Human Epidermal Cells Research Articles

Two Laminaria japonica Fermentation Broths Alleviate Oxidative Stress and Inflammatory Response Caused by UVB Damage: Photoprotective and Reparative Effects.

UVB radiation can induce oxidative stress and inflammatory response in human epidermal cells. We establish a UVB-induced damage model of human immortalized epidermal keratinocytes (HaCaT) to explore the protective and reparative effects of Laminaria japonica on UVB-damaged epidermal inflammation after fermentation by white Ganoderma lucidum (Curtis) P. Karst and Saccharomyces cerevisiae. Compared with unfermented Laminaria japonica, fermented Laminaria japonica possesses stronger in vitro free radical scavenging ability. Laminaria japonica white Ganoderma lucidum fermentation broth (LJ-G) and Laminaria japonica rice wine yeast fermentation broth (LJ-Y) can more effectively remove excess reactive oxygen species (ROS) in cells and increase the content of the intracellular antioxidant enzymes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO-1). In addition, fermented Laminaria japonica effectively reduces the content of pro-inflammatory factors ILs, TNF-α and MMP-9 secreted by cells. The molecular research results show that fermented Laminaria japonica activates the Nrf2 signaling pathway, increases the synthesis of antioxidant enzymes, inhibits the gene expression levels of pro-inflammatory factors, and alleviates cellular oxidative stress and inflammatory response caused by UVB radiation. Based on the above results, we conclude that fermented Laminaria japonica has stronger antioxidant and anti-inflammatory activity than unfermented Laminaria japonica, possesses good safety, and can be developed and used as a functional inflammation reliever. Fermented Laminaria japonica polysaccharide has a more slender morphological structure and more rockulose, with better moisturizing and rheological properties.

Comparative assessment of commercially available wound gels in ex vivo human skin reveals major differences in immune response-modulatory effects

Wound healing is a crucial process for maintaining the function of human skin as a protective barrier to pathogens and other external stress factors. Hydrogels—in combination with antimicrobials—are often used, as moist wound care has been widely accepted as standard therapy. Recently, we reported about immune response-modulatory effects of an octenidine-based hydrogel, however little is known about the mechanism of action of other hydrogels including antiseptic molecules or chlorine-based and chlorine-releasing agents, respectively. The aim of this study was the comparative assessment of commercially available wound gels (octenilin®, Prontosan®, Lavanid®, Betadona®, ActiMaris®, Microdacyn60®, VeriforteTMmed) with regard to their effects on the secretion of distinct cytokines (IL-6, IL-8, IL-10), matrix-metalloproteinases as well as their potential to cause alterations in skin structure and apoptosis. Hence, tape-stripped human ex vivo skin biopsies were treated topically with wound gels and cultured for 48 h. Enzyme-linked immunosorbent assays and an enzyme activity assay of culture supernatants revealed that octenilin® demonstrates significantly broader anti-inflammatory and protease-inhibitory capacities than other wound gels. Further, haematoxylin & eosin as well as caspase-3 staining of treated biopsies showed that octenilin® does not alter skin morphology and shows the least interfering effect on human epidermal cells compared to untreated controls. Overall, this study clearly demonstrates totally different effects for several commercially available hydrogels in our wound model, which gives also new insight into their tissue compatibility and mode of action.

592 Ribosome profiling reveals distinct translation termination kinetics and efficiency between Fischer rat thyroid and human bronchial epidermal cells

592 Ribosome profiling reveals distinct translation termination kinetics and efficiency between Fischer rat thyroid and human bronchial epidermal cells

Transcriptional programming of immunoregulatory responses in human Langerhans cells.

Human epidermal Langerhans cells (LCs) maintain immune homeostasis in the skin. To examine transcriptional programming of human primary LCs during homeostasis, we performed scRNA-seq analysis of LCs before and after migration from the epidermis, coupled with functional assessment of their regulatory T cell priming capabilities. The analysis revealed that steady-state LCs exist in a continuum of maturation states and upregulate antigen presentation genes along with an immunoregulatory module including the genes IDO1, LGALS1, LAMTOR1, IL4I, upon their migration. The migration-induced transition in genomic state is accompanied by the ability of LCs to more efficiently prime regulatory T cell responses in co-culture assays. Computational analyses of the scRNAseq datasets using SCENIC and Partial Information Decomposition in Context identified a set of migration-induced transcription factors including IRF4, KLF6 and RelB as key nodes within a immunoregulatory gene regulatory network. These findings support a model in which efficient priming of immunoregulatory responses by LCs is dependent on coordinated upregulation of a migration-coupled maturation program with a immunoregulation-promoting genomic module.

