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1.
PLoS One ; 13(9): e0203863, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30208100

RESUMO

The epidermis undergoes constant renewal during its lifetime. This is possible due to a special population of keratinocyte stem cells (KSCs) located at the basal layer. These cells are surrounded by their direct progeny, keratinocyte progenitors or transient amplifying cells (TAs), which arise from cell division. Skin is exposed every day to sun radiation; in particular, UVA radiation penetrates through the epidermis and induces damage to KSCs and TAs. Although keratinocytes in the basal layer are the most likely skin carcinomas and/or photoaging cells of origin, surprisingly few studies have addressed the specific responses of these cells to UV radiation. In this study, we showed for the first time that keratinocyte stem cells were more resistant to UVA irradiation than their direct progeny, transient amplifying cells. Using both the MTT assay and clonogenic assay, we found that KSCs were more photo-resistant compared to TAs after exposure to different doses of UVA (from 0 to 50 J/cm2). Moreover, KSCs had a greater ability to reconstruct human epidermis (RHE) after UVA exposure compared with TAs. Finally, investigations of DNA repair using the comet assay showed that DNA single-strand breaks and thymine dimers were repaired quicker and more efficiently in KSCs compared with TAs. In a previous work, we showed that the same stem cell population was more resistant to ionizing radiation, another carcinogenic agent. Collectively, our results combined with other observations demonstrate that keratinocyte stem cells, which are responsible for epidermal renewal throughout life, are equipped with an efficient arsenal against several genotoxic agents. Our future work will try to identify the factors or signaling pathways that are responsible for this differential photo-sensitivity and DNA repair capacity between KSCs and TAs.


Assuntos
Queratinócitos/efeitos da radiação , Células-Tronco/efeitos da radiação , Adulto , Diferenciação Celular/efeitos da radiação , Ensaio Cometa , Quebras de DNA de Cadeia Simples/efeitos da radiação , Dano ao DNA/genética , Reparo do DNA/genética , Derme/efeitos da radiação , Células Epidérmicas/efeitos da radiação , Epiderme/metabolismo , Epiderme/efeitos da radiação , Feminino , Humanos , Queratinócitos/metabolismo , Cultura Primária de Células , Dímeros de Pirimidina/metabolismo , Tolerância a Radiação/genética , Pele/efeitos da radiação , Células-Tronco/metabolismo , Raios Ultravioleta/efeitos adversos
2.
Photochem Photobiol ; 94(2): 343-350, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29110298

RESUMO

Photoprotection is essential to prevent the long-term deleterious effects of ultraviolet (UV), including skin cancer and photoaging. So far, there has been an increase in the use of natural bioactive phytochemicals for the development of more effective skin photoprotective agents. However, the molecular mechanisms underlying the photochemoprotection activity of such compounds remain largely unknown. The objective of this study was to investigate the effects of a Sechium edule fruit extract (SEE) in terms of photoprotection against UVA in primary human keratinocytes. We found that SEE protected keratinocytes against UVA-induced cytotoxicity, decreased the intracellular amounts of reactive oxygen species, and reduced oxidatively induced DNA lesions after UVA exposure. Furthermore, SEE decreased the induction of CPD lesions in UVA-irradiated keratinocytes and exhibited increased DNA repair of such photoproducts at 24 h postexposure. Finally, using DNA repair biochips, we demonstrated that SEE-treated keratinocytes had DNA enzymatic repair activities more efficient for abasic sites, CPD and thymine glycols. Therefore, the benefits of SEE against UVA could be explained by a combination of antioxidant activity, the reduction in DNA damage, and the enhancement of DNA repair capacities.


Assuntos
Cucurbitaceae/química , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Extratos Vegetais/farmacologia , Proteção Radiológica , Protetores contra Radiação/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Raios Ultravioleta/efeitos adversos , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Cultura Primária de Células
3.
Int J Mol Sci ; 18(2)2017 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-28134816

RESUMO

The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC) and Transitory Amplifying (TA) cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i) the rapid adhesion method on coated substrate and (ii) the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71). Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6high/CD71low) phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6high/CD71low cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6high/CD71low phenotype.


Assuntos
Antígenos CD/metabolismo , Células Epidérmicas , Integrina alfa6/metabolismo , Queratinócitos/citologia , Receptores da Transferrina/metabolismo , Células-Tronco/metabolismo , Adesão Celular , Separação Celular , Células Clonais , Colágeno Tipo I/metabolismo , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Humanos , Fenótipo , Regeneração , Temperatura
4.
Exp Dermatol ; 26(1): 51-57, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27306475

RESUMO

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.


Assuntos
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/genética
5.
Matrix Biol ; 47: 85-97, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25840344

RESUMO

Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors. The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging. By using prolonged culture as a proxy for cellular aging, we extended to 120 days the culture time of a skin equivalent model based on collagen-glycosaminoglycan-chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages. We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120 days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the expression of hyaluronan and its surface receptor CD44 drastically decreased as observed during chronological skin aging. Finally, we found that the level of p16INK4A expression significantly increased supporting cellular senescence process associated to our model. To conclude, the major morphological and ultrastructural epidermal modifications observed in both our extended culture skin equivalent model and skin biopsies from old donors validate the relevance of our model for studying chronological aging, understanding and elucidating age-related modifications of basic skin biological processes. In addition, our model provides a unique tool for identifying new targeted molecules intended at improving the appearance of aging skin.


Assuntos
Epiderme/fisiologia , Adulto , Idoso , Envelhecimento , Membrana Basal/fisiologia , Membrana Basal/ultraestrutura , Senescência Celular , Epiderme/ultraestrutura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Proteínas Filagrinas , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Antígeno Ki-67/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Técnicas de Cultura de Tecidos , Adulto Jovem
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