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Decoy receptor 3 (DcR3) is a soluble receptor for Fas ligand, LIGHT and TL1A, but it also exerts effector functions. Previously, we found that DcR3 is upregulated in the serum and lesional skin of patients with psoriasis and is upregulated by EGFR activation in proliferating primary human epidermal keratinocytes. However, the functional role of intracellular DcR3 in keratinocyte differentiation is still incompletely defined. Herein, primary cultured human epidermal keratinocytes were differentiated by phorbol 12-myristate 13-acetate (PMA) treatment, calcium treatment and cell confluence, which are three standard in vitro differentiation models. We found that the constitutive expression of the DcR3 gene and protein was progressively suppressed during terminal differentiation of keratinocytes. These changes were correlated with downregulation of EGFR activation during keratinocyte differentiation. EGFR inhibition by gefitinib further decreased confluence-induced suppression of DcR3 mRNA expression, and, vice versa, knocking down DcR3 expression attenuated EGFR and EGFR ligand expression as well as EGFR activation. Under conditions without a change in cell growth, DcR3 silencing reduced the expression of involucrin and transglutaminase 1 but enhanced the induction of the terminal differentiation markers keratin 10 and loricrin. Of note, DcR3 interacted with PKCα and PKCδ and enhanced PKC activity. In keratinocytes with PKCα and PKCδ silencing, differentiation markers were differentially affected. In conclusion, DcR3 expression in keratinocytes is regulated by EGFR and forms a positive feedback loop to orchestrate constitutive EGFR and PKC activity. During differentiation, DcR3 is downregulated and involved in modulating the pattern of terminal differentiation.

Thioredoxin-interacting protein (TXNIP), also known as Vitamin-D upregulated protein-1 (VDUP-1), interacts with thioredoxin to regulate redox responses and participates in diverse disorders including metabolic, cardiovascular, inflammatory and malignant diseases. Psoriasis is characterized by chronic skin inflammation and an aberrant pattern of keratinocyte differentiation. Clinically, psoriasis is associated with various cardiometabolic comorbidities but studies on TXNIP's biological role in skin disorders are limited. In this study, we investigated TXNIP expression in psoriasis and its regulation in normal human epidermal keratinocytes (NHEKs), and then explored how TXNIP regulated skin keratinocyte differentiation to determine its role in psoriasis pathogenesis. Our immunohistochemical study demonstrated extensive TXNIP expression in the upper and lower epidermis of psoriasis compared to predominant TXNIP expression in the basal layer of normal skin. 1, 25-dihydroxyvitamin D

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.

UV irradiation can injure the epidermis, resulting in sunburn, inflammation, and cutaneous tissue disorders. Previous studies demonstrate that EGFR in keratinocytes can be activated by UVB and contributes to inflammation. Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme and plays an essential role in DNA repair under moderate stress. In this study, we set out to understand how PARP-1 regulates UVB irradiation-induced skin injury and interplays with EGFR to mediate the inflammation response. We found that PARP-1 deficiency exacerbated the UVB-induced inflammation, water loss, and back skin damage in mice. In human primary keratinocytes, UVB can activate PARP-1 and enhance DNA damage upon PARP-1 gene silencing. Moreover, PARP-1 silencing and PARP inhibitor olaparib can suppress UVB-induced COX-2 and MMP-1 expression, but enhance TNF-α and IL-8 expression. In addition, EGFR silencing or EGFR inhibition by gefitinib can decrease UVB-induced COX-2, TNF-α, and IL-8 expression, suggesting EGFR activation via paracrine action can mediate UVB-induced inflammation responses. Immunoblotting data revealed that PARP-1 inhibition decreases UVB-induced EGFR and p38 activation. Pharmacological inhibition of p38 also dramatically led to the attenuation of UVB-induced inflammatory gene expression. Of note, genetic ablation of PARP-1 or EGFR can attenuate UVB-induced ROS production, and antioxidant NAC can attenuate UVB-induced EGFR-p38 signaling axis and PARP-1 activation. These data suggest the regulatory loops among EGFR, PARP-1, and ROS upon UVB stress. PARP-1 not only serves DNA repair function but also orchestrates interactions to EGFR transactivation and ROS production, leading to p38 signaling for inflammatory gene expression in keratinocytes.

