Impact of Skin Exposure to Benzo[a]pyrene in Rat Model: Insights into Epidermal Cell Function and Draining Lymph Node Cell Response.

The skin is a direct target of the air pollutant benzo[a]pyrene (BaP). While its carcinogenic qualities are well-studied, the immunotoxicity of BaP after dermal exposure is less understood. This study examines the immunomodulatory effects of a 10-day epicutaneous BaP application, in environmentally/occupationally relevant doses, by analyzing ex vivo skin immune response (skin explant, epidermal cells and draining lymph node/DLN cell activity), alongside the skin's reaction to sensitization with experimental hapten dinitrochlorobenzene (DNCB). The results show that BaP application disrupts the structure of the epidermal layer and promotes immune cell infiltration in the dermis. BaP exposure led to oxidative stress in epidermal cells, characterized by decreased reduced glutathione and increased AHR and Cyp1A1 expression. Production and gene expression of proinflammatory cytokines (TNF, IL-1ß) by epidermal cells decreased, while IL-10 response increased. Decreased spontaneous production of IFN-γ and IL-17, along with unchanged IL-10, was observed in DLC cells, whereas ConA-stimulated production of these cytokines was elevated. Local immunosuppression caused by BaP application seems to reduce the skin's response to an additional stimulus, evidenced by decreased effector activity of DLN cells three days after sensitization with DNCB. These findings provide new insight into the immunomodulatory effects and health risks associated with skin exposure to BaP.

Morroniside promotes skin wound re-epithelialization by facilitating epidermal stem cell proliferation through GLP-1R-mediated upregulation of ß-catenin expression.

Epidermal stem cells (EpSCs) play a vital role in skin wound healing through re-epithelialization. Identifying chemicals that can promote EpSC proliferation is helpful for treating skin wounds. This study investigates the effect of morroniside on cutaneous wound healing in mice and explores the underlying mechanisms. Application of 10‒50 µg/mL of morroniside to the skin wound promotes wound healing in mice. In vitro studies demonstrate that morroniside stimulates the proliferation of mouse and human EpSCs in a time- and dose-dependent manner. Mechanistic studies reveal that morroniside promotes the proliferation of EpSCs by facilitating the cell cycle transition from the G1 to S phase. Morroniside increases the expression of ß-catenin via the glucagon-like peptide-1 receptor (GLP-1R)-mediated PKA, PKA/PI3K/AKT and PKA/ERK signaling pathways, resulting in an increase in cyclin D1 and cyclin E1 expression, either directly or by upregulating c-Myc expression. This process ultimately leads to EpSC proliferation. Administration of morroniside to mouse skin wounds increases the phosphorylation of AKT and ERK, the expressions of ß-catenin, c-Myc, cyclin D1, and cyclin E1, as well as the proliferation of EpSCs, in periwound skin tissue, and accelerates wound re-epithelialization. These effects of morroniside are mediated by the GLP-1R. Overall, these results indicate that morroniside promotes skin wound healing by stimulating the proliferation of EpSCs via increasing ß-catenin expression and subsequently upregulating c-Myc, cyclin D1, and cyclin E1 expressions through GLP-1R signaling pathways. Morroniside has clinical potential for treating skin wounds.

Comparative Analysis of Olive-Derived Phenolic Compounds' Pro-Melanogenesis Effects on B16F10 Cells and Epidermal Human Melanocytes.

Olive leaf contains plenty of phenolic compounds, among which oleuropein (OP) is the main component and belongs to the group of secoiridoids. Additionally, phenolic compounds such as oleocanthal (OL) and oleacein (OC), which share a structural similarity with OP and two aldehyde groups, are also present in olive leaves. These compounds have been studied for several health benefits, such as anti-cancer and antioxidant effects. However, their impact on the skin remains unknown. Therefore, this study aims to compare the effects of these three compounds on melanogenesis using B16F10 cells and human epidermal cells. Thousands of gene expressions were measured by global gene expression profiling with B16F10 cells. We found that glutaraldehyde compounds derived from olive leaves have a potential effect on the activation of the melanogenesis pathway and inducing differentiation in B16F10 cells. Accordingly, the pro-melanogenesis effect was investigated by means of melanin quantification, mRNA, and protein expression using human epidermal melanocytes (HEM). This study suggests that secoiridoid and its derivates have an impact on skin protection by promoting melanin production in both human and mouse cell lines.

