Lesional Keratinocytes Research Articles

MiR-4516, a microRNA downregulated in psoriasis inhibits keratinocyte motility by targeting fibronectin/integrin α9 signaling

Psoriasis is recognized as a T cell mediated inflammatory hyperproliferative skin disorder. Several microRNAs (miRNAs) have been implicated in the pathogenesis of psoriasis however, understanding of their mechanistic involvement remains unclear. Previously, we have shown that PUVA induced miR-4516 downregulates signal transducer and activator of transcription 3 (STAT3) by direct binding to its 3′ untranslated region (3′UTR) and suppresses STAT3 downstream genes (Bcl xl, Cyclin D1). Here, we demonstrate for the first time that expression of miR-4516 is significantly downregulated in psoriatic skin. We additionally validated extracellular matrix protein fibronectin 1 (FN1) and integrin subunit α9 (ITGA9) as direct targets of miR-4516. Interestingly, ITGA9 expression was found to be increased in the suprabasal psoriatic epidermis. We further showed that ectopic expression of miR-4516 in human keratinocytes not only suppresses cell motility and proliferation via significant downregulation of genes orchestrating cytoskeletal reorganization (Rac1, RhoA, Cdc42), but also inhibits F-actin assembly and induces terminal differentiation. Collectively, our results provide evidence that loss of expression of miR-4516 in psoriatic skin might be contributing to accelerated migration, resistance to apoptosis and differentiation as seen in psoriasis lesional keratinocytes and also highlight its potential as a novel small molecule for therapeutic intervention in psoriasis.

Effects of RNA interference combined with ultrasonic irradiation and SonoVue microbubbles on expression of STAT3 gene in keratinocytes of psoriatic lesions.

The most effective sequence of small interfering RNA (siRNA) silencing STAT3 of psoriatic keratinocytes (KCs) was screened out, and the effects of the most effective siRNA combined with ultrasonic irradiation and SonoVue microbubbles on the expression of STAT3 of KCs and the dose- and time-response were investigated. Three chemically-synthetic siRNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs, and the effects on STAT3 expression were detected, then the most effective siRNA was selected for the subsequent experiments. The negative controls of siRNA (siRNA-NC) labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles were transfected into KCs, then the optimal parameters of ultrasonic irradiation were determined. The most effective siRNA carried by Li-pofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and SonoVue microbubbles was transfected into KCs, and the dose- and time-response of RNA interference was determined. The effect of RNA interference by the most effective siRNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles (LUS group) was compared with that only carried by Li-pofectamine 3000 (L group). The results showed that siRNA-3 achieved the highest silencing efficacy. 0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation. The siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression at mRNA and protein levels in dose- and time-dependent manners determined at 100 nmol/L with maximum downregulation on mRNA at 48 h, and on protein at 72 h after transfection. The LUS group achieved the highest silencing efficacy. It was concluded that siRNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoVue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs, and the optimized transfection condition and the sequence of siRNA-3 could serve for further research on gene therapy of psoriasis.

Significance of vascular endothelial growth factor and CD31 and morphometric analysis of microvessel density by CD31 receptor expression as an adjuvant tool in diagnosis of psoriatic lesions of skin.

Pathogenesis of psoriasis is a debated issue. Several mechanisms have been proposed to identify the etiology and pathogenesis so that specific treatments can be given to patients with psoriasis. (1) To compare pattern and distribution of vascular endothelial growth factor (VEGF) and CD31 in patients with psoriasis and other psoriasiform lesions of skin. (2) To study the correlation between VEGF and CD31 expression, clinical severity, and histopathology of psoriasiform lesions of skin. (3) Evaluation of microvessel density (MVD) by using computer-assisted quantitative image analysis in psoriatic skin lesions. This study was conducted on eighty cases, out of which forty were diagnosed cases of psoriasis and forty cases of clinically suspected psoriasiform lesions, submitted in the Department of Pathology, Pt. B.D. Sharma, University of Health Sciences, Rohtak, for histopathological examination. Histopathological sections were stained by routine hematoxylin and eosin staining, and these biopsies were further subjected to immunohistochemical staining with VEGF and CD31 as per standard technique. Assessment of various histopathological features revealed strong correlation between epidermal hyperplasia, suprapapillary thinning, and elongation of rete ridges. Suprabasilar keratinocytes in psoriatic lesions stained intensely for VEGF. The difference for number of microvessels and MVD in psoriasis and psoriasiform lesions was statistically significant. Correlation between intensity of VEGF staining by suprabasilar keratinocytes and MVD was found to be highly significant in psoriatic lesions. The present study concluded that psoriatic lesions exhibit potent angiogenic activity. Early lesions show increased MVD along with other histomorphological parameters such as hypogranulosis, parakeratosis and Munro's microabscesses. Overexpression of VEGF by suprabasilar keratinocytes correlated with increased MVD in papillary dermis.

