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1.
Nat Commun ; 12(1): 2183, 2021 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-33846348

RESUMEN

Here we show that FTO as an N6-methyladenosine (m6A) RNA demethylase is degraded by selective autophagy, which is impaired by low-level arsenic exposure to promote tumorigenesis. We found that in arsenic-associated human skin lesions, FTO is upregulated, while m6A RNA methylation is downregulated. In keratinocytes, chronic relevant low-level arsenic exposure upregulated FTO, downregulated m6A RNA methylation, and induced malignant transformation and tumorigenesis. FTO deletion inhibited arsenic-induced tumorigenesis. Moreover, in mice, epidermis-specific FTO deletion prevented skin tumorigenesis induced by arsenic and UVB irradiation. Targeting FTO genetically or pharmacologically inhibits the tumorigenicity of arsenic-transformed tumor cells. We identified NEDD4L as the m6A-modified gene target of FTO. Finally, arsenic stabilizes FTO protein through inhibiting p62-mediated selective autophagy. FTO upregulation can in turn inhibit autophagy, leading to a positive feedback loop to maintain FTO accumulation. Our study reveals FTO-mediated dysregulation of mRNA m6A methylation as an epitranscriptomic mechanism to promote arsenic tumorigenicity.


Asunto(s)
Adenosina/análogos & derivados , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Arsénico/toxicidad , Autofagia , Carcinogénesis/genética , Adenosina/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Secuencia de Bases , Carcinogénesis/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/patología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Epidermis/metabolismo , Ontología de Genes , Células HEK293 , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , FN-kappa B/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Estabilidad Proteica/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína Sequestosoma-1/metabolismo , Transcriptoma/genética , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo , Vacuolas/ultraestructura
2.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33802009

RESUMEN

Olfactory receptors (ORs) have diverse physiological roles in various cell types, beyond their function as odorant sensors in the olfactory epithelium. These previous findings have suggested that ORs could be diagnostic markers and promising therapeutic targets in several pathological conditions. In the current study, we sought to characterize the changes in the expression of ORs in the HaCaT human keratinocytes cell line exposed to ultraviolet (UV) light or inflammation, well-recognized stimulus for skin barrier disruption. We confirmed that major olfactory signaling components, including ORs, GNAL, Ric8b, and adenylate cyclase type 3, are highly expressed in HaCaT cells. We have also demonstrated that the 12 ectopic ORs detectable in HaCaT cells are more highly expressed in UV-irradiated or inflamed conditions than in normal conditions. We further assessed the specific OR-mediated biological responses of HaCaT cells in the presence of known odorant ligands of ORs and observed that specific ligand-activated ORs downregulate skin barrier genes in HaCaT cells. This study shows the potential of OR as a marker for skin barrier abnormalities. Further research is needed to explore how OR is implicated in the development and progression of barrier dysfunction.


Asunto(s)
Regulación de la Expresión Génica/efectos de la radiación , Inflamación/genética , Queratinocitos/efectos de la radiación , Receptores Odorantes/genética , Piel/efectos de la radiación , Rayos Ultravioleta , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Línea Celular , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Humanos , Inflamación/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Receptores Odorantes/metabolismo , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Piel/metabolismo , Piel/patología
3.
Int J Mol Sci ; 22(6)2021 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-33808676

RESUMEN

Melanin granules cluster within supra-nuclear caps in basal keratinocytes (KCs) of the human epidermis, where they protect KC genomic DNA against ultraviolet radiation (UVR) damage. While much is known about melanogenesis in melanocytes (MCs) and a moderate amount about melanin transfer from MC to KC, we know little about the fate of melanin once inside KCs. We recently reported that melanin fate in progenitor KCs is regulated by rare asymmetric organelle movement during mitosis. Here, we explore the role of actin, microtubules, and centrosome-associated machinery in distributing melanin within KCs. Short-term cultures of human skin explants were treated with cytochalasin-B and nocodazole to target actin filaments and microtubules, respectively. Treatment effects on melanin distribution were assessed by the Warthin-Starry stain, on centrosome-associated proteins by immunofluorescence microscopy, and on co-localisation with melanin granules by brightfield microscopy. Cytochalasin-B treatment disassembled supra-nuclear melanin caps, while nocodazole treatment moved melanin from the apical to basal KC domain. Centrosome and centriolar satellite-associated proteins showed a high degree of co-localisation with melanin. Thus, once melanin granules are transferred to KCs, their preferred apical distribution appears to be facilitated by coordinated movement of centrosomes and centriolar satellites. This mechanism may control melanin's strategic position within UVR-exposed KCs.


