Abstract: 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.-Singh, S. K., Kurfurst, R., Nizard, C., Schnebert, S., Perrier, E., Tobin, D. J. Melanin transfer in human skin cells is mediated by filopodia-a model for homotypic and heterotypic lysosome-related organelle transfer.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: Senile lentigo is a common component of photoaged skin. It is characterized by hyperpigmented macules which affect chronically irradiated skin mostly after the age of 50. This study was undertaken to assess the morphology of senile lentigo on the dorsum of the hands. A systematic comparison between lesional and perilesional skin using histology and transmission electron microscopy was done to determine whether melanocytes or keratinocytes are affected in the evolution of lesions and which tissue structure is modified. The histology study showed that lesional skin is characterized by a hyperpigmented basal layer and an elongation of the rete ridges, which seem to drive deeply into the dermis. The epidermis contained clusters of keratinocytes, which retained and accumulated the melanin pigment. Electron microscopy studies showed important modifications in the lesional skin ultrastructure in comparison with perilesional skin. In melanocytes from perilesional and lesional skin, we observed normal size melanosomes at all stages of maturation in the cytoplasm and in migration within dendrites. No pigment accumulation was observed. However, the morphology of melanocytes in lesional skin revealed an activated status with numerous mitochondria and a well-developed endoplasmic reticulum, which could reflect intense protein synthesis. In basal keratinocytes from lesional skin, we observed numerous melanosome complexes called polymelanosomes, which formed massive caps on the nuclei. Observations in colored semi-thin sections also revealed perturbed structures in the basal layer region, which could explain the skin perturbation. Indeed, we observed keratinocytes that presented important microinvaginations and pendulum melanocytes, which sank into the dermis, beneath the basal layer of keratinocytes. These cell modifications seemed to be due to a perturbation of the dermal-epidermal junction, which appeared disorganized and disrupted and could directly disturb the basal support of the cells.
Abstract: 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.
Abstract: Proteins are modified by reactive oxygen species, and oxidation of specific amino acid residues can impair their biological functions, leading to an alteration in cellular homeostasis. Oxidized proteins can be eliminated through either degradation or repair. Repair is limited to the reversion of a few modifications such as the reduction of methionine oxidation by the methionine sulfoxide reductase (Msr) system. However, accumulation of oxidized proteins occurs during aging, replicative senescence, or neurological disorders or after an oxidative stress, while Msr activity is impaired. In order to more precisely analyze the relationship between oxidative stress, protein oxidative damage, and MsrA, we stably overexpressed MsrA full-length cDNA in SV40 T antigen-immortalized WI-38 human fibroblasts. We report here that MsrA-overexpressing cells are more resistant than control cells to hydrogen peroxide-induced oxidative stress, but not to ultraviolet A irradiation. This MsrA-mediated resistance is accompanied by a decrease in intracellular reactive oxygen species and is partially abolished when cells are cultivated at suboptimal concentration of methionine. These results indicate that MsrA may play an important role in cellular defenses against oxidative stress, by catalytic removal of oxidant through the reduction of methionine sulfoxide, and in protection against death by limiting, at least in part, the accumulation of oxidative damage to proteins.
Abstract: Modification of proteins by reactive oxygen species is implicated in different disorders. The proteasome is a multicatalytic proteinase in charge of intracellular protein turnover and of oxidized proteins degradation. Consequently, proteasome function is very important in controlling the level of altered proteins in eukaryotic cells. Evidence for a decline in proteasome activity during skin photo-aging has been provided in Bulteau et al. in 2002. The ability of a lipid algae extract (Phaeodactylum tricornutum) to stimulate 20S proteasome peptidase activities was described by Nizard et al. in 2001. Furthermore, keratinocytes treated with Phaeodactylum tricornutum extract and then UVA and UVB irradiated, exhibited a sustained level of proteasome activity comparable to the one of nonirradiated cells. The level of modified proteins can be quantified by measurement of protein carbonyl content (Oxyblot technique), which has been shown to increase with aging and other disorders. In this paper, it is described that, in the presence of this lipid algae extract, the level of oxidized proteins is reduced, as assessed by the Oxyblot technique. These results are obtained both with culture of human keratinocytes and stratum corneum skin cells (obtained by stripping) from human volunteers. Altogether, these results argue for the presence of compounds in this algae extract that have a stimulating and/or protective effect on proteasome activity, resulting in a decreased level of protein oxidation.
