Search

IκBζ plays a key role in psoriasis by mediating IL-17A–driven effects, but the molecular mechanism by which IL-17A regulates IκBζ expression is not clarified. We sought to explore the molecular transformation in patients with psoriasis during anti-IL-17A (secukinumab) treatment with a focus on IκBζ. The study was an open-label, single-arm, single-center secukinumab treatment study that included 14 patients with plaque psoriasis. Skin biopsy specimens and blood samples were collected on days 0, 4, 14, 42, and 84 and processed for microarray gene expression analysis. Furthermore, in vitro experiments with human keratinocytes and synovial fibroblasts were conducted. Secukinumab improved clinical scores and histologic psoriasis features. Moreover, secukinumab altered the skin transcriptome. The major transcriptional shift appeared between day 14 and day 42 after treatment initiation, although 80 genes were differentially expressed already at day 4. Expression of nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor (IκB) ζ (NFKBIZ , the gene encoding IκBζ) was reduced already after 4 days of treatment in the skin. NFKBIZ expression correlated to Psoriasis Area and Severity Index score, and NFKBIZ mRNA levels in the skin decreased during anti–IL-17A treatment. Moreover, specific NFKBIZ signature genes were significantly altered during anti–IL-17A treatment. Finally, we identified NF-κB activator 1 (Act1), p38 mitogen-activated protein kinase (MAPK), Jun NH2-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) as key signaling pathways in NFKBIZ/ IκBζ regulation. Our results define a crucial role for IκBζ in the antipsoriatic effect of secukinumab. Because IκBζ signature genes were regulated already after 4 days of treatment, this strongly indicates that IκBζ plays a crucial role in the antipsoriatic effects mediated by anti–IL-17A treatment. [ABSTRACT FROM AUTHOR]

Culturability on plates, growth behaviour in liquid media and acidification activity of three Lactococcus lactis strains was negatively affected by carboxyfluorescein diacetate (cFDA), the corresponding succinimidyl ester cFDA-SE, propidium iodide (PI) and TOTO-1 iodide. Single staining with the vitality dye cFDA-SE decreased the reproductive capability and acidification activity of L. lactis cells more than cFDA. Since TOTO-1 proved to have a higher toxicity than PI, the application of PI was favoured over TOTO-1 as counterstain for cells labelled with one of the vitality dyes. The overall extent to which double staining with cFDA/cFDA-SE and PI impaired bacterial physiology was determined by the dye with the greater influence on L. lactis cells during single staining. The observed strain-dependent differences in sensitivity highlight the importance of studying the impact of fluorescent labelling on cell physiology before using the dyes in combination with flow cytometric cell sorting for physiological characterisation of subpopulations. [ABSTRACT FROM AUTHOR]

IL-17C is a member of the IL-17 family of cytokines. The expression of IL-17C has been demonstrated to be strongly induced by TNFα in human keratinocytes, and recently the level of IL-17C was found to be increased in the inflammatory skin disease psoriasis. However, little is known about the molecular mechanisms involved in the regulation of IL-17C. Here, we show that pretreatment of cultured human keratinocytes with the inhibitor of κB kinase 2 inhibitor, SC-514, resulted in a significant reduction in both IL-17C mRNA and protein expression, indicating the significance of this pathway in the regulation of IL-17C. NP-κB binding sites were identified upstream from the IL-17C gene, and by electrophoretic mobility shift assay NF-κB was shown to bind to all three identified binding sites. Moreover, NF-κB binding to these sites was inducible by TNFα. Supershift analysis revealed binding of the NF-κB subunits p65 and p50 to all three NP-κB binding sites. To determine the contribution of NF-κB in IL-17C expression, we conducted luciferase gene reporter experiments and demonstrated that a 3204-bp promoter fragment of IL-17C containing three putative NP-κB binding sites was strongly activated by TNFα. Interestingly, mutations of the three NP-κB binding sites revealed that one specific NP-κB binding site was crucial for the TNFα-mediated IL-17C induction because mutation of this specific site completely abolished TNFα-induced IL-17C promoter activation. We conclude that the activation of NP-κB (p65/p50) is crucial for the TNPα-induced stimulation of IL-17C expression in human keratinocytes. [ABSTRACT FROM AUTHOR]

