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BACKGROUND: Visual and molecular changes occurring upon aging are rather well characterized. Still, aging signs show great significant inter-individual variations, and little is known concerning the link between perceived age and cutaneous microcirculation. MATERIALS AND METHODS: To investigate this point, we recruited Caucasian women in their mid-50's to mid-70's and subsampled women looking older or younger than their age. We studied their facial skin color, as well as their microvascular reactivity to local heating assessed in the forearm skin. We also used skin biopsies from some of these women for gene expression or immunohistochemical analysis. RESULTS: Clinical and instrumental analysis of skin color revealed that subjects who look 5 years younger differ only by a higher glowing complexion. Our most striking result is that subjects looking 5 years younger than their age present a higher microcirculation reactivity in forearm skin. Transcriptome comparison of skin samples from women looking older or younger than their age revealed 123 annotated transcripts differentially expressed, among which MYL9 relates to microcirculation. MYL9 is downregulated in the group of women looking younger than their real age. Microscopy shows that the labeling of MYL9 and CD31 are altered and heterogeneous with age, as is the morphology of microvessels. CONCLUSION: Therefore, assessing generalized vascular reactivity in non-photo-exposed skin to focus on the intrinsic aging allows subtle discrimination of perceived age within elderly healthy subjects.
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Antebraço , Microcirculação , Envelhecimento da Pele , Pele/irrigação sanguínea , Idoso , Face , Feminino , Antebraço/irrigação sanguínea , HumanosRESUMO
MicroRNAs (miRNAs) are a class of short non-coding RNAs capable of repressing gene expression at the post-transcriptional level. miRNAs participate in the control of numerous cellular mechanisms, including skin homeostasis and epidermal differentiation. However, few miRNAs involved in these processes have been identified so far in human skin, and the gene networks they control remain largely unknown. Here, we focused on miR-23b-3p, a miRNA that is expressed during the late step of human keratinocyte differentiation. We report that miR-23b-3p silencing modulates epidermal differentiation in human skin reconstructs. The SMAD transcriptional corepressor TGIF1 was identified on bioinformatic analysis as a potential target of miR-23b-3p. Expression analysis and reporter gene assays confirmed direct regulation of TGIF1 expression by miR-23b-3p. Finally, we showed that miR-23-3p was able to activate TGF-ß signalling in human keratinocytes by increasing SMAD2 phosphorylation through TGIF1 repression. Taken together, these data identify miR-23b-3p as a new regulator of human epidermal differentiation in line with TGF-ß signalling.
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Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica , Inativação Gênica , Proteínas de Homeodomínio/farmacologia , Humanos , Queratinócitos/fisiologia , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Repressoras/farmacologia , Fator de Crescimento Transformador beta/genéticaRESUMO
UNLABELLED: Innate immunity is the first line of host defense against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus 38 (HPV38) E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor Toll-like receptor 9 (TLR9) in human foreskin keratinocytes (HFK), a key mediator of the antiviral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA downregulation by promoting accumulation of ΔNp73α, an antagonist of p53 and p73. Inhibition of ΔNp73α expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of ΔNp73α in this event. Chromatin immunoprecipitation experiments showed that ΔNp73α is part of a negative transcriptional regulatory complex with IκB kinase beta (IKKß) that binds to a NF-κB responsive element within the TLR9 promoter. In addition, the Polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex, leading to histone 3 trimethylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21(WAF1) and p27(Kip1), decreased CDK2-associated kinase activity, and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can downregulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation. IMPORTANCE: The mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the host immune system in order to guarantee the persistence of the infection. Their capability to evade the immune system is as important as their ability to promote cellular transformation. Therefore, understanding the viral mechanisms involved in viral persistence is a valid tool to evaluate their potential role in human carcinogenesis. Here, we show that E6 and E7 oncoproteins from the cutaneous HPV38 downregulate the expression of the double-stranded DNA sensor TLR9 of innate immunity. We also present evidence that the HPV38-mediated downregulation of TLR9 expression, in addition to its potential impact on the innate immune response, is linked to cell cycle deregulation.
