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Background: Aframomum sp. is a genus of plants in the Zingiberaceae family. It includes several species, some of which are used in cosmetics for their various properties, making them useful in skincare products, particularly for anti-aging, moisturizing, and brightening the skin. However, to date, there is no experimental evidence on its natural extracts obtained or modified using microorganisms (bio-fermentation) as an anti-aging agent. Objective: The present study aimed to evaluate the antiaging effect of a Bio-fermented Aframomum angustifolium (BAA) extract on 3D bioprinted skin equivalent. Methods: The consortium of microorganisms contained Komagataeibacter, Gluconobacter, Acetobacter, Saccharomyces, Torulaspora, Brettanomyces, Hanseniaspora, Leuconostoc, Lactobacillus, Schizosaccharomyces. It was developed on a media containing water, sugar, and infused black tea leaves. The seeds of Aframomum angustifolium previously grounded were mixed with the culture medium, and the ferments in growth; this fermentation step lasted 10 days. Then, the medium was collected and filtered (0.22 µm) to obtain the BAA extract. To enhance our comprehension of the impact of BAA extract on skin aging, we developed skin equivalents using bio-printing methods with the presence or absence of keratinocyte stem cells (KSC). These skin equivalents were derived from keratinocytes obtained from both a middle-aged donor, with and without KSC. Moreover, we examined the effects of treating the KSC-depleted skin equivalents with Bio-fermented Aframomum angustifolium (BAA) extract for 5 days. Skin equivalents containing KSC-depleted keratinocytes exhibited histological characteristics typical of aged skin and were compared to skin equivalents derived from young donors. Results: The BAA extract contained specific organic acids such as lactic, gluconic, succinic acid and polyphenols. KSC-depleted skin equivalents that were treated with BAA extract exhibited higher specular reflection, indicating better hydration of the stratum corneum, higher mitotic activity in the epidermis basal layer, improved dermal-epidermal connectivity, and increased rigidity of the dermal-epidermal junction compared to non-treated KSC-depleted equivalents. BAA extract treatments also resulted in changes at the dermis level, with an increase in total collagen and a decrease in global laxity, suggesting that this extract could help maintain youthful-looking skin. Conclusion: In summary, our findings indicated that BAA extract treatments have pleiotropic beneficial effects on skin equivalents and that the bio-fermentation provides new biological activities to this plant.
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In the interfollicular epidermis (IFE), stem cells (KSC) generate transit amplifying (TA) cells that, after symmetric divisions, produce differentiating daughters. Here, we isolated and characterized the highly proliferative interfollicular epidermal basal cell population "early" TA (ETA) cells, based on their capacity to adhere to type IV collagen. Proliferation and colony-forming efficiency in ETA cells are lower than in KSC but higher than in "late" TA (LTA). Stemness, proliferation, and differentiation markers confirmed that ETA cells display a unique phenotype. Skin reconstructs derived from ETA cells present different features (epidermal thickness, Ki67, and Survivin expression), as compared to skin equivalents generated from either KSC or LTA cells. The low-affinity neurotrophin receptor CD271, which regulates the KSC to TA cell transition in the human epidermis through an on/off switch control mechanism, is predominantly expressed in ETA cells. Skin equivalents generated from siRNA CD271 ETA cells display a more proliferative and less differentiated phenotype, as compared to mock-derived reconstructs. Consistently, CD271 overexpression in LTA cells generates a more proliferative skin equivalent than mock LTA cells. Finally, the CD271 level declines with cellular senescence, while it induces a delay in p16INK4 expression. We conclude that ETA cells represent the first KSC progenitor with exclusive features. CD271 identifies and modulates ETA cells, thus participating in the early differentiation and regenerative capacity of the human epidermis.
