RESUMEN
The retinal pigment epithelium (RPE) plays a fundamental role in maintaining visual function and dedifferentiation of RPE contributes to the pathophysiology of several ocular diseases. To identify microRNAs (miRNAs) that may be involved in RPE differentiation, we compared the miRNA expression profiles of differentiated primary human fetal RPE (hfRPE) cells to dedifferentiated hfRPE cells. We found that miR-204/211, the two most highly expressed miRNAs in the RPE, were significantly down-regulated in dedifferentiated hfRPE cells. Importantly, transfection of pre-miR-204/211 into hfRPE cells promoted differentiation whereas adding miR-204/211 inhibitors led to their dedifferentiation. Microphthalmia-associated transcription factor (MITF) is a key regulator of RPE differentiation that was also down-regulated in dedifferentiated hfRPE cells. MITF knockdown decreased miR-204/211 expression and caused hfRPE dedifferentiation. Significantly, co-transfection of MITF siRNA with pre-miR-204/211 rescued RPE phenotype. Collectively, our data show that miR-204/211 promote RPE differentiation, suggesting that miR-204/211-based therapeutics may be effective treatments for diseases that involve RPE dedifferentiation such as proliferative vitreoretinopathy.
Asunto(s)
Diferenciación Celular/fisiología , Regulación hacia Abajo/fisiología , MicroARNs/biosíntesis , Factor de Transcripción Asociado a Microftalmía/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , MicroARNs/genética , Factor de Transcripción Asociado a Microftalmía/genética , Epitelio Pigmentado de la Retina/citología , Vitreorretinopatía Proliferativa/genética , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/terapiaRESUMEN
PURPOSE: Phenotypic transformation of retinal pigment epithelial (RPE) cells contributes to the onset and progression of ocular proliferative disorders such as proliferative vitreoretinopathy (PVR). The formation of epiretinal membranes in PVR may involve an epithelial-mesenchymal transformation (EMT) of RPE cells as part of an aberrant wound healing response. While the underlying mechanism remains unclear, this likely involves changes in RPE cell gene expression under the control of specific transcription factors (TFs). Thus, the purpose of the present study was to identify TFs that may play a role in this process. METHODS: Regulatory regions of genes that are differentially regulated during phenotypic transformation of ARPE-19 cells, a human RPE cell line, were subjected to computational analysis using the promoter analysis and interaction network toolset (PAINT). The PAINT analysis was used to identify transcription response elements (TREs) statistically overrepresented in the promoter and first intron regions of two reciprocally regulated RPE gene clusters, across four species including the human genome. These TREs were then used to construct transcriptional regulatory network models of the two RPE gene clusters. The validity of these models was then tested using RT-PCR to detect differential expression of the corresponding TF mRNAs during RPE differentiation in both undifferentiated and differentiated ARPE-19 and primary chicken RPE cell cultures. RESULTS: The computational analysis resulted in the successful identification of specific transcription response elements (TREs) and their cognate TFs that are candidates for serving as nodes in a transcriptional regulatory network regulating EMT in RPE cells. The models predicted TFs whose differential expression during RPE EMT was successfully verified by reverse transcriptase polymerase chain reaction (RT-PCR) analysis, including Oct-1, hepatocyte nuclear factor 1 (HNF-1), similar to mothers against decapentaplegic 3 (SMAD3), transcription factor E (TFE), core binding factor, erythroid transcription factor-1 (GATA-1), interferon regulatory factor-1 (IRF), natural killer homeobox 3A (NKX3A), Sterol regulatory element binding protein-1 (SREBP-1), and lymphocyte enhancer factor-1 (LEF-1). CONCLUSIONS: These studies successfully applied computational modeling and biochemical verification to identify biologically relevant transcription factors that are likely to regulate RPE cell phenotype and pathological changes in RPE in response to diseases or trauma. These TFs may provide potential therapeutic targets for the prevention and treatment of ocular proliferative disorders such as PVR.
