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Hereditary papillary renal cell carcinoma (HPRC) is a rare inherited renal cancer syndrome characterized by bilateral and multifocal papillary type 1 renal tumors (PRCC1). Activating germline pathogenic variants of the MET gene were identified in HPRC families. We reviewed the medical and molecular records of a large French series of 158 patients screened for MET oncogenic variants. MET pathogenic and likely pathogenic variants rate was 12.4% with 40.6% among patients with familial PRCC1 and 5% among patients with sporadic PRCC1. The phenotype in cases with MET pathogenic and likely pathogenic variants was characteristic: PRCC1 tumors were mainly bilateral (84.3%) and multifocal (87.5%). Histologically, six out of seven patients with MET pathogenic variant harbored biphasic squamoid alveolar PRCC. Genetic screening identified one novel pathogenic variant MET c.3389T>C, p.(Leu1130Ser) and three novel likely pathogenic variants: MET c.3257A>T, p.(His1086Leu); MET c.3305T>C, p.(Ile1102Thr) and MET c.3373T>G, p.(Cys1125Gly). Functional assay confirmed their oncogenic effect as they induced an abnormal focus formation. The genotype-phenotype correlation between MET pathogenic variants and PRCC1 presentation should encourage to widen the screening, especially toward nonfamilial PRCC1. This precise phenotype also constitutes a strong argument for the classification of novel missense variants within the tyrosine kinase domain when functional assays are not accessible.
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Carcinoma de Células Renales , Neoplasias Renales , Síndromes Neoplásicos Hereditarios , Proteínas Proto-Oncogénicas c-met , Carcinoma de Células Renales/genética , Femenino , Células Germinativas/metabolismo , Humanos , Neoplasias Renales/genética , Masculino , Fenotipo , Proteínas Proto-Oncogénicas c-met/genéticaRESUMEN
BACKGROUND: In atopic dermatitis (AD), some studies have shown an association with increased cardiovascular disease in certain populations. However, other investigations found modest or no association. Despite conflicting results, molecular profiling studies in both AD skin and blood have demonstrated upregulation of atherosclerosis and cardiovascular risk-related markers. However, the underlying mechanisms connecting AD to vascular inflammation/atherosclerosis are unknown. In this study, we aim to determine factors associated with vascular inflammation/atherosclerosis in AD patients. METHODS: We used 18-FDG PET-CT to characterize vascular inflammation in AD patients and healthy subjects. In parallel, we assessed their skin and blood immune profiles to determine AD-related immune biomarkers associated with vascular inflammation. We also assessed levels of circulating microparticles, which are known to be associated with increased cardiovascular risk. RESULTS: We found significant correlations between vascular inflammation and Th2-related products in skin and blood of AD patients as well as atherosclerosis-related markers that were modulated by dupilumab. Circulating levels of endothelial microparticles were significantly higher in severe AD patients and tended to correlate with vascular inflammation assessed by PET-CT. CONCLUSION: Vascular inflammation in AD is associated with enhanced Th2 response and clinical severity, which may explain cardiovascular comorbidities observed in select AD populations. Larger prospective studies are needed to further evaluate vascular inflammation and cardiovascular events and mortality in AD patients. Finally, as dupilumab treatment demonstrated significant modulation of atherosclerosis-related genes in AD patients compared to placebo, these data suggest that modulation of vascular inflammation with systemic treatment should be explored in patients with AD.
