Nuclear IL-33 Plays an Important Role in EGFR-Mediated Keratinocyte Migration by Regulating the Activation of Signal Transducer and Activator of Transcription 3 and NF-κB.

Nuclear IL-33 levels are high at the epidermal edges of skin wounds and facilitate wound healing. However, IL-33-mediated regulation of keratinocyte (KC) biology during wound healing remains poorly understood. During skin-wound healing, KC migration and re-epithelialization are mediated predominantly by EGFR signaling activation and depend on the function of signal transducer and activator of transcription 3 (STAT3). We found that migrating KCs at the leading edges of mouse skin wounds exhibited concomitant induction and nuclear colocalization of IL-33 and phosphorylated STAT3. In cultured human KCs, activation of EGFR signaling caused rapid elevation of nuclear IL-33, which directly interacts with phosphorylated STAT3, promoting STAT3 activation. In vitro KC migration and wound-healing assays revealed that high nuclear IL-33 levels were required for KC migration and wound closure. KC mobility associated with a lack of suprabasal epidermal keratins and extracellular matrix degradation mediated by matrix metalloproteinases (MMPs) control cell migration at the intracellular and extracellular levels, respectively. In EGFR-activated KCs, nuclear IL-33 mediated keratin 1 and 10 downregulation and MMP9 upregulation by promoting STAT3 activation and limited MMP1, MMP3, and MMP10 induction by suppressing NF-κB transactivation. Thus, epidermal nuclear IL-33 is involved in KC migration and wound closure by regulating the STAT3 and NF-κB pathways.

Highly concentrated trehalose induces prohealing senescence-like state in fibroblasts via CDKN1A/p21.

Trehalose is the nonreducing disaccharide of glucose, evolutionarily conserved in invertebrates. The living skin equivalent (LSE) is an organotypic coculture containing keratinocytes cultivated on fibroblast-populated dermal substitutes. We demonstrated that human primary fibroblasts treated with highly concentrated trehalose promote significantly extensive spread of the epidermal layer of LSE without any deleterious effects. The RNA-seq analysis of trehalose-treated 2D and 3D fibroblasts at early time points revealed the involvement of the CDKN1A pathway, the knockdown of which significantly suppressed the upregulation of DPT, ANGPT2, VEGFA, EREG, and FGF2. The trehalose-treated fibroblasts were positive for senescence-associated ß-galactosidase. Finally, transplantation of the dermal substitute with trehalose-treated fibroblasts accelerated wound closure and increased capillary formation significantly in the experimental mouse wounds in vivo, which was canceled by the CDKN1A knockdown. These data indicate that high-concentration trehalose can induce the senescence-like state in fibroblasts via CDKN1A/p21, which may be therapeutically useful for optimal wound repair.

EGFR ligands synergistically increase IL-17A-induced expression of psoriasis signature genes in human keratinocytes via IκBζ and Bcl3.

Various epidermal growth factor receptor (EGFR) ligands are highly expressed in the epidermis of psoriasis lesions, and abnormal EGFR activation appears to be involved in the pathogenesis of psoriasis. However, how EGFR signaling contributes to the development of psoriasis is unclear. Interleukin (IL)-17A, a critical effector of the IL-23/IL-17A pathway, increases the expression of psoriasis signature genes in keratinocytes and plays an essential role in the pathogenesis of psoriasis by inducing IκBζ, a critical transcriptional regulator in psoriasis. In this study, we stimulated primary human keratinocytes with IL-17A and various EGFR ligands to investigate whether EGFR ligands regulate the expression of psoriasis signature genes. In cultured normal human keratinocytes and a living skin equivalent, EGFR ligands did not induce psoriasis-related gene expression, but significantly enhanced the IL-17A-mediated induction of various psoriasis signature genes, including antimicrobial peptides, cytokines, and chemokines. This was dependent on an EGFR activation-mediated synergistic increase in IL-17A-induced IκBζ expression and was partially mediated by the EGFR-dependent upregulation of Bcl3. Therefore, EGFR ligands can act as synergistic agents of IL-17A signaling by stimulating the epidermal production of psoriasis signature genes in psoriasis lesions. This study reveals a potential mechanism by which EGFR signaling contributes to the pathogenesis of psoriasis.