Asunto(s)
Melaninas/metabolismo , Piel/metabolismo , Actinas/metabolismo , Biomarcadores , Polaridad Celular , Células Cultivadas , Centrosoma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Citoesqueleto/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Hibridación in Situ , Queratinocitos/metabolismo , Melanocitos/metabolismo , Fenotipo
4.
DNA Cell Biol ; 40(3): 523-531, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33687273

RESUMEN

Antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as or ciRS-7) is an important member of the circular RNA family and is involved in the regulation of numerous biological functions. Keratinocytes and fibroblasts (FBs) affect melanogenesis through paracrine effects. However, whether ciRS-7 is involved in melanogenesis by regulating paracrine effects remains unclear. This study demonstrates for the first time that ciRS-7 is highly expressed in keratinocytes, FBs, and melanocytes (MCs). Ultraviolet B (UVB) irradiation promotes ciRS-7 expression in keratinocytes and FBs. Following inhibition of ciRS-7 expression in keratinocytes and FBs, the culture supernatant from these cells inhibited melanogenesis of MCs. Further analyses revealed that the expression and secretion of fibroblast growth factor 2 (FGF2) and phosphorylation of STAT3 and AKT in keratinocytes and FBs were significantly downregulated following inhibition of ciRS-7 expression, whereas the level of miR-7 was increased. Overexpression of miR-7 in keratinocytes and FBs significantly inhibited the expression of FGF2. In conclusion, our findings demonstrate that UVB-induced ciRS-7 triggers melanogenesis in MCs through regulation of the miR-7/STAT3 and AKT/FGF2 paracrine axis in both keratinocytes and FBs. ciRS-7 may serve as a regulator in the development of pigmented skin diseases.


Asunto(s)
Regulación de la Expresión Génica/efectos de la radiación , Queratinocitos/metabolismo , Melaninas/biosíntesis , Comunicación Paracrina/efectos de la radiación , ARN Largo no Codificante/metabolismo , Transducción de Señal/efectos de la radiación , Rayos Ultravioleta , Línea Celular Transformada , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT3/metabolismo
5.
Methods Mol Biol ; 2265: 173-183, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704714

RESUMEN

Most currently available three-dimensional melanoma models have either focused on simplicity or were optimized for physiological relevance. Accordingly, these paradigms have been either composed of malignant cells only or they were sophisticated human skin equivalents featuring multiple cell types and skin-like organization. Here, an intermediate spheroid-based assay system is presented, which uses tri-cultures of human CCD-1137Sk fibroblasts, HaCaT keratinocytes, and SK-MEL-28 melanoma cells. Being made of cell lines, these spheroids can be reliably reproduced without any special equipment using standard culture procedures, and they feature different aspects of skin and early stage melanoma. Therefore, this kind of model can be useful for lead-compound testing or addressing fundamental principles of early melanoma formation.


Asunto(s)
Antineoplásicos/farmacología , Técnicas de Cocultivo/métodos , Fibroblastos/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Esferoides Celulares/metabolismo , Línea Celular Tumoral , Docetaxel/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Melanoma/metabolismo , Melanoma/patología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología
6.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-33672928

