Decoy receptor 3 is involved in epidermal keratinocyte commitment to terminal differentiation via EGFR and PKC activation.

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.

Blimp-1 Upregulation by Multiple Ligands via EGFR Transactivation Inhibits Cell Migration in Keratinocytes and Squamous Cell Carcinoma.

B lymphocyte-induced maturation protein-1 (Blimp-1) 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 Blimp-1 expression and cellular functions in keratinocytes and cancer cells. Previously we demonstrated that EGF can upregulate Blimp-1 gene expression in keratinocytes, playing a negative role in regulation of cell migration and inflammation. Because it remains unclear if Blimp-1 can be regulated by other stimuli beyond EGF, here we further investigated multiple stimuli for their regulation of Blimp-1 expression in keratinocytes and squamous cell carcinoma (SCC). We found that PMA, TNF-α, LPS, polyIC, H2O2 and UVB can upregulate the protein and/or mRNA levels of Blimp-1 in HaCaT and SCC cells. Concomitant EGFR activation was observed by these stimuli, and EGFR inhibitor gefitinib and Syk inhibitor can block Blimp-1 gene expression caused by PMA. Reporter assay of Blimp-1 promoter activity further indicated the involvement of AP-1 in PMA-, TNF-α-, LPS- and EGF-elicited Blimp-1 mRNA expression. Confocal microscopic data indicated the nuclear loclization of Blimp-1, and such localization was not changed by stimuli. Moreover, Blimp-1 silencing enhanced SCC cell migration. Taken together, Blimp-1 can be transcriptionally upregulated by several stimuli in keratinocytes and SCC via EGFR transactivation and AP-1 pathway. These include growth factor PMA, cytokine TNF-α, TLR ligands (LPS and polyIC), and ROS insults (H2O2 and UVB). The function of Blimp-1 as a negative regulator of cell migration in SCC can provide a new therapeutic target in SCC.

PARP-1 involves in UVB-induced inflammatory response in keratinocytes and skin injury via regulation of ROS-dependent EGFR transactivation and p38 signaling.

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.

Melatonin Supplementation for Children With Atopic Dermatitis and Sleep Disturbance: A Randomized Clinical Trial.

IMPORTANCE: Sleep disturbance is common in children with atopic dermatitis (AD), but effective clinical management for this problem is lacking. Reduced levels of nocturnal melatonin were found to be associated with sleep disturbance and increased disease severity in children with AD. Melatonin also has sleep-inducing and anti-inflammatory properties and therefore might be useful for the management of AD. OBJECTIVE: To evaluate the effectiveness of melatonin supplementation for improving the sleep disturbance and severity of disease in children with AD. DESIGN, SETTING, AND PARTICIPANTS: This randomized clinical trial used a double-blind, placebo-controlled crossover design to study 73 children and adolescents aged 1 to 18 years with physician-diagnosed AD involving at least 5% of the total body surface area. The study was conducted at the pediatric department of a large tertiary care hospital in Taiwan from August 1, 2012, through January 31, 2013. Forty-eight children were randomized 1:1 to melatonin or placebo treatment, and 38 of these (79%) completed the cross-over period of the trial. Final follow-up occurred on April 13, 2013, and data were analyzed from January 27 to April 25, 2014. Analyses were based on intention to treat. INTERVENTIONS: Melatonin, 3 mg/d, or placebo for 4 weeks followed by a 2-week washout period and then crossover to the alternate treatment for 4 weeks. MAIN OUTCOMES AND MEASURES: The primary outcome was AD severity evaluated using the Scoring Atopic Dermatitis (SCORAD) index, with scores ranging from 0 to 103 and greater scores indicating worse symptoms. Secondary outcomes included sleep variables measured by actigraphy, subjective change in sleep and dermatitis, sleep variables measured by polysomnography, nocturnal urinary levels of 6-sulfatoxymelatonin, and serum IgE levels. RESULTS: After melatonin treatment among the 48 children included in the study, the SCORAD index decreased by 9.1 compared with after placebo (95% CI, -13.7 to -4.6; P < .001), from a mean (SD) of 49.1 (24.3) to 40.2 (20.9). Moreover, the sleep-onset latency shortened by 21.4 minutes after melatonin treatment compared with after placebo (95% CI, -38.6 to -4.2; P = .02). The improvement in the SCORAD index did not correlate significantly with the change in sleep-onset latency (r = -0.04; P = .85). No patient withdrew owing to adverse events, and no adverse event was reported throughout the study. CONCLUSIONS AND RELEVANCE: Melatonin supplementation is a safe and effective way to improve the sleep-onset latency and disease severity in children with AD. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01638234.

Atopic dermatitis, melatonin, and sleep disturbance.

BACKGROUND AND OBJECTIVES: Sleep disturbance is common in patients with atopic dermatitis (AD). However, studies have largely been questionnaire-based, and the pathophysiology remains unclear. The aims of this study were to determine objective characteristics of sleep disturbance in children with AD and explore contributing factors and clinical predictors. METHODS: Sleep parameters were measured by actigraphy and polysomnography in 72 patients with AD and 32 controls ages 1 to 18 years. Urinary 6-sulfatoxymelatonin levels, serum cytokines, and total and allergen-specific immunoglobulin E (IgE) levels were also measured. RESULTS: The patients with AD had significantly reduced sleep efficiency, longer sleep onset latency, more sleep fragmentation, and less nonrapid eye movement sleep. Results from actigraphy correlated well with those from polysomnography. The AD disease severity was associated with sleep disturbance (r = 0.55-0.7), and a Scoring Atopic Dermatitis index of ≥48.7 predicted poor sleep efficiency with a sensitivity of 83.3% and a specificity of 75% (area under the curve = 0.81, P = .001). Lower nocturnal melatonin secretion was significantly associated with sleep disturbance in the patients with AD. Other correlates of sleep disturbance included pruritus, scratching movements, higher total serum IgE levels, and allergic sensitization to dust mite and staphylococcal enterotoxins. CONCLUSIONS: Poor sleep efficiency is common in children with AD and can be predicted by the Scoring Atopic Dermatitis index. Melatonin and IgE might play a role in the sleep disturbance. Further studies are required to explore the mechanisms and clinical implications, and actigraphy could serve as a useful evaluating tool.