Effect of estrogen/progesterone ratio on the differentiation and the barrier function of epidermal keratinocyte and three-dimensional cultured human epidermis.

AIMS: 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. MATERIALS AND 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. KEY FINDINGS: 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. SIGNIFICANCE: 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.

Triphenyl phosphate-induced pericardial edema is associated with elevated epidermal ionocytes within zebrafish embryos.

Triphenyl phosphate (TPHP) is an organophosphate ester-based plasticizer and flame retardant. The objective of this study was to identify the potential role of epidermal ionocytes in mediating TPHP-induced pericardial edema within zebrafish embryos. Exposure to TPHP from 24 to 72 h post fertilization (hpf) resulted in a significant increase in pericardial edema and the number of ionocytes at 72 hpf relative to time-matched embryos treated with vehicle. In addition, co-exposure of embryos to mannitol (an osmotic diuretic) blocked TPHP-induced pericardial edema and effects on ionocyte abundance. However, knockdown of ATPase1a1.4 - an abundant Na+/K+-ATPase localized to epidermal ionocytes - mitigated TPHP-induced effects on ionocyte abundance but not pericardial edema, whereas co-exposure of embryos to ouabain - a Na+/K+-ATPase inhibitor - enhanced TPHP-induced pericardial edema but not ionocyte abundance. Overall, our findings suggest that TPHP may have multiple mechanisms of toxicity leading to an increase in ionocyte abundance and pericardial edema within developing zebrafish embryos.

Amphiregulin promotes hair regeneration of skin-derived precursors via the PI3K and MAPK pathways.

OBJECTIVES: There are significant clinical challenges associated with alopecia treatment, including poor efficiency of related drugs and insufficient hair follicles (HFs) for transplantation. Skin-derived precursors (SKPs) exhibit great potential as stem cell-based therapies for hair regeneration; however, the proliferation and hair-inducing capacity of SKPs gradually decrease during culturing. MATERIALS AND METHODS: We describe a 3D co-culture system accompanied by kyoto encyclopaedia of genes and genomes and gene ontology enrichment analyses to determine the key factors and pathways that enhance SKP stemness and verified using alkaline phosphatase assays, Ki-67 staining, HF reconstitution, Western blot and immunofluorescence staining. The upregulated genes were confirmed utilizing corresponding recombinant protein or small-interfering RNA silencing in vitro, as well as the evaluation of telogen-to-anagen transition and HF reconstitution in vivo. RESULTS: The 3D co-culture system revealed that epidermal stem cells and adipose-derived stem cells enhanced SKP proliferation and HF regeneration capacity by amphiregulin (AREG), with the promoted stemness allowing SKPs to gain an earlier telogen-to-anagen transition and high-efficiency HF reconstitution. By contrast, inhibitors of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways downstream of AREG signalling resulted in diametrically opposite activities. CONCLUSIONS: By exploiting a 3D co-culture model, we determined that AREG promoted SKP stemness by enhancing both proliferation and hair-inducing capacity through the PI3K and MAPK pathways. These findings suggest AREG therapy as a potentially promising approach for treating alopecia.

Exploring the dermotoxicity of the mycotoxin deoxynivalenol: combined morphologic and proteomic profiling of human epidermal cells reveals alteration of lipid biosynthesis machinery and membrane structural integrity relevant for skin barrier function.

Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most prevalent mycotoxins worldwide. Crop infestation results not only in food and feed contamination, but also in direct dermal exposure, especially during harvest and food processing. To investigate the potential dermotoxicity of DON, epidermoid squamous cell carcinoma cells A431 were compared to primary human neonatal keratinocytes (HEKn) cells via proteome/phosphoproteome profiling. In A431 cells, 10 µM DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements (OXPHOS regulation) and transport proteins (TOMM22; TOMM40; TOMM70A). Mitochondrial impairment was reflected in altered metabolic competence, apparently combined with interference of the lipid biosynthesis machinery. Functional effects on the cell membrane were confirmed by live cell imaging and membrane fluidity assays (0.1-10 µM DON). Moreover, a common denominator for both A431 and HEKn cells was a significant downregulation of the squalene synthase (FDFT1). In sum, proteome alterations could be traced back to the transcription factor Klf4, a crucial regulator of skin barrier function. Overall, these results describe decisive molecular events sustaining the capability of DON to impair skin barrier function. Proteome data generated in the study are fully accessible via ProteomeXchange with the accession numbers PXD011474 and PXD013613.

