Isolation of an "Early" Transit Amplifying Keratinocyte Population in Human Epidermis: A Role for the Low Affinity Neurotrophin Receptor CD271.

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.

E-cadherin mediates ultraviolet radiation- and calcium-induced melanin transfer in human skin cells.

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.

Prevention of dicarbonyl-mediated advanced glycation by glyoxalases: implication in skin aging.

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.

Binding of chondroitin 4-sulfate to cathepsin S regulates its enzymatic activity.

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.

Human skin keratinocytes, melanocytes, and fibroblasts contain distinct circadian clock machineries.

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.

Skin-protective biological activities of bio-fermented Aframomum angustifolium extract by a consortium of microorganisms.

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.

Glyoxal Induces Senescence in Human Keratinocytes through Oxidative Stress and Activation of the Protein Kinase B/FOXO3a/p27KIP1 Pathway.

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 B‒FOXO3a-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.

Reconstruction of functional human epidermis equivalent containing 5%IPS-derived keratinocytes treated with mitochondrial stimulating plant extracts.

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.

Melanin transfer in human skin cells is mediated by filopodia--a model for homotypic and heterotypic lysosome-related organelle transfer.

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.

Elemental and molecular imaging of human full thickness skin after exposure to heavy metals.

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.

The silver locus product (Silv/gp100/Pmel17) as a new tool for the analysis of melanosome transfer in human melanocyte-keratinocyte co-culture.

Melanosomes are melanocyte-specific lysosome-related organelles that are transferred to keratinocytes of the epidermis and anagen hair bulb. Transferred melanin forms supra-nuclear caps that protect epidermal keratinocytes against UV irradiation. The mechanism(s) responsible for melanosome transfer into keratinocytes and their subsequent intra-keratinocyte distribution has long remained one of the most enigmatic of heterotypic cell interactions. Although there have been many attempts to study this process, significant progress has been hindered by the absence of an adequate in vitro model. During our ongoing study of melanocyte-keratinocyte interactions in skin and hair follicle, we have developed a novel in vitro assay that exploits the specificity of Silv/Pmel17/gp100 expression for melanosome/melanin granules. Using matched cultures of keratinocytes and melanocytes isolated from normal healthy epidermis together with double immunofluorescence, we have determined that gp100 is a surprisingly useful tracker of transferred melanin. Moreover, transferred gp100 stained melanin granules emit a bright fluorescence signal, facilitating ready quantification of melanin transfer levels between melanocytes and keratinocytes. This quantitative approach was validated using known inducers and inhibitors of the melanocyte phenotype. This assay further confirmed that cytophagocytosis of melanocyte components (e.g. dendrite tips) by keratinocytes is one route for melanin incorporation into keratinocytes. Lastly, a role for the recently proposed filopodium as a direct conduit for melanin transfer was substantiated using this novel approach. In conclusion, this assay promises to significantly aid our investigations of the molecular basis of melanosome transfer and offers a new tool for the clinical evaluation of melanocyte modulators.

A preliminary attempt to establish multiple stress response profiles of human skin fibroblasts exposed to mild or severe stress during ageing in vitro.

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.

Impairment of methionine sulfoxide reductase during UV irradiation and photoaging.

During chronic UV irradiation, which is part of the skin aging process, proteins are damaged by reactive oxygen species resulting in the accumulation of oxidatively modified protein. UV irradiation generates irreversible oxidation of the side chains of certain amino acids resulting in the formation of carbonyl groups on proteins. Nevertheless, certain amino acid oxidation products such as methionine sulfoxide can be reversed back to their reduced form within proteins by specific repair enzymes, the methionine sulfoxide reductases A and B. Using quantitative confocal microscopy, the amount of methionine sulfoxide reductase A was found significantly lower in sun-exposed skin as compared to sun-protected skin. Due to the importance of the methionine sulfoxide reductase system in the maintenance of protein structure and function during aging and conditions of oxidative stress, the fate of this system was investigated after UVA irradiation of human normal keratinocytes. When keratinocytes are exposed to 15 J/cm(2) UVA, methionine sulfoxide reductase activity and content are decreased, indicating that the methionine sulfoxide reductase system is a sensitive target for UV-induced inactivation.