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Pontos de Checagem do Ciclo Celular/genética , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Receptor Toll-Like 9/biossíntese , Linhagem Celular , Proliferação de Células/genética , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste , Histonas/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Queratinócitos/metabolismo , Queratinócitos/virologia , Metilação , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno , RNA Viral/genética , Receptor Toll-Like 9/antagonistas & inibidores , Receptor Toll-Like 9/genética , Quinases Ativadas por p21/metabolismoRESUMO
The epidermis, the outermost layer of the skin, serves as a protective barrier against external factors. Epidermal differentiation, a tightly regulated process essential for epidermal homeostasis, epidermal barrier formation and skin integrity maintenance, is orchestrated by several players, including signaling molecules, calcium gradient and junctional complexes such as gap junctions (GJs). GJ proteins, known as connexins facilitate cell-to-cell communication between adjacent keratinocytes. Connexins can function as either hemichannels or GJs, depending on their interaction with other connexons from neighboring keratinocytes. These channels enable the transport of metabolites, cAMP, microRNAs, and ions, including Ca2+, across cell membranes. At least ten distinct connexins are expressed within the epidermis and mutations in at least five of them has been linked to various skin disorders. Connexin mutations may cause aberrant channel activity by altering their synthesis, their gating properties, their intracellular trafficking, and the assembly of hemichannels and GJ channels. In addition to mutations, connexin expression is dysregulated in other skin conditions including psoriasis, chronic wound and skin cancers, indicating the crucial role of connexins in skin homeostasis. Current treatment options for conditions with mutant or altered connexins are limited and primarily focus on symptom management. Several therapeutics, including non-peptide chemicals, antibodies, mimetic peptides and allele-specific small interfering RNAs are promising in treating connexin-related skin disorders. Since connexins play crucial roles in maintaining epidermal homeostasis as shown with linkage to a range of skin disorders and cancer, further investigations are warranted to decipher the molecular and cellular alterations within cells due to mutations or altered expression, leading to abnormal proliferation and differentiation. This would also help characterize the roles of each isoform in skin homeostasis, in addition to the development of innovative therapeutic interventions. This review highlights the critical functions of connexins in the epidermis and the association between connexins and skin disorders, and discusses potential therapeutic options.
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Chronic wounds represent a serious worldwide concern, being often associated with bacterial infections. As the prevalence of bacterial infections increase, it is crucial to search for alternatives. Essential oils (EOs) constitute a promising option to antibiotics due to their strong anti-inflammatory, analgesic, antioxidant and antibacterial properties. However, such compounds present high volatility. To address this issue, a drug delivery system composed of coaxial wet-spun fibers was engineered and different EOs, namely clove oil (CO), cinnamon leaf oil (CLO) and tea tree oil (TTO), were loaded. Briefly, a coaxial system composed of two syringe pumps, a coagulation bath of deionized water, a cylindrical-shaped collector and a coaxial spinneret was used. A 10 % w/v polycaprolactone (PCL) solution was combined with the different EOs at 2 × minimum bactericidal concentration (MBC) and loaded to a syringe connected to the inner port, whereas a 10 % w/v cellulose acetate (CA) solution mixed with 10 % w/v polyethylene glycol (PEG) at a ratio of 90:10 % v/v (to increase the fibers' elasticity) was loaded to the syringe connected to the outer port. This layer was used as a barrier to pace the release of the entrapped EO. The CA's inherent porosity in water coagulation baths allowed access to the fiber's core. CA was also mixed with 10 % w/v polyethylene glycol (PEG) at a ratio of 90:10 % v/v (CA:PEG), to increase the fibers' elasticity. Microfibers maintained their structural integrity during 28 days of incubation in physiological-like environments. They also showed high elasticities (maximum elongations at break >300 %) and resistance to rupture in mechanical assessments, reaching mass losses of only ≈ 2.29 % - 57.19 %. The EOs were released from the fibers in a prolonged and sustained fashion, in which ≈ 30 % of EO was released during the 24 h of incubation in physiological-like media, demonstrating great antibacterial effectiveness against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa, the most prevalent bacteria in chronic wounds. Moreover, microfibers showed effective antioxidant effects, presenting up to 59 % of reduction of 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity. Furthermore, the coaxial system was deemed safe for contact with fibroblasts and human keratinocytes, reaching metabolic activities higher than 80 % after 48 h of incubation. Data confirmed the suitability of the engineered system for potential therapeutics of chronic wounds.
