RESUMEN
Limbal epithelial stem cells are not only critical for corneal epithelial homeostasis but also have the capacity to change from a relatively quiescent mitotic phenotype to a rapidly proliferating cell in response to population depletion following corneal epithelial wounding. Pax6+/- mice display many abnormalities including corneal vascularization and these aberrations are consistent with a limbal stem cell deficiency (LSCD) phenotype. FoxC1 has an inhibitory effect on corneal avascularity and a positive role in stem cell maintenance in many tissues. However, the role of FoxC1 in limbal epithelial stem cells remains unknown. To unravel FoxC1's role(s) in limbal epithelial stem cell homeostasis, we utilized an adeno-associated virus (AAV) vector to topically deliver human FOXC1 proteins into Pax6 +/- mouse limbal epithelium. Under unperturbed conditions, overexpression of FOXC1 in the limbal epithelium had little significant change in differentiation (PAI-2, Krt12) and proliferation (BrdU, Ki67). Conversely, such overexpression resulted in a marked increase in the expression of putative limbal epithelial stem cell markers, N-cadherin and Lrig1. After corneal injuries in Pax6 +/- mice, FOXC1 overexpression enhanced the behavior of limbal epithelial stem cells from quiescence to a highly proliferative status. Overall, the treatment of AAV8-FOXC1 may be beneficial to the function of limbal epithelial stem cells in the context of a deficiency of Pax6 function.
Asunto(s)
Enfermedades de la Córnea , Epitelio Corneal , Limbo de la Córnea , Animales , Humanos , Ratones , Córnea , Enfermedades de la Córnea/metabolismo , Desbridamiento , Células Epiteliales , Epitelio Corneal/metabolismo , Limbo de la Córnea/metabolismo , Células MadreRESUMEN
A distinct boundary exists between the progenitor cells in the basal limbal epithelium and the more differentiated corneal epithelial basal cells. We have shown that reciprocal expression patterns of EphA2 and Ephrin-A1 are likely to contribute to normal limbal-corneal epithelial compartmentalization as well as play a role in response to injury. How this signaling axis is regulated remains unclear. We have demonstrated that microRNAs (miRNAs) play critical roles in corneal epithelial wound healing and several miRNAs (e.g. miR-210) have been predicted to target ephrins. Previous expression profiling experiments demonstrated that miR-210 is prominently expressed in corneal epithelial cells. RNA-seq data acquired from miR-210-depleted HCECs showed up-regulation of genes involved in cellular migration. In addition, miR-210 is decreased after corneal injury while EphA2 is increased. Moreover, antago-210-treated HCECs markedly enhanced wound closure in a scratch wound assay. Antago-210 treatment resulted in increased EphA2 protein levels as well as pS897-EphA2, the pro-migratory form of EphA2. As expected, Ephrin-A1 levels were reduced, while levels of a well-known target of miR-210, Ephrin-A3, were increased by antago-210 treatment. The increase in migration with antago-210 could be inhibited by Ephrin-A1 overexpression, Ephrin-A1-Fc treatment or siRNA depletion of EphA2. However, depletion of Ephrin-A3 did not have effects on the antago-210-induced increase in migration. In addition, Ephrin-A1 overexpression and siEphA2 dampened EGFR signaling, which is increased by antago-210. Our data clearly demonstrate a link between miR-210 and EphA2/Ephrin-A1 signaling that regulates, in part, corneal epithelial migration. This interaction might potentially control the limbal-corneal epithelial boundary.