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Células Epidérmicas , Queratinocitos , Humanos , Diferenciación Celular , Proliferación Celular , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Piel/metabolismoRESUMEN
Reconstructed human epidermis equivalents (RHE) have been developed as a clinical skin substitute and as the replacement for animal testing in both research and industry. KiPS, or keratinocytes derived from induced pluripotent stem cells (iPSCs) are frequently used to generate RHE. In this study, we focus on the mitochondrial performance of the KiPS derived from iPSCs obtained from two donors. We found that the KiPS derived from the older donor have more defective mitochondria. Treatment of these KiPS with a plant extract enriched in compounds known to protect mitochondria improved mitochondrial respiration and rendered them fully competent to derive high-quality RHE. Overall, our results suggest that improving mitochondrial function in KiPS is one of the key aspects to obtain a functional RHE and that our plant extracts can improve in this process.
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Queratinocitos , Extractos Vegetales , Animales , Células Epidérmicas , Epidermis/metabolismo , Humanos , Queratinocitos/metabolismo , Mitocondrias , Extractos Vegetales/metabolismo , Extractos Vegetales/farmacologíaRESUMEN
Senescence is a well-characterized cellular state associated with specific markers such as permanent cell proliferation arrest and the secretion of messenger molecules by cells expressing the senescence-associated secretory phenotype. The senescence-associated secretory phenotype composition depends on many factors such as the cell type or the nature of the stress that induces senescence. Because the skin constitutes a barrier with the external environment, it is particularly subjected to different types of stresses and consequently prone to premature cellular aging. The dicarbonyl compounds glyoxal (GO) and methylglyoxal are precursors of advanced glycation end products, whose presence marks normal and pathological aging. In this study, we show that GO treatment provokes oxidative stress by increasing ROS and advanced glycation end-products levels and induces senescence in human keratinocytes. Furthermore, GO-induced senescence bears a unique molecular progression profile: an early-stage senescence when protein kinase BâFOXO3a-p27KIP1 pathway mediates cell cycle arrest and a late-stage senescence maintained by the p16INK4/pRb pathway. Moreover, we characterized the resulting secretory phenotype during early-stage senescence by mass spectrometry. Our study provides evidence that GO can affect keratinocyte functions and act as a driver of human skin aging. Hence, senotherapeutics aimed at modulating GO-associated senescence phenotype hold promising potential.
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Glioxal , Proteínas Proto-Oncogénicas c-akt , Senescencia Celular/fisiología , Humanos , Queratinocitos , Estrés OxidativoRESUMEN
Compelling evidence suggests that heavy metals have potentially harmful effects on the skin. However, knowledge about cellular signaling events and toxicity subsequent to human skin cell exposure to metals is still poorly documented. The aim of this study was to focus on the interaction between four different heavy metals (lead, nickel, cadmium, and mercury) at doses mimicking chronic low-levels of environmental exposure and the effect on skin to get better insight into metal-cell interactions. We provide evidence that the two metals (lead and nickel) can permeate the skin and accumulate at high concentrations in the dermis. The skin barrier was disrupted after metal exposure and this was accompanied by apoptosis, DNA damage and lipid oxidation. Skin antioxidant enzymes such as glutathione peroxidase and methionine sulfoxide reductase are also heavy metal targets. Taken together, our findings provide insight into potential mechanisms of metal-induced oxidative stress production and the cellular consequences of these events.
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Cadmio/toxicidad , Plomo/toxicidad , Mercurio/toxicidad , Níquel/toxicidad , Piel/efectos de los fármacos , Adulto , Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Femenino , Humanos , Metales Pesados/toxicidad , Imagen Molecular , Estrés Oxidativo/efectos de los fármacos , Piel/diagnóstico por imagen , Piel/metabolismoRESUMEN
Optimal stress response (SR) is an essential aspect of the property of dynamic homeostasis of all biological systems, including cells in culture. Whereas severe stress can induce the so-called stress-induced premature senescence (SIPS), a model developed by Olivier Toussaint, mild stress can strengthen homeodynamics and can postpone senescence through the phenomenon of hormesis. We have attempted to establish multiple stress response profiles (SRP) of early passage young and late passage senescent human facial skin fibroblasts, FSF-1, exposed to either mild (41°C) and severe (43°C) heat shock for 1h, or to mild (2%) and severe (0%) serum deprivation for up to 48h. The results obtained show that FSF-1 cells exposed to two different intensities of stress from two different stressors separately have differential SRP to mild and severe stress, which also vary significantly between young and senescent cells. Establishing multiple and differential SRP to mild and severe stress may facilitate distinguishing between the mild stress-induced beneficial hormetic effects and the harmful effects of severe stress.
