Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance.
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ABSTRACT:
SUBMITTER: Naso F
PROVIDER: S-EPMC7156966 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Vertebrate vision relies on the daily phagocytosis and lysosomal degradation of photoreceptor outer segments (POS) within the retinal pigment epithelium (RPE). However, how these events are controlled by light is largely unknown. Here we show that the light-responsive miR-211 controls lysosomal biogenesis at the beginning of light-dark transitions in the RPE by targeting Ezrin, a cytoskeleton-associated protein essential for the regulation of calcium homeostasis. miR-211-mediated downregulation of Ezrin leads to Ca2+ influx resulting in the activation of calcineurin, which in turn activates TFEB, a master regulator of lysosomal biogenesis. Light-mediated induction of lysosomal biogenesis and function is impaired in the RPE from miR-211-/- mice that show severely compromised vision. Pharmacological restoration of lysosomal biogenesis through Ezrin inhibition rescued the miR-211-/- phenotype, pointing to a new therapeutic target to counteract retinal degeneration associated with lysosomal dysfunction.
| S-SCDT-EMBOJ-2019-102468 | biostudies-other
Project description:The discovery of a gene network regulating lysosomal biogenesis and its transcriptional regulator transcription factor EB (TFEB) revealed that cells monitor lysosomal function and respond to degradation requirements and environmental cues. We report the identification of transcription factor E3 (TFE3) as another regulator of lysosomal homeostasis that induced expression of genes encoding proteins involved in autophagy and lysosomal biogenesis in ARPE-19 cells in response to starvation and lysosomal stress. We found that in nutrient-replete cells, TFE3 was recruited to lysosomes through interaction with active Rag guanosine triphosphatases (GTPases) and exhibited mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1)-dependent phosphorylation. Phosphorylated TFE3 was retained in the cytosol through its interaction with the cytosolic chaperone 14-3-3. After starvation, TFE3 rapidly translocated to the nucleus and bound to the CLEAR elements present in the promoter region of many lysosomal genes, thereby inducing lysosomal biogenesis. Depletion of endogenous TFE3 entirely abolished the response of ARPE-19 cells to starvation, suggesting that TFE3 plays a critical role in nutrient sensing and regulation of energy metabolism. Furthermore, overexpression of TFE3 triggered lysosomal exocytosis and resulted in efficient cellular clearance in a cellular model of a lysosomal storage disorder, Pompe disease, thus identifying TFE3 as a potential therapeutic target for the treatment of lysosomal disorders.
| S-EPMC4696865 | biostudies-literature
Project description:BackgroundIn recent years, microRNA-211 (miR211) has been considered as a tumor suppressor in multiple malignancies. However, the function of miR211 in human osteosarcoma has not been explored intensively so far. In this study, the relationship between miR211 and EZRIN was analyzed in human osteosarcoma.MethodsThe expression levels of miR211 and EZRIN were measured in both human osteosarcoma cells and tissues. The direct regulatory relationship between miR211 and EZRIN was evaluated using dual-luciferase assay. The effect of miR211 and EZRIN overexpression on cell proliferation, migration/invasion, and apoptosis was detected.ResultsThe expression of miR211 was obviously lower in osteosarcoma tissues than paracancerous tissues. EZRIN was identified as the direct target of miR211, and up-regulation of miR211 increased the percentage of cell apoptosis, and suppressed cell proliferation as well as cell migration/invasion via directly regulating EZRIN.ConclusionsOur study indicated that miR211 has an important role in the development and progress of osteosarcoma, and it might become a novel target in the diagnosis and treatment of human osteosarcoma.
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Project description:MicroRNA (miRNA) expression in fetal human retinal pigment epithelium (hfRPE), retina, and choroid were pairwise compared to determine those miRNAs that are enriched by 10-fold or more in each tissue compared with both of its neighbors. miRs-184, 187, 200a/200b, 204/211, and 221/222 are enriched in hfRPE by 10- to 754-fold compared with neuroretina or choroid (P<0.05). Five of these miRNAs are enriched in RPE compared with 20 tissues throughout the body and are 10- to 20,000-fold more highly expressed (P<0.005). miR-204 and 211 are the most highly expressed in the RPE. In addition, expression of miR-204/211 is significantly lower in the NCI60 tumor cell line panel compared with that in 13 normal tissues, suggesting the progressive disruption of epithelial barriers and increased proliferation. We demonstrated that TGF-beta receptor 2 (TGF-betaR2) and SNAIL2 are direct targets of miR-204 and that a reduction in miR-204 expression leads to reduced expression of claudins 10, 16, and 19 (message/protein) consistent with our observation that anti-miR-204/211 decreased transepithelial resistance by 80% and reduced cell membrane voltage and conductance. The anti-miR-204-induced decrease in Kir7.1 protein levels suggests a signaling pathway that connects TGF-betaR2 and maintenance of potassium homeostasis. Overall, these data indicate a critical role for miR-204/211 in maintaining epithelial barrier function and cell physiology.