Asunto(s)
Movimiento Celular , Córnea/metabolismo , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Receptores de la Familia Eph/metabolismo , Humanos , MicroARNs/genética , RNA-Seq , Receptores de la Familia Eph/genéticaRESUMEN
Angiotensin converting enzyme 2 (ACE2) plays an important role in inflammation, which is attributable at least, in part, to the conversion of the pro-inflammatory angiotensin (Ang) II peptide into angiotensin 1-7 (Ang 1-7), a peptide which opposes the actions of AngII. ACE2 and AngII are present in many tissues but information on the cornea is lacking. We observed that mice deficient in the Ace2 gene (Ace2-/- ), developed a cloudy cornea phenotype as they aged. Haze occupied the central cornea, accompanied by corneal edema and neovascularization. In severe cases with marked chronic inflammation, a cell-fate switch from a transparent corneal epithelium to a keratinized, stratified squamous, psoriasiform-like epidermis was observed. The stroma contained a large number of CD11c, CD68, and CD3 positive cells. Corneal epithelial debridement experiments in young ACE2-deficient mice showed normal appearing corneas, devoid of haze. We hypothesized, however, that these mice are "primed" for a corneal inflammatory response, which once initiated, would persist. In vitro studies reveal that interleukins (IL-1a, IL-1b), chemokines (CCL2, CXCL8), and TNF-α, are all significantly elevated, resulting in a cytokine storm-like phenotype. This phenotype could be partially rescued by treatment with the AngII type 1 receptor (AT1R) antagonist, losartan, suggesting that the observed effect was mediated by AngII acting on its main receptor. Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes human ACE2 as the receptor for entry with subsequent downregulation of ACE2, corneal inflammation in Ace2-/- mice may have a similar mechanism with that in COVID-19 patients. Thus the Ace2-/- cornea, because of easy accessibility, may provide an attractive model to explore the molecular mechanisms, immunological changes, and treatment modalities in patients with COVID-19.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , Córnea/patología , Síndrome de Liberación de Citoquinas/fisiopatología , Modelos Animales de Enfermedad , Angiotensina II/metabolismo , Animales , COVID-19 , Células Cultivadas , Quimiocinas/metabolismo , Células Epiteliales/metabolismo , Humanos , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , SARS-CoV-2 , Células THP-1 , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Whereas much is known about the genes regulated by ΔNp63α in keratinocytes, how ΔNp63α is regulated is less clear. During studies with the hydroxylase, factor inhibiting hypoxia-inducible factor 1 (FIH-1), we observed increases in epidermal ΔNp63α expression along with proliferative capacity in a conditional FIH-1 transgenic mouse. Conversely, loss of FIH-1 in vivo and in vitro attenuated ΔNp63α expression. To elucidate the FIH-1/p63 relationship, BioID proteomics assays identified FIH-1 binding partners that had the potential to regulate p63 expression. FIH-1 interacts with two previously unknown partners, Plectin1 and signal transducer and activator of transcription 1 (STAT1) leading to the regulation of ΔNp63α expression. Two known interactors of FIH-1, apoptosis-stimulating of P53 protein 2 (ASPP2) and histone deacetylase 1 (HDAC1), were also identified. Knockdown of ASPP2 upregulated ΔNp63α and reversed the decrease in ΔNp63α by FIH-1 depletion. Additionally, FIH-1 regulates growth arrest and DNA damage-45 alpha (GADD45α), a negative regulator of ΔNp63α by interacting with HDAC1. GADD45α knockdown rescued reduction in ΔNp63α by FIH-1 depletion. Collectively, our data reveal that FIH-1 positively regulates ΔNp63α in keratinocytes via variety of signaling partners: (a) Plectin1/STAT1, (b) ASPP2, and (c) HDAC1/GADD45α signaling pathways.
Asunto(s)
Proteínas Portadoras/metabolismo , Proliferación Celular , Células Epiteliales/citología , Queratinocitos/citología , Proteínas de la Membrana/metabolismo , Oxigenasas de Función Mixta/metabolismo , Proteoma/metabolismo , Proteínas Represoras/metabolismo , Animales , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Queratinocitos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Oxigenasas de Función Mixta/genética , Proteoma/análisis , Proteínas Represoras/genéticaRESUMEN
The anterior surface of the eye functions as a barrier to the external environment and protects the delicate underlying tissues from injury. Central to this protection are the corneal, limbal and conjunctival epithelia. The corneal epithelium is a self-renewing stratified squamous epithelium that protects the underlying delicate structures of the eye, supports a tear film and maintains transparency so that light can be transmitted to the interior of the eye (Basu et al., 2014; Cotsarelis et al., 1989; Funderburgh et al., 2016; Lehrer et al., 1998; Pajoohesh-Ganji and Stepp, 2005; Parfitt et al., 2015; Peng et al., 2012b; Stepp and Zieske, 2005). In this review, dedicated to James Funderburgh and his contributions to visual science, in particular the limbal niche, corneal stroma and corneal stromal stem cells, we will focus on recent data on the identification of novel regulators in corneal epithelial cell biology, their roles in stem cell homeostasis, wound healing, limbal/corneal boundary maintenance and the utility of single cell RNA sequencing (scRNA-seq) in vision biology studies.