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Dermatitis Atópica , Eccema , Dermatitis Atópica/epidemiología , Humanos , Inflamación , Tomografía Computarizada por Tomografía de Emisión de Positrones , Índice de Severidad de la Enfermedad , PielRESUMEN
Abnormally enlarged early endosomes (EEs) are pathological features of neurodegenerative diseases, yet insight into the mechanisms and consequences of EE expansion remains elusive. Here, we report swollen apical EEs in the retinal pigment epithelium (RPE) of aged human donors and in the pigmented Abca4-/- mouse model of Stargardt early-onset macular degeneration. Using high-resolution live-cell imaging, we show that age-related and pathological accumulation of lipofuscin bisretinoids increases ceramide at the apical surface of the RPE, which promotes inward budding and homotypic fusion of EEs. These enlarged endosomes internalize the complement protein C3 into the RPE, resulting in the intracellular generation of C3a fragments. Increased C3a in turn activates the mechanistic target of rapamycin (mTOR), a regulator of critical metabolic processes such as autophagy. The antidepressant desipramine, which decreases ceramide levels by inhibiting acid sphingomyelinase, corrects EE defects in the RPE of Abca4-/- mice. This prevents C3 internalization and limits the formation of C3a fragments within the RPE. Although uncontrolled complement activation is associated with macular degenerations, how complement contributes to pathology in a progressive disease is not well understood. Our studies link expansion of the EE compartment with intracellular complement generation and aberrant mTOR activation, which could set the stage for chronic metabolic reprogramming in the RPE as a prelude to disease. The pivotal role of ceramide in driving EE biogenesis and fusion in the Abca4-/- mice RPE suggests that therapeutic targeting of ceramide could be effective in Stargardt disease and other macular degenerations.
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Complemento C3a/metabolismo , Endosomas/metabolismo , Degeneración Macular/congénito , Epitelio Pigmentado de la Retina/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transportadoras de Casetes de Unión a ATP/deficiencia , Anciano , Anciano de 80 o más Años , Animales , Ceramidas/genética , Ceramidas/metabolismo , Complemento C3a/genética , Modelos Animales de Enfermedad , Endosomas/genética , Endosomas/patología , Femenino , Humanos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Masculino , Ratones , Ratones Noqueados , Epitelio Pigmentado de la Retina/patología , Enfermedad de Stargardt , Porcinos , Serina-Treonina Quinasas TOR/genéticaRESUMEN
BACKGROUND: Moderate-to-severe atopic dermatitis (AD) is increasingly recognized as a systemic disease, largely due to proteomic blood studies. There are growing efforts to develop AD biomarkers using minimal tissues. OBJECTIVE: To characterize the AD skin proteomic signature and its relationship with the blood proteome and genomic skin profile in the same individuals. METHODS: We evaluated lesional and nonlesional biopsy samples and blood from 20 individuals with moderate-to-severe AD and 28 healthy individuals using Olink Proteomics (Uppsala, Sweden), using 10 µg/10 µL for skin and blood and RNA sequencing of the skin. RESULTS: The AD skin proteome demonstrated significant upregulation in lesional and even in nonlesional skin compared with controls in inflammatory markers (matrix metalloproteinase 12; T-helper cell [Th]2/interleukin [IL]-1 receptor-like 1[IL1RL1]/IL-33R, IL-13, chemokine [C-C motif] ligand [CCL] 17; Th1/C-X-C motif chemokine 10; Th17/Th22/PI3, CCL20, S100A12), and in cardiovascular-associated proteins (E-selectin, matrix metalloproteinases, platelet growth factor, myeloperoxidase, fatty acid binding protein 4, and vascular endothelial growth factor A; false discovery rate, <0.05). Skin proteins demonstrated much higher and significant upregulations (vs controls) compared with blood, suggesting a skin source for the inflammatory/cardiovascular profile. Gene and protein expressions were correlated (r = 0.410, P < .001), with commonly upregulated inflammatory and cardiovascular risk-associated products, suggesting protein translation in skin. LIMITATIONS: Our analysis was limited to 354 proteins. CONCLUSIONS: The AD skin proteome shows an inflammatory and cardiovascular signature even in nonlesional skin, emphasizing the need for proactive treatment. Skin proteomics presents a sensitive option for biomarker monitoring.
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Dermatitis Atópica/genética , Proteómica , Adolescente , Adulto , Anciano , Biomarcadores/sangre , Dermatitis Atópica/sangre , Dermatitis Atópica/patología , Femenino , Humanos , Inflamación/sangre , Masculino , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Piel/patología , Adulto JovenRESUMEN
Cerebellum development involves the coordinated production of multiple neuronal cell types. The cerebellar primordium contains two germinative zones, the rhombic lip (RL) and the ventricular zone (VZ), which generate different types of glutamatergic and GABAergic neurons, respectively. What regulates the specification and production of glutamatergic and GABAergic neurons as well as the subtypes for each of these two broad classes remains largely unknown. Here we demonstrate with conditional genetic approaches in mice that SMAD4, a major mediator of BMP and TGFß signaling, is required early in cerebellar development for maintaining the RL and generating subsets of RL-derived glutamatergic neurons, namely neurons of the deep cerebellar nuclei, unipolar brush cells, and the late cohort of granule cell precursors (GCPs). The early cohort of GCPs, despite being deficient for SMAD4, is still generated. In addition, the numbers of GABAergic neurons are reduced in the mutant and the distribution of Purkinje cells becomes abnormal. These studies demonstrate a temporally and spatially restricted requirement for SMAD4 in generating subtypes of cerebellar neurons.