RESUMEN

Extensive water loss and melanin hyperproduction can cause various skin disorders. Low-temperature argon plasma (LTAP) has shown the possibility of being used for the treatment of various skin diseases, such as atopic dermatitis and skin cancer. However, the role of LTAP in regulating skin moisturizing and melanogenesis has not been investigated. In this study, we aimed to determine the effect of LTAP on yes-associated protein (YAP), a major transcriptional coactivator in the Hippo signaling pathway that is involved in skin moisturizing and melanogenesis-regulating markers. In normal human epidermal keratinocytes (NHEKs), the human epidermal keratinocyte line HaCaT, and human dermal fibroblasts (HDFs), we found that LTAP exhibited increased expression levels of YAP protein. In addition, the expression levels of filaggrin (FLG), which is involved in natural moisturizing factors (NMFs), and hyaluronic acid synthase (HAS), transglutaminase (TGM), and involucrin (IVL), which regulate skin barrier and moisturizing, were also increased after exposure to LTAP. Furthermore, collagen type I alpha 1 and type III alpha 1 (COL1A1, COL3A1) were increased after LTAP exposure, but the expression level of matrix metalloproteinase-3 (MMP-3) was reduced. Moreover, LTAP was found to suppress alpha-melanocyte stimulating hormone (α-MSH)-induced melanogenesis in murine melanoma B16F10 cells and normal human melanocytes (NHEMs). LTAP regulates melanogenesis of the melanocytes through decreased YAP pathway activation in a melanocortin 1 receptor (MC1R)-dependent manner. Taken together, our data show that LTAP regulates skin moisturizing and melanogenesis through modulation of the YAP pathway, and the effect of LTAP on the expression level of YAP varies from cell to cell. Thus, LTAP might be developed as a treatment method to improve the skin barrier, moisture content, and wrinkle formation, and to reduce melanin generation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Argón/farmacología , Melaninas/metabolismo , Gases em Plasma/farmacología , Piel/efectos de los fármacos , Factores de Transcripción/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Melanocitos/citología , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Ratones , Receptor de Melanocortina Tipo 1/metabolismo , Piel/metabolismo , Envejecimiento de la Piel/efectos de los fármacos , Temperatura , alfa-MSH/metabolismo
7.
Mol Med Rep ; 23(5)2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33760111

RESUMEN

Cholesteatoma constitutes an acquired benign epidermal non­permanent bone lesion that is locally destructive and patients often relapse. Inflammasomes, which mediate the maturation and production of IL­18 and IL­1ß, resulting in pyroptosis, have been documented to serve a core function in multiple inflammatory conditions. Absent in melanoma 2 (AIM2) is an inflammasome that identifies cytoplasmic DNA and has previously been reported as a pivotal modulator of inflammatory responses. Therefore, the present study aimed to determine the expression levels of AIM2 in human cholesteatoma tissues, and elucidate its function in modulating cytokine production. The expression levels of IL­18, apoptosis­associated speck­like protein containing a CARD (ASC), IL­1ß, AIM2 and caspase­1 were markedly elevated in cholesteatoma tissues. Protein expression levels of AIM2, caspase­1 and ASC were localized in the cellular cytoplasm, primarily in the granular and prickle­cell layers in the cholesteatoma epithelium. Induction using IFN­Î³, as well as cytoplasmic DNA markedly activated the AIM2 inflammasome and elevated the release of IL­18 and IL­1ß in human cholesteatoma keratinocytes. IFN­Î³ was found to enhance poly(dA:dT)­induced pyroptosis of cells and cytokine production. The results of the present study revealed that AIM2 expressed in human cholesteatoma serves a vital function in the inflammatory response by initiating the inflammasome signaling cascade in cholesteatoma.


Asunto(s)
Neoplasias Óseas/genética , Colesteatoma/genética , Proteínas de Unión al ADN/genética , Interleucina-18/genética , Interleucina-1beta/genética , Animales , Apoptosis/efectos de los fármacos , Neoplasias Óseas/patología , Proteínas Adaptadoras de Señalización CARD/genética , Caspasa 1/genética , Colesteatoma/patología , Citocinas/biosíntesis , Citocinas/genética , Citoplasma/genética , ADN/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inflamasomas/genética , Interferón gamma/genética , Interleucina-18/biosíntesis , Interleucina-1beta/biosíntesis , Queratinocitos/metabolismo , Neoplasias/genética , Neoplasias/patología , Poli dA-dT/farmacología , Piroptosis/efectos de los fármacos , Piroptosis/genética
8.
Mol Med Rep ; 23(5)2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33760132