The galectin family comprises β-galactoside-binding proteins widely expressed in many organisms. There are at least 16 family members, which can be classified into three groups based on their carbohydrate-recognition domains. Pleiotropic functions of different galectins in physiological and pathological processes through extracellular or intracellular actions have been revealed. In the skin, galectins are expressed in a variety of cells, including keratinocytes, melanocytes, fibroblasts, dendritic cells, lymphocytes, macrophages and endothelial cells. Expression of specific galectins is reported to affect cell status, such as activation or death, and regulate the interaction between different cell types or between cells and the extracellular matrix. In vitro cellular studies, in vivo animal studies and studies of human clinical material have revealed the pathophysiologic roles of galectins in the skin. The pathogenesis of diverse non-malignant skin disorders, such as atopic dermatitis, psoriasis, contact dermatitis and wound healing, as well as skin cancers, such as melanoma, squamous cell carcinoma, basal cell carcinoma and cutaneous haematologic malignancy can be regulated by different galectins. Revelation of biological roles of galectins in skin may pave the way to future development of galectin-based therapeutic strategies for skin diseases.

Background Galectin-3 is widely expressed in many immunocytes and epithelial cells including skin keratinocytes. Galectin-3 can regulate immunological or inflammatory processes and plays a proinflammatory role in some disease models. Galectin-3 has a role in disorders related to ultraviolet (UV) photodamage such as apoptosis, skin squamous cell carcinoma and basal cell carcinoma. However, the evidence of galectin-3 in UVB-induced skin inflammation is still limited and the underlying molecular mechanism remains elusive. Objective We aimed to investigate the effects of galectin-3 in human epidermal keratinocytes and in mice after UVB irradiation. Methods Primary human epidermal keratinocytes with galectin-3 knockdown were used as the in vitro model. ELISA, QPCR, and western blotting were applied to evaluate the released cytokine, mRNA and protein expression. Histologic analysis, measurement of erythema and transepidermal water loss (TEWL) were applied to evaluate UVB-induced skin damage in galectin-3 knockout mice. Results In UVB-irradiated human keratinocytes, galectin-3 knockdown downregulated the UVB-induced ASC crosslinking, cleavage of caspase-1, and formation of active IL-1β. Galectin-3 knockdown also decreased UVB-induced production of reactive oxygen species, p38 phosphorylation, and COX2 expression in human keratinocytes. After four days of UVB irradiation, galectin-3 knockout mice showed reduced gross erythema, histologic features of tissue inflammation, quantified levels of erythema and TEWL compared to wild type mice. The skin tissue lysate also showed less expression of active IL-1β and COX2 in galectin-3 knockout mice. Conclusion Galectin-3 may play a positive regulatory role in UVB-induced skin inflammation.

Melanoma represents less than 5% of skin cancers, but is the most lethal, mainly because of its high-metastatic potential and resistance to various therapies. Therefore, it is important to develop effective treatments, especially chemotherapeutic drugs with cytotoxicity, anti-metastaticity, and few side effects. One such natural product is hispidulin, a flavone distributed in plants of the Asteraceae. Previous studies have demonstrated that hispidulin has various pharmacological benefits, such as anti-tumor, anti-inflammation, and anti-allergic effects. This study aims to explore the effects of hispidulin against melanoma in vitro and in vivo. Results revealed that hispidulin selectively decreased the cell viability of A2058 cells in a dose- and time-dependent manner. Hispidulin induced cells accumulated in the sub-G1 phase via activating caspase 8 and 9, increased cleaved caspase 3, and cleaved PARP expression. Hispidulin was able to decrease AKT and ERK phosphorylation, which facilitated cell growth and survival. Moreover, hispidulin promoted reactive oxygen species generation in cells and suppressed cell migration through downregulated matrix metalloproteinase-2 expression. Hispidulin significantly inhibited tumor growth in a xenograft model. Based on these results, hispidulin produces its anti-melanoma effects by inducing cancer cell apoptosis and reducing its migration. Therefore, we suggest hispidulin as a potent therapeutic candidate for melanoma treatment.

Solar ultraviolet (UV) light has been recognized as the important environmental hazard and contributes to diverse skin damage such as cell death, photoageing and even carcinogenesis. Revelation of harmful responses attributed to UVA radiation has promoted the development of photoprotective agents against UVA-induced skin damage. In the present study, we tried to evaluate the potential protective effects of a synthetic green fluorescent protein (GFP) chromophore derivative, 4-chlorobenzyldene-1, 2-dimethylimidazolinone (Cl-BDI, called TC-22) on UVA- and UVB- induced stress responses in skin. The HaCaT keratinocytes were used to evaluate the cellular effects. Zebrafish (Danio rerio), which is regarded as a useful and cost-effective alternative to some mammalian models, was applied as the in vivo animal model. In HaCaT keratinocytes, TC-22 was able to obviously decrease UVA-induced cell death. Dissection of the UVA-induced signalling pathways revealed that TC-22 could suppress the activation of JNK and caspase 3, but not of ERK and p38. Reduction of UVA-induced cleavage of caspase 3 and sub-G1 phase accumulation by pretreatment of TC-22 was also observed. In zebrafish, we showed that UVA irradiation could decrease the survival and hatching rate, suppress heart beats of embryos and enhance the pigmentation of larvae. Pretreatment of TC-22 could significantly reverse UVA-induced the suppression in hatching of eggs and heart beating of embryos and also lowered the UVA-induced pigmentation in zebrafish. Collectively, we demonstrate that TC-22, a GFP chromophore derivative, can ameliorate the UVA-induced stress responses in both epidermal keratinocytes and zebrafish, suggesting the potential use of TC-22 in photoprotection in the future.

