Normal Human Epidermal Keratinocytes Research Articles

Spleen tyrosine kinase regulates keratinocyte inflammasome activation and skin inflammation induced by UVB irradiation

UVB can induce inflammatory responses contributing to diverse skin damage. UVB-triggered inflammasome activation of human keratinocytes underlies UVB-induced skin sunburn reaction. Pleiotropic functions of spleen tyrosine kinase (Syk) have rendered it as a potential therapeutic target. In immunocytes, Syk modulates immunoreceptor signaling and NLRP3 inflammasome activation. In skin, Syk mediates EGFR signaling, regulates keratinocyte differentiation and is involved in inflammatory disorders. However, roles of Syk in UVB-induced inflammasome activation in keratinocytes remain elusive. We investigated roles of keratinocyte Syk in UVB-triggered photo-responses. Primary normal human epidermal keratinocytes (NHEKs) isolated from skin were used. Syk knockdown or Syk inhibitor R406 was applied to investigate functions of keratinocyte Syk in UVB photobiology. The possible in vivo role of Syk was evaluated by checking UVB-induced skin damage in R406-treated mice. UVB was able to induce Syk phosphorylation in NHEKs that could be regulated by reactive oxygen species (ROS) generation and EGFR. Syk knockdown or Syk inhibitor (R406) treatment reduced UVB-triggered apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) crosslinking, procaspase-1 cleavage, active IL-1β formation, and gasdermin D activation, indicating roles of Syk in UVB-triggered inflammasome activation in keratinocytes. UVB-induced production of IL-8, TNF-α, ROS, and phosphorylation of JNK and p38 were attenuated after Syk knockdown or inhibition. R406 ameliorated UVB-induced mouse skin damage, including erythema and transepidermal water loss (TEWL). Thus, Syk participated in UVB-induced inflammasome activation and inflammatory response in vitro and in vivo, suggesting potential photo-protective effects of Syk inhibition in UVB-induced skin inflammation.

Psoriasis-Associated Inflammatory Conditions Induce IL-23 mRNA Expression in Normal Human Epidermal Keratinocytes.

Psoriasis is a multifactorial, chronic inflammatory skin disease, the development of which is affected by both genetic and environmental factors. Cytosolic nucleic acid fragments, recognized as pathogen- and danger-associated molecular patterns, are highly abundant in psoriatic skin. It is known that psoriatic skin exhibits increased levels of IL-23 compared to healthy skin. However, the relationship between free nucleic acid levels and IL-23 expression has not been clarified yet. To examine a molecular mechanism by which nucleic acids potentially modulate IL-23 levels, an in vitro system was developed to investigate the IL-23 mRNA expression of normal human epidermal keratinocytes under psoriasis-like circumstances. This system was established using synthetic nucleic acid analogues (poly(dA:dT) and poly(I:C)). Signaling pathways, receptor involvement and the effect of PRINS, a long non-coding RNA previously identified and characterized by our research group, were analyzed to better understand the regulation of IL-23 in keratinocytes. Our results indicate that free nucleic acids regulate epithelial IL-23 mRNA expression through the TLR3 receptor and specific signaling pathways, thereby, contributing to the development of an inflammatory milieu favorable for the appearance of psoriatic symptoms. A moderate negative correlation was confirmed between the nucleic-acid-induced IL-23 mRNA level and the rate of its decrease upon PRINS overexpression.

Pyrroloquinoline Quinone Disodium (PQQ2Na) Has an NLRP Inflammasome-Induced Caspase-1 Release Influence in UVB-Irradiated but Not ATP-Treated Human Keratinocytes but Has No Influence in Increasing Skin Cell Mitochondrial Biogenesis in Either Human Keratinocytes or Fibroblasts.

