Advertisement

Overexpression of LEDGF/DFS70 Induces IL-6 via p38 Activation in HaCaT Cells, Similar to that Seen in the Psoriatic Condition

      Lens epithelium–derived growth factor (LEDGF)/dense fine speckles 70kDa protein (DFS70) is a transcription cofactor that enhances growth and is overexpressed in various cancers. In the epidermis, LEDGF/DFS70 localizes to the nucleus of keratinocytes (KCs) in the basal layers and to the cytoplasm of cells in the upper layers. However, the biological and pathological relevance of LEDGF/DFS70 in the epidermis is virtually unknown. Compared with normal epidermis, we detected strong nuclear staining of LEDGF/DFS70 in both the spinous and basal layers of the epidermis of psoriatic skin. To investigate the roles of LEDGF/DFS70 in the epidermis of psoriatic skin, we generated HaCaT cells that constitutively express enhanced green fluorescence protein (EGFP)-LEDGF (EGFP-LEDGF-HaCaT) or EGFP alone (EGFP-HaCaT) as a control. EGFP-LEDGF-HaCaT cells had increased expression of IL-6, which was attenuated by LEDGF-specific RNA interference and the p38-specific inhibitors SB-239063 and SB-203580. Furthermore, EGFP-LEDGF-HaCaT cells had increased expression of S100A7 and S100A9 and decreased expression of filaggrin. These findings are compatible with the expression pattern in psoriatic tissues. Taken together, these results strongly suggest that ectopic expression of LEDGF/DFS70 in KCs could be involved in the pathology of psoriasis vulgaris.

      Abbreviations

      DFS70
      dense fine speckles 70kDa protein
      EGFP
      enhanced green fluorescent protein
      KC
      keratinocyte
      LEDGF
      lens epithelium-derived growth factor
      MAPK
      mitogen-activated protein kinase
      PBS
      phosphate-buffered saline
      siRNA
      small interfering RNA
      STAT3
      signal transducer and activator of transcription 3

