Signaling Pathways In Keratinocytes Research Articles

P22 A single-cell map of the epigenomic landscape in keratinocytes exposed to the cytokine milieu of atopic dermatitis

Abstract Introduction and aims In atopic dermatitis (AD), induction of inflammatory responses regulated by cytokines such as of thymic stromal lymphopoietin exacerbate the disrupted integrity of the skin barrier. Cytokines, such as interleukin (IL)-4, IL-13 and IL-22, present in the skin milieu, bind to their corresponding receptors on the cell surface of keratinocytes, activating multiple signal transduction pathways that lead to changes in the gene expression profile of keratinocytes, in part, by modulating chromatin structure. Understanding the intricate interplay between cytokine-mediated signalling pathways in keratinocytes and the resulting epigenetic changes is essential for elucidating the pathogenesis of AD. In this study, we utilized high-throughput technology to examine the effects of cytokines on modulation of the epigenetic landscape at the keratinocyte single-cell level, and subsequent effects on the gene expression. Methods We utilized the reconstructed human epidermis model, an in vitro three-dimensional human epidermis model consisting of a polycarbonate membrane and keratinocytes mimicking the structure of native human epidermis. Following exposure to AD cytokine(s), the model gene expression and chromatin accessibility of target cells were analysed using single-cell multiomics approaches. Results Our preliminary data show that IL-4, IL-13 and IL-22 reduce the expression levels of the barrier-related gene filaggrin in keratinocytes. Conclusions Unlike genetic changes, epigenetic alterations are dynamic and can be restored to their normal state, which has major implications for the development of therapeutics for various diseases. This study will provide valuable insights into the epigenetic mechanisms underlying AD and present potential targets for epigenetic-related therapeutic interventions. These interventions can be used in combination with available compounds to reduce the recurrence of AD and improve the treatment efficiency.

Jingfang granules exert anti-psoriasis effect by targeting MAPK-mediated dendritic cell maturation and PPARγ-mediated keratinocytes cell cycle progression in vitro and in vivo

BackgroundJingfang granules (JFG), derived from JingFangBaiDu San (JFBDS), are a traditional herbal formulas used for the treatment of respiratory tract infections. They were initially prescribed to treat skin disease, such as psoriasis in Chinese Taiwan, but are not widely used for psoriasis treatment in mainland China because of the lack of anti-psoriasis mechanism research. PurposesThe present study was designed to evaluate the anti-psoriasis effect of JFG and reveal the correlated mechanisms of JFG in vivo and in vitro using network pharmacology, UPLC-Q-TOF-MS technology and molecular biotechnology methods. ResultsAn imiquimod-induced psoriasis-like murine model was used to verify the anti-psoriasis effect in vivo, with inhibition of lymphocytosis and CD3+CD19+B cell proliferation in the peripheral blood and prevention of the activation of CD4+IL17+T cells and CD11c+ MHC Ⅱ+ dendritic cells (DCs) in the spleen. Network pharmacology analysis demonstrated that the targets of the active components were significantly enriched in pathways involved in cancer, inflammatory bowel disease and rheumatoid arthritis, which were closely related to cell proliferation and immune regulation. The drug-component-target networks and molecular docking analysis demonstrated the active ingredients to be luteolin, naringin and 6′-feruloylnodakenin, which had a good binding affinity to PPARγ, p38a MAPK and TNF-a. Finally, UPLC-Q-TOF-MS analysis to validate the active ingredients in drug-containing serum and in vitro experiments showed that JFG inhibited the maturation and activation of BMDCs via the p38a MAPK signaling pathway and translocation of the agonist PPARγ into the nuclei to reduce the activity of NF-κB/STAT3 inflammatory signaling pathway in keratinocytes. ConclusionsOur study demonstrated that JFG improved psoriasis by inhibiting the maturation and activation of BMDCs and proliferation and inflammation of keratinocytes, which may facilitate the applications of JFG in anti-psoriasis therapy in clinical settings.