TSLP Impairs Epidermal Barrier Integrity by Stimulating the Formation of Nuclear IL-33/Phosphorylated STAT3 Complex in Human Keratinocytes.

Atopic dermatitis (AD) is an inflammatory skin disease characterized by skin barrier dysfunction. Although T helper type 2 cytokines downregulate the expression of epidermal barrier proteins, the signaling mechanism underlying these effects remains unclear. IL-33, a chromatin-associated cytokine, is highly expressed in the nuclei of epidermal keratinocytes in AD skin; however, it is unclear whether this expression promotes the development of AD. TSLP, an epithelial cells-derived pro‒T helper type 2 cytokine, is elevated in the epidermis of patients with AD. TSLP affects the pathogenesis of AD by activating T helper type 2 responses and impairing epidermal barrier integrity. In this study, we stimulated postconfluent human keratinocytes and living skin equivalent with TSLP to investigate the role of nuclear IL-33 in TSLP-induced epidermal barrier defects. We observed that TSLP reduced the levels of FLG, hBD2, S100A7, and claudin-1, which required nuclear IL-33 expression. Similar to the T helper type 2 cytokines IL-4, IL-13, and IL-31, TSLP was shown to upregulate IL-33 expression and triggered the formation of nuclear IL-33/phosphorylated signal transducer and activator of transcription 3 complex, which bound to the FLG promoter, thereby inhibiting transcription. Moreover, nuclear IL-33 acted as a cofactor of signal transducer and activator of transcription 3 in the TSLP-induced transcriptional repression of hBD2, S100A7, and claudin-1. Therefore, epidermal nuclear IL-33 may be a key regulator of TSLP-mediated epidermal barrier dysfunction.

Nuclear IL-33 Plays an Important Role in IL-31‒Mediated Downregulation of FLG, Keratin 1, and Keratin 10 by Regulating Signal Transducer and Activator of Transcription 3 Activation in Human Keratinocytes.

IL-33, a chromatin-associated multifunctional cytokine, is implicated in the pathogenesis of atopic dermatitis (AD), an inflammatory skin disorder characterized by skin barrier dysfunction. IL-33 accumulates in the nuclei of epidermal keratinocytes (KCs) in AD lesions. However, it is unclear whether nuclear IL-33 directly contributes to the pathogenesis of AD. IL-31, a pruritogenic cytokine primarily produced by T helper type 2 cells, is elevated in AD lesions and promotes AD development by suppressing KC differentiation and inducing itching. In this study, we investigated the involvement of nuclear IL-33 in IL-31‒mediated suppression of KC differentiation. In monolayer cultures and living skin equivalent, IL-31 increased the expression of full-length IL-33 and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in the nuclei of human KCs, which in turn downregulated the expression of differentiation markers. We found that IL-31 and IL-4/IL-13 use very similar mechanisms to inhibit KC differentiation: nuclear IL-33 combines with phosphorylated STAT3 and functions as a STAT3 transcription cofactor, promoting phosphorylated STAT3 binding to the FLG promoter to inhibit its transcription; moreover, the nuclear IL-33/phosphorylated STAT3 complex drives the downregulation of keratin 1 and keratin 10 by reducing the availability of the transcription factor RunX1. Therefore, nuclear IL-33 plays an important role in IL-31‒mediated differentiation suppression by regulating STAT3 activation in human KCs.

Nuclear IL-33 Plays an Important Role in the Suppression of FLG, LOR, Keratin 1, and Keratin 10 by IL-4 and IL-13 in Human Keratinocytes.

