Filaggrin (FLG) loss-of-function mutations and the T helper 2 (Th2) dominated cytokine milieu are assumed to cause an impaired skin barrier function in atopic dermatitis (AD), but this presumed mechanism is still largely hypothetical. Previous studies have used in vitro skin equivalents to provide experimental evidence for the role of FLG deficiency but different experimental setups and incomplete knockdown approaches make these data difficult to compare and interpret. Using 3D epidermal equivalents, we here addressed the question if FLG deficiency alters skin barrier function. We excluded interplay of FLG mutations with other AD-typical concomitant factors like inflammation or altered microbiome. We therefore used keratinocytes of ichthyosis vulgaris patients that innately carry homozygous FLG null mutations. This approach uses genetically defined cells without detectable filaggrin protein and avoids potential off-target effects of knockdown approaches. FLG did not alter constitutive or Th2-cytokine dependent expression of differentiation-associated proteins. We observed a decrease of tight junction protein expression, which, however, did not lead to alteration of the outside-in nor did it affect inside-out stratum corneum barrier as measured by permeability for low molecular weight tracers (Lucifer Yellow or biotin-SH). Although these findings do not completely rule out alterations in epidermal permeability for molecules with other biophysical properties, our study contradicts previous work that suggests an increased permeability for low molecular weight polar solutes in FLG deficient epidermis.
We recently showed that the activities of the serine proteases of the plasminogen system (plasmin and urokinase) are elevated in facial stratum corneum (SC) compared to forearm SC and correlated with TEWL. We also demonstrated that plasmin activity is increased on sunexposed SC. Moreover, elevated plasmin activity was associated with reduced corneocyte maturation. The protease/protease-inhibitor balance of the plasminogen system may be key for the SC maturation processes and optimal barrier function. Thus, our aim was to develop a specific dual plasmin and urokinase inhibitor for topical application of barrier-impaired skin. Conventional synthetic, characterization and modelling methods were used in the development of the dual competitive inhibitor benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide (BSFA) with Ki values 46 and 25 nM for plasmin and urokinase, respectively. The catalytic domain of both proteases is highly conserved and in both models BSFA showed a similar binding mode in silico. In a study on normal human keratinocytes stimulated with inflammatory cytokines (IL-1b, TNFa) the addition of BSFA (100mM) led to a down-regulation of both plasmin (40%) and urokinase (97%) activities. Additionally, an increased gene expression (4.5x) of transglutaminase 1 was induced by BSFA in the cytokine-stimulated keratinocytes. Finally, in a four week, vehicle-controlled study on 44 female Caucasian subjects the effect of a topically applied BSFA hydrogel formulation (10 ppm) was evaluated for basal function and recovery of the barrier of facial skin that was previously challenged by tape stripping. Compared to the vehicle treated group the BSFA treated group showed significantly improved SC barrier integrity, chemo-sensation to a lactic acid challenge and facial skin condition. Our data reveal that the topical application of an inhibitor of the plasminogen system is a novel approach for the treatment of barrier impaired skin and premature desquamation. Gaining a better understanding of the different processes and their regulations occurring during skin aging is fundamental to improve solutions to fight the signs of skin aging. The emergence of miRNA studies and their roles in post-transcriptional inhibition of gene expression highlights the importance of these small RNAs in the skin aging process. miRNA maturation is divided into two major steps: the primary miRNA is processed by Drosha to generate a pre-miRNA; and after exportation in the cytoplasm, Dicer allows the final maturation of miRNA. On senescent lung fibroblasts, it is proved that the expression of these two key markers decreases, involving the dysregulation of 20% of miRNAs. Among them, miRNA-19b expression was shown to decrease with aging except for the centenarians, who keep a high level of this miRNA, suggesting its association with longevity. Firstly, we evaluated the expression of Drosha and Dicer by qPCR in in vitro skin fibroblasts that were aged by replicative senescence. We observed the decrease in the expression of these two key markers, as we...