Heat promotes melanogenesis by increasing the paracrine effects in keratinocytes via the TRPV3/Ca2+/Hh signaling pathway

Global warming and rising temperature significantly increase the incidence of heat stress, which is known to affect the process of inflammation and aging. However, the effect of heat stress on skin melanogenesis is not fully known. We found that healthy foreskin tissues underwent significant pigmentation when exposed to 41°C. Furthermore, heat stress promoted melanogenesis in pigment cells by increasing the paracrine effects of keratinocytes. High-throughput RNA sequencing showed that heat stress activates the Hedgehog (Hh) signaling pathway in keratinocytes. The agonists of Hh signaling promote the paracrine effect of keratinocytes on melanogenesis. In addition, transient receptor potential vanilloid (TRPV) 3 agonists activate the Hh signaling in keratinocytes and augment its paracrine effect on melanogenesis. The heat-induced activation of Hh signaling is dependent on TRPV3-mediated Ca2+ influx. Heat exposure promotes melanogenesis by increasing the paracrine effects in keratinocytes via the TRPV3/Ca2+/Hh signaling pathway. Our findings provide insights into the mechanisms of heat-induced skin pigmentation.

High glucose-induced increasing reactive nitrogen species accumulation triggered mitochondrial dysfunction, inflammation, and apoptosis in keratinocytes

Growing evidence indicates that skin injuries are a common complication of diabetes. However, the cellular and molecular mechanisms of high glucose (HG) environment trigger nitrosative stress-mediated inflammation and apoptosis in keratinocytes remains unknown. Here we investigated whether reactive nitrogen species (RNS) induced by HG environment restrain antioxidant activity, and mitochondrial dysfunction leading to inflammation, and apoptosis via stress signaling pathways in keratinocytes. Our results established that the HG environment enhanced the production of nitric oxide (NO) and peroxynitrite anion (ONOO−) by inducible NO synthase (iNOS) in keratinocytes. Overproduction of RNS in HG environment suppress the antioxidants activity leading to mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential (ΔΨm), increase in mitochondrial mass, decrease in mitochondrial transcription factor A(TFAM), increase in mitochondrial DNA (mtDNA) displacement loop (D-loop) and decrease in glycolytic flux concentration, which was attenuated by pharmacological inhibitors of NO/ONOO−, Nω-Nitro-l-argininemethyl ester hydrochloride (NAME)/hydralazine hydrochloride (Hyd.HCl). Excess production of RNS in HG environment restrained 8-oxoguanine DNA glycosylase-1 (OGG1) expression and increased 8-hydroxydeoxyguanosine (8-OHdG) accumulations in DNA were regulated by NO or ONOO−. Further, HG-induced RNA production caused an increase in the production of inflammatory mediators accompanied by activation of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3 signaling cascade, lipid peroxidation (LPO), and protein carbonylation (PC) reactions followed by breakdown the cell-cell communication and apoptosis. Pre-treatment of cell with NAME/Hyd.HCl, diminished the expression of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3, inflammatory mediators, and attenuated apoptosis in keratinocytes. Together, our results indicated that excess production of RNS in HG environment triggered inflammation and apoptosis, mediated by activation of ERK1/2MAPK/Akt/tuberin-mTOR/IRF3 signaling cascades in keratinocytes.

Ligustrazine alleviates psoriasis-like inflammation through inhibiting TRAF6/c-JUN/NFκB signaling pathway in keratinocyte.