Effects of collagen type ⅩⅦ α1 on epidermal stem cells in aging skin and the microRNA intervention mechanism

Effects of collagen type ⅩⅦ α1 on epidermal stem cells in aging skin and the microRNA intervention mechanism

Human fetal skin derived merkel cells display distinctive characteristics in vitro and in bio-engineered skin substitutes in vivo.

Human skin contains specialized neuroendocrine Merkel cells responsible for fine touch sensation. In the present study, we performed in-depth analysis of Merkel cells in human fetal back skin. We revealed that these Merkel cells expressed cytokeratin 20 (CK20), were positive for the neuroendocrine markers synaptophysin and chromogranin A, and the mechanosensitive ion channel Piezo2. Further, we demonstrated that Merkel cells were present in freshly isolated human fetal epidermal cells in vitro, and in tissue-engineered human dermo-epidermal skin substitutes 4 weeks after transplantation on immune-compromised rats. Merkel cells retained the expression of CK20, synaptophysin, chromogranin A, and Piezo2 after isolation and in culture, and in the skin substitutes after transplantation. Interestingly, we observed that in fetal skin and in skin substitutes, only Merkel cells were positive for CK8, while in culture, also non-Merkel cells showed positivity for CK8. In summary, human fetal Merkel cells showed phenotypical features confirming their cell identity. This findings are of pivotal importance for the future application of fetal tissue-engineered skin in clinics.

High-Sensitivity Immunohistochemistry Method Using a Combination of Fluorescent Nanoparticles and Tyramide Linker

Immunohistochemistry (IHC) is a useful method for visualizing antigens in tissues using target-specific antibodies in drug discovery and clinical research. Color reactions with horseradish peroxidase enzymatic activity have been used for IHC. This method has a long history and is highly reliable when applied to in vitro diagnostics. However, an IHC method with high sensitivity is required for low-expression target molecules in antibody-drug conjugates and immunotherapy. Therefore, many studies have been conducted in recent years to improve the sensitivity using highly brilliant and light-resistant phosphor-integrated dots (PIDs). In this method, streptavidin-coated PIDs are linked via biotinylated antibodies and the target molecules are visualized (PID-AbL). PID-AbL is more sensitive than IHC with chromophores. Hence, this study aimed to improve the sensitivity and quantitative results of IHC by combining PIDs with a biotinylated tyramide linker (PID-TL). PID-TL is able to detect ultralow human epidermal growth factor receptor 2-expressed cells. Moreover, PID-TL may be applied to human tissue samples as well as in vitro diagnostic kits. This new approach may help detect low-expression target molecules for clinical research.

MXD4/MAD4 Regulates Human Keratinocyte Precursor Fate

Deciphering the pathways that regulate human epidermal precursor cell fate is necessary for future developments in skin repair and graft bioengineering. Among them, characterization of pathways regulating the keratinocyte (KC) precursor immaturity versus differentiation balance is required for improving the efficiency of KC precursor exvivo expansion. In this study, we show that the transcription factor MXD4/MAD4 is expressed at a higher level in quiescent KC stem/progenitor cells located in the basal layer of human epidermis than in cycling progenitors. In holoclone KCs, stable short hairpin-RNA‒mediated decreased expression of MXD4/MAD4 increases MYC expression, whose modulation increases the proliferation of KC precursors and maintenance of their clonogenic potential and preserves the functionality of these precursors in three-dimensional epidermis organoid generation. Altogether, these results characterize MXD4/MAD4 as a major piece of the stemness puzzle in the human epidermis KC lineage and pinpoint an original avenue for exvivo expansion of human KC precursors.

The molecular mechanisms of vulpinic acid induced programmed cell death in melanoma.