Asunto(s)
Enfermedades de la Córnea/metabolismo , Epitelio Corneal/metabolismo , Células Madre/citología , Cicatrización de Heridas , Autofagia , Enfermedades de la Córnea/patología , Epitelio Corneal/patología , HumanosRESUMEN
Corneal avascularity is critical for achieving transparency necessary for proper transmission of light to the lens and visual acuity. Although much is known about angiogenesis and angiostasis, the precise regulation of these processes in the cornea is unclear. MicroRNA (miR)-184, the most abundant corneal epithelial miRNA, has been suggested to function in corneal angiostasis by altering VEGF signaling; however, the mechanism(s) underlying this regulation have not been addressed. Using a combination of in vitro and in vivo assays to evaluate angiogenesis, we demonstrated that human limbal epithelial keratinocytes (HLEKs) engineered to overexpress miR-184 secreted lower amounts of angiogenic mitogens. Human dermal microvascular cells exposed to conditioned medium from miR-184-overexpressing HLEKs were less proliferative and failed to seal linear scratch wounds. The in vivo Matrigel plug assay showed that conditioned medium from miR-184-expressing HLEKs elicited a lesser degree of neovascularization compared with controls. We found that miR-184 directly targets and represses the proangiogenic factors, friend of Gata 2 (FOG2), platelet-derived growth factor (PDGF)-ß, and phosphatidic acid phosphatase 2b (PPAP2B). FOG2 regulates VEGF expression, whereas PDGF-ß and PPAP2B regulate Akt activity. By attenuating both VEGF and Akt signaling, miR-184 acts as a broad-spectrum negative regulator of corneal angiogenesis.-Park, J. K., Peng, H., Yang, W., Katsnelson, J., Volpert, O., Lavker, R. M. miR-184 exhibits angiostatic properties via regulation of Akt and VEGF signaling pathways.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Queratinocitos/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Inductores de la Angiogénesis , Animales , Proliferación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Proteínas Proto-Oncogénicas c-akt/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Genetic variations mapping to 3' untranslated regions (3'UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3'UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) - an autoimmune blistering skin condition with unique endemic patterns - we investigated whether nine 3'UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene's activity through the 3'UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3'UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus.
Asunto(s)
Regiones no Traducidas 3' , Predisposición Genética a la Enfermedad , Lectinas Tipo C/genética , MicroARNs/genética , Pénfigo/genética , Polimorfismo de Nucleótido Simple , Transactivadores/genética , Alelos , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Factor Activador de Células B/genética , Factor Activador de Células B/metabolismo , Secuencia de Bases , Sitios de Unión , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Estudios de Casos y Controles , Análisis Mutacional de ADN , Regulación de la Expresión Génica , Frecuencia de los Genes , Haplotipos , Humanos , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , MicroARNs/metabolismo , Mutación , Subfamília D de Receptores Similares a Lectina de las Células NK/genética , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Pénfigo/diagnóstico , Pénfigo/metabolismo , Pénfigo/patología , Receptores Inmunológicos , Transactivadores/metabolismoRESUMEN
The stem cell niche is thought to affect cell cycle quiescence, proliferative capacity, and communication between stem cells and their neighbors. How these activities are controlled is not completely understood. Here we define a microRNA family (miRs-103/107) preferentially expressed in the stem cell-enriched limbal epithelium that regulates and integrates these stem cell characteristics. miRs-103/107 target the ribosomal kinase p90RSK2, thereby arresting cells in G0/G1 and contributing to a slow-cycling phenotype. Furthermore, miRs-103/107 increase the proliferative capacity of keratinocytes by targeting Wnt3a, which enhances Sox9 and YAP1 levels and thus promotes a stem cell phenotype. This miRNA family also regulates keratinocyte cell-cell communication by targeting: (a) the scaffolding protein NEDD9, preserving E-cadherin-mediated cell adhesion; and (b) the tyrosine phosphatase PTPRM, which negatively regulates connexin 43-based gap junctions. We propose that such regulation of cell communication and adhesion molecules maintains the integrity of the stem cell niche ultimately preserving self-renewal, a hallmark of epithelial stem cells.
