RESUMO
Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco do Limbo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Diferenciação Celular , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismoRESUMO
Melanoma is a primary malignant tumor with high lethality, which occurs in the skin and eye tissues, while the molecular mechanisms of melanomagenesis remain largely unknown. Here, we show that death-associated protein-like 1 (DAPL1) expression is lower in melanoma tissues than in paracancerous tissues or nevus tissues, and Uveal melanoma patients with lower DAPL1 expression have a poorer survival rate than those with higher expression of DAPL1. Overexpression of DAPL1 inhibits proliferation of cultured melanoma cells, whereas knockdown of DAPL1 increases cell proliferation. Tumor transplantation experiment results also demonstrate that DAPL1 inhibits tumorigenesis of melanoma cells both in subretinal and subcutaneous tissues of nude mice in vivo. Finally, DAPL1 inhibits proliferation of melanoma cells by increasing the protein level of P21 via decreasing the ubiquitin mediated degradation of P21 and promoting its stability. Conversely, knockdown of P21 neutralizes the effects of inhibition of DAPL1 on melanoma cell proliferation and enhances the severity of melanoma tumorigenesis. These results suggest that DAPL1 is a novel melanoma tumor suppressor gene and thus a potential therapeutic target for melanoma.
RESUMO
Perturbation of solute carriers (SLCs) has been implicated in metabolic disorders and cancer, highlighting the potential for drug discovery and therapeutic opportunities. However, there is relatively little exploration of the clinical relevance and potential molecular mechanisms underlying the role of the SLC12 family in uveal melanoma (UVM). Here, we performed an integrative multiomics analysis of the SLC12 family in multicenter UVM datasets and found that high expression of SLC12A3 and SLC12A9 was associated with unfavorable prognosis. Moreover, SLC12A3 and SLC12A9 were highly expressed in UVM in vivo. We experimentally characterized the roles of these proteins in tumorigenesis in vitro and explored their association with the prognosis of UVM. Lastly, we identified the HCP5-miR-140-5p axis as a potential noncoding RNA pathway upstream of SLC12A3 and SLC12A9, which was associated with immunomodulation and may represent a novel predictor for clinical prognosis and responsiveness to checkpoint blockade immunotherapy. These findings may facilitate a better understanding of the SLCome and guide future rationalized development of SLC-targeted therapy and drug discovery for UVM.
Assuntos
Melanoma , MicroRNAs , Neoplasias Uveais , Humanos , Melanoma/genética , Melanoma/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Prognóstico , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Regulação para Cima , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismoRESUMO
We previously showed that increases in reactive oxygen species (ROS) generation upregulate NLRP3 inflammasome and inflammation through increases in both caspase-1 activity and rises in IL-1ß expression levels in animal models of dry eye (DE). As changes in microRNA (miRNAs) expression levels can modulate inflammasome function, we determine here if there is a relationship in DE between changes in miR-223 expression levels and NLRP3 activation induced in an intelligent controlled environmental system (ICES) in mice. In parallel, ROS, miR-223 and NLRP3 expression levels were assessed in conjunctival impression cytology and tear fluid samples obtained from DE patients and normal subjects. MiR-223 expression levels were modulated by transfection of either a mimic or its negative control (NC) in a human corneal epithelial cell line (HCECs) exposed to a 500 mOsm hyperosmotic medium for 4 h. The dual-luciferase reporter assay confirmed that miR-223 controls NLRP3 gene expression readout through directly interacting with the 3' UTR of its mRNA. Hyperosmolarity-induced NLRP3 activation was confirmed based on recruitment and colocalization of NLRP3 with ASC as well as increases in IL-1ß expression. The miR-223 expression level decreased by 55% in the conjunctiva and cornea of the murine DE model from the level in the control group (P ≤ 0.047), while NLRP3 protein expression rose by 30% (P ≤ 0.017). In DE patients, miR-223 expression decreased in conjunctival impression cytology samples (P = 0.002), whereas IL-1ß tear content rose significantly (P < 0.001).The relevance of this decline was confirmed by showing that exposure to a 500 mOsm stress decreased the miR-223 expression level whereas ROS generation as well as the NLRP3, and IL-1ß expression levels rose in HCECs (P ≤ 0.037). In contrast, miR-223 mimic transfection reduced the NLRP3 protein expression level by 30% (P = 0.037), whereas both ROS generation and IL-1ß secretion rose compared to their corresponding levels in the control group (P ≤ 0.043). Thus, miR-223 negatively regulates NLRP3 inflammasome activity via suppressing NLRP3 translation in DE. This inverse regulation between miR-223 and NLRP3 expression levels suggests that selective upregulation of miR-223 expression may be a novel option to suppress chronic inflammation in DE.