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Antioxidantes , Celulose , Óleos Voláteis , Poliésteres , Poliésteres/química , Antioxidantes/farmacologia , Antioxidantes/química , Óleos Voláteis/farmacologia , Óleos Voláteis/química , Celulose/química , Celulose/análogos & derivados , Celulose/farmacologia , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Antibacterianos/farmacologia , Antibacterianos/química , Humanos , Cicatrização/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Escherichia coli/efeitos dos fármacosRESUMO
Although p63 and MYC are important in the control of epidermal homeostasis, the underlying molecular mechanisms governing keratinocyte proliferation or differentiation downstream of these two genes are not completely understood. By analyzing the transcriptional changes and phenotypic consequences of the loss of either p63 or MYC in human developmentally mature keratinocytes, we have characterized the networks acting downstream of these two genes to control epidermal homeostasis. We show that p63 is required to maintain growth and to commit to differentiation by two distinct mechanisms. Knockdown of p63 led to down-regulation of MYC via the Wnt/ß-catenin and Notch signaling pathways and in turn reduced keratinocyte proliferation. We demonstrate that a p63-controlled keratinocyte cell fate network is essential to induce the onset of keratinocyte differentiation. This network contains several secreted proteins involved in cell migration/adhesion, including fibronectin 1 (FN1), interleukin-1ß (IL1B), cysteine-rich protein 61 (CYR61), and jagged-1 (JAG1), that act downstream of p63 as key effectors to trigger differentiation. Our results characterized for the first time a connection between p63 and MYC and a cell adhesion-related network that controls differentiation. Furthermore, we show that the balance between the MYC-controlled cell cycle progression network and the p63-controlled cell adhesion-related network could dictate skin cell fate.
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Diferenciação Celular/genética , Redes Reguladoras de Genes , Queratinócitos/citologia , Queratinócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Adesão Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Movimento Celular/genética , Proliferação de Células , Regulação para Baixo/genética , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-myc/deficiência , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/genética , Receptores Notch/metabolismo , Transdução de Sinais/genética , Transcriptoma/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
BACKGROUND: MicroRNAs (miRNAs), a group of short non-coding RNAs that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive. RESULTS: We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. We found that proliferative cells irradiated at 6 Gy display a global fall of miRNA expression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated by these responding miRNAs. CONCLUSIONS: Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern strongly related to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation response to ensure the short-term survival of irradiated keratinocytes.
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Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , MicroRNAs/genética , Diferenciação Celular/efeitos da radiação , Raios gama , Perfilação da Expressão Gênica , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , MicroRNAs/efeitos da radiação , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de CélulasRESUMO
The cutaneous beta human papillomavirus (beta HPV) types appear to be involved in skin carcinogenesis. However, only a few beta HPVs have been investigated so far. Here, we compared the properties of E6 and E7 oncoproteins from six uncharacterized beta HPVs (14, 22, 23, 24, 36, 49). Only HPV49 E6 and E7 immortalized primary human keratinocytes and efficiently deregulated the p53 and pRb pathways. Furthermore, HPV49 E6, similarly to E6 from the oncogenic HPV16, promoted p53 degradation.
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Betapapillomavirus/fisiologia , Transformação Celular Viral , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Betapapillomavirus/genética , Células Cultivadas , Humanos , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The disturbance of intercellular communication is one of the hallmarks of aging. The goal of this study is to clarify the impact of chronological aging on extracellular vesicles (EVs), a key mode of communication in mammalian tissues. We focused on epidermal keratinocytes, the main cells of the outer protective layer of the skin which is strongly impaired in the skin of elderly. EVs were purified from conditioned medium of primary keratinocytes isolated from infant or aged adult skin. A significant increase of the relative number of EVs released from aged keratinocytes was observed whereas their size distribution was not modified. By small RNA sequencing, we described a specific microRNA (miRNA) signature of aged EVs with an increase abundance of miR-30a, a key regulator of barrier function in human epidermis. EVs from aged keratinocytes were found to be able to reduce the proliferation of young keratinocytes, to impact their organogenesis properties in a reconstructed epidermis model and to slow down the early steps of skin wound healing in mice, three features observed in aged epidermis. This work reveals that intercellular communication mediated by EVs is modulated during aging process in keratinocytes and might be involved in the functional defects observed in aged skin.
