Activity In Human Keratinocytes Research Articles

Alternative autophagy dampens UVB-induced NLRP3 inflammasome activation in human keratinocytes

Sunlight exposure results in an inflammatory reaction of the skin commonly known as sunburn, which increases skin cancer risk. In particular, the ultraviolet B (UVB) component of sunlight induces inflammasome activation in keratinocytes to instigate the cutaneous inflammatory responses. Here, we explore the intracellular machinery that maintains skin homeostasis by suppressing UVB-induced inflammasome activation in human keratinocytes. We found that pharmacological inhibition of autophagy promoted UVB-induced NLRP3 inflammasome activation. Unexpectedly, however, gene silencing of Atg5 or Atg7, which are critical for conventional autophagy, had no effect, whereas gene silencing of Beclin1, which is essential not only for conventional autophagy but also for Atg5/Atg7-independent alternative autophagy, promoted UVB-induced inflammasome activation, indicating an involvement of alternative autophagy. We found that damaged mitochondria were highly accumulated in UVB-irradiated keratinocytes when alternative autophagy was inhibited, and they appear to be recognized by NLRP3. Overall, our findings indicate that alternative autophagy, rather than conventional autophagy, suppresses UVB-induced NLRP3 inflammasome activation through the clearance of damaged mitochondria in human keratinocytes, and illustrate a previously unknown involvement of alternative autophagy in inflammation. Alternative autophagy may be a new therapeutic target for sunburn and associated cutaneous disorders.

P10-04: Impact of persistent organic pollutants on EGFR activity in human keratinocytes: Clues for a direct interaction with the EGFR extracellular domain

P10-04: Impact of persistent organic pollutants on EGFR activity in human keratinocytes: Clues for a direct interaction with the EGFR extracellular domain

Scutellarein attenuates atopic dermatitis by selectively inhibiting transient receptor potential vanilloid 3 channels.

Atopic dermatitis (AD) is one of the most common chronic inflammatory cutaneous diseases with unmet clinical needs. As a common ingredient found in several medicinal herbs with efficacy on cutaneous inflammatory diseases, Scutellarein (Scu) has been shown to possess anti-inflammatory and anti-proliferative activities. We aimed to evaluate the therapeutic efficacy of Scu against AD and its underlying molecular mechanism. Efficacy of Scu on AD was evaluated in 2,4-dinitrofluorobenzene (DNFB) and carvacrol-induced dermatitis mouse models. Cytokine mRNA and serum IgE levels were examined using qPCR and ELISA, respectively. Voltage clamp recordings were used to measure currents mediated by transient receptor potential (TRP) channels. In silico docking, site-direct mutagenesis, and covalent modification were used to explore the binding pocket of Scu on TRPV3. Subcutaneous administration of Scu efficaciously suppresses DNFB and carvacrol-induced pruritus, epidermal hyperplasia and skin inflammation in wild type mice but has no additional benefit in Trpv3 knockout mice in the carvacrol model. Scu is a potent and selective TRPV3 channel allosteric negative modulator with an apparent affinity of 1.18 μM. Molecular docking coupled with site-direct mutagenesis and covalent modification of incorporated cysteine residues demonstrate that Scu targets the cavity formed between the pore helix and transmembrane helix S6. Moreover, Scu attenuates endogenous TRPV3 activity in human keratinocytes and inhibits carvacrol-induced proliferative and proinflammatory responses. Collectively, these data demonstrate that Scu ameliorates carvacrol-induced skin inflammation by directly inhibiting TRPV3, and TRPV3 represents a viable therapeutic target for AD treatment.

620 Inflammasome activation in human keratinocytes and mouse epidermis by ultraviolet radiation

Ultraviolet B radiation (UVB) is a ubiquitous environmental toxin that causes extensive skin damage resulting in inflammation, photoaging and cancer. UVB is known to induce inflammasome activation and a sterile inflammation associated with cytokine release (e.g. IL-1β, IL-18) and immune cell infiltration. We developed circularly permutated luciferase reporters for caspase-1 activation to assess inflammasome activation by UVB in vitro and in vivo. HaCaT keratinocytes transduced with caspase-1 reporters encoding either the caspase-7 or Gasdermin D cleavage sequence became active after exposure to UVB, with maximum caspase-1 activation 9 hours post-UVB with 15 mJ/cm2.