Assuntos
Síndromes do Olho Seco , MicroRNAs , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Caspase 1/genética , Caspase 1/metabolismo , Síndromes do Olho Seco/genética , Síndromes do Olho Seco/metabolismo , Células Epiteliais/metabolismo , Humanos , Inflamassomos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Camundongos , MicroRNAs/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The retinal pigment epithelium (RPE) forms a monolayer at the back of the vertebrate eye and is fundamental to retinal function and homoeostasis. During early development, RPE cells undergo rapid proliferation, but in the adult, they remain normally nonproliferative throughout life. Nevertheless, under pathological conditions such as in proliferative vitreoretinopathy or after retinal ablation, mature RPE cells can re-enter the cell cycle and form nodules or multiple cell layers. Here we show that Dapl1, whose human homolog represents a susceptibility locus for age-related macular degeneration (AMD), is highly up-regulated in quiescent but not proliferating RPE cells and that experimental overexpression of DAPL1 in proliferating RPE cells inhibits their proliferation. Consistent with this observation, the percent of Ki67-positive cells is significantly higher in E11.5 Dapl1 knockout mouse embryos compared to age-matched controls. In adult Dapl1-/- mice, which survive without showing any overt pathology, RPE overgrowth leads to multiple cell layers and/or cellular nodules. The antiproliferative effect of DAPL1 is associated with an increase in CDKN1A protein levels. Reduction of CDKN1A by siRNA in DAPL1-overexpressing RPE cells in vitro partially restores cell proliferation. Hence, we show that DAPL1 is a novel regulator of RPE cell proliferation that is important for the maintenance of the RPE as a monolayer. The findings suggest that DAPL1 dysregulation may be involved in abnormal RPE-related proliferative diseases and corresponding retinal dysfunctions in humans.
Assuntos
Degeneração Macular/genética , Proteínas de Membrana/metabolismo , Epitélio Pigmentado da Retina/citologia , Animais , Linhagem Celular , Proliferação de Células/genética , Suscetibilidade a Doenças , Humanos , Degeneração Macular/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Retina/citologia , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fatores de RiscoRESUMO
There is increasing evidence that the mechanisms protecting the retinal pigment epithelium (RPE) against oxidative stress are important for preventing retinal degenerative diseases. Little, however, is known about these mechanisms. Here we show that MITF, a transcription factor responsible for RPE development and function, regulates redox signaling by acting through PGC1α, a master regulator of mitochondrial biogenesis. Mitf deficiency in mice leads to significantly higher levels of reactive oxygen species (ROS) in both RPE and retina, suggesting that Mitf dysfunction might lead to oxidative damage in the RPE and, by extension, in the retina. Furthermore, overexpression of MITF in the human RPE cell line ARPE-19 indicates that MITF up-regulates antioxidant gene expression and mitochondrial biogenesis by regulating PGC1α and protects cells against oxidative stress. Our findings provide new insights into understanding the redox function of MITF in RPE cells and its potential contribution to prevention of RPE-associated retinal degenerations.
Assuntos
Antioxidantes/fisiologia , Fator de Transcrição Associado à Microftalmia/fisiologia , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/fisiologia , Animais , Western Blotting , Linhagem Celular , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/fisiologia , Ativação Transcricional , Regulação para CimaRESUMO
This Correspondence relates to the article by Dadras et al (A Novel Role for Microphthalmia-Associated Transcription Factor-Regulated Pigment Epithelium-Derived Factor during Melanoma Progression. Am J Pathol 2015, 185:252-265).