Abstract: BACKGROUND: Senile lentigo (SL) is a common component of photoaged skin. It is characterized by hyperpigmented macules which affect chronically irradiated skin mostly after 50 years of age. This study was undertaken to assess the basic morphology of SL on dorsum of hands. METHODS: A systematic comparison between lesional vs. perilesional skin using immunohistochemistry and electron microscopy was done to detect precursor lesions of SL and to determine whether melanocytes or keratinocytes were first affected in the evolution of lesions. RESULTS: In 12 cases studied, the main findings show that clusters of perilesional keratinocytes accumulate melanin in large melanosomial complexes, and that melanocytes counts are increased respective to total length of section in lesional skin, but the increment is probably due to the development of characteristic epidermal rete ridges. Melanocytes had overall a normal ultrastructure, with mostly quiescent features in perilesional skin and melanosomial transport seeming more active in lesional skin. CONCLUSIONS: Our data indicate that SL may represent a loss of epidermal melanin unit homeostasis due to chronic irradiation, where keratinocytic changes predominate over melanocytic changes. We hypothesize that abnormal pigment retention in keratinocytes is the primary defect in SL, which may partly explain the therapeutic effect of retinoids.
Abstract: Heat shock protein (HSP) 47 is a specific chaperone of procollagen. This heat shock protein is responsible for the correct three-dimensional organization of procollagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP 47 in aged, photoaged, and senescent fibroblasts and its modulation by a plant extract (Salix alba). The level of HSP 47 and/or procollagen expression in fibroblasts was measured by real-time RT-PCR (mRNA transcripts) and by flow cytometry (immunochemistry technique for measurement of arbitrary fluorescence intensity). Immunochemistry techniques and confocal microscopy were used to visualize the cellular localization of HSP 47 and procollagen. These parameters were compared with different age donors, nonsenescent, and senescent fibroblasts. Fibroblasts were irradiated by a noncytotoxic dose of UVA (6 J/cm(2)), and HSP 47 level was evaluated. S. alba extract was tested for its capacity to modulate HSP 47 expression. Colocalization of HSP 47 and procollagen was shown by confocal microscopy, indicating that HSP 47 could play a role of procollagen molecular chaperone in the cellular model. It was also shown that the HSP 47 level is decreased in old-donor cells, senescent, and irradiated cells. This decrease can be modulated by a S. alba extract (polyphenols rich) in a dose-dependent manner. The evaluation of HSP 47 expression in the experimental conditions can lead to a new approach of aging and photoaging, pointing out the implication of this chaperone in these pathophysiologic phenomena. Modulation of HSP 47 expression by this family of molecules could be of cosmetic and/or dermatologic interest.
Abstract: Ultraviolet A (UVA) (320-400 nm) radiation is known to cause cutaneous aging and skin cancer. We studied the effect of UVA (365 nm) radiation on the human epidermis by focusing on keratinocyte gap junction-mediated intercellular communication (GJIC). We observed a dose-dependent 10-fold decrease in GJIC induced by UVA in normal human keratinocytes. This decrease in GJIC was associated with time-dependent internalization of connexin43 (Cx43). UVA radiation also damaged the actin cytoskeleton, as shown by microfilament disappearance. Importantly, the decrease in GJIC was transient when keratinocytes were irradiated with 10 J/cm(2) UVA, with a return to baseline values after 8 h. Concomitantly, Cx43 was relocalized and the actin cytoskeleton was restored. UVA irradiation and 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment activated protein kinase C and reduced GJIC. However, Cx43 localization and phosphorylation were differently regulated by the two treatments. This suggests that at least two different pathways may mediate the observed fall in GJIC. These findings identify keratinocyte GJIC as a new UVA target that might sensitize human skin to photoaging and cancer formation.
Abstract: The major environmental influence for epidermal cells is sun exposure and the harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Therefore, the effects of UV-irradiation on proteasome have been investigated in human keratinocyte cultures. Human keratinocytes were irradiated with 10 J/cm(2) of UVA and 0.05 J/cm(2) of UVB and proteasome peptidase activities were measured in cell lysates using fluorogenic peptides. All three peptidase activities were decreased as early as 1 h and up to 24 h after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be a target for the UV-induced damage. Finally, extracts from irradiated keratinocytes were able to inhibit degradation by the proteasome, demonstrating the presence of endogeneous inhibitors, including 4-hydroxy-2-nonenal modified proteins, generated upon UV-irradiation.