Regulatory T cells negatively regulate neovasculature of airway remodeling via DLL4-Notch signaling.

Regulatory T cells (Treg) have been shown to prevent the development of allergic asthma; however, the role of Treg in asthma with established airway remodeling is unknown. To address this, we exploited an OVA-induced chronic asthma mouse model wherein Treg were adoptively transferred to the mice at chronic stage of the model. We found that among the structural alterations of airway remodeling, Treg selectively reduced the vessel numbers in both peritracheal and peribronchial regions and the lung parenchyma. Extracellular matrix deposition, mucus metaplasia, muscular hyperplasia, and vasodilation, as were also induced by chronic allergen challenge, were not affected by Treg. TUNEL staining of the lung sections revealed an increased endothelial cell (EC) apoptosis in mice receiving Treg transfers compared with their asthmatic counterparts. By using Matrigel angiogenesis assays, we showed that Treg inhibited EC angiogenesis both in vitro and in vivo. Treg preferentially expressed Notch ligand DLL4, and an anti-DLL4 blocking Ab abrogated the inhibitory effect of Treg on EC tube formation. In vivo, decreased airway and lung vessel numbers as well as ameliorated airway hyperresponsiveness after Treg transfers were reverted when Treg-derived DLL4 signal was blocked by the anti-DLL4 Ab. Our findings demonstrate a novel function of Treg whereby Treg down-regulate remodeling angiogenesis via proapoptotic DLL4-Notch signaling, and suggest a therapeutic potential of Treg in alleviating airway hyperresponsiveness of chronic asthma.

Tumor-associated macrophage-induced invasion and angiogenesis of human basal cell carcinoma cells by cyclooxygenase-2 induction.

Tumor-associated macrophages (TAMs) and cyclooxygenase-2 (COX-2) are associated with invasion, angiogenesis, and poor prognosis in many human cancers. However, the role of TAMs in human basal cell carcinoma (BCC) remains elusive. We found that the number of TAMs infiltrating the tumor is correlated with the depth of invasion, microvessel density, and COX-2 expression in human BCC cells. TAMs also aggregate near COX-2 expressing BCC tumor nests. We hypothesize that TAMs might activate COX-2 in BCC cells and subsequently increase their invasion and angiogenesis. TAMs are a kind of M2 macrophage derived from macrophages exposed to Th2 cytokines. M2-polarized macrophages derived from peripheral blood monocytes were cocultured with BCC cells without direct contact. Coculture with the M2 macrophages induced COX-2-dependent invasion and angiogenesis of BCC cells. Human THP-1 cell line cells, after treated with phorbol myristate acetate (PMA), differentiated to macrophages with M2 functional profiles. Coculture with PMA-treated THP-1 macrophages induced COX-2-dependent release of matrix metalloproteinase-9 and subsequent increased invasion of BCC cells. Macrophages also induced COX-2-dependent secretion of basic fibroblast growth factor and vascular endothelial growth factor-A, and increased angiogenesis in BCC cells.

Linoleic acid metabolite levels and transepidermal water loss in children with atopic dermatitis.

BACKGROUND: It has been suggested that atopic dermatitis (AD) is associated with impaired delta-6 desaturase activity and the subsequent altered composition of n-6 essential fatty acids (EFAs). OBJECTIVE: To investigate whether n-6 EFA deficiency accounts for AD by affecting transepidermal water loss or the immune response. METHODS: Serum levels of n-6 EFAs were measured using gas chromatography-mass spectrometry in a well-defined group of 35 children with AD (IgE level >150 U/mL); 35 age-matched children with allergic rhinitis, asthma, or both (IgE level >150 U/mL); and 31 nonatopic controls (IgE level <100 U/mL). Skin barrier function was evaluated by measuring transepidermal water loss and severity of AD by computing the Scoring Atopic Dermatitis (SCORAD) index. RESULTS: Atopic children had higher levels of linoleic acid (LA) and lower levels of its metabolites. Furthermore, gamma-linolenic acid to LA and dihommo-gamma-linolenic acid to LA ratios were significantly reduced in atopic patients. Transepidermal water loss and the SCORAD index were negatively correlated with serum levels of LA metabolites. There was no correlation between the SCORAD index and IgE level (P = .51) or between n-6 EFA concentrations and IgE level (P > .10). CONCLUSIONS: Deficits in n-6 EFAs were correlated with the severity of AD by affecting skin barrier function and cutaneous inflammation. The link between impaired n-6 EFA metabolism and IgE level could not be defined.

Characteristics of mycobacterial infection in patients with immunodeficiency and nuclear factor-kappaB essential modulator mutation, with or without ectodermal dysplasia.

Hypomorphic mutations of the nuclear factor kappaB essential modulator gene cause ectodermal dysplasia and immunodeficiency. Affected patients have increased susceptibility to mycobacterial disease including cutaneous manifestations. We describe clinical and histopathologic characteristics of 5 patients with nuclear factor kappaB essential modulator gene mutations and mycobacterial infections, two of whom had mycobacterial cutaneous infections.