Background B lymphocyte-induced maturation protein-1 (BLIMP1) is a transcriptional repressor, and plays a crucial role in the regulation of development and functions of various immune cells. Currently, there is limited understanding about the regulation of BLIMP1 expression in keratinocytes and crosstalk between EGFR and BLIMP1 in skin homeostasis. Objective The aim of the study was to investigate the regulation and functional link between EGFR and BLIMP1 in human epidermal keratinocytes. Methods Immunoblotting and Q-PCR were used to determine the molecular mechanism of BLIMP1 expression induced by EGFR in primary human epidermal keratinocytes (NHEK) and HaCaT cells. In functional assay, effects of BLIMP1 knockdown on EGFR-mediated cytokine production, differentiation, and migration in NHEK were evaluated by Q-PCR, ELISA, immunoblotting, and/or wound-healing assay. Results EGFR activation by EGFR ligands could upregulate the protein and mRNA levels of BLIMP1 in NHEK and HaCaT cells. This effect was dependent on PKC, p38, and ERK activation. Additionally, the stability of BLIMP1 protein was under the control of the proteasome and lysosome degradation systems. EGF could also upregulate BLIMP1 expression in skin squamous cell carcinomas. In addition, BLIMP1 knockdown enhanced the EGFR-mediated IL8, CXCL5 and IL6 gene expression and keratinocyte migration, but reduced the EGFR-mediated suppression of differentiation marker K10. Conclusions Our findings shed new insights into the regulation of BLIMP1 expression by EGFR-mediated gene transcription and proteasome/lysosome-mediated degradation in keratinocytes. Functionally, BLIMP1 is a negative regulator of EGF-induced inflammation and migration in keratinocytes, and exerts a gene-specific regulation on keratinocyte differentiation.

Tumor metastasis is the major obstacle for cancer treatment. Previous studies have shown that butein exhibits antiangiogenesis property and anticancer effects in different kinds of human cancer cells. However, the effects of butein on metastasis and energy metabolism of cancer cells are mostly unknown. This study showed that butein significantly inhibited invasion of cancer cells without acting in a cytotoxic fashion. It was further demonstrated that butien dramatically suppressed cancer metastasis by an in vivo CAM-intravasation model. Additionally, butein concentration-dependently repressed the expression and activity of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA). The study indicated that butein may repress MMP-9 and uPA proteolytic activities and subsequently inhibit cancer metastasis via Akt/mTOR/p70S6K translational machinery. Moreover, butein may partly suppress cancer metastasis by down-regulating ATP synthesis via both oxidative and glycolytic metabolism. The results suggest that butein is a potential antimetastatic agent worthy of further development for cancer treatment.

Zeaxanthin is the dihydroxy carotenoid and is distributed in our daily foods. Various natural carotenoids, including zeaxanthin, have been shown to inhibit proliferation of several types of cancer cells, but available data on the effect of zeaxanthin on skin fibroblasts and melanoma cells are limited. Platelet-derived growth factor (PDGF) functions as a chemotactic factor for dermal fibroblasts and plays an important role in the progression of melanoma. In this study, we investigated the effects of zeaxanthin on the migration of skin fibroblasts induced by PDGF-BB and melanoma cells. We demonstrated that zeaxanthin inhibited PDGF-BB-induced skin fibroblast migration on collagen and gelatin by a modified Boyden chamber system. The electric cell-substrate impedance sensing (ECIS) method also showed similar inhibitory effects of zeaxanthin on the migration of fibroblasts. In functional studies, zeaxanthin decreased melanoma- induced fibroblast migration in a non-contact coculture system and also the migration stimulated by melanoma-derived conditioned medium. Further analysis showed that zeaxanthin attenuated PDGF-BB and melanoma-conditioned medium induced phosphorylation of PDGFR-b and MAP kinase in a concentration-dependent manner in human skin fibroblasts. However, these effects did not result from direct interaction of zeaxanthin with PDGF-BB. Thus, our results provide the first evidence showing that zeaxanthin is an effective inhibitor of migration of stromal fibroblasts induced by PDGF-BB and melanoma cells and this effect may further support its antitumor potential.