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Vesículas Extracelulares , MicroRNAs , Idoso , Humanos , Animais , Camundongos , MicroRNAs/genética , Queratinócitos , Epiderme , Envelhecimento/genética , Mamíferos/genéticaRESUMO
Skin is a key organ maintaining internal homeostasis by performing many functions such as water loss prevention, body temperature regulation and protection from noxious substance absorption, microorganism intrusion and physical trauma. Skin ageing has been well studied and it is well known that physiological changes in the elderly result in higher skin fragility favouring the onset of skin diseases. For example, prolonged and/or high-intensity pressure may suppress local blood flow more easily, disturbing cell metabolism and inducing pressure injury (PI) formation. Pressure injuries (PIs) represent a significant problem worldwide and their prevalence remains too high. A higher PI prevalence is correlated with an elderly population. Newborn skin evolution has been less studied, but some data also report a higher PI prevalence in this population compared to older children, and several authors also consider this skin as physiologically fragile. In this review, we compare the characteristics of newborn and elderly skin in order to determine common features that may explain their fragility, especially regarding PI risk. We show that, despite differences in appearance, they share many common features leading to higher fragility to shear and pressure forces, not only at the structural level but also at the cellular and molecular level and in terms of physiology. Both newborn and elderly skin have: (i) a thinner epidermis; (ii) a thinner dermis containing a less-resistant collagen network, a higher collagen III:collagen I ratio and less elastin; (iii) a flatter dermal-epidermal junction (DEJ) with lower anchoring systems; and (iv) a thinner hypodermis, resulting in lower mechanical resistance to skin damage when pressure or shear forces are applied. At the molecular level, reduced expression of transforming growth factor ß (TGFß) and its receptor TGFß receptor II (TßRII) is involved in the decreased production and/or increased degradation of various dermal extracellular matrix (ECM) components. Epidermal fragility also involves a higher skin pH which decreases the activity of key enzymes inducing ceramide deficiency and reduced barrier protection. This seems to be correlated with higher PI prevalence in some situations. Some data also suggest that stratum corneum (SC) dryness, which may disturb cell metabolism, also increases the risk of PI formation. Besides this structural fragility, several skin functions are also less efficient. Low applied pressures induce skin vessel vasodilation via a mechanism called pressure-induced vasodilation (PIV). Individuals lacking a normal PIV response show an early decrease in cutaneous blood flow in response to the application of very low pressures, reflecting vascular fragility of the skin that increases the risk of ulceration. Due to changes in endothelial function, skin PIV ability decreases during skin ageing, putting it at higher risk of PI formation. In newborns, some data lead us to hypothesize that the nitric oxide (NO) pathway is not fully functional at birth, which may partly explain the higher risk of PI formation in newborns. In the elderly, a lower PIV ability results from impaired functionality of skin innervation, in particular that of C-fibres which are involved in both touch and pain sensation and the PIV mechanism. In newborns, skin sensitivity differs from adults due to nerve system immaturity, but the role of this in PIV remains to be determined.
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Úlcera por Pressão , Fator de Crescimento Transformador beta , Doenças Vasculares , Adolescente , Adulto , Idoso , Criança , Humanos , Recém-Nascido , Colágeno , Matriz Extracelular , Fenômenos Fisiológicos da Pele , Fator de Crescimento Transformador beta/metabolismoRESUMO
Chronological aging is characterized by an alteration in the genes' regulatory network. In human skin, epidermal keratinocytes fail to differentiate properly with aging, leading to the weakening of the epidermal function. MiR-30a is particularly overexpressed with epidermal aging, but the downstream molecular mechanisms are still uncovered. The aim of this study was to decipher the effects of miR-30a overexpression in the human epidermis, with a focus on keratinocyte differentiation. We formally identified the mitophagy receptor BNIP3L as a direct target of miR-30a. Using a 3D organotypic model of reconstructed human epidermis overexpressing miR-30a, we observed a strong reduction in BNIP3L expression in the granular layer. In human epidermal sections of skin biopsies from donors of different ages, we observed a similar pattern of BNIP3L decreasing with aging. Moreover, human primary keratinocytes undergoing differentiation in vitro also showed a decreased expression of BNIP3L with age, together with a retention of mitochondria. Moreover, aging is associated with altered mitochondrial metabolism in primary keratinocytes, including decreased ATP-linked respiration. Thus, miR-30a is a negative regulator of programmed mitophagy during keratinocytes terminal differentiation, impairing epidermal homeostasis with aging.
