RESUMO
A major goal in human genetics is to use natural variation to understand the phenotypic consequences of altering each protein-coding gene in the genome. Here we used exome sequencing1 to explore protein-altering variants and their consequences in 454,787 participants in the UK Biobank study2. We identified 12 million coding variants, including around 1 million loss-of-function and around 1.8 million deleterious missense variants. When these were tested for association with 3,994 health-related traits, we found 564 genes with trait associations at P ≤ 2.18 × 10-11. Rare variant associations were enriched in loci from genome-wide association studies (GWAS), but most (91%) were independent of common variant signals. We discovered several risk-increasing associations with traits related to liver disease, eye disease and cancer, among others, as well as risk-lowering associations for hypertension (SLC9A3R2), diabetes (MAP3K15, FAM234A) and asthma (SLC27A3). Six genes were associated with brain imaging phenotypes, including two involved in neural development (GBE1, PLD1). Of the signals available and powered for replication in an independent cohort, 81% were confirmed; furthermore, association signals were generally consistent across individuals of European, Asian and African ancestry. We illustrate the ability of exome sequencing to identify gene-trait associations, elucidate gene function and pinpoint effector genes that underlie GWAS signals at scale.
Assuntos
Bancos de Espécimes Biológicos , Bases de Dados Genéticas , Sequenciamento do Exoma , Exoma/genética , África/etnologia , Ásia/etnologia , Asma/genética , Diabetes Mellitus/genética , Europa (Continente)/etnologia , Oftalmopatias/genética , Feminino , Predisposição Genética para Doença/genética , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Hipertensão/genética , Hepatopatias/genética , Masculino , Mutação , Neoplasias/genética , Característica Quantitativa Herdável , Reino UnidoRESUMO
Interleukin 24 (IL-24) is a tumor-suppressing protein, which inhibits angiogenesis and induces cancer cell-specific apoptosis. We have shown that IL-24 regulates apoptosis through phosphorylated eukaryotic initiation factor 2 alpha (eIF2α) during endoplasmic reticulum (ER) stress in cancer. Although multiple stresses converge on eIF2α phosphorylation, the cellular outcome is not always the same. In particular, ER stress-induced apoptosis is primarily regulated through the extent of eIF2α phosphorylation and activating transcription factor 4 (ATF4) action. Our studies show for the first time that cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation is required for IL-24-induced cell death in a variety of breast cancer cell lines and this event increases ATF4 activity. We demonstrate an undocumented role for PKA in regulating IL-24-induced cell death, whereby PKA stimulates phosphorylation of p38 mitogen-activated protein kinase and upregulates extrinsic apoptotic factors of the Fas/FasL signaling pathway and death receptor 4 expression. We also demonstrate that phosphorylation and nuclear import of tumor suppressor TP53 occurs downstream of IL-24-mediated PKA activation. These discoveries provide the first mechanistic insights into the function of PKA as a key regulator of the extrinsic pathway, ER stress, and TP53 activation triggered by IL-24.
Assuntos
Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Interleucinas/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Interleukin-24 (IL-24), a member of the IL-10 cytokine family, is an immunomodulatory cytokine that also displays broad cancer-specific suppressor effects. The tumor suppressor activities of IL-24 include inhibition of angiogenesis, sensitization to chemotherapy, and cancer-specific apoptosis. We show that Sigma 1 Receptor (S1R), a ligand-regulated protein chaperone contributes to IL-24 induction of apoptosis. IL-24 generated from an adenovirus expressing IL-24 (Ad.IL-24) induces cancer-specific apoptosis by inducing an endoplasmic reticulum (ER) stress, reactive oxygen species production, and calcium mobilization. The present studies reveals that S1R is required for Ad.IL-24-induced cell death. We provide several lines of evidence to confirm a physical and functional interaction between IL-24 and S1R including: (a) S1R and IL-24 co-localize, as judged by immunocytochemical analysis studies; (b) S1R and IL-24 co-immunoprecipitate using either S1R or IL-24 antibody; (c) S1R agonist (+)-SKF10047 inhibits apoptosis by Ad.IL-24; (d) (+)-SKF10047-mediated inhibition of Ad.IL-24 results in: diminished ER stress protein expression; (e) Calcium mobilization; and (f) ROS production. Collectively, these data demonstrate that S1R interacts with IL-24 and suggest that IL-24:S1R interaction determines apoptosis induction by Ad.IL-24. These studies define Sigma 1 Receptor as a key initial mediator of IL-24 induction of cancer-specific killing. These findings have important implications for our understanding of IL-24 as a tumor suppressor protein as well as an immune modulating cytokine.
