Keratinocyte Terminal Differentiation Research Articles

P19 Defining the mechanism of skin barrier restoration by Lactobacillus rhamnosus

Abstract Introduction and aims We aim to investigate the lysate of Lactobacillus rhamnosus GG (LGG), a bacterium previously established as a gut probiotic, and to examine its postulated ability to enhance and restore human skin barrier function. Compromised skin barriers are implicated in various skin conditions including atopic dermatitis. Furthermore, skin barrier function decreases with age leading to dryness and sensitivity. Previous research has shown promising effects of LGG lysates on keratinocytes, including increasing transepidermal electrical resistance, increasing tight junction protein expression, cell migration promotion and mitigation of Staphylococcus aureus toxicity. This study aims to take a closer look at the molecular mechanisms behind the effect of LGG lysates on the skin, including terminal differentiation, lamin dispersal and nuclear degradation in keratinocytes. Methods Normal human epithelial keratinocytes and rat epithelial keratinocytes were grown and treated with the LGG lysate. Western blot analysis was used to determine the relative concentrations of tight junction proteins and other differentiation markers. A phosphoproteomic study was performed to identify key pathways activated by the LGG lysate. A Staphylococcus epidermidis lysate control was used to filter out any nonspecific bacterial effects. Immunostaining of nuclear proteins was used to visualize the effects of LGG lysate on lamin dispersal and nuclear degradation. short hairpin RNA filaggrin knockdown normal human keratinocytes were developed to create a ‘barrier impaired’ model to assess ability of the lysate to ‘rescue’ the skin barrier. Results The LGG lysate was shown to increase tight junction protein expression in normal human keratinocytes and increase keratinocyte terminal differentiation markers. We present Results that show decreased tight junction protein expression in filaggrin knockdown cells. Conclusions These Results show the potential for LGG lysate to enhance and restore skin barrier function and begin to suggest potential mechanisms for this effect.

OSBPL2 compound heterozygous variants cause dyschromatosis, ichthyosis, deafness and atopic disease syndrome

PurposeIn this study, we identified and diagnosed a novel inherited condition called Dyschromatosis, Ichthyosis, Deafness, and Atopic Disease (DIDA) syndrome. We present a series of studies to clarify the pathogenic variants and specific mechanism. MethodsExome sequencing and Sanger sequencing was conducted in affected and unaffected family members. A variety of human and cell studies were performed to explore the pathogenic process of keratosis. ResultsOur finding indicated that DIDA syndrome was caused by compound heterozygous variants in the oxysterol-binding protein-related protein 2 (OSBPL2) gene. Furthermore, our findings revealed a direct interaction between OSBPL2 and Phosphoinositide phospholipase C-beta-3 (PLCB3), a key player in hyperkeratosis. OSBPL2 effectively inhibits the ubiquitylation of PLCB3, thereby stabilizing PLCB3. Conversely, OSBPL2 variants lead to enhanced ubiquitination and subsequent degradation of PLCB3, leading to epidermal hyperkeratosis, characterized by aberrant proliferation and delayed terminal differentiation of keratinocytes. ConclusionsOur study not only unveiled the association between OSBPL2 variants and the newly identified DIDA syndrome but also shed light on the underlying mechanism.

Electrical Impedance Spectroscopy Quantifies Skin Barrier Function in Organotypic In Vitro Epidermis Models

3 D human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in pre-clinical investigative dermatology and regulatory toxicology. Here, we investigated the utility of electrical impedance spectroscopy (EIS) for non-invasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for seven consecutive days did not impact epidermal morphology and readouts showed comparable trends to HEEs measured only once. We determined two frequency ranges in the resulting impedance spectra: a lower frequency range termed EISdiff correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EISSC correlated with stratum corneum thickness. HEEs generated from CRISPR/Cas9 engineered keratinocytes that lack key differentiation genes FLG, TFAP2A, AHR or CLDN1 confirmed that keratinocyte terminal differentiation is the major parameter defining EISdiff. Exposure to pro-inflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EISdiff. This cytokine-associated decrease in EISdiff was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a non-invasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects and repair.

Growth modulatory effects of fenretinide encompass keratinocyte terminal differentiation: A favorable outcome for oral squamous cell carcinoma chemoprevention.

