RESUMO
The retinoblastoma (Rb) family of proteins are key regulators of cell cycle exit during development and their deregulation is associated with cancer. Rb is critical for normal retinal development and germline mutations lead to retinoblastoma making retinae an attractive system to study Rb family signaling. Rb coordinates proliferation and differentiation through the E2f family of transcription factors, a critical interaction for the role of Rb in retinal development and tumorigenesis. However, whether the roles of the different E2fs are interchangeable in controlling development and tumorigenesis in the retina or if they have selective functions remains unknown. In this study, we found that E2f family members play distinct roles in the development and tumorigenesis. In Rb;p107-deficient retinae, E2f1 and E2f3 inactivation rescued tumor formation but only E2f1 rescued the retinal development phenotype. This allowed the identification of key target genes for Rb/E2f family signaling contributing to tumorigenesis and those contributing to developmental defects. We found that Sox4 and Sox11 genes contribute to the developmental phenotype and Hells and Uhrf1 contribute to tumorigenesis. Using orthotopic human xenografts, we validated that upregulation of HELLS and UHRF1 is essential for the tumor phenotype. Also, these epigenetic regulators are important for the regulation of SYK.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Cromatina/genética , DNA Helicases/genética , Neoplasias da Retina/genética , Retinoblastoma/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA Helicases/metabolismo , Progressão da Doença , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F3/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Neoplasias da Retina/metabolismo , Neoplasias da Retina/patologia , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Quinase Syk , Transfecção , Ubiquitina-Proteína LigasesRESUMO
PURPOSE: Relatively little is known about the contribution of p53/Mdm2 pathway in apoptosis of retinal pigment epithelial (RPE) cells or its possible link to dysfunction of aging RPE or to related blinding disorders such as age-related macular degeneration (AMD). METHODS: Age-associated changes in p53 activation were evaluated in primary RPE cultures from human donor eyes of various ages. Apoptosis was evaluated by activation of caspases and DNA fragmentation. Gene-specific small interfering RNA was used to knock down expression of p53. RESULTS: We observed that the basal rate of p53-dependent apoptosis increased in an age-dependent manner in human RPE. The age-dependent increase in apoptosis was linked to alterations in several aspects of the p53 pathway. p53 phosphorylation Ser15 was increased through the stimulation of ATM-Ser1981. p53 acetylation Lys379 was increased through the inhibition of SIRT1/2. These two posttranslational modifications of p53 blocked the sequestration of p53 by Mdm2, thus resulting in an increase in free p53 and of p53 stimulation of apoptosis through increased expression of PUMA (p53 upregulated modulator of apoptosis) and activation of caspase-3. Aged RPE also had reduced expression of antiapoptotic Bcl-2, which contributed to the increase in apoptosis. Of particular interest in these studies was that pharmacologic treatments to block p53 phosphorylation, acetylation, or expression were able to protect RPE cells from apoptosis. CONCLUSIONS: Our studies suggest that aging in the RPE leads to alterations of specific checkpoints in the apoptotic pathway, which may represent important molecular targets for the treatment of RPE-related aging disorders such as AMD.