Malignant melanoma is an aggressive skin tumor with a rapidly increasing incidence and there is not yet a successful treatment strategy. Vulpinic acid (VA) is derived from secondary metabolites from lichen species. In the current study, we, for the first time, investigated the anti-cancer effects of VA and the underlying mechanism VA induced programmed cell death in melanoma. The anti-cancer effects of VA on melanoma cells were evaluated by the xCELLigence system, flow cytometry, caspase-3 activity and RT-PCR analysis. Our results showed that VA had a strong anti-proliferative effect on A-375 melanoma cells without damaging human epidermal melanocyte cells. Additionally, VA promoted apoptotic cell death through G2/M arrest and the activation of both intrinsic and extrinsic apoptosis pathways according to the analysis of 88 genes associated with apoptosis by qRT-PCR. Our findings suggest that VA could become an alternative topical and transdermal treatment strategy in the treatment of maligned melanoma cancer. However, further investigations are needed to assess the underlying molecular mechanism of VA mediated apoptotic cell death in the treatment of melanoma.

Toxicity Analysis of Nano-Minimum Quantity Lubrication Machining—A Review

The lubrication properties of nanoparticles are of great interest to the manufacturing industry and led to the development of the nano-minimum quantity lubrication (NMQL) cooling strategy. To evaluate the sustainability characteristics of nano-minimum quantity lubrication, apart from analyzing the benefits of increasing machining efficiency, it is also essential to evaluate the potential detrimental effects of nanoparticles on human health and the environment. Existing literature provides substantial data on the benefits of nano-minimum quantity lubrication machining. However, the current literature does not provide researchers in the machining sector a comprehensive analysis of the toxicity of the nanoparticles used in nano-minimum quantity lubrication. This study aims to provide a comprehensive review that addresses the toxicity levels of the most frequently used nanoparticles in NMQL machining. To understand the impacts of nanoparticles on the human body and the environment, in vitro studies that evaluate the nanoparticles’ toxicity on human cells and in vitro/in vivo studies on other living organisms are considered. The results from toxicity studies on each of the chosen nanoparticles are summarized and presented in chronological order. The reviewed studies indicate transition metal dichalcogenides (MoS2 and WS2) exhibit very low toxicity when compared to other nanoparticles. The toxicity of hBN and AL2O3 nanoparticles varies depending on their lengths and crystalline structures, respectively. In conclusion, a chart that maps the toxicity levels of nanoparticles on seven different human cell lines (human lung epithelial cells (A549), human bronchial epithelial cells (Nl-20), AGS human gastric cells, human epidermal cells (HEK), human liver-derived cells (HepG2), human endothelial cells and human peripheral cells), representing exposures by inhalation, ingestion and dermal contact, was developed for easy and quick insights. This is the first attempt in open literature to combine the results of the experimental investigations of nano-minimum quantity lubrication cooling and the toxicity studies of nanoparticles, allowing researchers to make informed decisions in the selection of the most sustainable nanoparticles in the nano-minimum quantity lubrication machining process.

A reductionist approach to determine the effect of cell-cell contact on human epidermal stem cell differentiation

The balance between stem cell renewal and differentiation is determined by the interplay between intrinsic cellular controls and extrinsic factors presented by the microenvironment, or ‘niche’. Previous studies on cultured human epidermis have utilised suspension culture and restricted cell spreading to investigate regulation of differentiation in single keratinocytes. However, keratinocytes are typically adherent to neighbouring cells in vivo. We therefore developed experimental models to investigate the combined effects of cell-ECM adhesion and cell-cell contact. We utilized lipid-modified oligonucleotides to form clusters of keratinocytes which were subsequently placed in suspension to induce terminal differentiation. In this experimental model cell-cell contact had no effect on suspension-induced differentiation of keratinocytes. We next developed a high-throughput platform for robust geometrical confinement of keratinocytes to hexagonal ECM-coated islands permitting direct cell-cell contact between single cells. As in the case of circular islands, differentiation was stimulated on the smallest single hexagonal islands. However, the percentage of involucrin-positive cells on small bowtie islands was significantly lower than on single islands, demonstrating that cell-cell contact reduced differentiation in response to decreased substrate adhesion. None of the small bowtie islands contained two involucrin-positive cells. Rather, if one cell was involucrin-positive the other was involucrin-negative. This suggests that there is intrinsic asymmetry in the effect of cell-cell contact in decreasing differentiation. Thus, our reductionist approaches provide new insights into the effect of the niche on keratinocyte differentiation. Statement of significanceStem cell behaviour is regulated by a combination of external signals, including the nature of the adhesive substrate and cell-cell interactions. An understanding of how different signals are integrated creates the possibility of developing new biomaterials to promote tissue regeneration and broaden our understanding of skin diseases such as eczema and psoriasis, in which stem cell proliferation and differentiation are perturbed. In this study we have applied two methods to engineer intercellular adhesion of human epidermal stem cells, one involving lipid-modified DNA and the other involving hexagonal micropatterns. We show that the effect of cell-cell adhesion depends on cell-substrate adhesion and uncover evidence that two cells in equivalent environments can nevertheless behave differently.

