RESUMO
The neocortex, the center for higher brain function, first emerged in mammals and has become massively expanded and folded in humans, constituting almost half the volume of the human brain. Primary microcephaly, a developmental disorder in which the brain is smaller than normal at birth, results mainly from there being fewer neurons in the neocortex because of defects in neural progenitor cells (NPCs). Outer radial glia (oRGs), NPCs that are abundant in gyrencephalic species but rare in lissencephalic species, are thought to play key roles in the expansion and folding of the neocortex. However, how oRGs expand, whether they are necessary for neocortical folding, and whether defects in oRGs cause microcephaly remain important questions in the study of brain development, evolution, and disease. Here, we show that oRG expansion in mice, ferrets, and human cerebral organoids requires cyclin-dependent kinase 6 (CDK6), the mutation of which causes primary microcephaly via an unknown mechanism. In a mouse model in which increased Hedgehog signaling expands oRGs and intermediate progenitor cells and induces neocortical folding, CDK6 loss selectively decreased oRGs and abolished neocortical folding. Remarkably, this function of CDK6 in oRG expansion did not require its kinase activity, was not shared by the highly similar CDK4 and CDK2, and was disrupted by the mutation causing microcephaly. Therefore, our results indicate that CDK6 is conserved to promote oRG expansion, that oRGs are necessary for neocortical folding, and that defects in oRG expansion may cause primary microcephaly.
Assuntos
Quinase 6 Dependente de Ciclina , Células Ependimogliais , Microcefalia , Neocórtex , Animais , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Células Ependimogliais/citologia , Células Ependimogliais/enzimologia , Furões , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Microcefalia/genética , Neocórtex/anormalidades , Neocórtex/enzimologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/enzimologia , Organoides/embriologiaRESUMO
Diabetic retinopathy (DR) is a major complication of diabetes and a leading cause of blindness and visual impairment. This study focuses on the function of lysine acetyltransferase 1 (KAT1) in the progression of DR and the epigenetic mechanism. A mouse model with DR was induced by streptozotocin (STZ). Abundantly expressed genes in STZ-induced mice were analyzed. KAT1 was found to be significantly downregulated in the retinal tissues of model mice. Retinal microvascular endothelial cells (RMECs) and retinal Müller cells (rMCs) were cultured in high-glucose medium for in vitro studies. Upregulation of KAT1 suppressed inflammation, neovascularization, and vascular leakage in mouse retinal tissues, and it reduced the activity and inflammatory responses in rMCs, as well as the proliferation and metastatic potential of RMECs. KAT1 activated the transcription activity of YTHDF2 through histone acetylation of the promoter, and YTHDF2 triggered the instability of ITGB1 mRNA to induce mRNA degradation in an m6A manner. The activities of rMCs and RMECs were increased by sh-YTHDF2 but suppressed by sh-ITGB1. The FAK/PI3K/AKT signaling pathway was suppressed upon ITGB1 silencing. Collectively, this study demonstrated that KAT1 triggers YTHDF2-mediated ITGB1 mRNA instability to alleviate the progression of DR.
