Modulation of Piezo1 influences human skin architecture and oxytocin expression.

OBJECTIVE: Throughout our existence, the skin senses and analyses the mechanical forces imposed by the environment. In response to these environmental forces, skin can deform itself and achieve a biological response. The subsequent cutaneous plasticity emerges from mechanical properties arising from the collective action of the skin cells, particularly keratinocytes, that govern the tensile strength via cell-to-cell adhesions and via cell-matrix adhesion structures. In addition to serving as force-bearing entities, keratinocytes respond to forces by activating signalling pathways to control their own fate and function. To detect and adapt to mechanical signals, keratinocytes possess a panel of sensory receptors and junctional intercellular structures. Mechanically activated ion channel Piezo1 has been described as a force sensor and as being involved in pleasant touch perception. In this study, relationships between Piezo1 modulation and oxytocin synthesis were investigated. METHODS: The expression of Piezo1 in the skin was studied and compared with the expression of TRPV1. Dooku1 antagonist and Jedi1 agonist were used to modulate Piezo1. The level of E-cadherin and oxytocin was monitored in ex vivo skin biopsies by immunodetection. RESULTS: Taken together, our results illustrate the major role of mechanosensitive ion channel Piezo1 in skin barrier integrity, and in peripheral oxytocin synthesis in the skin. CONCLUSION: In conclusion, this study highlights the relationships between pleasant touch, soft touch and local oxytocin synthesis.

OBJECTIF: Tout au long de notre existence, la peau détecte et analyse les forces mécaniques imposées par l'environnement. En réponse à ces forces environnementales, la peau peut se déformer et obtenir une réponse biologique. La plasticité cutanée qui s'ensuit émerge des propriétés mécaniques découlant de l'action collective des cellules cutanées, en particulier les kératinocytes, qui déterminent la résistance à la traction via les adhérences intercellulaires et les structures d'adhésion cellule-matrice. En plus de servir d'entités porteuses de force, les kératinocytes répondent aux forces en activant les voies de signalisation pour contrôler leur propre destin et leur propre fonction. Pour détecter et s'adapter aux signaux mécaniques, les kératinocytes possèdent un panel de récepteurs sensoriels et de structures intercellulaires jonctionnelles. Le canal ionique activé mécaniquement Piezo1 a été décrit comme un capteur de force et comme étant impliqué dans la perception d'un toucher agréable. Dans cette étude, les relations entre la modulation Piezo1 et la synthèse de l'ocytocine ont été étudiées. MÉTHODES: L'expression de Piezo1 dans la peau a été étudiée et comparée à l'expression de TRPV1. L'antagoniste Dooku1 et l'agoniste Jedi1 ont été utilisés pour moduler Piezo1. Le taux de cadhérine-E et d'ocytocine a été contrôlé dans des biopsies cutanées ex vivo par immunodétection. RÉSULTATS: Dans l'ensemble, nos résultats illustrent le rôle majeur du canal ionique mécanosensible Piezo1 dans l'intégrité de la barrière cutanée et dans la synthèse de l'ocytocine périphérique dans la peau. CONCLUSION: En conclusion, cette étude met en évidence les relations entre le toucher agréable, le toucher doux et la synthèse d'ocytocine locale.

Co-culture of iNeurons with primary human skin cells provides a reliable model to examine intercellular communication.

OBJECTIVE: The skin is a sensory organ, densely innervated with various types of sensory nerve endings, capable of discriminating touch, environmental sensations, proprioception, and physical affection. Neurons communication with skin cells confer to the tissue the ability to undergo adaptive modifications during response to environmental changes or wound healing after injury. Thought for a long time to be dedicated to the central nervous system, the glutamatergic neuromodulation is increasingly described in peripheral tissues. Glutamate receptors and transporters have been identified in the skin. There is a strong interest in understanding the communication between keratinocytes and neurons, as the close contacts with intra-epidermal nerve fibers is a favorable site for efficient communication. To date, various coculture models have been described. However, these models were based on non-human or immortalized cell line. Even the use of induced pluripotent stem cells (iPSCs) is posing limitations because of epigenetic variations during the reprogramming process. METHODS: In this study, we performed small molecule-driven direct conversion of human skin primary fibroblasts into induced neurons (iNeurons). RESULTS: The resulting iNeurons were mature, showed pan-neuronal markers, and exhibited a glutamatergic subtype and C-type fibers characteristics. Autologous coculture of iNeurons with human primary keratinocytes, fibroblasts, and melanocytes was performed and remained healthy for many days, making possible to study the establishment of intercellular interactions. CONCLUSION: Here, we report that iNeurons and primary skin cells established contacts, with neurite ensheathment by keratinocytes, and demonstrated that iNeurons cocultured with primary skin cells provide a reliable model to examine intercellular communication.

The impact of airborne ultrafine particulate matter on human keratinocyte stem cells.