Chronic arsenic exposure suppresses ATM pathway activation in human keratinocytes

An estimated 220 million people worldwide are chronically exposed to inorganic arsenic (iAs) primarily as a result of drinking iAs-contaminated water. Chronic iAs exposure is associated with a plethora of human diseases including skin lesions and multi-organ cancers. iAs is a known clastogen, inducing DNA double strand breaks (DSBs) in both exposed human populations and in vitro. However, iAs does not directly interact with DNA, suggesting that other mechanisms, such as inhibition of DNA repair and DNA Damage Response (DDR) signaling, may be responsible for iAs-induced clastogenesis. Recent RNA-sequencing data from human keratinocytes (HaCaT cells) indicate that mRNAs for phosphatases important for resolution of DDR signaling are induced as a result of chronic iAs exposure prior to epithelial to mesenchymal transition. Here, we report that phosphorylation of ataxia telengectasia mutated (ATM) protein at a critical site (pSer1981) important for DDR signaling, and downstream CHEK2 activation, are significantly reduced in two human keratinocyte lines as a result of chronic iAs exposure. Moreover, RAD50 expression is reduced in both of these lines, suggesting that suppression of the MRE11-RAD50-NBS1 (MRN) complex may be responsible for reduced ATM activation. Lastly, we demonstrate that DNA double strand break accumulation and DNA damage is significantly higher in human keratinocytes with low dose iAs exposure. Thus, inhibition of the MRN complex in iAs-exposed cells may be responsible for reduced ATM activation and reduced DSB repair by homologous recombination (HR). As a result, cells may favor error-prone DSB repair pathways to fix damaged DNA, predisposing them to chromosomal instability (CIN) and eventual carcinogenesis often seen resulting from chronic iAs exposure.

Biological Effects of Maslinic Acid on Human Epithelial Cells Used in Tissue Engineering.

In the present work, we evaluated the potential of maslinic acid (MA) to improve currently available keratinocyte culture methods for use in skin tissue engineering. Results showed that MA can increase cell proliferation and WST-1 activity of human keratinocytes after 24, 48, and 72 h, especially at the concentration of 5 μg/ml, without affecting cell viability. This effect was associated to a significant increase of KI-67 protein expression and upregulation of several genes associated to cell proliferation (PCNA) and differentiation (cytokeratins, intercellular junctions and basement membrane related genes). When human keratinocytes were isolated from skin biopsies, we found that MA at the concentration of 5 μg/ml significantly increased the efficiency of the explant and the cell dissociation methods. These results revealed the positive effects of MA to optimize human keratinocyte culture protocols for use in skin tissue engineering.

Restriction of viral gene expression and replication prevents immortalization of human keratinocytes by a beta-human papillomavirus

SignificanceHigh-risk (HR) human papillomaviruses (HPV) from the genus alpha cause anogenital and oropharyngeal cancers, whereas the contribution of HPV from the genus beta to the development of cutaneous squamous cell cancer is still under debate. HR-HPV genomes display potent immortalizing activity in human keratinocytes, the natural target cell for HPV. This paper shows that immortalization of keratinocytes by the beta-HPV49 genome requires the inactivation of the viral E8^E2 repressor protein and the presence of the E6 and E7 oncoproteins but also of the E1 and E2 replication proteins. This reveals important differences in the carcinogenic properties of HR-HPV and beta-HPV but also warrants further investigations on the distribution and mutation frequencies of beta-HPV in human cancers.

Nuclear IL-33 Plays an Important Role in IL-31‒Mediated Downregulation of FLG, Keratin 1, and Keratin 10 by Regulating Signal Transducer and Activator of Transcription 3 Activation in Human Keratinocytes

