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Microphthalmia-associated transcription factor (MITF) promotes differentiation of human retinal pigment epithelium (RPE) by regulating microRNAs-204/211 expression.

ABSTRACT: 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.

SUBMITTER: Adijanto J

PROVIDER: S-EPMC3370234 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Microphthalmia-associated transcription factor (MITF) promotes differentiation of human retinal pigment epithelium (RPE) by regulating microRNAs-204/211 expression.

Adijanto Jeffrey J Castorino John J JJ Wang Zi-Xuan ZX Maminishkis Arvydas A Grunwald Gerald B GB Philp Nancy J NJ

The Journal of biological chemistry 20120420 24

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-regulate ...[more]

PMID: 22523078

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Project description:BackgroundAge-related changes in the retina are often accompanied by visual impairment but their mechanistic details remain poorly understood.MethodologyProteomic studies were pursued toward a better molecular understanding of retinal pigment epithelium (RPE) aging mechanisms. RPE cells were isolated from young adults (3-4 month-old) and old (24-25 month-old) F344BN rats, and separated into subcellular fractions containing apical microvilli (MV) and RPE cell bodies (CB) lacking their apical microvilli. Proteins were extracted in detergent, separated by SDS-PAGE, digested in situ with trypsin and analyzed by LC MS/MS. Select proteins detected in young and old rat RPE were further studied using immunofluorescence and Western blot analysis.Principal findingsA total of 356 proteins were identified in RPE MV from young and 378 in RPE MV from old rats, 48% of which were common to each age group. A total of 897 proteins were identified in RPE CB from young rats and 675 in old CB, 56% of which were common to each age group. Several of the identified proteins, including proteins involved in response to oxidative stress, displayed both quantitative and qualitative changes in overall abundance during RPE aging. Numerous proteins were identified for the first time in the RPE. One such protein, collectrin, was localized to the apical membrane of apical brush border of proximal tubules where it likely regulates several amino acid transporters. Elsewhere, collectrin is involved in pancreatic β cell proliferation and insulin secretion. In the RPE, collectrin expression was significantly modulated during RPE aging. Another age-regulated, newly described protein was DJ-1, a protein extensively studied in brain where oxidative stress-related functions have been described.Conclusions/significanceThe data presented here reveals specific changes in the RPE during aging, providing the first protein database of RPE aging, which will facilitate future studies of age-related retinal diseases.

Project description:Microphthalmia-associated transcription factor (Mitf) is expressed in neural crest cell-derived melanocytes, and in the retinal pigment epithelium (RPE) during ocular development. Mutations in Mitf are associated with auditory/visual/pigmentary syndromes in humans. Mitf(mi/mi) mouse mutants lack pigmentation, and are microphthalmic, while Mitf(vit/vit) mouse mutants display abnormal RPE pigmentation, and progressive retinal degeneration. Microarray analysis was used to identify novel downstream gene targets/pathways in the RPE that are altered by mutations in the transcription factor Mitf. Using the Affymetrix platform, gene expression profiles were generated using the eyes of E13.5 mouse fetuses that were wildtype, heterozygous, or homozygous for the Mitf(mi) mutation. In a separate experiment, eyes from E13.5 mouse fetuses homozygous for the Mitf(vit) mutation were compared to eyes from the C57BL/6 control background strain. Statistical analyses were performed using robust multiarray average, mixed-effects ANOVA and random-variance t-tests. Altered expression of genes involved in pigment formation, melanosome biogenesis/transport, and redox homeostasis were observed. Twelve genes were commonly mis-regulated in the eyes of both Mitf mutants: 10 of these genes were downregulated in both mutants relative to controls, while 2 of the genes (Nramp1 (Slc11a1) and epoxide hydrolase) were downregulated in Mitf(mi/mi) mutants, and conversely, upregulated in Mitf(vit/vit) mutants. Quantitative RT-PCR and immunohistochemistry were used to confirm altered gene/protein expression. RPE expression of the Fe(+2) iron transporter Nramp1 (Slc11a1) has not previously been reported. Fe(+2) is an important co-factor utilized by the iron-dependent isomerohydrolase RPE65 in the retinoid visual cycle. However, excess accumulation of Fe(+2) in the RPE has recently been associated with oxidative damage and age-related macular degeneration. Abnormal pigmentation and increased activity of Slc11a1 in the RPE of Mitf(vit) mice may contribute to the pathology and progressive retinal degeneration observed in these mutants.

Project description:Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness among the elderly population in the industrialized world. One of the typical features of this pathology is the gradual death of retinal pigment epithelial (RPE) cells, which are essential for maintaining photoreceptor functions and survival. The etiology is multifactorial, and oxidative stress is clearly one of the key factors involved in disease pathogenesis (Plafker, Adv. Exp. Med. Biol. 664 (2010) 447-56; Qin, Drug Dev. Res. 68 (2007) 213-225). Recent work has revealed the presence of phosphorylated signaling proteins in the vitreous humour of patients affected by AMD or other retinal diseases. While the location of these signaling proteins is typically the cell membrane or intracellular compartments, vitreous samples were proven to be cell-free (Davuluri et al., Arch. Ophthalmol. 127 (2009) 613-21). To gain a better understanding of how these proteins can be shed into the vitreous, we used reverse phase protein arrays (RPMA) to analyze the protein and phosphoprotein content of exosomes shed by cultured ARPE-19 cells under oxidative stress conditions. Seventy two proteins were shown to be released by ARPE-19 cells and compartmentalized within exosomes. Forty one of them were selectively detected in their post-translationally modified form (i.e., phosphorylated or cleaved) for the first time in exosomes. Sets of these proteins were linked together reflecting activation of pathway units within exosomes. A subset of (phospho)proteins were altered in exosomes secreted by ARPE-19 cells subjected to oxidative stress, compared to that secreted by control/non stressed cells. Stress-altered exosome proteins were found to be involved in pathways regulating apoptosis/survival (i.e, Bak, Smac/Diablo, PDK1 (S241), Akt (T308), Src (Y416), Elk1 (S383), ERK 1/2 (T202/Y204)) and cell metabolism (i.e., AMPKα1 (S485), acetyl-CoA carboxylase (S79), LDHA). Exosomes may thus represent the conduit through which membrane and intracellular signaling proteins are released into the vitreous. Changes in their (phospho)protein content upon stress conditions suggest their possible role in mediating cell-cell signaling during physio-pathological events; furthermore, exosomes may represent a potential source of biomarkers.

Dataset Information

Microphthalmia-associated transcription factor (MITF) promotes differentiation of human retinal pigment epithelium (RPE) by regulating microRNAs-204/211 expression.

Publications

Microphthalmia-associated transcription factor (MITF) promotes differentiation of human retinal pigment epithelium (RPE) by regulating microRNAs-204/211 expression.

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