OBJECTIVE: Air pollution is today fully acknowledged to be a significant public health problem. Rapid urbanization exposed us to a variety of unhealthy ambient air pollutants at high concentrations. The emergence of airborne ultrafine particles has added an additional dimension to this already complex problem of air pollution. The skin has different functions, one of them being the protection against the deleterious effect of external agents. The aim of this study is to evaluate the impact of airborne ultrafine particles (UFP) pollution on skin aging and on keratinocyte differentiation. METHODS: Ex vivo human skin biopsies and cultured keratinocytes stem cells (KSC) were submitted to diesel exhaust-derived UFP. Reactive oxygen species (ROS) production was assessed with the MitoSOX™ probe. Keratinocyte stemness potential was evaluated by the immunodetection of keratin 15 (K15) and p63 (∆N isoforms). Effect of UFP on the epithelial niche maintenance was evaluated by immunodetection of Sox9. Reconstructed epidermis model was used to assess the impact of UFP on keratinocyte differentiation and aging. RESULTS: UFP exposure induced ROS production and disturbed K15, ∆Np63 and Sox9 expression in KSC or ex vivo skin. Finally, investigations on reconstructed epidermis revealed a phenotype marked by impaired keratinocyte differentiation. CONCLUSION: These results indicate that UFP pollution is a potent extrinsic factor of skin aging, affecting the keratinocyte stem cell potential and the skin renewal process.

OBJECTIF: La pollution de l'air est désormais pleinement reconnue comme un problème de santé publique important. L'urbanisation rapide nous a exposés à une variété de polluants atmosphériques ambiants malsains à des concentrations élevées. L'émergence de particules ultrafines en suspension dans l'air a ajouté une dimension supplémentaire à ce problème déjà complexe de la pollution de l'air. La peau exerce différentes fonctions, l'une d'elles étant la protection contre l'effet délétère d'agents extérieurs. L'objectif de cette étude est d'évaluer l'impact de la pollution par les particules ultrafines (UFP) aéroportées sur le vieillissement cutané et sur la différenciation des kératinocytes. MÉTHODES: Des biopsies de peau humaine ex vivo et des kératinocytes souches (KSC) en culture ont été mis en présence d'UFP provenant d'échappement de véhicule diesel. La production d'espèces réactives de l'oxygène (ROS) a été évaluée avec la sonde MitoSOX™. Le potentiel de souche des kératinocytes a été évalué par immunodétection de la kératine 15 (K15) et p63 (isoformes ∆N). L'effet des UFP sur la niche épithéliale a été évalué par immunodétection de Sox9. Un modèle d'épiderme reconstruit a été utilisé pour évaluer l'impact des UFP sur la différenciation et le vieillissement des kératinocytes. RÉSULTATS: L'exposition aux UFP a induit la production de ROS, a perturbé l'expression de K15, ∆Np63 et de Sox9 dans les KSC et dans la peau ex vivo. Enfin, des investigations sur des épidermes reconstruits ont révélé un phénotype marqué par une différenciation altérée des kératinocytes. CONCLUSION: Ces résultats indiquent que la pollution par les UFP est un facteur extrinsèque puissant du vieillissement cutané, affectant le potentiel des cellules souches de kératinocytes et le processus de renouvellement cutané.

miR-203 represses keratinocyte stemness by targeting survivin.

OBJECTIVE: The epidermis possesses the capacity to replace dying cells and to heal wounds, thanks to resident stem cells, which have self-renewal properties. In skin physiology, miRNAs have been shown to be involved in many processes, including skin and hair morphogenesis. Recently, differentiation of epidermal stem cells was shown to be promoted by the miR-203. The miR-203 is upregulated during epidermal differentiation and is of interest because of significant targets. METHODS: By utilizing a bioinformatic tool, we identified a target site for miR-203 in the survivin mRNA. Silencing miR-203 was managed with the use of antagomir; the silencing of survivin was performed with a siRNA. Survivin expression was determined by qPCR or immunofluorescence in cultured cells, and by immunohistochemistry in skin sections. Involucrin expression was used as marker of keratinocyte differentiation. A rice extract with previously demonstrated anti-aging properties was evaluated on miR-203 modulation. RESULTS: In this study, we identified a miR-203/survivin axis, important for epidermal homeostasis. We report that differentiation of keratinocyte is dependent on the level of miR-203 expression and that inhibition of miR-203 can increase the expression of survivin, an epidermal marker of stemness. CONCLUSION: In summary, our findings suggest that miR-203 target 3'UTR region of survivin mRNA and directly represses survivin expression in the epidermis. The rice extract was identified as modulator of miR-203 and pointed out as a promising microRNA-based strategy in treating skin changes occurring with aging.

Soothing Effect of Pogostemon cablin Extract (Patchouli), via Cannabinoid Receptor 2 Modulation in Human Skin.

