Epidermal Differentiation Markers Research Articles

237 Skin barrier integrity is enhanced after topical application of emulsions with an acidic pH

The pH of the stratum corneum is slightly acidic (mean 4.9) and tightly regulated. This is important for antimicrobial defense and the formation of the physical barrier. The acidic pH inhibits the growth of pathogenic Staphylococcus aureus and enables optimal activity of enzymes for lipid synthesis, epidermal differentiation and desquamation. Here we asked whether topical application of an oil-in-water emulsions adjusted to pH 4 or pH 5.8 and the untreated control influence biophysical markers and mechanical stability of the skin barrier, Stratum corneum morphology and inflammation. Twenty-four healthy volunteers aged 18 to 75 years were treated in a randomized way on the lower arms for 28 days; pH, TEWL, SC hydration, mechanical stability, corneocyte size and inflammation were recorded. After 28 days, skin surface pH was significantly more reduced by the pH 4 compared to the pH 5.8 emulsion. TEWL as a marker of skin barrier function was slightly reduced by both emulsions. SC hydration was significantly increased to the same level by both emulsions. The number of D-Squame® tape-strips to disrupt the skin barrier to a threefold increase in TEWL as a marker for skin integrity was significantly increased by both emulsions compared to untreated control, but slightly more by the pH 4 emulsion. Inflammation after tape-stripping as determined by clinical grading of erythema was considerable reduced by the pH 4 emulsion, and IL-1α protein expression was significantly reduced by both emulsions, but the reduction was higher with the pH 4 preparation. The size of corneocytes attached to the tape-strips as a marker for epidermal differentiation and desquamation was significantly increased after treatment with the pH 4 compared to the pH 5.8 emulsion and the control. In summary, treatment with a pH 4 emulsion positively influenced SC pH, TEWL, SC hydration, epidermal differentiation/desquamation resulting in increased mechanically stability and reduced inflammation against tape-stripping induced irritation.

647 Investigating re-epithelialization in a novel ex vivo human skin model

Although numerous wound healing models have been established, a model to study re-epithelialization in vitro, without disrupting the underlying dermal compartment, is lacking. Therefore, we established a new standardized human ex vivo model based on application of negative pressure, inducing blistering and consequently dermal epidermal separation. Effects of this treatment for epidermal regeneration and immune cells were investigated. Immunostaining revealed type IV collagen expression on the remaining dermal compartment, indicating that the basement membrane and thus the underlying dermis remained intact after removal of the blister roof epidermis. Keratinocytes were absent in the wounded area as evidenced by a lack of K14 staining. Four to six days after removal of the epidermis and culture, the wound was completely covered again with one to three-cell thick K14+ keratinocyte layers. Keratinocytes (∼20%) covering the initial wound bed expressed the endogenous cell proliferation marker Ki67, indicating that proliferation and migration were involved in wound closure. After eight to twelve days, a multi-layered epidermis was formed expressing several epidermal differentiation markers (e.g., K10, filaggrin, DSG-1, CDSN). While application of negative pressure did not affect the morphology, phenotype and density of Langerhans cells, we observed more T cells in the blister roof epidermis as compared to normal epidermis. Additionally, we identified several populations in blister roof epidermis and suction blister fluid that are absent in normal epidermis which correlated with their decrease in the dermis, indicating an influx upon pressure. Whether and how this contributes to the wound healing process is currently under investigation. This model recapitulates the main features of epithelial wound regeneration, and thus can be applied for testing wound healing therapies and investigating the underlying mechanisms in the future.

Epidermal autophagy and beclin 1 regulator 1 and loricrin: a paradigm shift in the prognostication and stratification of the American Joint Committee on Cancer stage I melanomas.