514 ILF2 contributes to hyperproliferation of keratinocytes and skin inflammation in a KLHDC7B-DT dependent manner in psoriasis

Background: The extensive involvement of interleukin enhancer binding factor 2 (ILF2) in RNA stability and inflammation response is well documented. Aberrant LncRNAs expression contributes to psoriasis development and progression. However, little is known about the role of ILF2 in psoriasis. Objectives: Aimed to explore the role of ILF2 and KLHDC7B-DT in psoriasis. Methods: Long noncoding RNA (lncRNA) expression in psoriatic tissues was measured by lncRNA microarray and qRT-PCR. Normal human epidermal keratinocytes (NHEKs), HaCaT cells, and Ker-CT cells stimulated by M5 (IL-17A, IL-22, IL-1 α, oncostatin M, and TNF-α) were used to establish a psoriasis model in vitro.

Giving life to robotic skins

The skin of humanoid robots often lacks human tactility and the inherent self-repair capability of biological tissues. Recently, researchers have grown a living, self-healing skin on a robot finger by subsequent culturing of human dermal and epidermal cells. Here, we highlight the significance of this study alongside challenges toward developing biohybrid robots equipped with sensate and adaptive living robotic skins.

Effect of a hyaluronic acid-based mesotherapeutic injectable on the gene expression of CLOCK and Klotho proteins, and environmentally induced oxidative stress in human skin cells.

Normal circadian rhythms are essential to the repair mechanisms of oxidative stress implicated in skin aging. Given reports that hyaluronic acid (HA) homeostasis exhibits a different profile in chronological skin aging, as compared to environmental or extrinsic aging, an improved understanding of the way HA interacts with its surroundings, and the impact of HA injectables in replacing lost HA and encouraging rejuvenation, is of key benefit to skin aging treatments. The objectives of these current studies were twofold. Firstly, to demonstrate the in vitro effects of two lightweight hyaluronic-based injectables on the expression of CLOCK protein in human skin fibroblasts, and their effects on Klotho protein expression as a marker for circadian rhythms in a combined human keratinocyte and Merkel cell model. Secondly, to ascertain whether these findings could be correlated with in vitro effects on various environmental oxidative stress aging markers (blue light, UVA/UVB, Urban Dust, and IR exposures). Oxidative stress studies were aimed to highlight possible protective effects through different challenge conditions in two models, ex vivo human skin explants and in vitro monolayer cultures of normal human dermal fibroblasts (NHDF). The protective effects of the test products were evaluated against an increase of cyclobutene pyrimidine dimers (CPDs) abundance within epidermal section of ex vivo skin explants after UVA/UVB radiation; effects of blue light on gene expression from NHDFs fibroblasts; effects of pollutants (Urban dust, UbD) on gene expression in NHDFs fibroblasts; and an increase of reactive oxygen species (ROS) production by NHDFs fibroblasts after infrared-A radiation. Gene expression was assayed and analyzed utilizing microfluidic TaqMan qPCR arrays. CLOCK expression was measured in young and senescing NHDFs by immunostaining, and Klotho and melatonin expression by immunostaining in Merkel cell-enriched normal adult human epidermal cell cultures. In an aging culture of mixed keratinocyte and Merkel skin cells, activation of Klotho expression was induced by the application of both HA test products. Moreover, the HA products increase Klotho protein expression in both Merkel cells and keratinocytes. The observed positive effect of the tested products on melatonin receptors 1A and 1B expression in aging Merkel cell culture and keratinocytes is also interesting. HA-Y (developed for patients 25+ years old) stimulated melatonin receptors type 1B expression in aging cell cultures more strongly than HA-S (developed for patients 35-65 years old). In age (stressed) cells, a lower expression of Klotho protein and melatonin receptors 1A and 1B is apparent. The addition of HA-Y and HA-S stimulates their expression thus providing a "protective" effect. The blue light irradiation at 40 J/cm2 performed in NHDF fibroblast cultures led to a modification of the expression of several genes, all involved in mechanisms known to be modulated in case of solar radiation stress. Although these are preliminary findings, they are the first we know of that demonstrate HA facial injectables having a benefit and possibilities beyond the "physical filling" of the skin. As regards the beneficial effects against blue light-induced oxidative stress, and a return to cellular homeostasis, there is a need to conduct further and more precise investigations into HA-S. Furthermore, the benefit of these HA injectables (Novacutan®) in the modulation of oxidative stressed circadian rhythms widens their potential benefit.