IntroductionPyrroloquinoline quinone is a bacterial-derived redox factor that has been shown to have numerous benefits in humans. Recently, a model for examining the ability of normal human epidermal keratinocytes (NHEKs) to demonstrate anti-inflammatory benefits via nod-like receptor protein (NLRP)-activated caspase-1 release was reported. The question of whether PQQ2Na might have anti-inflammatory benefits that function through NLRP-activated release of active caspase-1 has not been explored. In addition, it has been reported that PQQ2Na will induce mitochondrial biogenesis in humans when taken orally. Whether or not this effect occurs in skin cells is presently unknown.MethodsThe inflammation studies followed previously published methods that demonstrated both UVB and ATP were able to upregulate the NLRP-activated release of caspase-1 in NHEKs. In addition, NHEK and normal dermal human fibroblasts (NHDF) were treated with PQQ2Na to see if the molecule might stimulate mitochondrial biogenesis measured by increased expression of cyclooxygenase-1 (COX-1) and succinate dehydrogenase complex, subunit A (SDHA).ResultsAt non-cytotoxic concentrations between 5 µg/mL and 100 µg/mL in NHEKs and between 0.1 µg/mL and 5 µg/mL in fibroblasts, the PQQ2Na had no influence on cellular mitochondrial biogenesis. In ATP-activated NHEKs at concentrations of PQQ2Na between 0.05 µg/mL and 50 µg/mL, there was no influence of PQQ2Na on release of active caspase-1. In NHEKs irradiated with 60mJ/cm2 of UVB radiation as previously described and treated with 0.05 µg/mL to 50 µg/mL of PQQ2Na, the molecule showed a dose-dependent benefit at reducing the expression of active caspase-1 in the irradiated cells.DiscussionBenefits of PQQ2Na on various skin cell types which had not been investigated previously were addressed. Surprisingly, the PPQ2Na had no apparent influence on skin cell mitochondrial biogenesis. However, the molecule has a strong suppressing influence on UVB-induced active caspase-1 release in UVB-irradiated NHEKs.

The Effect of Capsaicin on Neuroinflammatory Mediators of Rosacea.

BackgroundRosacea is a chronic inflammatory skin disease with a pathophysiological mechanism that remains unclear. Recently, dysregulation of the sensory nerve system has been implicated in the development of this condition.ObjectiveThis study aimed to investigate the effect of capsaicin on neuroinflammatory mediators in rosacea. In addition, this study aimed to evaluate the attenuating effects of capsazepine, a transient receptor potential vanilloid type 1 (TRPV1) antagonist.MethodsWe obtained skin tissue from both rosacea patients and normal individuals for an in vivo study. In addition, normal human epidermal keratinocytes (NHEKs) were cultured, and treated with capsaicin and capsazepine for an in vitro study. Quantitative changes in neuroinflammatory mediators were evaluated by semi-quantitative reverse transcription-polymerase chain reaction (PCR), real-time PCR, enzyme-linked immunosorbent assay, and immunofluorescence staining.ResultsThe data showed the increase of TRPV1, TRPV4, cathelicidin (LL37) and tumor necrosis factor-α (TNF-α) in skin tissue by real-time PCR. In addition, the data showed that cathelicidin (LL37), kallikrein-5 (KLK-5), TNF-α, vascular endothelial growth factor (VEGF), interleukin (IL)-1α, IL-1β, IL-8, and protease-activated receptor 2 (PAR2) increased in capsaicin-treated NHEKs. Capsazepine attenuated the expression of TRPV1 and other mediators, except for IL-8, in capsaicin-treated NHEKs.ConclusionWe confirmed that TRPV1, TRPV4, cathelicidin (LL37) and TNF-α are increased in rosacea skin, and that capsaicin is associated with increase of neuroinflammatory mediators such as LL37, KLK-5, TNF-α, VEGF, IL-1α, IL-1β, IL-8, and PAR2. Modulators or inhibitors of neuroinflammatory mediators including TRPV1 could be potential therapeutic option in the treatment of patients with rosacea.

A combination of Vitreoscilla filiformis extract and Vichy volcanic mineralizing water strengthens the skin defenses and skin barrier.