IL-33 is a chromatin-associated multifunctional cytokine implicated in the pathogenesis of atopic dermatitis (AD), an inflammatory skin disorder characterized by skin barrier dysfunction. The previous reports show that IL-33 is highly detected in the nucleus of epidermal keratinocytes in AD lesions compared with that in unaffected or normal skin. However, it is unclear whether intracellular IL-33 directly contributes to the pathogenesis of AD. T helper type 2 cytokines IL-4 and IL-13 that are elevated in AD lesions suppress keratinocyte differentiation to impair skin barrier function. We investigated whether intracellular IL-33 is involved in IL-4 and IL-13 function. In monolayer culture and living skin equivalent analyses, IL-4 and IL-13 increased the expression of full-length IL-33 in the nucleus of keratinocytes by activating the MAPK/extracellular signal‒regulated kinase kinase/extracellular signal‒regulated kinase signaling pathway, which is necessary for the inhibition of differentiation markers FLG, LOR, keratin 1, and keratin 10. The nuclear IL-33 functions as a transcription cofactor of signal transducer and activator of transcription 3, increasing the binding of phosphorylated signal transducer and activator of transcription 3 to FLG promoter, thereby inhibiting its transcription, and it inhibits the expression of transcription factor RUNX1 by signal transducer and activator of transcription 3 and signal transducer and activator of transcription 6, thereby downregulating LOR, keratin 1, and keratin 10. Thus, the elevated nuclear IL-33 in the epidermis of AD lesions may be involved in the pathogenesis of AD by inhibiting keratinocyte differentiation and skin barrier function.

Suppression of IL-17A-induced CCL20 production by cytokine inducible SH2-containing protein 1 in epidermal keratinocytes.

BACKGROUND: Lesions of atopic dermatitis have fewer Th17 cells than those of psoriasis, resulting in frequent skin infections. Expression of CCL20, a chemokine that is important for recruiting Th17 cells, is suppressed in the lesions of atopic dermatitis. We previously reported that IL-4 induces the expression of cytokine-inducible SH2-containing protein 1 (CIS1), a member of the CIS/SOCS family, in epidermal keratinocytes. OBJECTIVE: To investigate whether CIS1 influences CCL20 production in epidermal keratinocytes. METHODS: Expression of CIS1 was examined in atopic dermatitis skin and in cultured keratinocytes. The effects of overexpression of CIS1 on CCL20 production by IL-17A, and on signaling pathways inhibited by CIS1, were assessed in vitro. RESULTS: Expression of CIS1 was enhanced in the basal layer of the lesional epidermis of skin with atopic dermatitis. When CIS1 was expressed in keratinocytes using adenoviral vectors, IL-17A-induced CCL20 expression, but not HBD2 or S100A7 expression, was significantly suppressed. TNF-α/IL-1-induced CCL20 production was not altered by CIS1. Overexpression of CIS1 attenuated IL-17A-induced ERK phosphorylation. ERK phosphorylation was mediated by the Act1 and Src family kinase pathways. CIS1 overexpression suppressed Src phosphorylation. Among the Src family kinases, the Yes kinase may have an important role because knockdown of Yes in epidermal keratinocytes resulted in suppression of ERK phosphorylation and CCL20 mRNA expression by IL-17A. CONCLUSION: CIS1 induced by Th2 cytokines has the ability to change the response of epidermal keratinocytes to IL-17A by suppression of Src family kinases.

House dust mite allergens induce interleukin 33 (IL-33) synthesis and release from keratinocytes via ATP-mediated extracellular signaling.