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Cerebelo/citología , Cerebelo/embriología , Neuronas/citología , Proteína Smad4/fisiología , Animales , Diferenciación Celular , Núcleos Cerebelosos/citología , Núcleos Cerebelosos/embriología , Cerebelo/fisiología , Ratones , Neuronas/fisiología , Células de Purkinje/citología , Células de Purkinje/fisiología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
The olfactory sensory epithelium and the respiratory epithelium are derived from the olfactory placode. However, the molecular mechanisms regulating the differential specification of the sensory and the respiratory epithelium have remained undefined. To address this issue, we first identified Msx1/2 and Id3 as markers for respiratory epithelial cells by performing quail chick transplantation studies. Next, we established chick explant and intact chick embryo assays of sensory/respiratory epithelial cell differentiation and analyzed two mice mutants deleted of Bmpr1a;Bmpr1b or Fgfr1;Fgfr2 in the olfactory placode. In this study, we provide evidence that in both chick and mouse, Bmp signals promote respiratory epithelial character, whereas Fgf signals are required for the generation of sensory epithelial cells. Moreover, olfactory placodal cells can switch between sensory and respiratory epithelial cell fates in response to Fgf and Bmp activity, respectively. Our results provide evidence that Fgf activity suppresses and restricts the ability of Bmp signals to induce respiratory cell fate in the nasal epithelium. In addition, we show that in both chick and mouse the lack of Bmp or Fgf activity results in disturbed placodal invagination; however, the fate of cells in the remaining olfactory epithelium is independent of morphological movements related to invagination. In summary, we present a conserved mechanism in amniotes in which Bmp and Fgf signals act in an opposing manner to regulate the respiratory versus sensory epithelial cell fate decision.
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Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Mucosa Olfatoria/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Células Receptoras Sensoriales/efectos de los fármacos , Animales , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/fisiología , Células CHO , Diferenciación Celular/genética , Células Cultivadas , Embrión de Pollo , Cricetinae , Cricetulus , Antagonismo de Drogas , Embrión de Mamíferos , Embrión no Mamífero , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/fisiología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Ratones , Modelos Biológicos , Mucosa Olfatoria/metabolismo , Mucosa Olfatoria/fisiología , Vías Olfatorias/efectos de los fármacos , Vías Olfatorias/metabolismo , Vías Olfatorias/fisiología , Codorniz/embriología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/fisiología , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
MET is a receptor tyrosine kinase that is activated in many cancers through various mechanisms. MET exon 14 (Ex14) skipping occurs in 3% of nonsmall cell lung tumors. However, the contribution of the regulatory sites lost upon this skipping, which include a phosphorylated serine (S985) and a binding site for the E3 ubiquitin ligase CBL (Y1003), remains elusive. Sequencing of 2808 lung tumors revealed 71 mutations leading to MET exon 14 skipping and three mutations affecting Y1003 or S985. In addition, MET exon 14 skipping and MET Y1003F induced similar transcriptional programs, increased the activation of downstream signaling pathways, and increased cell mobility. Therefore, the MET Y1003F mutation is able to fully recapitulate responses induced by MET exon 14 skipping, suggesting that loss of the CBL binding site is the main contributor of cell transformation induced by MET Ex14 mutations.