Probiotics are live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host. Semiactive, non-replicating bacteria or extracts used in dermocosmetics have interesting properties for skin quality. Vitreoscilla filiformis is cultured by a fermentation process to obtain an extract. It is considered as a probiotic fraction and topical application of this extract has shown activity to strengthen the skin physical barrier function and maintain good homeostasis of skin defenses. Vichy volcanic mineralizing water (VVMW) is a pure, highly mineralized water that has been shown to strengthen the skin against exposome aggressions. This manuscript reviews properties of probiotic fractions used in skin care, especially studies on an extract of V. filiformis grown in a medium containing VVMW (VfeV) and evaluated in combination with VVMW. Skin barrier function: In normal human epidermal keratinocyte cultures, the combination of 10% VVMW and 0.002% VfeV significantly increased transglutaminase, filaggrin, involucrin, claudin-1, and zonula occludens-1 in comparison with the controls. Antimicrobial peptide defenses: The combination of 16.7% VVMW and 0.1% VfeV increased the expression of β-defensin-4A and S100A7. Skin immune defense functions: In lipopolysaccharide-stimulated peripheral blood mononuclear cells, the combination of 16.7% VVMW and 0.1% VfeV down-regulated IL-8, TNF-α, IL-12/IL-23p40, and increased IL10 and IL-10/IL-12 ratio compared to the control. Additionally, the combination of 79% VVMW plus 5% VfeV protected Langerhans cells in skin explants exposed to ultraviolet radiation. In conclusion, the combination of VfeV plus VVMW has properties to strengthen the skin barrier by stimulating skin differentiation and tight junctions, biochemical defenses by stimulating antimicrobial peptides, and cellular immune defenses by increasing the IL-10/IL-12 ratio and by protecting Langerhans cells challenged by ultraviolet radiation.

Skin Barrier-enhancing, Antiwrinkle, and Antimelanogenic Effects of Probiotic Lysates Composed of Nucleotides

Several previous studies have investigated the skin aging prevention effects of ceramide, hyaluronic acid, and natural or fermented plant materials. Recently, oral administration and dermal application of probiotics or probiotic lysates have shown antiaging effects. The purpose of this study is to optimize the preparation of probiotic lysates with a high concentration of nucleotides and to confirm the effects of probiotic lysates on the skin. Probiotic lysates were prepared by heating at 121°C for various periods with adding of sodium hyaluronic acid. Probiotic lysates of <i>Bifidobacterium longum</i> HDB7072, <i>Lactobacillus paracasei</i> HDB1196, and <i>Lactobacillus acidophilus</i> HDB1014 were applied to normal human epidermal keratinocytes (NHEKs), fibroblast cells, and B16F1 cells, respectively. Cell viability, antioxidant effects, and mRNA expression were evaluated by using MTT assays, DPPH assays, and qRT-PCR. Probiotic lysates prepared by heating the culture medium at 121°C for 2 h with 0.5% sodium hyaluronic acid showed the highest nucleotide concentration. In the three tested skin cells, the cell viability of filtered lysates was similar or higher to that of unfiltered lysates. HDB7072 lysates increased filaggrin expression in NHEKs. HDB1196 lysates showed DPPH radical-scavenging and antiwrinkle effects through the downregulation of matrix metalloproteinase-1 and upregulation of collagen type 1 in fibroblasts. HDB1014 lysates had antioxidant and antimelanogenic effects in B16F1 cells. Cell wall-removed probiotic lysates could be used as novel ingredients to improve skin aging and skin barrier issues.