RESUMEN

Hair loss, including alopecia, is a common and distressing problem for men and women, and as a result, there is considerable interest in developing treatments that can prevent or reverse hair loss. Dermal papillae closely interact with epidermal cells and play a key role during hair follicle induction and hair morphogenesis. As dermal papilla cells (DPCs) lose their hair­inducing ability in monolayer cultures in vitro, it is difficult to obtain de novo hair follicle structures following DPC transplantation in vivo. The present study aimed to explore culture conditions to maintain DPC characteristics using conditioned media (CM) from the supernatant of cultured HaCaT keratinocyte cells supplemented with other components. Initially, it was observed that during passaging of in vitro monolayer DPC cultures, the Wnt/ß­catenin pathway was repressed, while the TGF­ß/Smad pathway was activated, and that HaCaT cells cultivated in 1% fetal bovine serum had higher levels of expression of Wnt3a and Wnt10b compared with normal keratinocytes. Culturing of high­passage (P7) DPCs in CM from HaCaT cells (HaCaT­CM) actively stimulated cell proliferation and maintained Sox2 and Versican expression levels. Supplementation of HaCaT­CM with SB431542 (SB, a TGF­ß receptor inhibitor), CHIR99021, (CHIR, a GSK3α/ß inhibitor and activator of Wnt signaling) and platelet­derived growth factor (PDGF)­AA further increased the expression levels of Sox2, Versican and alkaline phosphatase (ALP) in P7 DPCs. Three­dimensional culture of P7 DPCs using hanging drop cultures in HaCaT­CM supplemented with SB, CHIR and PDGF­AA resulted in larger cell aggregates and a further significant upregulation of Sox2, ALP and Versican expression levels. Taken together, these findings demonstrated that HaCaT­CM supplemented with SB, CHIR and PDGF­AA may preserve the hair­inducing ability of high­passage DPCs and may therefore be useful in reconstructing new hair follicles in vivo.


Asunto(s)
Alopecia/genética , Desdiferenciación Celular/efectos de los fármacos , Dermis/crecimiento & desarrollo , Factor de Crecimiento Derivado de Plaquetas/genética , Alopecia/tratamiento farmacológico , Alopecia/patología , Benzamidas/farmacología , Técnicas de Cultivo de Célula , Medios de Cultivo Condicionados , Dermis/citología , Dioxoles/farmacología , Cabello/crecimiento & desarrollo , Cabello/metabolismo , Folículo Piloso/citología , Folículo Piloso/crecimiento & desarrollo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Vía de Señalización Wnt/efectos de los fármacos
9.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-33670118

RESUMEN

Inherited ichthyoses represent a large heterogeneous group of skin disorders characterised by impaired epidermal barrier function and disturbed cornification. Current knowledge about disease mechanisms has been uncovered mainly through the use of mouse models or human skin organotypic models. However, most mouse lines suffer from severe epidermal barrier defects causing neonatal death and human keratinocytes have very limited proliferation ability in vitro. Therefore, the development of disease models based on patient derived human induced pluripotent stem cells (hiPSCs) is highly relevant. For this purpose, we have generated hiPSCs from patients with congenital ichthyosis, either non-syndromic autosomal recessive congenital ichthyosis (ARCI) or the ichthyosis syndrome trichothiodystrophy (TTD). hiPSCs were successfully differentiated into basal keratinocyte-like cells (hiPSC-bKs), with high expression of epidermal keratins. In the presence of higher calcium concentrations, terminal differentiation of hiPSC-bKs was induced and markers KRT1 and IVL expressed. TTD1 hiPSC-bKs showed reduced expression of FLG, SPRR2B and lipoxygenase genes. ARCI hiPSC-bKs showed more severe defects, with downregulation of several cornification genes. The application of hiPSC technology to TTD1 and ARCI demonstrates the successful generation of in vitro models mimicking the disease phenotypes, proving a valuable system both for further molecular investigations and drug development for ichthyosis patients.


Asunto(s)
Regulación de la Expresión Génica , Ictiosis/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Queratinocitos/metabolismo , Modelos Biológicos , Niño , Preescolar , Femenino , Humanos , Ictiosis/patología , Células Madre Pluripotentes Inducidas/patología , Lactante , Queratinocitos/patología , Masculino
10.
Methods Mol Biol ; 2269: 175-201, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687680

RESUMEN

Bench-to-bedside axis of therapeutic product development is currently being oriented towards minimum invasiveness on both ends-not only clinical application but harvesting of the starting biological material as well. This is particularly relevant for Advanced Therapy Medicinal Products and their specific legislative requirements, even more so in skin regeneration. It is precisely the skin equivalents and grafts that benefit from the minimum-to-noninvasive approach to a noteworthy extent, taking in account the sensitive nature of both skin harvesting and grafting.This chapter includes protocols for two separate steps of generating skin equivalent from the cells cultured from hair follicle outer root sheath. The first step is a non-pigmented epidermal equivalent generated from human keratinocytes from the outer root sheath named non-pigmented epidermal graft. The second step consists of co-cultivating human keratinocytes and human melanocytes from the outer root sheath, hereby producing a pigmented epidermal graft.