      Introduction

      Lens epithelium–derived growth factor (LEDGF)/dense fine speckles 70kDa protein (DFS70) was isolated as a transcription cofactor (
      • Ge H.
      • Si Y.
      • Roeder R.G.
      Isolation of cDNAs encoding novel transcription coactivators p52 and p75 reveals an alternate regulatory mechanism of transcriptional activation.
      ), a survival factor (
      • Singh D.P.
      • Ohguro N.
      • Chylack Jr, L.T.
      • et al.
      Lens epithelium derived growth factor: increased resistance to thermal and oxidative stresses.
      ), and a target of autoantibodies in atopic dermatitis (
      • Ochs R.L.
      • Muro Y.
      • Si Y.
      • et al.
      Autoantibodies to DFS70 kd/transcription coactivator p75 in atopic dermatitis and other conditions.
      ). LEDGF/DFS70 is induced by tumor necrosis factor-α, oxidative stress, and hyperthermia (
      • Shinohara T.
      • Singh D.P.
      • Fatma N.
      LEDGF, a survival factor, activates stress-related genes.
      ;
      • Nguyen T.A.
      • Boyle D.L.
      • Wagner L.M.
      • et al.
      LEDGF activation of PKC gamma and gap junction disassembly in lens epithelial cells.
      ). In addition, it has been linked to various diseases; anti-LEDGF/DFS70 autoantibodies have been detected in autoimmunity (
      • Muro Y.
      Autoantibodies in atopic dermatitis.
      ), and LEDGF/DFS70 has been shown to bind to HIV-1 integrase (
      • Ciuffi A.
      • Bushman F.D.
      Retroviral DNA integration: HIV and the role of LEDGF/p75.
      ). Furthermore, LEDGF/DFS70 has been implicated as having a key role in cancer and other multiplication diseases (
      • Ahuja H.G.
      • Hong J.
      • Aplan P.D.
      • et al.
      t(9;11)(p22;p15) in acute myeloid leukemia results in a fusion between NUP98 and the gene encoding transcriptional coactivators p52 and p75-lens epithelium-derived growth factor (LEDGF).
      ;
      • Daugaard M.
      • Kirkegaard-Sorensen T.
      • Ostenfeld M.S.
      • et al.
      Lens epithelium-derived growth factor is an Hsp70-2 regulated guardian of lysosomal stability in human cancer.
      ;
      • Huang T.S.
      • Myklebust L.M.
      • Kjarland E.
      • et al.
      LEDGF/p75 has increased expression in blasts from chemotherapy-resistant human acute myelogenic leukemia patients and protects leukemia cells from apoptosis in vitro.
      ). For example, there is an association between LEDGF/p75 overexpression and chemoresistance in acute myelogenous leukemia (
      • Huang T.S.
      • Myklebust L.M.
      • Kjarland E.
      • et al.
      LEDGF/p75 has increased expression in blasts from chemotherapy-resistant human acute myelogenic leukemia patients and protects leukemia cells from apoptosis in vitro.
      ), and LEDGF/DFS70 expression is increased in human prostate, breast, and bladder carcinomas and correlates with Hsp70-2 expression in invasive bladder cancer (
      • Daugaard M.
      • Kirkegaard-Sorensen T.
      • Ostenfeld M.S.
      • et al.
      Lens epithelium-derived growth factor is an Hsp70-2 regulated guardian of lysosomal stability in human cancer.
      ;
      • Mediavilla-Varela M.
      • Pacheco F.J.
      • Almaguel F.
      • et al.
      Docetaxel-induced prostate cancer cell death involves concomitant activation of caspase and lysosomal pathways and is attenuated by LEDGF/p75.
      ). Moreover, LEDGF was recently shown to interact with Cdc7–ASK (activator of S-phase kinase), which is essential for initiating DNA replication throughout S phase, and LEDGF/DFS70 was shown to stimulate its enzymatic activity (
      • Hughes S.
      • Jenkins V.
      • Dar M.J.
      • et al.
      Transcriptional co-activator LEDGF interacts with Cdc7-activator of S-phase kinase (ASK) and stimulates its enzymatic activity.
      ).
      Through studies on LEDGF/DFS70 and autoantibodies to this molecule (
      • Ogawa Y.
      • Sugiura K.
      • Watanabe A.
      • et al.
      Autoantigenicity of DFS70 is restricted to the conformational epitope of C-terminal alpha-helical domain.
      ;
      • Okamoto M.
      • Watanabe A.
      • Ogawa Y.
      • et al.
      Autoantibodies to DFS70/LEDGF are increased in alopecia areata patients.
      ;
      • Watanabe A.
      • Kodera M.
      • Sugiura K.
      • et al.
      Anti-DFS70 antibodies in 597 healthy hospital workers.
      ), we recently found that LEDGF/DFS70 is located predominantly in the nucleus of basal epidermal cells and translocate to the cytoplasm during their differentiation (
      • Sugiura K.
      • Muro Y.
      • Nishizawa Y.
      • et al.
      LEDGF/DFS70, a major autoantigen of atopic dermatitis, is a component of keratohyalin granules.
      ). We further conducted immunohistochemical analyses of LEDGF/DFS70 in various skin diseases and determined that LEDGF/DFS70 expression is deregulated in the nucleus of keratinocytes (KCs) in the spinous layers of psoriatic lesions. Therefore, we speculate that LEDGF/DFS70 may have pivotal roles in the nuclei of KCs in this disease.
      Psoriasis is a common skin disorder that is characterized by epidermal hyperplasia, T-cell recruitment, and changes in the endothelial vascular system (
      • Bos J.D.
      • de Rie M.A.
      • Teunissen M.B.
      • et al.
      Psoriasis: dysregulation of innate immunity.
      ;
      • Bowcock A.M.
      • Krueger J.G.
      Getting under the skin: the immunogenetics of psoriasis.
      ;
      • Krueger G.
      • Ellis C.N.
      Psoriasis – recent advances in understanding its pathogenesis and treatment.
      ). It has been suggested that psoriatic KCs have abnormal expression and/or activation of various transcription factors and deregulation of several signaling pathways (
      • McKenzie R.C.
      • Sabin E.
      Aberrant signalling and transcription factor activation as an explanation for the defective growth control and differentiation of keratinocytes in psoriasis: a hypothesis.
      ;
      • Johansen C.
      • Kragballe K.
      • Rasmussen M.
      • et al.
      The AP-1 DNA binding activity is decreased in lesional psoriatic skin compared to nonlesional psoriatic skin.
      ). For example, IL-6 levels are significantly increased in psoriatic lesion samples (
      • Ameglio F.
      • Bonifati C.
      • Fazio M.
      • et al.
      Interleukin-11 production is increased in organ cultures of lesional skin of patients with active plaque-type psoriasis as compared with nonlesional and normal skin. Similarity to interleukin-1 beta, interleukin-6 and interleukin-8.
      ). In addition, psoriatic KCs are characterized by activation of signal transducer and activator of transcription 3 (STAT3) (
      • Sano S.
      • Chan K.S.
      • Carbajal S.
      • et al.
      Stat3 links activated keratinocytes and immunocytes required for development of psoriasis in a novel transgenic mouse model.
      ). Moreover, the activity of mitogen-activated protein kinase (MAPK) p38 is increased in lesional psoriatic skin (
      • Johansen C.
      • Kragballe K.
      • Westergaard M.
      • et al.
      The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin.
      ;
      • Yu X.J.
      • Li C.Y.
      • Dai H.Y.
      • et al.
      Expression and localization of the activated mitogen-activated protein kinase in lesional psoriatic skin.
      ).
      IL-6 is a multifunctional cytokine, and one of its functions is to activate target genes involved in cell proliferation. It signals through a heterodimeric IL-6R/gp130 complex that triggers the activation of the Janus (JAK) kinases and downstream effectors, including STAT3. STAT3 participates in cellular transformation and oncogenesis (
      • Bromberg J.F.
      • Wrzeszczynska M.H.
      • Devgan G.
      • et al.
      Stat3 as an oncogene.
      ;
      • Hodge D.R.
      • Hurt E.M.
      • Farrar W.L.
      The role of IL-6 and STAT3 in inflammation and cancer.
      ). Activated STAT3, in turn, is transported into the nucleus, where it functions as a transcription factor that regulates genes involved in cell survival (
      • Battle T.E.
      • Frank D.A.
      The role of STATs in apoptosis.
      ).
      Although LEDGF/DFS70 is a well-characterized survival factor in KCs (
      • Shinohara T.
      • Singh D.P.
      • Fatma N.
      LEDGF, a survival factor, activates stress-related genes.
      ), the relationship between LEDGF/DFS70 and growth factors such as IL-6 has not been well characterized. Therefore, the aim of this study was to determine the mechanism by which this protein functions as a survival factor in psoriasis vulgaris. To facilitate these studies and investigate the relationship between LEDGF overexpression and IL-6 production and STAT3 activation, we generated HaCaT cells that constitutively express enhanced green fluorescence protein (EGFP)-LEDGF (EGFP-LEDGF-HaCaT) or EGFP alone (EGFP-HaCaT) as a control. We further examined whether IL-6 production in EGFP-LEDGF-HaCaT cells is inhibited by the p38-specific inhibitors SB-239063 and SB-203580 because p38 MAPKs were previously reported to regulate IL-6 expression (
      • Zarubin T.
      • Han J.
      Activation and signaling of the p38 MAP kinase pathway.
      ;
      • Cuenda A.
      • Rousseau S.
      p38 MAP-kinases pathway regulation, function and role in human diseases.
      ). Finally, we used western blotting to examine the expression of S100A7, S100A9, and filaggrin, which are known to be altered in psoriatic tissues, in EGFP-LEDGF-HaCaT cells compared with EGFP-HaCaT cells.