Histamine enhances ATP-induced itching and responsiveness to ATP in keratinocytes

Mechanical stimulation of cultured keratinocytes and a living epidermis increases intracellular calcium ion concentrations ([Ca2+]i) in stimulated cells. This action propagates a Ca2+ wave to neighboring keratinocytes via ATP/P2Y2 receptors. Recent behavioral, pharmacological studies revealed that exogenous ATP induces itching via P2X3 receptors in mice. We previously showed that alloknesis occurs when an external stimulus is applied to the skin with increased epidermal histamine in the absence of spontaneous pruritus. Based on these results, we investigated the effects of histamine at a concentration that does not cause itching on ATP-induced itching. The mean number of scratching events induced by the mixture of ATP and histamine increased by 28% over the sum of that induced by histamine alone or ATP alone. A317491, a P2X3 receptor antagonist, suppressed the mixture-induced scratching more often than the ATP-induced scratching. Next, we examined the ATP-induced [Ca2+]i change before and after histamine stimulation using normal human epidermal keratinocytes. Some cells did not respond to ATP before histamine stimulation but responded to ATP afterward, the phenomenon suppressed by chlorpheniramine maleate. These findings suggest that histamine enhances ATP-induced itching and that a potential mechanism could involve increased responsiveness to ATP in keratinocytes.

Levofloxacin loaded clove essential oil nanoscale emulsion as an efficient system against Pseudomonas aeruginosa biofilm

Biofilm formation by bacteria has become one of the major reasons for escalation of antimicrobial resistance. The present research focused on the development, optimization and evaluation of antibiofilm potential of clove oil nanoscale emulsion (o/w) loaded with levofloxacin (LFX-NE) against Pseudomonas aeruginosa biofilm. The optimized NE was selected through the construction of pseudo-ternary phase diagrams and was found to be stable. The particle size and drug content of LFX-NE was found to be 18.84 ± 0.5 nm and 99.2 ± 0.5%, respectively. It demonstrated high antibacterial activity with 16-fold and 8-fold reduction in MIC of LFX against P. aeruginosa and Escherichia coli and Klebsiella pneumoniae, respectively. LFX-NE was able to eradicate pre-formed biofilm of P. aeruginosa as evidenced by field emission scanning electron microscopy and confocal laser scanning microscopy. Antiquorum sensing study revealed inhibition of quorum sensing activity. LFX-NE was found to be non-irritant and cytocompatible as confirmed by HET-CAM test and safety study in normal human epidermal keratinocytes (NHEK) cell line. This study demonstrates the potential of LFX-NE in possible treatment of non-healing wounds inflicted with bacterial biofilm.

Update on the role of noncoding RNAs in vitiligo.

Update on the role of noncoding RNAs in vitiligo.

Methoxy-Monobenzoylmethane Protects Skin from UV-Induced Damages in a Randomized, Placebo Controlled, Double-Blinded Human In Vivo Study and Prevents Signs of Inflammation While Improving the Skin Barrier.