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Senescencia Celular , Fibroblastos/metabolismo , Respuesta al Choque Térmico , Piel/metabolismo , Línea Celular , Fibroblastos/patología , Humanos , Piel/patologíaRESUMEN
Compelling evidence suggests that volatile organic compounds (VOCs) have potentially harmful effects to the skin. However, knowledge about cellular signaling events and toxicity subsequent to VOC exposure to human skin cells is still poorly documented. The aim of this study was to focus on the interaction between 5 different VOCs (hexane, toluene, acetaldehyde, formaldehyde and acetone) at doses mimicking chronic low level environmental exposure and the effect on human keratinocytes to get better insight into VOC-cell interactions. We provide evidence that the proteasome, a major intracellular proteolytic system which is involved in a broad array of processes such as cell cycle, apoptosis, transcription, DNA repair, protein quality control and antigen presentation, is a VOC target. Proteasome inactivation after VOC exposure is accompanied by apoptosis, DNA damage and protein oxidation. Lon protease, which degrades oxidized, dysfunctional, and misfolded proteins in the mitochondria is also a VOC target. Using human skin explants we found that VOCs prevent cell proliferation and also inhibit proteasome activity in vivo. Taken together, our findings provide insight into potential mechanisms of VOC-induced proteasome inactivation and the cellular consequences of these events.
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Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Estrés Oxidativo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Compuestos Orgánicos Volátiles/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores , Daño del ADN , Glutatión/metabolismo , Humanos , Inmunofenotipificación , Oxidación-Reducción , Complejo de la Endopetidasa Proteasomal/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Compuestos Orgánicos Volátiles/análisisRESUMEN
Skin pigmentation is directed by epidermal melanin units, characterized by long-lived and dendritic epidermal melanocytes (MC) that interact with viable keratinocytes (KC) to contribute melanin to the epidermis. Previously, we reported that MC:KC contact is required for melanosome transfer that can be enhanced by filopodi, and by UVR/UVA irradiation, which can upregulate melanosome transfer via Myosin X-mediated control of MC filopodia. Both MC and KC express Ca2+ -dependent E-cadherins. These homophilic adhesion contacts induce transient increases in intra-KC Ca2+ , while ultraviolet radiation (UVR) raises intra-MC Ca2+ via calcium-selective ORAI1 ion channels; both are associated with regulating melanogenesis. However, how Ca2+ triggers melanin transfer remains unclear. Here we evaluated the role of E-cadherin in UVR-mediated melanin transfer in human skin cells. MC and KC in human epidermis variably express filopodia-associated E-cadherin, Cdc42, VASP and ß-catenin, all of which were upregulated by UVR in human MC in vitro. Knockdown of E-cadherin revealed that this cadherin is essential for UVR-induced MC filopodia formation and melanin transfer. Moreover, Ca2+ induced a dose-dependent increase in filopodia formation and melanin transfer, as well as increased ß-catenin, Cdc42, Myosin X and E-cadherin expression in these skin cells. Together, these data suggest that filopodial proteins and E-cadherin, which are upregulated by intracellular (UVR-stimulated) and extracellular Ca2+ availability, are required for filopodia formation and melanin transfer. This may open new avenues to explore how Ca2+ signalling influences human pigmentation.