      Results

      Strong nuclear staining of LEDGF/DFS70, pY-STAT3, and p-p38 in the epidermis of psoriatic lesions

      First, we conducted immunohistochemical analyses of LEDGF/DFS70, pY-STAT3, and phosphorylated p38 (p-p38) in psoriasis vulgaris skin lesions. Figure 1 shows that LEDGF/DFS70 and p-p38 are strongly expressed in the nuclei in both the spinous and basal layers of psoriatic skin (Figure 1a and e) compared with cytoplasmic staining in normal skin (Figure 1b and f), and this staining pattern was found in 90% of psoriatic skin (n=9 of 10). STAT3 activation was significantly greater in the nucleus of psoriatic KCs (Figure 1c) than in normal skin (Figure 1d).
      Figure thumbnail gr1
      Figure 1Immunohistochemistry of psoriatic and normal epidermis. The epidermis from psoriatic lesions from psoriasis patients (a, c, e) and normal skin from healthy donors (b, d, f) were stained with anti-LEDGF (a, b), anti-pY-STAT3 (c, d), and anti-p-p38 (e, f) antibodies. Scale bars=50μm. LEDGF, lens epithelium–derived growth factor; p-p38, phosphorylated p38; STAT3, signal transducer and activator of transcription 3.
      We next conducted immunocytochemical analyses of HaCaT cells under serum-free conditions. Serum starvation for 72hours increased the proportion of cells in the G0/G1 phase (
      • Qi L.
      • Allen R.R.
      • Lu Q.
      • et al.
      PAI-1 transcriptional regulation during the G0-->G1 transition in human epidermal keratinocytes.
      ). As shown in Supplementary Figure S1 online, LEDGF was located in the cytoplasm of HaCaT cells under serum-free conditions. Ki-67 is a cell cycle marker that is not expressed in the G0 phase (
      • Verheijen R.
      • Kuijpers H.J.
      • Schlingemann R.O.
      • et al.
      Ki-67 detects a nuclear matrix-associated proliferation-related antigen. I. Intracellular localization during interphase.
      ). However, LEDGF localized to the nucleus when cells were incubated in DMEM containing 10% fetal bovine serum. Our findings suggest that LEDGF localizes to the cytoplasm when cells are in the G0/G1 phase and localizes to the nucleus in proliferating cells in vitro. These findings are compatible with immunohistochemical analyses of LEDGF/DFS70 in psoriasis vulgaris skin lesions and normal epidermis specimens (Figure 1a and b).

      LEDGF production in transgenic EGFP-LEDGF-HaCaT cells

      To investigate the function of LEDGF/DFS70 in KCs, we generated HaCaT cells constitutively expressing EGFP-LEDGF (EGFP-LEDGF-HaCaT) or EGFP alone (EGFP-HaCaT). As shown in Figure 2c, microscopic analyses of EGFP-LEDGF-HaCaT cells that were incubated for 24hours showed that the signals for EGFP fluorescence (Figure 2a) colocalized with the blue fluorescence representing nuclear staining by 4,6-diamidino-2-phenylindole (Figure 2b). These data indicated that EGFP-LEDGF localized to both nuclei and cytoplasm in EGFP-LEDGF-HaCaT cells compared with EGFP-HaCaT cells (Figure 2d–f). After incubating for 72hours, the EGFP-LEDGF-HaCaT cells had reached 70% confluency and the expression of LEDGF mRNA and EGFP-LEDGF protein had increased, as determined by quantitative PCR and western blot analyses, respectively (Figure 2g and h).
      Figure thumbnail gr2
      Figure 2Increased expression of LEDGF in EGFP-LEDGF-HaCaT cells. After incubating for 24hours, (ac) non-fixed EGFP-LEDGF-HaCaT cells and (df) EGFP-HaCaT cells were immunolabeled with Blue, a nuclear-specific dye. Confocal images show (a, d) EGFP as green, (b, e) the nucleus as blue, and (c, f) merged EGFP (green fluorescence) and nuclear (blue fluorescence) staining. Scale bars=50μm. After 72hours of incubation, the LEDGF mRNA and protein levels in EGFP-LEDGF-HaCaT cells and EGFP-HaCaT cells were evaluated by (g) RT-PCR and (h) western blot, respectively. GAPDH was used to normalize the gene expression. β-Actin was used as an internal loading control for the cell lysate samples in the western blot analysis. Statistical significance between groups was assessed using paired Student's t-test (n=6). Error bars represent the SEM. *P<0.01. EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; LEDGF, lens epithelium-derived growth factor; RT-PCR, reverse transcription-PCR.

      LEDGF/DFS70 induced IL-6 mRNA and protein expression and phosphorylated STAT3

      Next, we investigated whether IL-6 was upregulated in EGFP-LEDGF-HaCaT cells when compared with EGFP-HaCaT cells using quantitative PCR and ELISAs. These analyses showed that EGFP-LEDGF-HaCaT cells had higher IL-6 expression at both the mRNA and protein levels (Figure 3a and b). We also performed western blot analyses to determine whether STAT3 phosphorylation increased. As shown in Figure 3c, STAT3 was more highly phosphorylated in EGFP-LEDGF-HaCaT cells than in EGFP-HaCaT cells. To ascertain whether LEDGF/DFS70 regulates IL-6 expression, we used small interference RNA (siRNA) to knock down LEDGF (siLED) or a control (siCD4) in EGFP-LEDGF-HaCaT cells and then examined the IL-6 mRNA and protein levels (Supplementary Figure S2 online). We validated the effect of LEDGF siRNA and determined that reduced LEDGF/DFS70 expression was associated with reduced IL-6 expression.
      Figure thumbnail gr3
      Figure 3Production of IL-6 mRNA and protein in EGFP-LEDGF-HaCaT cells. IL-6 mRNA and protein expression in EGFP-LEDGF-HaCaT cells and HaCaT cells was evaluated by (a) q-PCR and (b) ELISA, respectively. GAPDH was used to normalize the gene expression. (c) Western blotting for pY-STAT3 in EGFP-LEDGF-HaCaT and EGFP-HaCaT cell extracts. The statistical significance between groups was assessed using paired Student's t-test (n=4). Error bars represent the SEM. *P<0.01. EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; LEDGF, lens epithelium-derived growth factor; q-PCR, quantitative PCR; STAT3, signal transducer and activator of transcription 3.