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Proteínas do Olho/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Feminino , Humanos , MasculinoRESUMO
The T-box transcription factor TBX2 is known for its role as a critical regulator of melanoma cell proliferation, but its role in regulating melanogenesis has not been widely studied. Here we use a series of experiments to show in primary and immortalized mouse melanocytes that TBX2 acts as regulator of melanogenesis by repressing the expression of the gene encoding the melanosomal protein OCA2. We find that α-MSH or forskolin, both of which stimulate melanogenesis, also reduce TBX2 expression, and that specific knockdown of TBX2 increases melanogenesis. This effect primarily involves an increase in Oca2 expression as the combined knockdown of both Tbx2 and Oca2 interferes with the Tbx2 knockdown-mediated increase in melanogenesis. Standard chromatin immunoprecipitation and reporter assays suggest that TBX2 represses Oca2 at least in part directly. Hence, the results suggest that TBX2 may act as a nexus linking cell proliferation and melanogenesis.
Assuntos
Proteínas de Transporte/genética , Colforsina/farmacologia , Melaninas/biossíntese , Melanócitos/metabolismo , Proteínas de Membrana/genética , Proteínas com Domínio T/fisiologia , Animais , Linhagem Celular Transformada , Técnicas de Silenciamento de Genes , Melanócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas com Domínio T/genética , alfa-MSH/farmacologiaRESUMO
In the process of coal mining, prevention and control of water hazard is essential. It is the precondition for water hazard control to detect and determine the distribution of underground water-conducting channels. In urban environments, traditional methods such as active source seismic exploration and transient electromagnetic exploration commonly used in the field are difficult to carry out effectively due to various factors. In this paper, the microtremor survey method (MSM) and the opposing coils transient electromagnetic method (OCTEM) are adapted to conduct the surface exploration of the coal mine water-conducting channels in the urban environment. Combined with the detection results of the low-velocity area and the low-resistivity area, the distribution of water-conducting channels is preliminarily analyzed and determined, which is basically consistent with the drilling and coring results. It verifies the feasibility and accuracy of the comprehensive exploration method used in this paper.
Assuntos
Minas de Carvão , Carvão Mineral , Carvão Mineral/análise , Campos Eletromagnéticos , China , ÁguaRESUMO
Cells of the retinal pigment epithelium (RPE) play major roles in metabolic functions, maintenance of photoreceptor function, and photoreceptor survival in the retina. They normally form a stable monolayer, but migrate during disease states. Although growth factors produced by the RPE cells primarily control these cellular events, how these factors are regulated in RPE cells remain largely unknown. Here we show that the basic-helix-loop-helix-leucine zipper microphthalmia-associated transcription factor (MITF), which plays central roles in the development and function of a variety of cell types including RPE cells, upregulates the expression of a multifunctional factor PEDF in RPE cells. Consequently, the upregulation of PEDF impairs microtubule assembly and thus inhibits RPE cell migration. Conversely, specific knockdown of PEDF partially rescues the impairment of microtubule assembly and cell migration proceeds in MITF overexpressing stable cells. We conclude that MITF acts through PEDF to inhibit RPE cell migration and to play a significant role in regulating RPE cellular function. We suggest that MITF has a novel and important role in maintaining RPE cells as a stable monolayer and the down-regulation of PEDF that may contribute to retinal degenerative diseases.