Oral squamous cell carcinoma (OSCC) is world-wide health problem associated with high morbidity and mortality. From both the patient and socio-economic perspectives, prevention of progression of premalignant oral intraepithelial neoplasia (OIN) to OSCC is clearly the preferable outcome. Optimal OSCC chemopreventives possess a variety of attributes including high-tolerability, bioavailability, efficacy and preservation of an intact surface epithelium. Terminal differentiation, which directs oral keratinocytes leave the proliferative pool to form protective cornified envelopes, preserves the protective epithelial barrier while concurrently eliminating growth-aberrant keratinocytes. This study employed human premalignant oral keratinocytes and an OSCC cell line to evaluate the differentiation-inducing capacity of the synthetic retinoid, fenretinide (4HPR). Full thickness oral mucosal explants were evaluated for proof of concept differentiation studies. Results of this study characterize the ability of 4HPR to fulfill all requisite components for keratinocyte differentiation i.e. nuclear import via binding to CRABP-II (molecular modeling), binding to and subsequent activation of retinoic acid nuclear receptors (receptor activation assays), increased expression and translation of genes associated with keratinocyte differentiation (RT-PCR, immunoblotting) upregulation of a transglutaminase enzyme essential for cornified envelope formation (TGM3, functional assay) and augmentation of terminal differentiation in human oral epithelial explants (image-analyses quantified corneocyte desquamation). These data build upon the chemoprevention repertoire of 4HPR that includes function as a small molecule kinase inhibitor and inhibition of essential mechanisms necessary for basement membrane invasion. An upcoming clinical trial, which will assess whether a 4HPR-releasing mucoadhesive patch induces histologic, clinical and molecular regression in OIN lesions, will provide essential clinical insights.

The Aryl Hydrocarbon Receptor Regulates Epidermal Differentiation through Transient Activation of TFAP2A

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 additional insights into the molecular mechanism behind AHR-mediated barrier function and identifies potential targets for the treatment of skin barrier diseases.

Quantitative Analysis and Molecular Docking Simulation of Flavonols from Eruca sativa Mill. and Their Effect on Skin Barrier Function.

Eruca sativa is a commonly used edible plant in Italian cuisine. E. sativa 70% ethanol extract (ES) was fractionated with five organic solvents, including n-hexane (EHex), chloroform (ECHCl3), ethyl acetate (EEA), n-butyl alcohol (EBuOH), and water (EDW). Ethyl acetate fraction (EEA) had the highest antioxidant activity, which was correlated with the total polyphenol and flavonoid content. ES and EEA acted as PPAR-α ligands by PPAR-α competitive binding assay. EEA significantly increased cornified envelope formation as a keratinocyte terminal differentiation marker in HaCaT cells. Further, it significantly reduced nitric oxide and pro-inflammatory cytokines (IL-6 and TNF-α) in lipopolysaccharide-stimulated RAW 264.7 cells. The main flavonol forms detected in high amounts from EEA are mono-and di-glycoside of each aglycone. The main flavonol form of EEA is the mono-glycoside of each aglycone detected, and the most abundant flavonol mono-glycoside is kaempferol 3-glucoside 7.4%, followed by quercetin-3-glucoside 2.3% and isorhamnetin 3-glucoside 1.4%. Flavonol mono-glycosides were shown to be a potent PPAR-α ligand using molecular docking simulation and showed the inhibition of nitric oxide. These results suggest that the flavonol composition of E. sativa is suitable for use in improving skin barrier function and inflammation in skin disorders, such as atopic dermatitis.

AP-2α/AP-2β Transcription Factors Are Key Regulators of Epidermal Homeostasis

AP-2 transcription factors regulate ectodermal development, but their roles in epidermal homeostasis in adult skin are unknown. We find that AP-2α is the predominant AP-2 family member in adult epidermis, followed by AP-2β. Through inactivation of AP-2α, AP-2β, or both in keratinocytes, we assessed the effects of a gradient of epidermal AP-2 activity on skin function. We find that (i) loss of AP-2β in keratinocytes is compensated for by AP-2α, (ii) loss of AP-2α impairs terminal keratinocyte differentiation and hair morphogenesis, and (iii) the combined loss of AP-2α/AP-2β results in more severe skin and hair abnormalities. Keratinocyte differentiation defects precede progressive neutrophilic skin inflammation. Inducible inactivation of AP-2α/AP-2β in the adult phenocopies these manifestations. Transcriptomic analyses of epidermis lacking AP-2α or AP-2α/AP-2β in keratinocytes demonstrate a terminal keratinocyte differentiation defect with upregulation of alarmin keratins and of several immune pathway regulators. Moreover, our analyses suggest a key role of reduced AP-2α-dependent gene expression of CXCL14 and the keratin 15 gene K15 as an early pathogenic event toward the manifestation of skin inflammation. Thus, AP-2α and AP-2β are critical regulators of epidermal homeostasis in adult skin.