Asunto(s)
Epitelio/metabolismo , Redes Reguladoras de Genes/genética , Mesodermo/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Animales , Diferenciación Celular , Línea Celular Transformada , Pollos , Secuencia Conservada , Células Epiteliales/citología , Células Epiteliales/metabolismo , Evolución Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Modelos Genéticos , Familia de Multigenes , Filogenia , Epitelio Pigmentado Ocular/citología , Reproducibilidad de los Resultados , Elementos de Respuesta/genética , Especificidad de la Especie , Factores de Transcripción , Transcripción GenéticaRESUMEN
The historical roots of cell adhesion research stretch back over a hundred years, commencing with fundamental questions from the advent of experimental embryology in the late nineteenth century. The transition of embryology from a descriptive to an experimentally driven and mechanistic branch of the biological sciences included investigations focused on the interactions of the first cells of the newly developing embryo, the blastomeres, and followed the movement, interactions and fate of these cells as the tissues and organs of the growing embryo took form. Of special interest to early investigators were cell-cell contacts, which were obviously dynamic but of an obscure nature. This historical review, the first of a series, explores the early years of the cell adhesion field, including the work of Roux, Wilson, Galtsoff, Just and Holtfreter, and discusses the classical experiments, observations and conceptual developments that formed the cornerstone of cell adhesion research during its premolecular era.
Asunto(s)
Blastómeros/citología , Biología Celular/historia , Modelos Biológicos , Animales , Adhesión Celular , Diferenciación Celular , Embrión de Mamíferos/citología , Historia del Siglo XXRESUMEN
PURPOSE: MCT3 is a proton-coupled monocarboxylate transporter preferentially expressed in the basolateral membrane of the retinal pigment epithelium (RPE) and has been shown to play an important role in regulating pH and lactate concentrations in the outer retina. Decreased expression of MCT3 in response to trauma or disease could contribute to pathologic changes in the retina. The present study followed the expression of MCT3 after wounding and re-epithelialization of chick RPE explant and human fetal (hf) RPE cultures. METHODS: Immunofluorescence microscopy and immunoblotting were performed to determine changes in MCT expression after scratch wounding and re-epithelialization of chick RPE/choroid explant cultures and hfRPE cell monolayers. RESULTS: MCT3 expression and basolateral polarity were maintained in chick RPE/choroid explant cultures and hfRPE monolayers. Wounding resulted in loss of MCT3 and the upregulation of MCT4 expression in migrating cells at the edge of the wound. On re-epithelialization, MCT3 was detected in chick and hfRPE cells when cells became hexagonally packed and pigmented. However, in hfRPE cells, MCT4 was consistently expressed throughout the epithelial monolayer. RPE cells at the edges of chick explants and hfRPE cultures with a free edge expressed MCT4 but not MCT3. CONCLUSIONS: Wounding of RPE monolayers resulted in dedifferentiation of the cells at the edge of the wound, as evidenced by a loss of MCT3 and increased MCT4 expression. Collectively, these findings suggest that both cell-cell and cell-substrate interactions are essential in directing and maintaining differentiation of the RPE and expression of MCT3.
Asunto(s)
Transportadores de Ácidos Monocarboxílicos/metabolismo , Epitelio Pigmentado de la Retina/embriología , Cicatrización de Heridas/fisiología , Animales , Células Cultivadas , Embrión de Pollo , Coroides/embriología , Coroides/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Fluorescente , Regeneración/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Simportadores , Regulación hacia ArribaRESUMEN
Cell adhesion molecules are cell surface proteins that play critical roles in cell recognition and cell adhesion. These adhesion molecules, which include the cadherins, integrins, occludins, and a variety of immunoglobulin-like molecules, are essential for a wide variety of physiologic processes such as epithelial barrier function, tissue development, learning and memory, and immune responses. In light of the evidence that toxic metals can affect many of these processes, investigators have begun to examine the possibility that cell adhesion molecules may be targets for metal toxicity. This review summarizes the results of recent studies showing that certain cell adhesion molecules, particularly the cadherins family of Ca(2+)-dependent cell adhesion molecules and the immunoglobulin family of Ca(2+)-independent cell adhesion molecules, may be important early targets on which toxic metals such as a Cd, Hg, and Pb act to produce their toxic effects. These metals, and in some cases their organic compounds, can target cell adhesion molecules at multiple levels, including protein-protein interactions, post-translational modification, and transcriptional regulation. Moreover, by interfering with the normal function of the cadherin family of cell adhesion molecules, some of these metals may activate the beta-catenin nuclear signaling pathway. These studies have provided important new insights into the molecular mechanisms of metal toxicity and have opened several exciting avenues of research.