Assuntos
Movimento Celular/genética , Proteínas do Olho/fisiologia , Fator de Transcrição Associado à Microftalmia/fisiologia , Fatores de Crescimento Neural/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Serpinas/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Relação Dose-Resposta a Droga , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/genética , Proteínas do Olho/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteínas dos Microtúbulos/metabolismo , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/farmacologia , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , RNA Interferente Pequeno/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Serpinas/genética , Serpinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Tempo , TransfecçãoRESUMO
Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a hallmark of the pathogenesis of proliferative vitreoretinopathy (PVR) that can lead to severe vision loss. Nevertheless, the precise regulatory mechanisms underlying the pathogenesis of PVR remain largely unknown. Here, we show that the expression of death-associated protein-like 1 (DAPL1) is downregulated in PVR membranes and that DAPL1 deficiency promotes EMT in RPE cells in mice. In fact, adeno-associated virus (AAV)-mediated DAPL1 overexpression in RPE cells of Dapl1-deficient mice inhibited EMT in physiological and retinal-detachment states. In a rabbit model of PVR, ARPE-19 cells overexpressing DAPL1 showed reduced ability to induce experimental PVR, and AAV-mediated DAPL1 delivery attenuated the severity of experimental PVR. Furthermore, a mechanistic study revealed that DAPL1 promotes P21 phosphorylation and its stabilization partially through NFκB (RelA) in RPE cells, whereas the knockdown of P21 led to neutralizing effects on DAPL1-dependent EMT inhibition and enhanced the severity of experimental PVR. These results suggest that DAPL1 acts as a novel suppressor of RPE-EMT and has an important role in antagonizing the pathogenesis of experimental PVR. Hence, this finding has implications for understanding the mechanism of and potential therapeutic applications for PVR.
Assuntos
Proteínas de Membrana , Epitélio Pigmentado da Retina , Vitreorretinopatia Proliferativa , Animais , Camundongos , Coelhos , Transição Epitelial-Mesenquimal , Epitélio Pigmentado da Retina/metabolismo , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Proteínas de Membrana/metabolismoRESUMO
Purpose: To determine whether SIRT1 regulates high glucose (HG)-induced inflammation and cataract formation through modulating TXNIP/NLRP3 inflammasome activation in human lens epithelial cells (HLECs) and rat lenses. Methods: HG stress from 25 to 150 mM was imposed on HLECs, with treatments using small interfering RNAs (siRNAs) targeting NLRP3, TXNIP, and SIRT1, as well as a lentiviral vector (LV) for SIRT1. Rat lenses were cultivated with HG media, with or without the addition of NLRP3 inhibitor MCC950 or SIRT1 agonist SRT1720. High mannitol groups were applied as the osmotic controls. Real-time PCR, Western blots, and immunofluorescent staining evaluated the mRNA and protein levels of SIRT1, TXNIP, NLRP3, ASC, and IL-1ß. Reactive oxygen species (ROS) generation, cell viability, and death were also assessed. Results: HG stress induced a decline in SIRT1 expression and caused TXNIP/NLRP3 inflammasome activation in a concentration-dependent manner in HLECs, which was not observed in the high mannitol-treated groups. Knocking down NLRP3 or TXNIP inhibited NLRP3 inflammasome-induced IL-1ß p17 secretion under HG stress. Transfections of si-SIRT1 and LV-SIRT1 exerted inverse effects on NLRP3 inflammasome activation, suggesting that SIRT1 acts as an upstream regulator of TXNIP/NLRP3 activity. HG stress induced lens opacity and cataract formation in cultivated rat lenses, which was prevented by MCC950 or SRT1720 treatment, with concomitant reductions in ROS production and TXNIP/NLRP3/IL-1ß expression levels. Conclusions: The TXNIP/NLRP3 inflammasome pathway promotes HG-induced inflammation and HLEC pyroptosis, which is negatively regulated by SIRT1. This suggests viable strategies for treating diabetic cataract.