The inflammasome is a cytosolic multiprotein complex with two major functions: recognizing pathogen-associated molecular patterns and reacting to these through activation of the proinflammatory cytokines IL-1β and IL-18. In this study, we characterized the expression of inflammasome components in psoriatic skin and other common inflammatory skin diseases. Human skin biopsy specimens, cultured primary human keratinocytes, and peripheral blood mononuclear cells (PBMCs) were analyzed using quantitative reverse transcriptase-PCR (RT-PCR) and semiquantitative western blotting. mRNA expression of the inflammasome components NALP1, NALP3, ASC, caspase-1, caspase-4, and caspase-5 was detected in psoriatic skin. Interestingly, we found an extensive, 20-fold upregulation (P<0.01) of caspase-5 mRNA in lesional compared with nonlesional psoriatic skin, whereas caspase-1, caspase-4, and ASC (apoptosis-associated speck-like protein with CARD domain) mRNAs were upregulated by only 1.5- to 2.6-fold (P<0.01). Caspase-5 mRNA was not increased in biopsies from other inflammatory skin diseases, suggesting that this finding could be psoriasis specific. In vitro experiments revealed that caspase-5 mRNA was induced in primary keratinocytes as well as PBMCs stimulated with IFN-γ. Inhibition studies suggested that caspase-5 mRNA upregulation was mediated through the NF-κB pathway. Our findings suggest that caspase-5 and the inflammasome may have an important role in the inflammatory response in psoriasis. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinase (MAPK) AP kinase 2 (MK2) is a serine/threonine kinase that is phosphorylated and activated by p38 MAPK. MK2 regulates the expression of various proinflammatory cytokines including TNF-α, IL-1β, IL-6, and IL-8. Recently, MK2 was demonstrated to be activated in lesional psoriatic epidermis. This study investigates for the first time the role of MK2 in skin inflammation using the model of oxazolone-induced acute allergic contact dermatitis in mice. We show that oxazolone treatment leads to increased expression and sustained activation of both p38 MAPK and MK2. The inflammatory response was determined by ear thickness, myeloperoxidase activity, and histology after oxazolone challenge. Pretreatment with the p38 MAPK inhibitor SB202190 and genetic ablation of MK2 inhibit this inflammatory response. In particular, IL-1β and, to a smaller but significant extent, also TNF-α and IFN-γ expression were decreased in MK2 knockout mice compared with wild-type mice. These results indicate that MK2 is a potential target for the treatment of inflammatory skin diseases.Journal of Investigative Dermatology (2009) 129, 891–898; doi:10.1038/jid.2008.322; published online 6 November 2008 [ABSTRACT FROM AUTHOR]

Caspase-1 belongs to the group of inflammatory caspases and is the activating enzyme for the proinflammatory cytokine IL-18, a cytokine known to play an important role in the pathogenesis of psoriasis. The purpose of this study was to determine the expression of caspase-1 in psoriatic skin and the signaling mechanisms involved in stress-induced activation of caspase-1 and IL-18. Interestingly, increased caspase-1 activity in lesional compared with non-lesional psoriatic skin was seen. In vitro experiments in cultured human keratinocytes demonstrated anisomycin-induced, p38 mitogen-activated protein kinase (p38 MAPK)-dependent increased secretion of procaspase-1 and active caspase-1. Furthermore, anisomycin increased the mRNA expression of IL-18 through a p38 MAPK-dependent but caspase-1-independent mechanism, reaching a maximum level after 12 hours of stimulation. Finally, anisomycin caused a rapid (4 hours) increase in the secretion of proIL-18 and active IL-18. Secretion of active IL-18 was mediated through a p38 MAPK/caspase-1-dependent mechanism, whereas secretion of proIL-18 was mediated by a p38 MAPK-dependent but caspase-1-independent mechanism. These data demonstrate that the activity of caspase-1 is increased in psoriatic skin and that IL-18 secretion is regulated by a p38 MAPK/caspase-1-dependent mechanism, making caspase-1 a potential target in the treatment of psoriasis.Journal of Investigative Dermatology (2007) 127, 2857–2864; doi:10.1038/sj.jid.5700922; published online 28 June 2007 [ABSTRACT FROM AUTHOR]