Assuntos
Envelhecimento/fisiologia , Apoptose/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Epitélio Pigmentado da Retina/patologia , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose/metabolismo , Benzamidas/farmacologia , Western Blotting , Caspase 3/metabolismo , Proliferação de Células , Células Cultivadas , Fragmentação do DNA , Suscetibilidade a Doenças , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imidazóis/farmacologia , Marcação In Situ das Extremidades Cortadas , Pessoa de Meia-Idade , Naftóis/farmacologia , Fosforilação , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , RNA Interferente Pequeno/genética , Epitélio Pigmentado da Retina/metabolismo , Sirtuína 1/antagonistas & inibidores , Sirtuína 2/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
Neuronal differentiation with respect to the acquisition of synaptic competence needs to be regulated precisely during neurogenesis to ensure proper formation of circuits at the right place and time in development. This regulation is particularly important for synaptic triads among photoreceptors, horizontal cells (HCs), and bipolar cells in the retina, because HCs are among the first cell types produced during development, and bipolar cells are among the last. HCs undergo a dramatic transition from vertically oriented neurites that form columnar arbors to overlapping laminar dendritic arbors with differentiation. However, how this process is regulated and coordinated with differentiation of photoreceptors and bipolar cells remains unknown. Previous studies have suggested that the retinoblastoma (Rb) tumor suppressor gene may play a role in horizontal cell differentiation and synaptogenesis. By combining genetic mosaic analysis of individual synaptic triads with neuroanatomic analyses and multiphoton live imaging of developing HCs, we found that Rb plays a cell-autonomous role in the reorganization of horizontal cell neurites as they differentiate. Aberrant vertical processes in Rb-deficient HCs form ectopic synapses with rods in the outer nuclear layer but lack bipolar dendrites. Although previous reports indicate that photoreceptor abnormalities can trigger formation of ectopic synapses, our studies now demonstrate that defects in a postsynaptic partner contribute to the formation of ectopic photoreceptor synapses in the mammalian retina.
Assuntos
Diferenciação Celular/fisiologia , Dendritos/fisiologia , Neurogênese/fisiologia , Células Horizontais da Retina/citologia , Proteína do Retinoblastoma/metabolismo , Sinapses/fisiologia , Animais , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteína do Retinoblastoma/genéticaRESUMO
PURPOSE: Because recent studies indicate that blocking the interaction between p53 and Mdm2 results in the nongenotoxic activation of p53, the authors sought to investigate whether the inhibition of p53-Mdm2 binding activates p53 and sensitizes human retinal epithelial cells to apoptosis. METHODS: Apoptosis was evaluated by the activation of caspases and DNA fragmentation assays. The Mdm2 antagonist Nutlin-3 was used to dissociate p53 from Mdm2 and, thus, to increase p53 activity. Knockdown of p53 expression was accomplished by using p53 siRNA. RESULTS: ARPE-19 and primary RPE cells expressed high levels of the antiapoptotic proteins Bcl-2 and Bcl-xL. Exposure of these cells to camptothecin (CPT) or TNF-α/ cycloheximide (CHX) failed to induce apoptosis. In contrast, treatment with the Mdm2 antagonist Nutlin-3 in the absence of CPT or TNF-α/CHX increased apoptosis. Activation of p53 in response to Nutlin-3 also increased levels of Noxa, p53-upregulated modulator of apoptosis (PUMA), and Siva-1, decreased expression of Bcl-2 and Bcl-xL, and simultaneously increased caspases-9 and -3 activities and DNA fragmentation. Knockdown of p53 decreased the basal expression of p21Cip1 and Bcl-2, inhibited the Nutlin-3-induced upregulation of Siva-1 and PUMA expression, and consequently inhibited caspase-3 activation. CONCLUSIONS: These results indicate that the normally available pool of intracellular p53 is predominantly engaged in the regulation of cell cycle checkpoints by p21Cip1 and does not trigger apoptosis in response to DNA-damaging agents. However, the blockage of p53 binding to Mdm2 frees a pool of p53 that is sufficient, even in the absence of DNA-damaging agents, to increase the expression of proapoptotic targets and to override the resistance of RPE cells to apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Epitélio Pigmentado da Retina/patologia , Proteína Supressora de Tumor p53/metabolismo , Adulto , Western Blotting , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA , Humanos , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Epitélio Pigmentado da Retina/metabolismo , Transfecção , Proteína bcl-X/antagonistas & inibidoresRESUMO
Vascular endothelial growth factor (VEGF) appears to be an important mediator of pathologic retinal angiogenesis. In understanding the mechanisms of pathologic retinal neovascularization, we found that VEGF activates PLD1 in human retinal microvascular endothelial cells, and this event is dependent on Src. In addition, VEGF activates protein kinase C-gamma (PKCgamma) via Src-dependent PLD1 stimulation. Inhibition of Src, PLD1, or PKCgamma via pharmacologic, dominant negative mutant, or siRNA approaches significantly attenuated VEGF-induced human retinal microvascular endothelial cell migration, proliferation, and tube formation. Hypoxia also induced Src-PLD1-PKCgamma signaling in retina, leading to retinal neovascularization. Furthermore, siRNA-mediated down-regulation of VEGF inhibited hypoxia-induced Src-PLD1-PKCgamma activation and neovascularization. Blockade of Src-PLD1-PKCgamma signaling via the siRNA approach also suppressed hypoxia-induced retinal neovascularization. Thus, these observations demonstrate, for the first time, that Src-dependent PLD1-PKCgamma activation plays an important role in pathologic retinal angiogenesis.