IL-33, a chromatin-associated multifunctional cytokine, is implicated in the pathogenesis of atopic dermatitis (AD), an inflammatory skin disorder characterized by skin barrier dysfunction. IL-33 accumulates in the nuclei of epidermal keratinocytes (KCs) in AD lesions. However, it is unclear whether nuclear IL-33 directly contributes to the pathogenesis of AD. IL-31, a pruritogenic cytokine primarily produced by T helper type 2 cells, is elevated in AD lesions and promotes AD development by suppressing KC differentiation and inducing itching. In this study, we investigated the involvement of nuclear IL-33 in IL-31‒mediated suppression of KC differentiation. In monolayer cultures and living skin equivalent, IL-31 increased the expression of full-length IL-33 and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in the nuclei of human KCs, which in turn downregulated the expression of differentiation markers. We found that IL-31 and IL-4/IL-13 use very similar mechanisms to inhibit KC differentiation: nuclear IL-33 combines with phosphorylated STAT3 and functions as a STAT3 transcription cofactor, promoting phosphorylated STAT3 binding to the FLG promoter to inhibit its transcription; moreover, the nuclear IL-33/phosphorylated STAT3 complex drives the downregulation of keratin 1 and keratin 10 by reducing the availability of the transcription factor RunX1. Therefore, nuclear IL-33 plays an important role in IL-31‒mediated differentiation suppression by regulating STAT3 activation in human KCs.

Extraction of Antioxidant Compounds from Blueberry Fruit Waste and Evaluation of Their In Vitro Biological Activity in Human Keratinocytes (HaCaT)

The purpose of this study was to investigate the biological properties of an extract obtained from the waste of blueberry fruit. The study covered the optimization of extraction of antioxidants from blueberry pomace and the determination of antioxidant properties of the extract using HaCaT as the model organism. Research showed that the yield of antioxidants extraction from blueberry waste was dependent on the applied extraction conditions. Based on the mathematical models, the optimal conditions of extraction process in which the maximum quantity of antioxidant compounds is achieved from the waste mass unit, i.e., the relation of the waste mass to the volume of ethanol equal to 1:17.36, and process time equal to 1000 s. The obtained extract was characterized by high antioxidant activity, which was shaped by high content of polyphenols, mainly anthocyanins. Moreover, the extract showed a high ability to protect HaCaT cells from the occurrence of oxidative stress induced by H2O2. Cells treated with the extract and H2O2 generated a lower amount of ROS than cells treated with H2O2 only. The obtained results will be base of further studies on applying the extract in production of diet supplements and functional foods with increased antioxidant activity. Moreover, the main research material is blueberry pomace which is a troublesome waste material for juice producers. Consequently, according to a sustainable development idea, the study results will provide an opportunity to increase interest in the problem of rational use of the waste material to a certain extent.

493 Determination of the critical sources of cAMP for Crisaborole activity in human keratinocytes

Crisaborole is an FDA approved topical PDE4 inhibitor used to treat inflammatory diseases of the skin. The proposed mechanism of action of Crisaborole is the elevation of cyclic adenosine monophosphate (cAMP), which inhibits inflammatory pathways. Currently, the sources of cAMP responsible and cells affected by Crisaborole are not known. cAMP is produced by various adenylyl cyclases (ACs) and is degraded to AMP by phosphodiesterase (PDE) enzymes such as PDE4. Thus, for Crisaborole to increase cAMP levels, one or more adenylyl cyclases much be active. We have performed a comprehensive analysis using primary human keratinocytes to determine which adenylyl cyclases are required for the actions of Crisaborole in these cells. In addition to using pharmacological inhibitors to adenylyl cyclases, we generated adenylyl cyclase knockout human keratinocytes using the CRISPR/Cas9 system. We have learned that Crisaborole is able to inhibit the induction of inflammatory pathways in human keratinocytes and it appears that sAC, and not tmAC, is the primary source of Crisaborole-induced cAMP generation in human keratinocytes.

Effects of a complex mixture prepared from agrimonia, houttuynia, licorice, peony, and phellodendron on human skin cells