Assuntos
Retinopatia Diabética/enzimologia , Células Endoteliais/enzimologia , Células Ependimogliais/enzimologia , Histona Acetiltransferases/metabolismo , Integrina beta1/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenina/análogos & derivados , Adenina/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/patologia , Células Ependimogliais/patologia , Quinase 1 de Adesão Focal/metabolismo , Histona Acetiltransferases/genética , Integrina beta1/genética , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais , EstreptozocinaRESUMO
NADPH oxidase (NOX) is a main producers of reactive oxygen species (ROS) that may contribute to the early pathogenesis of diabetic retinopathy (DR). ROS has harmful effects on endogenous neuro-survival factors brain-derived neurotrophic factor (BDNF) and sirtuin 1 (SIRT1) are necessary for the growth and survival of the retina. The role of NOX isoforms NOX4 in triggering ROS in DR is not clear. Here we determine the protective effects of a plant-derived NOX inhibitor apocynin (APO) on NOX4-induced ROS production which may contribute to the depletion of survival factors BDNF/SIRT1 or cell death in the diabetic retinas. Human retinal Müller glial cells (MGCs) were treated with hypoxia mimetic agent cobalt chloride (CoCl2) in the absence or presence of APO. Molecular analysis demonstrates that NOX4 is upregulated in CoCl2-treated MGCs and in the diabetic retinas. Increased NOX4 was accompanied by the downregulation of BDNF/SIRT1 expression or in the activation of apoptotic marker caspase-3. Whereas, APO treatment downregulates NOX4 and subsequently upregulates BDNF/SIRT1 or alleviate caspase-3 expression. Accordingly, in the diabetic retina we found a positive correlation in NOX4 vs ROS (p = 0.025; R2 = 0.488) and caspase-3 vs ROS (p = 0.04; R2 = 0.428); whereas a negative correlation in BDNF vs ROS (p = 0.009; R2 = 0.596) and SIRT1 vs ROS (p = 0.0003; R2 = 0.817) respectively. Taken together, NOX4-derived ROS could be a main contributor in downregulating BDNF/SIRT1 expression or in the activation of caspase-3. Whereas, APO treatment may minimize the deleterious effects occurring due to hyperglycemia and/or diabetic mimic hypoxic condition in early pathogenesis of DR.
Assuntos
Acetofenonas/farmacologia , Diabetes Mellitus Experimental/enzimologia , Retinopatia Diabética/enzimologia , Células Ependimogliais/enzimologia , NADPH Oxidase 4/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Retina/enzimologia , Animais , Linhagem Celular , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/patologia , Células Ependimogliais/patologia , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Retina/patologiaRESUMO
Diabetic retinopathy (DR) is a retinal disease representing one of the main causes of vision loss in developed countries. In the early stage of DR, disruption of blood retinal barrier (BRB) is observed, and it will lead to vascular permeability and visual impairment. Therefore, protection against the breakdown of BRB may be useful strategy for prevention of DR. Matrix metalloproteinases (MMPs) plays an important role in the degradation of extracellular matrix proteins. In DR, they attribute to increased vascular permeability by degrading the junction proteins, such as occuldin and cadherin that are important to maintain the BRB junction complex. Müller cells constitute the main glial cells of the retina and are involved in many retinal functions. They are reported to be one of the MMP-producing cells in the retina. In this symposium review, I present the molecular mechanism of MMP expression in retinal Müller cells. In addition, I would like to introduce polymethoxylated flavones, nobiletin and the derivatives isolated from natural resource as novel MMP inhibitors, which may be applicable to prevention of DR.
Assuntos
Retinopatia Diabética/etiologia , Retinopatia Diabética/prevenção & controle , Células Ependimogliais/enzimologia , Flavonas/farmacologia , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/metabolismo , Fitoterapia , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Flavonas/isolamento & purificação , Flavonas/uso terapêutico , Humanos , Camundongos , Relação Estrutura-AtividadeRESUMO
Oxidative stress and inflammation are important pathogenic factors of diabetic retinopathy (DR). DR remains the most common ocular complication caused by diabetes mellitus (DM) and is the leading cause of visual impairment in working-aged people worldwide. Melatonin has attracted extensive attention due to its potent antioxidant and anti-inflammatory effects. In the present study, melatonin inhibited oxidative stress and inflammation by enhancing the expression and activity of silent information regulator factor 2-related enzyme 1 (Sirt1) both in in vitro and in vivo models of DR, and the Sirt1 inhibitor EX-527 counteracted melatonin-mediated antioxidant and anti-inflammatory effects on Müller cells. Moreover, melatonin enhanced Sirt1 activity through the maternally expressed gene 3 (MEG3)/miR-204 axis, leading to the deacetylation of the Sirt1 target genes forkhead box o1 (Foxo1) and nuclear factor kappa B (NF-κB) subunit p65, eventually contribute to the alleviation of oxidative stress and inflammation. The study revealed that melatonin promotes the Sirt1 pathway, thereby protecting the retina from DM-induced damage.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Retinopatia Diabética/tratamento farmacológico , Células Ependimogliais/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Melatonina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 1/metabolismo , Acetilação , Animais , Animais Recém-Nascidos , Retinopatia Diabética/enzimologia , Retinopatia Diabética/patologia , Ativação Enzimática , Células Ependimogliais/enzimologia , Células Ependimogliais/patologia , Proteína Forkhead Box O1/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismoRESUMO
Leptin, a hormone mainly produced by adipocytes in proportion to fat mass, is a key component in the regulation of energy homeostasis and reproductive, neuroendocrine, immune, and metabolic functions. Leptin binds to the leptin receptor, which is expressed throughout the central nervous system but particularly in neurons of several nuclei of the hypothalamus, such as the arcuate nucleus (ARC) and paraventricular nucleus (PVN). It has been found that nitric oxide (NO) plays an important role in mediating effects of leptin. Since PVN and ARC neurons are known to express leptin receptors, we investigated the effects of leptin on nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in the PVN and ARC of male Wistar rats. Our results have shown that systemic administration of leptin resulted in increased NADPH-d positive cell number in the PVN and ARC, suggesting that both the PVN and ARC may be important centers in the hypothalamus for the leptin action, mediated by increased NO production. In addition, we have also observed that hypothalamic tanycytes in the ventral portion of the third ventricle were NADPH-d positive. We speculate that leptin may affect the release of neurohormones and hypothalamic neurogenesis by activating nitric oxide synthase in hypothalamic tanycytes.