In atopic diseases, the epithelium releases cytokines and chemokines that initiate skin inflammation. Atopic dermatitis (AD) is characterized by a disrupted epidermal barrier and is triggered or exacerbated by environmental stimuli such as house dust mite (HDM) allergens. The proinflammatory cytokine interleukin 33 (IL-33) plays an important role in the pathogenesis of AD, but how IL-33 production in keratinocytes is elicited by HDM is unknown. To that end, here we stimulated monolayer-cultured human keratinocytes and human living skin equivalents with Dermatophagoides pteronyssinus HDM extract to investigate its effects on IL-33 production from keratinocytes. The HDM extract induced intracellular expression of IL-33 and modulated its processing and maturation, triggering rapid IL-33 release from keratinocytes. Group 1 HDM allergen but not group 2 HDM allergen elicited IL-33 production. An ATP assay of keratinocyte culture supernatants revealed an acute and transient accumulation of extracellular ATP immediately after the HDM extract stimulation. Using the broad-spectrum P2 antagonist suramin, the specific purinergic receptor P2Y2 (P2RY2) antagonist AR-C118925XX, and P2RY2-specific siRNA, we discovered that the HDM extract-induced IL-33 expression was mainly dependent on extracellular ATP/P2Y2 signaling mediated by transactivation of epidermal growth factor receptor, followed by activation of the ERK kinase signaling pathway. Moreover, HDM extract-induced release of 25-kDa IL-33 from the keratinocytes depended on an extracellular ATP/P2 signaling-mediated intracellular Ca2+ increase. Our study demonstrates the new mechanism controlling the induction and maturation of keratinocyte-produced IL-33 by HDM allergens, an innate immune process that might play a role in AD development or severity.

Heparinoid suppresses Der p-induced IL-1ß production by inhibiting ERK and p38 MAPK pathways in keratinocytes.

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro-inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen-induced interleukin (IL)-1ß release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase-1 release, suggesting that heparinoid did not affect HDM allergen-induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro-IL-1ß, but also suppressed IL-1ß messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signalling pathways, the activation of extracellular signal-regulated kinase and p38 pathways, which are required for IL-1ß expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL-1ß mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte-mediated skin inflammation.

Reduced-HMGB1 suppresses poly(I:C)-induced inflammation in keratinocytes.

BACKGROUND: High mobility group box 1 (HMGB1) is a nuclear protein that stabilizes DNA and facilitates gene transcription. Additionally, cell stress or death induces the release of HMGB1 outside the cell membrane, where HMGB1 functions as an alarmin, causing an inflammatory response in combination with other cytokines, damage-associated molecular patterns (DAMPs), and pathogen-associated molecular patterns (PAMPs). OBJECTIVE: To evaluate the effect of reduced-HMGB1 (previously termed chemoattractive-HMGB1) on polyinosine-polycytidylic acid [poly(I:C)]-induced inflammation in normal human keratinocytes (NHKs). METHODS: We focused on downstream components of the poly(I:C)-Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) pathways, including IκBα, nuclear factor (NF)-κB p65, mitogen-activated protein kinase (MAPK), and interferon regulatory factor 3 (IRF3), and assessed whether these pathways are involved in the suppression of poly(I:C)-induced inflammation in NHKs by HMGB1. An immunoprecipitation was performed to know whether HMGB1 could bind to poly(I:C), and immunofluorescence staining and flow cytometric analysis were performed to check whether reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis). RESULTS: Application of exogenous HMGB1 before, but not after, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs. In addition, reduced-HMGB1, but not disulfide-HMGB1, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs, suggesting the importance of the redox status of exogenous HMGB1. Pre-treatment with reduced-HMGB1 inhibited the phosphorylation of IκBα, NF-κB p65, and IRF3 induced by poly(I:C) stimulation in NHKs; however, phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was unaffected. Disulfide-HMGB1 formed a complex with poly(I:C), as did reduced- and oxidized-HMGB1, albeit to a lesser extent. Immunofluorescence staining and flow cytometric analysis indicated that reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis). CONCLUSION: These findings suggest that pre-treatment with reduced-HMGB1 ameliorates poly(I:C)-mediated inflammation in NHKs.

Bcl-3 induced by IL-22 via STAT3 activation acts as a potentiator of psoriasis-related gene expression in epidermal keratinocytes.