SummaryBackgroundThe updated American Joint Committee on Cancer (AJCC) staging criteria for melanoma remain unable to identify high‐risk stage I tumour subsets.ObjectivesTo determine the utility of epidermal autophagy and beclin 1 regulator 1 (AMBRA1)/loricrin (AMLo) expression as a prognostic biomarker for AJCC stage I cutaneous melanoma.MethodsPeritumoral AMBRA1 expression was evaluated in a retrospective discovery cohort of 76 AJCC stage I melanomas. AMLo expression was correlated with clinical outcomes up to 12 years in two independent powered, retrospective validation and qualification cohorts comprising 379 AJCC stage I melanomas.ResultsDecreased AMBRA1 expression in the epidermis overlying primary melanomas in a discovery cohort of 76 AJCC stage I tumours was associated with a 7‐year disease‐free survival (DFS) rate of 81·5% vs. 100% survival with maintained AMBRA1 (P < 0·081). Following an immunohistochemistry protocol for semi‐quantitative analysis of AMLo, analysis was undertaken in validation (n = 218) and qualification cohorts (n = 161) of AJCC stage I melanomas. Combined cohort analysis revealed a DFS rate of 98·3% in the AMLo low‐risk group (n = 239) vs. 85·4% in the AMLo high‐risk cohort (n = 140; P < 0·001). Subcohort multivariate analysis revealed that an AMLo hazard ratio (HR) of 4·04 [95% confidence interval (CI) 1·69–9·66; P = 0·002] is a stronger predictor of DFS than Breslow depth (HR 2·97, 95% CI 0·93–9·56; P = 0·068) in stage IB patients.ConclusionsLoss of AMLo expression in the epidermis overlying primary AJCC stage I melanomas identifies high‐risk tumour subsets independently of Breslow depth. What's already known about this topic? There is an unmet clinical need for biomarkers of early‐stage melanoma.Autophagy and beclin 1 regulator 1 (AMBRA1) is a proautophagy regulatory protein with known roles in cell proliferation and differentiation, and is a known tumour suppressor.Loricrin is a marker of epidermal terminal differentiation. What does this study add? AMBRA1 has a functional role in keratinocyte/epidermal proliferation and differentiation.The combined decrease/loss of peritumoral AMBRA1 and loricrin is associated with a significantly increased risk of metastatic spread in American Joint Committee on Cancer (AJCC) stage I tumours vs. melanomas, in which peritumoral AMBRA1 and loricrin are maintained, independently of Breslow depth. What is the translational message? The integration of peritumoral epidermal AMBRA1/loricrin biomarker expression into melanoma care guidelines will facilitate more accurate, personalized risk stratification for patients with AJCC stage I melanomas, thereby facilitating stratification for appropriate follow‐up and informing postdiagnostic investigations, including sentinel lymph node biopsy, ultimately resulting in improved disease outcomes and rationalization of healthcare costs.

Nicotinamide Metabolism Modulates the Proliferation/Differentiation Balance and Senescence of Human Primary Keratinocytes

Nicotinamide (NAM) is the main precursor of nicotinamide adenine dinucleotide (NAD+), a coenzyme essential for DNA repair, glycolysis, and oxidative phosphorylation. NAM has anti-aging activity on human skin, but theunderlying mechanisms of action are unclear. Using 3-dimensional organotypic skin models, we show thatNAM inhibits differentiation of the upper epidermal layers and maintains proliferation in the basal layer. In2-dimensional culture, NAM reduces the expression of early and late epidermal differentiation markers andincreases the proliferative capacity of human primary keratinocytes. This effect is characterized by elevated clonogenicity and an increased proportion of human primary keratinocyte stem cell (holoclones) compared tocontrols. By contrast, preventing the conversion of NAM to NAD+ using FK866 leads to premature humanprimary keratinocyte differentiation and senescence, together with a dramatic drop in glycolysis and cellular adenosine triphosphate levels while oxidative phosphorylation is moderately affected. All these effectsare rescued by addition of NAM, known to compete with FK866, which suggests that conversion to NAD+ is part of the mechanistic response. These data provide insights into the control of differentiation, proliferation, and senescence by NAM and NAD+ in skin. They may lead to new therapeutic advances for skin conditions characterized by dysregulated epidermal homeostasis and premature skin aging, such as photoaging.