Assuntos
Apoptose , Interleucinas/imunologia , Neoplasias/imunologia , Receptores sigma/imunologia , Sinalização do Cálcio , Caspase 3/imunologia , Linhagem Celular Tumoral/imunologia , Estresse do Retículo Endoplasmático , Humanos , Neoplasias/patologia , Espécies Reativas de Oxigênio/imunologia , Receptores sigma/agonistas , Receptor Sigma-1RESUMO
Introduction: Diabetic Retinopathy (DR) is a potentially blinding retinal disorder that develops through the pathogenesis of diabetes. The lack of disease predictors implies a poor prognosis with frequent irreversible retinal damage and vision loss. Extracellular Vesicles (EVs) present a novel opportunity for pre-symptomatic disease diagnosis and prognosis, both severely limited in DR. All biological fluids contain EVs, which are currently being studied as disease biomarkers. EV proteins derived from urine have emerged as potential noninvasive biomarkers. Methods: In this study, we isolated EVs from DR retinal tissue explants and from DR patients' urine, and characterized the vesicles, finding differences in particle number and size. Next, we performed proteomic analysis on human explanted DR retinal tissue conditioned media, DR retinal EVs and DR urinary EVs and compared to normal human retinal tissue, retinal EVs, and urinary EVs, respectively. Results: Our system biology analysis of DR tissue and EV expression profiles revealed biological pathways related to cell-to-cell junctions, vesicle biology, and degranulation processes. Junction Plakoglobin (JUP), detected in DR tissue-derived EVs and DR urinary EVs, but not in controls, was revealed to be a central node in many identified pathogenic pathways. Proteomic results were validated by western blot. Urinary EVs obtained from healthy donors and diabetic patient without DR did not contain JUP. Conclusion: The absence of JUP in healthy urinary EVs provide the basis for development of a novel Diabetic Retinopathy biomarker, potentially facilitating diagnosis.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Vesículas Extracelulares , Doenças Retinianas , Humanos , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/metabolismo , Proteômica , Retina/metabolismo , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Diabetes Mellitus/metabolismoRESUMO
Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.
Assuntos
Polaridade Celular/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Degeneração Macular/complicações , Degeneração Macular/metabolismo , Proteínas/metabolismo , Drusas Retinianas/complicações , Drusas Retinianas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Nicotina/farmacologia , Organoides/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Secretoma/metabolismoRESUMO
The mechanisms underlying retinal development have not been completely elucidated. Extracellular vesicles (EVs) are novel essential mediators of cell-to-cell communication with emerging roles in developmental processes. Nevertheless, the identification of EVs in human retinal tissue, characterization of their cargo, and analysis of their potential role in retina development has not been accomplished. Three-dimensional retinal tissue derived from human induced pluripotent stem cells (hiPSC) provide an ideal developmental system to achieve this goal. Here we report that hiPSC-derived retinal organoids release exosomes and microvesicles with small noncoding RNA cargo. EV miRNA cargo-predicted targetome correlates with Gene Ontology (GO) pathways involved in mechanisms of retinogenesis relevant to specific developmental stages corresponding to hallmarks of native human retina development. Furthermore, uptake of EVs by human retinal progenitor cells leads to changes in gene expression correlated with EV miRNA cargo predicted gene targets, and mechanisms involved in retinal development, ganglion cell and photoreceptor differentiation and function.