Assuntos
Células Ependimogliais/enzimologia , Leptina/farmacologia , NADPH Desidrogenase/metabolismo , Núcleo Hipotalâmico Paraventricular/enzimologia , Animais , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/enzimologia , Células Ependimogliais/citologia , Masculino , Neurônios/citologia , Neurônios/enzimologia , Óxido Nítrico/metabolismo , Núcleo Hipotalâmico Paraventricular/citologia , Ratos , Ratos Wistar , Receptores para Leptina/metabolismoRESUMO
Gelatinases are a class of matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) to regulate intercellular signaling and cell migration. Gelatinase activity is tightly regulated via proteolytic activation and through the expression of tissue inhibitors of matrix metalloproteinases (TIMPs). Gelatinase activity has been implicated in retinal pathophysiology in different animal models and human disease. However, the role of gelatinases in retinal regeneration remains uncertain. In this study we investigated the dynamic changes in gelatinase activity in response to excitotoxic damage and how this enzymatic activity influenced the formation of Müller glia progenitor cells (MGPCs) in the avian retina. This study used hydrogels containing a gelatinase-degradable fluorescent peptide to measure gelatinase activity in vitro and dye quenched gelatin to localize enzymatic activity in situ. These data were corroborated by using single cell RNA sequencing (scRNA-seq). Gelatinase mRNA, specifically MMP2, was detected in oligodendrocytes and Non-Astrocytic Inner Retinal Glia (NIRG). Total retinal gelatinase activity was reduced following NMDA-treatment, and sustained inhibition of MMP2 prior to damage or growth factor treatment increased the formation of proliferating MGPCs and c-fos signaling. We observed that microglia, Müller glia (MG), and NIRG cells were involved in regulating changes in gelatinase activity through TIMP2 and TIMP3. Collectively, these findings implicate MMP2 in reprogramming of Muller glia into MGPCs.
Assuntos
Reprogramação Celular/fisiologia , Células Ependimogliais/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Regeneração Nervosa/fisiologia , Retina/enzimologia , Animais , Proliferação de Células/fisiologia , Galinhas , Gelatinases/metabolismo , Células-Tronco/enzimologiaRESUMO
In response to retinal damage, the Müller glial cells (MGs) of the zebrafish retina have the ability to undergo a cellular reprogramming event in which they enter the cell cycle and divide asymmetrically, thereby producing multipotent retinal progenitors capable of regenerating lost retinal neurons. However, mammalian MGs do not exhibit such a proliferative and regenerative ability. Here, we identify Hippo pathway-mediated repression of the transcription cofactor YAP as a core regulatory mechanism that normally blocks mammalian MG proliferation and cellular reprogramming. MG-specific deletion of Hippo pathway components Lats1 and Lats2, as well as transgenic expression of a Hippo non-responsive form of YAP (YAP5SA), resulted in dramatic Cyclin D1 upregulation, loss of adult MG identity, and attainment of a highly proliferative, progenitor-like cellular state. Our results reveal that mammalian MGs may have latent regenerative capacity that can be stimulated by repressing Hippo signaling.