The p38 mitogen-activated protein kinase (MAPK) signaling pathway, which regulates the activity of different transcriptions factors including NF-κB, is activated in lesional psoriatic skin. The purpose of this study was to investigate the effect of fumaric acid esters (FAEs) on the p38 MAPK and the downstream kinases mitogen- and stress-activated protein kinase (MSK)1 and 2 in cultured human keratinocytes. Cell cultures were incubated with dimethylfumarate (DMF), methylhydrogenfumarate (MHF), or fumaric acid (FA) and then stimulated with IL-1β before kinase activation was determined by Western blotting. A significant inhibition of both MSK1 and 2 activations was seen after preincubation with DMF and stimulation with IL-1β, whereas MHF and FA had no effect. In addition, DMF decreased phosphorylation of NF-κB/p65 (Ser276), which is known to be transactivated by MSK1. Furthermore, incubation with DMF before stimulation with IL-1β resulted in a significant decrease in NF-κB binding to the IL-8 κB and the IL-20 κB-binding sites as well as a subsequent decrease in IL-8 and IL-20 mRNA expression. Our results suggest that DMF specifically inhibits MSK1 and 2 activations and subsequently inhibits NF-κB-induced gene–transcriptions, which are believed to be important in the pathogenesis of psoriasis. These effects of DMF explain the anti-psoriatic effect of FAEs.Journal of Investigative Dermatology (2007) 127, 2129–2137; doi:10.1038/sj.jid.5700859; published online 10 May 2007 [ABSTRACT FROM AUTHOR]

The activity of the p38 mitogen-activated protein kinases (MAPKs) is increased in lesional psoriatic skin, supporting a possible role of these kinases in the pathogenesis of psoriasis. Recently, increased focal activation of the downstream target mitogen- and stress-activated protein kinase 1 (MSK1) was demonstrated in psoriatic epidermis. The purpose of this study is to investigate MSK2 and the transcription factor cyclic adenosine monophosphate response element-binding protein (CREB) in psoriatic skin and in cultured normal human keratinocytes. In lesional psoriatic skin, significantly increased MSK2 (Ser196) and CREB (Ser133) activation was demonstrated by phospho blotting. Immunofluorescence staining of phosphorylated MSK2 (Ser196) revealed colocalization with phosphorylated MSK1 (Thr 581) in the epidermis. Keratinocyte cultures stimulated with anisomycin and IL-1β showed increased MSK2 (Ser196) and CREB (Ser133) phosphorylation. Such activation was abolished during preincubation with a p38 inhibitor. Keratinocytes transfected with small interfering RNA showed a stronger decrease in CREB phosphorylation in MSK1/2 double-transfected cells than in MSK1 and MSK2 single-transfected cells. This study demonstrate for the first time the expression of MSK2 in keratinocytes and increased MSK2 and CREB activation in lesional psoriatic skin. Our results indicate that the p38-MAPK/MSK1/MSK2 and CREB signalling pathway may play a role in the pathogenesis of psoriasis.Journal of Investigative Dermatology (2007) 127, 2012–2019; doi:10.1038/sj.jid.5700821; published online 12 April 2007 [ABSTRACT FROM AUTHOR]

Mitogen- and stress-activated protein kinase 1 (MSK1) is a downstream target of both the p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinases (MAPKs). MSK1 stimulates transcription of different pro-inflammatory genes through activation of transcription factors. The purpose of this study was to investigate the expression and activation of MSK1 in lesional psoriatic skin and its role in cytokine production in cultured normal human keratinocytes. Western blotting revealed a consistent and significant increase in phosphorylated (activated) MSK1(Ser376) in lesional psoriatic skin. Immunofluorescence staining revealed the phosphorylated MSK1(Thr581) to be localized in the basal layers of the epidermis in lesional psoriatic skin. No staining was found in non-lesional psoriatic skin. Cultured human keratinocytes incubated with anisomycin or IL-1β resulted in the phosphorylation of the p38 MAPK and MSK1(Ser376). MSK1(Ser376) phosphorylation was inhibited by pre-incubation with the p38 inhibitor SB 202190. Transfection of the keratinocytes with specific MSK1 small interfering RNA resulted in 80% reduction of MSK1 expression and 51, 40, and 31% decrease in IL-6, IL-8, and tumor necrosis factor-α protein production, respectively. This study demonstrates for the first time the expression of MSK1 in epidermal keratinocytes and increased activation focally in psoriatic epidermis. As MSK1 regulates the production of pro-inflammatory cytokines, it may play a role in the pathogenesis of psoriasis.Journal of Investigative Dermatology (2006) 126, 1784–1791. doi:10.1038/sj.jid.5700252; published online 16 March 2006 [ABSTRACT FROM AUTHOR]

