RESUMO
Neuron death due to deprivation of target-derived neurotrophic factors depends on protein synthesis regulated by transcription factor activity. We investigated the content and phosphorylation of activating transcription factor 2 (ATF-2) in axon-damaged retinal ganglion cells of neonatal rats. In the retina of neonatal rats, the ATF-2 protein is predominantly located in the nucleus of the ganglion cells. A gradual loss of the immunoreactivity for ATF-2 occurred after explantation. ATF-2 is phosphorylated early after explantation, with a peak within 3 hours, preceding the peak of cell death that occurs at 18 hours. Both the phosphorylation of ATF-2 and ganglion cell death were blocked by treatment with an inhibitor of c-Jun N-terminal kinase (JNK), whereas an inhibitor of p38 reduced only slightly the rate of ganglion cell death with no effect upon phosphorylation of ATF-2. Inhibitors of phosphatidyl inositol 3 kinase (PI-3K), protein kinase C (PKC) or extracellular regulated kinase (ERK) had no effect. Finally, the inhibitor of JNK blocked the upregulation of both c-Jun and Hrk in the GCL after retinal explantation. The data show that phosphorylation of ATF-2 by JNK is associated with retinal ganglion cell death after axon damage.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Degeneração Neural/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Células Cultivadas , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Fosforilação , RatosRESUMO
Receptors of the P2X7 type have been demonstrated in granulocytes, monocytes/macrophages, B and T lymphocytes, and have been involved in several cellular mechanisms including those related to inflammation and immunological response. This study attempted to investigate the role of these receptors on the inflammatory and fibrogenic response in the kidneys of unilateral ureteral obstruction (UUO), by using P2X7 knockout mice (-/-). C57Bl6 mice were submitted to left UUO and killed after 7 and 14 days. Histopathology using hematoxylin-eosin, periodic-acid Schiff and Sirius-red staining, immunohistochemistry for macrophages, myofibroblasts, transforming growth factor-beta (TGF-beta)1 and P2X7, and immunofluorescence for apoptotic cells (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling) were performed. Protocols were as follows: (1) control; (2) sham; (3) control P2X7 (-/-); (4) sham P2X7 (-/-); (5) UUO wild type (WT); (6) UUO P2X7 (-/-). Myofibroblasts and Sirius-red staining were significantly lower in UUO P2X7 (-/-) mice at days 7 and 14, compared to UUO WT. Kidneys from UUO P2X7 (-/-) mice showed reduced number of inflammatory cells at day 14 but not at day 7, compared to UUO WT. TGF-beta1 was less in UUO P2X7 (-/-) mice at days 7 and 14 when compared to UUO WT. Macrophage infiltration and tubular apoptosis were lower in UUO P2X7 (-/-) at day 14 but not at day 7, compared to UUO WT. P2X7 was expressed only in tubular epithelial cells at day 7 of UUO WT mice. These findings constitute the first evidence that P2X7 receptors are implicated in macrophage infiltration, collagen deposition and apoptosis in response to ureteral obstruction in mice.