IntroductionSun protection is important in skin care and requires special attention as inefficient protection might trigger skin pathologies including polymorphic light eruption (PLE). The reduce-improve-protect (RIP) concept to avoid the onset of ultraviolet (UV) irradiation-induced diseases or damage to human skin is important. Methoxy-monobenzoylmethane (MeO-MBM), which is neither a UVB nor a UVA filter, converts to the UV filter avobenzone under UV irradiation and further acts as a photoantioxidant during its conversion process and initially as an antioxidant material. The aim of this study was to understand the mechanisms by which MeO-MBM improves the condition of UV-stressed skin through its photoantioxidant properties. The improvement of the skin condition by the activity of MeO-MBM as active ingredient was also investigated.MethodsPotential molecular targets were identified by in silico docking to numerous cellular membrane receptors on the cell surface or nuclear membrane, followed by microarray analysis of 164 genes after MeO-MBM treatment of normal human epidermal keratinocytes (NHEK). We conducted randomized, double-blinded, intra-individual comparison vs. placebo studies on ten volunteers, aged between 34 and 65 years, to assess the effect of MeO-MBM in vivo. The effect after UV-induced inflammation was assessed in a protective and curative set-up with 2% MeO-MBM vs. 1% hydrocortisone and placebo based on the change in blood flow. The barrier function of the skin was assessed by the change in transepidermal water loss (TEWL), skin scaling and skin thickness after the treatment with MeO-MBM. Additionally, the effect of MeO-MBM after UV-induced stress on the activation of ferritin in human explants was determined ex vivo.ResultsA docking simulation of MeO-MBM showed a potential interaction with the retinoic acid receptor gamma and further revealed downregulation of proteins related to inflammation. In the protective treatment set-up, after 24 h MeO-MBM significantly reduced the delta blood flow compared to placebo, while this reduction was more prominent with hydrocortisone. In the curative treatment set-up, a greater reduction in delta blood flow was also observed with MeO-MBM compared to placebo and similar to hydrocortisone. Treatment with MeO-MBM revealed an improvement in skin barrier function as a result of decreased TEWL, reduced skin scaling and increased skin thickness. Immunohistochemistry staining of ferritin on human skin explants further showed that the treatment with MeO-MBM reduced the ferritin expression.ConclusionBased on these results, MeO-MBM is capable of exerting an anti-aging activity via the retinoic acid receptor gamma. Its anti-inflammatory and anti-oxidative activity manifested via the downregulation of multiple anti-inflammatory genes as well as the reduction of ferritin in skin tissue. This study shows that the multidimensional functionality of MeO-MBM offers an effective approach to combat acute and chronic deleterious effects of oxidative UV damage while simultaneously enhancing the skin barrier function.

MiR-181a Promotes Multiple Protumorigenic Functions by Targeting TGFβR3

Cutaneous squamous cell carcinoma (cSCC) comprises 15‒20% of all skin cancers and has a well-defined progression sequence from precancerous actinic keratosis to invasive cSCC. To identify targets for chemoprevention, we previously reported a cross-species analysis to identify the transcriptional drivers of cSCC development and identified miR-181a as a potential oncomiR. We show that the upregulation of miR-181a promotes multiple protumorigenic properties by targeting an understudied component of TGFβ signaling, TGFβR3. miR-181a and TGFβR3 are upregulated and downregulated, respectively, in cSCC. miR-181a overexpression (OE) and TGFβR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT cells and in primary normal human epidermal keratinocytes. In addition, OE of miR-181a or KD of TGFβR3 by short hairpin RNA enhances anchorage-independent survival. miR-181a OE or TGFβR3 KD enhances cellular migration and invasion and upregulation of epithelial‒mesenchymal transition markers. Luciferase reporter assays demonstrate that miR-181a directly targets the 3'-untranslated region of TGFβR3. miR-181a upregulates phosphorylated SMAD3 levels after TGFβ2 administration and results in elevated SNAIL and SLUG expression. Finally, we confirm invivo that miR-181a inhibition compromises tumor growth. Importantly, these phenotypes can be reversed with TGFβR3 OE or KD in the context of miR-181a OE or KD, respectively, further highlighting the physiologic relevance of this regulation in cSCC.

HPV16-LINC00393 Integration Alters Local 3D Genome Architecture in Cervical Cancer Cells.

High-risk human papillomavirus (hrHPV) infection and integration were considered as essential onset factors for the development of cervical cancer. However, the mechanism on how hrHPV integration influences the host genome structure remains not fully understood. In this study, we performed in situ high-throughput chromosome conformation capture (Hi-C) sequencing, chromatin immunoprecipitation and sequencing (ChIP-seq), and RNA-sequencing (RNA-seq) in two cervical cells, 1) NHEK normal human epidermal keratinocyte; and 2) HPV16-integrated SiHa tumorigenic cervical cancer cells. Our results reveal that the HPV-LINC00393 integrated chromosome 13 exhibited significant genomic variation and differential gene expression, which was verified by calibrated CTCF and H3K27ac ChIP-Seq chromatin restructuring. Importantly, HPV16 integration led to differential responses in topologically associated domain (TAD) boundaries, with a decrease in the tumor suppressor KLF12 expression downstream of LINC00393. Overall, this study provides significant insight into the understanding of HPV16 integration induced 3D structural changes and their contributions on tumorigenesis, which supplements the theory basis for the cervical carcinogenic mechanism of HPV16 integration.