Nuclear factor-κB (NF-κB) is an inducible nuclear transcription factor regulating a range of cellular processes. An imbalance of the DNA binding activity of NF-κB may, therefore, be part of the pathophysiological mechanisms in psoriasis. The purpose of this study was to determine the NF-κB DNA binding activity in psoriatic skin using three differentκB sites and to determine how DNA binding activity was modulated by the anti-psoriatic drug calcipotriol. By electrophoretic mobility shift assay, we demonstrated that the NF-κB DNA binding to the p53κB site was decreased, whereas the NF-κB DNA binding to the interleukin-8 (IL-8)κB site was increased in lesional psoriatic skin compared with non-lesional psoriatic skin. No regulation was seen on the NF-κB DNA binding to the major histocompatibility complex class IκB site. These changes were paralleled by a similar decrease in p53 expression and an increase in IL-8 expression in involved psoriatic skin compared with uninvolved skin as determined by quantitative RT-PCR. The alteration in NF-κB DNA binding activity was neither accompanied by any change in the expression of the inhibitorκB (IκB) kinases, IKKα, IKKβ, and IKKγ nor in the expression of the NF-κB inhibitor proteins, IκBα and IκBβ. Immunofluorescence analysis revealed that p65 was sequestered in the cytoplasm of keratinocytes, whereas p50 exhibited a cytoplasmic as well as a nuclear localization. Interestingly, this distribution of p50 and p65 was similar in lesional and non-lesional psoriatic skin. Topical application of calcipotriol to lesional psoriatic skin for 4 d resulted in increased NF-κB binding to the p53κB site and decreased NF-κB binding to the IL-8κB site. Taken together, our data demonstrate that the NF-κB DNA binding activity is regulated in a specific manner in psoriatic skin depending on theκB sites investigated, and that topical treatment of psoriatic skin normalizes the abnormal NF-κB binding activity seen in lesional psoriatic skin. [ABSTRACT FROM AUTHOR]

Investigate the mechanism by which 1alpha,25-dihydroxyvitamin D[sub3] modulates activator protein 1 DNA binding activity in cultured normal human keratinocytes. Characteristics of stratified epidermis; Findings of western blotting and transfection experiments regarding dihydroxyvitamin; Effect of anti-annexin II antibody on the 1alpha,25(OH)[sub2]D[sub3]-induced phosphorylation.

Summary1α,25(OH)2D3 and its analogs are potent mediators of keratinocyte differentiation in vitro. The precise mechanism of this action is still unknown. The nuclear transcription factor activator protein 1 seems to play an important role in keratinocyte differentiation. The purpose of this study was to investigate the effect of 1α,25(OH)2D3 on activator protein 1 DNA binding activity in cultured human keratinocytes. In a time-course study of human keratinocytes incubated with 1α,25(OH)2D3 (10-7-10-11 M) a significant dose-dependent increase in activator protein 1 DNA binding activity as determined by electrophoretic mobility shift assay was seen after 36 h. This increase was followed by a significant dose-dependent decrease in activator protein 1 DNA binding activity after 72 h. When differentiation was induced by raising the calcium concentration in the culture medium from 0.09 to 0.3 mM a similar increase in activator protein 1 DNA binding activity was observed after incubation for 48 h. Pharmacologic down-modulation of the protein kinase C activity with GF 109203X reversed the calcium-induced increase in activator protein 1 DNA binding activity and abolished keratinocyte differentiation as determined by a transglutaminase assay. In contrast, activator protein 1 DNA binding activity and keratinocyte differentiation were not affected when protein kinase C activity was down-modulated in the experiments with 1α,25(OH)2D3. The activator protein 1 complex in human keratinocytes consists of dimers of Fra-1, Fra-2, c-Jun, JunD, and c-Fos. Our results demonstrate that 1α,25(OH)2D3- and calcium-induced keratinocyte differentiation are accompanied by changes in activator protein 1 DNA binding activity. Protein kinase C activation appears to be essential for the calcium-dependent induction of keratinocyte differentiation, whereas a protein-kinase-C-independent activation of activator protein 1 DNA binding and keratinocyte differentiation is responsible for the 1α,25(OH)2D3-induced effects. [ABSTRACT FROM AUTHOR]