      Effects of MAPK inhibitors on increased IL-6 expression in EGFP-LEDGF-HaCaT cells

      As shown in Figure 3, LEDGF overexpression resulted in increased IL-6 expression at both the mRNA and protein levels, and IL-6 expression was regulated by p38 MAPKs (
      • Zarubin T.
      • Han J.
      Activation and signaling of the p38 MAP kinase pathway.
      ;
      • Cuenda A.
      • Rousseau S.
      p38 MAP-kinases pathway regulation, function and role in human diseases.
      ). Therefore, we examined the effects of several MAPK inhibitors to determine how specific signaling molecules and pathways affect IL-6 expression in EGFP-LEDGF-HaCaT cells. The cells were incubated for 24hours in the presence or absence of SP-600125 (10μM), U-0126 (10μM), SB-239063 (2μM), or SB-203580 (2μM). As shown in Figure 4a and b, SP-600125, a specific c-Jun N-terminal kinase inhibitor (
      • Bennett B.L.
      • Sasaki D.T.
      • Murray B.W.
      • et al.
      SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase.
      ), did not significantly affect IL-6 mRNA production, but increased IL-6 protein expression. U-0126, an extracellular signal-regulated kinase 1 and 2 inhibitor (
      • Duncia J.V.
      • Santella III, J.B.
      • Higley C.A.
      • et al.
      MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products.
      ), stimulated the production of both IL-6 mRNA and protein. However, SB-239063 and SB-203580, the specific inhibitors of p38 (
      • Kramer R.M.
      • Roberts E.F.
      • Um S.L.
      • et al.
      p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2.
      ;
      • Underwood D.C.
      • Osborn R.R.
      • Bochnowicz S.
      • et al.
      SB 239063, a p38 MAPK inhibitor, reduces neutrophilia, inflammatory cytokines, MMP-9, and fibrosis in lung.
      ), blocked both IL-6 expression and secretion (Figure 4a and b) (SB-203580, data not shown). To determine whether p-p38 increased in EGFP-LEDGF-HaCaT cells, we performed western blot analyses. Figure 4c shows that the p-p38 (Thr 180/Tyr 182) levels were greater in EGFP-LEDGF-HaCaT cells than in EGFP-HaCaT cells. In addition, this increased p-p38 expression was downregulated with SB-239063 (Figure 4d) and SB-203580 treatment (data not shown), as expected.
      Figure thumbnail gr4
      Figure 4Effects of MAPK inhibitors on IL-6 expression and secretion in EGFP-LEDGF-HaCaT cells. EGFP-LEDGF-HaCaT cells were incubated for 24hours in the presence or absence of SP-600125 (10μM), U0126 (10μM), or SB-239063 (2μM). (a) The IL-6 mRNA levels were evaluated by q-PCR. GAPDH was used to normalize the gene expression. (b) The amount of IL-6 secreted into the culture supernatant was determined by ELISA. (c) Lysates from EGFP-LEDGF-HaCaT cells and EGFP-HaCaT cells that were cultured for 72hours were examined by western blot analysis to detect the p-p38 levels. (d) Western blotting of p-p38 extracted from EGFP-LEDGF-HaCaT cells that were incubated for 24hours in the presence or absence of SB-239063 (2μM). Statistical significance between groups was assessed using paired Student's t-test (n=4). Error bars represent the SEM. *P<0.01; **P<0.05. EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; LEDGF, lens epithelium-derived growth factor; MAPK, mitogen-activated protein kinase; p-p38, phosphorylated p38; q-PCR, quantitative PCR.

      Decreased IL-6 levels in EGFP-LEDGF-HaCaT cells transfected with p38-specific siRNAs

      To ascertain whether p38 phosphorylation is involved in IL-6 expression in cells overexpressing LEDGF/DFS70, siRNA was used to knock down p38 mRNA (sip38) in EGFP-LEDGF-HaCaT cells, as shown in Figure 5a, and the IL-6 mRNA and protein levels were then measured. Figure 5c and d shows that the IL-6 mRNA and protein levels were reduced to approximately 60% compared with the controls when p38 protein and p-p38 protein levels were reduced by siRNA (Figure 5b).
      Figure thumbnail gr5
      Figure 5Decreased IL-6 levels in EGFP-LEDGF-HaCaT cells transfected with p38-specific siRNAs. EGFP-LEDGF-HaCaT cells were transfected with siRNAs specific for p38 (sip38) or CD4 (siCD4). After incubating the cells for 72hours, the (a) p38 mRNA levels, and (b) the p38 and p-p38 protein levels were evaluated by q-PCR and western blot analysis, respectively. (c) IL-6 mRNA and (d) IL-6 protein levels were also evaluated by q-PCR and ELISA, respectively. GAPDH was used to normalize the gene expression. β-Actin was used as an internal loading control for the cell lysates in the western blot analysis. Statistical significance between groups was assessed using paired Student's t-test (n=4). Error bars represent the SEM. *P<0.01. EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; LEDGF, lens epithelium-derived growth factor; p-p38, phosphorylated p38; q-PCR, quantitative PCR; siRNA, small interfering RNA.

      Effects of LEDGF-specific siRNAs on HSC-1 cells

      HSC-1 cells, a human skin squamous cell carcinoma line, were transfected with LEDGF siRNA (siLED) or a CD4 control siRNA (siCD4). As shown in Supplementary Figure S3c and d online, the IL-6 mRNA and protein levels were reduced upon siLED-mediated reduction of LEDGF mRNA when compared with the controls (Supplementary Figure S3a). Moreover, p-p38 levels as well as IL-6 expression were reduced by LEDGF siRNA (Supplementary Figure S3b).

      LEDGF/DFS70 altered the expression of S100A7, S100A9, and filaggrin

      Because we expected that EGFP-LEDGF-HaCaT cells may be a useful KC model for experimental psoriasis research, we performed additional experiments to characterize this model. S100A7 and S100A9 have been reported to be overexpressed in psoriasis, whereas filaggrin has been shown to be downregulated. To detect the levels of these molecules in our system, we performed western blotting as shown in Figure 6. The expression of the S100A7 and S100A9 proteins in EGFP-LEDGF-HaCaT cells was higher than that in EGFP-HaCaT cells. On the other hand, reduced filaggrin protein expression was observed in EGFP-LEDGF-HaCaT cells when compared with EGFP-HaCaT cells (Figure 6).
      Figure thumbnail gr6
      Figure 6Western blotting of EGFP-LEDGF-HaCaT cell lysates to examine S100A7, S100A9, and filaggrin expression. EGFP-LEDGF-HaCaT (EGFP-LEDGF) cells and EGFP-HaCaT (EGFP-vector) cells were incubated for 72hours and then analyzed by western blotting for S100A7, S100A9, filaggrin, and β-actin. β-Actin was used as an internal loading control. EGFP, enhanced green fluorescent protein; LEDGF, lens epithelium-derived growth factor.