Expression of Angiogenic Factors in Psoriasis Vulgaris.

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperproliferation, abnormal differentiation and inflammatory infiltration in the dermis. The dermal microvascular expansion associated with abnormal orientation and dilatation of capillaries in the biopsies of the psoriatic skin suggest that the disease is dependent on angiogenesis. To analyze and compare the immunohistochemical expression of angiogenic factors - Vascular Endothelial Growth Factor (VEGF), von Willebrand Factor (vWF) and CD 34 in skin biopsies of psoriasis cases with control skin samples; and to correlate the expression of angiogenic factors with Psoriasis Area and Severity Clinical Index (PASI SCORE). This was a prospective case control study conducted over a period of 15 months. Thirty-two psoriasis cases and thirty control skin samples were included in the study. Skin biopsy specimen was taken from clinically diagnosed psoriasis cases who did not receive any treatment. The diagnosis of psoriasis vulgaris was confirmed after microscopic examination. Immunohistochemical expression for VEGF, vWF and CD 34 was studied. VEGF expression in epidermis was significantly higher in cases when compared to control skin (p <0.01). CD 34 expression was significantly upregulated in cases when compared to controls (p<0.01). Von Willebrand factor expression was weak in both the cases and the controls. Significant correlation between the expression of VEGF and PASI score (r=0.944; p<0.01), and expression of CD 34 and PASI score was observed (r=0.942; p<0.01). In the present study, significant overexpression of VEGF and CD 34 was noted in cases when compared to controls. The keratinocytes in the psoriatic skin lesions were recognized as a source of pro-angiogenic cytokines namely the VEGF and other growth factors which promotes angiogenesis in psoriatic plaque. Angiogenesis plays an important role in genesis and development of psoriasis vulgaris. Therefore, development of targeted anti-angiogenic therapy might be beneficial for this chronic disabling dermatological disease.

MicroRNA-194 regulates keratinocyte proliferation and differentiation by targeting Grainyhead-like 2 in psoriasis

MicroRNAs (miRNAs) are currently emerged as important regulators in psoriasis. Psoriasis is characterized by hyperproliferation and impaired differentiation of keratinocytes in skin lesions. miR-194 is a well-known regulator of cell proliferation and differentiation. However, the role of miR-194 in psoriasis pathogenesis remains unclear. In this study we aimed to investigate the role of miR-194 in keratinocyte hyperproliferation and differentiation. We found that miR-194 was significantly downregulated in psoriasis lesional skin. Overexpression of miR-194 inhibited the proliferation and promoted the differentiation of primary human keratinocytes, whereas miR-194 suppression promoted the proliferation and inhibited their differentiation. Bioinformatic analysis predicted that the Grainyhead-like 2 (GRHL2) was a target gene of miR-194, which we further validated with a dual-luciferase reporter assay, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blot analysis. The effect of miR-194 on cell proliferation and differentiation was significantly reversed by overexpression of GRHL2. Moreover, the expression of miR-194 and GRHL2 was inversely correlated in psoriasis lesional skin. Taken together, our results suggest that miR-194 inhibits the proliferation and promotes the differentiation of keratinocytes through targeting GRHL2. The downregulation of miR-194 expression may contribute to the pathogenesis of psoriasis and targeting miR-194 may represent a novel and potential therapeutic strategy for psoriasis.

Randomized trial of calcipotriol combined with 5-fluorouracil for skin cancer precursor immunotherapy.