Assuntos
Catarata , Inflamassomos , Sirtuína 1 , Animais , Humanos , Ratos , Proteínas de Transporte , Furanos , Glucose/farmacologia , Inflamação , Proteína 3 que Contém Domínio de Pirina da Família NLR , Espécies Reativas de Oxigênio , Sirtuína 1/metabolismoRESUMO
The decreased antioxidant capacity in the retinal pigment epithelium (RPE) is the hallmark of retinal degenerative diseases including age-related macular degeneration (AMD). Nevertheless, the exact regulatory mechanisms underlying the pathogenesis of retinal degenerations remain largely unknown. Here we show in mice that deficiencies in Dapl1, a susceptibility gene for human AMD, impair the antioxidant capacity of the RPE and lead to age-related retinal degeneration in the 18-month-old mice homozygous for a partial deletion of Dapl1. Dapl1-deficiency is associated with a reduction of the RPE's antioxidant capacity, and experimental re-expression of Dapl1 reverses this reduction and protects the retina from oxidative damage. Mechanistically, DAPL1 directly binds the transcription factor E2F4 and inhibits the expression of MYC, leading to upregulation of the transcription factor MITF and its targets NRF2 and PGC1α, both of which regulate the RPE's antioxidant function. When MITF is experimentally overexpressed in the RPE of DAPL1 deficient mice, antioxidation is restored and retinas are protected from degeneration. These findings suggest that the DAPL1-MITF axis functions as a novel regulator of the antioxidant defense system of the RPE and may play a critical role in the pathogenesis of age-related retinal degenerative diseases.
Assuntos
Degeneração Macular , Degeneração Retiniana , Animais , Camundongos , Antioxidantes/metabolismo , Linhagem Celular , Degeneração Macular/genética , Degeneração Macular/patologia , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Propranolol hydrochloride is the first-line drug for the clinical treatment of hypertension, arrhythmia, and other diseases. However, with the increasing use of this drug, its safety and environmental health have received more and more attention. In this study, aquatic vertebrate zebrafish were used as a model to study the toxic effects and mechanisms of propranolol hydrochloride. It was revealed that zebrafish larvae exposed to propranolol hydrochloride showed aberrant head nerve development and locomotor disorders. Additionally, exposure to propranolol hydrochloride could induce oxidative stress, alter the activities of AChE and ATPase, and disrupt the expression of genes involved in neurodevelopment and neurotransmitter pathways. More interestingly, the expression of Parkinson's disease-related genes was altered in zebrafish treated with propranolol hydrochloride. We detected the expression of genes related to the Wnt signaling pathway and found that their expression appeared to be down-regulated. The phenotype of nerve developmental defects and locomotor disorders can be effectively rescued by astaxanthin and Wnt activators. Collectively, the results suggest that propranolol hydrochloride may induce neurotoxicity and abnormal movement behavior with PD-like symptoms in zebrafish larvae.
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Poluentes Químicos da Água , Peixe-Zebra , Animais , Larva , Embrião não Mamífero , Propranolol/toxicidade , Propranolol/metabolismo , Poluentes Químicos da Água/toxicidadeAssuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Neoplasias Cutâneas/metabolismo , Pele/metabolismo , Animais , Antígeno CD146/genética , Antígeno CD146/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Proteínas de Fluorescência Verde/metabolismo , Folículo Piloso/metabolismo , Melanócitos/citologia , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , CicatrizaçãoRESUMO
BACKGROUND: Oxidative stress is a common cause of neurodegeneration and plays a central role in retinal degenerative diseases. Heme oxygenase-1 (HMOX1) is a redox-regulated enzyme that is induced in neurodegenerative diseases and acts against oxidative stress but can also promote cell death, a phenomenon that is still unexplained in molecular terms. Here, we test whether HMOX1 has opposing effects during retinal degeneration and investigate the molecular mechanisms behind its pro-apoptotic role. METHODS: Basal and induced levels of HMOX1 in retinas are examined during light-induced retinal degeneration in mice. Light damage-independent HMOX1 induction at two different expression levels is achieved by intraocular injection of different doses of an adeno-associated virus vector expressing HMOX1. Activation of Müller glial cells, retinal morphology and photoreceptor cell death are examined using hematoxylin-eosin staining, TUNEL assays, immunostaining and retinal function are evaluated with electroretinograms. Downstream gene expression of HMOX1 is analyzed by RNA-seq, qPCR examination and western blotting. The role of one of these genes, the pro-apoptotic DNA damage inducible transcript 3 (Ddit3), is analyzed in a line of knockout mice. RESULTS: Light-induced retinal degeneration leads to photoreceptor degeneration and concomitant HMOX1 induction. HMOX1 expression at low levels before light exposure prevents photoreceptor degeneration but expression at high levels directly induces photoreceptor degeneration even without light stress. Photoreceptor degeneration following high level expression of HMOX1 is associated with a mislocalization of rhodopsin in photoreceptors and an increase in the expression of DDIT3. Genetic deletion of Ddit3 in knockout mice prevents photoreceptor cell degeneration normally resulting from high level HMOX1 expression. CONCLUSION: The results reveal that the expression levels determine whether HMOX1 is protective or deleterious in the retina. Furthermore, in contrast to the protective low dose of HMOX1, the deleterious high dose is associated with induction of DDIT3 and endoplasmic reticulum stress as manifested, for instance, in rhodopsin mislocalization. Hence, future applications of HMOX1 or its regulated targets in gene therapy approaches should carefully consider expression levels in order to avoid potentially devastating effects.