Assuntos
Inflamação/patologia , Inflamação/fisiopatologia , Receptores Purinérgicos P2/fisiologia , Obstrução Ureteral/patologia , Obstrução Ureteral/fisiopatologia , Actinas/genética , Actinas/metabolismo , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Apoptose/fisiologia , Atrofia/metabolismo , Atrofia/patologia , Atrofia/fisiopatologia , Colágeno/genética , Colágeno/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Fibrose/fisiopatologia , Regulação da Expressão Gênica/fisiologia , Inflamação/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Túbulos Renais/fisiopatologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7 , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Obstrução Ureteral/metabolismoRESUMO
Exclusion of the transcription factor Max from the nucleus of retinal ganglion cells is an early, caspase-independent event of programmed cell death following damage to the optic axons. To test whether the loss of nuclear Max leads to a reduction in neuroprotection, we developed a procedure to overexpress Max protein in rat retinal tissue in vivo. A recombinant adeno-associated viral vector (rAAV) containing the max gene was constructed, and its efficiency was confirmed by transduction of HEK-293 cells. Retinal ganglion cells were accessed in vivo through intravitreal injections of the vector in rats. Overexpression of Max in ganglion cells was detected by immunohistochemistry at 2 weeks following rAAV injection. In retinal explants, the preparation of which causes damage to the optic axons, Max immunoreactivity was increased after 30 h in vitro, and correlated with the preservation of a healthy morphology in ganglion cells. The data show that the rAAV vector efficiently expresses Max in mammalian retinal ganglion cells, and support the hypothesis that the Max protein plays a protective role for retinal neurons.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação Viral da Expressão Gênica , Vetores Genéticos , Parvoviridae , Células Ganglionares da Retina/metabolismo , Animais , Animais Recém-Nascidos , Axônios , Imuno-Histoquímica , Degeneração Neural/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Células Ganglionares da Retina/patologiaRESUMO
Exclusion of the transcription factor Max from the nucleus of retinal ganglion cells is an early, caspase-independent event of programmed cell death following damage to the optic axons. To test whether the loss of nuclear Max leads to a reduction in neuroprotection, we developed a procedure to overexpress Max protein in rat retinal tissue in vivo. A recombinant adeno-associated viral vector (rAAV) containing the max gene was constructed, and its efficiency was confirmed by transduction of HEK-293 cells. Retinal ganglion cells were accessed in vivo through intravitreal injections of the vector in rats. Overexpression of Max in ganglion cells was detected by immunohistochemistry at 2 weeks following rAAV injection. In retinal explants, the preparation of which causes damage to the optic axons, Max immunoreactivity was increased after 30 h in vitro, and correlated with the preservation of a healthy morphology in ganglion cells. The data show that the rAAV vector efficiently expresses Max in mammalian retinal ganglion cells, and support the hypothesis that the Max protein plays a protective role for retinal neurons.
Assuntos
Animais , Ratos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação Viral da Expressão Gênica , Vetores Genéticos , Parvoviridae , Células Ganglionares da Retina/metabolismo , Animais Recém-Nascidos , Axônios , Imuno-Histoquímica , Degeneração Neural/metabolismo , Proteínas Recombinantes/metabolismo , Células Ganglionares da Retina/patologiaRESUMO
Retinal cell differentiation leads to resistance to apoptosis induced by inhibition of protein synthesis, suggesting the accumulation of anti-apoptotic proteins. The redox factor/AP endonuclease Ref-1 (APE, APEX, HAP1) affects both DNA repair and the activity of various transcription factors, and controls sensitivity to genotoxic insults. We studied the expression of Ref-1 in the retina and brain of developing rats. Ref-1 immunoreactivity increased progressively within the nucleus of differentiating retinal cells, whereas it decreased in the developing hippocampal formation. During both natural and experimentally-induced cell death, Ref-1 disappeared from the nucleus of apoptotic cells. Degradation of Ref-1 in axotomized ganglion cells preceded the morphological characteristics of apoptosis. The sensitivity to apoptosis triggered by either thapsigargin or okadaic acid was the highest in photoreceptors, that contain the least Ref-1 among differentiated retinal cells. In both these differentiated cell types, inhibition of protein synthesis prevented the loss of Ref-1 and rescued the neurons. The data suggest that Ref-1 is an anti-apoptotic protein associated with cell differentiation in the retina.
Assuntos
Apoptose , Carbono-Oxigênio Liases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Endodesoxirribonucleases/metabolismo , Retina/citologia , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Diferenciação Celular , Células Cultivadas , Neurônios/citologia , RatosRESUMO
Programmed cell death by apoptosis plays a major role in neurogenesis. The sensitivity to apoptosis in developing nervous tissue is strongly dependent on cell interactions taking place within a highly structured environment, composed of various cell types at distinct stages of differentiation. In this article, we review evidence gathered both in vivo and in a histotypical retinal explant preparation in vitro that the bifunctional AP endonuclease/redox factor Ref-1 (HAP1, APE, APEX) may be an anti-apoptotic protein associated with cell differentiation in the developing retina.