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Project description:The plasma membrane contributes to the formation of autophagosomes, the double-membrane vesicles that sequester cytosolic cargo and deliver it to lysosomes for degradation during autophagy. In this study, we have identified a regulatory role for connexins (Cx), the main components of plasma membrane gap junctions, in autophagosome formation. We have found that plasma-membrane-localized Cx proteins constitutively downregulate autophagy through a direct interaction with several autophagy-related proteins involved in the initial steps of autophagosome formation, such as Atg16 and components of the PI(3)K autophagy initiation complex (Vps34, Beclin-1 and Vps15). On nutrient starvation, this inhibitory effect is released by the arrival of Atg14 to the Cx-Atg complex. This promotes the internalization of Cx-Atg along with Atg9, which is also recruited to the plasma membrane in response to starvation. Maturation of the Cx-containing pre-autophagosomes into autophagosomes leads to degradation of these endogenous inhibitors, allowing for sustained activation of autophagy.
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Project description:Autophagy is a cellular catabolic process that relies on the cooperation of autophagosomes and lysosomes. During starvation, the cell expands both compartments to enhance degradation processes. We found that starvation activates a transcriptional program that controls major steps of the autophagic pathway, including autophagosome formation, autophagosome-lysosome fusion, and substrate degradation. The transcription factor EB (TFEB), a master gene for lysosomal biogenesis, coordinated this program by driving expression of autophagy and lysosomal genes. Nuclear localization and activity of TFEB were regulated by serine phosphorylation mediated by the extracellular signal-regulated kinase 2, whose activity was tuned by the levels of extracellular nutrients. Thus, a mitogen-activated protein kinase-dependent mechanism regulates autophagy by controlling the biogenesis and partnership of two distinct cellular organelles.
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Project description:CREG1 is a small glycoprotein which has been proposed as a transcription repressor, a secretory ligand, a lysosomal, or a mitochondrial protein. This is largely because of lack of antibodies for immunolocalization validated through gain- and loss-of-function studies. In the present study, we demonstrate, using antibodies validated for immunofluorescence microscopy, that CREG1 is mainly localized to the endosomal-lysosomal compartment. Gain- and loss-of-function analyses reveal an important role for CREG1 in both macropinocytosis and clathrin-dependent endocytosis. CREG1 also promotes acidification of the endosomal-lysosomal compartment and increases lysosomal biogenesis. Functionally, overexpression of CREG1 enhances macroautophagy/autophagy and lysosome-mediated degradation, whereas knockdown or knockout of CREG1 has opposite effects. The function of CREG1 in lysosomal biogenesis is likely attributable to enhanced endocytic trafficking. Our results demonstrate that CREG1 is an endosomal-lysosomal protein implicated in endocytic trafficking and lysosomal biogenesis.Abbreviations: AIFM1/AIF: apoptosis inducing factor mitochondria associated 1; AO: acridine orange; ATP6V1H: ATPase H+ transporting V1 subunit H; CALR: calreticulin; CREG: cellular repressor of E1A stimulated genes; CTSC: cathepsin C; CTSD: cathepsin D; EBAG9/RCAS1: estrogen receptor binding site associated antigen 9; EIPA: 5-(N-ethyl-N-isopropyl)amiloride; ER: endoplasmic reticulum; GFP: green fluorescent protein; HEXA: hexosaminidase subunit alpha; IGF2R: insulin like growth factor 2 receptor; LAMP1: lysosomal associated membrane protein 1; M6PR: mannose-6-phosphate receptor, cation dependent; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; PDIA2: protein disulfide isomerase family A member 2; SQSTM1/p62: sequestosome 1; TF: transferrin; TFEB: transcription factor EB.