Asunto(s)
Dermis/metabolismo , Fibroblastos/metabolismo , Folículo Piloso/metabolismo , Queratinocitos/metabolismo , Melanocitos/metabolismo , Ingeniería de Tejidos , Técnicas de Cocultivo , Dermis/citología , Fibroblastos/citología , Folículo Piloso/citología , Humanos , Queratinocitos/citología , Melanocitos/citología
11.
Nat Commun ; 12(1): 1089, 2021 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-33597528

RESUMEN

Cell-to-cell communication can be inferred from ligand-receptor expression in cell transcriptomic datasets. However, important challenges remain: global integration of cell-to-cell communication; biological interpretation; and application to individual cell population transcriptomic profiles. We develop ICELLNET, a transcriptomic-based framework integrating: 1) an original expert-curated database of ligand-receptor interactions accounting for multiple subunits expression; 2) quantification of communication scores; 3) the possibility to connect a cell population of interest with 31 reference human cell types; and 4) three visualization modes to facilitate biological interpretation. We apply ICELLNET to three datasets generated through RNA-seq, single-cell RNA-seq, and microarray. ICELLNET reveals autocrine IL-10 control of human dendritic cell communication with up to 12 cell types. Four of them (T cells, keratinocytes, neutrophils, pDC) are further tested and experimentally validated. In summary, ICELLNET is a global, versatile, biologically validated, and easy-to-use framework to dissect cell communication from individual or multiple cell-based transcriptomic profiles.


Asunto(s)
Comunicación Celular/genética , Biología Computacional/métodos , Bases de Datos Factuales , Perfilación de la Expresión Génica/métodos , Transcriptoma/genética , Animales , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Linfocitos T/citología , Linfocitos T/metabolismo
12.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-33540902

RESUMEN

Human epidermal keratinocytes are constantly exposed to UV radiation. As a result, there is a significant need for safe and effective compounds to protect skin cells against this environmental damage. This study aimed to analyze the effect of phytocannabinoid-cannabinoid (CBD)-on the proteome of UVA/B irradiated keratinocytes. The keratinocytes were cultured in a three-dimensional (3D) system, designed to mimic epidermal conditions closely. The obtained results indicate that CBD protected against the harmful effects of UVA/B radiation. CBD decreased the expression of proinflammatory proteins, including TNFα/NFκB and IκBKB complex and decreased the expression of proteins involved in de novo protein biosynthesis, which are increased in UVA/B-irradiated cells. Additionally, CBD enhanced the UV-induced expression of 20S proteasome subunits. CBD also protected protein structures from 4-hydroxynonenal (HNE)-binding induced by UV radiation, which primarily affects antioxidant enzymes. CBD-through its antioxidant/anti-inflammatory activity and regulation of protein biosynthesis and degradation-protects skin cells against UVA/B-induced changes. In the future, its long-term use in epidermal cells should be investigated.


Asunto(s)
Cannabidiol/farmacología , Queratinocitos/efectos de los fármacos , Proteoma/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Rayos Ultravioleta , Aldehídos/farmacología , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Cannabidiol/química , Técnicas de Cultivo de Célula , Células Cultivadas , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Quinasa I-kappa B/metabolismo , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Estructura Molecular , Complejos Multiproteicos/metabolismo , FN-kappa B/metabolismo , Análisis de Componente Principal , Proteoma/efectos de la radiación , Transducción de Señal/efectos de la radiación , Factor de Necrosis Tumoral alfa/metabolismo
13.
Molecules ; 26(4)2021 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-33562140

RESUMEN

Crataegus laevigata belongs to the family Rosaceae, which has been widely investigated for pharmacological effects on the circulatory and digestive systems. However, there is limited understanding about its anti-oxidative stress and anti-inflammatory effects on skin. In this study, 70% ethanol C. laevigata berry extract (CLE) was investigated on lipopolysaccharide (LPS)-stimulated keratinocytes. The LPS-induced overproduction of reactive oxygen species (ROS) was suppressed by the treatment with CLE. In response to ROS induction, the overexpression of inflammatory regulating signaling molecules including mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1), nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB), and nuclear factor of activated T-cells (NFAT) were reduced in CLE-treated human keratinocytes. Consequently, CLE significantly suppressed the mRNA levels of pro-inflammatory chemokines and interleukins in LPS-stimulated cells. Our results indicated that CLE has protective effects against LPS-induced injury in an in vitro model and is a potential alternative agent for inflammatory treatment.


