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The Journal of Immunology, 2006, 176: 1431-1438.
Copyright © 2006 by The American Association of Immunologists

Protein Expression of TNF-{alpha} in Psoriatic Skin Is Regulated at a Posttranscriptional Level by MAPK-Activated Protein Kinase 21

Claus Johansen2,*, Anne Toftegaard Funding*, Kristian Otkjaer*, Knud Kragballe3,*, Uffe Birk Jensen4,{dagger}, Mogens Madsen5,{ddagger}, Lise Binderup6,§, Tine Skak-Nielsen{ddagger}, Marianne Scheel Fjording5,{ddagger} and Lars Iversen3,*

* Department of Dermatology and {dagger} Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark; {ddagger} Department of Biochemistry and Cell Biology and § Department of Biological Research, LEO Pharma, Ballerup, Denmark


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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-{alpha} protein expression, but similar TNF-{alpha} mRNA levels, in lesional compared with nonlesional psoriatic skin, demonstrating for the first time that TNF-{alpha} 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-1beta caused a significant activation of p38 MAPK and MK2 in cultured normal human keratinocytes. In addition, TNF-{alpha} protein levels were significantly up-regulated in keratinocytes stimulated with anisomycin or IL-1beta. This increase in TNF-{alpha} 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-{alpha}, 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-{alpha} protein expression in psoriatic skin, making MK2 a potential target in the treatment of psoriasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Psoriasis is an immune-mediated inflammatory skin disorder characterized by skin-infiltrating lymphocytes causing hyperproliferation and abnormal differentiation of the keratinocytes (1, 2). The initiation and persistence of the characteristic inflammatory processes in psoriasis seem to be triggered by a cytokine pattern belonging to the Th1 type. This pattern consists of signaling molecules such as TNF-{alpha}, IFN-{gamma}, IL-1, IL-2, IL-3, IL-6, IL-8, epidermal growth factor, and TGF-{alpha} (3, 4). Because of the strong antipsoriatic activity of TNF-{alpha} antagonists, the increased TNF-{alpha} expression is of particular interest. However, the intracellular signaling pathways implicated in this altered cytokine pattern remain to be determined.

The p38 MAPK is activated by cellular stress, such as anisomycin, heat shock, H2O2, and UV radiation; by several proinflammatory cytokines, including TNF-{alpha} and IL-1; and LPS (5, 6). There are four p38 MAPK isoforms: the {alpha} and beta isoforms, which are 75% homologous, and the {gamma} and {delta} isoforms, which are more distant relatives (7). We have recently demonstrated activation of p38{alpha}, -beta, and -{delta} in lesional psoriatic skin compared with nonlesional psoriatic skin, whereas p38{gamma} is not expressed in human epidermis (8). A major function of p38{alpha} and p38beta in inflammation is regulation of the expression of inflammatory cytokines (9). The mechanism by which p38 MAPK mediates its regulatory effects is through several p38 MAPK downstream kinases, including the MAPK-activated protein kinase 2 (MAPKAP kinase 2 (MK2)7) (10, 11, 12).

MK2 is a serine/threonine kinase that is phosphorylated and activated by the p38{alpha} and p38beta MAPKs (13). In its inactive form, MK2 is located in the nucleus. Upon activation by the p38{alpha} and -beta MAPKs, MK2 rapidly translocates from the nucleus to the cytoplasm and also cotransports p38 MAPK back to the cytoplasm (14, 15). MK2 is important in several p38-regulated pathways, because it is a direct substrate for p38 and has been shown to be involved in posttranscriptional regulation of TNF-{alpha} in macrophages and rheumatoid synovial cells, IL-6 in macrophages and HeLa cells, and IL-8 in HeLa cells (12, 16, 17, 18). Thus, several reports have observed that cells isolated from mice deleted for MK2 are deficient in the LPS-induced biosynthesis of several proinflammatory cytokines regulated by p38, including TNF-{alpha}, IL-6, and IL-1 (17, 19, 20). In addition, MK2–/– mice show increased stress resistance and survive LPS-induced endotoxic shock due to a reduction of ~90% in the production of TNF-{alpha} (19). In another study, MK2 was demonstrated to play an essential role in host defense against intracellular bacteria via regulation of TNF-{alpha} and IFN-{gamma} production (21). MK2 signaling is known to increase TNF-{alpha} through translational control via the AU-rich elements of the 3'-untranslated region of TNF-{alpha} mRNA (17), whereas in the case of IL-6 and IL-8, MK2 signaling leads to increased mRNA stability (12, 17).