Broad-spectrum cytotoxicity to cancer cells of BrevilaterinC from Brevibacillus laterosporus and its specific mechanism on human epidermal cancer cells.

Antimicrobial peptides (AMP) from Brevibacillus laterosporus have good prospects as clinical treatments for cancer. Nevertheless, details about their anticancer spectrum and mode of cytotoxicity remain poorly understood. A newly found AMP (namedBrevilaterin C) secreted by B. laterosporus S62-9 exhibited strong inhibition on almost cancer cell lines examined at a concentration of 8 µg/ml but was relatively safe for normal cells. We further systematically examined its cytotoxicity and mechanism toward human epidermal cancer cell A431. A dosage of 3 µg/ml of Brevilaterin C could significantly increase lactate dehydrogenase release of tumor cells. Moreover, it could remarkably increase the ratio of apoptosis and reactive oxygen species generation of A431, indicating effective induction of apoptosis. Moreover, the formation of JC-1 aggregates was effectively prevented by a low concentration of Brevilaterin C, indicating its effective induction of A431's apoptosis. Brevilaterin C exhibited broad-spectrum cytotoxicity to cancer cells, indicating a good potential prospect in the medical field.

Molecular mechanism of betulin palliative therapy for chronic obstructive pulmonary disease (COPD) based on P2X7 receptor target of gated ion channel

BackgroundThe aim of this study was to discover the molecular mechanism of betulin palliative therapy for chronic obstructive pulmonary disease (COPD) based on the P2X7 receptor target of gated ion channel.MethodsA COPD mouse model was developed. Changes in pulmonary ventilation function, pulmonary airway and vascular remodeling indicators, inflammatory cells, and inflammatory factors were determined after betulin intervention, and the pathological alterations of lung tissues were detected. An in vitro experimental model was constructed to observe the influence of betulin at varying concentrations on the proliferation of human bronchial epidermal cell line (16-HBE) cells and changes in inflammatory factors in cell supernatant. The expression levels of key proteins in 16-HBE cells transfected with overexpressed or silenced P2X7 genes were determined through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot.ResultsAfter betulin intervention, pulmonary ventilation function in the 20 mg/kg betulin and 40 mg/kg betulin groups was improved. Levels of white blood cells (WBCs), neutrophils (Ns), tumor necrosis factor (TNF), TNF-ɑ, interleukin (IL)-1β, and IL-6 in the 2 groups also decreased significantly (all P<0.05). The pathological changes in COPD mice were detected. After betulin intervention, the pathological injury of the lung was reduced, the pathological score decreased significantly, and the remodeling indicators of pulmonary airway and pulmonary vessels diminished remarkably (all P<0.05). Betulin had no effect on the proliferation of 16-HBE cells in vitro. After cigarette smoke extract (CSE) stimulation, the rate of survival for 16-HBE cells decreased significantly. After betulin treatment, the survival rate of 16-HBE cells augmented remarkably, and the levels of TNF-ɑ, IL-6, and IL-1β in cell supernatant reduced substantially (all P<0.05). 16-HBE with overexpression and knockdown of P2X7 was constructed. After being treated with betulin, the relative expression levels of messenger RNA (mRNA) of ERK, JNK, rho-associated protein kinase (ROCK), nuclear factor-κB (NF-κB), and p38 in 16-HBE cells with P2X7 overexpression or knockdown were decreased significantly (all P<0.05), but the above indicators were largely unchanged (all P>0.05).ConclusionsBetulin relieved lung pathological injury, ameliorated lung ventilation function, and diminished the level of inflammatory factors in COPD mice, playing a therapeutic role via the P2X7 signaling pathway.