Assuntos
Apoptose/fisiologia , Carbono-Oxigênio Liases/metabolismo , Diferenciação Celular , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Retina/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Carbono-Oxigênio Liases/genética , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Ratos , Retina/citologia , Retina/embriologia , Retina/metabolismoRESUMO
Programmed cell death in the form of apoptosis involves a network of metabolic events and may be triggered by a variety of stimuli in distinct cells. The nervous system contains several neuron and glial cell types, and developmental events are strongly dependent on selective cell interactions. Retinal explants have been used as a model to investigate apoptosis in nervous tissue. This preparation maintains the structural complexity and cell interactions similar to the retina in situ, and contains cells in all stages of development. We review the finding of nuclear exclusion of several transcription factors during apoptosis in retinal cells. The data reviewed in this paper suggest a link between apoptosis and a failure in the nucleo-cytoplasmic partition of transcription factors. It is argued that the nuclear exclusion of transcription factors may be an integral component of apoptosis both in the nervous system and in other types of cells and tissues.
Assuntos
Apoptose , Sistema Nervoso/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular , Sistema Nervoso/citologia , Sistema Nervoso/metabolismo , Membrana Nuclear/metabolismo , Ratos , Retina/citologia , Retina/crescimento & desenvolvimento , Retina/metabolismoRESUMO
Programmed cell death in the form of apoptosis involves a network of metabolic events and may be triggered by a variety of stimuli in distinct cells. The nervous system contains several neuron and glial cell types, and developmental events are strongly dependent on selective cell interactions. Retinal explants have been used as a model to investigate apoptosis in nervous tissue. This preparation maintains the structural complexity and cell interactions similar to the retina in situ, and contains cells in all stages of development. We review the finding of nuclear exclusion of several transcription factors during apoptosis in retinal cells. The data reviewed in this paper suggest a link between apoptosis and a failure in the nucleo-cytoplasmic partition of transcription factors. It is argued that the nuclear exclusion of transcription factors may be an integral component of apoptosis both in the nervous system and in other types of cells and tissues
Assuntos
Animais , Ratos , Apoptose , Tecido Nervoso/crescimento & desenvolvimento , Retina/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Animais Recém-Nascidos , Diferenciação Celular , Tecido Nervoso/citologia , Tecido Nervoso/metabolismo , Membrana Nuclear/metabolismo , Retina/citologia , Retina/metabolismoRESUMO
The mechanisms of apoptosis are strongly dependent on cell-cell interactions typical of organized tissues. Experimental studies of apoptosis using a histotypical preparation of retinal explants are reported in the present article. We found that various characteristics of apoptosis are selectively associated with retinal cell death depending on cell type, stage of maturation, and means of induction of apoptosis. Among these were: (1) the requirements of protein synthesis; (2) the role of cAMP; (3) the expression of certain apoptosis-associated proteins; and (4) the sensitivity to excitotoxicity, modulation of protein phosphatases and calcium mobilization. Dividing cells undergo apoptosis in response to several inducers in specific phases of the cell cycle, and in distinct regions within their pathway of interkinetic nuclear migration. Recent post-mitotic cells are selectively sensitive to apoptosis induced by blockade of protein synthesis, while both proliferating and differentiated cells are more resistant. We also studied the association of several proteins, some of which play critical roles in the cell cycle, with both differentiation and apoptosis in the retinal tissue. Detection of cell cycle markers did not support the hypothesis that retinal cells re-enter the cell cycle on their pathway to apoptosis, although some proteins associated with cell proliferation re-appeared in degenerating cells. The transcription factors c-Jun, c-Fos and c-Myc were found associated with apoptosis in retinal cells, but their sub-cellular location in apoptotic bodies is not consistent with their canonical functions in the control of gene expression. The bifunctional redox factor/AP endonuclease Ref-1 and the transcription factor Max are associated with progressive cell differentiation, and both are down-regulated during cell death in the retina. The data suggest that Ref-1 and Max may normally function as negative modulators of retinal apoptosis. The results indicate that nuclear exclusion of transcription factors and other important control proteins is a hallmark of retinal apoptosis. Histotypical explants may be a choice preparation for the experimental analysis of the mechanisms of apoptosis, in the context both of cell-cell interactions and of the dynamic behavior of developing cells within the organized retinal tissue.