The Activation of PPAR-α and Wnt/β-catenin by Luffa cylindrica Supercritical Carbon Dioxide Extract

Luffa cylindrica (LC) is a very fast-growing climber and its fruit have been considered as agricultural wastes. We conducted to check the comparative qualities of ethanol solvent extraction (LCE) and supercritical carbon dioxide extraction (LCS) of L. cylindrica fruit and seed. LCS had higher antioxidant and polyphenol contents than LCE. LCS were significantly increased peroxisome proliferator-activated receptor (PPAR)-a and involucrin expression as epidermal differentiation marker in 3D skin equivalent model. LCS also showed antimicrobial activity against Staphylococcus aureus, a causative bacteria in atopic dermatitis. In addition, LCS inhibited the adipocyte differentiation of 3T3-L1 cells. When treated with the extract at a concentration of 100 μg/mL, the Wnt/β-catenin pathway reporter luciferase activity of HEK 293-TOP cells was increased approximately by 2-folds compared to that of the untreated control group. These results indicate that L. cylindrica supercritical carbon dioxide extract may serve as a cosmeceutical for improving skin barrier function and the treatment of obesity.

Adiponectin Attenuates the Inflammation in Atopic Dermatitis-Like Reconstructed Human Epidermis.

BackgroundAtopic dermatitis (AD) is a chronic disorder, with a vicious cycle of repetitive inflammation and deterioration of the epidermal barrier function. Adiponectin, an adipokine, has anti-inflammatory effects on various metabolic and inflammatory disorders. Recently, its level was found to be reduced in serum and tissue samples from AD patients.ObjectiveWe aimed to investigate the effects of adiponectin on epidermal inflammation and barrier structures in AD skin.MethodsA three-dimensional in vitro epidermal equivalent model mimicking AD was obtained by adding an inflammatory substance cocktail to normal human epidermal equivalents (HEEs). The expression of epidermal differentiation markers, primary inflammatory mediators, and lipid biosynthetic enzymes was compared between adiponectintreated AD-HEEs, untreated control AD-HEEs, and normal HEEs.ResultsAdiponectin co-treatment 1) inhibited the increase in mRNA expression of major inflammatory mediators (carbonic anhydrase II, neuron-specific NEL-like protein 2, thymic stromal lymphopoietin, interleukin-8, tumor necrosis factor-alpha, and human beta-defensin-2) from keratinocytes in AD-inflammatory HEEs, 2) enhanced the expression of lipid biosynthetic enzymes (fatty acid synthase, HMG CoA reductase, and serine-palmitoyl transferase), and 3) promoted the expression of differentiation factors, especially filaggrin. We also found that the expression of adiponectin receptor-1 and -2 decreased in the epidermis of chronic AD lesion.ConclusionActivation of the adiponectin pathway is expected to enhance epidermal differentiation and barrier function as well as attenuate inflammatory response to AD as a therapeutic approach.

From stem cells protection to skin microbiota balance: Orobanche rapum extract, a new natural strategy.

SummaryBackgroundHealthy skin is a delicate balance between skin renewal and microbiota homeostasis, and its imbalance promotes premature aging and dermatological disorders. Skin stem cells are key actors in this process but their sensitivity to aging and external stressors such as UV reduces the skin renewal power. The skin microbiota has been recently described as active in the healthy skin, and its imbalance could trigger some disorders.AimsWe hypothesized that reactivation of stem cells and maintenance of microbiota could be a disruptive strategy for younger and healthier skin. We thus developed a new plant extract that restores the entire skin renewal process by sequential activation from stem cells stimulation to microbiota protection.MethodsWe studied stem cells comportment in the presence of Orobanche rapum extract by survivin immunocytochemistry and caspases 3 and 9 dosages. We also analyzed epidermal differentiation markers by immunohistochemistry and lipids organization by GC/MS At the clinical level, we investigated the impact of O. rapum extract on microbiota and on skin aspect.ResultsWe demonstrated an active protection of skin stem cells through the maintenance of their clone‐forming capacity and resistance to UV through the overexpression of survivin coupled to caspases inhibition. Furthermore, we showed the restoration of epidermal differentiation markers and ceramide biosynthesis favorable to orthorhombic organization. Clinical studies, including microbiota analysis, showed an active skin surface renewal coupled with microbiota protection.ConclusionWe evidenced that our active ingredient is able to stimulate skin rejuvenation while protecting the cutaneous microbiota, creating healthier skin and thereby beauty.