Combinational Anti-tumor Effects of Chemicals from Paeonia lutea Leaf Extract in Oral Squamous Cell Carcinoma Cells.

We identified chemical components that exhibited antitumor activity against oral squamous cell carcinoma (OSCC) cells and examined their effective concentrations and additive and/or synergistic effects in combinational usage on the proliferation, apoptosis and cell cycle of OSCC cells. Using high-performance liquid chromatography, nuclear magnetic resonance spectroscopy and electrospray ionization-mass spectrometry, we identified the main chemical components of the methanol extracts from Paeonia lutea. We investigated the pharmaceutical effects of those components on the proliferation, apoptosis, and cell cycle of an OSCC cell line, SAS, using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and caspase assays, as well as flow cytometry cell cycle analysis. We also examined the effects of those components on the mitogen-activated protein kinase signal transduction pathway by western blotting. Finally, the effects on normal human epidermal keratinocyte cells were also examined in similar experiments. Three chemicals have been identified in P. lutea leaves using high performance liquid chromatography: gallic acid methyl ester (GAME), pentagalloyl glucose (PGG) and paeoniflorin (PF). Both GAME and PGG significantly suppressed cell proliferation, and their combined effects were synergistic, while the effect of PF was minimal. However, those chemicals did not induce apoptosis. Cell cycle and western blotting analysis showed that the suppressive effects on cell proliferation resulted from G2 arrest and the suppression of phosphorylation of Akt/PKB. No effect was identified on normal human epidermal keratinocyte cells. These results indicate that GAME and PGG are the main chemical components of P. lutea leaves that have potential anti-cancer therapeutic effects.

Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

Multifaceted Analysis of IL-23A- and/or EBI3-Including Cytokines Produced by Psoriatic Keratinocytes.

Interleukin (IL) 23 (p19/p40) plays a critical role in the pathogenesis of psoriasis and is upregulated in psoriasis skin lesions. In clinical practice, anti-IL-23Ap19 antibodies are highly effective against psoriasis. IL-39 (p19/ Epstein-Barr virus-induced (EBI) 3), a newly discovered cytokine in 2015, shares the p19 subunit with IL-23. Anti-IL-23Ap19 antibodies may bind to IL-39; also, the cytokine may contribute to the pathogenesis of psoriasis. To investigate IL23Ap19- and/or EBI3-including cytokines in psoriatic keratinocytes, we analyzed IL-23Ap19 and EBI3 expressions in psoriasis skin lesions, using immunohistochemistry and normal human epidermal keratinocytes (NHEKs) stimulated with inflammatory cytokines, using quantitative real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and liquid chromatography-electrospray tandem mass spectrometry (LC-Ms/Ms). Immunohistochemical analysis showed that IL-23Ap19 and EBI3 expressions were upregulated in the psoriasis skin lesions. In vitro, these expressions were synergistically induced by the triple combination of tumor necrosis factor (TNF)-α, IL-17A, and interferon (IFN)-γ, and suppressed by dexamethasone, vitamin D3, and acitretin. In ELISA and LC-Ms/Ms analyses, keratinocyte-derived IL-23Ap19 and EBI3, but not heterodimeric forms, were detected with humanized anti-IL-23Ap19 monoclonal antibodies, tildrakizumab, and anti-EBI3 antibodies, respectively. Psoriatic keratinocytes may express IL-23Ap19 and EBI3 proteins in a monomer or homopolymer, such as homodimer or homotrimer.