Assuntos
Luz , Retina/metabolismo , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Fator de Transcrição CHOP/metabolismo , Animais , Estresse do Retículo Endoplasmático , Células Ependimogliais/metabolismo , Camundongos Knockout , Estresse Oxidativo/fisiologia , Rodopsina/farmacologiaRESUMO
Purpose: Retinal pigment epithelium (RPE) cell proliferation is precisely regulated to maintain retinal homoeostasis. Microphthalmia-associated transcription factor (MITF), a critical transcription factor in RPE cells, has two alternatively spliced isoforms: (+)MITF and (-)MITF. Previous work has shown that (-)MITF but not (+)MITF inhibits RPE cell proliferation. This study aims to investigate the role of long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) in regulating MITF splicing and hence proliferation of RPE cells. Methods: Mouse RPE, primary cultured mouse RPE cells, and different proliferative human embryonic stem cell (hESC)-RPE cells were used to evaluate the expression of (+)MITF, (-)MITF, and NEAT1 by reverse-transcription PCR (RT-PCR) or quantitative RT-PCR. NEAT1 was knocked down using specific small interfering RNAs (siRNAs). Splicing factor proline- and glutamine-rich (SFPQ) was overexpressed with the use of lentivirus infection. Cell proliferation was analyzed by cell number counting and Ki67 immunostaining. RNA immunoprecipitation (RIP) was used to analyze the co-binding between the SFPQ and MITF or NEAT1. Results: NEAT1 was highly expressed in proliferative RPE cells, which had low expression of (-)MITF. Knockdown of NEAT1 in RPE cells switched the MITF splicing pattern to produce higher levels of (-)MITF and inhibited cell proliferation. Mechanistically, NEAT1 recruited SFPQ to bind directly with MITF mRNA to regulate its alternative splicing. Overexpression of SFPQ in ARPE-19 cells enhanced the binding enrichment of SFPQ to MITF and increased the splicing efficiency of (+)MITF. The binding affinity between SFPQ and MITF was decreased after NEAT1 knockdown. Conclusions: NEAT1 acts as a scaffold to recruit SFPQ to MITF mRNA and promote its binding affinity, which plays an important role in regulating the alternative splicing of MITF and RPE cell proliferation.
Assuntos
Processamento Alternativo/genética , Proliferação de Células/fisiologia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Processamento Associado a PTB/metabolismo , RNA Longo não Codificante/fisiologia , RNA Mensageiro/genética , Epitélio Pigmentado da Retina/metabolismo , Animais , Contagem de Células , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Células-Tronco Embrionárias Humanas , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/citologiaRESUMO
Photoreceptor degeneration is a major cause of blindness and a considerable health burden during aging but effective therapeutic or preventive strategies have not so far become readily available. Here, we show in mouse models that signaling through the tyrosine kinase receptor KIT protects photoreceptor cells against both light-induced and inherited retinal degeneration. Upon light damage, photoreceptor cells upregulate Kit ligand (KITL) and activate KIT signaling, which in turn induces nuclear accumulation of the transcription factor NRF2 and stimulates the expression of the antioxidant gene Hmox1. Conversely, a viable Kit mutation promotes light-induced photoreceptor damage, which is reversed by experimental expression of Hmox1. Furthermore, overexpression of KITL from a viral AAV8 vector prevents photoreceptor cell death and partially restores retinal function after light damage or in genetic models of human retinitis pigmentosa. Hence, application of KITL may provide a novel therapeutic avenue for prevention or treatment of retinal degenerative diseases.