Conditional ablation of p130Cas/BCAR1 adaptor protein impairs epidermal homeostasis by altering cell adhesion and differentiation

Backgroundp130 Crk-associated substrate (p130CAS; also known as BCAR1) is a scaffold protein that modulates many essential cellular processes such as cell adhesion, proliferation, survival, cell migration, and intracellular signaling. p130Cas has been shown to be highly expressed in a variety of human cancers of epithelial origin. However, few data are available regarding the role of p130Cas during normal epithelial development and homeostasis.MethodsTo this end, we have generated a genetically modified mouse in which p130Cas protein was specifically ablated in the epidermal tissue.ResultsBy using this murine model, we show that p130Cas loss results in increased cell proliferation and reduction of cell adhesion to extracellular matrix. In addition, epidermal deletion of p130Cas protein leads to premature expression of “late” epidermal differentiation markers, altered membrane E-cadherin/catenin proteins localization and aberrant tyrosine phosphorylation of E-cadherin/catenin complexes. Interestingly, these alterations in adhesive properties in absence of p130Cas correlate with abnormalities in progenitor cells balance resulting in the amplification of a more committed cell population.ConclusionAltogether, these results provide evidence that p130Cas is an important regulator of epidermal cell fate and homeostasis.

Mutations in PERP Cause Dominant and Recessive Keratoderma

Investigation of genetic determinants of Mendelian skin disorders has substantially advanced understanding of epidermal biology. Here we show that mutations in PERP, encoding a crucial component of desmosomes, cause both dominant and recessive human keratoderma. Heterozygosity for a C-terminal truncation, which produces a protein that appears to be unstably incorporated into desmosomes, causes Olmsted syndrome with severe periorificial and palmoplantar keratoderma in multiple unrelated kindreds. Homozygosity for an N-terminal truncation ablates expression and causes widespread erythrokeratoderma, with expansion of epidermal differentiation markers. Both exhibit epidermal hyperproliferation, immature desmosomes lacking a dense midline observed via electron microscopy, and impaired intercellular adhesion upon mechanical stress. Localization of other desmosomal components appears normal, which is in contrast to other conditions caused by mutations in genes encoding desmosomal proteins. These discoveries highlight the essential role of PERP in human desmosomes and epidermal homeostasis and further expand the heterogeneous spectrum of inherited keratinization disorders.

Characterization of Epidermal Lipoxygenase Expression in Normal Human Skin and Tissue-Engineered Skin Substitutes.

Lipoxygenases (LOXs) are enzymes likely to be involved in corneocyte lipid envelope formation and skin barrier function. In humans, mutations in epidermis-type lipoxygenase 3 ( eLOX-3) and 12R-lipoxygenase ( 12R-LOX) genes are associated with autosomal recessive congenital ichthyosis (ARCI), whereas deletion of these genes in mice causes epidermal defects. LOXs also represent a matter of interest in psoriasis as well as in cancer research. However, their expression as well as the exact role of these enzymes in normal human skin have not been fully described. Our goal was to characterize the expression of epidermal LOXs in both normal human skin and Tissue-Engineered Skin Substitutes (TESS) and to consider TESS as a potential model for LOX functional studies. Staining for epidermal differentiation markers and LOXs was performed, in parallel, on normal human skin and TESS. Our results showed similar expression profiles in TESS when compared with native skin for e-LOX3, 12R-LOX, 12S-lipoxygenase (12S-LOX), and 15-lipoxygenase 2 (15-LOX-2) but not for 15-lipoxygenase 1 (15-LOX-1). Because of their appropriate epidermal differentiation and LOX expression, TESS represent an alternative model for future studies on LOX function.