N-Succinyl-S-Farnesyl-L-Cysteine (SFC): A Novel Isoprenylcysteine Analog with In Vitro Anti-Inflammatory Activity and Clinical Skin Protecting Properties

Over the past 15 years, small molecule isoprenylcysteine (IPC) analogs have been identified as a potential new class of topical anti-inflammatories. Clinical studies have demonstrated that IPCs are both safe and effective in promoting healthy skin when applied topically. This work aims to demonstrate N-Succinyl-S-farnesyl-L-cysteine (SFC) as a novel IPC molecule that provides a broad spectrum of benefits for skin. Human promyelocytic cell line HL-60, human dermal microvascular endothelial cells (HDMECs), human dermal fibroblasts (HDFs), and normal human epidermal keratinocytes (NHEKs) were exposed in culture to various inducers to trigger reactive oxygen species, cytokines, or collagenase production. A 49-subject randomized double-blind, vehicle-controlled, split face trial was performed with 1% SFC gel, or 5% niacinamide and vehicle applied for 12 weeks to evaluate anti-wrinkle and anti-aging endpoints. We demonstrated that SFC inhibited GPCR and TLR-induced pro-inflammatory cytokine release in NHEKs and HDMECs from several inflammatory inducers such as UVB, chemicals, cathelicidin, and bacteria. SFC successfully reduced GPCR-induced oxidation in differentiated neutrophils. Moreover, photoaging studies showed that SFC reduced UVA-induced collagenase (pro-MMP-1) production in HDFs. Clinical assessment of 1% SFC gel demonstrated improvement above the vehicle for wrinkle reduction, hydration, texture, and overall appearance of skin. N-Succinyl-S-farnesyl-L-cysteine (SFC) is a novel anti-inflammatory small molecule and is the first farnesyl-cysteine IPC shown to clinically improve appearance and signs of aging, while also having the potential to ameliorate inflammatory skin disorders.

SMG1 and CDK12 Link ΔNp63α Phosphorylation to RNA Surveillance in Keratinocytes.

The tumor suppressor p53-like protein p63 is required for self-renewal of epidermal tissues. Loss of p63 or exposure to ultraviolet (UV) irradiation triggers terminal differentiation in keratinocytes. However, it remains unclear how p63 diverts epidermal cells from proliferation to terminal differentiation, thereby contributing to successful tissue self-renewal. Here, we used bottom-up proteomics to identify the proteome at the chromatin in normal human epidermal keratinocytes following UV irradiation and p63 depletion. We found that loss of p63 increased DNA damage and that UV irradiation recruited the cyclin-dependent kinase CDK12 and the serine/threonine protein kinase SMG1 to chromatin only in the presence of p63. A post-translational modification analysis of ΔNp63α with mass spectrometry revealed that phosphorylation of T357/S358 and S368 was dependent on SMG1, whereas CDK12 increased the phosphorylation of ΔNp63α at S66/S68 and S301. Indirect phosphorylation of ΔNp63α in the presence of SMG1 enabled ΔNp63α to bind to the tumor suppressor p53-specific DNA recognition sequence, whereas CDK12 rendered ΔNp63α less responsive to UV irradiation and was not required for specific DNA binding. CDK12 and SMG1 are known to regulate the transcription and splicing of RNAs and the decay of nonsense RNAs, respectively, and a subset of p63-specific protein-protein interactions at the chromatin also linked p63 to RNA transcription and decay. We observed that in the absence of p63, UV irradiation resulted in more ORF1p. ORF1p is the first protein product of the intronless non-LTR retrotransposon LINE-1, indicating a derailed surveillance of RNA processing and/or translation. Our results suggest that p63 phosphorylation and transcriptional activation might correspond to altered RNA processing and/or translation to protect proliferating keratinocytes from increased genotoxic stress.