Skin functions as a neuro-immuno-endocrine tissue with well-defined neuronal networks and functions. The endocannabinoid system has been proven to be an important, homeostatic regulator for homeostatic and inflammatory events. The system comprises endogenous or exogenous ligands and receptors (CB1 and CB2). In the present study, we evaluated the soothing properties of a Pogostemon cablin (patchouli) extract. Agonist AM1241 and antagonist AM630 were used for CB2 receptor activation/inhibition. Expression of CB2 receptor and ß-endorphin was monitored by immunohistochemistry. Skin inflammation was induced with ultraviolet B (UVB) or lipopolysaccharide (LPS), and the following markers were used to highlight the anti-inflammatory properties of the extract: transient receptor potential vanilloid 1 (TRPV1), interleukin receptors 1 (IL1R1), and the interleukin 6 signal transducer (IL6ST). Our results demonstrated the implication of the CB2 receptor in the skin inflammation process. The expression of CB2 receptor and ß-endorphin was increased 48 hours after application of the extract. Furthermore, patchouli extract application helped to reduce IL1R1, IL6ST, and TRPV1 expression, in skin exposed to UVB or LPS. In conclusion, the application of the patchouli extract helps maintain skin integrity and reduce skin discomfort via modulation of CB2 receptor stimulation and the subsequent ß-endorphin release.

Modulating the expression of survivin and other basal epidermal proteins protects human skin from UVB damage and oxidative stress.

BACKGROUND: The chromosomal passenger complex (CPC) is an assembly made of four interacting proteins: survivin, borealin, INCENP, and aurora kinase B. CPC is the key regulatory complex responsible for the correct development of cellular mitosis, accompanying each step of the chromosomal segregation. This control of mitosis is particularly important in undifferentiated cells that must renew themselves and also further differentiate and specialize. The epidermis is a self-renewing tissue that needs to continuously generate new cells through proliferation and differentiation of progenitor cells. Both the mitosis supervision by the CPC and a correct extracellular environment are physiologically required for the homeostasis of the adult keratinocyte stem cells (KSCs) of the epidermis. KSCs are mainly found in the basal layer of the epidermis and are responsible for the replenishment and maintenance of the tissue, by compensating for the loss of terminally differentiated cells called corneocytes, especially during aging. AIM: The aim of our study was to investigate the implication of survivin in epidermal renewal and the relationships between survivin expression and UVB-induced DNA damage levels in cultured human keratinocytes and in skin biopsies. In parallel, the effects of a treatment by compound IV08.009 were studied. MATERIAL AND METHODS: Cultured human keratinocytes and skin biopsies were used in this study. KSCs-enriched fractions of keratinocytes were isolated from total keratinocytes by differential attachment to a type IV collagen matrix. Survivin expression levels were assessed by immunoblotting in cultured keratinocytes, and α6-integrin, ß1-integrin, keratin 15, and survivin were observed after immunodetection in skin biopsies cross sections. Comet assay, immunodetection of CPDs and of cleaved-caspase 3, and electron microscopy were used to characterize UVB-induced DNA damage. RESULTS: We demonstrated the ability of compound IV08.009 to efficiently protect ex vivo skin against basal UVB-induced damage. Moreover, comet assay studies demonstrated the efficacy of IV08.009 in protecting DNA damage from UVB stress. We found that IV08.009 protects skin from apoptosis induced by oxidative stress, ex vivo. Electron microscopy confirmed the protective efficiency of IV08.009 on cell ultrastructural damage induced by UVB exposure. CONCLUSION: Compound IV08.009 demonstrated to be effective in regulating survivin expression and in preserving the basal epidermis from stresses such as UVB and H2 O2 . These results suggest a protective activity of IV08.009 on the essential renewing potential of KSCs.

USP9x-mediated deubiquitination of EFA6 regulates de novo tight junction assembly.

In epithelial cells, the tight junction (TJ) functions as a permeability barrier and is involved in cellular differentiation and proliferation. Although many TJ proteins have been characterized, little is known about the sequence of events and temporal regulation of TJ assembly in response to adhesion cues. We report here that the deubiquitinating enzyme USP9x has a critical function in TJ biogenesis by controlling the levels of the exchange factor for Arf6 (EFA6), a protein shown to facilitate TJ formation, during a narrow temporal window preceding the establishment of cell polarity. At steady state, EFA6 is constitutively ubiquitinated and turned over by the proteasome. However, at newly forming contacts, USP9x-mediated deubiquitination protects EFA6 from proteasomal degradation, leading to a transient increase in EFA6 levels. Consistent with this model, USP9x and EFA6 transiently co-localize at primordial epithelial junctions. Furthermore, knockdown of either EFA6 or USP9x impairs TJ biogenesis and EFA6 overexpression rescues TJ biogenesis in USP9x-knockdown cells. As the loss of cell polarity is a critical event in the metastatic spread of cancer, these findings may help to understand the pathology of human carcinomas.