Given that TNF-{alpha} is known to play a pivotal role in psoriasis, as demonstrated by the successful treatment of psoriasis by anti-TNF-{alpha} Abs (22, 23, 24), and the fact that p38 is activated in lesional psoriatic skin, we examined the p38 substrate MK2 in psoriatic skin and its role in cytokine production. We demonstrate for the first time that the increased TNF-{alpha} protein expression in lesional psoriatic skin is associated with normal TNF-{alpha} mRNA expression. Furthermore, the MK2 activity was found to be significantly augmented in lesional psoriatic skin compared with nonlesional psoriatic skin. In vitro studies of cultured human keratinocytes, using small interfering RNA (siRNA) technology to inhibit MK2 expression, showed that inhibition of MK2 significantly decreased the anisomycin-induced production of the proinflammatory cytokines TNF-{alpha}, IL-6, and IL-8, identifying both p38 MAPK and MK2 as potential targets in the treatment of psoriasis and other inflammatory diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Biopsies

Keratome biopsies were obtained from lesional and nonlesional psoriatic skin as previously described (25). Briefly, keratome biopsies from lesional and nonlesional plaque-type psoriatic skin were taken from the center of a plaque from patients with moderate to severe chronic stable plaque psoriasis from either the upper or lower extremities. For each patient, biopsies were taken from only one anatomical site. Biopsies were taken as paired samples, and biopsies from nonlesional psoriatic skin were taken from the same body region as biopsies from lesional psoriatic skin at a distance of at least 5 cm from a lesional plaque. Informed consent was obtained from each patient. For Western blotting and kinase assay analysis, the biopsies were taken from the center of a plaque and from nonlesional skin from patients with moderate to severe chronic stable plaque psoriasis.

For immunofluorescence analysis, 4-mm punch biopsies were taken from the center of a chronic plaque and nonlesional psoriatic skin. These biopsies were embedded in paraffin. For quantitative RT-PCR, 4-mm punch biopsies from nonlesional skin, chronic plaques, and acute guttate skin lesions were taken, immediately snap-frozen in liquid nitrogen, and stored in liquid nitrogen until further use. The local ethical committee of Aarhus approved all described studies.

Cell cultures

Normal adult human keratinocytes were obtained by trypsinization of skin samples from patients undergoing plastic surgery as previously described (26). Second-passage keratinocytes were grown in keratinocyte serum-free medium (K-SFM; Invitrogen Life Technologies). Twenty-four hours before stimulation with either anisomycin or IL-1beta, the medium was changed to keratinocyte basal medium (KBM; the same as K-SFM, but without growth factors) in which the cells were stimulated. In some experiments keratinocytes were pretreated with the p38{alpha}beta inhibitor SB202190 (10 µM) for 30 min before stimulation. Cells were grown at 37°C in 5% CO2 in an incubator.

Western blot analysis

Total cell extracts were prepared from keratome biopsies. The biopsies were homogenized in a cell lysis buffer (20 mM Tris-base (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF) and left on ice for 30 min. Then the samples were centrifuged at 10,000 x g for 10 min at 4°C, after which the supernatant constituted the cell lysate. Equal protein amounts (as determined by Bradford assay) were separated by SDS-PAGE and blotted onto nitrocellulose membranes (27). Membranes were incubated with phospho-MK2 (recognizing isoforms 1 and 2 of human phospho-MK2; catalogue no. 3041), MK2 (recognizing isoform 1 and 2 of human MK2; catalogue no. 3042), phospho-p38 (catalogue no. 9211), or p38 (catalogue no. 9212) Abs (Cell Signaling Technology) and detected with anti-rabbit IgG-HRP (DakoCytomation) in a standard ECL reaction (Amersham Biosciences) according to the manufacturer’s instructions. Densitometric analysis of the band intensity was conducted using Kodak 1D Image analysis software.

In vitro kinase assays

The MK2 kinase activity was performed using anti-MK2 agarose-conjugated beads (Upstate Biotechnology). The agarose beads were washed twice with ice-cold cell lysis buffer (20 mM Trizma-base (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerolphosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, and 1 mM PMSF) and then immunoprecipitated with 200 µg of protein extract for 1.5 h at 4°C. Immunocomplexes were isolated and washed twice with ice-cold cell lysis buffer and once with ice-cold kinase buffer (20 mM MOPS (pH 7.2), 25 mM beta-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 30 mM MgCl2, 60 µM ATP, and 1 mM DTT). Immunocomplexes were then incubated for 30 min at 30°C with 30 µl of kinase buffer supplemented with 4 µCi of [{gamma}-32P]ATP and 2 µg of 27-kDa heat shock fusion protein (Hsp27). The reaction was stopped by the addition of Laemmli sample buffer and boiled for 5 min. Samples were analyzed by SDS-PAGE, and phosphorylated Hsp27 was detected by autoradiography.