Asunto(s)
Crataegus/química , Queratinocitos/efectos de los fármacos , Queratinocitos/patología , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/farmacología , Supervivencia Celular/efectos de los fármacos , Quimiocinas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/metabolismo , Inflamación/patología , Queratinocitos/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción NFATC/metabolismo , ARN Mensajero/genética , Factor de Transcripción AP-1/metabolismo
14.
ACS Appl Mater Interfaces ; 13(2): 2382-2398, 2021 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-33406837

RESUMEN

In this article, we describe a method of delivery of chondroitin sulfate to skin as nanoparticles and demonstrate its anti-inflammatory and antioxidant role using UV irradiation as a model condition. These nanoparticles, formed through electrostatic interactions of chondroitin sulfate with a skin-penetrating peptide, were found to be homogenous with positive surface charges and stable at physiological and acidic pH under certain conditions. They were able to enter into the human keratinocyte cell line (HaCaT), artificial skin membrane (mimicking the human skin), and mouse skin tissue unlike free chondroitin sulfate. The preapplication of nanoparticles also exhibited reduced levels of oxidative stress, cyclobutane pyrimidine dimer formation, TNF-α, and so on in UV-B-irradiated HaCaT cells. In an acute UV-B irradiation mouse model, their topical application resulted in reduced epidermal thickness and sunburn cells, unlike in the case of free chondroitin sulfate. Thus, a completely noninvasive method was used to deliver a bio-macromolecule into the skin without using injections or abrasive procedures.


Asunto(s)
Antiinflamatorios/administración & dosificación , Antioxidantes/administración & dosificación , Sulfatos de Condroitina/administración & dosificación , Portadores de Fármacos/química , Péptidos/química , Quemadura Solar/prevención & control , Administración Tópica , Animales , Antiinflamatorios/farmacocinética , Antiinflamatorios/uso terapéutico , Antioxidantes/farmacocinética , Antioxidantes/uso terapéutico , Línea Celular , Sulfatos de Condroitina/farmacocinética , Sulfatos de Condroitina/uso terapéutico , Portadores de Fármacos/metabolismo , Femenino , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Nanopartículas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Péptidos/metabolismo , Absorción Cutánea , Quemadura Solar/metabolismo , Quemadura Solar/patología , Rayos Ultravioleta/efectos adversos
15.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-33443188

RESUMEN

Dysregulation of inflammatory cytokines in keratinocytes promote the pathogenesis of the skin inflammation, such as allergic contact dermatitis (ACD). High-mobility group box 1 protein (HMGB1) has been implicated in the promotion of skin inflammation upon its extracellular release as a damage-associated molecular pattern molecule. However, whether and how HMGB1 in keratinocytes contributes to ACD and other skin disorders remain elusive. In this study, we generated conditional knockout mice in which the Hmgb1 gene is specifically deleted in keratinocytes, and examined its role in ACD models. Interestingly, the mutant mice showed exacerbated skin inflammation, accompanied by increased ear thickening in 2,4-dinitrofluorobenezene-induced ACDs. The mRNA expression of interleukin-24 (IL-24), a cytokine known to critically contribute to ACD pathogenesis, was elevated in skin lesions of the mutant mice. As with constitutively expressed, IL-4-induced Il24 mRNA, expression was also augmented in the Hmgb1-deficient keratinocytes, which would account for the exacerbation of ACD in the mutant mice. Mechanistically, we observed an increased binding of trimethyl histone H3 (lys4) (H3K4me3), a hallmark of transcriptionally active genes, to the promoter region of the Il24 gene in the hmgb1-deficient cells. Thus, the nuclear HMGB1 is a critical "gate keeper" in that the dermal homeostasis is contingent to its function in chromatin remodeling. Our study revealed a facet of nuclear HMGB1, namely its antiinflammatory function in keratinocytes for the skin homeostasis.