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Cadherinas/metabolismo , Calcio/farmacología , Melaninas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/efectos de la radiación , Rayos Ultravioleta , Adulto , Cadherinas/genética , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Células Epidérmicas , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Uniones Intercelulares , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Sistema de Señalización de MAP Quinasas , Masculino , Melanocitos/efectos de los fármacos , Melanocitos/metabolismo , Melanocitos/efectos de la radiación , Melanosomas/metabolismo , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Miosinas/metabolismo , Fosfoproteínas/metabolismo , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Seudópodos/efectos de la radiación , ARN Interferente Pequeño , Regulación hacia Arriba/efectos de la radiación , beta Catenina/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Few studies have evaluated the prevalence of skin tumours in the geriatric population and none have analysed different skin aging parameters for whole-body skin in this population. To evaluate the prevalence of skin tumours and global skin aging in a French cohort of elderly people. In total, 209 subjects, 105 women and 104 men (mean age: 77.5; range: 74-81 years), were enrolled from the PROOF (PROgnostic indicator OF cardiovascular and cerebrovascular events) cohort. SCINEXA (SCore for INtrinsic and EXtrinsic skin Aging) was used to assess the degree of skin aging and the prevalence of skin tumours. Some additional cutaneous parameters were also studied. Skin aging in women and men was compared. Mean global SCINEXA was 24.3 (SD: 4.7; range: 8.2-35.3). Solar elastosis and lax appearance were more severe in women (t test; p<0.0001), whereas pseudoscars (t test; p = 0.0312) and coarse wrinkles (t test; p = 0.0479) were more severe in men. Erythrosis coli (chi-square test; p <0.0001) was more frequent in men, whereas varicous veins (chi-square test; p = 0.0026) and eyelid xanthomas (chi-square test; p = 0.0282) were more frequent in women. Twelve patients presented with cutaneous carcinomas and two patients had early melanomas. This research describes in detail the main indices of skin aging in an old population and the differences related to gender. Moreover, it highlights the utility of systematic screening of old patients by dermatologists in order to diagnose skin cancers early.
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Carcinoma Basocelular/epidemiología , Carcinoma de Células Escamosas/epidemiología , Melanoma/epidemiología , Lesiones Precancerosas/epidemiología , Envejecimiento de la Piel , Neoplasias Cutáneas/epidemiología , Anciano , Anciano de 80 o más Años , Dermatitis/epidemiología , Enfermedades de los Párpados/epidemiología , Femenino , Francia/epidemiología , Humanos , Masculino , Prevalencia , Factores Sexuales , Várices/epidemiología , Xantomatosis/epidemiologíaRESUMEN
Intracellular autophagy (AP) is a stress response that is enhanced under conditions of limitation of amino acids, growth factors and other nutrients, and also when macromolecules become damaged, aggregated and fibrillated. Aging is generally accompanied by an increase in intracellular stress due to all the above factors. Therefore, we have compared the basal levels of AP in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from the photo-protected and photo-exposed area of the arms of 20 healthy persons of young and old ages. Immunofluorescence microscopy, employing antibodies against a specific intracellular microtubule-associated protein-1 light chain-3 (LC3) as a well established marker of AP, showed a 5-fold increase in the basal level of LC3 in near senescent human skin fibroblasts. However, no such age-related increase in LC3 fluorescence and AP could be detected in full thickness skin sections from the biopsies obtained from 10 healthy young (age 25 to 30 yr) and 10 old (age 60 to 65 yr) donors. Furthermore, there was no difference in the basal level of LC3 in the skin sections from photo-protected and photo-exposed areas of the arm. Thus, in normal conditions, the aging phenotype of the skin cells in culture and in the body appears to be different in the case of AP.