Effect of Lactic Fermentation Products on Human Epidermal Cell Differentiation, Ceramide Content, and Amino Acid Production.

INTRODUCTION: Lactic fermentation products (LFPs) are thought to affect "good" bacteria in the gut. We previously reported that oral administration of LFPs has beneficial therapeutic effects in a mouse model of atopic dermatitis. However, it is unclear how LFPs affect human epidermal cell differentiation, ceramide (Cer), and amino acid production. OBJECTIVE: The aim of this study was to determine the effects of LFPs on epidermal cell differentiation, by assessing amino acid and Cer production. METHODS: A 3-dimensional cultured human epidermis model and normal human epidermal keratinocytes were used. Cytotoxicity tests were performed using alamar Blue. Transepidermal water loss (TEWL) was used as an index to assess barrier function. Keratin 1 (K1), keratin 5 (K5), keratin 10 (K10), involucrin (INV), calpain 1, and transglutaminase (TGase) (markers of differentiation) and profilaggrin (proFLG) and bleomycin hydrolase (amino acid synthesis-related genes) expression levels were quantified by RT-PCR. In addition, TGase protein levels were measured by Western blotting. The intercellular lipid content of the stratum corneum was measured by high-performance thin-layer chromatography. Amino acids were quantified using an amino acid analyzer. Finally, bound water content in the stratum corneum was measured by differential scanning calorimetry. RESULTS: Cell viability did not change, but TEWL was significantly decreased in the cells treated with LFPs compared with the control cells. Treatment with LFPs significantly increased expression of the late-differentiation markers INV and TGase at the RNA level. Furthermore, TGase protein expression was significantly increased by treatment with LFPs. Treating a 3-dimensional cultured epidermis model with LFPs significantly increased the intercellular lipid content of the stratum corneum and production of the amino acid arginine (Arg). The amount of bound water in the stratum corneum was increased significantly in the LFP application group. CONCLUSION: Treatment with LFPs promotes human epidermal cell differentiation and increases the intercellular content of the free fatty acid, Chol, Cer [NS], Cer [AS], and Cer [AP]. This may result in improved skin barrier function. The increased amount of Arg observed in keratinocytes may help improve water retention.

A Predictive Self-Organizing Multicellular Computational Model of Infant Skin Permeability to Topically Applied Substances.

Computational models of skin permeability are typically based on assumptions of fixed geometry and homogeneity of the whole epidermis or of epidermal strata and are often limited to adult skin. Infant skin differs quantitatively from that of the adult in its structure and its functional properties, including its barrier function to permeation. To address this problem, we developed a self-organizing multicellular epidermis model of barrier formation with realistic cell morphology. By modulating the parameters relating to cell turnover reflecting those in adult or infant epidermis, we were able to generate accordingly two distinct models. Emerging properties of these models reflect the corresponding experimentally measured values of epidermal and stratum corneum thickness. Diffusion of an externally applied substance (e.g., caffeine) was simulated by a molecular exchange between the model agents, defined by the individual cells and their surrounding extracellular space. By adjusting the surface concentration and the intercellular exchange rate, the model can recapitulate experimental permeability data after topical exposure. By applying these parameters to an infant model, we were able to predict the caffeine concentration profile in infant skin, closely matching experimental results. This work paves the way for a better understanding of skin physiology and function during the first years of life.

Skin irritation and inhalation toxicity of biocides evaluated with reconstructed human epidermis and airway models.