Ligusticum chuanxiong Hort (Ligusticum; Apiaceae) (accepted name, Ligusticum striatum DC, on "The Plant List" for the latest version) is a Chinese herbal medicine (CHM) which mainly distributed in Sichuan Basin, China. Chuanxiong is the dried rhizome of Ligusticum chuanxiong Hort. Ligustrazine, also known as tetramethylpyrazine (TMP), is a main active fraction of chuanxiong. The aim of this study was to clarify the underlying mechanisms by which TMP protect against psoriasis-like inflammation in keratinocytes. Here, we demonstrated that TMP alleviated the severity and PASI scores of IMQ-induced psoriasis-like skin lesion in vivo. For the histopathology level, TMP inhibited the over-proliferation of keratinocytes in the epidermis and the substantial immune cells influx in dermis. For the mechanism of the ability of TMP on regulating inflammation, we confirmed that TMP regulate the TRAF6/c-JUN/NFκB signaling pathway through analyzing the proteomics profiling and verifying the expression of TRAF6, pho-c-Jun, pho-NFκB, so that the downstream psoriasis-relevant genes transcribed by c-JUN or NFκB were down-regulated. Furthermore, we predicted TRAF6 as the potential binding point of TMP. Accordingly, our study demonstrated that TMP regulated psoriasis-like inflammation through inhibiting TRAF6/c-JUN/NFκB signaling pathway in keratinocytes, which potentially provides evidence of the mechanism of TMP in the treatment and prevention of psoriasis.

Epidermal Fatty Acid‒Binding Protein Mediates Depilatory-Induced Acute Skin Inflammation

Depilatory creams are widely used to remove unwanted body hair, but people with sensitive skin are subject to depilatory-induced skin burn/inflammation. It remains unknown what makes their skin more sensitive than others. In this study, we show that epidermal fatty acid‒binding protein (E-FABP) expressed in the skin plays a critical role in promoting depilatory-induced acute skin inflammation in mouse models. Although a depilatory cream removed hair by breaking down keratin disulfide bonds, it activated cytosolic phospholipase A2, leading to activation of the arachidonic acid/E-FABP/peroxisome proliferator-activated receptor β signaling pathway in keratinocytes. Specifically, peroxisome proliferator-activated receptor β activation induced downstream targets (e.g., cyclooxygenase 2) and chemokine (e.g., CXCL1) production, which systemically mobilized neutrophils and recruited them to localize in the skin for acute inflammatory responses. Importantly, E-FABP deletion by CRISPR-Cas9 reduced cytosolic phospholipase A2/peroxisome proliferator-activated receptor β activation in keratinocytes, and genetic deletion of E-FABP protected mice from depilatory cream-induced neutrophil recruitment and skin inflammation. Our findings suggest E-FABP as a molecular sensor for sensitive skin by triggering depilatory-induced, lipid-mediated skin inflammatory responses.

Leucosceptoside A from Devil's Claw Modulates Psoriasis-like Inflammation via Suppression of the PI3K/AKT Signaling Pathway in Keratinocytes.

Psoriasis is a chronic inflammatory skin condition characterized by abnormal keratinocyte proliferation and differentiation that is accompanied with dysregulated immune response and abnormal vascularization. Devil’s claw (Harpagophytum procumbens (Burch.) DC. ex Meisn.) tubers extract has been used both systemically and topically for treatment of chronic inflammatory diseases such as arthritis, osteoporosis, inflammatory bowel disease, among others. However, its potential mechanisms of action against psoriasis remains poorly investigated. The human keratinocyte HaCaT cell line is a well-accepted in vitro model system for inflammatory skin disorders such as psoriasis. The present study involved an exploration of the effect of biotechnologically produced H. procumbens (HP) cell suspension extract and pure phenylethanoid glycosides verbascoside (VER) and leucosceptoside A (LEU) in interferon (IFN)-γ/interleukin (IL)-17A/IL-22-stimulated HaCaT cells as a model of psoriasis-like inflammation. Changes in key inflammatory signaling pathways related to psoriasis development were detected by reverse transcription polymerase chain reaction and western blotting. Treatment with LEU, but not VER and HP extract improved psoriasis-related inflammation via suppression of the PI3K/AKT signaling in IFN-γ/IL-17A/IL-22-stimulated HaCaT cells. Our results suggest that LEU may exhibit therapeutic potential against psoriasis by regulating keratinocyte differentiation through inhibition of the PI3K/AKT pathway.