IL-22 induces STAT3 phosphorylation and mediates psoriasis-related gene expression. However, the signaling mechanism leading from pSTAT3 to the expression of these genes remains unclear. We focused on Bcl-3, which is induced by STAT3 activation and mediates gene expression. In cultured human epidermal keratinocytes, IL-22 increased Bcl-3, which was translocated to the nucleus with p50 via STAT3 activation. The increases in CXCL8, S100As and human ß-defensin 2 mRNA expression caused by IL-22 were abolished by siRNA against Bcl-3. Although CCL20 expression was also augmented by IL-22, the knockdown of Bcl-3 increased its level. Moreover, the combination of IL-22 and IL-17A enhanced Bcl-3 production, IL-22-induced gene expression, and the expression of other psoriasis-related genes, including those encoding IL-17C, IL-19, and IL-36γ. The expression of these genes (except for CCL20) was also suppressed by the knockdown of Bcl-3. Bcl-3 overexpression induced CXCL8 and HBD2 expression but not S100As expression. We also compared Bcl-3 expression between psoriatic skin lesions and normal skin. Immunostaining revealed strong signals for Bcl-3 and p50 in the nucleus of epidermal keratinocytes from psoriatic skin. The IL-22-STAT3-Bcl-3 pathway may be important in the pathogenesis of psoriasis.

Epidermal keratinocytes sense dsRNA via the NLRP3 inflammasome, mediating interleukin (IL)-1ß and IL-18 release.

Skin epidermis, in addition to its barrier function, is able to actively sense harmful pathogens using pattern recognition receptors. In immune cells, the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome can mediate innate immunity against viral infection via a mechanism involving viral dsRNA recognition. Epidermal keratinocytes express NLRP3 inflammasome, which can sense contact sensitizers and mite allergens, leading to pro-interleukin (IL)-1ß and pro-IL-18 cleavage into their active forms. Skin often faces viral infection. However, it is unknown whether viral dsRNA can be detected by the keratinocyte NLRP3 inflammasome. We transfected polyinosinic:polycytidylic acid (poly I:C), a synthetic viral dsRNA analogue, into cultured primary human keratinocytes at the aid of Lipofectamine 2000, and found that transfected poly I:C activated caspase-1 and induced caspase-1-dependent release of IL-1ß and IL-18, which were suppressed on transfection with NLRP3 siRNA. The activation of keratinocyte NLRP3 inflammasome by transfected poly I:C was dependent on dsRNA-induced protein kinase (PKR) activation, and priming with type I interferons upregulated NLRP3 inflammasome activation through promoting PKR activation in poly I:C-transfected keratinocytes. In conclusion, the NLRP3 inflammasome can act as a sensor of dsRNA in epidermal keratinocytes, which may be important in both skin innate immune defense against viral infection and skin inflammation.

Vesicular LL-37 contributes to inflammation of the lesional skin of palmoplantar pustulosis.

"Pustulosis palmaris et plantaris", or palmoplantar pustulosis (PPP), is a chronic pustular dermatitis characterized by intraepidermal palmoplantar pustules. Although early stage vesicles (preceding the pustular phase) formed in the acrosyringium contain the antimicrobial peptides cathelicidin (hCAP-18/LL-37) and dermcidin, the details of hCAP-18/LL-37 expression in such vesicles remain unclear. The principal aim of the present study was to clarify the manner of hCAP-18/LL-37 expression in PPP vesicles and to determine whether this material contributed to subsequent inflammation of lesional skin. PPP vesicle fluid (PPP-VF) induced the expression of mRNAs encoding IL-17C, IL-8, IL-1α, and IL-1ß in living skin equivalents, but the level of only IL-8 mRNA decreased significantly upon stimulation of PPP vesicle with depletion of endogenous hCAP-18/LL-37 by affinity chromatography (dep-PPP-VF). Semi-quantitative dot-blot analysis revealed higher concentrations of hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09 µM). This concentration of hCAP-18/LL-37 in PPP-VF could upregulate expression of IL-17C, IL-8, IL-1α, and IL-1ß at both the mRNA and protein levels. Recombinant hCAP-18 was incubated with dep-PPP-VF. Proteinase 3, which converts hCAP-18 to the active form (LL-37), was present in PPP-VF. Histopathological and immunohistochemical examination revealed that early stage vesicles contained many mononuclear cells but no polymorphonuclear cells, and the mononuclear cells were CD68-positive. The epidermis surrounding the vesicle expresses monocyte chemotactic chemokine, CCL2. In conclusion, PPP-VF contains the proteinase required for LL-37 processing and also may directly upregulate IL-8 in lesional keratinocytes, in turn contributing to the subsequent inflammation of PPP lesional skin.

