RESUMO
Growth factors stimulate astroglial and neuronal proliferation and differentiation in culture. Estrogens markedly influence astroglia, and are key factors participating in neurodegeneration. The aim of the present study was to investigate interactions between estradiol (E2) and epidermal growth factor (EGF) during astroglia development, maturation and differentiation in culture. DNA or RNA labeling in 16 or 40 or 60 days in vitro (DIV) astrocyte cultures treated for 24 or 48 h with EGF and/or E2 was evaluated. A significant increase in DNA labeling in 16 DIV astrocyte cultures treated for 24 h with EGF (5 ng/ml) and E2 (1 nM) was found. EGF (5 or 10 ng/ml) addition in the last 24 h in 48 h E2 (1 or 5 nM)-treated astrocyte cultures at 16 DIV caused a slight, but significant increase in DNA labeling. No differences in RNA labeling were observed in 16 DIV astrocyte cultures treated for 24 or 48 h with EGF (5 or 10 ng/ml) in the presence of E(2) (1 or 5 nM). A significant stimulation in DNA labeling was shown in 40 DIV astrocyte cultures treated for 48 h with E2 (1 or 5 nM) in the presence of EGF (5 or 10 ng/ml) added in the last 24 h. In well differentiated astroglial cell cultures (60 DIV), DNA labeling was remarkably increased after 24 h treatment with 1 nM E2 or 5 ng/ml EGF. Co-addition of 1 nM E2 and 5 ng/ml EGF for 24 h reduced [methyl-(3)H]thymidine incorporation, when data are compared to E2- or EGF-treated cultures. Addition of EGF in the presence of E2 for 48 h or only in the last 24 h caused a significant decrease of [methyl-(3)H]thymidine incorporation in comparison with EGF-treated cultures at 60 DIV or with untreated cultures. Treatment of cultures for 24 h with EGF (5 or 10 ng/ml) alone or in combination with E2 (1 or 5 nM) induced a strong increase of RNA labeling in 60 DIV astrocyte cultures. Addition for 48 h of E2 (1 or 5 nM) or EGF (5 or 10 ng/ml) alone or in association stimulated significantly RNA labeling in astrocyte cultures at 60 DIV. When 60 DIV astrocyte cultures were treated for 48 h with E2 (1 or 5 nM) in the presence of EGF (5 or 10 ng/ml) added only in the last 24 h, a potentiating effect of RNA labeling was observed. The above results suggest that interaction between growth factors and estrogens may contribute to regulate astroglia development, maturation and differentiation.
Assuntos
DNA/biossíntese , Fator de Crescimento Epidérmico/metabolismo , Estradiol/metabolismo , RNA/biossíntese , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Diferenciação Celular , Divisão Celular , Células Cultivadas , Fator de Crescimento Epidérmico/farmacologia , Estradiol/farmacologia , Marcação por Isótopo , Timidina/farmacocinética , Uridina/farmacocinéticaRESUMO
Recent evidence indicates that astroglial-derived growth factors (GFs) participate in the development of luteinizing hormone-releasing hormone (LHRH) neurons, but it is still unknown whether LHRH neurons may exert a reciprocal modulation of glial cell function. Using immortalized hypothalamic LHRH (GT1-1) neurons in co-culture with glial cells, we have recently shown that basic fibroblast growth factor (bFGF) plays a prominent role in the glial-induced acquisition of the mature LHRH phenotype by GT1-1 cells. We have resorted to this model and combined biochemical and morphological approaches to study whether the response of glial cells to a number of GFs (including bFGF, insulin-like growth factor I, IGF-I, epidermal growth factor, EGF and insulin) expressed during LHRH neuron differentiation, is modulated by co-culture with pure LHRH neurons. Pre-treatment of hypothalamic astrocytes with an inactive ('priming') dose of bFGF for 12 h powerfully increased astroglia proliferative response to IGF-I (10 ng/ml), EGF (10 g/ml) and insulin (10 microg/ml), inducing a 65-100% increase in the [3H]thymidine incorporation compared to untreated cultures. When astroglial cells and developing GT1-1 neurons were co-cultured for 5 days in vitro (DIV), the [3H]thymidine incorporation was significantly higher than in astroglial cells cultured without neurons. Application of the different GFs to the co-culture for either 12 or 24 h further stimulated DNA synthesis to various extent according to the GF applied and the time of application. Localization of the proliferating cells by dual immunohistochemical staining, followed by cell counting and bromodeoxiuridine (BrdU) labeling index calculation, revealed that the incorporation of BrdU was restricted to the nuclei of LHRH-immunopositive neurons. Such changes were accompanied by extensive morphological alterations of astroglial and LHRH fiber networks, whereas neutralization of bFGF activity in GT1-1 neuron-glial co-cultures by a bFGF-antibody, dramatically counteracted the observed effects. The functional switch of astroglia proliferative response to GFs coupled to the potent morphological and functional modifications of developing glia and pure LHRH neurons observed in vitro, support a bidirectional interaction between immortalized LHRH neurons and astroglial cells and identify bFGF as a key player in this crosstalk.
Assuntos
Astrócitos/citologia , Astrócitos/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Hormônio Liberador de Gonadotropina/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Animais , Anticorpos/farmacologia , Astrócitos/química , Comunicação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Transformada , DNA/biossíntese , Fator 2 de Crescimento de Fibroblastos/imunologia , Imunofluorescência , Proteína Glial Fibrilar Ácida/análise , Hipotálamo/citologia , Testes de Neutralização , Ratos , Ratos Sprague-Dawley , Timidina/farmacocinética , TrítioRESUMO
Bidirectional communication between the neuroendocrine and immune systems during ontogeny plays a pivotal role in programming the development of neuroendocrine and immune responses in adult life. Signals generated by the hypothalamic-pituitary-gonadal axis (i.e. luteinizing hormone-releasing hormone, LHRH, and sex steroids), and by the hypothalamic-pituitary-adrenocortical axis (glucocorticoids (GC)), are major players coordinating the development of immune system function. Conversely, products generated by immune system activation exert a powerful and long-lasting regulation on neuroendocrine axes activity. The neuroendocrine-immune system is very sensitive to preperinatal experiences, including hormonal manipulations and immune challenges, which may influence the future predisposition to several disease entities. We review our work on the ongoing mutual regulation of neuroendocrine and immune cell activities, both at a cellular and molecular level. In the central nervous system, one chief compartment is represented by the astroglial cell and its mediators. Hence, neuron-glial signalling cascades dictate major changes in response to hormonal manipulations and pro-inflammatory triggers. The interplay between LHRH, sex steroids, GC and pro-inflammatory mediators in some physiological and pathological states, together with the potential clinical implications of these findings, are summarized. The overall study highlights the plasticity of this intersystem cross-talk for pharmacological targeting with drugs acting at the neuroendocrine-immune interface.