We previously showed that topical application of hexoses such as fructose accelerates stratum corneum barrier recovery after barrier disruption. We also showed that various hexoses and polyols interact with phospholipids and decrease the phase transition temperature from the liquid crystal to the crystal phase; i.e., they stabilize the fluid phase of lipids at low temperature. In the present study, we confirmed that topical application of xylitol aqueous solution on human skin after tape stripping accelerated barrier recovery. We next examined changes of lipid fluidity in an epidermal-equivalent model after the application of water and aqueous solutions of xylitol and fructose. For this purpose, we used Laurdan, an environmentally sensitive fluorescence dye, as an indicator of lipid fluidity and observed its emission spectrum with a two-photon microscope. Application of xylitol and/or fructose aqueous solution increased the lipid fluidity of the stratum granulosum layer compared to water application. These results indicate that topical application of certain hexoses or polyols increases the lipid fluidity at the uppermost layer of the stratum granulosum, accelerates the release of lipid from the stratum granular layer, and improves epidermal barrier homeostasis. 627Fluctuation of Caspase 14 caused by temperature and humidity unbalances the NMF production pathway and the process of keratinocyte enucleation
Cathelicidin antimicrobial peptide (CAMP) is a key antimicrobial peptide in skin. CAMP production is increased during epidermal differentiation and enriched in the stratum corneum. We recently identified a novel endoplasmic reticulum (ER) stress-mediated sphingosine-1-phosphate (S1P)-dependent mechanism of CAMP synthesis. In this study, we found that S1P synthesized by an isoform of sphingosine kinase (SPHK), SPHK1, serves as a signal for CAMP synthesis; and conversely, another isoform SPHK2 likely has a suppressor or no role in CAMP synthesis. Pertinently, prior studies showed that physiological ER stress is essential for normal epidermal differentiation. We here show that: increased ER stress occurs in differentiated cultured keratinocytes (KC); 2) increases in both CAMP and S1P production depend upon differentiation level of KC (proliferated
Cathelicidin antimicrobial peptide (CAMP) is a key antimicrobial peptide in skin. CAMP production is increased during epidermal differentiation and enriched in the stratum corneum. We recently identified a novel endoplasmic reticulum (ER) stress-mediated sphingosine-1-phosphate (S1P)-dependent mechanism of CAMP synthesis. In this study, we found that S1P synthesized by an isoform of sphingosine kinase (SPHK), SPHK1, serves as a signal for CAMP synthesis; and conversely, another isoform SPHK2 likely has a suppressor or no role in CAMP synthesis. Pertinently, prior studies showed that physiological ER stress is essential for normal epidermal differentiation. We here show that: increased ER stress occurs in differentiated cultured keratinocytes (KC); 2) increases in both CAMP and S1P production depend upon differentiation level of KC (proliferated
Ultraviolet rays, pollutants and the resulting oxidative stress all contribute significantly to the appearance of skin aging. Human skin cells' ability to repair oxidative damage steadily decreases over the years, leading to reactive oxygen species (ROS) accumulation. Over time, the presence of lipophilic antioxidants such as coenzyme Q10 (CoQ10) declines leading to an increased presence of the visible signs of aging. CoQ10 is required for ATP synthesis in the mitochondria and also functions as an antioxidant in cell membranes, suggesting the existence of non-mitochondrial sites of synthesis. In the present study, we aimed at developing an approach to modulate the expression of Ubiad1, a prenyltransferase previously described in the Zebra fish, by using a synthetic peptide. Our results showed that Ubiad1 is expressed in the epidermis, mainly located in the Golgi apparatus. In addition, downregulation of Ubiad1 also exhibited H 2 O 2 -mediated ROS accumulation. Modulation of Ubiad1 protects keratinocytes submitted to oxidative stress from ROS accumulation and lipid peroxidation, and helps to preserve plasma membrane mechanical properties. Our results present a new strategy involving endogenous coenzyme Q10 synthesis by the modulation of non-mitochondrial Ubiad1 enzyme, to preserve skin appearance from visible aging signs and confirms that the CoQ10 existing in cell membranes is synthetized out from the canonical mitochondrial pathway. 433Opsin photoreceptor expression in normal human keratinocytes and melanocytes The skin is a complex sensory system, reacting to the surrounding environment through an integrated network of cell receptors. Among them, G Protein-Coupled Receptors (GPCRs) represent a major way for cells to sense a large variety of environmental signals. Recent studies identified several extra-retinal opsins localized in human epidermal cells, three of them being sensitive to blue light (400-495 nm). As Light-Emitting Diode (LED)-based devices are increasingly part of our daily life, excessive exposure to artificial blue light can be considered as a source of indoor pollution, which has been shown to impact the circadian rhythm. The aim of the present study was to evaluate opsin expression in human skin. The localization of blue light-sensitive opsins was investigated in cultures of human keratinocytes and melanocytes and in human skin biopsies. Our results showed the expression and distribution of opsin 1 short-wavelength-sensitive, opsin 2 (Rhodopsin) and opsin 3 (Encephalopsin) within the epidermis, and their variation following exposure to blue light emitted from LEDs. In addition, our observations revealed a specific cellular localization of opsin 3 during mitosis. Taken together, these results emphasize the role of opsin photoreceptors in maintaining physiological conditions within the epidermis, and their implication in the skin's response to stressful conditions induced by blue light.
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