MiR-193b-3p\u2013ERBB4 axis regulates psoriasis pathogenesis via modulating cellular proliferation and inflammatory-mediator production of keratinocytes

Psoriasis is an auto-inflammatory skin disease characterized by abnormal activation of epidermal keratinocytes, aberrant neovascularization, and dysregulation of immune cells. MicroRNAs are small non-coding RNAs that mainly function in the post-transcriptional regulation of gene expression. Recently, accumulating evidence has demonstrated that expression of microRNAs is dysregulated in psoriasis patients and microRNAs play key roles in psoriasis pathogenesis. Downregulation of miR-193b-3p has been identified to be associated with psoriasis development. However, the precise functions and action mechanisms of miR-193b-3p in psoriasis pathogenesis remain unclear. In this study, we confirmed the downregulation of miR-193b-3p in psoriasis patients, psoriasis-like inflammatory cellular models, and an imiquimod (IMQ) -induced mouse model. A negative correlation was found between miR-193b-3p level and patient Psoriasis Area and Severity Index (PASI) score. Furthermore, miR-193b-3p suppressed proliferation, inflammatory-factor secretion, and the STAT3 and NF-κB signaling pathways in keratinocytes. Importantly, intradermal injection of agomiR-193b-3p blocked, whereas antagomiR-193b-3p augmented, the psoriasis-like inflammation in the IMQ-induced mouse model. Bioinformatics analysis and the dual-luciferase reporter assay showed that miR-193b-3p targets ERBB4 3ʹ untranslated region (UTR). In addition, ERBB4 induced proliferation, inflammatory-factor production, and the STAT3 and NF-κB pathways in keratinocytes. Most importantly, forced expression of ERBB4 could attenuate the effects of miR-193b-3p in keratinocytes, indicating that miR-193b-3p inhibits keratinocyte activation by directly targeting ERBB4. In conclusion, our findings demonstrated that the miR-193b-3p–ERBB4 axis underlies the hyperproliferation and aberrant inflammatory-factor secretion of psoriatic keratinocytes, providing a novel, microRNA-related causal mechanism and a potential therapeutic target in psoriasis.

Wound Healing Effect of Gintonin Involves Lysophosphatidic Acid Receptor/Vascular Endothelial Growth Factor Signaling Pathway in Keratinocytes.

Gintonin, a novel compound of ginseng, is a ligand of the lysophosphatidic acid (LPA) receptor. The in vitro and in vivo skin wound healing effects of gintonin remain unknown. Therefore, the objective of this study was to investigate the effects of gintonin on wound healing-linked responses, especially migration and proliferation, in skin keratinocytes HaCaT. In this study, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, Boyden chamber migration assay, scratch wound healing assay, and Western blot assay were performed. A tail wound mouse model was used for the in vivo test. Gintonin increased proliferation, migration, and scratch closure in HaCaT cells. It also increased the release of vascular endothelial growth factor (VEGF) in HaCaT cells. However, these increases, induced by gintonin, were markedly blocked by treatment with Ki16425, an LPA inhibitor, PD98059, an ERK inhibitor, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester), a calcium chelator, and U73122, a PLC inhibitor. The VEGF receptor inhibitor axitinib also attenuated gintonin-enhanced HaCaT cell proliferation. Gintonin increased the phosphorylation of AKT and ERK1/2 in HaCaT cells. In addition, gintonin improved tail wound healing in mice. These results indicate that gintonin may promote wound healing through LPA receptor activation and/or VEGF release-mediated downstream signaling pathways. Thus, gintonin could be a beneficial substance to facilitate skin wound healing.