Assuntos
Apoptose , Retina/crescimento & desenvolvimento , Animais , Ciclo Celular , Diferenciação Celular , Divisão Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Proto-Oncogênicas/metabolismo , Retina/citologia , Retina/metabolismo , Fatores de TranscriçãoRESUMO
In the retina of newborn rats there is evidence for two mechanisms of programmed cell death. Apoptosis of ganglion cells (RGCs) following axotomy depends on protein synthesis. In contrast, inhibition of protein synthesis leads to apoptosis in the neuroblastic layer (NBL). The induction of apoptosis following translational arrest suggests that post-translational modifications of apoptosis-associated proteins may be crucial to the cell death programs in the developing retina. We investigated the possible role of protein kinases upon apoptosis in retinal explants in vitro. An increase in the intracellular concentration of cAMP produced either by the adenylyl-cyclase activator forskolin (10 microM) or by 8-Br-cAMP (1 mM), prevented apoptosis induced in the NBL by inhibition of protein synthesis, but had no statistically significant effect upon RGC death. In contrast, neither 8-Br-cGMP (1 mM) nor the specific cGMP-phosphodiesterase inhibitor zaprinast (10-100 microM) had significant effects on apoptosis in the retina. The cAMP-phosphodiesterase inhibitors isobutylmethylxantine (IBMX, 0.1-1 mM) and Ro-201724 (50-200 microM) also prevented apoptosis in the NBL. The isoquinolinesulfonamide H89 (20 microM), a specific cAMP-dependent protein kinase inhibitor, partially reverted the protective effect of either forskolin or IBMX within the NBL. Neither 12-O-tetradecanoyl phorbol-13-acetate (TPA, 10 nM) nor bisindolylmaleimide (0.2-0.5 microM), respectively an activator and an inhibitor of protein kinase C had significant effects upon the retinal explants. The protein kinase inhibitor 2-aminopurine (2-AP, 10 mM) prevented apoptosis of axotomized ganglion cells and induced apoptosis in the NBL. Forskolin prevented the apoptosis induced by 2-AP in the NBL, whereas TPA had no effect. The effects of 2-AP were, however, not dependent on inhibition of protein synthesis. The data indicate that modulation of the activity of both cAMP-dependent protein kinase and several protein kinases sensitive to 2-aminopurine selectively affect apoptosis in distinct cell layers of the developing retina.
Assuntos
Envelhecimento/fisiologia , Apoptose/fisiologia , Proteínas Quinases/fisiologia , Retina/fisiologia , 2-Aminopurina/farmacologia , Animais , Animais Recém-Nascidos/fisiologia , Antimetabólitos/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Embrião de Mamíferos/fisiologia , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Proteínas Quinases , Ratos , Retina/citologiaRESUMO
Studies of programmed cell death in the developing retina in vitro are currently reviewed. The results of inhibiting protein synthesis in retinal explants indicate two mechanisms of apoptosis. One mechanism depends on the synthesis of positive modulators ('killer proteins'), while a distinct, latent mechanism appears to be continuously blocked by negative modulators. Extracellular modulators of apoptosis include the neurotrophic factors NT-4 and BDNF, while glutamate may have either a positive or a negative modulatory action on apoptosis. Several protein kinases selectively modulate apoptosis in distinct retinal layers. Calcium and nitric oxide were also shown to affect apoptosis in the developing retinal tissue. The protein c-Jun was found associated with apoptosis in various circumstances, while p53 seems to be selectively expressed in some instances of apoptosis. The results indicate that the sensitivity of each retinal cell to apoptosis is controlled by multiple, interactive, cell type- and context-specific mechanisms. Apoptosis in the retina depends on a critical interplay of extracellular signals delivered through neurotrophic factors, neurotransmitters and neuromodulators, several signal transduction pathways, and the expression of a variety of genes.