Eccrine sweat contains IL-1α, IL-1ß and IL-31 and activates epidermal keratinocytes as a danger signal.

Eccrine sweat is secreted onto the skin's surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1ß, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1ß by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin's surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal.

Interactions between myofibroblast differentiation and epidermogenesis in constructing human living skin equivalents.

BACKGROUND: During skin wounding and healing, skin homeostasis is interrupted. How the altered epithelial-mesenchymal interactions influence scar formation and epidermogenesis should be investigated using three-dimensional models that are similar to in vivo structures. OBJECTIVE: In this study, we assessed the effects of epithelial-mesenchymal interactions on myofibroblast differentiation and how myofibroblasts influence epidermogenesis using a human living skin equivalent (LSE) model. METHODS: We constructed a fibroblast-populated type I collagen gel upon which LSEs were formed by seeding with normal human keratinocytes. Samples of the collagen gel and LSEs were collected at different time points. Myofibroblast differentiation, epidermal differentiation, and proliferation status were investigated immunohistochemically. Several measures were taken to suppress α-smooth muscle actin (α-SMA) expression to determine the effects of myofibroblasts on epidermogenesis, including the addition of basic fibroblast growth factor or a transformation growth factor-ß (TGF-ß) kinase inhibitor to the culture medium and the inclusion of an amniotic membrane (AM) in the dermal matrix. RESULTS: The myofibroblast/fibroblast ratio in the fibroblast-populated collagen gel kept rising during culture. In the LSEs, most fibroblasts were α-SMA-negative, except for those along the dermal-epidermal junction. The suppression of α-SMA expression enhanced epidermal differentiation and decreased TGF-ß1 expression in the epidermis. The inhibition of TGF-ß kinase completely suppressed α-SMA expression in the dermal matrix. CONCLUSIONS: Epidermogenesis suppressed α-SMA expression in the fibroblast-rich dermal matrix, except near the dermal-epidermal junction. The α-SMA-positive cells at the dermal-epidermal junction contributed to the hyperproliferative phenotype of the epidermis. In contrast, the hyperproliferative epidermis expressed more TGF-ß1, which is responsible for myofibroblast differentiation.

Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33 ng/ml) enhanced LEC migration by 2-fold and increased tube length by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions.

Mite allergen is a danger signal for the skin via activation of inflammasome in keratinocytes.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disorder caused by multiple factors. Among them, house dust mite (HDM) allergens are important in the development of AD. In airway allergy, HDM allergens activate innate immunity. However, information regarding the activation of innate immunity by HDM allergens in the skin is limited. OBJECTIVES: The inflammasome is a key regulator of pathogen recognition and inflammation. We investigated whether HDM allergens activate the inflammasome in epidermal keratinocytes. METHODS: Keratinocytes were stimulated with Dermatophagoides pteronyssinus, and the activation of caspase-1 and secretion of IL-1ß and IL-18 were examined. Formation of the inflammasome was studied by analyzing the subcellular distributions of inflammasome proteins. The importance of specific inflammasome proteins was studied by knocking down their expression through transfection of keratinocytes with lentiviral particles carrying short hairpin RNAs (shRNAs). RESULTS: D pteronyssinus activated caspase-1 and induced caspase-1-dependent release of IL-1ß and IL-18 from keratinocytes. Moreover, D pteronyssinus stimulated assembly of the inflammasome by recruiting apoptosis-associated specklike protein containing a caspase-recruitment domain (ASC), caspase-1, and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin-domain containing 3 (NLRP3) to the perinuclear region. Finally, infection with lentiviral particles carrying ASC, caspase-1, or NLRP3 shRNAs suppressed the release of IL-1ß and IL-18 from the keratinocytes. Activation of the NLRP3 inflammasome by D pteronyssinus was dependent on cysteine protease activity. CONCLUSION: House dust mite allergens are danger signals for the skin. In addition, HDM-induced activation of the NLRP3 inflammasome may play a pivotal role in the pathogenesis of AD.