Biocides are widely used in household products. Humans are exposed to biocides through dermal, inhalational, and oral routes. However, information on the dermal and inhalational toxicity of biocides is limited. We evaluated the effects of biocides on the skin and airways using the reconstructed human epidermis model KeraSkin™ and the airway model SoluAirway™. We determined the irritancy of 11 commonly used biocides (1,2-benzisothiazol-3(2H)-one [BIT], 2-phenoxyethanol [PE], zinc pyrithione, 2-bromo-2-nitropropane-1,3-diol, 3-iodoprop-2-ynyl N-butylcarbamate [IPBC], 2-octyl-1,2-thiazol-3-one, 2,2-dibromo-2-cyanoacetamide, 4-chloro-3-methylphenol [CC], 2-phenylphenol, deltamethrin, and 4,5-dichloro-2-octyl-1,2-thiazol-3-one) in the KeraSkin™ and SoluAirway™ by viability and histological examinations. BIT and CC were found to cause skin irritation at the approved concentrations or at the concentration close to approved limit while the others were non-irritants within the approved concentration. These results were confirmed via histology, wherein skin irritants induced erosion, vacuolation, and necrosis of the tissue. In the SoluAirway™, most of the biocides decreased cell viability even within the approved limits, except for PE, IPBC, and deltamethrin, suggesting that the airway may be more vulnerable to biocides than the skin. Taken together, our result indicates that some biocides can induce toxicity in skin and airway. Further studies on the dermal and inhalational toxicity of biocides are warranted.

Isolindleyin exerts anti-melanogenic effects in human epidermal melanocytes via direct binding to tyrosinase.

To overcome dermatological concerns causing abnormally excessive melanin synthesis, highly effective and safe skin depigmentation compounds have been identified in the cosmetic and pharmaceutical industries. Among several methods used to achieve skin depigmentation, inhibition of tyrosinase is one of the most effective, since tyrosinase is a crucial enzyme in melanogenesis. Herein, isolindleyin, a novel inhibitor of human tyrosinase, was introduced and evaluated for its anti-melanogenic effects in human epidermal melanocytes. The results revealed that isolindleyin was directly bound to tyrosinase and it suppressed melanin synthesis. The binding mode between isolindleyin and the active sites of human tyrosinase was investigated using computational molecular docking at the atomic level. Isolindleyin binding was found to be stabilized by hydrophobic interactions between His 367 and Val 377 and by hydrogen bonds between Ser 380 and Asn 364. The results of this study revealed the anti-melanogenic effects of isolindleyin that could contribute toward overcoming dermatological concerns that cause abnormally excessive melanin synthesis.

JAK1/2 inhibition impairs the development and function of inflammatory dendritic epidermal cells in atopic dermatitis.

BACKGROUND: Janus kinase (JAK) inhibitors are a new class of therapeutic compounds for dermatological diseases. In atopic dermatitis (AD), data of clinical phase III trials show rapid improvement of pruritus and significant reduction of inflammation within the first weeks with a favorable safety profile. However, their mode of action in AD is not fully understood. OBJECTIVES: In our study, we investigate the effect of different JAK inhibitors on cell differentiation, phenotype, and function of inflammatory dendritic epidermal cells (IDECs). METHODS: We analyzed the JAK expression in IDEC from ex vivo skin and in vitro generated IDECs using flow cytometry and PCR. Further, we studied in vitro the effect of different JAK inhibitors on IDEC cell differentiation, phenotype, and maturation. RESULTS: IDECs express JAK1 and JAK2 ex vivo and in vitro. We found that JAK1 and JAK2 were upregulated during the differentiation from monocytes to IDECs. Conversely, JAK2 inhibition by ruxolitinib (JAK1/2 inhibitor) or BMS-911543 (JAK2 inhibitor) abrogated the differentiation from monocytes into IDECs. Differentiated IDECs can redifferentiate into a more monocyte-like phenotype in the presence of ruxolitinib or BMS-911543. Furthermore, we showed that concomitant inhibition of JAK1/2 rather than blocking JAK1 or JAK2 alone, impaired maturation and the release of proinflammatory cytokines on lipopolysaccharide stimulation. CONCLUSIONS: Our results suggest that inhibition of JAK1/2 impairs IDEC differentiation and function. We provide new insight into the mode of action of JAK inhibitors in AD and highlight the role of JAK1/2 inhibitors for the treatment of patients with AD.

Coptis chinensis Franch Directly Inhibits Proteolytic Activation of Kallikrein 5 and Cathelicidin Associated with Rosacea in Epidermal Keratinocytes.