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MicroRNAs , Mitofagia , Envelhecimento/genética , Proteínas Reguladoras de Apoptose/metabolismo , Epiderme/metabolismo , Humanos , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Chronological aging is defined as a time-dependent decline of tissue homeostasis which severely impacts skin. Understanding the mechanisms of skin aging is an active research area limited by the lack of relevant in vitro models. Being a component of aging, replicative or stress-induced senescence is repeatedly used to mimic skin aging in vitro, thus presenting only a partial view of the complexity of aging. Herein, we aimed to clarify whether primary normal human dermal fibroblasts retained age-related characteristics when cultured in 2D monolayer, and could be used as a relevant model for aging research. We compared three groups of fibroblasts isolated from different aged donors. We observed strongly decreased population doubling capacities, a reduced clonogenic ability, an impairment in extracellular matrix production together with modifications of respiratory metabolism with an increase in age. These disruptions were particularly marked when comparing fibroblasts isolated from old individuals (over 70 years old) to those isolated from young individuals (18-37 years old), while cells from middle-aged donors exhibited an intermediate profile. These alterations of cell features can be related to the signs of dermis aging, thus showing that cultured primary cells indeed retain some characteristics of the original tissue from which they were extracted.
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Derme , Envelhecimento da Pele , Adolescente , Adulto , Idoso , Envelhecimento/metabolismo , Derme/metabolismo , Fibroblastos/metabolismo , Humanos , Pessoa de Meia-Idade , Pele/metabolismo , Adulto JovemRESUMO
Sarcoidosis is a multisystem disease characterized by the development and accumulation of granulomas, the hallmark of an inflammatory process induced by environmental and/or infectious and or genetic factors. This auto-inflammatory disease mainly affects the lungs, the gateway to environmental aggressions and viral infections. We have shown previously that genetic predisposition to sarcoidosis occurring in familial cases is related to a large spectrum of pathogenic variants with, however, a clustering around mTOR (mammalian Target Of Rapamycin)-related pathways and autophagy regulation. The context of the COVID-19 pandemic led us to evaluate whether such genetic defects may increase the risk of a severe course of SARS-CoV2 infection in patients with sarcoidosis. We extended a whole exome screening to 13 families predisposed to sarcoidosis and crossed the genes sharing mutations with the list of genes involved in the SARS-CoV2 host-pathogen protein-protein interactome. A similar analysis protocol was applied to a series of 100 healthy individuals. Using ENRICH.R, a comprehensive gene set enrichment web server, we identified the functional pathways represented in the set of genes carrying deleterious mutations and confirmed the overrepresentation of autophagy- and mitophagy-related functions in familial cases of sarcoidosis. The same protocol was applied to the set of genes common to sarcoidosis and the SARS-CoV2-host interactome and found a significant enrichment of genes related to mitochondrial factors involved in autophagy, mitophagy, and RIG-I-like (Retinoic Acid Inducible Gene 1) Receptor antiviral response signaling. From these results, we discuss the hypothesis according to which sarcoidosis is a model for studying genetic abnormalities associated with host response to viral infections as a consequence of defects in autophagy and mitophagy processes.
