Changes in mRNA expression precede changes in microRNA expression in lesional psoriatic skin during treatment with adalimumab.

BACKGROUND: Tumour necrosis factor (TNF)-α inhibition is an effective treatment for moderate to severe plaque-type psoriasis. A change in the cytokine expression profile occurs in the skin after 4 days of treatment, preceding any clinical or histological improvements. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, but miRNA expression has never been studied in psoriatic skin during treatment. OBJECTIVE: To investigate changes in miRNA expression in psoriatic skin during adalimumab treatment and to compare results with changes in miRNA expression in a mouse model of Aldara-induced psoriasis-like skin inflammation. METHODS: Punch biopsies were obtained from nonlesional and lesional psoriatic skin during adalimumab treatment. In the mouse model of Aldara-induced skin inflammation, biopsies were obtained from TNF-α knockout (KO), IL-17A KO and wild-type mice. miRNA expression levels were analysed with microarray, reverse transcriptase quantitative polymerase chain reaction and in situ hybridization. RESULTS: In psoriatic skin, no changes in miRNA expression were seen 4 days after treatment initiation. After 14 days of treatment, the expression of several miRNAs was normalized towards the level seen in nonlesional skin before treatment. miR-23b expression increased after 14 days of treatment and remained high for 84 days, despite unaltered levels at baseline. In the mouse model of Aldara-induced skin inflammation, the level of miR-146a increased, whereas no regulation was seen for miR-203, miR-214-3p, miR-125a, miR-23b or let-7d-5p. CONCLUSIONS: This study demonstrates that the changes seen in the cytokine expression levels after 4 days of treatment with adalimumab are not facilitated by early changes in miRNA expression.

The expression of dual-specificity phosphatase 1 mRNA is downregulated in lesional psoriatic skin.

BACKGROUND: The p38 mitogen-activated protein kinase (MAPK) plays an important role in inflammatory processes and displays increased activity in psoriasis. Dual-specificity phosphatase 1 (DUSP1) is an important negative regulator of p38 MAPK activity. OBJECTIVES: To study mRNA expression of DUSP1 in normal human epidermal keratinocytes (NHEKs) stimulated with proinflammatory cytokines and to investigate DUSP1 in psoriatic skin. METHODS: NHEKs were cultured in vitro and punch biopsies were obtained from the skin of patients with psoriasis vulgaris and atopic dermatitis. mRNA expression was analysed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS: In NHEKs, interleukin (IL)-1ß induced DUSP1 mRNA expression in a rapid and time-dependent manner through the p38 MAPK/mitogen- and stress-activated kinase (MSK) signalling pathway. DUSP1 mRNA expression was demonstrated to be significantly downregulated in psoriatic skin lesions compared with paired samples of nonlesional psoriatic skin. This was in contrast to atopic dermatitis. The downregulation of DUSP1 mRNA in lesional psoriatic skin was not explained by the difference in the mRNA expression of the potential DUSP1 transcript stability-affecting proteins Hu antigen R or tristetraprolin. Furthermore, DUSP1 mRNA expression was shown not to increase during the early course of treatment with the antitumour necrosis factor-α antibody adalimumab. CONCLUSIONS: In lesional psoriatic skin, the p38 MAPK negative feedback mechanism provided by DUSP1 seems to be inhibited. Downregulation of DUSP1 may contribute to the sustained inflammatory response seen in psoriasis.

STAT1 expression and activation is increased in lesional psoriatic skin.

BACKGROUND: The JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling pathway is known to play an important role in many cellular processes including inflammation. The activation of STAT1 is dependent on tyrosine 701 and serine 727 phosphorylation, which leads to the formation of the STAT dimer and modulation of STAT1 activity, respectively. OBJECTIVE: To determine STAT1 expression and activation in psoriatic skin. METHODS: Biopsies were collected from patients with psoriasis. mRNA expression was evaluated by quantitative polymerase chain reaction, whereas the protein and phosphorylation level of STAT1 were evaluated by Western blotting. STAT1 localization was determined by immunofluorescence analysis and STAT1-induced transcriptional activity was analysed in cultured human keratinocytes using a reporter assay. RESULTS: The expression of STAT1 was demonstrated to be significantly increased at both mRNA and protein level in lesional psoriatic skin. In addition, the phosphorylation level of STAT1(Tyr701) and STAT1(Ser727) was significantly increased in lesional compared with nonlesional psoriatic skin. Luciferase assays showed a significant induction of the STAT1-induced transcriptional activity when cultured human keratinocytes were stimulated with either interferon (IFN)-α or IFN-γ. STAT1(Ser727) phosphorylation induced by IFN-α, IFN-γ or ultraviolet B was mediated by a protein kinase C (PKC)-δ- and p38 mitogen-activated protein kinase-dependent mechanism in human keratinocytes, whereas IFN-α-induced STAT1(Tyr701) phosphorylation was mediated by a PKC-δ-dependent mechanism. CONCLUSIONS: This study demonstrates for the first time that the phosphorylation level of STAT1(Tyr701) and STAT1(Ser727) is increased in lesional psoriatic skin. In addition, specific signalling pathways leading to this phosphorylation have been identified. Together, our data indicate an important role of STAT1 in the pathogenesis of psoriasis.