Mutations in γ-secretase subunit–encoding PSENEN gene alone may not be sufficient for the development of acne inversa

BackgroundThe effects of PSENEN mutations in patients with acne inversa (AI) are poorly understood. Hyperproliferation of follicular keratinocytes and resulting occlusion may constitute the initial pathophysiology. ObjectiveTo investigate the effects of PSENEN knockdown on γ-secretase subunits, biological behaviors, and related signaling pathways in keratinocytes. MethodsHaCaT cells were divided into an experimental group (PSENEN knock down), a negative control group, and a blank control group. Whole transcriptome sequencing was used to measure differences in mRNA expression of the whole genome; real-time PCR and Western blotting were performed to determine the interference efficiency and the effects of interference on the components of γ-secretase and related molecules. CCK-8 was used to measure cell proliferation, and flow cytometry was used to measure apoptosis and the cell cycle. ResultsA comparison of five healthy controls with three patients with PSENEN mutation (c.66delG, c.279delC, c.229_230insCACC) revealed decreased expression of mRNA and protein in skin lesions of the experimental group. In this group, expression of the other components of γ-secretase presenilin C-terminal fragment decreased, expression of immature nicastrin increased, expression of mature nicastrin decreased, and expression of anterior pharynx defective-1 remained unchanged. KEGG analysis revealed that differentially expressed molecules were enriched in m-TOR signaling pathways. Subsequent verification confirmed that differences in PI3K-AKT-mTOR signaling pathway molecules, cell proliferation, apoptosis, cell cycle and the expression levels of Ki-67, KRT1, and IVL between the groups were not statistically significant. ConclusionsPSENEN mutations alone may be insufficient to cause the development of AI, or they may only induce a mild phenotype of AI.

MicroRNA-125a Correlates with Decreased Psoriasis Severity and Inflammation and Represses Keratinocyte Proliferation

Background: Psoriasis has a complex etiology related to inflammation and dysregulated immune system. MicroRNA (miR)-125a is a miRNA intimately related to inflammation and immunity; therefore, we presumed that it might play a role in the pathogenesis of psoriasis. This study aimed to investigate the correlation of miR-125a with disease severity and inflammation in psoriasis patients, and the effect of miR-125a on proliferation, apoptosis as well as its target signaling pathway in keratinocytes. Methods: Sixty psoriasis patients were consecutively recruited, then lesional and non-lesional skin tissue samples were collected. miR-125a in lesional and non-lesional skin tissues, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-17 mRNA expressions in lesional skin tissues were detected. Then, miR-125a overexpression, control overexpression, miR-125a knockdown and control knockdown plasmids were transfected into HaCaT cells. Subsequently, cell proliferation, apoptosis, IL-23R, JAK2, and STAT3 expressions were assessed. Results: miR-125a was reduced in lesional skin tissue compared with non-lesional skin tissue (p < 0.001), and it distinguished lesional skin tissue from non-lesional skin tissue with a high area under curve of 0.917 (95% CI 0.866–0.968). Negative association of miR-125a in lesional skin tissue with lesional body surface area (p = 0.037) and psoriasis area and severity index score (p < 0.001) was found. Additionally, miR-125a was negatively correlated with TNF-α (p = 0.001), IL-1β (p = 0.014), and IL-17 (p = 0.003) in lesional skin tissue. In cellular experiments, miR-125a overexpression inhibited proliferation and promoted apoptosis, while miR-125a knockdown enhanced proliferation and repressed apoptosis in HaCaT cells. Additionally, miR-125a negatively regulated the IL-23R/JAK2/STAT3 pathway in HaCaT cells. Conclusion: miR-125a could facilitate the disease monitoring and probably has the potential to be a therapeutic target in psoriasis.

An Autocrine Circuit of IL-33 in Keratinocytes Is Involved in the Progression of Psoriasis

IL-33 is constitutively expressed in the skin. Psoriasis is a common skin inflammatory disease. The roles of IL-33 in psoriasis have not been well-elucidated. We identified that keratinocytes (KCs) are the predominant cells expressing IL-33 and its receptor, suppression of tumorigenicity 2, in the skin. KCs actively released IL-33 on psoriasis inflammatory stimuli and induced psoriasis-related cytokine, chemokine, and inflammatory molecules genes transcription in KCs in an autocrine manner. IL-33‒specific deficiency in KCs ameliorated imiquimod-induced psoriatic dermatitis. In addition, intradermal injection of recombinant IL-33 alone induced psoriasis-like dermatitis, which is attributed to the transcriptional upregulation of genes enriched in IL-17, TNF, and chemokine signaling pathway in KCs on recombinant IL-33 stimulation. Our data demonstrate that the autocrine circuit of IL-33 in KCs promotes the progression of psoriatic skin inflammation, and IL-33 is a potential therapeutic target for psoriasis.