Ethnopharmacological relevance Soy has been used in traditional Chinese medicine for thousands of years for its health and nutritional benefits, as well as to treat and care for the skin. Advanced skin care research has shown that soy isoflavone and genistein are effective in reducing damage to the skin from the sun. Aim of the study To study the protective effects of isoflavone extract from soybean cake against the UVB-induced skin damage. Materials and methods The in vitro effects and possible mechanisms of soybean extract on UVB protection were determined in HaCaT cells. In the in vivo study, ICR-Foxn/ nu mice were irradiated with UVB. The epidermal thickness, catalase and the expressions of cyclooxygenase-2 (COX-2) and proliferating cell nuclear antigen (PCNA) were detected to evaluate the antioxidant and anti-inflammatory activities of the isoflavone extract. Results Our in vitro studies showed that UVB-induced HaCaT cell death and the phosphorylation of p38, JNK, and ERK1/2 decreased in the presence of isoflavone extract. In the in vivo studies, we found that the topical application of isoflavone extract before UVB irritation decreased the epidermal thickness and the expressions of COX-2 and PCNA and increased catalase concentration. These results showed anti-photoaging effect of isoflavone extract from soybean cake involved the inhibition of UVB-induced apoptosis and inflammation. Conclusions It is shown that isoflavone extract from soybean cake may be functional cosmeceutical candidate for skin photoaging.

Chrysin (5,7-dihydroxyflavone), a natural flavonoid occurring in various plants and foods such as propolis and honey, reportedly opposes inflammation and carcinogenesis, but has rarely been applied in skin care. This study, therefore, aimed to explore the roles of chrysin in protection against UV-induced damage in HaCaT keratinocytes. Results showed that chrysin can attenuate apoptosis, reactive oxygen species (ROS) production, and cyclooxygenase 2 (COX-2) expression induced by UVB and UVA. Chrysin predominantly reversed the down-regulation of aquaporin 3 (AQP-3) by UVB. It predominantly reversed JNK activation and also mildly inhibited p38 activation triggered by UVA and UVB. Animal studies revealed that chrysin's topical application demonstrated efficient percutaneous absorption and no skin irritation. Overall, results demonstrated significant benefits of chrysin on the protection of keratinocytes against UVA- and UVB-induced injuries and suggested its potential use in skin photoprotection.

Cornification, the terminal differentiation of keratinocytes, is a special form of programmed cell death in the skin. In this article, we report that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) can induce the expression of the keratinocyte differentiation markers involucrin and type 1 transglutaminase in normal human epidermal keratinocytes. The induction of differentiation occurs mainly under the activation of caspases 3 and 8, and apoptosis can also be triggered. Inhibition of these apoptotic caspases attenuates both apoptosis and differentiation of keratinocytes caused by TRAIL but barely affects the induction of differentiation caused by calcium and phorbol 12-myristate 13-acetate. Differential regulation of extracellular signal–regulated kinase and p38 activation by TRAIL is also observed. Moreover, the degradation of p63 is induced by TRAIL-elicited caspase activation. However, the existence of p63 is essential for the initiation of keratinocyte differentiation by TRAIL because knockdown of ΔNp63 decreases TRAIL-induced differentiation. Taken together, our results suggest that TRAIL can be an inducer of both differentiation and apoptosis in human keratinocytes, and that caspases critically mediate these processes. This study identifies a new role of apoptotic caspases for terminal differentiation of keratinocytes and further elucidates the molecular pathways involved in this unique model of cell death.

Soy isoflavones, found in soybean and soybean products, have been reported to possess many physiological activities such as antioxidant activity, inhibition of cancer cell proliferation, reduction of cardiovascular risk, prevention of osteoporosis and alleviation of postmenopausal syndrome. In our previous study, soy isoflavone extract ISO-1 (containing 12 soy isoflavones) from soybean cake was demonstrated to prevent skin damage caused by UVB exposure. In this study, soy isoflavone extract from soybean cake was further purified and evaluated for the protective effects on UVB-induced damage. The results revealed that Fraction 3, which contains the aglycone group (daidzein, genistein and glycitein) and acetylglucoside group (acetyldaidzin, acetylgenistin and acetylglycitin) of soy isoflavones, could inhibit UVB-induced death of human keratinocytes and reduce the level of desquamation, transepidermal water loss (TEWL), erythema and epidermal thickness in mouse skin. Furthermore, topical application of Fraction 3 increased the activity of catalase and suppressed cyclooxygenase-2 (COX-2) and proliferating cell nuclear antigen (PCNA) expression in mice exposed to UVB. In addition, in comparison with ISO-1 and genistein, the Fraction 3 possessed much greater protective effects on both UVB-induced oxidative stress and keratinocyte death than other fractions. Therefore, the soy isoflavone extract Fraction 3 from soybean cake is a desirable anti-photoaging agent for skin care.