Assuntos
Endotélio Vascular/metabolismo , Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Retina/metabolismo , Neovascularização Retiniana , Western Blotting , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Imunofluorescência , Humanos , Fosforilação , Retina/patologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
To investigate the mechanisms underlying 15(S)-HETE-induced angiogenesis, we have studied the role of the small GTPase, Rac1. We find that 15(S)-HETE activated Rac1 in human retinal microvascular endothelial cells (HRMVEC) in a time-dependent manner. Blockade of Rac1 by adenovirus-mediated expression of its dominant negative mutant suppressed HRMVEC migration as well as tube formation and Matrigel plug angiogenesis. 15(S)-HETE stimulated Src in HRMVEC in a time-dependent manner and blockade of its activation inhibited 15(S)-HETE-induced Rac1 stimulation in HRMVEC and the migration and tube formation of these cells as well as Matrigel plug angiogenesis. 15(S)-HETE stimulated JNK1 in Src-Rac1-dependent manner in HRMVEC and adenovirus-mediated expression of its dominant negative mutant suppressed the migration and tube formation of these cells and Matrigel plug angiogenesis. 15(S)-HETE activated ATF-2 in HRMVEC in Src-Rac1-JNK1-dependent manner and interference with its activation via adenovirus-mediated expression of its dominant negative mutant abrogated migration and tube formation of HRMVEC and Matrigel plug angiogenesis. In addition, 15(S)-HETE-induced MEK1 stimulation was found to be dependent on Src-Rac1 activation. Blockade of MEK1 activation inhibited 15(S)-HETE-induced JNK1 activity and ATF-2 phosphorylation. Together, these findings show that 15(S)-HETE activates ATF-2 via the Src-Rac1-MEK1-JNK1 signaling axis in HRMVEC leading to their angiogenic differentiation.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Neovascularização Patológica/induzido quimicamente , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Colágeno , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Laminina , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteoglicanas , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Transdução de Sinais/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
PURPOSE: The cytotoxic effects of oxidative stress, which play an important role in ocular diseases, are well known. In this study, we investigated the effect of non-lethal doses of oxidative stress on various cell functions, namely cell viability, cell attachment and cell migration in a widely used retinal pigment epithelium (RPE) cell line (ARPE-19). METHODS: A single exposure to various concentrations of hydrogen peroxide (H(2)O(2)) was used to establish a dose response for H(2)O(2)-induced cell death. Other cellular responses, such as changes in cell attachment and migration, were monitored after exposure to increasing doses. Finally, the effects of preconditioning cells with increasing non-lethal doses of H(2)O(2), with and without a subsequent exposure to lethal doses of H(2)O(2), were determined. RESULTS: The optimum dose for inducing cell death in ARPE-19 cells was between 900 and 1000 microm H(2)O(2). Preconditioning the cells with 1, 10 and 50 microm of H(2)O(2) provided a dose-dependent protection against cell death induced by a lethal dose (900-1000 microm) of H(2)O(2). Preconditioning with higher doses caused cells to become more susceptible to the cytotoxic effects of the lethal dose. Although H(2)O(2) increased cell attachment in lower doses, it induced a dose-dependent inhibition of cell attachment to the substrate in higher doses. H(2)O(2) did not affect cell migration in sub-lethal doses. CONCLUSION: Preconditioning RPE cells with limited exposure to non-lethal oxidative stress confers significant protection against subsequent H(2)O(2)-induced cell death. It also affects cell attachment in a dose-specific manner. This finding may help in understanding the pathogenesis of diseases in which oxidative stress plays an important role and in determining the suitability of certain treatment strategies, in particular RPE transplantation in the treatment of age-related macular degeneration.