RNA isolation

Punch biopsies were transferred to 1 ml of –80°C cold RNAlater-ICE (Ambion). Samples were kept at –80°C until 24 h before RNA purification, at which time they were transferred to –20°C. Upon RNA purification, biopsies were removed from RNAlater-ICE and transferred to 175 µl of SV RNA lysis buffer supplemented with 2-ME (SV Total RNA Isolation System; Promega) and homogenized. RNA purification, including DNase treatment of the samples, was completed according to the manufacturer’s instructions (SV Total RNA Isolation System; Promega). The RNA was stored until further use at –80°C.

Quantitative RT-PCR

For RT we used TaqMan RT reagents (Applied Biosystems). Primers and probes were purchased from Applied Biosystems. TNF-{alpha} mRNA expression was analyzed using TaqMan 20X Assays-On-Demand expression assay mix (assay ID: Hs00174128_m1). The probe was a FAM-labeled minor groove binder probe with a nonfluorescent quencher. As housekeeping gene, we used ribosomal protein, large, P0 (RPLP0).

RPLP0 mRNA expression was determined using TaqMan 20X Assays-On-Demand expression assay mix (assay ID: Hs99999902_m1). The probe was a FAM-labeled minor groove binder probe with a nonfluorescent quencher.

PCR Master Mix was TaqMan 2x Universal PCR Master Mix, No AmpErase (Applied Biosystems). Each gene was analyzed in triplicate.

The real-time PCR machine was a Rotorgene-3000 (Corbett Research). Reactions were run once. Relative gene expression levels were determined using the relative standard curve method as outlined in User Bulletin 2 (ABI PRISM 7700 sequencing detection system; Applied Biosystems). Briefly, a standard curve for each gene was made of 4-fold serial dilutions of total RNA from a punch biopsy from a psoriatic plaque. The curve was then used to calculate the relative amounts of target mRNA in the samples.

ELISA

The TNF-{alpha}, IL-6, IL-8, and IL-10 levels in the keratinocyte culture supernatants were measured as follows. TNF-{alpha} was measured using a TNF-{alpha} DuoSet ELISA Development kit (R&D Systems; catalogue no. DY210) according to the manufacturer’s protocol. IL-6 and IL-8 were measured using an ELISA kit from BioSource International (catalogue nos. CHC1263 and CHC1304, respectively), according to the manufacturer’s protocol. IL-10 was measured by a sandwich ELISA using a combination of an mAb (catalogue no. MAB217; R&D Systems) and a biotinylated polyclonal Ab (catalogue no. BAF217; R&D Systems), both against IL-10. The final result was determined using an ELISA reader (Laboratory Systems; iEMS Reader MF) at 450 nm. All measurements were performed twice.

Small iRNA transfection

MK2 siRNA against MK2-1 (accession no. NM_004759) and MK2-2 (accession no. NM_32960) were designed by Qiagen. The following probes were designed encoding the desired strands: MK2-1, r(CCAUCAUCGAUGACUACAA)dTdT (sense) and r(UUGUAGUCAUCGAUGAUGG)dCdG (antisense); and MK2-2, r(ACGAGCAGAUCAAGAUAAA)dTdT (sense) and r(UUUAUCUUGAUCUGCUCGU)dAdG (antisense). In this study cultured human keratinocytes were transfected with 75 nM MK2-1 and 75 nM MK2-2 siRNA. Nonsilencing control siRNA is an irrelevant siRNA with random nucleotides and no known specificity. Transfections of the keratinocytes were made according to the manufacturer’s protocol (Qiagen) using RNAiFect transfection reagent. A fluorescein-labeled nontarget siRNA control was used to monitor transfection efficiency. MK2 siRNA transfection caused no unintentional activation of the IFN response as determined by ELISA.

Immunofluorescence analysis

Four-micrometer sections of paraffin-embedded tissue samples from lesional and nonlesional psoriatic skin were used. The samples were deparaffinized and then heated at 95°C for 10 min in 10 mM sodium citrate buffer (pH 6.0) for Ag unmasking. The samples were then blocked for 1 h in blocking buffer (PBS containing 0.3% Triton X-100, 0.5% skimmed milk powder, and 1% fish gelatin) before being incubated with anti-phospho-MK2 Ab in blocking buffer overnight at 4°C. The samples were washed and incubated with AlexaFluor 594 secondary Ab (Molecular Probes) for 2 h, washed, and incubated with anti-keratin 14 Ab (mAb LL002; from Dr. I. Leigh, Queen Mary, University of London, London, U.K.) directly conjugated to AlexaFluor 488 (Molecular Probes). Nuclear staining was performed by embedding samples in Prolong Gold antifade reagent with DAPI (Molecular Probes). Samples were viewed using an epifluorescence microscope (Leica).

