Erratum to “Neutrophil-dominant psoriasis-like skin inflammation induced by epidermal-specific expression of Raf in mice” [J. Dermatol. Sci. 58 (2010) 28–35]
Article Outline
- Abstract
- 1. Introduction
- 2. Materials and methods
- 3. Results
- 3.1. Morphological changes of K14-Raf:ER mouse skin following a single application of 4OHT
- 3.2. Expression of phospho-Erk1/2 in the epidermis of K14-Raf:ER mice and in psoriatic epidermis
- 3.3. Production of cytokines and chemokines by K14-Raf:ER keratinocytes
- 3.4. Induction of cytokines and chemokines in K14-Raf:ER mouse skin
- 3.5. The differentiation potential of CD4-positive regional lymph node cells of K14-Raf:ER mice
- 3.6. The effect of an anti-Gr-1 antibody on the skin of K14-Raf:ER mice
- 4. Discussion
- Acknowledgements
- Appendix A. Supplementary data
- References
- Copyright
Abstract
Background
Raf is one of the downstream effectors of Ras GTPases. The induction of Raf in the epidermis causes the proliferation of keratinocytes and epidermal hyperplasia. However, skin inflammation accompanying Ras-induced epidermal reactions has not been fully delineated.
Objective
The aim of this study was to characterize inflammatory reactions induced by epidermal-specific Raf expression and to elucidate its role in skin inflammation.
Methods
K14-Raf:ER transgenic mice, in which the 4-hydroxytamoxifen (4OHT)-responsive mutant estrogen receptor (ER) ligand binding domain-Raf fusion gene was expressed under control of the keratin 14 promoter, were used to characterize inflammatory reactions induced by Raf expression in the epidermis.
Results
A single topical application of 4OHT induced the expression of phosphorylated extracellular signal-related kinase 1/2 and elicited neutrophil-dominant inflammatory infiltrates in the skin. The Raf expression also rapidly induced the production of several cytokines and chemokines, including VEGF and CXCL1, by keratinocytes and in mouse skin in vivo. Furthermore, CD4-positive cells from regional lymph nodes had the potential to differentiate into IFNγ- and IL17-producing cells. Treatment with an anti-Gr-1 antibody diminished the Raf-induced cutaneous inflammation and partially reversed the epidermal hyperplasia and hyperkeratosis.
Conclusion
Activation of the Raf signaling pathway is involved in the epidermal hyperplasia and the neutrophil-dominant cutaneous inflammatory reactions which are characteristics of psoriasis.
Keywords: Raf, Skin inflammation, Neutrophil, Transgenic mice, Psoriasis
1. Introduction
Ras GTPase functions during signal transduction that regulates cell proliferation and differentiation [1]. The activation of Ras then signals through three major cascades, Raf/mitogen-activated protein kinase (MAPK), phosphatidylinositol-3′ kinase (PI3K)/Akt, and Ral guanine nucleotide dissociation stimulator (RalGDS)/Ral [2]. Of those, the downstream effector pathway of Raf/MAPK plays the main role in regulating the growth and differentiation of epidermal keratinocytes [3], [4], [5]. Further, the Ras effector pathway is up-regulated in skin carcinogenesis [6], [7] and in the hyperplastic epidermis of psoriasis, a common inflammatory skin disorder [8].
The major characteristics of psoriasis are epidermal hyperplasia and inflammatory infiltrates, but it is still unknown which is the primary event for the pathogenesis of the disease [9]. In studies using animal models, the epidermal-specific expression of integrin β1 [10], TGF-β [11], Stat3 [12] or VEGF [13] causes epidermal hyperplasia which mimics psoriatic skin. In contrast, evidence that psoriasis is improved by bone marrow transplantation [14] or by new biologics, such as antibodies to TNF-α [15] or the IL-12/IL-23 p40 subunit [16], suggests the importance of immunological abnormalities in the disorder accompanied by the activation of Th1 cells [17], Th17 cells [18] and/or CD11c-positive dendritic cells (Tip-DC in mice) [19]. However, it has not been delineated whether the activation of signal transduction systems that regulate keratinocyte proliferation is involved in the induction of the cutaneous inflammation and/or its exacerbation in psoriasis, or in turn, whether the inflammatory reactions affect the progression of epidermal hyperplasia in psoriasis.
In the present study, we analyzed the immunological phenotype of a mouse model with reversible epidermal-specific expression of Raf, the major signaling molecule in the downstream effector pathway of Ras. We show here evidence that the activation of Raf plays a key role in the induction of psoriasis-like epidermal hyperplasia and in immunological responses involving Th1 and Th17 cells.