Abstract 3082: Epidermal-specific overexpression of T-cell protein tyrosine phosphatase in mouse attenuates chemically-induced skin carcinogenesis

Abstract Tyrosine phosphorylation signaling, which is regulated by the counter-activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is critical in maintaining cellular homeostasis. The aberrant increase of tyrosine phosphorylation by mutation and/or overexpression of PTKs can contribute to skin carcinogenesis. While PTKs have been extensively studied in skin carcinogenesis, PTPs have not been studied due to their inactivation by environmental toxicants. Our recent studies showed that T-cell protein tyrosine phosphatase (TC-PTP; encoded by Ptpn2) deficiency in mouse epidermis significantly increased skin tumor formation, demonstrating TC-PTP deficiency predisposes mice to skin carcinogenesis. To further examine the tumor suppressive role of TC-PTP in skin carcinogenesis, we generated epidermal-specific TCPTP-overexpressing (K5HA.Ptpn2) transgenic mice. TC-PTP overexpression led to sensitization to tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA)-induced apoptosis both in vivo epidermis and in vitro keratinocytes. TC-PTP overexpression in epidermis significantly reduced epidermal thickness and hyperproliferation compared to wild-type control following treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, TC-PTP overexpression significantly induced epidermal differentiation following TPA treatment as evidenced by the increased expression of epidermal differentiation markers loricrin, involucrin, keratin 10, and transglutaminase 5 in comparison to control. Two-stage skin carcinogenesis analysis using a DMBA/TPA regimen clearly revealed that K5HA.Ptpn2 mice exhibited delayed tumor development and significantly reduced tumor numbers compared to control mice. Taken together, our results suggest that TC-PTP is a potential therapeutic target for the prevention of skin cancer given its ability to promote epidermal apoptosis and differentiation and inhibit epidermal proliferation. Citation Format: Mihwa Kim, Liza D. Morales, Iksoon Jang, Andrew Tsin, Dae Joon Kim. Epidermal-specific overexpression of T-cell protein tyrosine phosphatase in mouse attenuates chemically-induced skin carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3082.

MiR‑339‑5p negatively regulates loureirin A‑induced hair follicle stem cell differentiation by targeting DLX5.

Our previous study indicated that loureirin A induces hair follicle stem cell (HFSC) differentiation through Wnt/β-catenin signaling pathway activation. However, if and how microRNAs (miRNAs/miRs) modulate loureirin A-induced differentiation remains to be elucidated. In the present study, HFSCs were separated from the vibrissae of rats and identified by CD34 and keratin, type 1 cytoskeletal (K)15 expression. Microarray-based miRNA profiling analysis revealed that miR-339-5p was downregulated in loureirin A-induced HFSC differentiation. miR-339-5p overexpression by transfection with miR-339-5p mimics markedly inhibited the expression of K10 and involucrin, which are markers of epidermal differentiation, whereas inhibition of miR-339-5p by miR-339-5p inhibitor transfection promoted the expression of K10 and involucrin. These results suggest that miR-339-5p is a negative regulator of HFSC differentiation following induction by loureirin A. These findings were confirmed by a luciferase assay. Homeobox protein DLX-5 (DLX5) was identified as a direct target of miR-339-5p. Furthermore, it was demonstrated that miR-339-5p inhibited DLX5. Overexpression of miR-339-5p by mimic transfection significantly inhibited protein Wnt-3a (Wnt3a) expression, while inhibition of miR-339-5p by inhibitor transfection significantly increased the expression of Wnt3a. Furthermore, small interfering RNA targeting DLX5 was transfected into HFSCs, and western blot analysis revealed that Wnt3a, involucrin and K10 expression was significantly downregulated. Taken together, these results suggest that miR-339-5p negatively regulated loureirin A-induced HFSC differentiation by targeting DLX5, resulting in Wnt/β-catenin signaling pathway inhibition. This may provide a possible therapeutic target for skin repair and regeneration.

