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Alternative splicing (AS) of messenger RNAs occurs in ∼95% of multi-exon human genes and generates diverse RNA and protein isoforms. We investigated AS events associated with human epidermal differentiation, a process crucial for skin function. We identified 6,413 AS events, primarily involving cassette exons. We also predicted 34 RNA-binding proteins (RBPs) regulating epidermal AS, including 19 previously undescribed candidate regulators. From these results, we identified FUS as an RBP that regulates the balance between keratinocyte proliferation and differentiation. Additionally, we characterized the function of a cassette exon AS event in MAP3K7, which encodes a kinase involved in cell signaling. We found that a switch from the short to long isoform of MAP3K7, triggered during differentiation, enforces the demarcation between proliferating basal progenitors and overlying differentiated strata. Our findings indicate that AS occurs extensively in the human epidermis and has critical roles in skin homeostasis.

The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multi-omics analyses using human skin keratinocytes revealed that, upon ligand activation, AHR binds open chromatin to induce expression of transcription factors (TFs), e.g., Transcription Factor AP-2α (TFAP2A), as a swift response to environmental stimuli. The terminal differentiation program including upregulation of barrier genes, filaggrin and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides novel insights into the molecular mechanism behind AHR-mediated barrier function and potential novel targets for the treatment of skin barrier diseases.

Introduction: In skin traumas, such as burns, epidermal homeostasis is affected, often requiring clinical approaches. Different therapeutic strategies can be used including transplantation, besides the use of synthetic or natural materials with allogeneic cells. In this context, tissue engineering is an essential tool for skin regeneration, and using mesenchymal stem cells (MSC) from the umbilical cord appears to be a promising strategy in regenerative medicine due to its renewal and differentiation potential and hypo immunogenicity. We evaluated the transdifferentiation of MSC from umbilical cord into keratinocytes in three-dimensional (3D) in vitro skin models, using dermal equivalents composed by type I collagen with dermal fibroblasts and a commercial porcine skin decellularized matrix, both cultured at air-liquid interface (ALI). Methods: The expression of epidermal proteins cytokeratins (CK) 5, 14 and 10, involucrin and filaggrin was investigated by real-time PCR and immunofluorescence, in addition to the activity of epidermal kallikreins (KLK) on the hydrolysis of fluorogenic substrates. Results and discussion: The cultivation of MSCs with differentiation medium on these dermal supports resulted in organotypic cultures characterized by the expression of the epidermal markers CK5, CK14, CK10 and involucrin, mainly on the 7th day of culture, and filaggrin at 10th day in ALI. Also, there was a 3-fold increase in the KLK activity in the epidermal equivalents composed by MSC induced to differentiate into keratinocytes compared to the control (MSC cultivated in the proliferation medium). Specifically, the use of collagen and fibroblasts resulted in a more organized MSC-based organotypic culture in comparison to the decellularized matrix. Despite the non-typical epithelium structure formed by MSC onto dermal equivalents, the expression of important epidermal markers in addition to the paracrine effects of these cells in skin may indicate its potential use to produce skin-based substitutes.

The skin of mammals is a multilayered and multicellular tissue that forms an environmental barrier with key functions in protection, regulation, and sensation. While animal models have long served to study the basic functions of the skin in vivo, new insights are expected from in vitro models of human skin development. Human pluripotent stem cells (PSCs) have proven to be invaluable tools for studying human development in vitro. To understand the mechanisms regulating human skin homeostasis and injury repair at the molecular level, recent efforts aim to differentiate PSCs towards skin epidermal keratinocytes, dermal fibroblasts, and skin appendages such as hair follicles and sebaceous glands. Here, we present an overview of the literature describing strategies for human PSC differentiation towards the components of skin, with a particular focus on keratinocytes. We highlight fundamental advances in the field employing patient-derived human induced PSCs (iPSCs) and skin organoid generation. Importantly, PSCs allow researchers to model inherited skin diseases in the search for potential treatments. Skin differentiation from human PSCs holds the potential to clarify human skin biology.

Sorting transmembrane cargo is essential for tissue development and homeostasis. However, mechanisms of intracellular trafficking in stratified epidermis are poorly understood. Here, we identify an interaction between the retromer endosomal trafficking component, VPS35, and the desmosomal cadherin, desmoglein-1 (Dsg1). Dsg1 is specifically expressed in stratified epidermis and, when properly localized on the plasma membrane of basal keratinocytes, promotes stratification. We show that the retromer drives Dsg1 recycling from the endo-lysosomal system to the plasma membrane to support human keratinocyte stratification. The retromer-enhancing chaperone, R55, promotes the membrane localization of Dsg1 and a trafficking-deficient mutant associated with a severe inflammatory skin disorder, enhancing its ability to promote stratification. In the absence of Dsg1, retromer association with and expression of the glucose transporter GLUT1 increases, exposing a potential link between Dsg1 deficiency and epidermal metabolism. Our work provides evidence for retromer function in epidermal regeneration, identifying it as a potential therapeutic target.