Active ingredients derived from natural sources are widely utilized in many industries. Cosmetic active ingredients are largely derived from various plants. In this study, we examined whether a mixture of plant extracts obtained from agrimonia, houttuynia, licorice, peony, and phellodendron (hereafter AHLPP), which are well-known for their effects on skin, could affect skin barrier function, inflammation, and aging in human skin cells. We also determined whether AHLPP extracts sterilized using γ-irradiation (to avoid preservatives) retained their skin cell regulating activity. The AHLPP mixture could downregulate representative pro-inflammatory cytokines including IL 1-β and IL 7. Procollagen peptide synthesis was also increased by AHLPP treatment along with mRNA upregulation of barrier proteins such as filaggrin and desmoplakin. The AHLPP mixture showed an anti-aging effect by significantly upregulating telomerase activity in human keratinocytes. We further observed TERT upregulation and CDKN1B downregulation, implying a weakening of pro-aging signal transduction. Co-cultivation of a hydrogel polymer containing the AHLPP mixture with human skin cells showed an alteration in skin-significant genes such as FLG, which encodes filaggrin. Thus, the AHLPP mixture with or without γ-irradiation can be utilized for skin protection as it alters the expression of some significant genes in human skin cells.

637 Age and insulin-like growth factor-1 (IGF-1) status impact translesion synthesis (TLS) pathway activation in human keratinocytes and skin epidermis

Several studies have indicated that UV-induced DNA photoproducts are removed from epidermal genomic DNA at a slower rate in geriatric skin than in young adult skin. Such a situation may result in a greater need for the translesion synthesis (TLS) pathway of DNA replication, in which specialized DNA polymerases are recruited to sites of DNA damage to replicate across DNA adducts in either an error-free or error-prone manner. Here we show that skin epidermis from geriatric individuals (>65 years of age) exhibits higher levels of mono-ubiquitinated PCNA, a biochemical marker of TLS pathway activation, than the skin of young adults (aged 21-29) following exposure to 700 J/m2 of UVB light.

Beta HPV Type 15 Can Interfere With NF-κB Activity and Apoptosis in Human Keratinocytes

E7 protein from cutaneous as well as mucosal HPV types can alter NF-κB activity. Conflicting literature data show a HPV-induced up- or down-regulation of the NF-κB pathway in different cell lines. In a previous study we detected the expression of E7 gene of HPV15 in a subungual tumor of a patient affected by incontinentia pigmenti (IP). IP is a rare X-linked genodermatosis in which the IKKγ gene is altered. From observations in transgenic IKKγ defective mice, it was suggested that IKK-deficient cells may undergo rapid hyper-proliferation and apoptosis/necrosis, leading to increased pro-inflammatory cytokine production in the neighboring IKK-positive cells. The objective of this study was to ascertain if beta HPV 15 can alter apoptosis and NF-κB pathway in normal and IKKγ-deficient keratinocytes. The human immortalized keratinocyte cell line (HaCaT), and human primary keratinocyte (HPK) cells were transduced with a retrovirus expressing E6–E7 proteins of HPV 15 and IKKγ was successful silenced mimicking the HPV15 infection and IP. HPV15 E6–E7 gene expression improved NF-κB activity in human keratinocytes even when IKKγ was silenced by siRNA. In IKKγ silenced keratinocyte cells, TNF-α-induced apoptosis was strongly reduced by the expression of HPV15 E6–E7 genes. Beta HPV15 exerted this anti-apoptotic activity by decreasing pro-apoptotic BAK and cleaved Caspase 3 proteins. In conclusion, we can speculate that presence of persistent infection by beta papillomavirus might influence the biological fate of IP by altering NF-κB activation and apoptosis in IKKγ mutated cells, favoring their survival and possibly the development of tumors in the late stage of disease. Taken together, our data reinforce the importance of host genetic background in the pathogenesis of HPV-associated skin lesions.

Phospholipase A2 from bee venom increases poly(I:C)-induced activation in human keratinocytes.

Bee venom (BV) induces skin inflammation, characterized by erythema, blisters, edemas, pain and itching. Although BV has been found to have an inhibitory effect on toll-like receptors (TLRs), we here show that BV enhances keratinocyte responses to polyinosinic-polycytidylic acid [poly(I:C)], a ligand for TLR3. Our results revealed that the enhanced TLR activity was primarily induced by secretory phospholipase A2 (sPLA2), a component of BV (BV-sPLA2). PLA2 mediates the hydrolysis of membrane phospholipids into lysophospholipids and free fatty acids. We demonstrated that BV-sPLA2 increased the intracellular uptake of poly(I:C), phosphorylation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs), and poly(I:C)-mediated interleukin 8 production in human keratinocytes. We further showed that the enzymatic activity of BV-sPLA2 was essential for the increased uptake of poly(I:C). These findings suggest that BV-sPLA2 may induce a modification of the cell membrane structure, leading to enhanced poly(I:C) uptake in keratinocytes. BV-sPLA2 might be able to promote wound healing by enhancing TLR3 responses.