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Autofagia , Senescencia Celular , Fibroblastos/citología , Envejecimiento de la Piel , Adulto , Anciano , Envejecimiento , Células Cultivadas , Femenino , Humanos , Masculino , Proteínas Asociadas a Microtúbulos/análisis , Persona de Mediana EdadRESUMEN
Repeated exposure to mild heat shock (HS) has been shown to induce a wide range of health promoting hormetic effects in various biological systems, including human cells undergoing aging in vitro. In order to understand how cells distinguish between mild and severe stress, we have investigated the extent of early and immediate HS response by analyzing the nuclear translocation of the transcription factor heat shock factor-1 (HSF1), in serially passaged normal adult human facial skin fibroblasts exposed to mild (41 °C) or severe (43 °C) HS. Cells respond differently when exposed to mild and severe HS at different passage levels in terms of the extent of HSF1 translocation. In early passage young cells there was a 5-fold difference between mild and severe HS in the extent of HSF1 translocation. However, in near senescent late passage cells, the difference between mild and severe stress in terms of the extent of HSF1 translocation was reduced to less than 2-fold. One of the reasons for this age-related attenuation of heat shock response is due to the fact there was a higher basal level of HSF1 in the nuclei of late passage cells, which is indicative of increased intrinsic stress during cellular aging. These observations are consistent with previously reported data that whereas repeated mild stress given at younger ages can slow down aging and increase the lifespan, the same level of stress given at older ages may not provide the same benefits. Therefore, elucidating the early and immediate steps in the induction of stress response can be useful in deciding whether a particular level of stress is potentially hormetically beneficial or not.
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Skin aging is the result of intrinsic chronological aging and photoaging, due to UV exposure, that both share important histological modifications and molecular features, including alterations of proteins. One of the main damage is glycation that occurs when reducing sugars react non-enzymatically with proteins. This reaction also happens when the dicarbonyl compounds GO (glyoxal) and MG (methylglyoxal), which are glucose derivatives, react with proteins. These compounds can be detoxified by the glyoxalase system composed of two enzymes, Glo1 (glyoxalase I) and Glo2 (glyoxalase II). The aims of the present mini-review are to briefly summarize our current knowledge of the biological roles of these enzymes in aging and then discuss the relevance of studying the role of glycation and of detoxifying systems in human skin aging.
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Lactoilglutatión Liasa/metabolismo , Envejecimiento de la Piel/fisiología , Animales , Glicosilación , Humanos , Piruvaldehído/metabolismoRESUMEN
Cutaneous aging translates drastic structural and functional alterations in the extracellular matrix (ECM). Multiple mechanisms are involved, including changes in protease levels. We investigated the age-related protein expression and activity of cysteine cathepsins and the expression of two endogenous protein inhibitors in young and aged Caucasian women skin epidermis. Immunofluorescence studies indicate that the expression of cathepsins K, S and V, as well as cystatins A and M/E within keratinocytes is reduced in photoprotected skin of aged women. Furthermore, the overall endopeptidase activity of cysteine cathepsins in epidermis lysates decreased with age. Albeit dermal elastic fiber and laminin expression is reduced in aged skin, staining of nidogen-1, a key protein in BM assembly that is sensitive to proteolysis by cysteine, metallo- and serine proteases, has a similar pattern in both young and aged skin. Since cathepsins contribute to the hydrolysis and turnover of ECM/basement membrane components, the abnormal protein degradation and deposition during aging process may be related in part to a decline of lysosomal/endosomal cathepsin K, S and V activity.