As a negative control, sections were incubated with blocking buffer without primary Ab or with the respective preimmune sera.

Statistical analysis

For statistical analysis, Student’s t test was performed. To test for normal distribution, a probability test was made. A value of p < 0.05 was regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TNF-{alpha} mRNA and protein expression in psoriatic skin

In accordance with previous studies, we found that the TNF-{alpha} protein level was significantly increased in lesional psoriatic skin compared with nonlesional psoriatic skin (p < 0.01). In Fig. 1A, the results from six psoriatic patients are depicted, showing a mean 4.5-fold increase in the TNF-{alpha} protein content in lesional compared with nonlesional psoriatic skin (Fig. 1A). The TNF-{alpha} protein level in normal skin was comparable to that in nonlesional psoriatic skin. Guttate/acute psoriasis is made up of small lesions, which do not provide enough material to perform Western blot analysis. Therefore, TNF-{alpha} protein expression was not determined in these lesions.


Figure 1
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  		FIGURE 1. TNF-{alpha} expression in psoriatic skin. A, The TNF-{alpha} protein level is significantly increased in lesional psoriatic skin compared with nonlesional psoriatic skin. TNF-{alpha} ELISA was performed on cell extracts from keratome biopsies from normal skin (three different subjects) and nonlesional and lesional psoriatic skin (six different psoriatic patients). Each value was determined twice. B, The TNF-{alpha} mRNA expression was not altered among normal skin, nonlesional, lesional, and guttate psoriatic skin. Punch biopsies from normal skin (three different subjects) and paired punch biopsies from nonlesional, lesional, and guttate psoriatic skin (seven psoriatic patients) were analyzed for TNF-{alpha} mRNA by quantitative RT-PCR. The TNF-{alpha} mRNA expression was normalized to RPLP0.

 
Quantitative RT-PCR analysis was performed to investigate whether the increased TNF-{alpha} protein level was paralleled by an increased accumulation of the corresponding mRNA in psoriatic skin. Interestingly, no difference in TNF-{alpha} mRNA expression was found among normal skin, nonlesional psoriatic skin, chronic plaques, and acute guttate psoriatic skin lesions (Fig. 1B), demonstrating a posttranscriptional regulation of TNF-{alpha} expression in psoriatic skin.

Immunofluorescence staining of activated MK2 in psoriatic skin

Due to the expression profile of TNF-{alpha} in psoriatic skin and the fact that MK2 is known to regulate TNF-{alpha} expression at a posttranscriptional level, we examined MK2 activation and localization in lesional and nonlesional psoriatic skin. Single positive cells strongly stained for phospho-MK2 were found in lesional psoriatic skin (Fig. 2, G and H), but not in nonlesional psoriatic skin (Fig. 2, C and D). These positively stained cells were scattered throughout the basal and suprabasal layers of the epidermis, and phospho-MK2 was mainly located in the cytoplasm of these cells (Fig. 2, G–I). Double staining with keratin 14, a specific keratinocyte marker, showed that phospho-MK2-positive cells also stained positively for keratin 14 (Fig. 2, H and I).


Figure 2
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  		FIGURE 2. Localization of activated MK2 in lesional and nonlesional psoriatic skin. Increased activation of MK2 was found in lesional psoriatic skin, as determined by immunofluorescence staining of nonlesional (A–D) and lesional (E–I) psoriatic skin using a phospho-MK2-specific primary Ab recognizing phospho-MK2 isoforms 1 and 2. Nuclear staining was performed using 4',6-diamido-2-phenylindole hydrochloride (blue; A and E). Green (AlexaFluor 488) demonstrates keratin 14 (B and F), and red (AlexaFluor 594) demonstrates activated MK2 (C and G). Yellow indicates colocalization (H and I). These results were conducted on biopsies from six different patients.