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Neoplasias Pulmonares , Proteínas Proto-Oncogénicas c-met , Humanos , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Neoplasias Pulmonares/genética , Mutación , Exones/genética , Sitios de Unión , Ubiquitinas/genética , Ligasas/metabolismoRESUMEN
Importance: The pathogenesis of eosinophilic cellulitis (EC) is poorly understood, limiting available treatment options. The current treatment paradigm focuses on delayed type 2 hypersensitivity reaction to various triggers. Objective: To gain further insight into the nature of EC inflammation and into the cellular signal transduction pathways that are activated in the context of EC. Design, Setting, and Participants: This case series was conducted in Lyon, France, from January 2018 to December 2021. Analysis of archival skin biopsy samples from patients with EC and from healthy control participants was performed using histology, Janus kinase (JAK)-signal transducer and activator of transcription (STAT) immunohistochemistry, and gene profiling. Data analysis was conducted between January 2020 and January 2022. Main Outcomes and Measures: Pruritus (visual analog score), percentage of body surface area with lesional skin, and RNA transcripts of inflammatory biomarkers from the skin (threshold cycle) were assessed in 1 index patient with refractory EC who received oral JAK1/JAK2 inhibitor baricitinib (4 mg/d). Results: This study included samples from 14 patients with EC (7 men and 7 women) and 8 healthy control participants (4 men and 4 women). The mean (SD) age of patients was 52 (20) years. Marked type 2 inflammation (chemokines CCL17, CCL18, and CCL26 and interleukin 13) with preferential activation of the JAK1/JAK2-STAT5 pathways in EC lesions was observed. In the 1 index patient with refractory EC, complete clinical remission of skin lesions was observed after 1 month of treatment with baricitinib. Conclusions and Relevance: These findings suggest that EC is a type 2 inflammatory disease with preferential activation of the JAK1/JAK2-STAT5 pathways. In addition, these results suggest the potential of treatment approaches targeting JAK1/JAK2 for patients with EC.
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Factor de Transcripción STAT5 , Transducción de Señal , Femenino , Humanos , Persona de Mediana Edad , Inflamación , Janus Quinasa 1/metabolismo , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT5/metabolismo , Masculino , Adulto , AncianoRESUMEN
In hereditary papillary renal cell carcinoma (HPRCC), the MET receptor tyrosine kinase (RTK) mutations recorded to date are located in the kinase domain and lead to constitutive MET activation. This contrasts with MET mutations recently identified in non-small cell lung cancer (NSCLC), which lead to exon 14 skipping and deletion of a regulatory domain: in this latter case, the mutated receptor still requires ligand stimulation. Sequencing of MET in samples from 158 HPRCC and 2808 NSCLC patients revealed ten uncharacterized mutations. Four of these, all found in HPRCC and leading to amino acid substitutions in the N-lobe of the MET kinase, proved able to induce cell transformation, further enhanced by HGF stimulation: His1086Leu, Ile1102Thr, Leu1130Ser, and Cis1125Gly. Similar to the variant resulting in MET exon14 skipping, the two N-lobe MET variants His1086Leu, Ile1102Thr further characterized were found to require stimulation by HGF in order to strongly activate downstream signaling pathways and epithelial cell motility. The Ile1102Thr mutation displayed also transforming potential, promoting tumor growth in a xenograft model. In addition, the N-lobe-mutated MET variants were found to trigger a common HGF-stimulation-dependent transcriptional program, consistent with an observed increase in cell motility and invasion. Altogether, this functional characterization revealed that N-lobe variants still require ligand stimulation, in contrast to other RTK variants. This suggests that HGF expression in the tumor microenvironment is important for tumor growth. The sensitivity of these variants to MET TKIs opens the way for use of targeted therapies for patients harboring the corresponding mutations.