GDU-952, a novel AhR agonist ameliorates skin barrier abnormalities and immune dysfunction in DNFB-induced atopic dermatitis in mice

The aryl hydrocarbon receptor (AhR) is widely expressed in the skin. It controls immune-mediated skin responses to various external environmental signals, promote terminal differentiation of epidermal keratinocytes and participates the maintenance of the skin barrier function. As a therapeutic target, AhR activation modulates many diseases progression driven by immune/inflammatory processes such as atopic dermatitis (AD) and psoriasis. In this study, we revealed that GDU-952 is a novel AhR agonist, which is able to decreases IgE serum levels, to inhibit pro-inflammatory cytokines such as IL-6 and TNF-α and to induce immunoregulatory effects through restoring Th1/Th2 immune balance and promoting CD4+FOXP3+regulatory T (Treg) populations in AD skin lesions. Furthermore, GDU-952 can strengthen the skin barrier function through upregulating epidermal differentiation-related and tight junction proteins. This may alleviate AD symptoms, such as dermatitis scores, epidermal hyperplasia and mast cell infiltration. These results offer a rationale for further preclinical/clinical studies to evaluate the possible use of GDU-952 in the management of AD.

PGC-1s shape epidermal physiology by modulating keratinocyte proliferation and terminal differentiation.

Skin plays central roles in systemic physiology, and it undergoes significant functional changes during aging. Members of the peroxisome proliferator-activated receptor-gamma coactivator (PGC-1) family (PGC-1s) are key regulators of the biology of numerous tissues, yet we know very little about their impact on skin functions. Global gene expression profiling and gene silencing in keratinocytes uncovered that PGC-1s control the expression of metabolic genes as well as that of terminal differentiation programs. Glutamine emerged as a key substrate promoting mitochondrial respiration, keratinocyte proliferation, and the expression of PGC-1s and terminal differentiation programs. Importantly, gene silencing of PGC-1s reduced the thickness of a reconstructed living human epidermal equivalent. Exposure of keratinocytes to a salicylic acid derivative potentiated the expression of PGC-1s and terminal differentiation genes and increased mitochondrial respiration. Overall, our results show that the PGC-1s are essential effectors of epidermal physiology, revealing an axis that could be targeted in skin conditions and aging.

Heme Oxygenase-1 Is Upregulated during Differentiation of Keratinocytes but Its Expression Is Dispensable for Cornification of Murine Epidermis.

The epidermal barrier of mammals is initially formed during embryonic development and continuously regenerated by the differentiation and cornification of keratinocytes in postnatal life. Cornification is associated with the breakdown of organelles and other cell components by mechanisms which are only incompletely understood. Here, we investigated whether heme oxygenase 1 (HO-1), which converts heme into biliverdin, ferrous iron and carbon monoxide, is required for normal cornification of epidermal keratinocytes. We show that HO-1 is transcriptionally upregulated during the terminal differentiation of human keratinocytes in vitro and in vivo. Immunohistochemistry demonstrated expression of HO-1 in the granular layer of the epidermis where keratinocytes undergo cornification. Next, we deleted the Hmox1 gene, which encodes HO-1, by crossing Hmox1-floxed and K14-Cre mice. The epidermis and isolated keratinocytes of the resulting Hmox1f/f K14-Cre mice lacked HO-1 expression. The genetic inactivation of HO-1 did not impair the expression of keratinocyte differentiation markers, loricrin and filaggrin. Likewise, the transglutaminase activity and formation of the stratum corneum were not altered in Hmox1f/f K14-Cre mice, suggesting that HO-1 is dispensable for epidermal cornification. The genetically modified mice generated in this study may be useful for future investigations of the potential roles of epidermal HO-1 in iron metabolism and responses to oxidative stress.

Take a breath: oxygen sensing of epidermal differentiation.

