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In response to corneal injury, an activation of corneal epithelial stem cells and their direct progeny the early transit amplifying (eTA) cells to rapidly proliferate is critical for proper re-epithelialization. Thus, it is important to understand how such stem/eTA cell activation is regulated. Angiotensin-converting enzyme 2 (ACE2) is predominantly expressed in the stem/eTA-enriched limbal epithelium but its role in the limbal epithelium was unclear. Single cell RNA sequencing (scRNA-seq) suggested that Ace2 involved the proliferation of the stem/eTA cells. Ace2 was reduced following corneal injury. Such reduction enhanced limbal epithelial proliferation and downregulated LCN2, a negative regulator of proliferation in a variety of tissues, via upregulating TGFA and consequently activating epidermal growth factor receptor (EGFR). Inhibition of EGFR or overexpression of LCN2 reversed the increased proliferation in limbal epithelial cells lacking ACE2. Our findings demonstrate that after corneal injury, ACE2 is downregulated, which activates limbal epithelial cell proliferation via a TGFA/EGFR/LCN2 pathway.
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Intricate signaling systems are required to maintain homeostasis and promote differentiation in the epidermis. Receptor tyrosine kinases are central in orchestrating these systems in epidermal keratinocytes. In particular, EPHA2 and EGFR transduce distinct signals to dictate keratinocyte fate, yet how these cell communication networks are integrated has not been investigated. Our work shows that loss of EPHA2 impairs keratinocyte stratification, differentiation, and barrier function. To determine the mechanism of this dysfunction, we drew from our proteomics data of potential EPHA2 interacting proteins. We identified EGFR as a high-ranking EPHA2 interactor and subsequently validated this interaction. We found that when EPHA2 is reduced, EGFR activation and downstream signaling are intensified and sustained. Evidence indicates that prolonged SRC association contributes to the increase in EGFR signaling. We show that hyperactive EGFR signaling underlies the differentiation defect caused by EPHA2 knockdown because EGFR inhibition restores differentiation in EPHA2-deficient 3-dimensional skin organoids. Our data implicate a mechanism whereby EPHA2 restrains EGFR signaling, allowing for fine tuning in the processes of terminal differentiation and barrier formation. Taken together, we purport that crosstalk between receptor tyrosine kinases EPHA2 and EGFR is critical for epidermal differentiation.
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Diferenciación Celular , Epidermis , Receptores ErbB , Queratinocitos , Receptor EphA2 , Transducción de Señal , Receptor EphA2/metabolismo , Receptor EphA2/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Humanos , Queratinocitos/metabolismo , Queratinocitos/citología , Epidermis/metabolismo , Células Cultivadas , Ratones , Animales , Células Epidérmicas/metabolismo , Células Epidérmicas/citología , Proteómica/métodosRESUMEN
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.
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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.
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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
INTRODUCTION: Adding dexrazoxane to the treatment during neoadjuvant/adjuvant anthracycline-based chemotherapy in patients with breast cancer prevents the development of heart failure. In this study, we investigated whether dexrazoxane has a protective effect on arrhythmia resulting from chemotherapy. METHODS: Patients with breast cancer who received neoadjuvant/adjuvant anthracycline-based chemotherapy in the medical oncology polyclinic between 2017 and 2020 were included in the study. To investigate the effect of dexrazoxane on arrhythmia, this retrospective study included 70 patients, whose 12-lead surface electrocardiograms (ECGs) and echocardiography were obtained before receiving anthracycline-based treatment and after receiving four cycles of chemotherapy. Thirty-two patients received anthracycline only, and 38 patients received anthracycline and dexrazoxane. Arrhythmia parameters such as QT interval, QTc interval, Tp-e interval, Tp-e/QT, Tp-e/QTc and frontal QRS-T angle were calculated from 12-lead ECGs. RESULTS: Arrhythmia parameters such as frontal QRS-T angle , QT , QTc and heart rate were significantly increased after chemotherapy in both the groups that received dexrazoxane and did not receive dexrazoxane (P < .05). Contrary to the ECG parameters, ejection fraction was decreased in the dexrazoxane group (60.5 ± 2.2 vs 60.1 ± 2.0; P = .038) and the other group (60.4 ± 1.3 vs 60.0 ± 2.6; P = .043) after the chemotherapy. CONCLUSION: This study demonstrated that dexrazoxane may not have a protective effect on ECG parameters which are predictors of arrhythmia, at breast cancer patients who received anthracyclines.