Assuntos
Apoptose , Precondicionamento Isquêmico , Estresse Oxidativo/fisiologia , Epitélio Pigmentado da Retina/citologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Peróxido de Hidrogênio/toxicidade , Oxidantes/toxicidadeRESUMO
During neurogenesis, the progression from a progenitor cell to a differentiated neuron is believed to be unidirectional and irreversible. The Rb family of proteins (Rb, p107, and p130) regulates cell-cycle exit and differentiation during retinogenesis. Rb and p130 are redundantly expressed in the neurons of the inner nuclear layer (INL) of the retina. We have found that in the adult Rb;p130-deficient retinae p107 compensation prevents ectopic proliferation of INL neurons. However, p107 is haploinsufficient in this process. Differentiated Rb(-/-);p107(+/-);p130(-/-) horizontal interneurons re-entered the cell cycle, clonally expanded, and formed metastatic retinoblastoma. Horizontal cells were not affected in Rb(+/-);p107(-/-);p130(-/-) or Rb(-/-);p107(-/-);p130(+/-), retinae suggesting that one copy of Rb or p130 was sufficient to prevent horizontal proliferation. We hereby report that differentiated neurons can proliferate and form cancer while maintaining their differentiated state including neurites and synaptic connections.
Assuntos
Interneurônios/fisiologia , Neoplasias da Retina/patologia , Retinoblastoma/secundário , Células-Tronco/fisiologia , Animais , Neoplasias da Medula Óssea/patologia , Neoplasias da Medula Óssea/secundário , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Diferenciação Celular , Divisão Celular , Interneurônios/metabolismo , Metástase Linfática , Camundongos , Retina/patologia , Retinoblastoma/patologia , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/fisiologia , Proteína p107 Retinoblastoma-Like/genética , Proteína p107 Retinoblastoma-Like/fisiologia , Células-Tronco/metabolismoRESUMO
PURPOSE: To examine for the expression of 15-lipoxygenase 1 (15-LOX1) and 15-LOX2 in human retinal microvascular endothelial cells (HRMVECs) and study the role of arachidonic acid metabolites of these enzymes in angiogenesis. METHODS: Quantitative RT-PCR and reverse-phase HPLC analyses were used to determine 15-LOX1/2 expression and their arachidonic acid metabolites in HRMVECs. The role of MEK1 in 15(S)-HETE-induced angiogenesis was studied using HRMVEC migration, tube formation, and basement membrane matrix plug angiogenesis. RESULTS: HRMVECs expressed both 15-LOX1 and 15-LOX2. Hypoxia induced the expression of 15-LOX1 and the production of its arachidonic acid metabolites 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 15(S)-HETE stimulated HRMVEC migration and tube formation as potently as 20 ng/mL fibroblast growth factor-2 (FGF-2). In addition, 15(S)-HETE stimulated the phosphorylation of ERK1/2, JNK1, p38 MAPK, and MEK1 in a time-dependent manner in these cells. Inhibition of MEK1 by pharmacologic and dominant-negative mutant approaches attenuated 15(S)-HETE-induced phosphorylation of ERK1/2 and JNK1 but not p38 MAPK. Blockade of ERK1/2 and JNK1 activation suppressed 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. Inhibition of p38 MAPK attenuated 15(S)-HETE-induced HRMVEC migration only. Inhibition of MEK1 also blocked 15(S)-HETE-induced HRMVEC migration and tube formation and basement membrane matrix plug angiogenesis. CONCLUSIONS: These results suggest that hypoxia, through the induction of 15-LOX1 expression, leads to the production of 15(S)-HETE in HRMVECs. In addition, 15(S)-HETE, through MEK1-dependent activation of ERK1/2 and JNK1, stimulates the angiogenic differentiation of HRMVECs and basement membrane matrix plug angiogenesis.