2. Materials and methods
2.1. Materials
Anti-Gr-1 antibody, a hybridoma supernatant (RB6-8C5), was obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University, Japan. Anti-phosphorylated extracellular signal-related kinase 1/2 (phospho-Erk1/2) antibody, phycoerythrin (PE)-labeled anti-Gr-1 antibody, PE-labeled anti-I-A/I-E antibody, and anti-β-actin (I-19) antibody were purchased from Cell Signaling Technology Inc. (Beverly, MA), BioLegend Inc. (San Diego, CA), Pharmingen (San Diego, CA), and Santa Cruz Biotechnology Inc. (Santa Cruz, CA), respectively; 4-hydroxytamoxifen (4OHT) was from Sigma–Aldrich (St. Louis, MO); dispase II was from Godo Shusei Co. Ltd. (Tokyo, Japan); Cnt-07 medium was from CELLnTEC Advanced Cell Systems (Bern, Switzerland); and ProLongGold antifade reagent was from Molecular Probes (Carlsbad, CA).
2.2. Animals
All studies involving animals were designed in accordance with the International Guiding Principles for Biomedical Research Involving Animals published by the Council for the International Organization of Medical Science and were reviewed and approved by the Animal Use and Care Committee of the Hyogo College of Medicine. K14-Raf:ER mice were kindly provided by Dr. Paul A. Khavari, Program in Epithelial Biology, Stanford University [3]. Those mice were backcrossed more than 10 times with C57BL/6 mice to generate K14-Raf:ER mice with a C57BL/6 background. The PCR primers 5′-CCGAAGATCAACCGGAGCGCTTCCGA-3′ and 5′-AACCAGCTCCCTGTCTGCCAGGTTGG-3′ were used for genotyping those mice as described previously [3]. To activate the Raf protein in adult K14-Raf:ER mouse epidermis, 0.1
ml 10mg/ml 4OHT (dissolved in ethanol) was topically applied to shaved areas of mouse dorsal skin. 4OHT-treated wild-type mice or mock-treated K14-Raf:ER mice were used as controls. For sampling of specimens, mice were euthanized with an intraperitoneal injection of 75mg/kg sodium pentobarbital.
2.3. Histology, immunohistochemistry and immunofluorescence
Excised skins were fixed in 10% formaldehyde in phosphate-buffered saline (PBS), and then were embedded in paraffin. Those tissues were sectioned at 4-μm thickness and deparaffinized sections were subjected to hematoxylin and eosin stainings or were used for immunohistochemistry. After informed consent was obtained, human skin samples were also used for immunohistochemistry. Sections for immunohistochemistry were deparaffinized with xylene, followed by antigen unmasking using 10mM sodium citrate buffer, pH 6.0. After blocking endogenous peroxidase with 3% H2O2 in methanol for 10min, sections were treated with 10% horse serum for 1h and then with anti-phospho-Erk1/2 antibody (1:200 dilution) for 1h at room temperature. Those sections were washed 3 times with PBS and were then incubated for 10min with a biotin-conjugated universal secondary antibody, followed by rinsing with PBS. Those sections were treated with a streptavidin/peroxidase complex for 5min, followed by rinsing again, and phospho-Erk1/2 was visualized with diaminobenzidine.
For immunofluorescence, cryosections of skin specimens were allowed to air dry for 30min and were fixed with cold acetone for 10min. Sections were blocked with 3% bovine serum albumin for 1h and were treated with a PE-labeled anti-Gr-1 antibody (1:100 dilution), or PE-labeled anti-I-A/I-E (1:50 dilution) for 1h at room temperature. After washing with PBS, sections were mounted in the ProLongGold antifade reagent and fluorescence images were examined using a Model IX81 laser scanning confocal microscope (Olympus, Tokyo, Japan).