Electrical stimulation promotes the proliferation of human keratinocytes, increases the production of keratin 5 and 14, and increases the phosphorylation of ERK1/2 and p38 MAP kinases.

Effective wound healing remains a significant clinical challenge in reducing patient morbidity and improving quality of life. Wound healing is a complex process involving the endogenous electrical field. The electrical field can contribute to wound healing by activating keratinocytes to promote reepithelialization. The objective of this study was to determine the effects of exogenous electrical stimulation (ES) on human keratinocyte viability and proliferation and on production of IL-6, IL-8, and keratins (K5 and K14) and to investigate the activated signalling pathways in keratinocytes exposed to ES. Keratinocytes were cultured under ES at different intensities for 6 or 24 hr. Cell proliferation, cytokines and growth factors, K5 and K14, as well as phosphorylated ERK1/2 and p38 MAP kinases, were evaluated. The results showed that the keratinocytes exposed to ES between 100 and 150 mV/mm for 6 or 24 hr showed a significantly increased proliferation rate. However, a 24 hr exposure to 200 mV/mm revealed no significant effect in cell growth. ES at 100 and 200 mV/mm for 6 hr increased the secretion of epidermal growth factor and vascular endothelial growth factor, and the production of K5 and K14. K14 was more sensitive than K5 to ES. However, ES down-regulated the secretions of IL-6 and IL-8. Finally, ES increased the phosphorylation of ERK1/2 and p38 MAP kinases. Overall results suggested that ES can be useful in supporting skin wound healing by activating keratinocytes.

Sam68 is required for the growth and survival of nonmelanoma skin cancer.

Although targeting DNA repair signaling pathways has emerged as a promising therapeutic for skin cancer, the relevance of DNA damage responses (DDR) in the development and survival of nonmelanoma skin cancer (NMSC), the most common type of skin cancer, remains obscure. Here, we report that Src‐associated substrate during mitosis of 68 kDa (Sam68), an early signaling molecule in DDR, is elevated in skin tumor tissues derived from NMSC patients and skin lesions from Gli2‐transgenic mice. Downregulation of Sam68 impacts the growth and survival of human tumor keratinocytes and genetic ablation of Sam68 delays the onset of basal cell carcinomas (BCC) in Gli2‐transgenic mice. Moreover, Sam68 plays a critical role in DNA damage‐induced DNA repair and nuclear factor kappa B (NF‐κB) signaling pathways in keratinocytes, hence conferring keratinocyte sensitivity to DNA damaging agents. Together, our data reveal a novel function of Sam68 in regulating DDR in keratinocytes that is crucial for the growth and survival of NMSC.

Cell responses to the impact of physiological doses of ultraviolet radiation and their modulation by plant polyphenols

In this study it was investigated the responses of cultured human cells – keratinocytes and fibroblasts to physiological doses of ultraviolet radiation (UVR) applied with or without plant polyphenolic compounds: rutin, quercetin, taxofolin, silybin and baikalein. Experimental data obtained in this work indicate the presence of synergism in the action of physiological doses of UVR and plant polyphenolic compounds on inflammatory signaling pathways in keratinocytes and fibroblasts. It is concluded that the identified synergies can contribute to the adaptation of the skin to subsequent exposure to UVR, and thus be one of the photoprotective mechanisms of the plant polyphenolic compounds.