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Catepsina K/metabolismo , Catepsinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Epidermis/enzimología , Adolescente , Adulto , Anciano , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Cistatinas/metabolismo , Exposición a Riesgos Ambientales , Epidermis/fisiología , Epidermis/efectos de la radiación , Femenino , Expresión Génica , Humanos , Queratinocitos/enzimología , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Envejecimiento de la Piel/efectos de la radiación , Luz Solar , Adulto Joven , KalininaRESUMEN
Human cysteine cathepsin S (catS) participates in distinct physiological and pathophysiological cellular processes and is considered as a valuable therapeutic target in autoimmune diseases, cancer, atherosclerosis, and asthma. We evaluated the capacity of negatively charged glycosaminoglycans (heparin, heparan sulfate, chondroitin 4/6-sulfates, dermatan sulfate, and hyaluronic acid) to modulate the activity of catS. Chondroitin 4-sulfate (C4-S) impaired the collagenolytic activity (type IV collagen) and inhibited the peptidase activity (Z-Phe-Arg-AMC) of catS at pH 5.5, obeying a mixed-type mechanism (estimated Ki = 16.5 ± 6 µM). Addition of NaCl restored catS activity, supporting the idea that electrostatic interactions are primarly involved. Furthermore, C4-S delayed in a dose-dependent manner the maturation of procatS at pH 4.0 by interfering with the intermolecular processing pathway. Binding of C4-S to catS was demonstrated by gel-filtration chromatography, and its affinity was measured by surface plasmon resonance (equilibrium dissociation constant Kd = 210 ± 40 nM). Moreover, C4-S induced subtle conformational changes in mature catS as observed by intrinsic fluorescence spectroscopy analysis. Molecular docking predicted three specific binding sites on catS for C4-S that are different from those found in the crystal structure of the cathepsin K-C4-S complex. Overall, these results describe a novel glycosaminoglycan-mediated mechanism of catS inhibition and suggest that C4-S may modulate the collagenase activity of catS in vivo.
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Catepsinas/metabolismo , Sulfatos de Condroitina/metabolismo , Sitios de Unión , Catepsinas/antagonistas & inhibidores , Cromatografía en Gel , Colágeno Tipo IV/metabolismo , Cumarinas/metabolismo , Dipéptidos/metabolismo , Humanos , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Resonancia por Plasmón de SuperficieRESUMEN
Application of hormesis in aging research and interventions is becoming increasingly attractive and successful. The reason for this is the realization that mild stress-induced activation of one or more stress response (SR) pathways, and its consequent stimulation of repair mechanisms, is effective in reducing the age-related accumulation of molecular damage. For example, repeated heat stress-induced synthesis of heat shock proteins has been shown to have a variety of anti-aging effects on growth and other cellular and biochemical characteristics of normal human skin fibroblasts, keratinocytes and endothelial cells undergoing aging in vitro. Therefore, searching for potential hormetins - conditions and compounds eliciting SR-mediated hormesis - is drawing attention of not only the researchers but also the industry involved in developing healthcare products, including nutriceuticals, functional foods and cosmeceuticals. Here we present the example of a skin care cosmetic as one of the first successful product developments incorporating the ideas of hormesis. This was based on the studies to analyse the molecular effects of active ingredients extracted from the roots of the Chinese herb Sanchi (Panax notoginseng) on gene expression at the level of mRNAs and proteins in human skin cells. The results showed that the ginsenosides extracted from Sanchi induced the transcription of stress genes and increased the synthesis of stress proteins, especially the heat shock protein HSP1A1 or Hsp70, in normal human keratinocytes and dermal fibroblasts. Furthermore, this extract also has significant positive effects against facial wrinkles and other symptoms of facial skin aging as tested clinically, which may be due to its hormetic mode of action by stress-induced synthesis of chaperones involved in protein repair and removal of abnormal proteins. Acceptance of such a hormesis-based product by the wider public could be instrumental in the social recognition of the concept of hormesis as the beneficial effects of mild stress of choice, and will encourage the development of novel health care products with physical, nutritional and mental hormetins.
RESUMEN
Cathepsin S (catS), which is expressed in normal human keratinocytes and localized close to the dermal-epidermal junction (DEJ) degrades some of major basement membrane (BM) constituents. Among them, catS readily hydrolyzed in a time and dose dependent manner human nidogen-1 (nid-1) and nidogen-2, which are key proteins in the BM structure. CatS preferentially cleaved nid-1 at both acid and neutral pH. Hydrolysis of nid-1 was hampered in murine ctss(-/-) spleen lysates pretreated with inhibitors of other classes of proteases. Nid-1 was cleaved within its G2 and G3 globular domains that are both involved in interactions with other BM components. Binding assays with soluble and immobilized ligands indicated that catS altered the formation of complexes between nid-1 and other BM components. Assuming that the cleavage of nid-1 impairs its ability to crosslink with BM partners and perturbs the viscoelastic properties of BM matrix, these data indicate that catS may participate in BM proteolysis, in addition to already identified proteases.