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Autofagia , COVID-19/fisiopatologia , Sarcoidose/fisiopatologia , COVID-19/enzimologia , Genômica , Humanos , Mitofagia , Proteínas Serina-Treonina Quinases , Sarcoidose/enzimologia , Sequenciamento do ExomaRESUMO
The skin is a sentinel organ making easily visible the passing of time. Chronological and environmental aging weakens skin structure and functions. The skin barrier, the elastic and mechanical properties of the cutaneous tissue as well as its vascular reactivity are impacted by aging. The barrier dysfunction in aged skin is caused by defects in epidermal keratinocytes renewal and differentiation notably linked to abnormal expression of microRNAs regulating cell death and autophagy. An abnormal balance between synthesis and degradation of matrix proteins modifies the mechanical properties of the dermis in aged skin. Finally, a reduction of the vascular reactivity linked to endothelial dysfunctions is observed in elderly people. These biological processes can be targeted by therapeutic approaches either topical or systemic, especially using anti-oxydants or senolytics. These anti-aging strategies might contribute to restore, at least in part, the functional integrity of aged skin.
TITLE: Vieillissement et intégrité de la peau - De la biologie cutanée aux stratégies anti-âge. ABSTRACT: La peau est un organe sentinelle, soumis au vieillissement chronologique et environnemental qui fragilise sa structure et ses fonctions. La fonction barrière de la peau, ses propriétés élastiques et de résistance, ainsi que sa réactivité vasculaire sont atteintes par le vieillissement dans les compartiments épidermiques, dermiques et vasculaires. Les progrès de la recherche ont permis de révéler des processus biologiques sous-jacents, qui peuvent être ciblés par des approches médicamenteuses topiques ou globales à base notamment d'anti-oxydants ou de sénolytiques. Ces stratégies anti-âge pourront contribuer à restaurer, au moins en partie, l'intégrité fonctionnelle de la peau âgée.
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Envelhecimento/fisiologia , Envelhecimento da Pele/fisiologia , Fenômenos Fisiológicos da Pele , Terapias em Estudo/tendências , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/efeitos dos fármacos , Cosmecêuticos/uso terapêutico , Fármacos Dermatológicos/uso terapêutico , Epiderme/efeitos dos fármacos , Epiderme/fisiologia , Humanos , Microvasos/efeitos dos fármacos , Microvasos/fisiologia , Microvasos/fisiopatologia , Pele/irrigação sanguínea , Pele/efeitos dos fármacos , Pele/patologia , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/patologia , Fenômenos Fisiológicos da Pele/efeitos dos fármacos , Terapias em Estudo/métodosRESUMO
Although it is well established that 5 to 15% of radiotherapy patients exhibit severe side-effects in non-cancerous tissues, the molecular mechanisms involved are still poorly known, and the links between cellular and tissue radiosensitivity are still debated. We here studied fibroblasts from non-irradiated skin of patients with severe sequelae of radiotherapy, to determine whether specific basal cell activities might be involved in susceptibility to side-effects in normal tissues. Compared to control cells, patient fibroblasts exhibited higher radiosensitivity together with defects in DNA repair. Transcriptome profiling of dermal fibroblasts from 16 radiotherapy patients with severe side-effects and 8 healthy individuals identified 540 genes specifically deregulated in the patients. Nuclear factor of activated T cells 2 (NFATC2) was the most differentially expressed gene, poorly expressed at both transcript and protein level, whereas the NFATC2 gene region was hypermethylated. Furthermore, NFATC2 expression correlated with cell survival after irradiation. Finally, silencing NFATC2 in normal cells by RNA interference led to increased cellular radiosensitivity and defects in DNA repair. This study demonstrates that patients with clinical hypersensitivity also exhibit intrinsic cellular radiosensitivity in their normal skin cells. It further reveals a new role for NFATC2 as a potential regulator of cellular sensitivity to ionizing radiation.