Abstract: Proteins are modified by reactive oxygen species and oxidation of specific aminoacid residues can impair their biological functions leading to an alteration of cellular homeostasis. Oxidized proteins can be rapidly eliminated through either degradation or repair. Degradation of oxidized proteins is achieved by the proteasome, a multicatalytic proteolytic system, while repair is limited to few modifications such as methionine oxidation that can be reversed by the Msr system. This system is composed of MsrA and MsrB, which reduce the methionine-S-sulfoxide and the methionine-R-sulfoxide, respectively. However, accumulation of oxidized proteins occurs during aging, replicative senescence, neurological disorders or after an oxidative stress while MsrA activity is impaired. MsrA was shown to be involved in antioxidant defense in several organisms such as bacteria, yeast, drosophila as well as mammals. To analyse more precisely the relationship between oxidative stress and MsrA, we stably overexpressed the MsrA full-length cDNA in SV40 T antigen-immortalized human fibroblasts WI38. We showed that MsrA overexpressing cells are more resistant than control cells to hydrogen peroxyde-induced oxidative stress at concentrations from 250 up to 500 mM. Moreover, in the transfected cells, Msr activity is stimulated by addition of H2O2 while an H2O2-dose dependent inhibition of the Msr activity is observed in control cells For higher concentration of hydrogen peroxide (750 mM), the overexpression of MsrA is no more able to protect cells from oxidative damage and apoptosis. These results suggest that MsrA may play an important role in cellular defenses against oxidative stress and in protection against apoptosis by limiting, at least in part, the accumulation of oxidatively modified proteins.
Abstract: Proteins are modified by reactive oxygen species and oxidation
of specific aminoacid residues can impair their biological
functions leading to an alteration of cellular homeostasis.
Oxidized proteins can be rapidly eliminated through either
degradation or repair. Degradation of oxidized proteins is
achieved by the proteasome, a multicatalytic proteolytic system,
while repair is limited to few modifications such as methionine
oxidation that can be reversed by the Msr system. This system
is composed of MsrA and MsrB, which reduce the methionine-Ssulfoxide
and the methionine-R-sulfoxide, respectively.
However, accumulation of oxidized proteins occurs during aging,
replicative senescence, neurological disorders or after an
oxidative stress while MsrA activity is impaired. MsrA has been
shown to be involved in antioxidant defense in several
organisms such as bacteria, yeast, drosophila as well as
mammals. To analyse more precisely the relationship between
oxidative stress and MsrA, we stably overexpressed the MsrA
full-length cDNA in SV40 T antigen-immortalized human
fibroblasts WI38. We showed that MsrA overexpressing cells are
more resistant than control cells to hydrogen peroxyde-induced
oxidative stress at concentrations from 250 up to 500 µM.
Moreover, in the transfected cells, Msr activity is stimulated by
addition of H2O2 while an H2O2-dose dependent inhibition of
the Msr activity is observed in control cells For higher
concentration of hydrogen peroxide (750 µM), the
overexpression of MsrA is no more able to protect cells from
oxidative damage and apoptosis. These results suggest that
MsrA may play an important role in cellular defenses against
oxidative stress and in protection against apoptosis by limiting,
at least in part, the accumulation of oxidatively modified proteins.
Abstract: HSP 47 is a specific chaperone of pro-collagen. This heat shock protein is responsible for the correct 3-dimentional organization of pro-collagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP47 in aged, photoaged and senescent fibroblasts and its modulation by a plant extract (Salix Alba).
We measured the level of HSP47 and/or pro-collagen expression in fibroblasts by real time RT-PCR (mRNA transcripts) and by flow cytometry (immuno-chemistry technique for measurement of arbitrary fluorescence intensity). We used immuno-chemistry techniques and confocal microscopy to vizualized the cellular localization of HSP 47 and pro-collagen.
These parameters were compared with different age-donors, non senescent and senescent fibroblasts. Fibroblasts were irradiated by a non cytotoxic dose of UVA (6 J/cm2 ) and HSP 47 level was evaluated. Salix Alba extract was tested for its capacity to modulate HSP 47 expression.
We showed colocalization of HSP47 and procollagen by confocal microscopy, indicating that HSP47 could play a role of procollagen molecular chaperone in our cellular model. We showed that HSP47 level is decreased in old-donor cells, senescent and irradiated cells. This decrease can be modulated by a salix alba extract (polyphenols rich) in a dose dependent manner.
The evaluation of HSP 47 expression in our experimental conditions can lead to a new approach of aging and photoaging pointing out the implication of this chaperone in these pathophysiologic phenomenons. Modulation of HSP 47 expression by molecules family could be of cosmetic and/or dermatologic interest.