 
MK2 is activated in lesional psoriatic skin

TNF-{alpha} has been demonstrated to be regulated at a posttranscriptional level by MK2 (19). We therefore analyzed paired keratome biopsies taken from nonlesional and lesional psoriatic skin for MK2 activity. By Western blot analysis using an Ab recognizing the phosphorylated form of MK2, we demonstrated that the phosphorylated form of MK2 was significantly increased in lesional compared with nonlesional psoriatic skin (3.2-fold; p < 0.05). No change was seen in the total protein level of MK2 (Fig. 3A). The increased level of phosphorylated MK2 in psoriatic skin was paralleled by an increased kinase activity of MK2 in lesional psoriatic skin compared with nonlesional psoriatic skin, as measured by Hsp27 phosphorylation induced by immunoprecipitated MK2 in a kinase assay (2.8-fold; p < 0.05; Fig. 3B).


Figure 3
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  		FIGURE 3. MK2 activity in psoriatic skin. A, Whole-cell protein extracts were prepared from lesional and nonlesional psoriatic skin and analyzed by Western blotting. The proteins were separated on an 8–16% gradient gel and blotted to a nitrocellulose membrane. After blotting, the membrane was probed with an Ab recognizing the phosphorylated form of both MK2 isoforms 1 and 2. These experiments demonstrated increased levels of the phosphorylated form of MK2 in lesional compared with nonlesional psoriatic skin. B, Whole-cell protein extracts (200 µg of protein) from lesional and nonlesional psoriatic skin were prepared for immunoprecipitation of MK2 (isoforms 1 and 2). Enzyme activities were determined by an in vitro kinase assay, using Hsp27 as the substrate. Kinase reactions were prepared for SDS-PAGE, and phosphorylated Hsp27 was detected by autoradiography. Increased kinase activity of MK2 was found in lesional compared with nonlesional psoriatic skin.

 
Anisomycin and IL-1beta activate p38 and MK2, leading to increased TNF-{alpha} protein expression

To further analyze the p38/MK2 signaling pathway in epidermis, we used cultured normal human keratinocytes stimulated with anisomycin, a well-characterized p38 activator, or IL-1beta. Within 5 min, anisomycin induced rapid activation/phosphorylation of both p38 and MK2, as determined by Western blotting. After 3 h, anisomycin-induced MK2 phosphorylation had almost returned to the basal level, whereas p38 was still clearly activated even after 3 h (Fig. 4A). IL-1beta also induced a rapid phosphorylation of both p38 and MK2. The IL-1beta-induced phosphorylation of MK2 returned to the basal level after 1 h, whereas the IL-1beta-induced phosphorylation of p38 returned to the basal level after 3 h (Fig. 4A). Equal protein loading was determined by assessing the total protein amount of p38.


Figure 4
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  		FIGURE 4. Effects of anisomycin and IL-1beta on MK2, p38, and TNF-{alpha}. A, Cultured human keratinocytes were simulated with anisomycin (0.5 µg/ml) or IL-1beta (10 ng/ml) for the indicated times. The whole-cell extracts were isolated, and the proteins were separated by SDS-PAGE on an 8–16% gradient gel. After electroblotting, the separated proteins were probed with anti-phospho-MK2 (recognizing the phosphorylated form of MK2 isoforms 1 and 2), anti-phospho-p38, and anti-p38. Both anisomycin and IL-1beta led to increased phosphorylation of MK2 and p38 in a time-dependent manner. B, Cultured human keratinocytes were stimulated with anisomycin or IL-1beta. The supernatant was isolated at the indicated times, and the TNF-{alpha} protein content was analyzed by ELISA. Results represent the mean ± SD from four separate experiments. TNF-{alpha} protein production in keratinocytes was significantly increased by anisomycin and IL-1beta after 12 and 24 h of stimulation. All measurements were performed twice. *, p < 0.01 compared with vehicle-treated cells.

 
It has been demonstrated that inhibition of the p38/MK2 signaling pathway leads to decreased TNF-{alpha} protein production in macrophages (18, 28). To determine whether the anisomycin- and IL-1beta-induced phosphorylation of p38 and MK2 led to increased TNF-{alpha} protein production in cultured human keratinocytes, TNF-{alpha} expression was analyzed by an ELISA. The level of TNF-{alpha} was significantly increased (p < 0.01) after stimulation with anisomycin or IL-1beta for 12 and 24 h. At 24 h, anisomycin-induced TNF-{alpha} production had increased ~15-fold, whereas IL-1beta-induced TNF-{alpha} production had increased ~5-fold compared with vehicle-treated cells (Fig. 4B).