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Neoplasias de la Mama , Dexrazoxano , Antraciclinas/efectos adversos , Arritmias Cardíacas/etiología , Arritmias Cardíacas/prevención & control , Neoplasias de la Mama/tratamiento farmacológico , Dexrazoxano/uso terapéutico , Femenino , Humanos , Terapia Neoadyuvante/efectos adversos , Estudios RetrospectivosRESUMEN
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.
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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.
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Lipoproteínas HDL , Nanopartículas del Metal , Colesterol , OroRESUMEN
OBJECTIVE: Transarterial radioembolisation (TARE) is a promising technique for unresectable primary tumours of the liver. We present our clinical experience and the response to treatment and survival data of patients with hepatocellular carcinoma (HCC) who were treated with Y-90 radioembolisation in our hospital's angiography department. MATERIAL AND METHODS: The data of all the patients with HCC referred to our department for Y-90 treatment were analysed retrospectively. The patients were selected according to the treatment protocol criteria, and lung shunt fraction was evaluated using macroaggregated albumin scintigraphy before radioembolisation. Patients with compatible blood tests and lung shunt fraction rates were chosen for treatment with Y-90 TARE. RESULTS: Twenty-four patients were suitable for Y-90 treatment. The patients were treated with 137 ± 44.6 (80-245) Gy Y-90 glass microspheres. The treatment results were evaluated using modified RECIST criteria, and the partial response, complete response, stable disease and progression rates were found to be 54.2, 16.7, 20.8 and 8.3%, respectively. The median survival rate following treatment was 10 months. Higher alpha-fetoprotein (AFP) levels were related to decreased survival, and posttreatment AFP levels had a significant effect on mortality rates. Higher survival rates were detected in the patients who were treated more selectively than the group treated via a lobar approach. CONCLUSION: Y-90 microsphere radioembolisation is a safe method and may be helpful in treating patients with unresectable hepatocellular tumours. More favourable results were obtained in the patients treated using the more selective approach. AFP levels before and after treatment could predict survival rates.
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Carcinoma Hepatocelular , Embolización Terapéutica , Neoplasias Hepáticas , Carcinoma Hepatocelular/diagnóstico por imagen , Carcinoma Hepatocelular/terapia , Humanos , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/terapia , Microesferas , Estudios Retrospectivos , Resultado del Tratamiento , Radioisótopos de Itrio/uso terapéuticoRESUMEN
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: Soft tissue sarcomas are associated with a poor prognosis and low chemotherapeutic efficiency. Pazopanib is an orally available multi-tyrosine kinase inhibitor that was explored in patients with non-adipocytic advanced soft tissue sarcomas. The aim of this retrospective study was to evaluate the real life data of single-agent pazopanib efficacy and safety for soft tissue sarcomas in the Turkish population. MATERIALS AND METHODS: We evaluated a total of 103 patients (41 males, 62 females) who received pazopanib for advanced non-adipocytic soft tissue sarcomas diagnosis in eight centers of Turkey, retrospectively. The pazopanib dose was 800 mg once daily. Progression-free survival, overall survival, and adverse events were analyzed. RESULTS: The median age was 50 years (range, 38-58). Majority of the patients had leimyosarcoma (41%). Median progression-free survival was 4.3 months, and the median overall survival was 10.1 months. The main common toxicities were fatigue, anorexia, weight loss, nausea, hypertension, and grade ≥3 toxicities were fatigue, anorexia, weight loss, and liver disorder. CONCLUSION: Pazopanib is an efficient and tolerable agent and is well tolerated in good performance status patients with relapsed, advanced non-adipocytic soft tissue sarcomas.
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Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Sarcoma/tratamiento farmacológico , Neoplasias de los Tejidos Blandos/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Adulto , Femenino , Humanos , Indazoles , Leiomiosarcoma/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Supervivencia sin Progresión , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Pirimidinas/administración & dosificación , Pirimidinas/efectos adversos , Estudios Retrospectivos , Sulfonamidas/administración & dosificación , Sulfonamidas/efectos adversos , Análisis de SupervivenciaRESUMEN
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.
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Enfermedades de la Córnea/metabolismo , Epitelio Corneal/metabolismo , Células Madre/citología , Cicatrización de Heridas , Autofagia , Enfermedades de la Córnea/patología , Epitelio Corneal/patología , HumanosRESUMEN
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.