      Discussion

      We previously reported that LEDGF/DFS70 is predominantly localized in the nucleus of basal epidermal cells and translocates into the cytoplasm during differentiation (
      • Sugiura K.
      • Muro Y.
      • Nishizawa Y.
      • et al.
      LEDGF/DFS70, a major autoantigen of atopic dermatitis, is a component of keratohyalin granules.
      ). In this study we report that LEDGF/DFS70 as well as p-STAT3 and p-p38 localize to the nucleus in both the spinous and basal layers of psoriatic skin (Figure 1).
      LEDGF/DFS70 is a nuclear protein that shows a characteristic heterogeneous distribution in interphase cells and is intimately associated with condensed chromosomes during mitosis (
      • Ochs R.L.
      • Muro Y.
      • Si Y.
      • et al.
      Autoantibodies to DFS70 kd/transcription coactivator p75 in atopic dermatitis and other conditions.
      ;
      • Maertens G.
      • Cherepanov P.
      • Pluymers W.
      • et al.
      LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells.
      ). LEDGF/DFS70 interacts with Cdc7–ASK, a key kinase in S phase, and stimulates its enzymatic activity in the nucleus (
      • Hughes S.
      • Jenkins V.
      • Dar M.J.
      • et al.
      Transcriptional co-activator LEDGF interacts with Cdc7-activator of S-phase kinase (ASK) and stimulates its enzymatic activity.
      ). In view of these reports, LEDGF/DFS70 may also function as a chromatin-associated protein in the nuclei of proliferating KCs, such as in KCs in psoriatic lesions.
      It has not been determined what induces LEDGF/DFS70 to localize to the nucleus in KCs of psoriatic lesions. However, we show that LEDGF/DFS70 localizes to the cytoplasm in quiescent HaCaT cells after 3 days of starvation but localizes to the nucleus in proliferating cells (Supplementary Figure S1 online). Therefore, it is very likely that LEDGF/DFS70 localizes to the nucleus of KCs in psoriatic lesions because these KCs are actively proliferating and differentiating from the suprabasal layer in the normal epidermis.
      Previous studies have shown that LEDGF/DFS70 acts as a survival factor (
      • Singh D.P.
      • Ohguro N.
      • Chylack Jr, L.T.
      • et al.
      Lens epithelium derived growth factor: increased resistance to thermal and oxidative stresses.
      ). However, it was not known whether LEDGF/DFS70 overexpression induces growth factors or cytokines that direct cell growth until a recent report demonstrated that vascular endothelial growth factor C was induced by LEDGF/DFS70 in lung cancer cells (
      • Cohen B.
      • Addadi Y.
      • Sapoznik S.
      • et al.
      Transcriptional regulation of vascular endothelial growth factor C by oxidative and thermal stress is mediated by lens epithelium-derived growth factor/p75.
      ). We showed that IL-6 was significantly upregulated via the p38 MAPK pathway and that STAT3 was activated in HaCaT cells constitutively expressing LEDGF/DFS70 (Figures 3 and 4). Our findings that IL-6 was induced through LEDGF/DFS70 in EGFP-LEDGF HaCaT cells was further supported by the fact that IL-6 was reduced upon RNA interference-mediated knockdown of LEDGF in these cells. In addition, phosphorylated STAT3 in EGFP-LEDGF-HaCaT cells was decreased upon siRNA-mediated knockdown of IL-6 (data not shown). Our findings strongly suggested that the overexpression of LEDGF/DFS70 in the nucleus of psoriatic KCs could activate the IL-6/STAT3 pathway.
      MAPKs are known to be key regulators of the expression of many cytokines (
      • Arthur J.S.
      • Darragh J.
      Signaling downstream of p38 in psoriasis.
      ;
      • Gazel A.
      • Nijhawan R.I.
      • Walsh R.
      • et al.
      Transcriptional profiling defines the roles of ERK and p38 kinases in epidermal keratinocytes.
      ). We found that inhibitors and siRNAs specific to p38 MAPKs significantly attenuated the IL-6 mRNA and protein levels in EGFP-LEDGF-HaCaT cells (Figure 5). Previous reports showed that p38 MAPKs regulate IL-6 (
      • Zarubin T.
      • Han J.
      Activation and signaling of the p38 MAP kinase pathway.
      ;
      • Cuenda A.
      • Rousseau S.
      p38 MAP-kinases pathway regulation, function and role in human diseases.
      ), which is consistent with our data. However, it remains to be determined how LEDGF/DFS70 induces p38 phosphorylation. Recent work has shown that LEDGF can stimulate the enzymatic activity of Cdc7–ASK, increasing the phosphorylation of MCM2 in vitro (
      • Hughes S.
      • Jenkins V.
      • Dar M.J.
      • et al.
      Transcriptional co-activator LEDGF interacts with Cdc7-activator of S-phase kinase (ASK) and stimulates its enzymatic activity.
      ). In our model system, LEDGF/DFS70 may also interact with Cdc7–ASK, increasing MCM2 phosphorylation, and their interaction may be relevant to activated p38 MAPK in these cells. We hope to determine this mechanism in future studies.
      We showed that LEDGF/DFS70 overexpression increased the expression of psoriasis-associated molecules, including S100A7 and S100A9, and reduced the expression of filaggrin (Figure 6). S100A7 is a small calcium-binding protein, and S100A7 mRNA is expressed in psoriatic skin lesions (
      • Madsen P.
      • Rasmussen H.H.
      • Leffers H.
      • et al.
      Molecular cloning, occurrence, and expression of a novel partially secreted protein “psoriasin” that is highly up-regulated in psoriatic skin.
      ). S100A9 forms a noncovalent heterodimer with S100A8, the calgranulin, and is highly expressed in psoriatic skin (
      • Broome A.M.
      • Ryan D.
      • Eckert R.L.
      S100 protein subcellular localization during epidermal differentiation and psoriasis.
      ). On the other hand, the filaggrin protein is downregulated in psoriasis (
      • Iizuka H.
      • Takahashi H.
      • Honma M.
      • et al.
      Unique keratinization process in psoriasis: late differentiation markers are abolished because of the premature cell death.
      ). The expression of these proteins in psoriatic lesions is similar to that observed in EGFP-LEDGF-HaCaT cells in this study. Therefore, EGFP-LEDGF-HaCaT cells may be a useful psoriatic KC in vitro model.
      In summary, we showed that LEDGF/DFS70 localized to the nucleus of KCs not only in the spinous layer but also in the basal layer of psoriatic skin. We also demonstrated that LEDGF/DFS70 regulated the IL-6/STAT3 signaling pathway via p38 phosphorylation, S100A7, S100A9, and filaggrin in LEDGF-transgenic HaCaT cells. These findings suggest that LEDGF/DFS70 may have a pivotal role in activating psoriatic KCs in vivo.