Skin serves as a barrier to protect our body from injury, pathogens and trans-epidermal water loss. It is the only tissue directly exposed to oxygen besides lungs. Air exposure is an essential step of in vitro generation skin graft. However, the role of oxygen in this process remains hitherto unclear. Teshima et al. unveiled the impact of the hypoxia-inducible factor (HIF) pathway on epidermal differentiation in three-dimensional skin models. The authors of this work describe how air-lifting of organotypic epidermal cultures impairs HIFs activity, leading to a proper terminal differentiation of keratinocytes and stratification.

CYSRT1: An Antimicrobial Epidermal Protein that Can Interact with Late Cornified Envelope Proteins

Late cornified envelope (LCE) proteins are small cationic epidermal proteins with antimicrobial properties, and the combined deletion of LCE3B and LCE3C genes is a risk factor for psoriasis that affects skin microbiome composition. In a yeast two-hybrid screen, we identified CYSRT1 as an interacting partner of members of all LCE groups except LCE6. These interactions were confirmed in a mammalian cell system by coimmunoprecipitation. CYSRT1 is a protein of unknown function that is specifically expressed in cutaneous and oral epithelia and spatially colocalizes with LCE proteins in the upper layers of the suprabasal epidermis. Constitutive CYSRT1 expression is present in fully differentiated epidermis and can be further induced invivo by disruption of the skin barrier upon stratum corneum removal. Transcriptional regulation correlates to keratinocyte terminal differentiation but not to skin bacteria exposure. Similar to LCEs, CYSRT1 was found to have antibacterial activity against Pseudomonas aeruginosa. Comparative gene sequence analysis and protein amino acid alignment indicate that CYSRT1 is highly conserved among vertebrates and has putative antimicrobial activity. To summarize, we identified CYSRT1 in the outer skin layer, where it colocalizes with LCE proteins and contributes to the constitutive epidermal antimicrobial host defense repertoire.

Lead optimization of aryl hydrocarbon receptor ligands for treatment of inflammatory skin disorders

Therapeutic aryl hydrocarbon receptor (AHR) modulating agents gained attention in dermatology as non-steroidal anti-inflammatory drugs that improve skin barrier properties. By exploiting AHR’s known ligand promiscuity, we generated novel AHR modulating agents by lead optimization of a selective AHR modulator (SAhRM; SGA360). Twenty-two newly synthesized compounds were screened yielding two novel derivatives, SGA360f and SGA388, in which agonist activity led to enhanced keratinocyte terminal differentiation. SGA388 showed the highest agonist activity with potent normalization of keratinocyte hyperproliferation, restored expression of skin barrier proteins and dampening of chemokine expression by keratinocytes upon Th2-mediated inflammation in vitro. The topical application of SGA360f and SGA388 reduced acute skin inflammation in vivo by reducing cyclooxygenase levels, resulting in less neutrophilic dermal infiltrates. The minimal induction of cytochrome P450 enzyme activity, lack of cellular toxicity and mutagenicity classifies SGA360f and SGA388 as novel potential therapeutic AHR ligands and illustrates the potential of medicinal chemistry to fine-tune AHR signaling for the development of targeted therapies in dermatology and beyond.

Potential Role of the Epidermal Differentiation Complex in the Pathogenesis of Psoriasis

The skin is the largest barrier organ of the human body and serves to protect the internal structure of the body from the harmful environment. The epidermis forms the outermost layer and is exposed to the environment. Keratinocytes are important constituent cells of the epidermis and alter their morphology and structural integrity through a highly complex differentiation process referred to as cornification. Abnormalities in the process of epidermal cornification can lead to skin barrier dysfunction. The epidermal differentiation complex (EDC) is a gene cluster located within a 2 Mb region of human chromosome 1q21. EDC is responsible for epithelial tissue development and for properties of the stratum corneum. One of the most important features of psoriasis is the abnormal terminal differentiation of keratinocytes. However, the relationship between EDC and the occurrence of psoriasis is still unclear. In this review, we summarize current knowledge regarding the physiological functions of EDC and discuss its possible contributions to the pathogenesis of psoriasis.

174 Defining the composition and function of Lamin ribonuclear bodies during keratinocyte terminal differentiation and nuclear degradation

Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, requiring degradation of organelles, including the nucleus. However, the underlying molecular mechanisms are not well defined. We have identified the myosin II-mediated dispersal of an internal fragment of Lamin A (LMNA) during end-stage terminal differentiation prior to nuclear degradation. non-membrane-bound LMNA bodies were distinct from keratohyalin granules and lamellar bodies and associated both with RNA and with components of the nuclear envelope.