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Exon skipping mutations of the MET receptor tyrosine kinase (METex14), increasingly reported in cancers, occur in 3-4% of non-small-cell lung cancer (NSCLC). Only 50% of patients have a beneficial response to treatment with MET-tyrosine kinase inhibitors (TKIs), underlying the need to understand the mechanism of METex14 oncogenicity and sensitivity to TKIs. Whether METex14 is a driver mutation and whether it requires hepatocyte growth factor (HGF) for its oncogenicity in a range of in vitro functions and in vivo has not been fully elucidated from previous preclinical models. Using CRISPR/Cas9, we developed a METex14/WT isogenic model in nontransformed human lung cells and report that the METex14 single alteration was sufficient to drive MET-dependent in vitro anchorage-independent survival and motility and in vivo tumorigenesis, sensitising tumours to MET-TKIs. However, we also show that human HGF (hHGF) is required, as demonstrated in vivo using a humanised HGF knock-in strain of mice and further detected in tumour cells of METex14 NSCLC patient samples. Our results also suggest that METex14 oncogenicity is not a consequence of an escape from degradation in our cell model. Thus, we developed a valuable model for preclinical studies and present results that have potential clinical implication.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/patología , Exones , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Pulmonares/patología , Mutación/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , RatonesRESUMEN
Although targeted therapies have increased the life expectancy of patients with druggable molecular alterations directly involved in tumor development, the efficacy of these therapies is limited by acquired resistances leading to treatment failure. Most targeted therapies, including ones exploiting therapeutic antibodies and kinase inhibitors, are directed against receptor tyrosine kinases (RTKs) or major signaling hubs. Resistances to these therapies arise when inhibition of these targets is bypassed through activation of alternative signaling pathways. In recent years, activation of the receptor tyrosine kinase MET has been shown to promote resistance to various targeted therapies. This casts MET as important actor in resistance. In this review, we describe how the MET receptor triggers resistance to targeted therapies against RTKs such as EGFR, VEGFR, and HER2 and against signaling hubs such as BRAF. We also describe how MET can be its own resistance factor, as illustrated by on-target resistance of lung tumors harboring activating mutations causing MET exon 14 skipping. Interestingly, investigation of all these situations reveals functional physiological relationships between MET and the target of the therapy to which the cancer becomes resistant, suggesting that resistance stems from preexisting mechanisms. Identification of MET as a resistance factor opens the way to co-treatment strategies that are being tested in current clinical trials.
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Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Exones/efectos de los fármacos , Humanos , Terapia Molecular Dirigida/métodosRESUMEN
Epithelial to mesenchymal transition (EMT) is a biological process involved in tissue morphogenesis and disease that causes dramatic changes in cell morphology, migration, proliferation, and gene expression. The retinal pigment epithelium (RPE), which supports the neural retina, can undergo EMT, producing fibrous epiretinal membranes (ERMs) associated with vision-impairing clinical conditions, such as macular pucker and proliferative vitreoretinopathy (PVR). We found that co-treatment with TGF-ß and TNF-α (TNT) accelerates EMT in adult human RPE stem cell-derived RPE cell cultures. We captured the global epigenomic and transcriptional changes elicited by TNT treatment of RPE and identified putative active enhancers associated with actively transcribed genes, including a set of upregulated transcription factors that are candidate regulators. We found that the vitamin B derivative nicotinamide downregulates these key transcriptional changes, and inhibits and partially reverses RPE EMT, revealing potential therapeutic routes to benefit patients with ERM, macular pucker and PVR.
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Epigenómica , Membrana Epirretinal/prevención & control , Transición Epitelial-Mesenquimal , Modelos Biológicos , Niacinamida/uso terapéutico , Epitelio Pigmentado de la Retina/patología , Células Madre/metabolismo , Transcriptoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Elementos de Facilitación Genéticos/genética , Membrana Epirretinal/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Niacinamida/genética , Niacinamida/farmacología , Fenotipo , Células Madre/efectos de los fármacos , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Hepatocyte growth factor receptor (MET) tyrosine kinase inhibitors (MET TKIs) have been found to have efficacy against advanced NSCLC with mutations causing MET exon 14 skipping (METex14 mutations), but primary resistance seems frequent, as response rates are lower than those for targeted TKIs of other oncogene-addicted NSCLCs. Given the known interplay between MET and phosphoinositide 3-kinases (PI3K), we hypothesized that in METex14 NSCLC, PI3K pathway alterations might contribute to primary resistance to MET TKIs. We reviewed clinical data from 65 patients with METex14 NSCLC, assessing PI3K pathway alterations by targeted next-generation sequencing (mutations) and immunohistochemistry (loss of phosphatase and tensin homolog [PTEN]). Using a cell line derived from a patient with primary resistance to a MET TKI and cell lines harboring both a METex14 mutation and a PI3K pathway alteration, we assessed sensitivity to MET TKIs used alone or with a PI3K inhibitor and investigated relevant signaling pathways. We found a phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutation in two of 65 samples (3%) and loss of PTEN in six of 26 samples (23%). All three of the MET TKI-treated patients with a PI3K pathway alteration had been found to have progressive disease at first assessment. Likewise, MET TKIs had no effect on the proliferation of METex14-mutated cell lines with a PI3K pathway alteration, including the PTEN-lacking patient-derived cell line. Treatment combining a MET TKI with a PI3K inhibitor caused inhibition of both PI3K and MAPK signaling and restored sensitivity to MET TKIs. PI3K pathway alterations are common in METex14 NSCLC and may confer primary resistance to MET TKIs. In preclinical models, PI3K inhibition restores sensitivity to MET TKIs.