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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
Autophagy is a multistage catabolic process that mediates stress responses. However, the role of autophagy in epidermal proliferation, particularly under conditions when the epidermis becomes "activated" (hyperproliferative), remains unclear. We have shown that inhibition of Beclin 1, a key activator in the initiation phase of autophagy, attenuates imiquimod (IMQ)-induced epidermal hyperplasia in adult mice as well as naturally occurring hyperproliferation in neonatal mouse epidermis. Inhibition of Beclin 1 did not change the levels of several key inflammatory molecules or the numbers of immune cells in lesional skins. This indicates that autophagy does not affect inflammatory regulators in IMQ-treated mouse skin. Bioinformatic analysis combined with gene expression quantitative assays, revealed that a deficiency in autophagy decreases the expression of PDZ Binding Kinase (PBK), a regulator of the cell cycle, in mouse epidermis and human epidermal keratinocytes (HEKs). Interestingly, the decrease in PBK results in inhibition of proliferation in HEKs and such reduced proliferation can be rescued by activation of p38, the downstream signaling of PBK. Collectively, autophagy plays a positive role in epidermal proliferation, which is in part via regulating PBK expression.
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Autofagia/fisiología , Proliferación Celular/fisiología , Epidermis/fisiología , Animales , Autofagia/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Epidermis/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Humanos , Hiperplasia/inducido químicamente , Hiperplasia/fisiopatología , Imiquimod/farmacología , Inflamación/fisiopatología , Queratinocitos/efectos de los fármacos , Queratinocitos/fisiología , Ratones , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Piel/efectos de los fármacos , Piel/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.
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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
microRNAs regulate numerous biological processes, making them potential therapeutic agents. Problems with delivery and stability of these molecules have limited their usefulness as treatments. We demonstrate that synthetic high-density lipoprotein nanoparticles (HDL NPs) topically applied to the intact ocular surface are taken up by epithelial and stromal cells. microRNAs complexed to HDL NPs (miR-HDL NPs) are similarly taken up by cells and tissues and retain biological activity. Topical treatment of diabetic mice with either HDL NPs or miR-HDL NPs significantly improved corneal re-epithelialization following wounding compared with controls. Mouse corneas with alkali burn-induced inflammation, topically treated with HDL NPs, displayed clinical, morphological and immunological improvement. These results should yield a novel HDL NP-based eye drop for patients with compromised wound healing ability (diabetics) and/or corneal inflammatory diseases (e.g. dry eye).
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Biotin identification (BioID) proteomics facilitates the unbiased detection of protein interaction neighborhoods in live cells. The BioID technique relies on the covalent biotin alteration of vicinal proteins by a modified bacterial biotin ligase. The biotin ligase is fused to a protein of interest to identify putative protein-protein interactions. Here, we describe the adaptation of this technique for use in three-dimensional epidermal cultures. Due to the covalent biotin modification of proteins, our protocol allows for the complete solubilization of the total cellular protein content in differentiated keratinocytes. Thus, a comprehensive network of potential interactors of a protein of interest can be mapped.
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Biotina/química , Proteómica/métodos , Piel/citología , Humanos , Técnicas de Cultivo de Órganos/métodos , Mapeo de Interacción de Proteínas , Piel/metabolismoRESUMEN
A 58-year old patient with a history of subungual malign melanoma was referred to our department for a 18F-FDG positron emission tomography (PET)/computed tomography (CT) whole body scan. An unexpected 18F-FDG uptake in left ventricule which mimicked either trombus or physiological papillary muscle was detected. Filling defect of intravenous contrast in CT images was also demonstrated in left ventricule cavity. Magnetic resonance imaging scan confirmed cardiac mass with metastatic features of malign melanoma in left ventricule
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Purpose: Single-cell RNA-sequencing (scRNA-seq) was used to interrogate the relatively rare stem (SC) and early transit amplifying (TA) cell populations in limbal/corneal epithelia from wild-type and autophagy-compromised mice. Methods: We conducted scRNA-seq on ocular anterior segmental tissue from wild-type and beclin 1-deficient (beclin1+/-) mice, using a 10X Gemomics pipeline. Cell populations were distinguished by t-distributed stochastic neighbor embedding. Seurat analysis was conducted to compare gene expression profiles between these two groups of mice. Differential protein expression patterns were validated by immunofluorescence staining and immunoblotting. Results: Unbiased clustering detected 10 distinct populations: three clusters of mesenchymal and seven clusters of epithelial cells, based on their unique molecular signatures. A discrete group of mesenchymal cells expressed genes associated with corneal stromal SCs. We identified three limbal/corneal epithelial cell subpopulations designated as stem/early TA, mature TA, and differentiated corneal epithelial cells. Thioredoxin-interacting protein and PDZ-binding kinase (PBK) were identified as novel regulators of stem/early TA cell quiescence. PBK arrested corneal epithelial cells in G2/M phase of the cell cycle. Beclin1+/- mice displayed a decrease in proliferation-associated (Ki67, Lrig1) and stress-response (H2ax) genes. The most increased gene in beclin1+/- mice was transcription factor ATF3, which negatively regulates limbal epithelial cell proliferation. Conclusions: Establishment of a comprehensive atlas of genes expressed by stromal and epithelial cells from limbus and cornea forms the foundation for unraveling regulatory networks among these distinct tissues. Similarly, scRNA-seq profiling of the anterior segmental epithelia from wild-type and autophagy-deficient mice provides new insights into how autophagy influences proliferation in these tissues.