A novel, topical, nonsteroidal, TRPV1 antagonist, PAC-14028 cream improves skin barrier function and exerts anti-inflammatory action through modulating epidermal differentiation markers and suppressing Th2 cytokines in atopic dermatitis

BackgroundAlthough it is established that epidermal barrier disturbance and immune dysfunction resulting in IgE sensitization are critical factors in the development of cutaneous inflammation, the pathogenesis and targeted therapy of atopic dermatitis (AD)-specific pathways have still been unknown. ObjectiveTaking into account the fact that Th2 cytokines in AD have both unique and overlapping functions including increased epidermal thickening, inflammation, and decreased expressing of the barrier proteins keratinocyte differentiation, we sought to clarify our hypothesis that TRPV1 antagonist plays a critical role in skin barrier function and can be a therapeutic target for AD. MethodsAD-like dermatitis was induced in hairless mice by repeated oxazolone (Ox) challenges to hairless mice. The functional studies concerning skin barrier function, anti-inflammatory action, and molecular mechanism by TRPV1 antagonism were conducted by histopathological assays, ELISA, qPCR, western blotting, and skin blood flow measurement. ResultsTopically administered TRPV1 antagonist, PAC-14028 (Asivatrep: C21H22F5N3O3S), improved AD-like dermatitis and skin barrier functions, and restored the expression of epidermal differentiation markers. In addition, the PAC-14028 cream significantly inhibited cutaneous inflammation by decreasing the expression of serum IgE, and the epidermal expression of IL-4, and IL-13 in Ox-AD mice. These results may provide a novel insight into the molecular mechanism of PAC-14028 cream involved in anti-inflammatory effects and skin barrier functions by suppressing the multiple signaling pathways including IL-4/-13-mediated activation of JAK/STAT, TRPV1, and neuropeptides. ConclusionPAC-14028 cream can be a potential therapeutic tool for the treatment of chronic inflammation and disrupted barrier function in patients with AD.

756 Generalized ichthyotic peeling skin syndrome due to FLG2 mutations

Peeling skin syndrome is a group of recessive skin fragility genodermatoses with superficial peeling of the skin as the characteristic clinical feature, that may be accompanied by ichthyosis and/or inflammation. Here we present two siblings with non-syndromic generalized ichthyotic peeling skin syndrome and a homozygous nonsense mutation in FLG2, a novel peeling skin syndrome gene, resulting in loss of functional filaggrin-2. The level of separation was in the lowest part of the stratum corneum. Desmosomal proteins of the upper epidermal layers, keratin 2 and differentiation markers were reduced in patient skin and corneocytes showed abnormal morphology. These results suggest a crucial role for filaggrin-2 in cell-cell contact homeostasis in the stratum corneum and granulosum, corneocyte stress resilience, and normal cornification.

Blockade of TNF receptor superfamily 1 (TNFR1)–dependent and TNFR1-independent cell death is crucial for normal epidermal differentiation

A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD-like ICE)-inhibitory protein plays a crucial role in prevention of keratinocytes from TNF-α-dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE-inhibitory protein in TNF-α-independent cell death remains unclear. We investigated contribution of TNF-α-dependent and TNF-α-independent signals to the development of dermatitis in epidermis-specific Cflar-deficient (CflarE-KO) mice. We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of CflarE-KO;Tnfrsf1a+/- and CflarE-KO;Tnfrsf1a-/- mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand to block TNF-α-independent cell death of CflarE-KO;Tnfrsf1a-/- mice. CflarE-KO;Tnfrsf1a-/- mice were born but experienced severe dermatitis and succumbed soon after birth. CflarE-KO;Tnfrsf1a+/- mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from CflarE-KO;Tnfrsf1a-/- mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand substantially prolonged survival of CflarE-KO;Tnfrsf1a-/- mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of CflarE-KO;Tnfrsf1a-/- mice. Treatment of primary keratinocytes with IL-6 and, to a lesser extent, IL-17A suppressed expression of epidermal differentiation markers. TNF receptor superfamily 1 (TNFR1)-dependent or TNFR1-independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1-dependent and TNFR1-independent cell death might be an alternative strategy to treat skin diseases when treatment with anti-TNF-α antibody alone is not sufficient.

