c-fos mRNA and Fos protein without change of c-jun and related genes mRNA concentration was observed after short term treatment with 17 beta-estradiol (E2). A significant decrease of c-fos, c-jun and jun-D proto-oncogene mRNA levels were found after prolonged hormonal treatment. The exposure to the hormone did not determine any change in N-myc expression. Since the three protooncogene mRNAs are rapidly induced following estrogen treatment in other cell systems and target tissues, it is concluded that the estrogen-induced differentiation of neuroblastoma cells is correlated to a pattern of expression of early genes that might be peculiar for the activity of this hormone in neural cells.

Several lines of evidence support the hypothesis of a role played by estrogens in the manifestation of affective disorders in women. The analysis of the mechanism of action of a number of antidepressant drugs clearly demonstrated the involvement of the catecholaminergic system in the etiology of these complex behavioral pathologies. The present in vitro study was therefore undertaken to investigate the presence of a functional link between estrogen and catecholamine metabolism in cells of neural origin. The model system utilized was a human neuroblastoma cell line which was obtained by stable transfection of the estrogen receptor cDNA (SK-ER3). The present study shows that in SK-ER3 activation of the estrogen receptor correlates with a marked decrease in monoamine oxidase A activity. This effect is observed following treatment with a physiological concentration of 17 beta-estradiol and can be blocked by the specific antagonist of the steroid receptor, ICI 182,780. Dibutyryl cyclic AMP acting, like estrogens, on the state of differentiation of SK-ER3 cells did not affect monoamine oxidase A activity. The present study provides strong evidence of a strict relationship between estrogen receptor and monoamine oxidase A activity in human cells of neural origin, thus favoring the hypothesis of an antidepressive effect of estrogens exerted via inhibition of the monoamine oxidative pathway.

The neuroblastoma cell line SK-ER3, which is stably transfected with the estrogen receptor (ER), was used to study the effect of insulin and insulin-like growth factors (IGF-I and IGF-II) on growth and morphological differentiation induced by estrogens. The data demonstrate that insulin and related growth factors control the growth and morphological differentiation of the cell line expressing the ER, but not of the parental cell line. Effects elicited by the growth factors in SK-ER3 cells can be blocked by ER antagonists. Transient transfection studies further confirm an effect of the IGFs in modulation of ER-activated promoters. The results presented support the hypothesis of the existence of cross-talk between membrane and intracellular receptors and provide evidence for physiological consequences of the activation of such a pathway of communication. The present study is of particular interest with regard to the theory of prenatal involvement of the ER in maturation of nerve cells. It could, in fact, be hypothesized that IGF-I and IGF-II, present in high concentrations in the developing brain, might activate the ER expressed in several embryonic brain nuclei.

In order to assess the neuronal-like properties of a human neuroblastoma cell line obtained by stable transfection of the estrogen receptor (SK-ER3) a series of quantitative measurements of the activity of two neurotransmitter-related enzymes: tyrosine hydroxylase (TH) and monamine oxidase (MAO), and of catecholamine concentrations were performed. When compared to the parental SK-N-BE cell line, the stably transfected SK-ER3 cells show a more pronounced dopaminergic phenotype. The immunoreactivity to a TH antibody is in fact increased and the ratio between dopamine and noradrenaline concentrations is elevated. Treatment with estradiol further enhances the expression of this phenotype. Interestingly, in the transfected cell line MAO-A activity is decreased and further reduced by estrogen treatment. This finding substantiated by previous reports indicates that our model system might represent an interesting tool for the study of the pharmacological treatments of estrogen-induced pathological responses of nervous cells.