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Neoplasias Pulmonares , Fosfatidilinositol 3-Quinasas , Resistencia a Antineoplásicos/genética , Exones , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Membrane-anchored full-length MET stimulated by its ligand HGF/SF induces various biological responses, including survival, growth, and invasion. This panel of responses, referred to invasive growth, is required for embryogenesis and tissue regeneration in adults. On the contrary, MET deregulation is associated with tumorigenesis in many kinds of cancer. In addition to its well-documented ligand-stimulated downstream signaling, the receptor can be cleaved by proteases such as secretases, caspases, and calpains. These cleavages are involved either in MET receptor inactivation or, more interestingly, in generating active fragments that can modify cell fate. For instance, MET fragments can promote cell death or invasion. Given a large number of proteases capable of cleaving MET, this receptor appears as a prototype of proteolytic-cleavage-regulated receptor tyrosine kinase. In this review, we describe and discuss the mechanisms and consequences, both physiological and pathological, of MET proteolytic cleavages. [BMB Reports 2019; 52(4): 239-249].
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Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Proto-Oncogénicas c-met/fisiología , Animales , Apoptosis , Calpaína/metabolismo , Caspasas/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/fisiología , Humanos , Ligandos , Necrosis/metabolismo , Neoplasias/metabolismo , Proteolisis , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Proliferative vitreoretinopathy (PVR) is a metaplasia in the vitreous of the eye manifested by the transformation of retinal pigment epithelial (RPE) cells and the development of contracting epiretinal membranes (ERM), which lead to retinal detachment and vision loss. While TGFß1 and TNFα have been associated with PVR, here we show that these cytokines act synergistically to induce an aggressive contraction phenotype on adult human (ah)RPE. Connected RPE detach upon contraction and form motile membranes that recruit more cells. TGFß1 and TNFα (TNT)-induced contracting membranes uniquely express muscle and extracellular rearrangement genes. Whole transcriptome RNA sequencing of patient-dissected PVR membranes showed activation of the p38-MAPK signaling pathway. Inhibition of p38 during TNT treatment blocks ahRPE transformation and membrane contraction. Furthermore, TNT-induced membrane contractility can be reversed by p38 inhibition after induction. Therefore, targeting the p38-MAPK pathway may have therapeutic benefits for patients with PVR even after the onset of contracting ERMs.