Malassezia pachydermatis up-regulates AhR related CYP1A1 gene and epidermal barrier markers in human keratinocytes.

Cytochrome P450 CYP1A1 and CYP1B1 enzymes are regulated by the aryl hydrocarbon receptor (AhR), a transcription factor activated by a variety of ligands among which Malassezia metabolites. In this study, we analyzed the modulation of CYP1A1, CYP1B1, and AhR in human keratinocytes infected with different strains of Malassezia pachydermatis, as well as the upregulation of some genes involved in the epidermal homeostasis. We demonstrated that all the strains induced AhR activation and its nuclear translocation in HaCaT cells infected for 24h, compared to untreated cells. The expression of CYP1A1 and CYP1B1, prototypical markers of the AhR signaling pathway, were upregulated with the level of CYP1A1 mRNA approximately 100-fold greater than that for CYP1B1. Filaggrin, involucrin, and TGaseI, proteins involved in epidermal differentiation, were all modulated by Malassezia pachydermatis strains, with the strongest induction observed for filaggrin. By contrast, quinone oxidoreductase 1 (NQO1), which is part of the antioxidant defense system involved in detoxification, was not modulated in our experimental model. In conclusions, our findings suggest that Malassezia pachydermatis infection of human keratinocytes induces activation of the AhR, and increases the expression of its responsive genes and markers of epidermal differentiation, paving the way for occurrence/exacerbation of pathological skin conditions.

Phosphodiesterase 4B plays a role in benzophenone-3-induced phototoxicity in normal human keratinocytes

Benzophenone-3 (BP-3), which is extensively used in organic sunscreen, has phototoxic potential in human skin. Phosphodiesterase 4B (PDE4B) has a well-established role in inflammatory responses in immune cells. Currently, it is unknown if PDE4B is associated with BP-3-induced phototoxicity in normal human keratinocytes (NHKs). We found that BP-3 significantly increased PDE4B expression in ultraviolet B (UVB)-irradiated NHKs. Notably, BP-8, a sunscreen agent that shares the 2-hydroxy-4-methoxyphenyl methanone moiety with BP-3, also upregulated PDE4B expression in NHKs. Upon UVB irradiation, BP-3 upregulated the expression of pro-inflammatory factors, such as prostaglandin endoperoxide synthase 2, tumor necrosis factor α, interleukin 8, and S100A7, and downregulated the level of cornified envelope associated proteins, which are important in the development of the epidermal permeability barrier. The additive effects of UVB-activated BP-3 on the expression of both pro-inflammatory mediators and cornified envelope associated proteins were antagonized by treatment with the PDE4 inhibitor rolipram. The BP-3 and UVB co-stimulation-induced PDE4B upregulation and its association with the upregulation of pro-inflammatory mediators and the downregulation of epidermal differentiation markers were confirmed in a reconstituted three dimensional human epidermis model. Therefore, PDE4B has a role in the mechanism of BP-3-induced phototoxicity.