      Materials and Methods

      Human skin tissues

      Normal human skin was obtained from the scalp of a 16-year-old patient with a benign scalp tumor and from the left anterior arm of a 37-year-old healthy individual. Psoriatic skin was obtained from psoriatic lesions in 10 psoriasis patients (mean age 56 years; range 35–78). All skin tissue samples were taken after written informed consent had been obtained. This study was approved by the ethics committee of Nagoya University, Graduate School of Medicine, and was conducted according to the Declaration of Helsinki Principles.

      Antibodies and reagents

      An antiserum against LEDGF/DFS70, named No. 6369 antiserum, was acquired from the serum bank in our laboratory (
      • Sugiura K.
      • Muro Y.
      • Futamura K.
      • et al.
      The unfolded protein response is activated in differentiating epidermal keratinocytes.
      ). The following polyclonal antibodies were purchased from commercial sources: anti-p-p38 (Thr 180/Tyr 182; Santa Cruz Biotechnology, Santa Cruz, CA), anti-STAT3 (Ab-705; Signalway Antibody, Pearland, TX), anti-STAT3 (phospho-Tyr705; Signalway Antibody), anti-S100A7 (Abnova Corporation, Taipei, Taiwan), anti-p38 (Abnova Corporation), and anti-β-actin (Sigma Aldrich, St Louis, MO). The following monoclonal antibodies were purchased from commercial sources: anti-S100A9 (Abnova Corporation), anti-filaggrin (Abcam, Cambridge, UK), and anti-Ki-67 (Dako Santa Barbara, CA). SP-600125, U-0126, and SB-239063 were purchased from Sigma Aldrich. SB-203580 was also purchased from Enzo Life Science (Farmingdale, NY). The sequences of the Taqman probes for real-time quantitative PCR are shown in Supplementary Table S1a online. The TaqMan Probes were purchased from TaqMan Gene Expression Assays provided by Applied Biosystems (Foster City, CA). siRNA sequences for LEDGF, p38, and CD4 are summarized in Supplementary Table S1b online.

      Cell culture and stable cell lines

      The KC cell line HaCaT was kindly provided by N Fusenig (German Cancer Research Center, Heidelberg, Germany) (
      • Boukamp P.
      • Petrussevska R.T.
      • Breitkreutz D.
      • et al.
      Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.
      ). The skin squamous cell carcinoma cell line, HSC-1, was obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). LEDGF complementary DNA was isolated by reverse transcription–PCR from HeLa complementary DNA libraries and inserted into the PstI sites of pEGFP-C1 (Clontech, Mountain View, CA), creating EGFP-LEDGF. The plasmid was verified by sequence analysis to confirm the absence of mutations. HaCaT and HSC-1 cells were cultured in DMEM containing 1.8mM calcium supplemented with 10% fetal calf serum at 37°C with 5% CO2. For stable transfections, HaCaT cells were cultured in 96-well dishes and then transfected with 2μg of the EGFP-LEDGF or EGFP-empty vector plasmids that are geneticin (G418) resistant using Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA). After 2 days, the cells were subcultured in the presence of 1mgml–1 of G418 (Roche Applied Science, Mannheim, Germany). After approximately 2 weeks, single G418-resistant colonies were obtained by serial dilution in 96-well dishes. The colonies were maintained and analyzed individually for expression of EGFP-LEDGF or the EGFP-empty vector.

      Real-time quantitative PCR

      HaCaT cells overexpressing LEDGF and empty vector-transfected cells were seeded at 10,000cellscm–2 and cultured for 72hours in DMEM containing 1.8mM calcium and 350μgml–1 G418 supplemented with 10% fetal bovine serum at 37°C with 5% CO2. For the study involving the MAPK inhibitors, EGFP-LEDGF-transfected HaCaT cells were cultured in the presence or absence of the following inhibitors when they reached 60% confluency: SP-600125 (10μM), U-0126 (10μM), SB-239063 (2μM), or SB-203580 (2μM) with 350μgml–1 of G418. Total RNA was extracted from the cells at different time points using an RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Complementary DNA was synthesized from 250ng of total RNA using a random primer procedure and the Takara RNA PCR kit, version 3.0 (Takara, Otsu, Japan), according to the manufacturer’s instructions. Real-time quantitative PCR was performed using a Sequence Detector System (Mx3000P Real-Time PCR System and software; Stratagene, La Jolla, CA). Amplification was performed in a final volume of 25μl that contained 10ng of complementary DNA from the reverse transcription reaction, the primers and TaqMan probes, and 12.5μl TaqMan Universal PCR Master Mix (Roche, Branchburg, NJ). The gene expression values were normalized to glyceraldehyde 3 phosphate dehydrogenase to correct for minor variations in mRNA extraction and reverse transcription. Each experiment was performed at least three times.

      Western blotting

      Western blotting was performed as previously described (
      • Sugiura K.
      • Muro Y.
      Anti-annexin V antibodies and digital ischemia in patients with scleroderma.
      ). In brief, strips of membrane were incubated with anti-LEDGF, anti-STAT3, anti-pY-STAT3, anti-p38, anti p-p38, anti-S100A7, anti-S100A9, anti-profilaggrin, or anti-β-actin antibodies. The antibody–antigen complexes were detected with horseradish peroxidase–conjugated goat anti-mouse or anti-rabbit IgG (Dako, Glostrup, Denmark) in a dilution of 1:1,000, followed by enhanced chemiluminescence western blotting substrate (GE Healthcare Bio-Sciences, Little Chalfont, UK), as described by the manufacturer.

      ELISA for IL-6

      To determine the level of secretory IL-6, cells were cultured in six-well plates (density at 3.0 × 105 cells per cm2) for 72 and 96hours after being transfected with the indicated siRNAs. The medium was collected and stored at -80°C. The IL-6 content in the cultured medium was determined by ELISA using a kit purchased from R&D Systems (Minneapolis, MN).