Assuntos
Endotélio Vascular/metabolismo , Ácidos Hidroxieicosatetraenoicos/biossíntese , Hipóxia/metabolismo , MAP Quinase Quinase 1/fisiologia , Neovascularização Retiniana/metabolismo , Vasos Retinianos/citologia , Adenoviridae/genética , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Membrana Basal , Movimento Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Humanos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Retinoblastomas initiate in the developing retina in utero and are diagnosed during the first few years of life. We have recently generated a series of knockout mouse models of retinoblastoma that recapitulate the timing, location, and progression of human retinoblastoma. One of the most important benefits of these preclinical models is that we can study the earliest stages of tumor initiation and expansion. This is not possible in human retinoblastoma because tumors initiate in utero and are not diagnosed until they are at an advanced stage. We found that mouse retinoblastoma cells exhibit a surprising degree of differentiation, which has not been previously reported for any neural tumor. Early-stage mouse retinoblastoma cells express proteins found normally in retinal plexiform layers. They also extend neurites and form synapses. All of these features, which were characterized by immunostaining, Golgi-Cox staining, scanning electron microscopy, and transmission electron microscopy, suggest that mouse retinoblastoma cells resemble amacrine/horizontal cells from the retina. As late-stage retinoblastoma cells expand and invade the surrounding tissue, they lose their differentiated morphology and become indistinguishable from human retinoblastomas. Taken together, our data suggest that neuronal differentiation is a hallmark of early-stage retinoblastoma and is lost as cells become more aggressive and invasive. We also show that rosette formation is not a hallmark of retinoblastoma differentiation, as previously believed. Instead, rosette formation reflects extensive cell-cell contacts between retinoblastoma cells in both early-stage (differentiated) and late-stage (dedifferentiated) tumors.
Assuntos
Neurônios/patologia , Retinoblastoma/patologia , Sinapses/patologia , Animais , Câmara Anterior/patologia , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos , Camundongos Knockout , Invasividade Neoplásica , Neuritos/patologia , Neurônios/ultraestrutura , Retina/patologia , Retinoblastoma/genética , Retinoblastoma/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinapses/ultraestrutura , Corpo Vítreo/patologiaRESUMO
The retinoblastoma gene (Rb) regulates neural progenitor cell proliferation and cell fate specification and differentiation. For the developing mouse retina, two distinct functions of Rb have been described: regulation of retinal progenitor cell proliferation and rod photoreceptor development. Cells that would normally become rods fail to mature and remain as immature cells in the outer nuclear layer in the adult. By using Chx10-Cre;Rb(Lox/-) mice, we generated a chimeric retina with alternating apical-basal stripes of wild-type and Rb-deficient tissue. This provides a unique model with which to study synaptogenesis at the outer plexiform layer within regions that lack differentiated rods. In regions where rods failed to differentiate, the outer plexiform layer (OPL) was disrupted. Horizontal cells formed, and their somata were appropriately aligned, but their neurites did not project laterally. Instead many horizonal cell neurites extended apically, forming ectopic synapses with photoreceptors at all levels of the outer nuclear layer. These ectopic photoreceptor terminals contained synaptic ribbons, horizontal cell processes with synaptic vesicles, and a single mitochrondrion characteristic of rod spherules. Rb-deficient bipolar cells differentiated normally, extended dendrites to the OPL, and formed synapses that were indistinguishable from adjacent wild-type cells. In contrast to OPL-positioned synapses, ectopic synapses did not contain bipolar dendrites. This finding suggests that horizontal cells and photoreceptors can form stable synapses that are devoid of bipolar dendrites outside the normal boundaries of the OPL. Finally, analysis of P4, P7, P12, and P15 retinae suggests that the apical horizontal cell processes result from their failure to establish their normal lateral projections during development.