Rosacea is a common and chronic inflammatory skin disease that is characterized by dysfunction of the immune and vascular system. The excessive production and activation of kallikerin 5 (KLK5) and cathelicidin have been implicated in the pathogenesis of rosacea. Coptis chinensis Franch (CC) has been used as a medicinal herb in traditional oriental medicine. However, little is known about the efficacy and mechanism of action of CC in rosacea. In this study, we evaluate the effect of CC and its molecular mechanism on rosacea in human epidermal keratinocytes. CC has the capacity to downregulate the expression of KLK5 and cathelicidin, and also inhibits KLK5 protease activity, which leads to reduced processing of inactive cathelicidin into active LL-37. It was determined that CC ameliorates the expression of pro-inflammatory cytokines through the inhibition of LL-37 processing. In addition, it was confirmed that chitin, an exoskeleton of Demodex mites, mediates an immune response through TLR2 activation, and CC inhibits TLR2 expression and downstream signal transduction. Furthermore, CC was shown to inhibit the proliferation of human microvascular endothelial cells induced by LL-37, the cause of erythematous rosacea. These results demonstrate that CC improved rosacea by regulating the immune response and angiogenesis, and revealed its mechanism of action, indicating that CC may be a useful therapeutic agent for rosacea.

Agonism of Gpr40 Protects the Capacities of Epidermal Stem Cells (ESCs) Against Ultraviolet-B (UV-B).

INTRODUCTION: Skin damage due to overexposure to ultraviolet B (UV-B) radiation can lead to the development of cancers and reduce the skin's functionality as a vital protective barrier. Epidermal stem cells (ESCs) are pluripotent cells responsible for skin regeneration and healing. Upon exposure to UV-B radiation, ESCs produce excess amounts of reactive oxygen species (ROS) and inflammatory cytokines. However, the functional protection of ESCs is not fully explored. G-protein coupled G protein-coupled receptor 40 (Gpr40) is a free fatty acid receptor that is emerging as a potential treatment target for various diseases. Gpr40 has been found to be expressed in various cell types. METHODS: ESCs were exposed to UV-B at the intensities of 25, 50, and 100 mJ/cm2 for 24 h using TL 20 W/12 RS UV lamps. ESCs were treated with UV-B at 50 mJ/cm2 in the presence or absence of 25 or 50 µM of the Gpr40 agonist GW9508 for 24 h. The gene expression of the Wnt1 pathway and proinflammatory cytokines were evaluated. To antagonize Gpr40 expression, ESCs were treated with 10 µM GW1100. RESULTS: Our findings demonstrate that Gpr40 agonism can reduce the production of ROS as well as the expression of interleukins 1ß and 8, two key proinflammatory cytokines. We demonstrate that agonism of Gpr40 can rescue the reduction in integrin ß1 and Krt19 induced by UV-B exposure, thereby improving the capacities of ESCs to resist UV-B damage. Moreover, we show that the effects of Gpr40 agonism observed in our experiments are mediated through the Wnt/ß-catenin canonical signaling pathway, as evidenced by the expression of Wnt1 and cyclin D1. CONCLUSION: Our findings present evidence of the role of Gpr40 agonism in mediating the protective capacities of ESCs against insult from UV-B radiation.

Coenzyme Q10 Efficacy Test for Human Skin Equivalents Using a Pumpless Skin-On-A-Chip System.

A human skin equivalent (HSE) composed of the epidermis and dermis is cultured using a pumpless skin-on-a-chip system to supply cultures the desired flow rate using gravity flow without a pump or an external tube connection. Coenzyme Q10 efficacy is tested by adjusting its concentration, as it is known to have anti-aging and antioxidant effects in culture solutions. The relationship between the contraction rate of a full-thickness human skin equivalent and secreted transforming growth factor (TGF) ß-1 is analyzed via enzyme-linked immunosorbent assay (ELISA). Following hematoxylin and eosin (H&E) staining, an image of the skin equivalent is analyzed to measure the epidermal layer's thickness. The cell density and differentiation of the dermis layer are investigated. Gene and protein expression in the dermal and epidermal layers are quantitatively analyzed using quantitative real time polymerase chain reaction (qPCR) and immunohistochemical staining. As the coenzyme Q10 treatment concentration increased, the number of cells per unit area and the thickness of the epidermal layer increased, the expression level of filaggrin increased, and the contraction rate of full-thickness HSE was proportional to the amount of TGF ß-1 secreted.