Assuntos
Comunicação Celular , Vesículas Extracelulares/metabolismo , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/metabolismo , Retina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Retina/citologiaRESUMO
Purpose: Extracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study. Methods: Adult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Immunogold transmission electron microscopy (TEM) was used to image EV ultrastructure and marker protein localization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released by adult retina, and internalized by isolated retinal cells. Mass spectrometry was used to characterize the proteomes of adult retina and EVs. Results: Adult neural retina released EVs at a rate of 1.42 +/- 0.08 × 108/mL over 5 days, with diameters ranging from 30 to 910 nm. The canonical EV markers CD63 and Tsg101 localized to retinal EVs. Adult retinal and neuronal mRNA species present in both retina and EVs included rhodopsin and the neuronal nuclei marker NeuN. Fluorescently labeled RNA in retinal cells was enclosed in EVs, transported to, and uptaken by co-cultured adult retinal cells. Proteomic analysis revealed 1696 protein species detected only in retinal cells, 957 species shared between retina and EVs, and 82 detected only in EVs. Conclusions: The adult neural retina constitutively releases EVs with molecular cargo capable of intercellular transport and predicted involvement in biological processes including retinal physiology, mRNA processing, and transcription regulation within the retinal microenvironment.
Assuntos
Vesículas Extracelulares/metabolismo , Proteínas do Olho/metabolismo , Neurônios/metabolismo , Transporte Proteico/fisiologia , Transporte de RNA/fisiologia , Retina/metabolismo , Animais , Camundongos , Microscopia Eletrônica de Transmissão , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: Vitamin D receptor (VDR) activities have been noted for a number of B cell malignancies which showed varying sensitivities to vitamin D3 (1,25-dihydroxyvitamin D3, VD3, calcitriol) and its synthetic analogs. The objective of this study was to address the potential effects of VD3 and vitamin D3 analogs (VDAs) on the growth of Hodgkin's lymphoma (HL), a malignant pathology of B cell origin, in vitro. RESULTS: Immunofluorescence staining showed the expression of VDR by primary Hodgkin's (H) and Reed-Sternberg (RS)-HRS-tumor cells in HL histological sections. Western blot analyses revealed expression of VDR in the HL cell lines Hs445, HDLM2, KMH2, and L428. One-way analysis of variance (ANOVA) on data obtained from water-soluble tetrazolium 1 (WST-1) cell proliferation assay showed decreased cell growth in HDLM2 and L428, 72 h after treatment with 10 µM of either VD3 of VDAs. Western blot analyses showed that treatment of L428 cells with the VDAs (calcipotriol and EB1089) resulted in modest increases in nuclear accumulation of VDR (nuVDR) compared to either dimethyl sulfoxide (DMSO) or VD3 treatments. nuVDR for DMSO control and VD3 was comparable. These results suggest that VD3 or VDAs may affect growth of HL.
Assuntos
Calcitriol/análogos & derivados , Colecalciferol/farmacologia , Regulação Neoplásica da Expressão Gênica , Receptores de Calcitriol/genética , Vitamina D/análogos & derivados , Calcitriol/metabolismo , Calcitriol/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colecalciferol/metabolismo , Dimetil Sulfóxido/farmacologia , Doença de Hodgkin/metabolismo , Doença de Hodgkin/patologia , Humanos , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/metabolismo , Vitamina D/metabolismo , Vitamina D/farmacologiaRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
A range of cell types, including embryonic stem cells, neurons and astrocytes have been shown to release extracellular vesicles (EVs) containing molecular cargo. Across cell types, EVs facilitate transfer of mRNA, microRNA and proteins between cells. Here we describe the release kinetics and content of EVs from mouse retinal progenitor cells (mRPCs). Interestingly, mRPC derived EVs contain mRNA, miRNA and proteins associated with multipotency and retinal development. Transcripts enclosed in mRPC EVs, include the transcription factors Pax6, Hes1, and Sox2, a mitotic chromosome stabilizer Ki67, and the neural intermediate filaments Nestin and GFAP. Proteomic analysis of EV content revealed retinogenic growth factors and morphogen proteins. mRPC EVs were shown to transfer GFP mRNA between cell populations. Finally, analysis of EV mediated functional cargo delivery, using the Cre-loxP recombination system, revealed transfer and uptake of Cre+ EVs, which were then internalized by target mRPCs activating responder loxP GFP expression. In summary, the data supports a paradigm of EV genetic material encapsulation and transfer within RPC populations. RPC EV transfer may influence recipient RPC transcriptional and post-transcriptional regulation, representing a novel mechanism of differentiation and fate determination during retinal development.