Asunto(s)
Ensamble y Desensamble de Cromatina , Dermatitis Alérgica por Contacto/metabolismo , Proteína HMGB1/metabolismo , Histonas/metabolismo , Interleucinas/metabolismo , Queratinocitos/metabolismo , Animales , Dermatitis Alérgica por Contacto/genética , Dermatitis Alérgica por Contacto/prevención & control , Dinitrofluorobenceno/toxicidad , Modelos Animales de Enfermedad , Oído/patología , Eliminación de Gen , Regulación de la Expresión Génica/genética , Proteína HMGB1/deficiencia , Proteína HMGB1/genética , Inflamación/genética , Inflamación/metabolismo , Interleucina-4/farmacología , Interleucinas/genética , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Piel/inmunología , Piel/metabolismo , Piel/patología , Quimera por Trasplante
16.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33467646

RESUMEN

Achilles tendon ruptures are very common tendon ruptures and their incidence is increasing in modern society, resulting in work incapacity and months off sport, which generate a need for accelerated and successful therapeutic repair strategy. Platelet-rich plasma (PRP) is emerging as adjuvant human blood-derived constructs to assist Achilles tendon rupture treatment. However, myriad PRP preparation methods in conjunction with poor standardization in the modalities of their applications impinge on the consistent effectiveness of clinical and structural outcomes regarding their therapeutic efficacy. The purpose of this review is to provide some light on the application of PRP for Achilles tendon ruptures. PRP has many characteristics that make it an attractive treatment. Elements such as the inclusion of leukocytes and erythrocytes within PRP, the absence of activation and activation ex vivo or in vivo, the modality of application, and the adjustment of PRP pH can influence the biology of the applied product and result in misleading therapeutic conclusions. The weakest points in demonstrating their consistent effectiveness are primarily the result of myriad PRP preparation methods and the poor standardization of modalities for their application. Selecting the right biological scaffold and applying it correctly to restitutio ad integrum of ruptured Achilles tendons remains a daunting and complex task.


Asunto(s)
Tendón Calcáneo/lesiones , Tendón Calcáneo/cirugía , Colágeno/química , Queratinocitos/citología , Plasma Rico en Plaquetas/metabolismo , Rotura/cirugía , Traumatismos de los Tendones/cirugía , Ciclo Celular , Movimiento Celular , Proliferación Celular , Receptores ErbB/metabolismo , Humanos , Integrina beta1/metabolismo , Queratinocitos/metabolismo , Ligandos , FN-kappa B/metabolismo , Receptor IGF Tipo 1/metabolismo , Transducción de Señal , Cicatrización de Heridas
17.
Mol Carcinog ; 60(3): 172-178, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33482042

RESUMEN

Although beta 2 adrenergic receptors (ß2 ADR) are present in the keratinocytes, their role in cutaneous squamous cell tumorigenesis needs to be ascertained. For the first time, we report here that selective ß2 ADR antagonists by inhibiting ß2 ADR actions significantly retarded the progression of ultraviolet B (UVB) induced premalignant cutaneous squamous cell lesions. These antagonists acted by inhibiting vascular endothelial growth factor-A (VEGF) mediated angiogenesis to prevent UVB radiation-induced squamous cell carcinoma of the skin.


Asunto(s)
Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Neoplasias de Células Escamosas/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Rayos Ultravioleta/efectos adversos , Agonistas de Receptores Adrenérgicos beta 1/farmacología , Animales , Butoxamina/farmacología , Humanos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Masculino , Ratones Endogámicos , Neoplasias Inducidas por Radiación/irrigación sanguínea , Neoplasias Inducidas por Radiación/tratamiento farmacológico , Neoplasias Inducidas por Radiación/etiología , Neoplasias de Células Escamosas/irrigación sanguínea , Neoplasias de Células Escamosas/etiología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/etiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Xamoterol/farmacología
18.
Nat Chem Biol ; 17(3): 280-290, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33462494

RESUMEN

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.