Finite Element Analysis for Predicting Skin Pharmacokinetics of Nano Transdermal Drug Delivery System Based on the Multilayer Geometry Model.

BACKGROUND: Skin pharmacokinetics is an indispensable indication for studying the drug fate after administration of transdermal drug delivery systems (TDDS). However, the heterogeneity and complex skin structured with stratum corneum, viable epidermis, dermis, and subcutaneous tissue inevitably leads the drug diffusion coefficient (Kp) to vary depending on the skin depth, which seriously limits the development of TDDS pharmacokinetics in full thickness skin. METHODS: A multilayer geometry skin model was established and the Kp of drug in SC, viable epidermis, and dermis was obtained using the technologies of molecular dynamics simulation, in vitro permeation experiments, and in vivo microdialysis, respectively. Besides, finite element analysis (FEA) based on drug Kps in different skin layers was applied to simulate the paeonol nanoemulsion (PAE-NEs) percutaneous dynamic penetration process in two and three dimensions. In addition, PAE-NEs skin pharmacokinetics profile obtained by the simulation was verified by in vivo experiment. RESULTS: Coarse-grained modeling of molecular dynamic simulation was successfully established and the Kp of PAE in SC was 2.00×10-6 cm2/h. The Kp of PAE-NE in viable epidermis and in dermis detected using penetration test and microdialysis probe technology, was 1.58×10-5 cm2/h and 3.20×10-5 cm2/h, respectively. In addition, the results of verification indicated that PAE-NEs skin pharmacokinetics profile obtained by the simulation was consistent with that by in vivo experiment. DISCUSSION: This study demonstrated that the FEA combined with the established multilayer geometry skin model could accurately predict the skin pharmacokinetics of TDDS.

Quercetin promotes human epidermal stem cell proliferation through the estrogen receptor/ß-catenin/c-Myc/cyclin A2 signaling pathway.

Skin epidermal stem cells (EpSCs) play an important role in wound healing. Quercetin is a phytoestrogen reported to accelerate skin wound healing, but its effect on EpSCs is unknown. In this study, we investigated the effect of quercetin on human EpSC proliferation and explored the underlying mechanisms. We found that quercetin at 0.1~1 µM significantly promoted EpSC proliferation and increased the number of cells in S phase. The pro-proliferative effect of quercetin on EpSCs was confirmed in cultured human skin tissue. Mechanistic studies showed that quercetin significantly upregulated the expressions of ß-catenin, c-Myc, and cyclins A2 and E1. Inhibitor for ß-catenin or c-Myc significantly inhibited quercetin-induced EpSC proliferation. The ß-catenin inhibitor XAV-939 suppressed quercetin-induced expressions of ß-catenin, c-Myc, and cyclins A2 and E1. The c-Myc inhibitor 10058-F4 inhibited the upregulation of c-Myc and cyclin A2 by quercetin. Pretreatment of EpSCs with estrogen receptor (ER) antagonist ICI182780, but not the G protein-coupled ER1 antagonist G15, reversed quercetin-induced cell proliferation and upregulation of ß-catenin, c-Myc, and cyclin A2. Collectively, these results indicate that quercetin promotes EpSC proliferation through ER-mediated activation of ß-catenin/c-Myc/cyclinA2 signaling pathway and ER-independent upregulation of cyclin E1 and that quercetin may accelerate skin wound healing through promoting EpSC proliferation. As EpSCs are used not only in clinic to treat skin wounds but also as seed cells in skin tissue engineering, quercetin is a useful reagent to expand EpSCs for basic research, skin wound treatment, and skin tissue engineering.

Employment of cytology for in vitro skin irritation test using a reconstructed human epidermis model, Keraskin™.