Assuntos
Diferenciação Celular/genética , Vias Neurais/anormalidades , Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/anormalidades , Proteína do Retinoblastoma/genética , Sinapses/patologia , Animais , Animais Recém-Nascidos , Quimera/anormalidades , Quimera/crescimento & desenvolvimento , Coristoma/genética , Coristoma/metabolismo , Coristoma/patologia , Dendritos/patologia , Dendritos/ultraestrutura , Modelos Animais de Doenças , Deleção de Genes , Camundongos , Camundongos Knockout , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/ultraestrutura , Terminações Pré-Sinápticas/patologia , Terminações Pré-Sinápticas/ultraestrutura , Células Bipolares da Retina/patologia , Células Bipolares da Retina/ultraestrutura , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Células-Tronco/citologia , Células-Tronco/metabolismo , Sinapses/ultraestruturaRESUMO
BACKGROUND: Retinal pigment epithelium cells play an important role in the pathogenesis of age related macular degeneration. Their morphological, molecular and functional phenotype changes in response to various stresses. Functional profiling of genes can provide useful information about the physiological state of cells and how this state changes in response to disease or treatment. In this study, we have constructed a functional profile of the genes expressed by the ARPE-19 cell line of retinal pigment epithelium. METHODS: Using Affymetrix MAS 5.0 microarray analysis, genes expressed by ARPE-19 cells were identified. Using GeneChip annotations, these genes were classified according to their known functions to generate a functional gene expression profile. RESULTS: We have determined that of approximately 19,044 unique gene sequences represented on the HG-U133A GeneChip, 6,438 were expressed in ARPE-19 cells irrespective of the substrate on which they were grown (plastic, fibronectin, collagen, or Matrigel). Rather than focus our subsequent analysis on the identity or level of expression of each individual gene in this large data set, we examined the number of genes expressed within 130 functional categories. These categories were selected from a library of HG-U133A GeneChip annotations linked to the Affymetrix MAS 5.0 data sets. Using this functional classification scheme, we were able to categorize about 70% of the expressed genes and condense the original data set of over 6,000 data points into a format with 130 data points. The resulting ARPE-19 Functional Gene Expression Profile is displayed as a percentage of ARPE-19-expressed genes. CONCLUSION: The Profile can readily be compared with equivalent microarray data from other appropriate samples in order to highlight cell-specific attributes or treatment-induced changes in gene expression. The usefulness of these analyses is based on the assumption that the numbers of genes expressed within a functional category provide an indicator of the overall level of activity within that particular functional pathway.
Assuntos
Proteínas do Olho/genética , Perfilação da Expressão Gênica , Expressão Gênica/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Epitélio Pigmentado Ocular/metabolismo , Linhagem Celular , Humanos , Sensibilidade e EspecificidadeRESUMO
In order to form and maintain a protective barrier for photoreceptors, the retinal pigment epithelium relies on integrin signaling and related pathways to form adhesion complexes, undergo cell spreading, and establish a confluent cellular monolayer. Polyamines are multifunctional polycations that are essential for cell attachment and spreading, although their exact mechanisms of action are as yet unclear. We report new immunocytochemical evidence suggesting that in the cells of retinal pigment epithelium and also the intestinal epithelium, polyamines are present in a population of intracellular vesicles that appear transiently during initial stages of cell spreading. In newly attached cells with minimal spreading, the vesicles are seen near the nucleus, whereas in more highly spread cells, the vesicles are localized to the plasma membrane, near, but not precisely co-localized with an enzyme marker for adhesion complexes, focal adhesion kinase. We also observe pronounced nuclear staining in newly attached cells that have not spread, whereas this staining is decreased in cells that have spread. Nuclear staining has been previously reported in other cell types and has been attributed to DNA binding of polyamines, which is known to stabilize chromatin structure. We hypothesize that the appearance of polyamine vesicles near focal adhesions of cells undergoing attachment and spreading may reflect the mechanism by which polyamine pools are targeted to appropriate interaction sites necessary for the assembly of adhesion complexes. Alternatively, the vesicles could represent the mechanism by which polyamines are removed from the nucleus and possibly released from the cell.