Abstract: Modification of proteins by reactive oxygen species is implicated in different disorders. The proteasome is a multicatalytic proteinase in charge of intracellular protein turnover and of oxidized proteins degradation. Consequently, proteasome function is very important in controlling the level of altered proteins in eukaryotic cells. Evidence for a decline in proteasome activity during skin photo-aging has been provided by us in Bulteau et al. (2002). We previously described the ability of a lipid algae extract (Phaeodactylum tricornutum) to stimulate 20S proteasome peptidase activities (Nizard et al. 2002). Furthermore, keratinocytes treated with Phaeodactylum tricornutum extract and then UVA and UVB irradiated, exhibited a sustained level of proteasome activitiy comparable to the one of non-irradiated cells. The level of modified proteins can be quantified by measurement of protein carbonyl content (Oxyblot technique), which has been shown to increase with aging and other disorders. Here, we describe that, in the presence of this lipid algae extract, the level of oxidized proteins is reduced as assessed by the Oxyblot technique. These results are obtained both with culture human keratinocytes and stratum corneum skin cells (obtained by stripping) from human volunteers. Altogether, these results argue for the presence of compounds in this algae extract that have a stimulating and/or protective effect on proteasome activity resulting in a decrease level of protein oxidation.
Abstract: HSP 47 is a specific chaperone of pro-collagen. This heat shock protein is responsible for the correct 3-dimentional organization of pro-collagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP47 in aged, photoaged and senescent fibroblasts and its modulation by a plant extract (Salix Alba).
We measured the level of HSP47 and/or pro-collagen expression in fibroblasts by real time RT-PCR (mRNA transcripts) and by flow cytometry (immuno-chemistry technique for measurement of arbitrary fluorescence intensity). We used immuno-chemistry techniques and confocal microscopy to vizualized the cellular localization of HSP 47 and pro-collagen. These parameters were compared with different age-donors, non senescent and senescent fibroblasts. Fibroblasts were irradiated by a non cytotoxic dose of UVA (6 J/cm2 ) and HSP 47 level was evaluated. Salix Alba extract was tested for its capacity to modulate HSP 47 expression.
We showed colocalization of HSP47 and procollagen by confocal microscopy, indicating that HSP47 could play a role of procollagen molecular chaperone in our cellular model. We showed that HSP47 level is decreased in old-donor cells, senescent and irradiated cells. This decrease can be modulated by a salix alba extract (polyphenols rich) in a dose dependent manner.
The evaluation of HSP 47 expression in our experimental conditions can lead to a new approach of aging and photoaging pointing out the implication of this chaperone in these pathophysiologic phenomenons. Modulation of HSP 47 expression by molecules family could be of cosmetic and/or dermatologic interest.
Abstract: The proteasome is a multicatalytic proteinase in charge of intracellular protein turnover and of oxidized proteins degradation. Consequently, proteasome function is very important in controlling the level of altered proteins in eukaryotic cells. Evidence for a decline in proteasome activity during aging and upon oxidative stress has been provided previously. However, no data are available on the status of skin cells proteasome exposed to ultraviolet radiations which are known to induce damages of cellular components such as DNA, proteins, and lipids. We have reported (Bulteau et al. ,(in press) that 10 J/cm2 UVA-and 0.05 J/cm2 UVB-irradiation induced a decrease of proteasome activities and an increase of modified proteins (e. g. oxidized, ubiquitinated and modified by the lipid peroxydation product 4-hydroxy-2-nonenal).
We describe here the ability of an apolar algae extract (Phaeodactylum Tricornutum) to stimulate 20S proteasome peptidase activities. Moreover, the observed decline of proteasome peptidase activities following UVA (10 J/cm2) and UVB (0,05 J/cm2) irradiation of keratinocytes is abolished when the algae extract is added to the culture medium. Furthermore, keratinocytes treated with Phaeodactylum Tricornutum extract and then irradiated, exhibited a sustained level of proteasome peptidase activities comparable to the one of non irradiated cells. Finally, in the presence of this algae extract the level of oxidized proteins is reduced as assessed by the Oxyblot technique. Altogether, these results argue for the presence of a compound in this algae extract that have a stimulating and protective effect on proteasome activity and whose characterization is under progress.
Abstract: It is well established that the proteasome is responsible for the degradation of oxidized proteins. The effects of UV irradiation on proteasome has been investigated in human keratinocytes cultures. The effects of an apolar algae extract (Pht) on proteasome were performed.