Asunto(s)
Células Epiteliales/metabolismo , MicroARNs/metabolismo , Células Madre/metabolismo , Animales , Cadherinas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Uniones Comunicantes/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Limbo de la Córnea/citología , Masculino , Ratones Endogámicos BALB C , MicroARNs/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Células Madre/efectos de los fármacos , Proteína Wnt3A/farmacologíaRESUMEN
The longstanding concept that corneal epithelial stem cells reside mainly in the limbus is supported by the absence of major corneal epithelial differentiation markers, that is, K3 and K12 keratins, in limbal basal cells (these markers are expressed, however, in corneal basal cells, thus distinguishing the mode of keratin expression in corneal epithelium from that of all other stratified epithelia), the centripetal migration of corneal epithelial cells, the exclusive location of slow-cycling cells in the limbal basal layer, the superior in vitro proliferative potential of limbal epithelial cells, and the transplanted limbal cells' ability to reconstitute corneal epithelium in vivo (reviewed in refs 1-4). Moreover, previous data indicate that corneal and conjunctival epithelia represent two separate cell lineages (reviewed in refs 1-4). Majo et al. suggested, however, that corneal and conjunctival epithelia are equipotent, and that identical oligopotent stem cells are present throughout the corneal, limbal and conjunctival epithelia. We point out here that these suggestions are inconsistent with many known growth, differentiation and cell migration properties of the anterior ocular epithelia.
Asunto(s)
Movimiento Celular , Epitelio Corneal/citología , Limbo de la Córnea/citología , Células Madre/citología , Animales , Bovinos , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Conjuntiva/citología , Células Caliciformes/citología , Humanos , Ratones , Modelos Biológicos , Conejos , Reproducibilidad de los Resultados , Ovinos , PorcinosRESUMEN
Factor inhibiting hypoxia-inducible factor 1 (FIH-1; official symbol HIF1AN) is a hydroxylase that negatively regulates hypoxia-inducible factor 1α but also targets other ankyrin repeat domain-containing proteins such as Notch receptor to limit epithelial differentiation. We show that FIH-1 null mutant mice exhibit delayed wound healing. Importantly, in vitro scratch wound assays demonstrate that the positive role of FIH-1 in migration is independent of Notch signaling, suggesting that this hydroxylase targets another ankyrin repeat domain-containing protein to positively regulate motogenic signaling pathways. Accordingly, FIH-1 increases epidermal growth factor receptor (EGFR) signaling, which in turn enhances keratinocyte migration via mitogen-activated protein kinase pathway, leading to extracellular signal-regulated kinase 1/2 activation. Our studies identify leucine-rich repeat kinase 1 (LRRK1), a key regulator of the EGFR endosomal trafficking and signaling, as an FIH-1 binding partner. Such an interaction prevents the formation of an EGFR/LRRK1 complex, necessary for proper EGFR turnover. The identification of LRRK1 as a novel target for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration.
Asunto(s)
Receptores ErbB/metabolismo , Queratinocitos/citología , Oxigenasas de Función Mixta/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Epitelio/embriología , Humanos , Inmunohistoquímica , Queratinocitos/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Oxigenasas de Función Mixta/genética , Unión Proteica , Transducción de Señal , Cicatrización de HeridasRESUMEN
Notch plays a critical role in the transition from proliferation to differentiation in the epidermis and corneal epithelium. Furthermore, aberrant Notch signaling is a feature of diseases like psoriasis, eczema, nonmelanoma skin cancer, and melanoma where differentiation and proliferation are impaired. Whereas much is known about the downstream events following Notch signaling, factors responsible for negatively regulating Notch receptor signaling after ligand activation are incompletely understood. Notch can undergo hydroxylation by factor-inhibiting hypoxia-inducible factor 1 (FIH-1); however, the biological significance of this phenomenon is unclear. Here we show that FIH-1 expression is up-regulated in diseased epidermis and corneal epithelium. Elevating FIH-1 levels in primary human epidermal keratinocytes (HEKs) and human corneal epithelial keratinocytes (HCEKs) impairs differentiation in submerged cultures and in a "three-dimensional" organotypic raft model of human epidermis, in part, via a coordinate decrease in Notch signaling. Knockdown of FIH-1 enhances keratinocyte differentiation. Loss of FIH-1 in vivo increased Notch activity in the limbal epithelium, resulting in a more differentiated phenotype. microRNA-31 (miR-31) is an endogenous negative regulator of FIH-1 expression that results in keratinocyte differentiation, mediated by Notch activation. Ectopically expressing miR-31 in an undifferentiated corneal epithelial cell line promotes differentiation and recapitulates a corneal epithelium in a three-dimensional raft culture model. Our results define a previously unknown mechanism for keratinocyte fate decisions where Notch signaling potential is, in part, controlled through a miR-31/FIH-1 nexus.
