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.

Basal keratinocytes may divide frequently during skin lifespan, and signs of deterioration could appear such as loss of protein factors required for correct mitosis. Our findings suggest that mitotic abnormalities can be prevented by the modulation of CRM1 and survivin. We demonstrated the ability of compound 'IV08.009' to efficiently protect cultured keratinocytes from mitotic abnormalities.

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.

No abstract

Opsin photoreceptors are responsible for the absorption of light, transduce information about daily lighting conditions and provide vision. Opsins are also involved in diverse non-visual functions such as the circadian clock. The non-visual opsin 3, also known as encephalopsin, is expressed in skin and its functions remain poorly defined.The expression of opsin 3 in human skin tissue was evaluated by immunohistochemistry, in keratinocytes and melanocytes by immunodetection. Opsin 3 and β-tubulin colocalization was performed during the keratinocyte mitotic phase. Opsin 3-siRNA transfection was used in primary keratinocytes and successful transfection was confirmed by immunodetection.These results show that opsin 3 was detected in the epidermis of human skin, both in keratinocytes and melanocytes. Opsin 3 colocalize with β-tubulin during the mitotic phase and participate in keratinocyte cell division. Opsin 3 protein expression was reduced after transfection with opsin 3-siRNA; inhibition of opsin 3-siRNA reduces keratinocyte cell proliferation and promotes the apparition of binucleated cells.This study highlights an unconventional function of opsin 3 in extra-ocular cells. Opsin 3 appears to be involved in the regulation of late cytokinesis in keratinocytes, and therefore in the maintenance of epidermal homeostasis.

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.

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.

Abstract:The significance of Coenzyme Q10 (CoQ10) as an anti-oxidant barrier of the skin, as well as a key component in anti-aging strategies for skin care products, has been firmly established. Biosynthesis of CoQ10 in the mitochondria is well known, but there is only limited information on the non-mitochondrial synthesis of CoQ10 in the skin. Recent findings in zebrafish identified that a tumor suppressor, Ubiad1, is also a key enzyme in the non-mitochondrial synthesis of CoQ10. The purpose of this study was to investigate expression of Ubiad1 in human skin, and its implication in the skin's cutaneous response to oxidative stress. We observed Ubiad1 localization in the epidermis, particularly a subcellular localization in the Golgi apparatus. Ubiad1 modulation by a pentapeptide was associated with an observed reduction in ROS/RNS stresses (−44%/−19% respectively), lipid peroxidation (−25%) and preservation of membrane fluidity under stress conditions. Electron microscopy of keratinocytes revealed a significant degree of stimulation of the Golgi complex, as well as significantly improved mitochondrial morphology. Given the importance of CoQ10 in mitigating the visible signs of skin aging, our findings identify Ubiad1 as an essential component of the defensive barriers of the epidermis.