Human keratinocytes were irradiated by 10 J/cm2 of UVA and 0.05 J/cm2 of UVB. Proteasome peptidase activities and levels of modified proteins, were measured in cell lysates. 2D gel pattern of proteasome subunits were performed. Lysates ablility to inhibit FITC-casein degradation by the proteasome were assessed. Peptidase activities and levels of modified proteins were measured in Pht-treated cells lysates.
After cells irradiation, peptidases activities were decreased and increased levels of modified proteins were also observed. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be a target for the UV-induced damage. Finally, extracts from irradiated keratinocytes were able to inhibit FITC-casein degradation by the proteasome, demonstrating the presence of endogeneous inhibitors generated upon UV irradiation. We describe also here the ability of an algae extract (Pht) to stimulate 20S proteasome peptidase activities in human keratinocytes and to reduce level of oxydized proteins.
Proteasome is a new target of UV radiations. Pht algae extract has stimulating effects on proteasome which can be of skin care interest.
Abstract: The major environmental influence for skin cells is sun exposure, and the combined effect of aging and sun damage has been referred as to photoaging. Photoaging induces morphological changes such as wrinkling and sagging due to general alteration of all the epidermal and dermal components of the skin including the cutaneous cells (Bisset et al., 1992). The harmful effect of UV radiation on skin is related to the generation of reactive oxygen species (hydroxyl radical, superoxide ion and singlet oxygen) that requires the absorption of photons by endogeneous photosensitizer molecules (Tyrrell and Keyse, 1990). Indeed there is accumulating evidence for the damaging effects of reactive oxygen species generated after UVA and UVB radiation, on cellular components including proteins (Sander et al., 2002; Bulteau et al., 2002). In addition, accumulation of oxidatively modified proteins has been described as a hallmark of cellular aging and these modifications are strongly believed to be implicated in the aging process (Levine and Stadtman, 2001). This accumulation of altered proteins with age raises the problem of the efficacy of the proteasomal system, a major intracellular proteolytic system which is responsible for the degradation of oxidized proteins. Indeed, impairment of proteasome function has been implicated in the age-related accumulation of oxidized proteins in epidermal cells (Bulteau et al., 2000; Petropoulos et al., 2000). Protein oxidative damage and the proteasomal system will be first described and recent evidence arguing for UV irradiation induced accumulation of oxidized proteins and decrease of proteasome activity will be then presented.
Abstract: Cellular aging is associated with the accumulation of modified macromolecules. The major environmental influence for epidermal cells is sun exposure, and the combined effect of aging and sun damage has been referred as photo-aging. The harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Impairment of proteasome function has been implicated in the age-related decline of protein turnover and accumulation of oxidized proteins in epidermis. Therefore, the effects of UV irradiation on proteasome has been investigated both in vitro and in human keratinocytes cultures. In this study, 20S proteasome purified from human placenta was irradiated by 10 J/ cm2 of UVA and 0.05 J /cm2 of UVB, assayed for the three classical peptidase activities with fluorogenic peptides and analysed by 2D gel electrophoresis. The present data show that only the peptidylglutamyl-peptide hydrolase activity of the active form of the proteasome is decreased upon UV irradiation and that the 2D gel pattern is modified. These results indicate that the proteasome can be a target of UV irradiation in vitro. Human keratinocyte cultures were irradiated by 10 J/ cm2 of UVA and 0.05 J /cm2 of UVB and harvested at different times after irradiation. Proteasome peptidase activities were measured in cell lysates using fluorogenic peptides in the absence and in the presence of the proteasome inhibitor MG 132. All three peptidases activities measured were decreased 7 hours after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxydation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be itself a target for the UV-induced damages to the keratinocytes. Finally, 20S proteasome content was found to decrease by about 25 % in irradiated cells 24 hours after irradiation which suggests that proteasome expression may be down regulated after UV irradiation.
Abstract: Cellular aging is associated with the accumulation of modified macromolecules. The major environmental influence for epidermal cells is sun exposure, and the combined effect of aging and sun damage has been referred as photo-aging. The harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Impairment of proteasome function has been implicated in the age-related decline of protein turnover and accumulation of oxidized proteins in epidermis. Therefore, the effects of UV irradiation on proteasome has been investigated in human keratinocytes cultures. In this study, keratinocytes were irradiated by 10 J/ cm2 of UVA and 0.05 J /cm2 of UVB and collected at different times after irradiation. Proteasome peptidase activities were measured in cell lysates using fluorogenic peptides. All three peptidases activities measured were decreased 7 hours after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxydation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be a target for UV-induced damages. Finally, 20S proteasome content was found to decrease by about 25 % in irradiated cells 24 hours after irradiation which suggests that proteasome expression may be down regulated after UV irradiation.