Proteasome and photoaging: the effects of UV irradiation.

Cellular aging is characterized by the accumulation of oxidatively modified proteins that result, at least in part, from impaired degradation of abnormal proteins. The proteasome is the major intracellular proteolytic system implicated in the removal of abnormal and oxidized proteins. In human epidermal cells, previous studies have evidenced that proteasome function is decreased during aging as well as upon UV irradiation, which is the main component of photoaging. The age-related decline of proteasome activity has been reported to be due to either or both decreased proteasome subunits expression and content, inactivation upon alteration of proteasome subunits, and accumulation of endogenous inhibitors, such as highly oxidized and cross-linked proteins. To gain further insight in the mechanisms that might be implicated in the decreased activity of the proteasome upon photoaging, purified 20S human proteasome has been exposed to UVA- and UVB-irradiation. The effect of such an irradiation on proteasome peptidase activities has been monitored and shown to promote a stimulation or an inhibition of the peptidase activities depending on whether the proteasome is under its latent or a nonphysiological active form. Analysis of the patterns of proteasome subunits by 2D gel electrophoresis has revealed modification for several subunits for UV-irradiated proteasome only in its irreversibly activated form, compared with nonirradiated and irradiated latent forms, indicating that the 20S proteasome is rather resistant to UV irradiation.

The use of antisense strategy to modulate human melanogenesis.

BACKGROUND AND OBJECTIVES: Skin without significant dyschromia is an aesthetic goal of people worldwide. Current options for lightening skin could have significant drawbacks. The antisense strategy may be a viable alternative. The reactions in melanogenesis are catalyzed mainly by tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2. Activation of tyrosinase is associated with phosphorylation by protein kinase C-betaI (PKC-betaI) and formation of a complex between phosphorylated tyrosinase and TRP-1. The aim of this study was to use 2 antisense oligonucleotides to modulate the synthesis of the tyrosinase/TRP-1 complex, PKC-beta, or both by interacting with the targeted mRNA, thus whitening skin by interfering with melanogenesis at the translational level. METHODS/STUDY DESIGN: In the in vitro study, the effect of the antisense oligonucleotides was evaluated by measuring the rate at which dihydroxyphenylalanine (DOPA) oxidase transforms L-DOPA to DOPAchrome in the pathway for melanin biosynthesis. A reduction in the reaction rate compared to the controls corresponded to a decrease in the enzyme activity and, consequently, to a reduction of the formation of melanin pigments. To evaluate the in vivo lightening effect of the antisense oligonucleotides, 30 Asian women volunteers with pigmented spots on both hands applied the test product twice daily for 8 weeks. The test product was applied to 2 marked-off areas of the hand: a pigmented spot (to evaluate the effect of the test product on the color of the spot) and a nonpigmented spot area (to evaluate the effect of the test product on normal skin pigmentation). The lightening effect was evaluated by comparing chromametric and mexametric parameters before treatment, after 4 weeks, and after 8 weeks. RESULTS: In vitro DOPA-oxidase activity was inhibited by 13% in melanocytes treated with the antisense sequence for PKC-BI alone, by 16% with the antisense sequence for TRP-1 alone, and by 36% with the association of 2 sequences. The inhibiting effect with both sequences required the specific sequences with nonreversed polarities. In vivo clinical results showed statistically significant whitening in both pigmented spots and nonpigmented spots when the test product was applied twice daily for 8 weeks by up to 30 Asian women. CONCLUSIONS: The association of TRP-1 and PKC-betaI antisense molecules significantly increased the inhibition of tyrosinase activity on human melanocytes. Antisense oligonucleotides are a new generation of active cosmetic ingredients that offer unprecedented specificity, biological stability, and safety in lightening skin. This is the first report of positive results in a cosmetic based on the use of these new active agents.