Assuntos
Células Fotorreceptoras de Vertebrados/efeitos da radiação , Degeneração Retiniana/prevenção & controle , Fator de Células-Tronco/fisiologia , Animais , Modelos Animais de Doenças , Heme Oxigenase-1/análise , Luz , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/fisiologia , Proteínas Proto-Oncogênicas c-kit/fisiologia , Degeneração Retiniana/etiologia , Degeneração Retiniana/genética , Transdução de SinaisRESUMO
Oxidative damage is one of the major contributors to retinal degenerative diseases such as age-related macular degeneration (AMD), while RPE mediated antioxidant defense plays an important role in preventing retinopathies. However, the regulatory mechanisms of antioxidant signaling in RPE cells are poorly understood. Here we show that transcription factor MITF regulates the antioxidant response in RPE cells, protecting the neural retina from oxidative damage. In the oxidative stress-induced retinal degeneration mouse model, retinal degeneration in Mitf+/- mice is significantly aggravated compared to WT mice. In contrast, overexpression of Mitf in Dct-Mitf transgenic mice and AAV mediated overexpression in RPE cells protect the neural retina against oxidative damage. Mechanistically, MITF both directly regulates the transcription of NRF2, a master regulator of antioxidant signaling, and promotes its nuclear translocation. Furthermore, specific overexpression of NRF2 in Mitf+/- RPE cells activates antioxidant signaling and partially protects the retina from oxidative damage. Taken together, our findings demonstrate the regulation of NRF2 by MITF in RPE cells and provide new insights into potential therapeutic approaches for prevention of oxidative damage diseases.
Assuntos
Degeneração Macular , Degeneração Retiniana , Animais , Degeneração Macular/genética , Camundongos , Fator de Transcrição Associado à Microftalmia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/metabolismoRESUMO
BACKGROUND: There are many advantages to the application of complete mitochondrial (mt) genomes in the accurate reconstruction of phylogenetic relationships in Metazoa. Although over one thousand metazoan genomes have been sequenced, the taxonomic sampling is highly biased, left with many phyla without a single representative of complete mitochondrial genome. Sipuncula (peanut worms or star worms) is a small taxon of worm-like marine organisms with an uncertain phylogenetic position. In this report, we present the mitochondrial genome sequence of Phascolosoma esculenta, the first complete mitochondrial genome of the phylum. RESULTS: The mitochondrial genome of P.esculenta is 15,494 bp in length. The coding strand consists of 32.1% A, 21.5% C, 13.0% G, and 33.4% T bases (AT = 65.5%; AT skew = -0.019; GC skew = -0.248). It contains thirteen protein-coding genes (PCGs) with 3,709 codons in total, twenty-two transfer RNA genes, two ribosomal RNA genes and a non-coding AT-rich region (AT = 74.2%). All of the 37 identified genes are transcribed from the same DNA strand. Compared with the typical set of metazoan mt genomes, sipunculid lacks trnR but has an additional trnM. Maximum Likelihood and Bayesian analyses of the protein sequences show that Myzostomida, Sipuncula and Annelida (including echiurans and pogonophorans) form a monophyletic group, which supports a closer relationship between Sipuncula and Annelida than with Mollusca, Brachiopoda, and some other lophotrochozoan groups. CONCLUSION: This is the first report of a complete mitochondrial genome as a representative within the phylum Sipuncula. It shares many more similar features with the four known annelid and one echiuran mtDNAs. Firstly, sipunculans and annelids share quite similar gene order in the mitochondrial genome, with all 37 genes located on the same strand; secondly, phylogenetic analyses based on the concatenated protein sequences also strongly support the sipunculan + annelid clade (including echiurans and pogonophorans). Hence annelid "key-characters" including segmentation may be more labile than previously assumed.