203 Constitutive expression of heme oxygenase-1 in differentiated keratinocytes is abolished in a new mouse model for the study of epidermal iron metabolism and redox balance

Cornification of epidermal keratinocytes is associated with the breakdown of organelles and other cell components. The degradation of hemoproteins, such as cytochrome c and cytochrome P450 family members, releases heme, a potent source of oxidative stress. Heme oxygenase 1 (HO-1) converts heme into ferrous iron, carbon monoxide and biliverdin, a strong antioxidant. Here, we show by single cell transcriptomics and immunohistochemistry that HO-1 is upregulated during terminal differentiation of human keratinocytes, leading to the accumulation of HO-1 mRNA and protein in the granular layer of the epidermis.

217 Distinctive and shared signatures of Th1 and Th2 cytokines on the lipid pathways involved in the epidermal barrier function

Abnormalities of the permeability barrier and impaired epidermal differentiation characterize both psoriasis and atopic dermatitis, which are driven by immune responses mediated by Th1 and Th2 cytokines. The spectrum of interference of the Th1 and Th2 type cytokines with normal terminal differentiation of keratinocytes is not fully characterized. Our aim was to identify specific and common features of epidermal perturbation associated with each group of cytokines. Immortalized human keratinocytes (Ker-CT) were treated with Th1 and Th2 cytokines in pro-differentiative culture conditions with high calcium concentration.

MAB21L4 Deficiency Drives Squamous Cell Carcinoma via Activation of RET.

Downregulation of RET mediated by MAB21L4-CacyBP interaction is required to induce epidermal differentiation and suppress carcinogenesis, suggesting RET inhibition as a potential therapeutic approach in squamous cell carcinoma.

Expression of BMP4 and FOXN1 in orthokeratinized odontogenic cyst compared to odontogenic keratocyst suggests an epidermal phenotype

ABSTRACT Odontogenic keratocysts (OKC) and orthokeratinized odontogenic cysts (OOC) are odontogenic cysts that share histological and immunohistochemical similarity with epidermal appendages and cutaneous cystic lesions despite exhibiting contrasting biological behavior. In epidermal appendages, BMP4 induces expression of FOXN1, which participates in terminal differentiation of keratinocytes and control of proliferation. We compared BMP4 and FOXN1 expression in OOC and OKC to investigate their role in the epithelial differentiation of these cysts. BMP4 and FOXN1 expression was assessed using immunohistochemistry in 20 primary sporadic OKC and compared to 16 OOC. BMP4 epithelial expression was detected in 81.25% OOC compared to 35% in OKC, while its expression in connective tissue was observed in 65% OKC and 75% OOC. FOXN1 was detected in 75% OOC vs. 30% OKC. The “triple positive” phenotype, i.e., BMP4 epithelial and connective tissue positivity and FOXN1 epithelial positivity, was seen in 56.25% OOC compared to 10% OKC. The greater expression of BMP4 and FOXN1 in OOC suggests greater activation of this pathway in OOC, which suggests a role in its more mature epithelium; it also resembles an epidermal phenotype.

Epigenetic Mechanisms of Epidermal Differentiation.

Keratinocyte differentiation is an essential process for epidermal stratification and stratum corneum formation. Keratinocytes proliferate in the basal layer of the epidermis and start their differentiation by changing their functional or phenotypical type; this process is regulated via induction or repression of epidermal differentiation complex (EDC) genes that play a pivotal role in epidermal development. Epidermal development and the keratinocyte differentiation program are orchestrated by several transcription factors, signaling pathways, and epigenetic regulators. The latter exhibits both activating and repressive effects on chromatin in keratinocytes via the ATP-dependent chromatin remodelers, histone demethylases, and genome organizers that promote terminal keratinocyte differentiation, and the DNA methyltransferases, histone deacetylases, and Polycomb components that stimulate proliferation of progenitor cells and inhibit premature activation of terminal differentiation-associated genes. In addition, microRNAs are involved in different processes between proliferation and differentiation during the program of epidermal development. Here, we bring together current knowledge of the mechanisms controlling gene expression during keratinocyte differentiation. An awareness of epigenetic mechanisms and their alterations in health and disease will help to bridge the gap between our current knowledge and potential applications for epigenetic regulators in clinical practice to pave the way for promising target therapies.