Assuntos
Células Epiteliais/metabolismo , Intestinos/citologia , Epitélio Pigmentado Ocular/citologia , Poliaminas/metabolismo , Actinas/metabolismo , Actinas/fisiologia , Animais , Adesão Celular , Núcleo Celular/metabolismo , Células Cultivadas , Células Epiteliais/fisiologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Imuno-Histoquímica , Proteínas Tirosina Quinases/metabolismo , CoelhosRESUMO
Mice represent a valuable species for studies of development and disease. With the availability of transgenic models for retinal degeneration in this species, information regarding development and structure of mouse retina has become increasingly important. Of special interest is the differentiation and synaptogenesis of photoreceptors since these cells are predominantly involved in hereditary retinal degenerations. Thus, some of the keys to future clinical management of these retinal diseases may lie in understanding the molecular mechanisms of outer retinal development. In this study, we describe the expression of markers for photoreceptors (recoverin), horizontal cells (calbindin), bipolar cells (protein kinase C; PKC) and cytoskeletal elements pivotal to axonogenesis (beta-tubulin and actin) during perinatal development of mouse retina. Immunocytochemical localization of recoverin, calbindin, PKC and beta-tubulin was monitored in developing mouse retina (embryonic day (E) 18.5 to postnatal day (PN) 14), whereas f-actin was localized by Phalloidin binding. Recoverin immunoreactive cells, presumably the photoreceptors, were observed embryonically (E 18.5) and their number increased until PN 14. Neurite projections from the immunoreactive cells towards the outer plexiform layer (OPL) were noted at PN 0 and these processes reached the OPL at PN 7 coincident with histological evidence for the differentiation of the OPL. Outer segments, all the cell bodies in the ONL, as well as the OPL were immunoreactive to recoverin at PN 14. Calbindin immunoreactive horizontal cells were also present in E 18.5 retinas. These cells became progressively displaced proximally as the ONL developed. A calbindin immunoreactive plexus was seen in the OPL at PN 7. PKC immunoreactive bipolar cells developed postnatally, becoming distinguished at PN 7. Both beta-tubulin and actin immunoreactive cells were present in the IPL as early as E 18.5; however, appearance of processes labeled with these markers in the OPL was delayed until PN 7, concurrent with the first appearance of photoreceptor neurites, development of the horizontal cell plexus, and development of synaptophysin immunoreactivity at this location. These results provide a developmental timeframe for the expression of recoverin, calbindin, synaptophysin, beta-tubulin and actin. Our findings suggest that the time between PN 3 and PN 7 represents a critical period during which elements of the OPL are assembled.
Assuntos
Proteínas do Olho , Lipoproteínas , Proteínas do Tecido Nervoso , Retina/crescimento & desenvolvimento , Envelhecimento , Animais , Animais Recém-Nascidos , Calbindinas , Proteínas de Ligação ao Cálcio/metabolismo , Embrião de Mamíferos , Feminino , Hipocalcina , Imuno-Histoquímica , Masculino , Camundongos , Faloidina/farmacocinética , Proteína Quinase C/metabolismo , Recoverina , Retina/citologia , Retina/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Sinaptofisina/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
PURPOSE: To measure the concentrations of polyamines, determine their cellular and subcellular localization, and analyze effects of their depletion in developing rabbit retina. METHODS: Isolated retinas at different developmental stages were analyzed for polyamine content by high-performance liquid chromatography (HPLC). An antibody against polyamines was used to localize endogenous stores in both freshly harvested retinas and neonatal retinal explants. To determine the effects of polyamine depletion on immature retina, neonatal explants were cultured in the presence or absence of alpha-difluoromethylornithine (DFMO), an inhibitor of the polyamine synthetic enzyme ornithine decarboxylase (ODC). Similar studies were also performed on dissociated cell cultures. Tissue was assessed using standard histologic stains as well as cell-specific markers (peanut agglutinin for cone photoreceptors and calbindin for horizontal cells). RESULTS: Retinal polyamine content was highest at birth, remained relatively high during the first postnatal week, and then steadily decreased to adult levels. At all ages analyzed, spermine concentration was higher than putrescine or spermidine; however, the differential was greatest in the adult. Polyamine immunoreactivity was localized to distal processes of both rods and cones during development. Strong immunoreactivity was maintained in adult cone inner and outer segments; comparatively weak staining was observed in the adult rods. Heavy staining of ganglion cells was present throughout development but was localized in the cytoplasm in immature cells and in the nucleus in the adult. Amacrine cells stained only in the adult. Polyamine depletion caused a disruption of immature cones, evident in the loss of their somata in the outer nuclear layer, in their processes in the outer plexiform layer in retinal explants, and in their decreased association with horizontal cells in dissociated cell culture. CONCLUSIONS: The relatively high concentrations of polyamines in neonatal retina and their discrete localization in developing photoreceptor outer segments and ganglion cells suggests an important role for these compounds in development. The disruption of cone-specific markers in polyamine-depleted retinas indicates a specific reliance on polyamines for expression of normal cone morphology or morphologic development. These developmental effects may involve polyamine-sensitive ion channels, which are known to exist in retina, or direct interactions with specialized cytoskeletal elements within outer segments.