Activation of a protumorigenic IFNγ/STAT1/IRF‐1 signaling pathway in keratinocytes following exposure to solar ultraviolet light

In this study, we evaluated the role of signal transducer and activator of transcription 1 (STAT1) in response to acute solar ultraviolet (SUV) radiation in mouse epidermis. Analysis of the epidermis from SUV-irradiated mice revealed rapid phosphorylation of STAT1 (pSTAT1) on both tyrosine (tyr701) and serine (ser727) residues and increased levels of IRF-1 while later timepoints showed increased levels of unphosphorylated STAT1 (uSTAT1). STAT1 activation led to upregulation of several proinflammatory chemokine mRNAs in epidermis including Cxcl9, Cxcl10, and Ccl2, as well as, the immune checkpoint inhibitor Pd-l1. In addition, mRNA and protein levels of cyclooxygenase-2 (Cox-2/COX2) were upregulated in epidermis following exposure to SUV. Mice with keratinocyte-specific STAT1 deletion did not exhibit increased IRF-1 or proinflammatory gene expression in epidermis. Furthermore, epidermal COX-2 induction after SUV exposure was significantly reduced in mice with keratinocyte-specific deletion of STAT1. Additionally, SUV irradiation rapidly upregulated interferon gamma (IFNγ) mRNA in the epidermis and that skin resident epidermal CD3 + T-cells were the source of IFNγ production. IFNγ receptor-deficient mice confirmed dependency of STAT1 activation, proinflammatory gene expression and COX-2 upregulation in the epidermis on paracrine IFNγ signaling. Furthermore, keratinocyte-specific STAT1-deficiency reduced proliferation and hyperplasia due to SUV irradiation and this was associated with decreased immune infiltration of mast cells in the dermis. Collectively, the current results demonstrate that exposure to SUV leads to upregulation of IFNγ and downstream pSTAT1/IRF-1/uSTAT1 signaling in the epidermis. Further study of this pathway could lead to identification of novel targets for the prevention of nonmelanoma skin cancer.

TNIP1 Regulates Cutibacterium acnes-Induced Innate Immune Functions in Epidermal Keratinocytes.

Human skin cells recognize the presence of the skin microbiome through pathogen recognition receptors. Epidermal keratinocytes are known to activate toll-like receptors (TLRs) 2 and 4 in response to the commensal Cutibacterium acnes (C. acnes, formerly known as Propionibacterium acnes) bacterium and subsequently to induce innate immune and inflammatory events. These events may lead to the appearance of macroscopic inflammatory acne lesions in puberty: comedos, papules and, pustules. Healthy skin does not exhibit inflammation or skin lesions, even in the continuous presence of the same microbes. As the molecular mechanism for this duality is still unclear, we aimed to identify factors and mechanisms that control the innate immune response to C. acnes in keratinocytes using a human immortalized keratinocyte cell line, HPV-KER, normal human keratinocytes (NHEK) and an organotypic skin model (OSM). TNIP1, a negative regulator of the NF-κB signaling pathway, was found to be expressed in HPV-KER cells, and its expression was rapidly induced in response to C. acnes treatment, which was confirmed in NHEK cells and OSMs. Expression changes were not dependent on the C. acnes strain. However, we found that the extent of expression was dependent on C. acnes dose. Bacterial-induced changes in TNIP1 expression were regulated by signaling pathways involving NF-κB, p38, MAPKK and JNK. Experimental modification of TNIP1 levels affected constitutive and C. acnes-induced NF-κB promoter activities and subsequent inflammatory cytokine and chemokine mRNA and protein levels. These results suggest an important role for this negative regulator in the control of bacterially induced TLR signaling pathways in keratinocytes. We showed that all-trans retinoic acid (ATRA) induced elevated TNIP1 expression in HPV-KER cells and also in OSMs, where TNIP1 levels increased throughout the epidermis. ATRA also reduced constitutive and bacterium-induced levels of TNFα, CCL5 and TLR2, while simultaneously increasing CXCL8 and TLR4 expression. Based on these findings, we propose that ATRA may exhibit dual effects in acne therapy by both affecting the expression of the negative regulator TNIP1 and attenuating TLR2-induced inflammation. Overall, TNIP1, as a possible regulator of C. acnes-induced innate immune and inflammatory events in keratinocytes, may play important roles in the maintenance of epidermal homeostasis.