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The nevoid basal cell carcinoma syndrome (NBCCS), also called Gorlin syndrome is an autosomal dominant disorder whose incidence is estimated at about 1 per 55,600-256,000 individuals. It is characterized by several developmental abnormalities and an increased predisposition to the development of basal cell carcinomas (BCCs). Cutaneous fibroblasts from Gorlin patients have been shown to exhibit an increased sensitivity to ionizing radiations. Mutations in the tumor suppressor gene PTCH1, which is part of the Sonic Hedgehog (SHH) signaling pathway, are responsible for these clinical manifestations. As several genetic mutations in the DNA repair genes are responsible of photo or radiosensitivity and high predisposition to cancers, we hypothesized that these effects in Gorlin syndrome might be due to a defect in the DNA damage response (DDR) and/or the DNA repair capacities. Therefore, the objective of this work was to investigate the sensitivity of skin fibroblasts from NBCCS patients to different DNA damaging agents and to determine the ability of these agents to modulate the DNA repair capacities. Gorlin fibroblasts showed high radiosensitivity and also less resistance to oxidative stress-inducing agents when compared to control fibroblasts obtained from healthy individuals. Gorlin fibroblasts harboring PTCH1 mutations were more sensitive to the exposure to ionizing radiation and to UVA. However, no difference in cell viability was shown after exposure to UVB or bleomycin. As BER is responsible for the repair of oxidative DNA damage, we decided to assess the BER pathway efficacy in Gorlin fibroblasts. Interestingly, a concomitant decrease of both BER gene expression and BER protein activity was observed in Gorlin fibroblasts when compared to control. Our results suggest that low levels of DNA repair within Gorlin cells may lead to an accumulation of oxidative DNA damage that could participate and partly explain the radiosensitivity and the BCC-prone phenotype in Gorlin syndrome.
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BACKGROUND: The general population is constantly exposed to low levels of radiation through natural, occupational or medical irradiation. Even if the biological effects of low-level radiation have been intensely debated and investigated, the molecular mechanisms underlying the cellular response to low doses remain largely unknown. RESULTS: The present study investigated the role of GATA3 protein in the control of the cellular and molecular response of human keratinocytes exposed to a 1 cGy dose of X-rays. Chromatin immunoprecipitation showed GATA3 to be able to bind the promoter of 4 genes responding to a 1 cGy exposure. To go further into the role of GATA3 after ionizing radiation exposure, we studied the cellular and molecular consequences of radiation in GATA3 knock-down cells. Knock-down was obtained by lentiviral-mediated expression of an shRNA targeting the GATA3 transcript in differentiated keratinocytes. First, radiosensitivity was assessed: the toxicity, in terms of immediate survival (with XTT test), associated with 1 cGy radiation was found to be increased in GATA3 knock-down cells. The impact of GATA3 knock-down on the transcriptome of X-ray irradiated cells was also investigated, using oligonucleotide microarrays to assess changes between 3 h and 72 h post-irradiation in normal vs GATA3 knock-down backgrounds; transcriptome response was found to be completely altered in GATA3 knock-down cells, with a strong induction/repression peak 48 h after irradiation. Functional annotation revealed enrichment in genes known to be involved in chaperone activity, TGFbeta signalling and stress response. CONCLUSION: Taken together, these data indicate that GATA3 is an important regulator of the cellular and molecular response of epidermal cells to very low doses of radiation.
Assuntos
Fator de Transcrição GATA3/metabolismo , Perfilação da Expressão Gênica , Queratinócitos/efeitos da radiação , Regiões Promotoras Genéticas , Células Cultivadas , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Queratinócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Tolerância a Radiação/genética , Radiação Ionizante , Transdução GenéticaRESUMO
The aim of the present study was to evaluate the impact of the microenvironment produced by dermal microvascular endothelial cells, secondary to a pro-inflammatory challenge, on 2D culture models using dermal fibroblasts and in 3D reconstructed skin model using dermal fibroblasts and keratinocytes from healthy donors. We hypothesized that specific microvascular endothelial low grade inflammation could change fibroblasts phenotype and be involved in extracellular matrix (ECM) modification and skin alteration. Following IFNγ, TNFα, IL-1ß pro-inflammatory stress on Human Dermal Endothelial Cells (HDMEC) we observed the increased release of Chemokine ligand 2 (CCL2), IL-6 and IL-8 but not VEGF-A in the conditioned medium (CM). The subsequent addition of this endothelial pro-inflammatory CM in dermal fibroblasts revealed an upregulation of IL6, IL8 and CCL2 but no NF-κB gene expression. The resulting ECM formation was impaired with a reduction of the collagen 1 network and a decrease in COL1A1 gene expression in 2D and 3D models. Collagen 1 and pro-LOX protein expression were significantly reduced confirming an impairment of the collagen network related to endothelial inflammation secretion. To conclude, this work showed that, without any immune cells, the endothelial secretion in response to a pro-inflammatory stress is able to activate the fibroblasts that will maintain the pro-inflammatory environment and exacerbate ECM degradation.