Assuntos
Poliaminas Biogênicas/fisiologia , Células Fotorreceptoras Retinianas Cones/crescimento & desenvolvimento , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Poliaminas Biogênicas/análise , Calbindinas , Técnicas de Cultura de Células , Cromatografia Líquida de Alta Pressão , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Técnicas Imunoenzimáticas , Inibidores da Ornitina Descarboxilase , Coelhos , Retina/química , Retina/efeitos dos fármacos , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Cones/química , Proteína G de Ligação ao Cálcio S100/metabolismo , Frações SubcelularesRESUMO
The purpose of our studies was to evaluate different strategies for possible neuroprotection in glutamate-induced neurotoxicity in the retina. In a first set of experiments we attempted to determine if dextrorphan antagonism of glutamate action on NMDA receptors would protect against excitotoxic injury associated with secondary damage seen after surgical laser treatment in retina. In a second set of experiments, the effects of different calcium channel blockers in an in-vitro model of N-methyl-D-aspartate (NMDA)-induced retinal ganglion cell excitotoxicity that utilized rabbit retinal explants were evaluated. Dextrorphan infusion prior to laser treatment of rabbit retina produced a significant decrease in the area of neural retinal damage. We attribute the apparent dextrorphan protection to attenuation of glutamate mediated excitotoxicity secondary to laser induced cell death. Preincubation of rabbit retinal explants with verapamil, nimodipine or omega-conotoxin MVIIA did not cause a significant change in NMDA induced cell death in the ganglion cell layer.
Assuntos
Dextrometorfano/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/metabolismo , Retina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Morte Celular/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/efeitos dos fármacos , Fotocoagulação a Laser , N-Metilaspartato/farmacologia , Coelhos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/fisiologia , Retina/patologia , Retina/efeitos da radiação , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Verapamil/farmacologiaRESUMO
PURPOSE: Migration of retinal pigment epithelial (RPE) cells can be triggered by disruption of the RPE monolayer or injury to the neural retina. Migrating cells may re-establish a confluent monolayer, or they may invade the neural retina and disrupt visual function. The purpose of this study was to examine the role of endogenous polyamines in mechanisms of RPE migration. METHODS: Endogenous polyamine levels were determined in an immortalized RPE cell line, D407, using HPLC. Activities of the two rate-limiting enzymes for polyamine synthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (SAMdc), were measured by liberation of ((14)CO(2))(.) Migration was assessed in confluent cultures by determining the number of cells migrating into a mechanically denuded area. All measurements were obtained both in control cultures and in cultures treated with synthesis inhibitors that deplete endogenous polyamines. Subcellular localization of endogenous polyamines was determined using a polyamine antibody. RESULTS: The polyamines, spermidine and spermine, as well as their precursor, putrescine, were normal constituents of RPE cells. The two rate-limiting synthetic enzymes were also present, and their activities were stimulated dramatically by addition of serum to the culture medium. Cell migration was similarly stimulated by serum exposure. When endogenous polyamines were depleted, migration was blocked. When polyamines were replenished through uptake, migration was restored. Polyamine immunoreactivity was limited to membrane patches in quiescent cells. In actively migrating and dividing cells, immunoreactivity was enhanced throughout the cytoplasm. CONCLUSIONS: Polyamines are essential for RPE migration. Pharmacologic manipulation of the polyamine pathway could provide a therapeutic strategy for regulating anomalous migration.