Actinic keratosis is a precursor to cutaneous squamous cell carcinoma. Long treatment durations and severe side effects have limited the efficacy of current actinic keratosis treatments. Thymic stromal lymphopoietin (TSLP) is an epithelium-derived cytokine that induces a robust antitumor immunity in barrier-defective skin. Here, we investigated the efficacy of calcipotriol, a topical TSLP inducer, in combination with 5-fluorouracil (5-FU) as an immunotherapy for actinic keratosis. The mechanism of calcipotriol action against skin carcinogenesis was examined in genetically engineered mouse models. The efficacy and safety of 0.005% calcipotriol ointment combined with 5% 5-FU cream were compared with Vaseline plus 5-FU for the field treatment of actinic keratosis in a randomized, double-blind clinical trial involving 131 participants. The assigned treatment was self-applied to the entirety of the qualified anatomical sites (face, scalp, and upper extremities) twice daily for 4 consecutive days. The percentage of reduction in the number of actinic keratoses (primary outcome), local skin reactions, and immune activation parameters were assessed. Calcipotriol suppressed skin cancer development in mice in a TSLP-dependent manner. Four-day application of calcipotriol plus 5-FU versus Vaseline plus 5-FU led to an 87.8% versus 26.3% mean reduction in the number of actinic keratoses in participants (P < 0.0001). Importantly, calcipotriol plus 5-FU treatment induced TSLP, HLA class II, and natural killer cell group 2D (NKG2D) ligand expression in the lesional keratinocytes associated with a marked CD4+ T cell infiltration, which peaked on days 10-11 after treatment, without pain, crusting, or ulceration. Our findings demonstrate the synergistic effects of calcipotriol and 5-FU treatment in optimally activating a CD4+ T cell-mediated immunity against actinic keratoses and, potentially, cancers of the skin and other organs. ClinicalTrials.gov NCT02019355. Not applicable (investigator-initiated clinical trial).

Adenosine A2A and A2B Receptors Differentially Modulate Keratinocyte Proliferation: Possible Deregulation in Psoriatic Epidermis

Adenosine is a potent regulator of inflammation and immunity, but the role of adenosine receptors in keratinocytes remains controversial. We determined that in addition to A2B receptors, human epidermal keratinocytes also express A2A receptors, although to a lower extent. Through the use of selective adenosine receptor agonists and antagonists, we showed that physiological concentrations of adenosine activate A2B receptors in normal human keratinocytes, inducing cell cycle arrest through the increase of intracellular calcium but not through cAMP signaling. In contrast, the selective activation of A2A receptors by CGS-21680 induces keratinocyte proliferation via p38-mitogen-activated protein kinase activation. Adenosine and selective A2A and A2B agonists presented anti-inflammatory profiles independent of adenosine receptors but mediated by membrane phosphatase activation. Finally, keratinocyte exposure to diverse inflammatory cytokines altered adenosine receptor expression by reducing A2B and increasing A2A, a pattern also observed in psoriatic epidermis. Because increased epidermal turnover and inflammatory response are characteristics of psoriatic disease, further studies are needed to assess the role and consequences of the altered adenosine receptor expression in lesional and nonlesional psoriatic keratinocytes.

Expression of caveolin-1 in keratinocytes and its correlation with cellular proliferation and apoptosis in condyloma acuminatum skin lesions

Objective To investigate the expression of caveolin-1 in keratinocytes of condylomata acuminatum(CA)skin lesions, and to explore its correlation with keratinocyte proliferation and apoptosis. Methods Tissue specimens were obtained from lesions of 40 patients with CA (CA group)and normal skin of 10 healthy human controls(control group), then fixed with formalin and embedded with paraffin. An immunohistochemical analysis was done using the EnVision system to measure the expressions of caveolin-1 (expressed as absorbance value)and proliferating cell nuclear antigen (PCNA)in keratinocytes, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to evaluate apoptosis of keratinocytes in these tissue specimens. The proliferative index and apoptotic index of cells were calculated. Independent samples t test was conducted to compare these parameters between the two groups, and Pearson correlation analysis to assess the relationship of caveolin-1 expression with proliferative index and apoptotic index. Three small interfering RNAs (siRNAs)were designed targeting the caveolin-1 gene, and transfected into HaCaT cells separately by using liposomes. Then, fluorescence-based quantitative PCR (qPCR)was performed to detect the expression of caveolin-1, so as to find the most efficient siRNA. Cultured HaCaT cells were divided into 3 groups: experimental group transfected with caveolin-1 siRNA, negative control group transfected with nonspecific siRNA, and blank control group receiving no treatment. Western blot analysis and immunofluorescence assay were carried out to quantify the protein expression of caveolin-1 in HaCaT cells at 48 hours after transfection, MTS assay was performed to evaluate proliferative activity of HaCaT cells at 24, 48 and 72 hours, and flow cytometry (FCM)to detect cell apoptosis at 48 hours. Results Compared with the control group, the CA group showed significantly decreased caveolin-1 expression (0.042±0.021 vs. 0.181±0.075, t = 5.843, P 0.05). Conclusion The expression of caveolin-1 is decreased in keratinocytes of CA skin lesions, which may be associated with accelerated proliferation and decreased apoptosis of keratinocytes. Key words: Condylomata acuminata; Caveolin 1; Keratinocytes; Apoptosis; Cell proliferation