Assuntos
Apoptose/fisiologia , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Técnicas de Cultura , Degeneração Retiniana/metabolismo , Transdução de Sinais/fisiologia , Animais , Camundongos , RatosRESUMO
Studies of programmed cell death in the developing retina in vitro are currently reviewed. The results of inhibiting protein synthesis in retinal explants indicate two mechanisms of apoptosis. One mechanism depends on the synthesis of positive modulators ('killer proteins'), while a distinct, latent mechanism appears to be continuously blocked by negative modulators. Extracellular modulators of apoptosis include the neurotrophic factors NT-4 and BDNF, while glutamate may have either a positive or a negative modulatory action on apoptosis. Several protein kinases selectively modulate apoptosis in distinct retinal layers. Calcium and nitric oxide were also shown to affect apoptosis in the developing retianl tissue. The protein c-Jun was found associated with apoptosis in various circumstances, while p53 seems to be selectively expressed in some instances of apoptosis. The results indicate that the sensitivity of each retinal cell to apoptosis is controlled by multiple, interactive, cell type- and context-specific mechanisms. Apoptosis in the retina depends on a critical interplay of extracellular signals delivered through neurotrophic factors, neurotransmitters and neuromodulators, several signal transduction pathways, and the expression of a variety of genes.
Assuntos
Camundongos , Ratos , Animais , Apoptose/fisiologia , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Técnicas de Cultura , Técnicas In Vitro , Degeneração Retiniana/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Hepatic injury elicits an excessive deposition of extracellular matrix probably due to a loss of control mechanisms in mesenchymal cells in fibrotic lesions, or a local activity of growth factors. To study collagen synthesis in an in vitro model of fibrotic lesions, we isolated liver connective tissue cells (LCTC) from murine schistosomal granulomas in C3H/HeN mice. Collagen was quantified in culture supernatants using a sirius red dye assay. LCTC and skin fibroblasts (SF) secreted similar amounts of collagen per cell and secretion was inversely proportional to the cell density. Cells cultured at low density (10,000 cells/cm2) secreted two- to three-times more collagen per cell when compared to cells grown in high-density cultures (60,000 cells/cm2). Collagen secretion was stimulated by transforming growth factor-beta (TGF-beta) in both cell lines, but the response of LCTC was detected from 1 ng/ml on, while SF responded only to higher concentrations (2.5 and 5 ng/ml). These data do not support the hypothesis that cells from fibrotic livers have lost the normal control mechanisms and suggest that their control is disturbed locally by the presence of peptide growth factors during the development of fibrosis.
Assuntos
Colágeno/biossíntese , Tecido Conjuntivo/metabolismo , Granuloma/metabolismo , Fígado/metabolismo , Esquistossomose/metabolismo , Animais , Tecido Conjuntivo/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Granuloma/patologia , Fígado/patologia , Hepatopatias Parasitárias/metabolismo , Hepatopatias Parasitárias/patologia , Camundongos , Camundongos Endogâmicos C3H , Esquistossomose/patologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Hepatic injury elicits an excessive deposition of extracellular matrix probably due to a loss of control mechanisms in mesenchymal cells in fibrotic lesions, or a local activity of growth factors. To study collagen synthesis in an in vitro model of fibrotic lesions, we isolated liver connective tissue cells (LCTC) from murine schistosomal granulomas in C3H/HeN mice. Collagen was quantified in culture supernatants using a sirius red dye assay. LCTC and skin fibroblasts (SF) secreted similar amounts of collagen per cell and secretion was inversely proportional to the cell density. Cells cultured at low density (10,000 cells/cm2) secreted two- to three-times more collagen per cell when compared to cells grown in high-density cultures (60,000 cells/cm2). Collagen secretion was stimulated by transforming growth factor-beta (TGF-beta) in both cell lines, but the response of LCTC was detected from 1 ng/ml on, while SF responded only to higher concentrations (2.5 and 5 ng/ml). These data do not support the hypothesis that cells from fibrotic livers have lost the normal control mechanisms and suggest that their control is disturbed locally by the presence of peptide growth factors during the development of fibrosis.