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BACKGROUND: Artificial visible light is everywhere in modern life. Social communication confronts us with screens of all kinds, and their use is on the rise. We are therefore increasingly exposed to artificial visible light, the effects of which on skin are poorly known. OBJECTIVE: The purpose of this study was to model the artificial visible light emitted by electronic devices and assess its effect on normal human fibroblasts. METHODS: The spectral irradiance emitted by electronic devices was optically measured and equipment was developed to accurately reproduce such artificial visible light. Effects on normal human fibroblasts were analyzed on human genome microarray-based gene expression analysis. At cellular level, visualization and image analysis were performed on the mitochondrial network and F-actin cytoskeleton. Cell proliferation, ATP release and type I procollagen secretion were also measured. RESULTS: We developed a device consisting of 36 LEDs simultaneously emitting blue, green and red light at distinct wavelengths (450â¯nm, 525â¯nm and 625â¯nm) with narrow spectra and equivalent radiant power for the three colors. A dose of 99â¯J/cm2 artificial visible light was selected so as not to induce cell mortality following exposure. Microarray analysis revealed 2984 light-modulated transcripts. Functional annotation of light-responsive genes revealed several enriched functions including, amongst others, the "mitochondria" and "integrin signaling" categories. Selected results were confirmed by real-time quantitative PCR, analyzing 24 genes representing these two categories. Analysis of micro-patterned culture plates showed marked fragmentation of the mitochondrial network and disorganization of the F-actin cytoskeleton following exposure. Functionally, there was considerable impairment of cell growth and spread, ATP release and type I procollagen secretion in exposed fibroblasts. CONCLUSION: Artificial visible light induces drastic molecular and cellular changes in normal human fibroblasts. This may impede normal cellular functions and contribute to premature skin aging. The present results extend our knowledge of the effects of the low-energy wavelengths that are increasingly used to treat skin disorders.
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PURPOSE: Gorlin syndrome (or basal-cell nevus syndrome) is a cancer-prone genetic disease in which hypersusceptibility to secondary cancer and tissue reaction after radiation therapy is debated, as is increased radiosensitivity at cellular level. Gorlin syndrome results from heterozygous mutations in the PTCH1 gene for 60% of patients, and we therefore aimed to highlight correlations between intrinsic radiosensitivity and PTCH1 gene expression in fibroblasts from adult patients with Gorlin syndrome. METHODS AND MATERIALS: The radiosensitivity of fibroblasts from 6 patients with Gorlin syndrome was determined by cell-survival assay after high (0.5-3.5 Gy) and low (50-250 mGy) γ-ray doses. PTCH1 and DNA damage response gene expression was characterized by real-time polymerase chain reaction and Western blotting. DNA damage and repair were investigated by γH2AX and 53BP1 foci assay. PTCH1 knockdown was performed in cells from healthy donors by using stable RNA interference. Gorlin cells were genotyped by 2 complementary sequencing methods. RESULTS: Only cells from patients with Gorlin syndrome who presented severe deficiency in PATCHED1 protein exhibited a significant increase in cellular radiosensitivity, affecting cell responses to both high and low radiation doses. For 2 of the radiosensitive cell strains, heterozygous mutations in the 5' end of PTCH1 gene explain PATCHED1 protein deficiency. In all sensitive cells, DNA damage response pathways (ATM, CHK2, and P53 levels and activation by phosphorylation) were deregulated after irradiation, whereas DSB repair recognition was unimpaired. Furthermore, normal cells with RNA interference-mediated PTCH1 deficiency showed reduced survival after irradiation, directly linking this gene to high- and low-dose radiosensitivity. CONCLUSIONS: In the present study, we show an inverse correlation between PTCH1 expression level and cellular radiosensitivity, suggesting an explanation for the conflicting results previously reported for Gorlin syndrome and possibly providing a basis for prognostic screens for radiosensitive patients with Gorlin syndrome and PTCH1 mutations.