PPARγ mediates innate immunity by regulating the 1α,25-dihydroxyvitamin D3 induced hBD-3 and cathelicidin in human keratinocytes.

BACKGROUND: Production of antimicrobial peptides (AMPs) is the primary mechanism by which skin innate immunity protects against infection. Hormonally active vitamin D3 (1α,25-dihydroxyvitamin D3; 1,25D3) is a vital regulator of skin innate immunity, and has been shown to increase the expression and function of AMPs. OBJECTIVE: PPARγ is a ligand-activated nuclear receptor and plays a role in keratinocyte differentiation and cutaneous homeostasis. In this study, we investigate whether 1,25D3-activated PPARγ signaling regulates AMP expression in keratinocytes. METHODS: Subconfluent keratinocytes were treated with 1,25D3 for the indicated times. The mRNA and protein levels of AMPs were detected by RT-PCR and Western blot, and the DNA binding activation of PPARγ, VDRE and AP-1 was investigated by EMSA. To examine the role of PPARγ, the recombinant adenovirus carrying a dominant-negative form of PPARγ (dn-PPARγ) was constructed and transfected into keratinocytes. RESULTS: We show here that 1,25D3 significantly enhances hBD-3 and cathelicidin expression in keratinocytes. Expression of dn-PPARγ did not affect binding to the vitamin D-responsive element (VDRE), which is crucial for cathelicidin induction by VD3; however, it did decrease 1,25D3 induction of both hBD-3 and cathelicidin. Inhibition of the p38, ERK, and JNK signaling pathways blocked hBD-3 expression, whereas only p38 inhibition suppressed cathelicidin induction. dn-PPARγ had no effect on ERK and JNK activity, but inhibited p38 phosphorylation and suppressed 1,25D3-induced AP-1 activation via effects on Fra1 and c-Fos proteins. CONCLUSIONS: In conclusion, PPARγ regulates the 1,25D3-induced hBD-3 and cathelicidin expression in keratinocytes through the regulation of AP-1 and p38 activity.

E2 Polyubiquitin-conjugating enzyme Ubc13 in keratinocytes is essential for epidermal integrity.

The E2 polyubiquitin-conjugating enzyme Ubc13 is a mediator of innate immune reactions. Ubc13 mediates the conjugation of keratin (K)63-linked polyubiquitin chains onto TNF receptor-associated factor 6 and IKKγ during NF-κB activation. In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains function in nonproteasomal biological processes. Although Ubc13 has been shown to be critical for Toll-like receptor (TLR) and IL-1 receptor signaling, the function of Ubc13 in the epidermis has not been studied. We generated keratinocyte-specific Ubc13-deficient mice (Ubc13(flox/flox)K5-Cre). At birth, the skin of the Ubc13(flox/flox)K5-Cre mice was abnormally shiny and smooth; in addition, the mice did not grow and died by postnatal day 2. Histological analysis showed atrophy of the epidermis with keratinocyte apoptosis. Immunohistochemical analyses revealed reduced proliferation, abnormal differentiation, and apoptosis of keratinocytes in the Ubc13(flox/flox)K5-Cre mouse epidermis. In culture, Ubc13(flox/flox)K5-Cre keratinocyte growth was impaired, and spontaneous cell death occurred. Moreover, the deletion of Ubc13 from cultured Ubc13(flox/flox) keratinocytes by means of an adenoviral vector carrying Cre recombinase also resulted in spontaneous cell death. Therefore, Ubc13 is essential for keratinocyte growth, differentiation, and survival. Analyses of intracellular signaling revealed that the IL-1 and TNF-induced activation of JNK, p38, and NF-κB pathways was impaired in Ubc13(flox/flox)K5-Cre keratinocytes. In conclusion, Ubc13 appears to be essential for epidermal integrity in mice.