Relaxing Effect of Novel Cosmetic Ingredient using Lactobacillus gasseri HDB1102 on Skin Problems Caused by Particulate Matter

In this study, we developed Dermabiotics HDB1102 using <i>Lactobacillus gasseri</i> HDB1102 to relieve skin irritation caused by particulate matter (PM). <i>L. gasseri</i> HDB1102 was provided from cell bank and identified by 16S ribosomal RNA gene sequencing. Dermabiotics HDB1102 was manufactured by heating, centrifuging, and filtering culture medium of <i>L. gasseri</i> HDB1102. When 0-2.5%(v/v) Dermabiotics HDB1102 was treated, cytotoxicity on normal human epidermal keratinocytes (NHEKs) and human fibroblast was not observed by using MTT assay. The mRNA expression levels of cytochrome P450 1A1 (CYP1A1), interleukin (IL)-1β, and IL-8 on Dermabiotics HDB1102 treated cells decreased compared to PM-treated cells. Conversely, the mRNA expressions of aquaporin-3 (AQP-3), CD-44, and collagen type 1 (COL-1) on Dermabiotics HDB1102 treated cells were dose-dependent higher than those of non-treated cells. These results indicated that Dermabiotics HDB1102 have anti-inflammatory, moisturizing, and anti-wrinkle effects and could be used as a potential cosmetic ingredient to alleviate skin symptoms caused by PM.

Evaluation of Anti-Photoaging Effects of a Novel Cosmeceutical Containing a Retinoids Mixture Using In Vitro Cell Models

Physiological ageing due to the passing of time and prolonged exposure to harmful sun rays generate wrinkles and reduce skin elasticity. These visible and clinical signs can be prevented or reversed by known strategies, such as the daily use of cosmetic products with antioxidant combinations or retinoids. A new dermocosmetic formulation enriched with a complex of retinoids, called RETINOIDS SERUM, was investigated through in vitro assays using human skin cells. The experiments were carried out to assess the anti-ageing activity in normal human dermal fibroblasts (NHDF) and epidermal keratinocytes (HaCaT). After the preliminary MTT assay, the proliferation together with the synthesis of collagen and elastin fibers was performed on NHDF cells after 24 h treatment with the two non-cytotoxic concentrations. Using UVB-irradiated HaCaT cells, the measurement of matrix metalloproteinase-1 (MMP-1) levels was also investigated. In vitro studies show that the dermocosmetic product improves collagen and elastin synthesis and the renewal of dermal fibroblasts. Moreover, a reduction in the MMP-1 secretion was also highlighted in UVB-irradiated HaCaT cells. These results suggest that the cosmetic formulation containing functional compounds such as retinoids can be useful to prevent the natural sign of ageing.

Nicotinamide Attenuates UV-Induced Stress Damage in Human Primary Keratinocytes from Cancerization Fields

UVB radiation directly damages DNA, increases ROS and nitric oxide (NO) release, and promotes inflammation leading to genomic instability and cell death. Nicotinamide (NAM) is the precursor of NAM adenine dinucleotide, essential for cell energy production and DNA damage repair. NAM protects HaCat cells from UV-induced impairment; however, little is known about its effects on human primary keratinocytes and those isolated from field cancerization (i.e., field cancerization human primary keratinocytes [FC-HPKs]). We examined the role of NAM against UV-induced oxidative stress damages in FC-HPKs, isolated from precancerous lesions and skin cancers, and in normal human epidermal keratinocytes. Cells were treated for 18, 24, and 48 hours with NAM (5, 25, and 50 μM, respectively) before UVB irradiation. FC-HPK showed four-fold higher basal ROS levels than normal human epidermal keratinocytes; NAM downregulated ROS production only in irradiated FC-HPKs, which showed a greater sensibility to UV rays. UV exposure increased OGG1, inducible nitric oxide synthase, and IL-1β expression, an effect counteracted by NAM pretreatment. Intracellular nitric oxide production and DNA damages were inhibited by NAM exposure before irradiation. Collectively, our findings indicate that pretreatment with 25 μM NAM 24 hours before UVB irradiation effectively prevents oxidative stress formation, DNA damage, and inflammation in both normal human epidermal keratinocytes and FC-HPKs.