Spleen tyrosine kinase (SYK) is a potential target for the treatment of cutaneous lupus erythematosus patients.

Spleen tyrosine kinase (SYK) is a protein kinase involved in cell proliferation and the regulation of inflammatory pathways. Due to the increasing evidence that kinase inhibitors have potential as specific anti-inflammatory drugs, we have investigated the potential for SYK inhibition as a therapeutic target in autoimmune diseases, particularly cutaneous lupus erythematosus (CLE). Skin samples of patients with different CLE subtypes and appropriate controls were analysed for the expression of SYK and SYK-associated pro-inflammatory mediators via gene expression analysis and immunohistochemistry. The functional role of SYK in keratinocytes was investigated invitro, using LE-typical pro-inflammatory stimuli and a selective inhibitor of SYK. SYK-associated genes are strongly upregulated in CLE skin lesions. Importantly, phosphorylated SYK (pSYK) is strongly expressed by several immune cell types and also keratinocytes in CLE skin. In vitro, immunostimulatory nucleic acids are capable of inducing SYK phosphorylation in keratinocytes leading to the induction of pro-inflammatory cytokines, while small-molecule SYK inhibition decreases the expression of these proteins. The results demonstrate that pSYK is expressed by immune cells and keratinocytes in skin lesions of CLE patients. LE-typical stimuli induce the expression of pSYK invitro. Small-molecule SYK inhibition leads to a reduction of pSYK expression and downregulation of pro-inflammatory cytokines in keratinocytes. We therefore believe that pSYK provides a potential future drug target for the treatment of patients who suffer from CLE and related skin disorders. Specifically, our study reveals evidence supporting the use of topical SYK inhibitors in treating lupus.

Tofacitinib attenuates pathologic immune pathways in patients with psoriasis: A randomized phase 2 study

Tofacitinib is an oral Janus kinase inhibitor being investigated for psoriasis. We sought to elucidate the molecular mechanisms underlying the clinical efficacy of tofacitinib in patients with psoriasis. Twelve patients with plaque psoriasis were randomized (3:1) to receive 10 mg of tofacitinib or placebo twice daily for 12 weeks. Biopsy specimens were taken from nonlesional (baseline) and lesional (baseline, days 1 and 3, and weeks 1, 2, 4, and 12) skin. Biopsy specimens were examined for psoriatic epidermal features (thickness, Ki67(+) keratinocytes and keratin 16 [KRT16] mRNA expression, and phosphorylated signal transducer and activator of transcription [pSTAT](+) nuclei) and T-cell and dendritic cell (DC) subsets by using immunohistochemistry, and mRNA transcripts were quantified by using a microarray. In lesional skin keratinocyte pSTAT1 and pSTAT3 staining was increased at baseline but reduced after 1 day of tofacitinib (baseline, median of 1290 pSTAT1(+) cells/μm(2); day 1, median of 332 pSTAT1(+) cells/μm(2); and nonlesional, median of 155 pSTAT1(+) cells/μm(2)). Epidermal thickness and KRT16 mRNA expression were significantly and progressively reduced after days 1 and 3 of tofacitinib administration, respectively (eg, KRT16 decreased 2.74-fold, day 3 vs baseline, P = .016). Decreases in DC and T-cell numbers were observed after weeks 1 and 2, respectively. At week 4, significant decreases in IL-23/TH17 pathways were observed that persisted through week 12. Improvements in clinical and histologic features were strongly associated with changes in expression of psoriasis-related genes and reduction in IL-17 gene expression. Tofacitinib has a multitiered response in patients with psoriasis: (1) rapid attenuation of keratinocyte Janus kinase/STAT signaling; (2) removal of keratinocyte-induced cytokine signaling, leading to reductions in pathologic DC and T-cell numbers to nonlesional levels; and (3) inhibition of the IL-23/TH17 pathway.

TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) has been reported to induce keratinocyte apoptosis in vitro by engaging its sole receptor of fibroblast growth factor-inducible 14 (Fn14). In this study, we explored the role of TWEAK/Fn14 pathway in the growth of psoriatic keratinocytes that is, however, characterized by suppressed apoptotic cell death. Skin tissues from the patients with psoriasis or healthy donors were determined for TWEAK and Fn14 expression, and primary keratinocytes were evaluated under the stimulation of psoriatic proinflammatory cytokines or plus TWEAK. The results showed that both TWEAK and Fn14 were highly expressed in psoriatic skins. Moreover, the stimulation of psoriatic cytokines enhanced Fn14 expression by keratinocytes in vitro, which expressed TNF receptor 2 predominantly and proliferated increasingly with the addition of TWEAK. Furthermore, TWEAK stimulation enhanced the synthesis of survivin, inhibitor of apoptosis protein 2 and cellular FLICE-inhibitory protein in lesional keratinocytes. Therefore, TWEAK/Fn14 interaction prefers to enhance proliferation but not apoptosis of keratinocytes under psoriatic inflammation. The activation of nuclear factor-κB signalling-dependent anti-apoptotic proteins and biased expression of TNF receptors may be responsible for such a novel principle in keratinocytes under psoriatic inflammation.

Effects of adalimumab, etanercept and ustekinumab on the expression of psoriasin (S100A7) in psoriatic skin

BackgroundPsoriasis is a chronic inflammatory skin disease. It is characterized by immune cell activation and altered epidermal differentiation. S100A7 (psoriasin) is overexpressed in psoriasis, suggesting a determinant role of this protein in inflammation and keratinocyte differentiation. ObjectiveThe purpose of this study was to investigate the expression of S100A7 in the skin from psoriatic patients undergoing biological therapy with adalimumab, etanercept or ustekinumab. MethodsS100A7 expression and distribution were analyzed by immunohistochemistry. ResultsS100A7, overexpressed in epidermal keratinocytes of psoriatic lesions, was downregulated, under the biological therapy with adalimumab, etanercept or ustekinumab, only in patients achieving a PASI score<15. ConclusionsDysregulation of S100A7 may represent a non-negligible player in the maintenance of psoriasis and the relative epidermal changes. Blockage of S100A7 may represent an additional therapeutic approach in the treatment of psoriasis.

Dexamethasone but not tacrolimus suppresses TNF-α-induced thymic stromal lymphopoietin expression in lesional keratinocytes of atopic dermatitis model

BackgroundThymic stromal lymphopoietin (TSLP) initiates the Th2-type allergic inflammation, and is thought to play an important role in the pathogenesis of atopic dermatitis (AD). TNF-α is a key cytokine which is involved in the pathophysiology of various inflammatory diseases, and the expression level is elevated in the sera and skin of patients with AD. In addition, TNF-α has been reported to induce TSLP expression in epidermal keratinocytes. Topical glucocorticoids and calcineurin inhibitors are safe and effective agents for AD, but the effects of these agents on TNF-α-induced TSLP expression are not fully understood. ObjectiveTo investigate whether the glucocorticosteroid dexamethasone and the calcineurin inhibitor tacrolimus could affect TSLP expression induced by TNF-α in lesional keratinocytes of AD. MethodsThe effects of topical dexamethasone and tacrolimus on TSLP expression were evaluated in an AD mouse model induced by repeated 2,4,6-trinitro-1-chlorobenzene application. Co-immunostaining for TSLP and TNF-α was performed using skin samples from AD patients and the mouse model. Normal human epidermal keratinocytes (NHEKs) were cultured with dexamethasone or tacrolimus in the presence of TNF-α to analyze TSLP expression. ResultsTopical application of dexamethasone but not tacrolimus repressed TSLP expression in the mouse model. TSLP and TNF-α showed similar distribution pattern in epidermal keratinocytes of AD lesions and the mouse model. TSLP expression was induced by TNF-α via NF-κB in a dose-dependent and an autocrine and/or paracrine manner in NHEKs, which was significantly suppressed by dexamethasone but not by tacrolimus. Similarly to TSLP expression, IL-6, TNF-α, IL-8, and IL-36γ expression induced by TNF-α were significantly suppressed by dexamethasone but not by tacrolimus in NHEKs. ConclusionDexamethasone but not tacrolimus suppresses the TSLP expression induced by TNF-α in lesional keratinocytes of AD model. Our observations uncover the unreported functional difference between topical glucocorticosteroids and calcineurin inhibitors in cutaneous inflammatory diseases.