TRPA1 promotes melanosome phagocytosis in keratinocytes via PAR-2/CYLD axis

BackgroundKeratinocytes are recipients of melanosomes. Although the chemical basis of melanogenesis is well documented, the molecular mechanism of melanosome transfer must be elucidated. TRPA1 is a member of the transient receptor potential A subfamily. Previous studies have shown that inhibition of TRPA1 activity reduces melanin synthesis in human epidermal melanocytes; however, the mechanism remains unknown. ObjectiveThis study aimed to investigate the roles and mechanism(s) of action of TRPA1 in keratinocytes. MethodsThe correlation between TRPA1 expression levels and the ability of keratinocytes to phagocytize melanosomes was examined using melanin silver staining. TRPA1 depleted human epidermal keratinocytes and keratinocyte cell lines HaCaT were established using adenovirus-expressing shRNAs against TRPA1. The effects of TRPA1 on keratinocytes and HaCaT cells were determined using cell-based analyses, including light stimulation, calcium imaging, melanin phagocytosis, immunoblotting, and co-immunoprecipitation assays. The degree of epidermal pigmentation was determined in a guinea pig model. ResultsTRPA1 mediated the phagocytic activity of keratinocytes. TRPA1 knockdown markedly suppressed melanosome transport to keratinocytes. Mechanistically, TRPA1 was required for PAR-2-induced melanosome phagocytosis in keratinocytes. Furthermore, TRPA1 activation indirectly stabilized microtubules by promoting the competitive binding of CYLD and acetylated α-tubulin. In addition, bortezomib (PS-341), a proteasome inhibitor, increased TRPA1 and CYLD expression and promoted phagocytic activity both in vitro and in vivo. ConclusionsOur findings firstly suggest that TRPA1 promotes melanosome transport in keratinocytes and reveal that TRPA1 is a regulator of PAR-2 activation and microtubule stability via the PAR-2/CYLD axis.

Study on the effects of alternating capacitive electric fields with different frequencies on promoting wound healing

Some researches to facilitate wound healing by using electrical stimulation are based on electric current stimulation, which may cause secondary damage and the imbalance of the microenvironment in vivo. In this study, alternating capacitive electric field (ACEF) was applied via a self-designed system so as to avoid direct contact with cells and to maintain stable microenvironment for cell growth. The influences of 58 ​mV/mm ACEFs with various frequencies of 10, 60 and 110 ​Hz on epidermal cells, fibroblasts and macrophages which involve in wound healing were comprehensively explored. The results suggested that ACEFs of 10, 60 and 110 ​Hz all significantly promoted the proliferation of human dermal fibroblasts (HDFs) and human epidermal keratinocyte cell line (HaCaT) cells, and 60 ​Hz ACEF furtherly accelerated the migration of these two kinds of cells. Moreover, ACEFs of all different studied frequencies facilitated M2-type polarization of macrophages, and YAP/TAZ expression of macrophages were enhanced under the stimulations of 10 and 60 ​Hz ACEFs. The enhancements in cell activity, migration rate and M2-type polarizability indicated that 58 ​mV/mm ACEFs especially at 60 ​Hz possessing potentially affirmative applications for wound healing without the risks of secondary damage and microenvironment imbalance.

ILF2 Contributes to Hyperproliferation of Keratinocytes and Skin Inflammation in a KLHDC7B-DT-Dependent Manner in Psoriasis.