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Autofagia/fisiología , Epitelio Corneal/citología , Limbo de la Córnea/citología , Células Madre Mesenquimatosas/citología , ARN/genética , Transcriptoma/genética , Animales , Beclina-1/fisiología , Biomarcadores/metabolismo , Recuento de Células , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Epitelio Corneal/metabolismo , Femenino , Inmunohistoquímica , Limbo de la Córnea/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARNRESUMEN
EphA2 receptor tyrosine kinase is activated by ephrin-A1 ligand, which harbors a glycosylphosphatidylinositol anchor that enhances lipid raft localization. Although EphA2 and ephrin-A1 modulate keratinocyte migration and differentiation, the ability of this cell-cell communication complex to localize to different membrane regions in keratinocytes remains unknown. Using a combination of biochemical and imaging approaches, we provide evidence that ephrin-A1 and a ligand-activated form of EphA2 partition outside of lipid raft domains in response to calcium-mediated cell-cell contact stabilization in normal human epidermal keratinocytes. EphA2 transmembrane domain swapping with a shorter and molecularly distinct transmembrane domain of EphA1 resulted in decreased localization of this receptor tyrosine kinase at cell-cell junctions and increased expression of ephrin-A1, which is a negative regulator of keratinocyte migration. Accordingly, altered EphA2 membrane distribution at cell-cell contacts limited the ability of keratinocytes to seal linear scratch wounds in vitro in an ephrin-A1-dependent manner. Collectively, these studies highlight a key role for the EphA2 transmembrane domain in receptor-ligand membrane distribution at cell-cell contacts that modulates ephrin-A1 levels to allow for efficient keratinocyte migration with relevance for cutaneous wound healing.
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Efrina-A1/metabolismo , Efrina-A2/genética , Epidermis/metabolismo , Regulación de la Expresión Génica , Queratinocitos/patología , ARN/genética , Heridas y Lesiones/genética , Western Blotting , Comunicación Celular , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Efrina-A2/biosíntesis , Epidermis/patología , Humanos , Recién Nacido , Queratinocitos/metabolismo , Masculino , Reacción en Cadena de la Polimerasa , Receptor EphA2 , Transducción de Señal , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patologíaRESUMEN
We have shown that microRNAs-103 and -107 (miRs-103/107) positively regulate end-stage autophagy by ensuring dynamin activity in cultured keratinocytes. Most work in end-stage autophagy has been conducted using in vitro model systems. In vivo regulation of end-stage autophagy in epidermis remains unknown. Here, we used antagomirs to subcutaneously knock down miR-107 in the skin; conversely, we delivered miR-107 mimic subcutaneously via in vivo transfection to increase this miR. We found that antagomir-107 treatment in epidermis: (i) depleted endogenous miR-107; (ii) increased GFP-LC3 puncta in epidermal basal layers of GFP-LC3 transgenic mice, indicative of an accumulation of autophagosomes; (iii) inhibited LC3 turnover and increased p62, suggesting an inhibition of autophagy flux; and (iv) increased phosphorylated dynamin (p-dynamin, an inactive form), a key enzyme in end-stage autophagy. Conversely, miR-107 mimic treatment in mouse epidermis: decreased GFP-LC3 puncta in basal layer, as well as p62 protein levels; and diminished p-dynamin, indicative of activation of this enzyme. In human epidermal keratinocytes, antagos-103/107 cause the formation of large vacuoles and an increase in p-dynamin, which can be rescued by inhibition of protein kinase C pathway. Collectively, these results suggest that the miR-103/107 family has a critical role in regulating end-stage autophagy in mouse epidermis via PLD1/2-protein kinase C-dynamin pathway.