Pyridoxine (VB6) is a vitamin that is essential to maintain the homeostasis of the human body by contributing to various metabolic reactions. In the skin, although some studies have shown that VB6 is involved in regulating homeostasis through the attenuation of intracellular oxidative stress, there are few reports regarding the effects of VB6 on the prevention or improvement of skin aging. Thus, we conducted this study to determine the potential anti-skin pigmentation effect of VB6 focusing on the phagocytosis of melanosomes (MSs) by keratinocytes. The phagocytosis of MSs by keratinocytes is activated by oxidative stress and is an important factor of skin pigmentation and the eventual appearance of pigmented spots. First, we confirmed the antioxidant property of VB6 that enhanced the expression of several intracellular antioxidants via nuclear erythroid factor 2-related factor 2 (Nrf2). Although the incorporation of fluorescent beads (FBs), which are used as pseudo-MSs, into keratinocytes was increased under higher oxidation conditions caused by UVB and by the depletion of intracellular glutathione, treatment with VB6 suppressed the increased incorporation of FBs into those keratinocytes via Nrf2 activation. Furthermore, VB6 restored the decreased expression of differentiation marker proteins in keratinocytes caused by FB incorporation. Taken together, the results show that VB6 has the potential to prevent the appearance of pigmented spots by suppressing the activation of phagocytosis in keratinocytes caused by oxidative stress, and by restoring the differentiation of keratinocytes disrupted by FB incorporation.

In the interfollicular epidermis (IFE), stem cells (KSC) generate transit amplifying (TA) cells that, after symmetric divisions, produce differentiating daughters. Here, we isolated and characterized the highly proliferative interfollicular epidermal basal cell population \"early\" TA (ETA) cells, based on their capacity to adhere to type IV collagen. Proliferation and colony-forming efficiency in ETA cells are lower than in KSC but higher than in \"late\" TA (LTA). Stemness, proliferation, and differentiation markers confirmed that ETA cells display a unique phenotype. Skin reconstructs derived from ETA cells present different features (epidermal thickness, Ki67, and Survivin expression), as compared to skin equivalents generated from either KSC or LTA cells. The low-affinity neurotrophin receptor CD271, which regulates the KSC to TA cell transition in the human epidermis through an on/off switch control mechanism, is predominantly expressed in ETA cells. Skin equivalents generated from siRNA CD271 ETA cells display a more proliferative and less differentiated phenotype, as compared to mock-derived reconstructs. Consistently, CD271 overexpression in LTA cells generates a more proliferative skin equivalent than mock LTA cells. Finally, the CD271 level declines with cellular senescence, while it induces a delay in p16INK4 expression. We conclude that ETA cells represent the first KSC progenitor with exclusive features. CD271 identifies and modulates ETA cells, thus participating in the early differentiation and regenerative capacity of the human epidermis.

The replication of varicella-zoster virus (VZV) in skin is critical to its pathogenesis and spread. Primary infection causes chickenpox, which is characterised by centrally distributed skin blistering lesions that are rich in infectious virus. Cell-free virus in the cutaneous blistering lesions not only spreads to cause further cases, but infects sensory nerve endings, leading to the establishment of lifelong latency in sensory and autonomic ganglia. The reactivation of virus to cause herpes zoster is again characterised by localised painful skin blistering rash containing infectious virus. The development of in vitro and in vivo models of VZV skin replication has revealed aspects of VZV replication and pathogenesis in this important target organ and improved our understanding of the vaccine strain vOKa attenuation. In this review, we outline the current knowledge on VZV interaction with host signalling pathways, the viral association with proteins associated with epidermal terminal differentiation, and how these interconnect with the VZV life cycle to facilitate viral replication and shedding.

OBJECTIVE: Estrogen (E) and progesterone (P) are the major female hormones and are secreted with changing concentration ratios throughout the menstrual cycle. These hormones have been studied individually regarding their physiological function in the skin, but their concentration ratio (E/P) and its effect on the skin has not yet been investigated. The purpose of this study was to clarify the effect of the E/P ratio on skin barrier function. The menstrual cycle was divided into the menstrual, follicular, ovulation, and luteal phases.
METHODS: The E/P concentration ratios corresponding with each phase were added to a three-dimensional epidermal model or normal human epidermal keratinocytes for 5 days. Gene and protein expression levels of several markers of cell differentiation, including loricrin (LOR) and transglutaminase (TGase), were quantified by real-time PCR and western blotting, respectively. Transepidermal water loss (TEWL) of the three-dimensional epidermal model was measured, and ceramide content was quantified by thin-layer chromatography.
RESULTS: Gene expression of the epidermal differentiation markers, LOR and TGase, increased when applying the concentration ratio of E/P associated with the menstrual and luteal phases. The LOR protein level decreased from menstrual to luteal phases, and the TGase protein level increased from menstrual to luteal phases. During the same phases, ceramide NS increased and TEWL decreased.
CONCLUSIONS: Skin barrier function was improved by culturing cells at specific E/P concentration ratios, which would, therefore, be considered beneficial for female skin. This suggests that dysregulated E/P concentration ratios may be the cause of certain skin problems.