Anisomycin- and IL-1beta-induced TNF-{alpha} production is dependent on p38 MAPK activation

Previous results have demonstrated p38 MAPK to be involved in the regulation of TNF-{alpha} in monocytes and macrophages (28, 29, 30). To examine whether anisomycin- and IL-1beta-induced TNF-{alpha} production in cultured human keratinocytes was dependent on p38 MAPK activation, we preincubated keratinocytes with the specific p38{alpha} and p38beta MAPK inhibitor SB202190 (10 µM) for 30 min before stimulation for 5 min with either anisomycin or IL-1beta. Both anisomycin- and IL-1beta-induced p38 activations were significantly (p < 0.05) reduced by SB202190, as determined by Western blotting (Fig. 5A). Measuring TNF-{alpha} protein expression by ELISA, we demonstrated that anisomycin-induced TNF-{alpha} protein production was significantly inhibited by preincubation of keratinocytes with SB202190 (p < 0.01), whereas IL-1beta-induced TNF-{alpha} protein expression was only moderately, and not significantly, inhibited by SB202190 (Fig. 5B).


Figure 5
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  		FIGURE 5. Effect of SB202190 on TNF-{alpha} protein production. A, Anisomycin- and IL-1beta-induced phosphorylation of p38 was inhibited by the p38 inhibitor, SB202190. Whole-cell extracts were isolated from keratinocytes preincubated with or without SB202190 (10 µM) for 30 min before being stimulated with anisomycin (0.5 µg/ml) or IL-1beta (10 ng/ml) for an additional 5 min. Proteins were separated on an 8–16% gradient gel before being electroblotted onto a nitrocellulose membrane. Active p38 was detected by an anti-phospho-p38 Ab. B, The TNF-{alpha} protein production induced by anisomycin and IL-1beta in keratinocytes was inhibited by SB202190. Cultured keratinocytes were preincubated with SB202190 for 30 min and then stimulated with anisomycin (0.5 µg/ml) or IL-1beta (10 ng/ml) for 24 h. The supernatant was isolated, and the TNF-{alpha} protein content was determined by ELISA. Results represent the mean ± SD from four separate experiments. All measurements were performed twice. *, p < 0.01 compared with vehicle-treated keratinocytes; **, p < 0.01 compared with keratinocytes stimulated with anisomycin and without SB202190.

 
MK2 is involved in the anisomycin- and IL-1beta-induced TNF-{alpha} protein production

Having shown the decisive role of p38 in anisomycin-induced TNF-{alpha} protein production, we then examined whether MK2 was involved in the induction of TNF-{alpha}. This was achieved using siRNA technology to silence MK2 gene expression in cultured human keratinocytes. In keratinocytes transfected with MK2 siRNA target sequences for 48 h, the MK2 protein content was reduced by 85 ± 6% compared with keratinocytes transfected with control siRNA (Fig. 6A).


Figure 6
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  		FIGURE 6. MK2-dependent TNF-{alpha} protein production. Keratinocytes were transfected with nonspecific control siRNA or specific siRNA for MK2. A, Transfection of keratinocytes with MK2 siRNA led to a pronounced inhibition of MK2 protein production. Keratinocytes were cultured for 48 h before whole-cell extracts were isolated. Western blot analysis was performed using an anti-MK2 Ab recognizing both MK2 isoforms 1 and 2. Equal loading was confirmed by incubation with an anti-MSK1 Ab. B and C, The anisomycin- and IL-1beta-induced TNF-{alpha} protein production was significantly inhibited by MK2 siRNA. Twenty-four hours after transfection, the medium was changed from K-SFM to KBM, in which keratinocytes were cultured for another 24 h before being stimulated with anisomycin (0.5 µg/ml) or IL-1beta (10 ng/ml). Twenty-four hours after stimulation, the supernatant was isolated, and the TNF-{alpha} protein content was determined by ELISA. Results represent the mean ± SD from experiments conducted on six different keratinocyte cultures. All measurements were performed twice. *, p < 0.01 compared with vehicle-treated keratinocytes; **, p < 0.01 compared with keratinocytes transfected with nonspecific control siRNA and stimulated with anisomycin (B) or IL-1beta (C).

 
To analyze the role of MK2 in TNF-{alpha} protein production, we transfected cultured normal human keratinocytes with MK2 siRNA for 48 h, and then the keratinocytes were stimulated with either anisomycin or IL-1beta for another 24 h. Stimulating keratinocytes transfected with control siRNA with anisomycin or IL-1beta resulted in significantly increased TNF-{alpha} protein production of ~12-fold (p = 0.00079) and 4-fold (p = 0.0047), respectively, compared with vehicle-treated cells (Fig. 6, B and C). When keratinocytes were transfected with MK2 siRNA, anisomycin-induced TNF-{alpha} protein production was significantly diminished, with an inhibition of ~55% (p = 0.00031) compared with keratinocytes transfected with control siRNA (Fig. 6B); in contrast to p38 MAPK inhibition, IL-1beta-induced TNF-{alpha} production was also significantly reduced with an inhibition of ~41% (p = 0.0017; Fig. 6C).