Abstract: The proteasome is a multicatalytic proteinase in charge of intracellular protein turnover and of oxidized proteins degradation. Consequently, proteasome function is very important in controlling the level of altered proteins in eukaryotic cells. Evidence for a decline in proteasome activity during aging and upon oxidative stress has been provided previously. However, no data are available on the status of skin cells proteasome exposed to ultraviolet radiations which are known to induce damages of cellular components such as DNA, proteins, and lipids. We have reported (cf. our other poster abstract) that UVA-and UVB-irradiation induced a decrease of proteasome activities and an increase of modified proteins (e. g. oxidized, ubiquitinated and modified by the lipid peroxydation product 4-hydroxy-2-nonenal). We describe here the ability of an apolar algae extract (Phaeodactylum Tricornutum) to stimulate 20S proteasome peptidase activities both in vitro and within human keratinocytes. Moreover, the observed decline of proteasome peptidase activities following UVA (10 J/cm2) and UVB (0,05 J/cm2) irradiation of keratinocytes is abolished when the algae extract is added to the culture medium. Furthermore, keratinocytes treated with Phaeodactylum Tricornutum extract and then irradiated, exhibited a sustained level of proteasome peptidase activities comparable to the one of non irradiated cells. Finally, in the presence of this algae extract the level of oxidized proteins is reduced as assessed by the Oxyblot technique. Altogether, these results argue for the presence of a compound in this algae extract that have a stimulating and protective effect on proteasome activity and whose characterization is under progress.
Abstract: Cellular aging is associated with the accumulation of modified macromolecules. The major environmental influence for epidermal cells is sun exposure, and the combined effect of aging and sun damage has been referred as photo-aging. The harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Impairment of proteasome function has been implicated in the age-related decline of protein turnover and accumulation of oxidized proteins in epidermis. Therefore, the effects of UV irradiation on proteasome has been investigated both in vitro and in human keratinocytes cultures. In this study, 20S proteasome purified from human placenta was irradiated by 10 J/ cm2 of UVA and 0.05 J /cm2 of UVB, assayed for the three classical peptidase activities with fluorogenic peptides and analysed by 2D gel electrophoresis. The present data show that only the peptidylglutamyl-peptide hydrolase activity of the active form of the proteasome is decreased upon UV irradiation and that the 2D gel pattern is modified. These results indicate that the proteasome can be a target of UV irradiation in vitro. Human keratinocyte cultures were irradiated by 10 J/ cm2 of UVA and 0.05 J /cm2 of UVB and harvested at different times after irradiation. Proteasome peptidase activities were measured in cell lysates using fluorogenic peptides in the absence and in the presence of the proteasome inhibitor MG 132. All three peptidases activities measured were decreased 7 hours after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxydation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be itself a target for the UV-induced damages to the keratinocytes. Finally, 20S proteasome content was found to decrease by about 25 % in irradiated cells 24 hours after irradiation which suggests that proteasome expression may be down regulated after UV irradiation.
Abstract: Skin aging is a multifactorial phenomenon involving programmed aging and oxidative stress which are linked to the darkening of skin tone and the appearance of fine lines and then wrinkles. The epidermis plays a central role in the skin and is mainly composed of keratinocytes.
Keratinocyte communication plays an important part in maintaining epidermal homeostasis and functionality. Gap junctions mediate essential communication signals within clusters of keratinocytes and are demonstrated here to be involved in the aging process. Gap junction intercellular communication is shown to be down regulated in aged keratinocytes using scrape loading and lucifer yellow cell microinjection. By flow cytometry, this can be related to a decrease in the level of connexin 43, the main gap junction protein expressed in human keratinocytes. A mixture of _ 3, 4, 6 fatty acids and hydroxylated carotenoids from microalgae origin restores keratinocyte communication and the connexin 43 level.
Furthermore, free radicals and their molecular damage also contribute to the deterioration of the cells’ main biological functions. A Sanguisorba officinalis extract is a powerful inhibitor of superoxide anion and oxygen reactive species as demonstrated by Electronic Paramagnetic Resonance, in vitro tests and on keratinocyte cultures. A topical emulsion including the fatty acids mixture and Sanguisorba extract was tested in vivo on human volunteers and was shown to improve skin radiance, giving a smoothing effect and scavenging free radicals.