MicroRNA let-7b inhibits keratinocyte differentiation by targeting IL-6 mediated ERK signaling in psoriasis

BackgroundThe extensive involvement of microRNA (miRNA) in the pathophysiology of psoriasis is well documented. However, in order for this information to be useful in therapeutic manipulation of miRNA levels, it is essential that detailed functional mechanisms are elucidated. This study aimed to explore the effects of IL-6 targeting by let-7b and ERK1/2 mediated signaling on keratinocyte differentiation in psoriasis.MethodsFollowing imiquimod cream (IMQ) application to let-7bTG (keratinocyte-specific let-7b overexpression mouse) and control mice for 7 days, we analyzed erythema, scaling and thickening of skin. A dual luciferase reporter assay and bioinformatics was carried out to detect target gene of let-7b. Additionally, the differentiation markers were measured. Immunohistochemistry analyses demonstrate a relationship of let-7b with IL-6 and ERK signaling.Resultswe found let-7bTG inhibits acanthosis and reduces the disease severity by treatment with IMQ compared to wild-type mice. Further study illustrated that let-7b promotes differentiation of keratinocytes in vivo and in vitro. Using bioinformatics and reporter gene assays, we found that IL-6 is a target gene of let-7b. In psoriasis, high expression levels of IL-6 lead to increased acivation of p-ERK1/2. High levels of let-7bTG transgene expression suppresses IL-6 expression and leads to increased keratinocyte differentiation. Moreover, let-7b acts as an upstream negative regulator of the ERK signaling pathway in keratinocytes of psoriasis.ConclusionsOur result reveals a previously unknown mechanism for regulation of IL-6 levels during psoriasis by let-7b and highlights a critical role for the ERK1/2 signaling pathway in epidermal differentiation during psoriasis.Trial registrationThe ethical approval for this study was from the Affiliated Hospital of Medical University of Anhui _ Fast_ PJ2017–11–14.

Abstract 3556: Sam68 is required for the growth and survival of non-melanoma skin cancer

Abstract Skin cancer is the most commonly diagnosed malignancy in the United States, and the incidence of skin cancer has increased dramatically over the last few decades. Non-melanoma skin cancer (NMSC), which includes squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), is the most common type of skin cancer with substantial associated morbidity and mortality. Whereas targeting DNA repair signaling pathways has emerged as a promising therapeutic for skin cancer, the relevance of DNA damage responses (DDR) in the development and survival of non-melanoma skin cancer (NMSC) remains obscure. Here we report that Src-associated substrate during mitosis of 68 kDa (Sam68), an early signaling molecule in DDR, is elevated in skin tumor tissues derived from NMSC patients and skin lesions from Gli2-transgenic mice. Downregulation of Sam68 impacts the growth and survival of human tumor keratinocytes and genetic ablation of Sam68 delays the onset of basal cell carcinomas (BCC) in Gli2-transgenic mice. Moreover, Sam68 plays a critical role in DNA damage-induced DNA repair and nuclear factor kappa B (NF-κB) signaling pathways in keratinocytes, hence conferring keratinocyte sensitivity to DNA damaging agents. Together our data reveal a novel function of Sam68 in regulating DDR in keratinocytes that is crucial for the growth and survival of NMSC. Citation Format: Kai Fu, Fengyi Wan. Sam68 is required for the growth and survival of non-melanoma skin cancer [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 3556.

444 Demethylzeylasteral inhibits the skin trafficking of CD8+ T cells in patients with vitiligo through the inactivation of IFN-γ-JAK-STAT1-CXCL10 signaling pathway in keratinocytes

444 Demethylzeylasteral inhibits the skin trafficking of CD8+ T cells in patients with vitiligo through the inactivation of IFN-γ-JAK-STAT1-CXCL10 signaling pathway in keratinocytes