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Project description:Cutaneous melanoma is the deadliest form of skin neoplasm and its high mortality rates could be averted by early accurate detection. While the detection of melanoma is currently reliant upon melanin visualisation, research into melanosome biogenesis, as a key driver of pathogenesis, has not yielded technology that can reliably distinguish between atypical benign, amelanotic and melanotic lesions. The endosomal-lysosomal system has important regulatory roles in cancer cell biology, including a specific functional role in melanosome biogenesis. Herein, the involvement of the endosomal-lysosomal system in melanoma was examined by pooled secondary analysis of existing gene expression datasets. A set of differentially expressed endosomal-lysosomal genes was identified in melanoma, which were interconnected by biological function. To illustrate the protein expression of the dysregulated genes, immunohistochemistry was performed on samples from patients with cutaneous melanoma to reveal candidate markers. This study demonstrated the dysregulation of Syntenin-1, Sortilin and Rab25 may provide a differentiating feature between cutaneous melanoma and squamous cell carcinoma, while IGF2R may indicate malignant propensity in these skin cancers.
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Project description:We have shown that single nucleotide polymorphisms (SNPs) in microRNA (miRNA) genes, miRNA target genes, and miRNA biogenesis genes minimally contribute to colon cancer risk. It is possible that these SNPs alter survival. We analyzed 565 SNPs in or adjacent to microRNAs, target genes, or biogenesis genes, using 1,115 cases and 1,173 controls; 837 cases had survival information. We tested SNPs for associations with colorectal cancer (CRC) survival using a Cox proportional hazard model adjusting for age, study center, gender, AJCC disease stage, and MSI tumor status. Multiple comparison adjustments were made using the step-down Bonferroni correction. SNPs associated with survival (Praw < 0.05) also were assessed with messenger RNA (mRNA). Seven of the 565 SNPs analyzed were associated significantly with CRC survival after adjustment for multiple comparisons. Six of these increased risk of dying, and one, rs12140 (ADAMTS1) decreased risk of dying from CRC (HRR = 0.44, 95% CI (0.24, 0.83; PHolm = 0.011). Six SNPs altered colon cancer risk and five were associated with altered mRNA expression across genotypes. One SNP, rs2059691 (PRKRA), was associated with increased mRNA expression and worse survival, and one SNP, rs6598964 (LIN28A), decreased risk of developing colon cancer [OR = 0.77 95% CI (0.61, 0.98)] and increased risk of dying from CRC (HRR = 2.26 95% CI (1.52, 3.36). PHolm = 0.003). The few SNPs associated with CRC survival, colon cancer risk, or with mRNA expression, resided in genes that influence metastasis and angiogenesis. © 2017 Wiley Periodicals, Inc.
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Project description:Host cytoskeletal proteins of the ezrin-moesin-radixin (EMR) family have been shown to modulate single-stranded RNA virus infection through regulating stable microtubule formation. Antibody engagement of CD81, a key receptor for hepatitis C virus (HCV) entry, induces ezrin phosphorylation. Here we tested the role of EMR proteins in regulating HCV infection and explored potential therapeutic targets. We show that HCV E2 protein induces rapid ezrin phosphorylation and its cellular redistribution with F-actin by way of spleen tyrosine kinase (SYK). Therapeutically blocking the functional roles of SYK or F-actin reorganization significantly reduced Huh7.5 cell susceptibility to HCV J6/JFH-1 infection. Using gene regulation, real-time quantitative polymerase chain reaction, western blot, and fluorescent microscopy analysis, we found that proteins of the EMR family differentially regulate HCV infection in the J6/JFH-1/Huh7.5 cell system. Moesin and radixin, but not ezrin, expression were significantly decreased in chronic HCV J6/JFH-1-infected Huh7.5 cells and HCV-infected patient liver biopsies compared to controls. The decreases in moesin and radixin in HCV J6/JFH-1-infected Huh7.5 cells were associated with a significant increase in stable microtubules. Ezrin knockdown inhibited immediate postentry events in HCV infection. Overexpression of moesin or radixin significantly reduced HCV protein expression. In contrast, transient knockdown of moesin or radixin augmented HCV infection. Making use of the Con1 HCV replicon system, we tested the effect of EMR proteins on HCV replication. We found that transient knockdown of moesin increased HCV RNA expression while overexpression of EMR showed no significant effect on HCV replication.Our findings demonstrate the important role of EMR proteins during HCV infection at the postentry level and highlight possible novel targets for HCV treatment.
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