Asunto(s)
Proteínas de Ciclo Celular/genética , Epidermis/efectos de los fármacos , Repitelización/efectos de los fármacos , Úlcera Cutánea/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/genética , Heridas no Penetrantes/tratamiento farmacológico , Animales , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epidermis/patología , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Cultivo Primario de Células , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/antagonistas & inhibidores , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Repitelización/genética , Úlcera Cutánea/genética , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Transcripción Genética , Heridas no Penetrantes/genética , Heridas no Penetrantes/metabolismo , Heridas no Penetrantes/patología
19.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-33477820

RESUMEN

Although the role of platelet-rich plasma (PRP) in tissue regeneration has been confirmed in many studies, the mechanism of this process is still not fully understood. Human keratinocytes (HaCaT) cells were used as an experimental model for studies on the effects of PRP on cell proliferation, migration, collagen biosynthesis, prolidase activity, and its expression and anabolic signaling. The activation of epidermal growth factor receptor (EGFR), ß1-integrin, and insulin-like growth factor-1 receptor (IGF-1R) by PRP were investigated by western blot and immunocytochemistry. It has been found that PRP induced keratinocytes migration and proliferation through activation of cell cycle progression and EGFR downstream signaling. Similar biological effects were achieved by an addition to the culture medium of prolidase (PEPD), a ligand of EGFR (PRP is a rich source of PEPD-2 ng/mL). PRP-dependent stimulation of collagen biosynthesis was accompanied by an increase in the expression of NF-κß, IGF-1R-downstream signaling proteins, and PEPD activity. The data suggest that PRP activates a complex of growth factors and adhesion receptors that stimulate cell proliferation, migration, and collagen biosynthesis. PRP induces PEPD-dependent human keratinocyte proliferation through activation of the EGFR receptor. Our study provides a novel mechanism of PRP-dependent wound healing.


Asunto(s)
Dipeptidasas/genética , Integrina beta1/genética , Plasma Rico en Plaquetas/metabolismo , Ciclo Celular/genética , División Celular/efectos de los fármacos , División Celular/genética , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Receptores ErbB/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Piel/efectos de los fármacos , Piel/metabolismo , Cicatrización de Heridas/efectos de los fármacos
20.
Mol Immunol ; 131: 180-190, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33423764

RESUMEN

Exposure to ultraviolet B (UVB) from sunlight causes DNA damage, serious cellular inflammation and aging, and even cell death in the skin, commonly known as sunburn, leading to cutaneous tissue disorders. DNA damage can be sensed as a danger-associated molecular pattern (DAMP) by the innate immune system. It has not been studied, however, whether cGAS-STING activation is involved in the apoptosis induced by UVB irradiation or by cisplatin treatment. Here we report the findings that within hours of DNA damages keratinocytes show an innate immune response, which involves the activation of cGAS-STING; a cytosolic DNA receptor, cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase), cyclic GMP-AMP (cGAMP) synthase, and DNA sensing adaptor, STING (protein stimulator of interferon genes). Either UVB irradiation or cisplatin treatment can cause DNA damages, releasing fragmented DNA from nucleus and/or mitochondria. Roles of cGAS-STING were examined in the HaCaT cells with DNA damages caused by UVB irradiation or cisplatin treatment. Silencing STING by siRNA rescued HaCaT cells from UVB or cisplatin-induced apoptosis. NF-κB, one of the major downstream components of STING pathway, which usually regulates the classical STING apoptotic pathway, was translocated to nucleus in the HaCaT cells irradiated with UVB. This translocation was attenuated by STING silencing. Treatment with BAY, an inhibitor of NF-κB pathway, blocked UVB-induced apoptosis. cGAS-STING-mediated production of IFNß was induced by nuclear translocation of interferon regulatory factor 3 (IRF3). UVB irradiation inceased the nuclear translocation of IRF3, accompanied by enhanced expression level of IFNß mRNA. The nuclear translocation of IRF3 and expression of IFNß mRNA were attenuated by STING silencing. Treatment with MRT67307, an inhibitor of TBK1-IRF3-IFNß pathway, blocked UVB-induced apoptosis. Therefore, we conclude that NF-κB pathway and IFNß pathway residing in the downstream of STING are resposible for apoptosis of UVB-irradiated or cisplatin-treated HaCaT cells.


Asunto(s)
Apoptosis/genética , Daño del ADN/fisiología , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Transducción de Señal/fisiología , Humanos , Inmunidad Innata/fisiología , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Queratinocitos/metabolismo , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo
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