      RNA interference experiments

      For the RNA interference experiments, HaCaT cells overexpressing LEDGF were transfected with 5nM siRNA specific for CD4, LEDGF, or p38 using HiPerFect transfection reagent (Qiagen) according to the manufacturer's protocol.

      Immunohistochemistry

      Immunohistochemistry on human skin was performed as described with slight modifications (
      • Sugiura K.
      • Muro Y.
      • Nishizawa Y.
      • et al.
      LEDGF/DFS70, a major autoantigen of atopic dermatitis, is a component of keratohyalin granules.
      ). The Vectastain Elite ABC-PO (rabbit IgG) kit (Vector Laboratories, Burlingame, CA) was used for staining. Thin sections (6μm) were cut from samples embedded in paraffin blocks. The sections were immersed in a 0.4% pepsin solution for 30minutes and soaked for 20minutes at room temperature in 0.3% H2O2/methanol to block endogenous peroxidase activity. After being washed in PBS with 0.01% Triton X-100, the sections were incubated for 30minutes in PBS with 4% BSA followed by an overnight incubation at 4°C with the primary antibodies (10ngml–1) in PBS containing 1% BSA. After a washing in PBS, the thin sections were stained with avidin-conjugated goat anti-rabbit immunoglobulin secondary antibodies for 1hour at room temperature and washed in PBS. After a washing, the tissue sections were immersed in Vectastain Elite ABC Reagent for 30minutes and then washed in PBS. The antibody complexes were visualized by adding 1% H2O2. Rabbit pre-immune serum (pre-immune no. 6369) (
      • Sugiura K.
      • Muro Y.
      • Nishizawa Y.
      • et al.
      LEDGF/DFS70, a major autoantigen of atopic dermatitis, is a component of keratohyalin granules.
      ) corresponding to 10ngml–1 was used as a negative control.

      Statistical analysis

      An unpaired t-test was used to analyze the real-time PCR and ELISA results. P<0.01 and P<0.05 were considered statistically significant.

      ACKNOWLEDGMENTS

      This work was supported by a grant-in-aid for Scientific Research, (C) 20591319 (to KS), from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