Asunto(s)
Queratinocitos/citología , Queratinocitos/fisiología , MicroARNs/metabolismo , Oxigenasas de Función Mixta/metabolismo , Psoriasis/metabolismo , Proteínas Represoras/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular Transformada , Células Epidérmicas , Epidermis/fisiología , Células Epiteliales/citología , Células Epiteliales/fisiología , Epitelio Corneal/citología , Epitelio Corneal/fisiología , Femenino , Humanos , Ratones , Ratones Transgénicos , Oxigenasas de Función Mixta/genética , Técnicas de Cultivo de Órganos , Fenotipo , Psoriasis/genética , Psoriasis/patología , Receptores Notch/metabolismo , Proteínas Represoras/genética , Transducción de Señal/fisiologíaRESUMEN
Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Vibrio vulnificus/clasificación , Vibrio vulnificus/patogenicidad , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Células CHO , Clonación Molecular , Técnicas de Cocultivo , Cricetinae , Cricetulus , Femenino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Vibriosis/microbiología , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , VirulenciaRESUMEN
Corneal epithelium relies on abundant glycogen stores as its primary energy source. MicroRNA-31 (miR-31), a corneal epithelial-preferred miRNA, negatively regulates factor inhibiting hypoxia-inducible factor-1 (FIH-1). Since HIF-1α is involved in anaerobic energy production, we investigated the role that miR-31 and FIH-1 play in regulating corneal epithelial glycogen. We used antagomirs (antago) to reduce the level of miR-31 in primary human corneal epithelial keratinocytes (HCEKs), and a miR-31-resistant FIH-1 to increase FIH-1 levels. Antago-31 raised FIH-1 levels and significantly reduced glycogen stores in HCEKs compared to irrelevant-antago treatment. Similarly, HCEKs retrovirally transduced with a miR-31-resistant FIH-1 had markedly reduced glycogen levels compared with empty vector controls. In addition, we observed no change in a HIF-1α reporter or known genes downstream of HIF-1α indicating that the action of FIH-1 and miR-31 on glycogen is HIF-1α-independent. An enzyme-dead FIH-1 mutation failed to restore glycogen stores, indicating that FIH-1 negatively regulates glycogen in a hydroxylase-independent manner. FIH-1 overexpression in HCEKs decreased AKT signaling, activated GSK-3ß, and inactivated glycogen synthase. Treatment of FIH-1-transduced HCEKs with either a myristolated Akt or a GSK-3ß inhibitor restored glycogen stores, confirming the direct involvement of Akt/GSK-3ß signaling. Silencing FIH-1 in HCEKs reversed the observed changes in Akt-signaling. Glycogen regulation in a HIF-1α-independent manner is a novel function for FIH-1 and provides new insight into how the corneal epithelium regulates its energy requirements.
Asunto(s)
Epitelio Corneal/metabolismo , Glucógeno/metabolismo , Queratinocitos/metabolismo , MicroARNs/fisiología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Animales , Células Cultivadas , Epitelio Corneal/efectos de los fármacos , Femenino , Glucógeno Sintasa/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Queratinocitos/efectos de los fármacos , Ratones , Oxigenasas de Función Mixta/metabolismo , Oligorribonucleótidos/farmacología , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Single-cell RNA-sequencing (scRNA-seq) is a powerful technique that can barcode individual cells and thus used to obtain a gene expression profile for every individual cell within a tissue. This makes scRNA-seq an excellent method for characterizing rare cell populations such as stem cells. We describe how scRNA-seq can be utilized to examine limbal epithelial stem cell population as well as investigate the contribution of autophagy to the function of limbal epithelial stem cells. To accomplish this, we used the Beclin1 heterozygous (Beclin1 het) mouse, a well-established model of autophagy deficiency. We provide a protocol that we developed for the isolation of viable, single-cell suspensions of limbal/corneal tissues, as well as the analysis of scRNA-seq data.
