Abstract: 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.
Abstract: 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.
Abstract: The phosphorylated form of histone H2AX, gammaH2AX, is a component of the DNA repair system. Most studies have focused on the role of gammaH2AX during cell transformation and human cancer, but little is known about its role in keratinocytes and the skin during UV irradiation. We analyzed the response to UV irradiation focusing on the phosphorylation of histone H2AX both in vitro, in keratinocyte cultures and in artificial epidermis, and then in vivo, in human skin. Acute UVB irradiation of human keratinocytes increased the phosphorylation of H2AX in a dose-dependent manner; two types of gammaH2AX response were observed either in vitro or in vivo. After a low nonapoptotic UVB irradiation, cells contained phosphorylated H2AX and arrested their cell cycle to repair the DNA damages. For a stronger and proapoptotic UVB irradiation, keratinocytes dramatically increased the phosphorylation of H2AX and committed apoptosis. Our results indicate that gammaH2AX constitutes a highly sensitive marker relevant for studying subapoptotic doses as well as proapoptotic doses of UVB in human skin.
Abstract: 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.
Abstract: Aquaporins (AQPs) are proteins that facilitate the transport of water across cell membranes. AQP3 expression is related to the expressions of other epidermal proteins involved in water maintenance (ie, CD44, claudin-1, and filaggrin). The expressions of AQP3 water channels are strongly affected by age and chronic sun exposure, and a defective osmotic equilibrium could occur in the epidermis, which would account for the skin dryness observed in older people and skin areas most exposed to sunlight. We investigated active ingredients that are able to increase AQP3 levels in order to improve hydration in human skin keratinocytes. We selected an ethanolic/water (70/30 v/v) extract of Ajuga turkestanica, a plant from Central Asia, as the hydrating agent. After 17 days of treatment every 2 days with this extract (2.5 microg/mL) in vitro, AQP3 expression measured at the protein level in human reconstructed epidermis was significantly increased. Water transport through both aquaporins and aquaglyceroporins and glycerol transport through aquaglyceroporins alone are important to skin hydration. The distribution and the variability of aquaporins in human skin cells suggest that these channels may have important roles in skin physiology. AQPs appear to be key protein targets to improve the resistance and quality of the skin surface as well as to improve aging and sun exposure-induced dryness as shown by their roles in 1) hydrating the living layers of the epidermis where the keratinocyte differentiation takes place and 2) barrier formation and recovery.
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: Lysyl oxidase initiates the enzymatic stage of collagen and elastin cross-linking. Among five isoforms comprising the lysyl oxidase family, LOX is the better studied. LOX is associated to an antitumor activity in ras-transformed fibroblasts, and its expression is down-regulated in many carcinomas. The aim of this work was to shed light on LOX functions within the epidermis by studying its expression in human basal and squamous cell carcinomas and analyzing the effect of its enzymatic activity inhibition and protein absence on human keratinocytes behavior in a skin equivalent. In both carcinomas, LOX expression by epidermal tumor cells was lacking, while it was up-regulated around invading tumor cells in association with the stromal reaction. Lysyl oxidase activity inhibition using beta-aminoproprionitrile in a skin equivalent model prepared with both primary human keratinocytes and HaCaT cell line affected keratin 10 and filaggrin expression and disorganized the collagen network and the basement membrane. In spite of all these changes, no invasion phenotype was observed. Modelization of the invasive phenotype was only noticed in the skin equivalent developed with LOX antisense HaCaT cell line, where the protein LOX is specifically absent. Our results clearly indicate that lysyl oxidase enzymatic activity is essential not only for the integrity maintenance of the dermis but also for the homeostasis of the epidermis. Moreover, LOX protein plays a role in the skin carcinomas and invasion but not through its enzymatic activity.
Abstract: More than other tissues, skin is exposed to numerous external stresses generating ROS that, in addition to endogenous oxygen radicals, cause keratinocyte alterations and contribute in part to photocarcinogenesis and aging. Recent evidence suggests a differentiation-dependent susceptibility of keratinocytes to apoptosis. We explored hydrogen peroxide-induced cell death in normal human keratinocytes according to their differentiation. On H(2)O(2)-exposed skin explants, caspase-3 was strongly activated in basal keratinocytes double stained with beta(1) integrin, whereas DNA fragmentation occurred in suprabasal cells only without caspase-3 activation. In addition, isolated basal keratinocytes, selected by adhesion to type IV collagen, were more sensitive than nonadherent cells to H(2)O(2)-induced apoptosis with regard to mitochondrial transmembrane potential (Deltapsi(mt)) collapse and membrane integrity. Similarly, necrotic/late apoptotic cells were present at low levels only in the adherent epidermal population. Furthermore, in primary cultures of undifferentiated keratinocytes H(2)O(2)-induced cell death appeared via a mitochondrial failure. Deltapsi(mt) collapse was associated with a strong early activation of the initiatory caspase-8, then the executive caspase-3, and, to a lesser extent, the inflammatory caspase-1. Finally, undifferentiated basal cells possess a higher sensitivity than differentiated suprabasal cells to H(2)O(2)-induced cell death, and apoptosis in human keratinocytes occurs via different pathways depending on the cell's differentiation state.
