RESUMO
The enzyme poly(ADP-ribose)polymerase (PARP) has a leader role in the DNA damage survey mechanisms by its nick-sensor function, but it is also involved in the early events of the programmed cell death, particularly during inflammatory injury, as a coactivator of NF-kB. In the present study, we evaluated the PARP involvement in the mechanisms of protection and/or cell death in rat astroglial cell cultures during the early phase of proinflammatory commitment after lipopolysaccharide and interferon gamma treatment. According with the recent findings that PARP-1 phosphorylation by MAPK/ERK-2 pathway seems to modulate PARP activation, in time course experiments we demonstrated that a very early PARP activation and expression is able to trigger a cell death pathway, DNA damage independent, during strong proinflammatory insults, maintaining its role of guardian of the genome stability only during the normal cell cycling.
Assuntos
Astrócitos/citologia , Morte Celular , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Western Blotting , Linhagem Celular , Interferon gama/farmacologia , L-Lactato Desidrogenase/metabolismo , Lipopolissacarídeos/farmacologia , Ratos , Ratos WistarRESUMO
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
Post-translational modifications of chromatin-bound proteins play an important role in the regulation of eukaryotic gene expression. Processes such as acetylation, methylation, phosphorylation and ADP-ribosylation may alter the interaction of these proteins with DNA and consequently affect chromatin conformation and the binding of enzymes and other molecules involved in the regulation of gene expression. In the present study the process of ADP-ribosylation of chromosomal proteins (histone and non-histone proteins) in some rat brain regions during postnatal development was investigated; also the effect of epidermal growth factor (EGF) on this process in fetal brain slices was studied. It has been found that the process of ADP-ribosylation of total histones extracted from rat cerebral cortex and cerebellum at 1, 10 and 30 days of age, increases from 1 to 10 days of age (i.e. the period of maximal cell proliferation) and decreases thereafter, while the process of ADP-ribosylation of non-histone proteins (NHPs) sharply decreases during the same developmental period. The addition of EGF to fetal brain slices causes a significant increase of ADP-ribosylation of total histones (particularly of the histone H1 fraction) and also of NHPs and microsomal proteins. This result is in agreement with the effect of EGF as a mitogen factor, previously shown in astroglial cell cultures.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Envelhecimento/metabolismo , Encéfalo/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Histonas/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Embrião de Mamíferos , Técnicas In Vitro , Processamento de Proteína Pós-Traducional , Ratos , Ratos EndogâmicosRESUMO
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 plays a pivotal role in health and disease. Signals generated by the hypothalamic-pituitary-gonadal (HPG) axis (i.e. luteinizing hormone-releasing hormone, LHRH, and sex steroids) are major players coordinating the development immune system function. Conversely, products generated by immune system activation exert powerful and longlasting effects on HPG axis activity. In the central nervous system (CNS), one chief neuroendocrine-immune (NEI) compartment is represented by the astroglial cell population and its mediators. Of special interest, the major supporting cells of the brain and the thymus, astrocytes and thymic epithelial cells, share a similar origin and a similar set of peptides, transmitters, hormones and cytokines functioning as paracrine/autocrine regulators. This may explain some fundamental analogies in LHRH regulation of both cell types during ontogeny and in adult life. Hence, the neuropeptide LHRH significantly modulates astrocyte and thymic cell development and function. Here we focus this work on LHRH neuron-glial signaling cascades which dictate major changes during LHRH neuronal differentiation and growth as well as in response to hormonal manipulations and pro-inflammatory challenges. The interplay between LHRH, growth factors, estrogens and pro-inflammatory mediators will be discussed, and the potential physiopathological implications of these findings summarized. The overall study highlights the plasticity of this intersystem cross-talk and emphasize neuron-glial interactions as a key regulatory level of neuroendocrine axes activity.