Background: The extensive involvement of interleukin enhancer binding factor 2 (ILF2) in RNA stability and the inflammatory response is well documented. Aberrant long noncoding RNA (lncRNA) expression contributes to the pathogenesis of psoriasis. However, little is known about the role of ILF2 in psoriasis. Objective: To investigate the role of ILF2 and KLHDC7B-DT in psoriasis. Methods: LncRNA expression in psoriatic tissues was measured by lncRNA microarray and qRT–PCR. Normal human epidermal keratinocytes (NHEKs), HaCaT cells, and Ker-CT cells stimulated with M5 (IL-17A, IL-22, IL-1α, oncostatin M, and TNF-α) were used to establish a psoriasis model in vitro. Fluorescence in situ hybridization was used to detect the distribution of KLHDC7B-DT and ILF2 in keratinocytes. The proliferative effects of KLHDC7B-DT and ILF2 on keratinocytes were demonstrated by EdU assay and flow cytometry. ELISA was used to detect the secretion levels of cytokines. RNA pull-down and RNA immunoprecipitation (RIP) were used to detect the direct binding of KLHDC7B-DT with ILF2. Western blotting was used to detect the proteins related to STAT3/JNK signalling pathways. Results: ILF2 and KLHDC7B-DT were significantly overexpressed in psoriatic tissues and M5-induced keratinocytes. KLHDC7B-DT promoted the proliferation of keratinocytes and induced the secretion of IL-6 and IL-8. KLHDC7B-DT could directly bind to ILF2 and activate the STAT3 and JNK signalling pathways. KLHDC7B-DT expression was regulated by ILF2. M5-induced proliferation and inflammatory cytokine secretion in keratinocytes was inhibited after ILF2 knockdown. Furthermore, we found that ILF2 promoted keratinocyte proliferation and the inflammatory response in a KLHDC7B-DT-dependent manner. Conclusions: ILF2 and KLHDC7B-DT are involved in the hyperproliferation of keratinocytes and skin inflammation in psoriasis. In addition, ILF2 functions in a KLHDC7B-DT-dependent manner.

Expression of Retinal G Protein-Coupled Receptor, a Member of the Opsin Family, in Human Skin Cells and Its Mediation of the Cellular Functions of Keratinocytes.

Background: Photoreceptive proteins play critical physiological roles in human skin cells. The retinal G protein-coupled receptor (RGR) is a photoisomerase in the human retina, but its expression and cellular functions in human skin cells have not been reported. Objectives: We aimed to detect RGR expression in various skin cells and evaluate its regulation of the cellular functions of keratinocytes. Methods: The expression, distribution, and subcellular location of the RGR in normal human epidermal keratinocytes and cells with pathological conditions including psoriasis, seborrheic keratosis, and squamous cell carcinoma were determined using microscopic tools (immunohistochemical staining, immunofluorescence staining, and immunoelectron microscopy) and Western blotting (WB). The protein levels of the RGR in primary human melanocytes, keratinocytes, and fibroblasts isolated from the neonatal foreskin were measured by WB. The expression and subcellular localization of the RGR in these cells were detected by immunofluorescence staining under a fluorescence microscope and laser scanning confocal microscope. Additionally, the levels of RGR expression in normal keratinocytes exposed to ultraviolet (UV)-A or total ultraviolet radiation (UVR) in the presence or absence of all-trans-retinal were measured by WB. Furthermore, the effects of the RGR on human keratinocyte functions including proliferation, migration, and apoptosis were evaluated using the Cell Counting Kit 8, wound healing, and Transwell assays after reducing the RGR mRNA level in keratinocytes using small interfering RNA technology. Results: The RGR was primarily located in the epidermal basal and spinous layers and skin appendages. Its expression increased in psoriatic lesions, seborrheic keratosis, and squamous cell carcinoma. Confocal microscopy showed that the RGR was located in the cell membrane and nucleus of keratinocytes, melanocytes, and fibroblasts. Keratinocytes had a higher expression of the RGR than melanocytes and fibroblasts, as well as nuclear expression, according to nuclear/cytoplasmic fractionation. Colloidal gold immunoelectron microscopy technology further confirmed that the RGR is mainly located in the nucleoplasm and mitochondria and is scattered in the cytoplasm and other organelles in the epidermal keratinocytes. Notably, RGR knockdown in keratinocytes led to the inhibition of cell proliferation and migration, augmenting cell apoptosis. Conclusions: This study is the first to demonstrate the presence of RGR in the human skin. Our findings indicate that the RGR may play a critical role in the physiological function of epidermal keratinocytes.