Assuntos
Vesículas Extracelulares/metabolismo , Retina/metabolismo , Células-Tronco/metabolismo , Animais , Astrócitos/metabolismo , Diferenciação Celular , Células Cultivadas , Vesículas Extracelulares/fisiologia , Regulação da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , MicroRNAs/genética , Neurônios/metabolismo , Proteômica , RNA Mensageiro/metabolismo , Retina/fisiologia , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: The cardiac glycoside digitoxin preferentially inhibits the growth of breast cancer cells and targets the Erk pathway. Digitoxin alters the expression of genes that mediate calcium metabolism and IAP genes. PURPOSE: Since the optimal treatment for cancer involves the use of agents in combination, we assessed the growth inhibitory effects of digitoxin combined with agents that alter calcium metabolism, thapsigargin, a sarcoplasmic/ER Ca(2+)-ATPase inhibitor, and the statin simvastatin, as well as digitoxin's effect on the IAP pathway of apoptosis. METHODS: To reveal signaling pathways, we treated human cancer cells with digitoxin, alone or combined with thapsigargin or simvastatin, and measured cell growth using the MTT and colony formation assays. We used histology and Western blot analysis of HEK293 cells to assay effects on IAPs. RESULTS: Digitoxin inhibited the growth of breast, colon and ovarian cancer cells. Consistent with an effect on calcium metabolism, digitoxin exhibited synergy with thapsigargin and simvastatin on ER-negative breast cancer cells. Digitoxin activates expression of Erk pathway genes and suppresses expression of IAP genes. The growth inhibitory effects on HEK293 cells are not blocked by the pancaspase inhibitor zVAD-FMK, indicating that digitoxin may act by a caspase independent pathway of apoptosis. Furthermore, digitoxin does not have an effect on XIAP protein, a major anti-apoptotic protein. CONCLUSION: Digitoxin appears to act through the Erk and stress response pathways and is worthwhile to study to prevent and treat cancer. Our findings warn of possible safety issues for cardiac patients who take a combination of digitoxin and statins.
Assuntos
Apoptose/efeitos dos fármacos , Digitoxina/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Sinvastatina/farmacologia , Tapsigargina/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Sinergismo Farmacológico , Células HEK293 , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
The deterioration of retinal tissue in advanced stages of retinitis pigmentosa and age-related macular degeneration and the lack of signaling cues for laminar regeneration are significant challenges highlighting the need for a tissue engineering approach to retinal repair. In this study, we fabricated a biodegradable thin-film polycaprolactone (PCL) scaffold with varying surface topographies using microfabrication techniques. Mouse retinal progenitor cells (mRPCs) cultured on PCL scaffolds exhibited enhanced potential to differentiate toward a photoreceptor fate in comparison to mRPCs cultured on control substrates, suggesting that PCL scaffolds are promising as substrates to guide differentiation of mRPCs toward a photoreceptor fate in vitro before transplantation. When cocultured with the retinal explants of rhodopsin null mice, mRPC/PCL constructs showed increased mRPC integration rates compared to directly applied dissociated mRPCs. Moreover, these mRPC/PCL constructs could be delivered into the subretinal space of rhodopsin null mice with minimal disturbance of the host retina. Whether cocultured with retinal explants or transplanted into the subretinal space, newly integrated mRPCs localized to the outer nuclear layer and expressed appropriate markers of photoreceptor fate. Thus, the PCL scaffold provides a platform to guide differentiation and organized delivery of mRPCs as a practical strategy to repair damaged retina.