Dimethylfumarate inhibits MIF-induced proliferation of keratinocytes by inhibiting MSK1 and RSK1 activation and by inducing nuclear p-c-Jun (S63) and p-p53 (S15) expression.

OBJECTIVE: Dimethylfumarate (DMF) is used in the treatment of psoriasis. Macrophage migration inhibitory factor (MIF) is elevated in patients with severe psoriasis. We studied the effect of DMF on the MIF-induced activation of the mitogen- and stress-activated kinase 1 (MSK1) and p90 kDa ribosomal S6 kinase (RSK1) signaling pathways which regulate the proliferation of human keratinocytes via transcription factors. METHODS: The effects of DMF on the MIF-induced activation of MSK1, RSK1, cAMP-responsive element-binding protein (CREB), Cox-2 and c-Jun, JunB and p53 were studied by Western blotting using phospho-specific antibodies. RESULTS: DMF inhibited the MIF-induced phosphorylation of MSK1, RSK1, CREB and JunB, and reduced Cox-2 expression and the proliferation of cultured human keratinocytes. The expression of p-p53 (S15) was induced simultaneously with the inhibition of Cox-2. Addition of DMF before MIF induced nuclear expression of p-c-Jun (S63) and c-Jun. Transfection with small interfering MSK1 and RSK1 RNA before MIF incubation stimulated p-p53 (S15) and nuclear p-c-Jun (S63) similarly to DMF. CONCLUSION: Our results indicate that the specific inhibitory effects of DMF on RSK1 and MSK1 activation together with the induction of p-c-Jun (S63) and p-p53 (S15) lead to the inhibition of keratinocyte proliferation, partly explaining the anti-psoriatic effect of DMF.

The expression and phosphorylation of eukaryotic initiation factor 4E are increased in lesional psoriatic skin.

BACKGROUND: Overexpression of the eukaryotic initiation factor (eIF) 4E results in increased translation of mRNAs encoding proteins involved in cell cycle control, proliferation, apoptosis and angiogenesis. Phosphorylation of eIF4E is conducted by MAP kinase interacting serine/threonine kinase 1 and 2, and phosphorylation of eIF4E has previously been associated with increased release of proinflammatory cytokines from keratinocytes. The actions of eIF4E are counteracted by the eIF4E-binding protein 1 (4E-BP1). OBJECTIVES: To characterize the mRNA and protein expression of eIF4E, as well as the phosphorylation of eIF4E in psoriatic skin. METHODS: Biopsies were collected from patients with psoriasis. mRNA expression and protein levels of eIF4E were evaluated by quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively. eIF4E distribution was determined by immunofluorescence analysis. RESULTS: We found a significant increase in mRNA expression and protein level of eIF4E in lesional as compared with nonlesional psoriatic skin. Immunofluorescence analysis demonstrated that eIF4E was located throughout the epidermis and was primarily cytoplasmic in distribution. The level of phosphorylated eIF4E protein was found to be strongly upregulated, and 4E-BP1 expression was also increased. CONCLUSIONS: We have demonstrated for the first time that the level of total and phosphorylated eIF4E and the expression of 4E-BP1 are increased in lesional psoriatic skin. As eIF4E-regulated proteins have been reported to be upregulated in psoriasis, it appears that the increase in eIF4E is only incompletely counteracted by 4E-BP1. Therefore, eIF4E might contribute to the pathogenesis of psoriasis.

Characterization of the interleukin-17 isoforms and receptors in lesional psoriatic skin.

BACKGROUND: Th17 cells are a lineage of proinflammatory T helper cells producing interleukin (IL)-17. The importance of Th17 cells in inflammation and autoimmunity has now been recognized. The IL-17 cytokine family consists of six isoforms (IL-17A-IL-17F) whereas five members of the IL-17 receptor (IL-17R) family have been identified (IL-17RA-IL-17RE). OBJECTIVES: To characterize the expression of the IL-17 isoforms and receptors in lesional and nonlesional psoriatic skin. Methods Keratome and punch biopsies taken from patients with psoriasis were examined by enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction in order to measure the IL-17 isoforms and receptors. RESULTS: We demonstrated significantly increased mRNA expression of IL-17A, IL-17C and IL-17F in psoriatic skin. In contrast, the mRNA expression of IL-17B and IL-17D was significantly decreased in lesional compared with nonlesional skin, while IL-17E mRNA was undetectable. The increased mRNA expression of IL-17A, IL-17C and IL-17F was paralleled by an increased protein accumulation of these cytokines in psoriatic skin. Analysis of the IL-17R mRNA expression revealed significantly impaired mRNA expression of IL-17RB, IL-17RC, IL-17RD and IL-17RE in lesional psoriatic skin, whereas the mRNA expression of IL-17RA was similar in lesional and nonlesional psoriatic skin. CONCLUSIONS: This study characterizes the mRNA profile of the IL-17 isoforms and receptors in psoriatic skin lesions. Furthermore, we demonstrate for the first time augmented protein levels of IL-17A, IL-17C and IL-17F in psoriatic skin lesions, indicating a possible role for IL-17C in addition to IL-17A and IL-17F in the pathogenesis of psoriasis.