RESUMEN
microRNA-205 (miR-205) and miR-184 coordinately regulate the lipid phosphatase SHIP2 for Akt survival signaling in keratinocytes. As the PI3K-Akt pathway has also been implicated in regulating the actin cytoskeleton and cell motility, we investigated the role that these 2 miRNAs play in keratinocyte migration. We used antagomirs (antago) to reduce the levels of miR-205 and miR-184 in primary human epidermal keratinocytes (HEKs) and corneal epithelial keratinocytes (HCEKs) as well as direct SHIP2 silencing using siRNA oligos. Treatment of HEKs and HCEKs with antago-205 increased SHIP2 levels and impaired the ability of these cells to seal linear scratch wounds compared with untreated or irrelevant-antago treatments. In contrast, AKT signaling was enhanced and wounds sealed faster in HCEKs where miR-184 was suppressed, enabling miR-205 to inhibit SHIP2. Similar increases in migration were observed following direct SHIP2 silencing in HEKs. Furthermore, down-regulation of miR-205 resulted in an increase in Rho-ROCKI activity, phosphorylation of the actin severing protein cofilin, and a corresponding diminution of filamentous actin. The connection among miR-205, RhoA-ROCKI-cofilin inactivation, and the actin cytoskeleton represents a novel post-translational mechanism for the regulation of normal human keratinocyte migration.
Asunto(s)
Movimiento Celular/fisiología , Regulación de la Expresión Génica/genética , Cabello/metabolismo , Queratinocitos/citología , MicroARNs/fisiología , Piel/metabolismo , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , Cartilla de ADN , Citometría de Flujo , Cabello/citología , Hibridación in Situ , Ratones , Piel/citologíaRESUMEN
Despite their potential to regulate approximately one-third of the whole genome, relatively few microRNA (miRNA) targets have been experimentally validated, particularly in stratified squamous epithelia. Here we demonstrate not only that the lipid phosphatase SHIP2 is a target of miRNA-205 (miR-205) in epithelial cells, but, more importantly, that the corneal epithelial-specific miR-184 can interfere with the ability of miR-205 to suppress SHIP2 levels. This is the first example of a miRNA negatively regulating another to maintain levels of a target protein. Interfering with miR-205 function by using a synthetic antagomir, or by the ectopic expression of miR-184, leads to a coordinated damping of the Akt signaling pathway via SHIP2 induction. This was associated with a marked increase in keratinocyte apoptosis and cell death. Aggressive squamous cell carcinoma (SCC) cells exhibited elevated levels of miR-205. This was associated with a concomitant reduction in SHIP2 levels. Partial knockdown of endogenous miR-205 in SCCs markedly decreased phosphorylated Akt and phosphorylated BAD levels and increased apoptosis. We were able to increase SHIP2 levels in SCC cells after inhibition of miR-205. Therefore, miR-205 might have diagnostic value in determining the aggressivity of SCCs. Blockage of miR-205 activity with an antagomir or via ectopic expression of miR-184 could be novel therapeutic approaches for treating aggressive SCCs.
Asunto(s)
Queratinocitos/fisiología , MicroARNs/genética , MicroARNs/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Apoptosis/fisiología , Carcinoma de Células Escamosas/fisiopatología , Supervivencia Celular/fisiología , Regulación hacia Abajo/genética , Epitelio Corneal/citología , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Queratinocitos/citología , Riñón/citología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño , Factor de Transcripción STAT1/genética , Neoplasias Cutáneas/fisiopatología , TransfecciónRESUMEN
Angiotensin converting enzyme 2 (ACE2), a component of the renin-angiotensin system (RAS), has been identified as the receptor for the SARS-CoV-2. Several RAS components including ACE2 and its substrate Ang II are present in both eye and skin, two stratified squamous epithelial tissues that isolate organisms from external environment. Our recent findings in cornea and others in both skin and eye suggest contribution of this system, and specifically of ACE2 in variety of physiological and pathological responses of these organ systems. This review will focus on the role RAS system plays in both skin and cornea, and will specifically discuss our recent findings on ACE2 in corneal epithelial inflammation, as well as potential implications of ACE2 in patients with COVID-19.