Skin irritation tests using reconstructed human epidermis (RhE) employ viability as an endpoint, but color interference or borderline results are often problematic. We examined whether the cytology of cells from treated RhE could determine skin irritancy. Six chemicals (three irritants; DnP, 1-B, PH, three non-irritants; DP, APA, HS) were evaluated in a RhE, Keraskin™. DP, HS, and PH were clearly classified with viability, but DnP, 1-B, and APA were often falsely determined, due to borderline values falling near the cutoff, 50%. In histology, the tissues treated with DnP, 1-B, and PH showed erosion of the stratum corneum, vacuolization, and necrosis in the basal layer. DP- and HS-treated tissues showed relatively normal morphology but APA induced necrosis similar to irritants. Cytology revealed that DnP, 1-B or PH depleted cells and induced irregular and abnormal cell shapes. In contrast, relatively regular and normal shapes and clear distinction between the nucleus and cytoplasm was observed for DP, APA and HS. To further confirm it, additional 10 substances, including false positives from OECD TG 439, were tested. Overall (16 substances in total), cytology: total area predicted the skin irritancy of test chemicals with the highest accuracy (87.5%) followed by cytology: cell count (81.3%), histology (75%) and viability (68.8%), confirming the utility of cytology as an alternative endpoint in the skin irritation test using RhE.

BC-Box Motif in SOCS6 Induces Differentiation of Epidermal Stem Cells into GABAnergic Neurons.

The BC-box motif in suppressor of cytokine signaling 6 (SOCS6) promotes the neuronal differentiation of somatic stem cells, including epidermal stem cells. SOCS6 protein belongs to the group of SOCS proteins and inhibits cytokine signaling. Here we showed that epidermal stem cells were induced to differentiate into GABAnergic neurons by the intracellular delivery of a peptide composed of the amino-acid sequences encoded by the BC-box motif in SOCS6 protein. The BC-box motif (SLQYLCRFVI) in SOCS6 corresponded to the binding site of elongin BC. GABAnergic differentiation mediated by the BC-box motif in SOCS6 protein was caused by ubiquitination of JAK2 and inhibition of the JAK2-STAT3 pathway. Furthermore, GABAnergic neuron-like cells generated from epidermal stem cells were transplanted into the brain of a rodent ischemic model. Then, we demonstrated that these transplanted cells were GAD positive and that the cognitive function of the ischemic model rodents with the transplanted cells was improved. This study could contribute to not only elucidating the mechanism of GABAnergic neuronal differentiation but also to neuronal regenerative medicine utilizing GABAnergic neurons.

Triterpenoid corosolic acid modulates global CpG methylation and transcriptome of tumor promotor TPA induced mouse epidermal JB6 P+ cells.

Epigenetic regulation is one of the driving forces in the process of carcinogenesis. Corosolic acid (CA); triterpenoid abundantly found in Lagerstroemia speciosa L. is known to modulate various cellular process including cellular oxidative stress and signaling kinases in various diseases, including skin cancer. Genetic mutations in early stages of skin cancer are well-documented, the epigenetic alterations remain elusive. In the present study, we identified the transcriptomic gene expression changes with RNAseq and genome-wide DNA CpG methylation changes with DNA methylseq to profile the early stage transcriptomic and epigenomic changes using tumor promoter TPA-mediated mouse epidermal epithelial JB6 P+ cells. JB6 P+ cells were treated with TPA and Corosolic acid by 7.5uM optimized by MTS assay. Differentiated expressed genes (DEGs) and Differentially methylated genes (DMRs) were analyzed by R software. Ingenuity Pathway Analysis (IPA) was employed to understand the differential regulation of specific pathways. Novel TPA induced differentially overexpressed genes like tumor promoter Prl2c2, small prolin rich protein (Sprr2h) was reported which was downregulated by corosolic acid treatment. Several cancer related pathways were identified by Ingenuity Pathways Analysis (IPA) including p53, Erk, TGF beta signaling pathways. Moreover, differentially methylated regions (DMRs) in genes like Dusp22 (Dual specificity protein phosphatase 22), Rassf (tumor suppressor gene family, Ras association domain family) in JB6 P+ cells were uncovered which are altered by TPA and are reversed by CA treatment. Interestingly, genes like CDK1 (Cyclin-dependent kinases 1) and RASSF2 (Ras association domain family member 2) observed to be differentially methylated and expressed which was further modulated by corosolic acid treatment, validated by qPCR. Given study indicated gene expression changes to DNA CpG methylation epigenomic changes modulated various molecular pathways in TPA-induced JB6 cells and revealed that CA can potentially reverse these changes which deciphering novel molecular targets for future prevention of early stages of skin cancer studies in human.