251 Morphogenetic events in hair follicle heterotypic spheroids

The long-term goal of the study is to generate human hair follicle (HF) germ-like structures in vitro. In this part of the work, we examined aggregates assembled using dermal papilla cells (DPCs) and keratinocytes (KCs) suspensions (mixed aggregates) and DPCs spheroids covered by KCs (coated aggregates) cultured in hanging drop model. RT qPCR analysis revealed upregulation of HF markers (Lef1, P cadherin, EdaR, CK75 and Wnt5a) in mixed aggregates in contradiction to coated ones. Lef1 and P cadherin positive KCs were identified at DPCs and KCs borderline. Any morphogenetic events had not been observed in coated aggregates. Downregulation of DPCs hair inducing abilities trough the passaging decreased the size of mixed aggregates and the number of proliferative cells within. Accordingly, DPCs and KCs interplay is essential for proper self-assembly initiation while DPCs hair inducing abilities controls the amount of cells involved in this process. Mixed aggregates structure and morphology had been investigated. Versican positive DPCs formed round-shaped structures; KCs generated multilayered trabeculae embedded into DPCs spheroids. CK5 and CK6 positive KCs were found in an external layer of trabeculae, surrounding the epithelial compartment and adjacent to DPCs. We did not found any markers of epidermal differentiation including loricrin, CK 1, and CK10. DPCs and KCs were positive for CD44, hyaluronic acid (HA) receptor. We showed that addition of 0.5 mg/ml HA to cell suspension prior to aggregate formation increased the size of aggregates and the number of proliferative cells. However HA did not maintain DPCs specific markers during 2D cultivation. Therefore HA influences size of aggregates not by upregulation of DPCs hair inducing abilities. CD44 staining detected microvillus-type plasma membrane protrusions on the DPCs in mixed aggregates. Microvilli have been shown to be involved in HA metabolism: synthesis and digestion. Accordingly, the results demonstrated the considerable impact of HA in aggregate formation, self-organization and HF development.

Abstract LB-033: The transcriptional landscape of p53 signaling pathway

Abstract Although recent cancer genomics studies have identified a large number of genes that were mutated in human cancers, p53 remains as the most frequently mutated gene. To further elucidate the p53-signalling network, we performed transcriptome analysis on 24 tissues in p53+/+ or p53-/- mice after whole-body X-ray irradiation. Here we found transactivation of a total of 3,551 genes in one or more of the 24 tissues only in p53+/+ mice, while 2,576 genes were downregulated. p53 mRNA expression level in each tissue was significantly associated with the number of genes upregulated by irradiation. Annotation using TCGA (The Cancer Genome Atlas) database revealed that p53 negatively regulated mRNA expression of several cancer therapeutic targets or pathways such as BTK, SYK, and CTLA4 in breast cancer tissues. In addition, stomach exhibited the induction of Krt6, Krt16, and Krt17 as well as loricrin, an epidermal differentiation marker, after the X-ray irradiation only in p53+/+ mice, implying a novel mechanism to protect damaged tissues by rapid induction of differentiation. Our comprehensive transcriptome analysis elucidated tissue specific roles of p53 and its signalling networks in DNA-damage response that will enhance our understanding of cancer biology. Citation Format: Koichi Matsuda, Chizu Tanikawa. The transcriptional landscape of p53 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-033. doi:10.1158/1538-7445.AM2017-LB-033

Abstract LB-136: Characterization of residual Basal Cell Carcinoma after vismodegib treatment

Abstract We have generated a mouse model of Superficial Basal Cell Carcinoma (BCC) to study the effect of the Smoothened inhibitor vismodegib in vivo. Despite the potency of vimodegib in blocking Hedgehog (Hh) signaling and efficacy in treatment of BCC, residual disease persists in the mouse model. To better understand the Biology of BCC and the potential mechanisms maintaining BCC during treatment, we profiled isolated untreated as well as residual tumors. We found that treated tumors change their transcriptional program to resemble the cells of skin and hair follicle structures like the Interfollicular Epidermis (IFE) and Isthmus, both of which harbor stem cell compartments. Consistent with these findings, residual disease lacks expression of epidermal differentiation markers and initiates growth upon cessation of treatment. While Wnt signaling is clearly required for BCC initiation in mouse models, we show that Wnt signaling is down-regulated in untreated full-blown disease and subsequently strongly induced in the treated tumors. We hypothesize that re-initiation of the Wnt pathway maintains BCC and study the effect of blocking both Wnt and Hh pathways with combined treatment of vismodegib and Wnt inhibitors. Citation Format: Brian Biehs, Gerrit J. Dijkgraaf, Bruno Alicke, Franklin Peale, Stephen E. Gould, Frederic J. de Sauvage. Characterization of residual Basal Cell Carcinoma after vismodegib treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-136. doi:10.1158/1538-7445.AM2017-LB-136