IL-20 is a novel member of the IL-10 cytokine family with pleiotropic effects. Current knowledge of what triggers and regulates IL-20 gene expression is sparse. The aim of this study was to investigate the regulation of IL-20 expression in cultured normal human keratinocytes. The expression of IL-20 was rapidly induced by proinflammatory stimuli, in particular IL-1β, IL-6, and UVB irradiation. Using kinase inhibitors and small-interfering RNA, we discovered that the p38 mitogen-activated protein kinase (MAPK) as well as inhibitory κB kinase -NF-κB signaling pathways are crucial for IL-20 expression. By electrophoretic mobility shift assay two κB-binding sites were identified upstream from the start codon in the IL-20 gene. Supershift analysis revealed binding of the p50/p65 heterodimer. Furthermore, the p38 MAPK was shown to exert its effects on IL-20 expression through activation of the downstream kinase mitogen- and stress-activated kinase 1 (MSK1), indicating transactivation of NF-κB driven IL-20 messenger RNA transcription as an important mechanism of action. IL-20 is assumed to be a key cytokine in the pathogenesis of psoriasis and possibly cancer, and therefore the p38 MAPK, MSK1, and NF-κB may be important new molecular targets for the modulation of IL-20 expression in these diseases.Journal of Investigative Dermatology (2007) 127, 1326–1336. doi:10.1038/sj.jid.5700713; published online 25 January 2007 [ABSTRACT FROM AUTHOR]

Abnormal epidermal proliferation and differentiation characterize the inflammatory skin disease psoriasis. Here we demonstrate that expression of PPARδ mRNA and protein is markedly upregulated in psoriatic lesions and that lipoxygenase products accumulating in psoriatic lesions are potent activators of PPARδ. The expression levels of NF-κB p50 and p65 were not significantly altered in lesional compared with nonlesional psoriatic skin. In the basal layer of normal epidermis both p50 and p65 were sequestered in the cytoplasm, whereas p50, but not p65, localized to nuclei in the suprabasal layers, and this distribution was maintained in lesional psoriatic skin. In normal human keratinocytes PPAR agonists neither impaired IL-1β-induced translocation of p65 nor IL-1β-induced NF-κB DNA binding. We show that PPARδ physically interacts with the N-terminal Rel homology domain of p65. Irrespective of the presence of agonists none of the PPAR subtypes decreased p65-mediated transactivation in keratinocytes. In contrast p65, but not p50, was a potent repressor of PPAR-mediated transactivation. The p65-dependent repression of PPARδ- but not PPARα- or PPARγ-mediated transactivation was partially relieved by forced expression of the coactivators p300 or CBP. We suggest that deficient NF-κB activation in chronic psoriatic plaques permitting unabated PPARδ-mediated transactivation contributes to the pathologic phenotype of psoriasis. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptors (PPARs) are pleiotropic regulators of growth and differentiation of many cell types. We have performed a comprehensive analysis of the expression of PPARs, transcriptional cofactors, and marker genes during differentiation of normal human keratinocytes using a combination of reverse transcriptase polymerase chain reaction, Northern and Western blotting, and immunohistochemistry. PPARδ was the predominant PPAR subtype in human keratinocytes and highly expressed in basal cells and suprabasal cells. Induction of PPARα and PPARγ expression was linked to differentiation, and accordingly, expression of PPARα and PPARγ was in essence confined to suprabasal cells. Differentiation was not accompanied by significant changes in the expression of the coactivators CREB-binding protein, p300, steroid receptor coactivator 1, or the corepressors nuclear receptor corepressor and silence mediator for retinoid and thyroid hormone receptors. We critically evaluated the effects of selective PPAR ligands and a synthetic fatty acid analog, tetradecylthioacetic acid. Tetradecylthioacetic acid activated all human PPAR subtypes in the ranking order PPARδ >> PPARα > PPARγ. All selective PPAR ligands marginally induced transglutaminase-1 expression with the PPARδ-selective ligand L165041 being the most potent. The PPARα- and PPARγ-selective ligands Wy14643 and BRL49653 had negligible effect on involucrin expression, whereas a dose-dependent induction was observed with L165041. Simultaneous addition of L165041 and BRL49653 synergistically induced strong involucrin expression. Additionally, L165041 potently induced CD36 mRNA expression. Administration of tetradecylthioacetic acid resulted in a dramatic decrease in proliferation and a robust upregulation of the expression of involucrin and transglutaminase. Our results indicate that tetradecylthioacetic acid may affect keratinocyte gene... [ABSTRACT FROM AUTHOR]