Plexin-B1 and semaphorin 4D cooperate to promote cutaneous squamous cell carcinoma cell proliferation, migration and invasion

Background objectivesSemaphorin 4D (Sema4D) and its receptor, Plexin-B1, are involved in the pathogenesis of squamous cell carcinoma (SCC) by mediating angiogenesis or perineural invasion through the interaction between Sema4D expression on SCC cells and Plexin-B1 expression on endothelial cells or nerves. Plexin-B1 was also recently found to be expressed on SCC cells. Plexin-B1 expression on several types of tumor cells could mediate various, and occasionally opposing, effects, including tumor cell survival, proliferation, angiogenesis, invasion, and metastasis. However, whether Sema4D exerts paracrine or autocrine effects on SCC via Plexin-B1 remains unclear. ObjectivesThe aim of this study is to explore the effects of Sema4D/Plexin-B1 interaction on SCC via Plexin-B1 expressed on the tumor cells. MethodsIn the present study, we detected the expression of Plexin-B1 and Sema4D in cutaneous SCC (cSCC) tissues and in the cSCC cell line A431 and analyzed the effects of the Sema4D/Plexin-B1 interaction on cSCC cell proliferation, migration, and invasion, as well as on the signaling pathway downstream of Plexin-B1. ResultsWe observed significantly increased Plexin-B1 and Sema4D expression in keratinocytes in cSCC lesions and in A431 cells compared with that in normal skin tissue and in non-malignant keratinocytes. Plexin-B1 silencing reduced the growth, proliferation, migration, and invasion of A431 cells and inhibited the phosphorylation of Akt and extracellular signal-regulated protein kinase (Erk). Soluble recombinant Sema4D promoted the growth, proliferation, migration, and invasion of A431 cells; Akt and Erk phosphorylation is also involved in these processes with a Plexin-B1 dependent manner. ConclusionPlexin-B1 induces cSCC cell proliferation, migration, and invasion by interacting with Sema4D. Plexin-B1 might serve as a useful biomarker and/or as a novel therapeutic target for cSCC.

Gamma-interferon-inducible lysosomal thiol reductase is upregulated in human melanoma (TUM2P.1038)

Abstract T cell-mediated immunity has the ability to produce durable anti-melanoma responses resulting in improved survival of patients with advanced melanoma. Antigen presentation in the tumor microenvironment directs anti-melanoma T cell responses. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens to CD4+ T cells. However, GILT expression in melanoma has not been defined. We evaluated GILT and MHC class II expression in human primary and metastatic melanomas and nevi using immunohistochemistry. Both GILT and MHC class II expression were increased in melanocytes of primary and metastatic melanomas compared with nevi. GILT expression was increased in antigen presenting cells of primary and metastatic melanomas compared to nevi, whereas MHC class II had equivalent high expression in antigen presenting cells of all melanocytic lesions. Keratinocytes in primary melanoma lesions had increased GILT and MHC class II expression compared with nevi. Increased GILT expression in melanocytes, antigen presenting cells and keratinocytes of melanoma demonstrates that changes in the antigen presentation pathway occur in the tumor microenvironment. GILT expression in melanoma is anticipated to result in improved presentation of melanoma antigens and more effective anti-melanoma T cell responses and may be a biomarker of immune recognition of melanoma.

Wnt/β-Catenin and Wnt5a/Ca2+ Pathways Regulate Proliferation and Apoptosis of Keratinocytes in Psoriasis Lesions