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Enzima Convertidora de Angiotensina 2/metabolismo , Epitelio Corneal/enzimología , Receptores de Coronavirus/metabolismo , Piel/enzimología , Autofagia , COVID-19/enzimología , COVID-19/virología , Humanos , Inflamación/enzimología , Sistema Renina-Angiotensina/fisiología , Cicatrización de HeridasRESUMEN
PURPOSE: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. METHODS: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. RESULTS: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. CONCLUSION: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.
Asunto(s)
Autofagia , Epitelio Corneal , Animales , Células Cultivadas , Cilios , RatonesRESUMEN
Medicine has been a great beneficiary of the nanotechnology revolution. Nanotechnology involves the synthesis of functional materials with at least one size dimension between 1 and 100 nm. Advances in the field have enabled the synthesis of bio-nanoparticles that can interface with physiological systems to modulate fundamental cellular processes. One example of a diverse acting nanoparticle-based therapeutic is synthetic high-density lipoprotein (HDL) nanoparticles (NP), which have great potential for treating diseases of the ocular surface. Our group has developed a spherical HDL NP using a gold nanoparticle core. HDL NPs: (i) closely mimic the physical and chemical features of natural HDLs; (ii) contain apoA-I; (iii) bind with high-affinity to SR-B1, which is the major receptor through which HDL modulates cell cholesterol metabolism and controls the selective uptake of HDL cargo into cells; (iv) are non-toxic to cells and tissues; and (v) can be chemically engineered to display nearly any surface or core composition desired. With respect to the ocular surface, topical application of HDL NPs accelerates re-epithelization of the cornea following wounding, attenuates inflammation resulting from chemical burns and/or other stresses, and effectively delivers microRNAs with biological activity to corneal cells and tissues. HDL NPs will be the foundation of a new class of topical eye drops with great translational potential and exemplify the impact that nanoparticles can have in medicine.
Asunto(s)
Lipoproteínas HDL , Nanopartículas del Metal , Colesterol , OroRESUMEN
BACKGROUND & AIMS: MicroRNA-21 negatively regulates several targets, thereby affecting tumorigenesis. However, its mechanism of action in human hepatocellular carcinoma is poorly understood, and no direct evidence has shown a correlation between microRNA-21 function and phenotype. In this study, we investigate the function of microRNA-21 as a potent oncomir and probe the relationship between microRNA-21, its targets, and phenotypic alterations. METHODS: We designed a set of rescue experiments using different combinations of anti-microRNA-21, siRNA, and a negative control to modulate the protein level of microRNA-21 targets and resulting phenotypic alterations. MicroRNA-21 was suppressed using anti-microRNA-21 to further uncover its effect on several critical signaling pathways. RESULTS: We demonstrate that hepatocellular carcinoma is characterized by elevated levels of microRNA-21 and marked reductions of PTEN, PDCD4, and RECK expression. Silencing of PTEN and PDCD4 to prevent their induction by anti-microRNA-21 treatment led to decreased apoptosis and increased invasion, while silencing of RECK only led to increased invasion. Moreover, knockdown of microRNA-21 resulted in alterations of the Akt signaling pathway, the expression of p21 and MMP families, which are associated with apoptosis, and the cell cycle or invasiveness of cancer cells. CONCLUSIONS: MicroRNA-21 simultaneously regulates multiple programs that enhance cell proliferation, apoptosis or tumor invasiveness by targeting PTEN, PDCD4, and RECK in hepatocellular carcinomas. Targeting of microRNA-21 is sufficient to limit tumor cell proliferation and invasion in a manner that is likely to involve associated changes in multiple targets, suggesting that suppression of microRNA-21 may be a novel approach for the treatment of hepatocellular carcinoma.