The Petroleum Ether Extract from Hypericum perforatum Root Cultures Exhibits Potent Antiproliferative Activity in Human Keratinocytes and Fibroblasts.

The hyperforin content of Hypericum perforatum herb was repeatedly reported to be responsible for a multitude of pharmacological activities. Our recent report about the hyperforin accumulation in in vitro root cultures of H. perforatum provides an alternative perspective to achieve constant product quality and to serve the rapidly growing market. In this study, the antiproliferative effect of a petroleum ether extract from the in vitro root cultures was investigated. When normalized to 1 µM hyperforin content, the extract reduced the viability of human keratinocytes (HaCaT) and human dermal fibroblast monolayers to 33 and 36%, respectively, after 72 h of incubation. A cytotoxicity assay and live-dead cell staining confirmed that the extract lacked a cytotoxic effect and that the reduction in cell viability was mainly due to the antiproliferative activity. Application of the 1 µM hyperforin-normalized extract to a 3D artificial skin construct significantly reduced the proliferation of HaCaT in the presence of fibroblasts. This effect was proved by the reduction in thickness of the epidermal construct from 100 µm (control) to 17 µm (treated). Notably, 1 µM pure hyperforin lacked effectiveness in both monolayer cultures and 3D artificial skin constructs. Nor were fractions of the extract containing colupulone and xanthones active. The combination of these constituents also failed to reassemble the antiproliferative activity, which indicates a synergistic role of yet unidentified components present in the extract. Our findings may introduce H. perforatum root cultures as a novel lead system for the treatment of hypertrophic scars.

Red light-emitting diode irradiation regulates oxidative stress and inflammation through SPHK1/NF-κB activation in human keratinocytes

Oxidative stress, in which the amount of oxidants exceeds the capacity of antioxidant defense system, is a well-accepted pathogenesis of several human diseases. Light-emitting diode irradiation (LEDI) is an efficient strategy to counteract this condition. The biological effect of phototherapy, using visible light, has attracted recent attention especially in dermatological practice. However, little is known about the molecular mechanism of the anti-oxidant and anti-inflammatory effects of red light irradiation. We evaluated these effects of LEDI in HaCaT human keratinocyte cells under phorbol-12-myristate-13-acetate (PMA) induced reactive oxygen species (ROS). Microarray analysis revealed changes in 309 genes after LEDI. LEDI at 625 nm produced ROS scavenging and anti-inflammatory effects. One of the most important genes identified by microarray analysis was sphingosine kinase-1 (SPHK1), which is a key molecule in sphingolipid metabolism. SPHK1 knock-down drastically reduced ROS scavenging efficiency as well as expression levels of inflammation-related proteins in PMA-treated HaCaT cells. These results not only indicate the potential for the clinical application of 625-nm LEDI in treating skin disorders via ROS and/or inflammation, but also suggest SPHK1 as a potential therapeutic target in phototherapy.

1412 AMP-IBP5, an antimicrobial peptide derived from insulin-like growth factor-binding protein 5, triggers human keratinocyte activation

Besides exhibiting direct microbicidal properties, antimicrobial peptides also display various immunomodulatory functions, including keratinocyte production of cytokines and chemokines, proliferation, migration and wound healing. Recently, a novel antimicrobial peptide named AMP- IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of well-known antimicrobial peptides, such as human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue is still not well understood. In the current study, we investigated whether AMP-IBP5 might trigger human keratinocyte activation and clarified the underlying molecular mechanism. The results revealed that among various cytokines, chemokines and growth factors tested in normal human keratinocytes, AMP-IBP5 selectively increased the production of interleukin (IL-8) and vascular endothelial growth factor (VEGF). Moreover, AMP-IBP5 markedly enhanced keratinocyte migration (assessed by in vitro wound closure assay,) and proliferation (analyzed using BrdU incorporation and XTT assays). AMP-IBP5-induced keratinocyte activation was mediated by Mas-related gene (Mrg) X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. Indeed, we confirmed that AMP-IBP5 induced ERK, JNK, p38 and IB phosphorylation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP. In conclusion, these observations suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of human keratinocytes.