Assuntos
Reprogramação Celular , Células Ependimogliais/citologia , Células Ependimogliais/enzimologia , Mamíferos/metabolismo , Neuroglia/citologia , Neuroglia/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Retina/citologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Ciclina D1/metabolismo , Ciclina D3/metabolismo , Via de Sinalização Hippo , Camundongos , Células-Tronco/metabolismo , Proteínas de Sinalização YAPRESUMO
De novo serine synthesis plays important roles in normal mitochondrial function and cellular anti-oxidative capacity. It is reported to be mainly activated in glial cells of the central nervous system, but its role in retinal Müller glia remains unclear. In this study, we inhibited de novo serine synthesis using CBR-5884, a specific inhibitor of phosphoglycerate dehydrogenase (PHGDH, a rate limiting enzyme in de novo serine metabolism) in MIO-M1 cells (immortalized human Müller cells) and huPMCs (human primary Müller cells) under mild oxidative stress. Alamar blue and LDH (lactate dehydrogenase) assays showed significantly reduced metabolic activities and increased cellular damage of Müller cells, when exposed to CBR-5884 accompanied by mild oxidative stress; however, CBR-5884 alone had little effect. The increased cellular damage was partially reversed by supplementation with exogenous serine/glycine. HSP72 (an oxidative stress marker) and reactive oxygen species (ROS) levels were significantly increased; glutathione and NADPH/NADP+ levels were pronouncedly reduced under PHGDH inhibition accompanied by oxidative stress. JC-1 staining and Seahorse respiration experiments showed that inhibition of de novo serine synthesis in Müller cells can also increase mitochondrial stress and decrease mitochondrial ATP production. qPCR and Western blot demonstrated an increased expression of HSP60 (a key mitochondrial stress-related gene), and this was further validated in human retinal explants. Our study suggests that de novo serine synthesis is important for Müller cell survival, particularly when they are exposed to mild oxidative stress, possibly by maintaining mitochondrial function and generating glutathione and NADPH to counteract ROS.
Assuntos
Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Mitocôndrias/patologia , Estresse Oxidativo , Serina/biossíntese , Trifosfato de Adenosina/metabolismo , Idoso , Chaperonina 60/metabolismo , Células Ependimogliais/enzimologia , Glutationa/metabolismo , Proteínas de Choque Térmico HSP72/metabolismo , Humanos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , NADP/metabolismo , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Fosfoglicerato Desidrogenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Polyunsaturated fatty acids (PUFA) and their cytochrome P450 (CYP450) metabolites have been linked to angiogenesis and vessel homeostasis. However, the role of individual CYP isoforms and their endogenous metabolites in those processes are not clear. Here, we focused on the role of Cyp2c44 in postnatal retinal angiogenesis and report that Cyp2c44 is highly expressed in Müller glial cells in the retina. The constitutive as well as inducible postnatal genetic deletion of Cyp2c44 resulted in an increased vessel network density without affecting vessel radial expansion during the first postnatal week. This phenotype was associated with an increased endothelial cell proliferation and attenuated Notch signaling. LC-MS/MS analyses revealed that levels of hydroxydocosahexaenoic acids (HDHA), i.e., 10-, 17- and 20-HDHA were significantly elevated in retinas from 5day old Cyp2c44-/- mice compared to their wild-type littermates. Enzymatic activity assays revealed that HDHAs were potential substrates for Cyp2c44 which could account for the increased levels of HDHAs in retinas from Cyp2c44-/- mice. These data indicate that Cyp2c44 is expressed in the murine retina and, like the soluble epoxide hydrolase, is expressed in Müller glia cells. The enhanced endothelial cell proliferation and Notch inhibition seen in retinas from Cyp2c44-deficient mice indicate a role for Cyp2c44-derived lipid mediators in physiological angiogenesis.