Assuntos
Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Poliésteres/farmacologia , Retina/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Alicerces Teciduais/química , Animais , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Células-Tronco/efeitos dos fármacosRESUMO
BACKGROUND: The triterpene glycoside actein from the herb black cohosh preferentially inhibits the growth of breast cancer cells and activates the ER stress response. The ER IP3 receptor and Na,K-ATPase form a signaling microdomain. Since actein is lipophilic, its action may be limited by bioavailability. PURPOSE: To develop actein to prevent and treat cancer, we examined the primary targets and combinations with chemotherapy agents, as well as the ability of nanoparticles to enhance the activity. MATERIALS AND METHODS: To reveal signaling pathways, we treated human breast and colon cancer, as well as 293T and 293T (NF-κB), cells with actein, and measured effects using the MTT, luciferase promoter, Western blot and histology assays. To assess effects on calcium release, we preloaded cells with the calcium sensitive dye Fura-2. To enhance bioavailability, we conjugated actein to nanoparticle liposomes. RESULTS: Actein strongly inhibited the growth of human breast cancer cells and induced a dose dependent release of calcium into the cytoplasm. The ER IP3 receptor antagonist heparin blocked this release, indicating that the receptor is required for activity. Heparin partially blocked the growth inhibitory effect, while the MEK inhibitor U0126 enhanced it. Consistent with this, actein synergized with the ER mobilizer thapsigargin. Further, actein preferentially inhibited the growth of 293T (NF-κB) cells. Nanoparticle liposomes increased the growth inhibitory activity of actein. CONCLUSIONS: Actein alters the activity of the ER IP3 receptor and Na,K-ATPase, induces calcium release and modulates the NF-κB and MEK pathways and may be worthwhile to explore to prevent and treat breast cancer.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Cálcio/metabolismo , Cimicifuga/química , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Extratos Vegetais/farmacologia , Saponinas/farmacologia , Triterpenos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Feminino , Heparina/farmacologia , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Lipossomos , NF-kappa B/metabolismo , Fitoterapia , Extratos Vegetais/uso terapêutico , Saponinas/uso terapêutico , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/metabolismo , Tapsigargina/farmacologia , Triterpenos/uso terapêuticoRESUMO
Previous studies indicate that extracts and purified components from Garcinia species inhibit the growth of human colon cancer cells. Garcinia benzophenones activate the expression of genes in the endoplasmic reticulum and cellular energy stress (mTOR) pathways. This study examines the growth inhibitory and synergistic effects of Garcinia benzophenones, alone or combined with chemopreventive agents, on human colon cancer cells. To find optimal combination treatments, HT29 colon cancer cells were treated with benzophenones alone, or combined with chemopreventive agents, and cell growth measured using the MTT assay. To reveal effects on signaling pathways, we assessed effects of the MEK inhibitor U0126 and the ER IP3 receptor antagonist heparin, as well as effects on the phosphorylation of 4E-BP-1 (mTOR pathway), using Western blot analysis. New and known benzophenones from Garcinia intermedia inhibited the growth of human colon cancer cells; an alcohol extract of Garcinia xanthochymus, as well as purified guttiferones (guttiferone E and xanthochymol), preferentially inhibited the growth of colon cancer versus nonmalignant intestinal epithelial cells. Guttiferone E exhibited synergy with the NSAID sulindac sulfide and xanthochymol, with the spice turmeric. Guttiferone A did not alter phosphorylation of 4E-BP-1, indicating that the mTORC1 pathway is not involved in its action. The effects of xanthochymol were enhanced by U0126, at low doses, and were blocked by heparin, indicating that the MEK pathway is involved, while the ER IP3 receptor is critical for its action. These studies indicate the potential of benzophenones, alone or combined with sulindac sulfide or turmeric, to prevent and treat colon cancer.
Assuntos
Antineoplásicos Fitogênicos/química , Benzofenonas/química , Garcinia/química , Regulação Neoplásica da Expressão Gênica , Extratos Vegetais/química , Sulindaco/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Benzofenonas/isolamento & purificação , Benzofenonas/farmacologia , Butadienos/farmacologia , Celecoxib , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Curcuma , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Heparina/farmacologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Nitrilas/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Extratos Vegetais/farmacologia , Pirazóis/farmacologia , Transdução de Sinais , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Sulindaco/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Electrical activity is abundant in early retinal development, and electrical stimulation has been shown to modulate embryonic stem cell differentiation towards a neuronal fate. The goal of this study was to simulate in vitro retinal developmental electrical activity to drive changes in mouse retinal progenitor cell (mRPC) gene expression and morphology. We designed a biomimetic electrical stimulation protocol based on spontaneous waves present during retinal development, and applied it to retinal progenitor cells (RPCs) over 3 days of culture. Analysis of protein localization and calcium dynamics, indicate that mRPCs undergo functional neuronal maturation. Our findings suggest that this type of electrical stimulation may be utilized for application in neural tissue engineering and open possibilities for understanding mechanisms guiding active electric membrane development and functional organization during early retinogenesis.