Claus Johansen

Protein Expression of TNF-α in Psoriatic Skin Is Regulated at a Posttranscriptional Level by MAPK-Activated Protein Kinase 2

Alterations in specific signal transduction pathways may explain the increased expression of proinflammatory cytokines seen in inflammatory diseases such as psoriasis. We reveal increased TNF-α protein expression, but similar TNF-α mRNA levels, in lesional compared with nonlesional psoriatic skin, demonstrating for the first time that TNF-α expression in lesional psoriatic skin is regulated posttranscriptionally. Increased levels of activated MAPK-activated protein kinase 2 (MK2) together with increased MK2 kinase activity were found in lesional compared with nonlesional psoriatic skin. Immunohistochemical analysis showed that activated MK2 was located in the basal layers of the psoriatic epidermis, whereas no positive staining was seen in nonlesional psoriatic skin. In vitro experiments demonstrated that both anisomycin and IL-1β caused a significant activation of p38 MAPK and MK2 in cultured normal human keratinocytes. In addition, TNF-α protein levels were significantly up-regulated in keratinocytes stimulated with anisomycin or IL-1β. This increase in TNF-α protein expression was completely blocked by the p38 inhibitor, SB202190. Transfection of cultured keratinocytes with MK2-specific small interfering RNA led to a significant decrease in MK2 expression and a subsequent significant reduction in the protein expression of the proinflammatory cytokines TNF-α, IL-6, and IL-8, whereas no change in the expression of the anti-inflammatory cytokine IL-10 was seen. This is the first time that MK2 expression and activity have been investigated in an inflammatory disease such as psoriasis. The results strongly suggest that increased activation of MK2 is responsible for the elevated and posttranscriptionally regulated TNF-α protein expression in psoriatic skin, making MK2 a potential target in the treatment of psoriasis.

Tumor Necrosis Factor α-Mediated Induction of Interleukin 17C in Human Keratinocytes Is Controlled by Nuclear Factor κB

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. NF-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 NF-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 NF-κB binding sites was strongly activated by TNFα. Interestingly, mutations of the three NF-κB binding sites revealed that one specific NF-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 NF-κB (p65/p50) is crucial for the TNFα-induced stimulation of IL-17C expression in human keratinocytes.

Activator protein 1 DNA binding activity is decreased in lesional psoriatic skin compared with nonlesional psoriatic skin

Backgroundu2002 Psoriasis is a common benign skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. The transcription factor activator protein 1 (AP‐1) is known to play an important role in cell proliferation and differentiation.

MK2 regulates the early stages of skin tumor promotion.

The association between inflammation and tumorigenesis is well recognized. Mitogen-activated protein kinase-activated protein kinase-2 (MK2) is known to play a pivotal role in inflammatory processes. Here, we studied the effect of MK2-deficiency and tumor necrosis factor (TNF)-alpha-deficiency on skin tumor development in mice using the two-stage chemical carcinogenesis model. We found that MK2(-/-) mice developed significantly fewer skin tumors compared with both TNF-alpha(-/-) and wild-type mice when induced by initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). The TPA-induced inflammatory response was reduced in both, TNF-alpha(-/-) mice and MK2(-/-) mice, but most pronounced in TNF-alpha(-/-) mice, indicating that a reduced inflammatory response was not the only explanation for the inhibited tumorigenesis seen in MK2(-/-) mice. Interestingly, increased numbers of apoptotic cells were detected in the epidermis of MK2(-/-) mice compared with TNF-alpha(-/-) and wild-type mice, suggesting an additional role of MK2 in the regulation of apoptosis. This was further supported by: (i) increased levels of the tumor suppressor protein p53 in MK2(-/-) mice after DMBA/TPA treatment compared with controls, (ii) reduced phosphorylation (activation) of the negative p53 regulator, murine double minute 2 in MK2(-)(/-) mouse keratinocytes in vitro and (iii) a significant decrease in the DMBA/TPA induced apoptosis in cultured MK2(-/-) keratinocytes transfected with p53 small interfering RNA. Taken together, these findings demonstrate a dual role of MK2 in the early stages of tumor promotion through regulation of both the inflammatory response and apoptosis of DNA-damaged cells. These results also identify MK2 as a putative target for future skin carcinoma therapy.