      REFERENCES

        • Ahuja H.G.
        • Hong J.
        • Aplan P.D.
        • et al.
        t(9;11)(p22;p15) in acute myeloid leukemia results in a fusion between NUP98 and the gene encoding transcriptional coactivators p52 and p75-lens epithelium-derived growth factor (LEDGF).
        Cancer Res. 2000; 60: 6227-6229
        • Ameglio F.
        • Bonifati C.
        • Fazio M.
        • et al.
        Interleukin-11 production is increased in organ cultures of lesional skin of patients with active plaque-type psoriasis as compared with nonlesional and normal skin. Similarity to interleukin-1 beta, interleukin-6 and interleukin-8.
        Arch Dermatol Res. 1997; 289: 399-403
        • Arthur J.S.
        • Darragh J.
        Signaling downstream of p38 in psoriasis.
        J Invest Dermatol. 2006; 126: 1689-1691
        • Battle T.E.
        • Frank D.A.
        The role of STATs in apoptosis.
        Curr Mol Med. 2002; 2: 381-392
        • Bennett B.L.
        • Sasaki D.T.
        • Murray B.W.
        • et al.
        SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase.
        Proc Natl Acad Sci USA. 2001; 98: 13681-13686
        • Bos J.D.
        • de Rie M.A.
        • Teunissen M.B.
        • et al.
        Psoriasis: dysregulation of innate immunity.
        Br J Dermatol. 2005; 152: 1098-1107
        • Boukamp P.
        • Petrussevska R.T.
        • Breitkreutz D.
        • et al.
        Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.
        J Cell Biol. 1988; 106: 761-771
        • Bowcock A.M.
        • Krueger J.G.
        Getting under the skin: the immunogenetics of psoriasis.
        Nat Rev Immunol. 2005; 5: 699-711
        • Bromberg J.F.
        • Wrzeszczynska M.H.
        • Devgan G.
        • et al.
        Stat3 as an oncogene.
        Cell. 1999; 98: 295-303
        • Broome A.M.
        • Ryan D.
        • Eckert R.L.
        S100 protein subcellular localization during epidermal differentiation and psoriasis.
        J Histochem Cytochem. 2003; 51: 675-685
        • Ciuffi A.
        • Bushman F.D.
        Retroviral DNA integration: HIV and the role of LEDGF/p75.
        Trends Genet. 2006; 22: 388-395
        • Cohen B.
        • Addadi Y.
        • Sapoznik S.
        • et al.
        Transcriptional regulation of vascular endothelial growth factor C by oxidative and thermal stress is mediated by lens epithelium-derived growth factor/p75.
        Neoplasia. 2009; 11: 921-933
        • Cuenda A.
        • Rousseau S.
        p38 MAP-kinases pathway regulation, function and role in human diseases.
        Biochim Biophys Acta. 2007; 1773: 1358-1375
        • Daugaard M.
        • Kirkegaard-Sorensen T.
        • Ostenfeld M.S.
        • et al.
        Lens epithelium-derived growth factor is an Hsp70-2 regulated guardian of lysosomal stability in human cancer.
        Cancer Res. 2007; 67: 2559-2567
        • Duncia J.V.
        • Santella III, J.B.
        • Higley C.A.
        • et al.
        MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products.
        Bioorg Med Chem Lett. 1998; 8: 2839-2844
        • Gazel A.
        • Nijhawan R.I.
        • Walsh R.
        • et al.
        Transcriptional profiling defines the roles of ERK and p38 kinases in epidermal keratinocytes.
        J Cell Physiol. 2008; 215: 292-308
        • Ge H.
        • Si Y.
        • Roeder R.G.
        Isolation of cDNAs encoding novel transcription coactivators p52 and p75 reveals an alternate regulatory mechanism of transcriptional activation.
        EMBO J. 1998; 17: 6723-6729
        • Hodge D.R.
        • Hurt E.M.
        • Farrar W.L.
        The role of IL-6 and STAT3 in inflammation and cancer.
        Eur J Cancer. 2005; 41: 2502-2512
        • Huang T.S.
        • Myklebust L.M.
        • Kjarland E.
        • et al.
        LEDGF/p75 has increased expression in blasts from chemotherapy-resistant human acute myelogenic leukemia patients and protects leukemia cells from apoptosis in vitro.
        Mol Cancer. 2007; 6: 31
        • Hughes S.
        • Jenkins V.
        • Dar M.J.
        • et al.
        Transcriptional co-activator LEDGF interacts with Cdc7-activator of S-phase kinase (ASK) and stimulates its enzymatic activity.
        J Biol Chem. 2010; 285: 541-554
        • Iizuka H.
        • Takahashi H.
        • Honma M.
        • et al.
        Unique keratinization process in psoriasis: late differentiation markers are abolished because of the premature cell death.
        J Dermatol. 2004; 31: 271-276
        • Johansen C.
        • Kragballe K.
        • Rasmussen M.
        • et al.
        The AP-1 DNA binding activity is decreased in lesional psoriatic skin compared to nonlesional psoriatic skin.
        Br J Dermatol. 2004; 151: 600-607
        • Johansen C.
        • Kragballe K.
        • Westergaard M.
        • et al.
        The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin.
        Br J Dermatol. 2005; 152: 37-42
        • Kramer R.M.
        • Roberts E.F.
        • Um S.L.
        • et al.
        p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2.
        J Biol Chem. 1996; 271: 27723-27729
        • Krueger G.
        • Ellis C.N.
        Psoriasis – recent advances in understanding its pathogenesis and treatment.
        J Am Acad Dermatol. 2005; 53: S94-S100
        • Madsen P.
        • Rasmussen H.H.
        • Leffers H.
        • et al.
        Molecular cloning, occurrence, and expression of a novel partially secreted protein “psoriasin” that is highly up-regulated in psoriatic skin.
        J Invest Dermatol. 1991; 97: 701-712
        • Maertens G.
        • Cherepanov P.
        • Pluymers W.
        • et al.
        LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells.
        J Biol Chem. 2003; 278: 33528-33539
        • McKenzie R.C.
        • Sabin E.
        Aberrant signalling and transcription factor activation as an explanation for the defective growth control and differentiation of keratinocytes in psoriasis: a hypothesis.
        Exp Dermatol. 2003; 12: 337-345
        • Mediavilla-Varela M.
        • Pacheco F.J.
        • Almaguel F.
        • et al.
        Docetaxel-induced prostate cancer cell death involves concomitant activation of caspase and lysosomal pathways and is attenuated by LEDGF/p75.
        Mol Cancer. 2009; 8: 68
        • Muro Y.
        Autoantibodies in atopic dermatitis.
        J Dermatol Sci. 2001; 25: 171-178
        • Nguyen T.A.
        • Boyle D.L.
        • Wagner L.M.
        • et al.
        LEDGF activation of PKC gamma and gap junction disassembly in lens epithelial cells.
        Exp Eye Res. 2003; 76: 565-572
        • Ochs R.L.
        • Muro Y.
        • Si Y.
        • et al.
        Autoantibodies to DFS70 kd/transcription coactivator p75 in atopic dermatitis and other conditions.
        J Allergy Clin Immunol. 2000; 105: 1211-1220
        • Ogawa Y.
        • Sugiura K.
        • Watanabe A.
        • et al.
        Autoantigenicity of DFS70 is restricted to the conformational epitope of C-terminal alpha-helical domain.
        J Autoimmun. 2004; 23: 221-233
        • Okamoto M.
        • Watanabe A.
        • Ogawa Y.
        • et al.
        Autoantibodies to DFS70/LEDGF are increased in alopecia areata patients.
        J Autoimmun. 2004; 23: 257-266
        • Qi L.
        • Allen R.R.
        • Lu Q.
        • et al.
        PAI-1 transcriptional regulation during the G0-->G1 transition in human epidermal keratinocytes.
        J Cell Biochem. 2006; 99: 495-507
        • Sano S.
        • Chan K.S.
        • Carbajal S.
        • et al.
        Stat3 links activated keratinocytes and immunocytes required for development of psoriasis in a novel transgenic mouse model.
        Nat Med. 2005; 11: 43-49
        • Shinohara T.
        • Singh D.P.
        • Fatma N.
        LEDGF, a survival factor, activates stress-related genes.
        Prog Retin Eye Res. 2002; 21: 341-358
        • Singh D.P.
        • Ohguro N.
        • Chylack Jr, L.T.
        • et al.
        Lens epithelium derived growth factor: increased resistance to thermal and oxidative stresses.
        Invest Ophthalmol Vis Sci. 1999; 40: 1444-1451
        • Sugiura K.
        • Muro Y.
        Anti-annexin V antibodies and digital ischemia in patients with scleroderma.
        J Rheumatol. 1999; 26: 2168-2172
        • Sugiura K.
        • Muro Y.
        • Futamura K.
        • et al.
        The unfolded protein response is activated in differentiating epidermal keratinocytes.
        J Invest Dermatol. 2009; 129: 2126-2135
        • Sugiura K.
        • Muro Y.
        • Nishizawa Y.
        • et al.
        LEDGF/DFS70, a major autoantigen of atopic dermatitis, is a component of keratohyalin granules.
        J Invest Dermatol. 2007; 127: 75-80
        • Underwood D.C.
        • Osborn R.R.
        • Bochnowicz S.
        • et al.
        SB 239063, a p38 MAPK inhibitor, reduces neutrophilia, inflammatory cytokines, MMP-9, and fibrosis in lung.
        Am J Physiol Lung Cell Mol Physiol. 2000; 279: L895-L902
        • Verheijen R.
        • Kuijpers H.J.
        • Schlingemann R.O.
        • et al.
        Ki-67 detects a nuclear matrix-associated proliferation-related antigen. I. Intracellular localization during interphase.
        J Cell Sci. 1989; 92: 123-130
        • Watanabe A.
        • Kodera M.
        • Sugiura K.
        • et al.
        Anti-DFS70 antibodies in 597 healthy hospital workers.
        Arthritis Rheum. 2004; 50: 892-900
        • Yu X.J.
        • Li C.Y.
        • Dai H.Y.
        • et al.
        Expression and localization of the activated mitogen-activated protein kinase in lesional psoriatic skin.
        Exp Mol Pathol. 2007; 83: 413-418
        • Zarubin T.
        • Han J.
        Activation and signaling of the p38 MAP kinase pathway.
        Cell Res. 2005; 15: 11-18