Assuntos
Família 2 do Citocromo P450/metabolismo , Células Ependimogliais/enzimologia , Neovascularização Fisiológica , Retina/fisiologia , Animais , Proliferação de Células , Família 2 do Citocromo P450/deficiência , Família 2 do Citocromo P450/genética , Ácidos Docosa-Hexaenoicos/metabolismo , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Retina/citologiaRESUMO
The visual system produces visual chromophore, 11-cis-retinal from dietary vitamin A, all-trans-retinol making this vitamin essential for retinal health and function. These metabolic events are mediated by a sequential biochemical process called the visual cycle. Retinol dehydrogenases (RDHs) are responsible for two reactions in the visual cycle performed in retinal pigmented epithelial (RPE) cells, photoreceptor cells and Müller cells in the retina. RDHs in the RPE function as 11-cis-RDHs, which oxidize 11-cis-retinol to 11-cis-retinal in vivo. RDHs in rod photoreceptor cells in the retina work as all-trans-RDHs, which reduce all-trans-retinal to all-trans-retinol. Dysfunction of RDHs can cause inherited retinal diseases in humans. To facilitate further understanding of human diseases, mouse models of RDHs-related diseases have been carefully examined and have revealed the physiological contribution of specific RDHs to visual cycle function and overall retinal health. Herein we describe the function of RDHs in the RPE and the retina, particularly in rod photoreceptor cells, their regulatory properties for retinoid homeostasis and future therapeutic strategy for treatment of retinal diseases.
Assuntos
Oxirredutases do Álcool/metabolismo , Células Ependimogliais/enzimologia , Doenças Retinianas/enzimologia , Epitélio Pigmentado da Retina/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Retinaldeído/metabolismo , Visão Ocular , Vitamina A/metabolismo , Oxirredutases do Álcool/genética , Animais , Predisposição Genética para Doença , Humanos , Mutação , Oxirredução , Fenótipo , Doenças Retinianas/genética , Doenças Retinianas/fisiopatologia , Doenças Retinianas/terapia , Epitélio Pigmentado da Retina/fisiopatologiaRESUMO
PURPOSE: To investigate the effect of thrombin on the proliferation of human Müller glial cells (MCs) and define the possible signaling mechanisms involved in this process. METHODS: Protease-activated receptor (PARs 1-4) expression was analyzed using RT-PCR and Western blot in the MIO-M1 Müller cell line (MC). Müller cell proliferation was assessed by the MTS reduction method. Wound healing and immunoreactivity to Ki67 antigen were used to dissociate proliferation and migration. Cell migration was examined using transwell migration assays. The involvement of extracellular signal-regulated kinase (ERK1/2) phosphorylation/activation in thrombin-induced human MC proliferation was determined by Western blot. Intracellular pathways involved in ERK1/2 activation were analyzed by pharmacologic inhibition. RESULTS: We first demonstrated that human MCs express PARs 1 to 4. Our results show that thrombin dose-dependently stimulates MC proliferation by 44%, with a calculated Ec50 of 0.86 nM. Müller cell maximal proliferation required sustained thrombin treatment for 72 hours, in contrast to our previous findings in RPE cells showing maximal thrombin-induced proliferation at 24-hour stimulation. We demonstrate that thrombin induces MC cell proliferation through the Ras-independent activation of the Raf/MEK/ERK cascade, under the control of protein kinase C (PKC)-ζ. CONCLUSIONS: The breakdown of blood-retina barrier (BRB) exposes MCs to thrombin contained in serum. Our findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.
Assuntos
Células Ependimogliais/enzimologia , Hemostáticos/farmacologia , Proteína Quinase C/fisiologia , Trombina/farmacologia , Análise de Variância , Linhagem Celular , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Ependimogliais/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores Ativados por Proteinase/metabolismo , Vitreorretinopatia Proliferativa/enzimologia , Vitreorretinopatia Proliferativa/etiologia , Cicatrização/fisiologiaRESUMO
Resistance to insulin action is a key cause of diabetic complications, yet much remains unknown about the molecular mechanisms that contribute to the defect. Glucose-induced insulin resistance in peripheral tissues such as the retina is mediated in part by the hexosamine biosynthetic pathway (HBP). Glucosamine (GAM), a leading dietary supplement marketed to relieve the discomfort of osteoarthritis, is metabolized by the HBP, and in doing so bypasses the rate-limiting enzyme of the pathway. Thus, exogenous GAM consumption potentially exacerbates the resistance to insulin action observed with diabetes-induced hyperglycemia. In the present study, we evaluated the effect of GAM on insulin action in retinal Müller cells in culture. Addition of GAM to Müller cell culture repressed insulin-induced activation of the Akt/mTORC1 signaling pathway. However, the effect was not recapitulated by chemical inhibition to promote protein O-GlcNAcylation, nor was blockade of O-GlcNAcylation sufficient to prevent the effects of GAM. Instead, GAM induced ER stress and subsequent expression of the protein Regulated in DNA Damage and Development (REDD1), which was necessary for GAM to repress insulin-stimulated phosphorylation of Akt on Thr308. Overall, the findings support a model whereby GAM promotes ER stress in retinal Müller cells, resulting in elevated REDD1 expression and thus resistance to insulin action.