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Membrana Epirretinal/genética , Desprendimiento de Retina/genética , Factor de Crecimiento Transformador beta1/genética , Factor de Necrosis Tumoral alfa/genética , Vitreorretinopatía Proliferativa/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Adulto , Anciano , Anciano de 80 o más Años , Movimiento Celular , Membrana Epirretinal/metabolismo , Membrana Epirretinal/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Desprendimiento de Retina/metabolismo , Desprendimiento de Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Transducción de Señal , Imagen de Lapso de Tiempo , Transcriptoma , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/patología , Cuerpo Vítreo/metabolismo , Cuerpo Vítreo/patología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Importance: Molecular profiling of skin biopsies is the criterion standard for evaluating the cutaneous atopic dermatitis (AD) phenotype. However, skin biopsies are not always feasible in children. A reproducible minimally invasive approach that can track cutaneous disease in pediatric longitudinal studies or clinical trials is lacking. Objective: To assess a minimally invasive approach using tape strips to identify skin biomarkers that may serve as a surrogate to biomarkers identified using whole-tissue biopsies. Design, Setting, and Participants: This cross-sectional study of 51 children younger than 5 years recruited children with moderate to severe AD and children without AD from the dermatology outpatient clinics at a children's hospital. Sixteen tape strips were serially collected from the nonlesional and lesional skin of 21 children who had AD and were less than 6 months from disease initiation and from the normal skin of 30 children who did not have AD between January 22, 2016, and April 20, 2018. Main Outcomes and Measures: Gene and protein expression were evaluated using quantitative real-time polymerase chain reaction and immunohistochemistry. Results: A total of 51 children younger than 5 years were included in the study; 21 children had moderate to severe AD with less than 6 months of disease duration, and 30 children did not have AD. Of the 21 children with AD, the mean (SD) age was 1.7 (1.7) years, and most were male (15 [71.4%] and white (15 [71.4%]). Of the 30 children without AD, the mean (SD) age was 1.8 (2.0) years, and most were female (20 [66.7%]) and white (22 [73.3%]). Seventy-seven of 79 evaluated immune and barrier gene products were detected (gene detection rate, 97%) in 70 of 71 tape strips (sample detection rate, 99%), with 53 of 79 markers differentiating between children with lesional and/or nonlesional AD from children without AD. Many cellular markers of T cells (CD3), AD-related dendritic cells (Fc ε RI and OX40 ligand receptors), and key inflammatory (matrix metallopeptidase 12), innate (interleukin 8 [IL-8] and IL-6), helper T cell 2 (TH2; IL-4, IL-13, and chemokines CCL17 and CCL26), and TH17/TH22 (IL-19, IL-36G, and S100A proteins) genes were significantly increased in lesional and nonlesional AD compared with tape strips from normal skin. For example, IL-4 mean (SE) for lesional was -15.2 (0.91) and normal was -19.5 (0.48); P < .001. Parallel decreases occurred in epidermal barrier gene products (FLG, CLDN23, and FA2H) and negative immune regulators (IL-34 and IL-37). For example, the decrease for FLG lesional was mean (SE) -2.9 (0.42) and for normal was 2.2 (0.45); P < .001. Associations were found between disease severity or transepidermal water loss and TH2 (IL-33 and IL-4R) and TH17/TH22 (IL-36G and S100As) products in lesional and nonlesional AD skin (evaluated using the SCORing Atopic Dermatitis, Eczema Area and Severity Index, and Pruritus Atopic Dermatitis Quickscore tools). Conclusions and Relevance: In this study, tape strips provide a minimally invasive alternative for serially evaluating AD-associated cutaneous biomarkers and may prove useful for tracking pediatric AD therapeutic response and predicting future course and comorbidities.
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Dermatitis Atópica/diagnóstico , Piel/patología , Edad de Inicio , Biomarcadores/análisis , Biomarcadores/metabolismo , Biopsia/métodos , Preescolar , Estudios Transversales , Dermatitis Atópica/inmunología , Dermatitis Atópica/patología , Estudios de Factibilidad , Femenino , Proteínas Filagrina , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa , Índice de Severidad de la Enfermedad , Piel/inmunología , Pérdida Insensible de Agua/inmunologíaRESUMEN
Cajal-Retzius (CR) cells, the earliest-born neurons in the neocortex, arise from discrete sources within the telencephalon, including the dorsal midline and the pallial-subpallial boundary (PSB). In particular, the cortical hem, a region of high bone morphogenetic proteins (BMPs) and Wnt (wingless-type MMTV integration site family) expression but lacking in Foxg1 (forkhead box G1) is a major source of CR neurons. Whether CR cells from distinct origins arise from disparate developmental processes or share a common mechanism is unclear. To elucidate the molecular basis of CR cell development, we assessed the role of both Foxg1 and dorsal midline signaling in the production of cortical hem- and PSB-derived CR cells. We demonstrate that the loss of Foxg1 results in the overproduction of both of these CR populations. However, removal of Foxg1 at embryonic day 13, although expanding the number of CR cells with a PSB phenotype, does not result in an expansion of BMPs or Wnts in the dorsomedial signaling center. Conversely, loss of the dorsal midline ligands as observed in Gli3 (glioma-associated oncogene homolog 3) mutants results in the loss of the cortical hem-derived CR character but does not affect the specification of PSB-derived CR cells. Hence, our findings demonstrate that, although the specification of cortical hem-derived CR cells is dependent on signaling from the dorsal midline, Foxg1 functions to repress the generation of both cortical hem- and PSB-derived CR cells.