Assuntos
Estrogênios/fisiologia , Hormônio Liberador de Gonadotropina/fisiologia , Substâncias de Crescimento/fisiologia , Neuroglia , Neurônios , Reprodução , Animais , Astrócitos , Células Cultivadas , Fator 2 de Crescimento de Fibroblastos/fisiologia , Imunidade , Sistemas NeurossecretoresRESUMO
Luteinizing hormone-releasing hormone (LHRH) neurons play a pivotal role in the neuroendocrine control of mammalian reproduction. Astrocytes were shown to be involved in the regulation of LHRH neuronal function, but little is known about the contribution of astroglial-derived factors in the regulation of LHRH neuron development. In order to gain insight into the mechanisms regulating the development of these cells, at morphological and biochemical levels we characterized the neurotrophic effects exerted by young astrocytes (maintained in culture for 8 days in vitro) and old astrocytes (maintained 26 days) on the differentiation, proliferation, and phenotypic expression of immortalized hypothalamic LHRH (GT(1-1)) neurons in vitro. Culturing GT(1-1) cells in the presence of young glia for different time intervals caused a marked acceleration in the acquisition of their neuronal phenotype. At all times examined, GT(1-1) cells cocultured with young glia exhibited a significantly greater extension of processes/cell, larger number of processes/cell and greater surface area of growth cones than GT(1-1) cells grown over nonglial adhesive substrates (polylysine). By contrast, when GT(1-1) neurons were cocultured with old glia, the length of neuronal processes and the growth cone surface area were significantly lower than in control GT(1-1) neurons cultured in the absence of glia. At 3 days in vitro (DIV), GT(1-1) neurons cocultured with young glia exhibited a 50% lower incorporation of [(3)H]thymidine than GT(1-1) neurons cultured without glia. By contrast, in the presence of old glia [(3)H]thymidine incorporation was significantly higher in cells cocultured with glia than in GT(1-1) neurons cultured alone. Localization of the proliferating cells by dual immunohistochemical staining revealed that the incorporation of bromodeoxiuridine (BrdU) was restricted to nuclei of GT(1-1) neurons when these were cocultured with young glia, but associated with both neurons and astrocytes in the presence of old glia. At the functional level, coculture of GT(1-1) neurons with young glia increased the spontaneous release of LHRH as compared to GT(1-1) neurons grown in the absence of glia. By contrast, in the presence of old glia LHRH release in the medium was significantly lower than in controls. Conditioned medium of young glia (ACM-Y) induced significant neurotrophic and functional effects on GT(1-1) cells, but these effects were 50% less potent than the coculture itself. Heat denaturation of ACM-Y totally abolished its neurotrophic and functional properties, indicating that they involved a peptide factor. Suppression of bFGF activity in ACM-Y reduced its neurotrophic activity by approximately 40%, but did not affect its LHRH release-promoting effects. By contrast, neutralization of endogenous bFGF activity in GT(1-1) neurons cocultured with young glia counteracted both neurotrophic and functional effects of young glia. Treatment of old glia with bFGF rescued its neurotrophic and functional effects on GT(1-1) cells. Moreover, the ACM of aged bFGF-treated old glia was the most powerful neurotrophic stimulus for GT(1-1) neurons. These results suggest that: 1) soluble peptidic factors, including bFGF, and mechanism(s) requiring coculture are responsible for the highly potent neurotrophic and functional effects of young glia; 2) the inhibitory effects of old glia on neurite outgrowth and LHRH release are mediated in part by soluble inhibitory molecules and in part by factors requiring coculture with old glia; 3) old glia may revert to a growth-supporting state when treated with bFGF and this functional shift involves a diffusible molecule with potent neurotrophic and functional effects on immortalized LHRH neurons. (c) 2000 Wiley-Liss, Inc.
Assuntos
Astrócitos/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Hormônio Liberador de Gonadotropina/metabolismo , Neuroglia/fisiologia , Neurônios/fisiologia , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Senescência Celular/fisiologia , Técnicas de Cocultura , Imuno-Histoquímica , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
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.