Assuntos
Células Ependimogliais/metabolismo , Glucosamina/farmacologia , Antagonistas da Insulina/farmacologia , Retina/metabolismo , Fatores de Transcrição/metabolismo , Acetilglucosamina/metabolismo , Animais , Células Cultivadas , Estresse do Retículo Endoplasmático , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/enzimologia , Insulina/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Retina/citologia , Retina/enzimologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Treonina/metabolismo , Fatores de Transcrição/biossínteseRESUMO
OBJECTIVE: To explore the potential regulatory mechanism of MMP9 in the development of DR. METHODS: Plasmids pcDNA-MMP9 and pcDNA-Ang2 were transfected into primary rat retinal Müller cells (RMCs) using Lipofectamine 2000. Cell viability and apoptosis were respectively determined by MTT assay and flow cytometry. Moreover, the interaction between MMP9 and Ang2 was explored. Besides, RMCs were treated with MMP-9 under normal glucose and high glucose condition for 2d. Besides, the expression levels of apoptotic proteins, like MMP9, Ang2, Bax2, Bcl2, cleaved PARP and cleaved caspase3 were determined by Western blot. RESULTS: The cell viability of siRNA-MMP9 group was significantly increased while decreased in MMP9 overexpression group when compared to control group, respectively. The apoptotic cells in MMP9 overexpression group significantly increased while decreased in siRNA-MMP9 group when compared with control group. MMP9 expression was significantly regulated by Ang2 whereas no significant changes occurred in Ang2 expression when MMP9 expression changed. Moreover, MMP9 expression in HG group significantly increased while there were no significant differences between NG group and control group. Besides, the expression of Bax2, Bcl2, cleaved PARP and cleaved caspase3 in HG group increased while there were no significant differences between NG group and control group. CONCLUSION: Our findings indicate that MMP9 may play an important role via inducing cell apoptosis in the development of DR via regulating by Ang2 or targeting apoptotic proteins, such as Bax2, Bcl2, cleaved PARP and cleaved caspase3.
Assuntos
Retinopatia Diabética/enzimologia , Células Ependimogliais/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Angiopoietina-2/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Sobrevivência Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Citometria de Fluxo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
PURPOSE: In ischemic proliferative retinopathies, Müller glial cells (MGCs) acquire migratory abilities. However, the mechanisms that regulate this migration remain poorly understood. In addition, proliferative disorders associated with enhanced activities of matrix metalloproteinases (MMPs) also involve insulin-like growth factor (IGF)-1 participation. Therefore, the main interest of this work was to investigate the IGF-1 effect on the extracellular proteolytic activity in MGCs. METHODS: Cell culture supernatants and cell lysates of the human MGC line MIO-M1 stimulated with IGF-1 were analyzed for MMP-2 by zymographic and Western blot analysis. The MGCs' motility was evaluated by scratch wound assay. The MMP-2, ß1-integrin, and focal adhesions were detected by confocal microscopy. The localization of active MMPs and actin cytoskeleton were evaluated by in situ zymography. RESULTS: The IGF-1 induced the activation of canonical signaling pathways through the IGF-1R phosphorylation. Culture supernatants showed a relative decrease in the active form of MMP-2, correlating with an increased accumulation of MMP-2 protein in the MGCs' lysate. The IGF-1 effect on MMP-2 was abolished by an IGF-1R blocking antibody, αIR3, as well as by the PI3-kinase inhibitor, LY294002. The IGF-1 increased the migratory capacity of MGCs, which was blocked by the GM6001 MMP inhibitor, LY294002 and αIR3. Finally, IGF-1 induced the intracellular distribution of MMP-2 toward cellular protrusions and the partial colocalization with ß1-integrin and phospo-focal adhesion kinase signals. Gelatinase activity was concentrated along F-actin filaments. CONCLUSIONS: Taken together, these data indicate that IGF-1, through its receptor activation, regulates MGCs' motility by a mechanism that involves the MMP-2 and PI3K signaling pathway.