Background/Aims: Wnt5a is overexpressed in psoriasis lesions, however the mechanism by which Wnt5a is involved in the pathogenesis of psoriasis is not clear. To address this, the expression of Wnt5a in psoriatic lesions and its effect on keratinocyte cell proliferation and apoptosis was examined in vitro. Methods: The expression levels of WNT5A, and genes encoding its receptors frizzled2 (FZD2) and frizzled5 (FZD5) were examined in samples obtained from individuals with psoriasis and healthy controls. Knockdown of Wnt5a with short interfering (si)RNAs was performed in cultured HaCaT keratinocytes and normal human keratinocytes (NHK), and the expression of Wnt5a, protein kinase C (PKC), and β-catenin were determined, and cell cycle activity, proliferation and apoptosis were assessed. Results: The expression of WNT5A, FZD2 and FZD5 mRNA and protein were increased in psoriatic lesions. Wnt5a knockdown suppressed proliferation and induced apoptosis in HaCaT and NHK cells. Additionally, expression of PCNA, MKI67, CCND1, BCL2, CTNNB1, and genes encoding PKC and survivin were downregulated, whereas CASP3 was upregulated. The mRNA levels of the Wnt pathway inhibitors DKK1 and SFRP1 were upregulated, Western blotting analyses demonstrated reduction in β-catenin and PKC protein levels. Conclusion: Knockdown of Wnt5a suppresses the proliferation of keratinocytes and induces apoptosis by inhibiting the Wnt/β-catenin or Wnt5a/Ca²⁺ pathways.

Identification of Somatic KRAS Mutation in a Korean Baby with Nevus Sebaceus Syndrome

Identification of Somatic <i>KRAS</i> Mutation in a Korean Baby with Nevus Sebaceus Syndrome

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-1β 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-1β 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.

Expression of peroxisome proliferator-activated receptor β/δ in psoriatic epidermal keratinocytes

Objective To measure the expression of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) in epidermal keratinocytes from patients with psoriasis,and to investigate its regulatory factors.Methods Tissue specimens were obtained from both lesional and non-lesional skin of 20 patients with psoriasis as well as from normal skin of 15 human controls.An immunohistochemical method was used to examine the expression of PPARβ/δ in these tissue specimens.Epidermal keratinocytes were isolated from these tissue specimens and subjected to a primary culture.After several passages of subculture,non-lesional psoriatic keratinocytes were stimulated with different concentrations of GW501516 (an agonist of PPARβ/δ,0-100 ng/ml) and Ca2+ (0-3.0 mmol/L).Reverse transcription-PCR and Western blot were performed to measure the mRNA and protein expressions of PPARβ/δ in the primary keratinocytes and stimulated keratinocytes respectively.Results Immunohistochemistry showed that the expression intensity of PPARβ/δ was significantly higher in lesional psoriatic skin than in normal control skin (t =19.28,P ＜ 0.01) and non-lesional psoriatic skin (t =23.26,P ＜ 0.01).Increased mRNA and protein levels of PPARβ/δ were observed in lesional psoriatic keratinocytes as compared to normal control keratinocytes (both P ＜0.01) and non-lesional psoriatic keratinocytes (both P ＜ 0.01).Among these stimulated non-lesional psoriatic keratinocytes,those treated with GW501516 at 10 ng/ml and those with Ca2+ of 1.0 mmol/L showed the strongest expression of PPARβ/δ (both P ＜ 0.01).Conclusions The expression of PPARβ/δ,which is higher in lesional psoriatic skin,can be enhanced by GW501516 and Ca2+ in keratinocytes. Key words: Psoriasis; Keratinocytes; PPARβ/δ

Thymic stromal lymphopoietin links keratinocytes and dendritic cell–derived IL-23 in patients with psoriasis

Thymic stromal lymphopoietin (TSLP) is a major proallergic cytokine that promotes TH2 responses through dendritic cell (DC) activation. Whether it also plays a role in human autoimmune inflammation and associated pathways is not known. In this study we investigated the potential role of several epithelium-derived factors, including TSLP, in inducing IL-23 production by human DCs. We further dissected the role of TSLP in patients with psoriasis, an IL-23-associated skin autoimmune disease. The study was performed in human subjects using primary cells and tissue samples from patients with psoriasis and healthy donors. We analyzed the production of IL-23 in vitro by blood and skin DCs. We studied the function for TSLP and its interaction with other components of the inflammatory microenvironment in situ and ex vivo. We found that TSLP synergized with CD40 ligand to promote DC activation and pathogenic IL-23 production by primary blood and skin DCs. In situ TSLP was strongly expressed by keratinocytes of untreated psoriatic lesions but not in normal skin. Moreover, we could demonstrate that IL-4, an important component of the TH2 inflammation seen in patients with atopic dermatitis, inhibited IL-23 production induced by TSLP and CD40 ligand in a signal transducer and activator of transcription 6-independent manner. Our results identify TSLP as a novel player within the complex psoriasis cytokine network. Blocking TSLP in patients with psoriasis might contribute to decreasing DC activation and shutting down the production of pathogenic IL-23.