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Membrana Basal/metabolismo , Catepsinas/química , Moléculas de Adhesión Celular/química , Glicoproteínas de Membrana/química , Proteínas de Unión al Calcio , Moléculas de Adhesión Celular/metabolismo , Dermis/metabolismo , Epidermis/metabolismo , Humanos , Hidrólisis , Queratinocitos/citología , Ligandos , Glicoproteínas de Membrana/metabolismo , Modelos Genéticos , Pichia/metabolismo , Unión Proteica , Proteolisis , Reología/métodos , Piel/patología , Resonancia por Plasmón de SuperficieRESUMEN
Skin acts as a barrier between the environment and internal organs and performs functions that are critical for the preservation of body homeostasis. In mammals, a complex network of circadian clocks and oscillators adapts physiology and behavior to environmental changes by generating circadian rhythms. These rhythms are induced in the central pacemaker and peripheral tissues by similar transcriptional-translational feedback loops involving clock genes. In this work, we investigated the presence of functional oscillators in the human skin by studying kinetics of clock gene expression in epidermal and dermal cells originating from the same donor and compared their characteristics. Primary cultures of fibroblasts, keratinocytes, and melanocytes were established from an abdominal biopsy and expression of clock genes following dexamethasone synchronization was assessed by qPCR. An original mathematical method was developed to analyze simultaneously up to nine clock genes. By fitting the oscillations to a common period, the phase relationships of the genes could be determined accurately. We thereby show the presence of functional circadian machinery in each cell type. These clockworks display specific periods and phase relationships between clock genes, suggesting regulatory mechanisms that are particular to each cell type. Taken together, our data demonstrate that skin has a complex circadian organization. Oscillators are present not only in fibroblasts but also in epidermal keratinocytes and melanocytes and are likely to act in coordination to drive rhythmic functions within the skin.
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Relojes Circadianos/genética , Fibroblastos/fisiología , Regulación de la Expresión Génica , Queratinocitos/fisiología , Melanocitos/fisiología , Piel/citología , Proteínas CLOCK/genética , Células Cultivadas , HumanosRESUMEN
Transfer of the melanocyte-specific and lysosome-related organelle, the melanosome, from melanocytes to keratinocytes is crucial for the protection of the skin against harmful ultraviolet radiation (UVR)--our main physiological cutaneous stressor. However, this commonplace event remains a most enigmatic process despite several early hypotheses. Recently, we and others have proposed a role for filopodia in melanin transfer, although conclusive experimental proof remained elusive. Using known filopodial markers (MyoX/Cdc42) and the filopodial disrupter, low-dose cytochalasin-B, we demonstrate here a requirement for filopodia in melanosome transfer from melanocytes to keratinocytes and also, unexpectedly, between keratinocytes. Melanin distribution throughout the skin represents the key phenotypic event in skin pigmentation. Melanocyte filopodia were also necessary for UVR-stimulated melanosome transfer, as this was also inhibited by MyoX knockdown and low-dose cytochalasin-B. Knockdown of keratinocyte MyoX protein, in its capacity as a phagocytosis effector, resulted in the inhibition of melanin uptake by keratinocytes. This indicates a central role for phagocytosis by keratinocytes of melanocyte filopodia. In summary, we propose a new model for the regulation of pigmentation in human skin cells under both constitutive and facultative (post-UVR) conditions, which we call the "filopodial-phagocytosis model." This model also provides a unique and highly accessible way to study lysosome-related organelle movement between mammalian cells.