Assuntos
Células Ependimogliais/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Metaloproteinase 2 da Matriz/metabolismo , Western Blotting , Linhagem Celular , Movimento Celular/fisiologia , Ativação Enzimática/fisiologia , Células Ependimogliais/enzimologia , Células Ependimogliais/metabolismo , Imunofluorescência , Humanos , Integrina beta1/fisiologia , Metaloproteinase 2 da Matriz/fisiologia , Microscopia Confocal , Transdução de Sinais/fisiologiaRESUMO
Fasting down-regulates the hypothalamus-pituitary-thyroid (HPT) axis activity through a reduction of TRH synthesis in neurons of the parvocellular paraventricular nucleus of the hypothalamus (PVN). These TRH neurons project to the median eminence (ME), where TRH terminals are close to the cytoplasmic extensions of ß2 tanycytes. Tanycytes express pyroglutamyl peptidase II (PPII), the TRH-degrading ectoenzyme that controls the amount of TRH that reaches the anterior pituitary. We tested the hypothesis that regulation of ME PPII activity is another mechanism by which fasting affects the activity of the HPT axis. Semiquantitative in situ hybridization histochemistry data indicated that PPII and deiodinase 2 mRNA levels increased in tanycytes after 48 hours of fasting. This increase was transitory, followed by an increase of PPII activity in the ME, and a partial reversion of the reduction in PVN pro-TRH mRNA levels and the number of TRH neurons detected by immunohistochemistry. In fed animals, adrenalectomy and corticosterone treatment did not change ME PPII activity 72 hours later. Methimazole-induced hypothyroidism produced a profound drop in tanycytes PPII mRNA levels, which was reverted by 3 days of treatment with T4. The activity of thyroliberinase, the serum isoform of PPII, was increased at most fasting time points studied. We conclude that delayed increases in both the ME PPII as well as the thyroliberinase activities in fasted male rats may facilitate the maintenance of the deep down-regulation of the HPT axis function, despite a partial reactivation of TRH expression in the PVN.
Assuntos
Aminopeptidases/genética , Células Ependimogliais/enzimologia , Jejum/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Hipotálamo/metabolismo , Eminência Mediana/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , RNA Mensageiro/metabolismo , Hormônio Liberador de Tireotropina/metabolismo , Adrenalectomia , Aminopeptidases/efeitos dos fármacos , Aminopeptidases/metabolismo , Animais , Antitireóideos/farmacologia , Corticosterona/farmacologia , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/metabolismo , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hipotireoidismo , Iodeto Peroxidase/genética , Masculino , Metimazol/farmacologia , Núcleo Hipotalâmico Paraventricular/citologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Precursores de Proteínas/genética , Ácido Pirrolidonocarboxílico/metabolismo , RNA Mensageiro/efeitos dos fármacos , Ratos , Hormônio Liberador de Tireotropina/efeitos dos fármacos , Hormônio Liberador de Tireotropina/genética , Tiroxina/farmacologia , Iodotironina Desiodinase Tipo IIRESUMO
Identification of the signaling pathways that influence the reprogramming of Müller glia into neurogenic retinal progenitors is key to harnessing the potential of these cells to regenerate the retina. Glucocorticoid receptor (GCR) signaling is commonly associated with anti-inflammatory responses and GCR agonists are widely used to treat inflammatory diseases of the eye, even though the cellular targets and mechanisms of action in the retina are not well understood. We find that signaling through GCR has a significant impact upon the ability of Müller glia to become proliferating Müller glia-derived progenitor cells (MGPCs). The primary amino acid sequence and pattern of GCR expression in the retina is highly conserved across vertebrate species, including chickens, mice, guinea pigs, dogs and humans. In all of these species we find GCR expressed by the Müller glia. In the chick retina, we find that GCR is expressed by progenitors in the circumferential marginal zone (CMZ) and is upregulated by Müller glia in acutely damaged retinas. Activation of GCR signaling inhibits the formation of MGPCs and antagonizes FGF2/MAPK signaling in the Müller glia. By contrast, we find that inhibition of GCR signaling stimulates the formation of proliferating MGPCs in damaged retinas, and enhances the neuronal differentiation while diminishing glial differentiation. Given the conserved expression pattern of GCR in different vertebrate retinas, we propose that the functions and mechanisms of GCR signaling are highly conserved and are mediated through the Müller glia. We conclude that GCR signaling directly inhibits the formation of MGPCs, at least in part, by interfering with FGF2/MAPK signaling.