Anisomycin-induced production of the proinflammatory cytokines IL-6 and IL-8 is mediated through a MK2-dependent mechanism

In addition to TNF-{alpha}, IL-6 and IL-8 have been described to be regulated at a posttranscriptional level through the p38/MK2 signaling pathway (12). Therefore, the anisomycin-induced IL-6, IL-8, and IL-10 protein production in human keratinocytes transfected with or without MK2 siRNA was determined. As depicted in Fig. 7, A and B, protein production of the proinflammatory cytokines IL-6 and IL-8 was significantly diminished, with an inhibition of ~32% (p = 0.037) and 50% (p = 0.0091), respectively (Fig. 7, A and B). In contrast, protein expression of the anti-inflammatory cytokine IL-10 was not inhibited by MK2 siRNA, although IL-10 protein expression was significantly induced by anisomycin (p = 0.0034; Fig. 7C).


Figure 7
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  		FIGURE 7. Inhibition of IL-6 and IL-8 protein production by MK2 siRNA. Transfection of keratinocytes with MK2 siRNA significantly inhibited the anisomycin-induced IL-6 and IL-8 protein production, but not the IL-10 protein production. Keratinocytes were transfected with nonspecific control siRNA or MK2-specific siRNA. Twenty-four hours after transfection, the medium was changed to KBM, and keratinocytes were cultured for another 24 h before being stimulated with anisomycin (0.5 µg/ml). Twenty-four hours after stimulation, the supernatants were isolated, and the IL-6 (A), IL-8 (B), and IL-10 (C) protein contents were determined by ELISA. Results represent the mean ± SD from experiments conducted on three (A), five (B), and three (C) different keratinocyte cultures, respectively. All measurements were performed twice. *, p < 0.01 compared with vehicle-treated keratinocytes; **, p < 0.05 compared with keratinocytes transfected with nonspecific control siRNA and stimulated with anisomycin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The proinflammatory cytokine TNF-{alpha} is a major mediator of inflammation. TNF-{alpha} is not only essential in the pathogenesis of psoriasis. Increased levels of TNF-{alpha} have also been found in rheumatoid arthritis and Crohn’s disease (31, 32, 33). Furthermore, a key role for this cytokine in the pathogenesis of psoriasis, rheumatoid arthritis, and Crohn’s disease has been demonstrated by the successful treatment of these diseases with TNF-{alpha} antagonists (24, 34, 35). In this study we showed that the protein expression of TNF-{alpha} was significantly augmented in lesional psoriatic skin compared with nonlesional psoriatic skin. This is in accordance with previous studies demonstrating increased immunoreactivity and bioactivity of TNF-{alpha} in psoriatic skin (36) together with increased TNF-{alpha} expression in psoriatic skin, as determined by immunohistochemistry (37). Interestingly, the increased TNF-{alpha} protein expression was not paralleled by increased TNF-{alpha} mRNA expression in either acute or chronic lesional psoriatic skin compared with nonlesional psoriatic skin. These results demonstrate for the first time that TNF-{alpha} is regulated at a posttranscriptional level in psoriatic skin. Previous reports have demonstrated that the p38/MK2 signaling pathway plays a pivotal role in the synthesis of proinflammatory cytokines such as TNF-{alpha}, IL-1, IL-6, and IL-8 at a posttranscriptional level (12, 17, 19, 29). We recently demonstrated increased kinase activity of the p38 MAPK in lesional compared with nonlesional psoriatic skin (8). Therefore, we examined the activity of MK2, a downstream target for p38 MAPK, and found an increased level of the phosphorylated form of MK2 together with increased kinase activity of MK2 in lesional compared with nonlesional psoriatic skin. In some patients there was a weak positive band for phospho-MK2 in nonlesional psoriatic skin. This may illustrate that the p38 MAPK/MK2 signaling pathway plays a role in maintaining homeostasis in normal skin, whereas it leads to inflammation when it becomes overactivated. Furthermore, detectable basal levels of both TNF-{alpha} mRNA and protein were demonstrated in nonlesional psoriatic skin and normal skin (Fig. 1, A and B). Activated MK2 was selectively expressed in single keratinocytes in the basal and suprabasal layers of lesional psoriatic epidermis, whereas no positive cells were found in nonlesional psoriatic epidermis. Activated MK2 was mainly located in the cytoplasm of the specifically stained cells, in accordance with previous reports demonstrating that MK2 is translocated from the nucleus to the cytoplasm when activated (15).