Enhancing cell longevity for cosmetic application: a complementary approach.

BACKGROUND AND OBJECTIVES: Cell longevity is linked to sirtuins (silent information regulators), which belong to a family of enzymes implicated in gene silencing, apoptosis, fatty acid metabolism, and regulation of cellular life spans of organisms. Sirtuins are associated with genes that coordinate and optimize the functions of cells as cells struggle to survive in a stressful environment, as it is the case for skin cells. This study focuses on 1) yeast Kluyveromyces biopetides in stimulating the expression of sirtuin in human cutaneous cells and 2) the benefit for the skin of an active skin care product containing yeast Kluyveromyces biopetides. METHODS: Silent mating type information regulation 2 homolog 1 (SIRT1) was investigated by immunostaining, Westem blotting, and cytometry on normal human skin cells in culture and on healthy skin samples ex vivo. SIRT7 are mammalian versions of the yeast SIR2 gene. Cellular integrity and aging was followed by comet assays measuring DNA fragmentation and beta galactosidase activity (a marker of senescence). The test product was yeast Kluyveromyces biopeptides. Thirty-three female subjects aged 37 to 64 years (mean 51.6 years) enrolled in the study. Subjects applied a formulation enriched in 1% of the yeast biopeptides SIRT1 activator once daily to the face and neck for 4 weeks. Dermatologists used a graded scale (1-9) to score fine lines and wrinkles, hydration, pigment color intensity, complexion radiance, skin density, firmness, complexion homogeneity, and texture of the skin before and after the first application and again after 4 weeks of use. A Pixel Skin method, based on an analysis of the gray-level variance and surface of imperfections (age-related parameters) from numerical pictures of the faces, was used to objectively measure the skin care efficacy. RESULTS: The yeast Kluyveromyces biopeptides 1) significantly increased SIRT1 expression in normal human dermal skin fibroblasts in vitro (+172%) and in epidermal cells of healthy human skin ex vivo and 2) decreased cell senescence and DNA fragmentation induced by ultraviolet-B (UVB) stress. At the end of the study, facial improvements could be seen on fine lines and wrinkles, hydration, pigmented spot color intensity, complexion radiance, firmness, complexion homogeneity, and texture. Improvement in hydration was significant immediately after the first application. Skin-pixel measurement and analysis show a significant reduction of the gray variance linked to pixel heterogeneity (-4.2%) and a significant reduction of the surface of skin imperfections (-30.4%). All the indicators from clinical evaluation to the objective measurements of the skin show a significant improvement of the aged skin. CONCLUSION: These results demonstrate the efficacy of the yeast Kluyveromyces biopeptides in activating SIRT 1 of human skin cells, improving their DNA resistance and senescence, and of a formulation enriched in this ingredient in treating multiple skin aging signs.

Oxidative damage and impairment of protein quality control systems in keratinocytes exposed to a volatile organic compounds cocktail.

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.

Algae extract-mediated stimulation and protection of proteasome activity within human keratinocytes exposed to UVA and UVB irradiation.

Sun exposure is the major environmental influence for epidermal cells; the harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that alter cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of most of oxidized proteins and that impairment of proteasome function is a hallmark of cellular aging. In a previous study, we investigated the effects of UV irradiation on proteasomes in human keratinocyte cultures and showed that all three peptidase activities were decreased 24 h after irradiation of the cells. Increased levels of oxidatively modified proteins were observed in irradiated cells and were found to act as endogenous inhibitors of the proteasome. We report here on the stimulating and protective effects of an algae extract, prepared from Phaeodactylum tricornutum, on proteasome peptidase activities of human keratinocytes exposed to UVA and UVB irradiation. In addition, preserving proteasome function resulted in lowering the extent of the irradiation-induced protein oxidative damage, opening up new strategies for protection of epidermal cells against the detrimental effects of UV irradiation.