Assuntos
Células Ependimogliais/metabolismo , Receptores de Glucocorticoides/metabolismo , Retina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Dexametasona/farmacologia , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/enzimologia , Células Ependimogliais/patologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , N-Metilaspartato/farmacologia , Retina/efeitos dos fármacos , Retina/patologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Müller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Müller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Müller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.
Assuntos
Acetiltransferases/metabolismo , Células Ependimogliais/enzimologia , Enzimas Multifuncionais/metabolismo , Retinol O-Graxo-Aciltransferase/metabolismo , Animais , Catálise , Bovinos , Galinhas , Opsinas dos Cones/metabolismo , Ésteres/química , Ácidos Graxos/química , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Cinética , Camundongos , Opsinas/metabolismo , Retina/metabolismoRESUMO
The radial glial cell (RGC) is a glial cell type in the central nervous system of all vertebrates. Adult teleost fish have abundant RGCs in the brain in contrast to mammals. Adult fish RGCs have many important functions, including forming a structural scaffold to guide neuronal migration and serving as the progenitor cells in the brain to generate neurons. The role of the RGC in adult neurogenesis explains the high regenerative capacity of adult fish brain. There is increasing evidence from several species that some glial cells produce or metabolize steroids. It is now well-known that teleost RGCs express aromatase and produce estrogens from androgen precursors, which may be important for local neuroendocrine functions and regulation of neurogenesis. The question of whether RGCs are capable of de novo steroid synthesis from cholesterol remains unanswered. However, the expression of steroidogenic acute regulatory protein, and the key enzyme cytochrome P450 17alpha-hydroxylase in primary cultures of goldfish RGCs indicate the potential to produce 17α-hydroxy-pregnenolone and thus other steroid intermediates. The possibility of synthesizing additional non-estrogenic steroids may indicate new functions for the RGC.
Assuntos
Células Ependimogliais/enzimologia , Peixes/metabolismo , Neurogênese/fisiologia , Esteroides/biossíntese , Animais , Aromatase/metabolismo , Neurônios/citologia , Neurônios/fisiologiaRESUMO
In many blinding diseases of the retina, loss of function and thus severe visual impairment results from apoptotic cell death of damaged photoreceptors. In an attempt to survive, injured photoreceptors generate survival signals to induce intercellular protective mechanisms that eventually may rescue photoreceptors from entering an apoptotic death pathway. One such endogenous survival pathway is controlled by leukemia inhibitory factor (LIF), which is produced by a subset of Muller glia cells in response to photoreceptor injury. In the absence of LIF, survival components are not activated and photoreceptor degeneration is accelerated. Although LIF is a crucial factor for photoreceptor survival, the detailed mechanism of its induction in the retina has not been elucidated. Here, we show that administration of tumor necrosis factor-alpha (TNF) was sufficient to fully upregulate Lif expression in Muller cells in vitro and the retina in vivo. Increased Lif expression depended on p38 mitogen-activated protein kinase (MAPK) since inhibition of its activity abolished Lif expression in vitro and in vivo. Inhibition of p38 MAPK activity reduced the Lif expression also in the model of light-induced retinal degeneration and resulted in increased cell death in the light-exposed retina. Thus, expression of Lif in the injured retina and activation of the endogenous survival pathway involve signaling through p38 MAPK.