TARC augments TNF-α-induced CTACK production in keratinocytes

Abstract:u2002 Thymus‐ and activation‐regulated chemokine (TARC)/CCL17 and cutaneous T cell‐attracting chemokine (CTACK)/CCL27 are both pivotal mediators of the inflammatory reaction of atopic dermatitis (AD). TARC attracts CCR4 positive T cells known to be mainly of Th2 subtype whereas CTACK attracts skin‐homing T cells of both Th1 and Th2 subtype that express CCR10. We found that CTACK can be induced in cultured human keratinocytes by tumor necrosis factor‐α (TNF‐α), but not by TARC alone. However, if the keratinocytes were preincubated with TNF‐a for 6u2003h, TARC was able to augment the CTACK‐inducing effect of TNF‐a. Performing immunohistochemical stainings, reverse‐transcription polymerase chain reaction (RT‐PCR), and Western blotting, we found that TNF‐a‐induced CCR4 mRNA production, but that stimulated as well as non‐stimulated keratinocytes expressed CCR4. In order to see if these results had any clinical relevance, we investigated the plasma concentrations of TARC and CTACK from 48 patients suffering from AD. This revealed that TARC and CTACK concentrations in plasma correlate with each other. Surprisingly, p‐CTACK correlated inversely with SCORAD scores of the patients, which most likely is due to the treatment the patients received. Our results suggest that the primary Th2‐dominated inflammatory reaction in AD induced by TARC leads to an augmented skin‐specific inflammatory reaction through CTACK.

Ustekinumab in the treatment of refractory chronic cutaneous lupus erythematosus: a case report.

© 2013 The Authors. doi: 10.2340/00015555-1467 Journal Compilation

Lysophosphatidylcholine induces keratinocyte differentiation and upregulation of AP-1- and NF-κb DNA-binding activity

Lysophosphatidylcholine (lysoPC) is generated by the action of phospholipase A2 on membrane phosphatidylcholine, the most abundant cellular phospholipid. In vitro, lysoPC has pro-inflammatory properties, as it upregulates the expression of adhesion molecules and is a chemoattractant to monocytes and T lymphocytes. It upregulates the expression of a variety of genes including genes encoding growth factors and cyclooxygenase-2 and modulates other cellular responses like proliferation and differentiation. A role for lysoPC as an intracellular messenger transducing signals from membrane-associated receptors has also been suggested. However, the mechanisms behind the diverse actions of lysoPC are poorly understood. In this study we found that lysoPC in non-toxic concentrations caused increased activator protein-1 (AP-1) DNA-binding activity and transglutaminase-1 expression in cultured human keratinocytes. The effects on transglutaminase-1 and AP-1 were dependent on protein kinase C and mitogen-activated protein kinase kinase. In addition, lysoPC caused a rapid and transient increase in DNA-binding activity of nuclear factor-kappaB.

IL-20, IL-21 and p40: potential biomarkers of treatment response for ustekinumab.

Although biological drugs in psoriasis treatment show clinical efficacy, there are still a proportion of patients in whom little treatment response is obtained. The aim of this study was to identify molecular biomarkers for treatment response and to investigate the molecular effects of ustekinumab treatment of psoriasis. The mRNA expression of various genes in skin biopsies was analysed by quantitative polymerase chain reaction (qPCR). At baseline, there was no significant clinical difference be-tween responders and non-responders. Ten patients were clinical responders, with a mean baseline Psoriasis Area and Severity Index (PASI) score of 15.4 and a mean percentage improvement of 89.6%. No significant reduction in PASI during treatment was seen among the 5 non-responders. In the responder group, ustekinumab therapy reduced the mRNA expression of the majority of the studied genes in lesional psoriatic skin. IL-20, IL-21 and p40 mRNA expression in lesional psoriatic skin at baseline were significantly upregulated by factors of 2.7, 2.4 and 2.3, respectively, among non-responders compared with responders. The mRNA levels of p40, IL-20 and IL-21 at baseline may serve as potential predictors of treatment response to ustekinumab treatment.

Role of p38 Mitogen-activated Protein Kinase Isoforms in Murine Skin Inflammation Induced by 12-O-tetradecanoylphorbol 13-acetate

p38 mitogen-activated protein kinase plays a pivotal role in skin inflammation. The purpose of this study was to investigate the role of the various p38 isoforms. p38β/δ-knockout-C57BL/6 mice were generated, studied in a 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced skin inflammation model and compared with wild-type mice. The inflammatory response was determined by ear thickness, myeloperoxidase activity and histology. mRNA and protein expression of interleukin (IL)-1β and IL-6 was determined by quantitative real-time reverse transcription PCR and enzyme-linked immunoassay. In both groups application of TPA resulted in a significant increase in inflammation, and pretreatment with the p38α/β inhibitor, SB202190 resulted in a significant inhibition. A significantly slower onset but prolonged duration of the response was seen in p38β/δ knockout mice. This was paralleled by a significant, but transient, lower IL-1β and IL-6 protein expression in p38β/δ knockout mice. Although the p38α isoform is important, our data also demonstrate an important role of the p38β and/or δ isoforms in the regulation of TPA-induced skin inflammation.

Inflammation-Induced Alterations in the Skin Barrier Function: Implications in Atopic Dermatitis

The pathogenesis of atopic dermatitis (AD) is very complex, but best characterized by an inflammatory reaction in the skin and a disrupted skin barrier. Until recently, these two factors have been studied as separate entities; however, it has been shown that inflammatory cytokines can regulate filaggrin, a very important component of the skin barrier, as well as proteins involved in the processing and maturation of filaggrin. Therefore, inflammation itself may be able to induce a functional skin barrier dysfunction and thereby aggravate the eczematous reaction in AD.