To further characterize the p38 MAPK/MK2 signaling pathway in epidermal keratinocytes, in vitro studies were conducted with cultured normal human keratinocytes. MK2 has previously been examined in different cell types and cell lines; however, this is the first time that MK2 has been examined in keratinocytes. Both anisomycin and IL-1beta caused activation of p38 MAPK and MK2, which led to augmented TNF-{alpha} protein expression. The anisomycin-induced TNF-{alpha} protein expression was significantly inhibited by the p38 MAPK inhibitor SB202190, demonstrating that anisomycin increases TNF-{alpha} by a p38 MAPK-dependent mechanism. In contrast, the IL-1beta-induced TNF-{alpha} protein expression was only moderately inhibited by SB202190, which could be explained by the fact that anisomycin is known to be a specific p38 MAPK and JNK activator (38), whereas IL-1beta, in addition to activation of p38 MAPK and JNK, activates several other kinases, including the MAPKs ERK1 and -2 (39). Because previous reports have demonstrated that MK2 is also a downstream target of ERK1 and -2 in vitro (40, 41), the IL-1beta-induced activation of MK2 in keratinocytes may also be mediated by these pathways.

Because specific inhibitors to MK2 are not commercially available, siRNA technology was used to modulate MK2 expression in this study. We demonstrated that specific MK2 siRNA inhibited MK2 expression in cultured human keratinocytes by ~85%. MK2 siRNA significantly inhibited both anisomycin- and IL-1beta-induced TNF-{alpha} protein expression. The use of siRNA technology led to only partial inhibition of the expression of the specific target protein; this may explain why the anisomycin- and IL-1beta-induced TNF-{alpha} protein expression was only partially inhibited by ~55 and 41%, respectively. These results demonstrate that MK2 is a more specific target than the p38 MAPK in the inhibition of IL-1beta-induced TNF-{alpha} protein expression, probably because a number of upstream signaling pathways, including ERK1 and -2 and p38 MAPK, are integrated into the MK2. Interestingly, we also showed that not only TNF-{alpha}, but also the anisomycin-induced protein expression of IL-6 and IL-8, was inhibited by MK2 siRNA in cultured human keratinocytes, whereas the anisomycin-induced protein expression of the anti-inflammatory cytokine IL-10 was not inhibited, although IL-10 expression was significantly induced by anisomycin. This strongly indicates that MK2 specifically regulates the translation of proinflammatory cytokines.

This present study is unique because it demonstrates that TNF-{alpha} expression is regulated at a posttranscriptional level in psoriasis. Furthermore, we characterize for the first time the localization, expression, and activity of MK2 in lesional and nonlesional psoriatic skin and identify MK2 as the key regulator of TNF-{alpha} expression in lesional psoriatic skin as well as in cultured normal human keratinocytes. In vitro data also indicate that MK2 integrate different upstream signaling pathways and induce the expression of predominantly proinflammatory cytokines. These findings are significant because they increase our understanding of how TNF-{alpha} expression is regulated in inflammatory conditions such as psoriasis. Based on these findings, we suggest that MK2 may be a new and promising target for specific anti-inflammatory therapy.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Leo Research Foundation, the Novo Nordisk Foundation, the Danish Psoriasis Foundation, and the Danish Research Agency. Back

2 Address correspondence and reprint requests to Dr. Claus Johansen, Department of Dermatology, Research Lab S, Aarhus University Hospital, P.P. Orumsgade 11, 8000 Aarhus C, Denmark. E-mail address: clausoglotte{at}hotmail.com Back

3 Current address: Department of Dermatology, Aarhus University Hospital, Research Lab S, P.P. Orumsgade 11, 8000 Aarhus C, Denmark. Back

4 Current address: Department of Clinical Genetics, Aarhus University Hospital, Wilhelm Meyers Allé, Bygn 240, 8000 Aarhus C, Denmark. Back

5 Current address: Department of Biochemistry and Cell Biology, LEO Pharma, Industriparken 55, 2750 Ballerup, Denmark. Back

6 Current address: Department of Biological Research, LEO Pharma, Industriparken 55, 2750 Ballerup, Denmark. Back

7 Abbreviations used in this paper: MK2, MAPK-activated protein kinase 2; Hsp27, 27-kDa heat shock protein; KBM, keratinocyte basal medium; RPLP0, ribosomal protein, large, P0; siRNA, small interfering RNA; K-SFM, keratinocyte serum-free medium. Back

Received for publication June 27, 2005. Accepted for publication November 14, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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