Asunto(s)
Diferenciación Celular/fisiología , Factores de Transcripción Forkhead/fisiología , Neocórtex/citología , Neocórtex/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuronas/citología , Neuronas/fisiología , Transducción de Señal/fisiología , Animales , Proteínas Morfogenéticas Óseas/biosíntesis , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neocórtex/embriología , Embarazo , Telencéfalo/citología , Telencéfalo/embriología , Telencéfalo/metabolismo , Telencéfalo/fisiología , Proteínas Wnt/biosíntesisRESUMEN
An adult human retinal pigment epithelial layer (ahRPE) model derived from stem cells isolated from native RPE monolayers (ahRPE-SCs) exhibits key physiological characteristics of native tissue and therefore provides the means to create a human "disease in a dish" model to study RPE diseases. Traditionally, RPE lines are established from whole globes dedicated to research. Here we describe a new technique for establishing primary RPE lines from the posterior poles of globes used for corneal transplants. Since tissues from corneal transplants are derived from younger and healthier donors than those used for research, we have hypothesized that RPE cells isolated from corneal transplantation globes will result in improved primary RPE line establishment. Our new procedure increases the rate of establishing successful RPE cultures and improves the total cell number yield. Use of this advanced methodology can provide a new source of high-quality primary RPE line cultures. © 2018 by John Wiley & Sons, Inc.
Asunto(s)
Trasplante de Córnea , Modelos Biológicos , Epitelio Pigmentado de la Retina/citología , Células Madre/citología , Adulto , Proliferación Celular , Separación Celular , Impedancia Eléctrica , Células Epiteliales/citología , Humanos , Fenotipo , Células Madre/metabolismoRESUMEN
Holoprosencephaly (HPE) is a devastating forebrain abnormality with a range of morphological defects characterized by loss of midline tissue. In the telencephalon, the embryonic precursor of the cerebral hemispheres, specialized cell types form a midline that separates the hemispheres. In the present study, deletion of the BMP receptor genes, Bmpr1b and Bmpr1a, in the mouse telencephalon results in a loss of all dorsal midline cell types without affecting the specification of cortical and ventral precursors. In the holoprosencephalic Shh(-/-) mutant, by contrast, ventral patterning is disrupted, whereas the dorsal midline initially forms. This suggests that two separate developmental mechanisms can underlie the ontogeny of HPE. The Bmpr1a;Bmpr1b mutant provides a model for a subclass of HPE in humans: midline inter-hemispheric HPE.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Holoprosencefalia/metabolismo , Mutación/genética , Transducción de Señal , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/deficiencia , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/genética , Encéfalo/embriología , Encéfalo/metabolismo , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Holoprosencefalia/embriología , Holoprosencefalia/genética , Masculino , Ratones , Ratones Transgénicos , FenotipoRESUMEN
Sonic hedgehog (SHH) is required to generate ventral cell types throughout the central nervous system. Its role in directly specifying ventral cells, however, has recently been questioned because loss of the Shh gene has little effect on ventral development if the Gli3 gene is also mutant. Consequently, another ventral determinant must exist. Here, genetic evidence establishes that FGFs are required for ventral telencephalon development. First, simultaneous deletion of Fgfr1 and Fgfr3 specifically in the telencephalon results in the loss of differentiated ventromedial cells; and second, in the Fgfr1;Fgfr2 double mutant, ventral precursor cells are lost, mimicking the phenotype obtained previously with a loss of SHH signalling. Yet, in the Fgfr1;Fgfr2 mutant, Shh remains expressed, as does Gli1, the transcription of which depends on SHH activity, suggesting that FGF signalling acts independently of SHH to generate ventral precursors. Moreover, the Fgfr1;Fgfr2 phenotype, unlike the Shh phenotype, is not rescued by loss of Gli3, further indicating that FGFs act downstream of Shh and Gli3 to generate ventral telencephalic cell types.