Beneficial effects of melittin on ovalbumin-induced atopic dermatitis in mouse

Atopic dermatitis (AD) is an inflammatory skin disease characterized by intense pruritus and relapsable eczematous lesions. The hallmarks of AD are defects in the epidermal barrier and immunoglobulin E (IgE)-mediated sensitization to several environmental allergens, as well as an immune disorder mediated by an imbalance toward T-helper-2 response. Melittin, a major component of bee venom, has been studied in various inflammatory diseases. However, the beneficial effects of melittin on mouse with AD-like symptoms have not been explored. Therefore, we investigated the anti-allergic effects of melittin. AD was induced by ovalbumin (OVA) patch. After agent treatment, skin tissues and sera were extracted from the sacrificed mice were used to demonstrate the effects of melittin through various molecular biological methods. The results showed that OVA-induced skin thickening and inflammatory infiltration were decreased in the melittin-treated group. Melittin prevented OVA-induced filaggrin deficiency and imbalanced inflammatory mediators. Furthermore, melittin inhibited IL-4/IL-13-induced filaggrin downregulation through the blockade of STAT3 activation in human keratinocytes. In summary, this study has shown that melittin ameliorated OVA-induced AD-like symptoms from various perspectives. The findings of this study may be the first evidence of the anti-inflammatory effects of melittin on OVA-induced AD.

20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, a metabolite of ginsenoside Rb1, enhances the production of hyaluronic acid through the activation of ERK and Akt mediated by Src tyrosin kinase in human keratinocytes.

The aim of the present study was to determine the mechanisms through which 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol (20GPPD) promotes the production of hyaluronic acid (HA) in human keratinocytes. 20GPPD is the primary bioactive metabolite of Rb1, a major ginsenoside found in ginseng (Panax ginseng). We sought to elucidate the underlying mechanisms behind the 20GPPD-induced production of HA. We found that 20GPPD induced an increase in HA production by elevating hyaluronan synthase 2 (HAS2) expression in human keratinocytes. The phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was also enhanced by 20GPPD in a dose-dependent manner. The pharmacological inhibition of ERK (using U0126) or Akt (using LY294002) suppressed the 20GPPD-induced expression of HAS2, whereas treatment with an epidermal growth factor receptor (EGFR) inhibitor (AG1478) or an intracellular Ca2+ chelator (BAPTA/AM) did not exert any observable effects. The increased Src phosphorylation was also confirmed following treatment with 20GPPD in the human keratinocytes. Following pre-treatment with the Src inhibitor, PP2, both HA production and HAS2 expression were attenuated. Furthermore, the 20GPPD-enhanced ERK and Akt signaling decreased following treatment with PP2. Taken together, our results suggest that Src kinase plays a critical role in the 20GPPD-induced production of HA by acting as an upstream modulator of ERK and Akt activity in human keratinocytes.

The δ-Opioid Receptor Affects Epidermal Homeostasis via ERK-Dependent Inhibition of Transcription Factor POU2F3

Neuropeptides and their receptors are present in human skin, and their importance for cutaneous homeostasis and during wound healing is increasingly appreciated. However, there is currently a lack of understanding of the molecular mechanisms by which their signaling modulates keratinocyte function. Here, we show that δ-opioid receptor (DOPr) activation inhibits proliferation of human keratinocytes, resulting in decreased epidermal thickness in an organotypic skin model. DOPr signaling markedly delayed induction of keratin intermediate filament (KRT10) during in vitro differentiation and abolished its induction in the organotypic skin model. This was accompanied by deregulation of involucrin (IVL), loricrin, and filaggrin. Analysis of the transcription factor POU2F3, which is involved in regulation of KRT10, IVL, and profilaggrin expression, revealed a DOPr-mediated extracellular signal-regulated kinase (ERK)-dependent downregulation of this factor. We propose that DOPr signaling specifically activates the ERK 1/2 mitogen-activated protein kinase pathway to regulate keratinocyte functions. Complementing our earlier studies in DOPr-deficient mice, these data suggest that DOPr activation in human keratinocytes profoundly influences epidermal morphogenesis and homeostasis.