Myricetin is a flavonoid similar to quercetin, which is commonly found in natural foods such as berries, vegetables, teas, wine, and herbs. It is considered to be an antioxidant which is capable of quenching photoaging-causing free radicals within the skin. In this study, we investigated the mechanisms underlying protective effect of myricetin on ultraviolet-B (UVB)-induced damage to keratinocytes. We found that myricetin concentration-dependently attenuated UVB-induced keratinocyte death as determined by a cell viability assay. Pretreatment with myricetin also reduced the UVB-induced malondialdehyde level. Moreover, UVB-induced H2O2 generation in keratinocytes was inhibited by myricetin according to flow cytometry, suggesting that myricetin can act as a free radical scavenger when keratinocytes experience photodamage. Furthermore, UVB-induced activation of c-jun-NH2 terminal kinase (JNK) in keratinocytes was inhibited by myricetin. UVB-induced pre-G1 phase arrest leading to apoptotic changes in keratinocytes was blocked by myricetin. Taken together, the protective mechanisms of keratinocyte by myricetin against UVB-induced photodamage occur by the inhibition of UVB-induced intracellular hydrogen peroxide production, lipid peroxidation and JNK activation. Therefore, myricetin is suitable for further development as an anti-aging agent for skin care.

IL-17 has an important role in the immunopathogenesis of autoimmune diseases, and spleen tyrosine kinase (Syk) has been implicated as a critical molecule in the signaling pathways of various immunoreceptors. Chemokine (C-C motif) ligand 20 (CCL20) interacts with chemokine (C-C motif) receptor 6 to recruit IL-17-producing cells into the skin to promote progression of psoriasis. Herein we investigate how Syk regulates IL-17 signaling to affect CCL20 expression in primary human epidermal keratinocytes. We found that IL-17 can induce CCL20 expression and activate TAK, IKK, NF-κB, c-Jun N-terminal kinase, and Syk. Data of TAK inhibitor and Syk small interfering RNA (siRNA) indicate Syk being an upstream molecule of TAK in IL-17-elicited signaling. The promoter activity assay combined with site-directed mutagenesis showed that IL-17-elicited CCL20 upregulation is depending on the Syk-mediated NF-κB pathway. Immunoprecipitation also indicated the interaction of Syk with signal molecules of IL-17R, such as TRAF6 and Act1, under IL-17A stimulation. However, the essential signaling events including TRAF6 interaction with Act1 and TRAF6 polyubiquitination under IL-17A stimulation were diminished by Syk siRNA and pharmacologically inhibiting Syk. Taken together, we identify Syk as an upstream signaling molecule in IL-17A-induced Act1-TRAF6 interaction in keratinocytes, and inhibition of Syk can attenuate CCL20 production, which highlights Syk as a potential therapeutic target for inflammatory skin diseases such as psoriasis.

Spleen tyrosine kinase (Syk), a nonreceptor tyrosine kinase, was initially identified as a crucial regulator in proximal immunoreceptor signaling. Additional studies have revealed its pleiotropic roles, and drugs targeting Syk are under development for inflammatory diseases. Syk expression in the skin has been detected, but its functions in the skin are still unknown. Here, we found that Syk phosphorylation and expression in primary human keratinocytes decreased gradually along with terminal differentiation. Human skin specimens showed similar in vivo patterns. Syk inhibitors or knockdown of Syk increased the expression of differentiation markers under in vitro differentiation models. Furthermore, EGFR activation prominently induced Syk phosphorylation, which could be inhibited by the EGFR inhibitor gefitinib or knockdown of EGFR. The Src inhibitor also partially attenuated EGF-induced phosphorylation of Syk. However, Syk inhibition suppressed EGF-induced phosphorylation of EGFR. Immunoprecipitation and confocal microscopy further revealed the increased molecular interaction between EGFR and Syk after EGF stimulation. This study unravels the role of Syk in EGFR-mediated signaling and reveals regulatory roles of Syk in keratinocyte differentiation, suggesting the clinical potential of topical or systemic Syk inhibitors in the treatment of skin diseases with aberrant differentiation.