2.4. Cell culture
Dorsal skins taken from euthanized newborn mice were incubated while floating on PBS containing 250U/ml dispase II overnight at 4°C. Epidermal sheets were then separated from the dermis with forceps and were trypsinized to isolate keratinocytes. Cells were seeded into 6-well plates at an initial seeding density of 1×104cells per cm2 and were cultured in Cnt-07 medium supplemented with antibiotics (100U/ml penicillin, 100μg/ml streptomycin, and 0.25μg/ml amphotericin B) at 37°C in a humidified atmosphere of 5% CO2 in air. Two days after the plating, 0, 10 or 100nM 4OHT was added to the medium. After 24h, the culture media were collected for measurement of the concentrations of cytokines or chemokines and proteins were extracted with lysis buffer (50mM HEPES pH 7.5, 150mM NaCl, 5mM EDTA, and 0.9% NP40) containing protease inhibitors, and were separated on 10% SDS-PAGE gels and then transferred to PVDF membranes. Western blotting was performed using an Amersham ECL Western Blotting System and an anti-phospho-Erk1/2 antibody (1:1000 dilution) according to the manufacturer's instructions. Membranes were reprobed with anti-β-actin (I-19) as a loading control.
2.5. Measurement of cytokines and chemokines
Skin specimens excised from mice were homogenized with a BeadSmash homogenizer (Wakenyaku Co. Ltd., Kyoto, Japan) using a Bio-Plex Cell Lysis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer's instructions. The concentrations of cytokines or chemokines in the supernatants from the skin samples or in the culture media were measured using a Bio-Plex Protein Array System (Bio-Rad Laboratories Inc.).
From cells isolated from the inguinal and axillary lymph nodes, CD4-positive cells were enriched using a magnetic cell sorting system as described previously [20]. Those cells were pooled and incubated with immobilized anti-CD3 for 24 or 48h, after which the concentrations of IFN-γ, IL-4 or IL-17 in the medium were measured using a Bio-Plex Protein Array System.
2.6. Statistical analysis
Experiments for measurement of cytokines and chemokines were repeated 3 times. Data are expressed as means±standard deviation of triplicate experiments and significance of differences were assessed using one-way analysis of variance (ANOVA) followed by Scheffe's test for cultured cells or with the unpaired Student's t-test for skin samples.
3. Results
3.1. Morphological changes of K14-Raf:ER mouse skin following a single application of 4OHT
In K14-Raf:ER transgenic mice, topical treatment of the dorsal skin once with 1mg 4OHT induced redness of the treated skin within 2 days, and thickening and hyperkeratosis of the treated area at 7 days, but those changes were transient and had subsided at 14 days (Fig. 1A).
Fig. 1.
Alterations in gross appearance (A) and histology (B) of K14-Raf:ER skin induced by a single topical application of 4OHT. K14-Raf:ER dorsal skin was shaved and then treated once with 1
mg 4OHT. Pictures of the skin were taken at days 0, 2, 7 and 14 and skin specimens for histology were harvested at days 0, 2, 4, 7 and 21. Images in the lowest panels (labeled with asterisks * and **) are higher magnifications of the indicated areas of the image at day 7. Arrowheads indicate the infiltration of neutrophils into the epidermis and horny layers. Bars: 100μm in 0, 2, 4, 7 and 21; 20μm in the lowest panels.
When the skin of K14-Raf:ER transgenic mice was treated with 4OHT, cutaneous hyperplasia began quickly and infiltrations of inflammatory cells in the dermis were noted at days 2 and 4 (Fig. 1B). At 7 days after treatment, epidermal hyperplasia with elongated rete ridges and hyperkeratosis lacking granular layers and dilated blood vessels in the papillary dermis were evident. Neutrophils had infiltrated the stratum corneum and formed microabscesses, as seen in the histology of psoriasis. Those histological changes were completely reversed at 4 weeks after the application of 4OHT (data not shown). In contrast, those morphological changes were not evident in K14-Raf:ER mice treated with ethanol (vehicle) or in wild-type mice treated with 4OHT (data not shown).
3.2. Expression of phospho-Erk1/2 in the epidermis of K14-Raf:ER mice and in psoriatic epidermis
The expression of phospho-Erk1/2, downstream intermediates of the Raf signaling pathway, was examined in K14-Raf:ER mouse epidermis by immunohistochemistry. As shown in Fig. 2A, a single topical application of 4OHT induced phospho-Erk1/2 expression within 8h along with early hyperplasia of the epidermis. The expression of phospho-Erk1/2 increased progressively for at least 6 days (144h) after the treatment, at which time the epidermis had markedly thickened. Thus, a single topical application of 4OHT to the K14-Raf:ER transgenic mice enabled us to analyze the reversible and transient responses of Raf signaling in the skin. Phospho-Erk1/2 was also induced in keratinocytes cultured from K14-Raf:ER mice as early as 4h after treatment with 10 or 100nM 4OHT in a dose-responsive manner (data not shown).
Fig. 2.
(A) The expression of phospho-Erk1/2 in the epidermis of K14-Raf:ER mice treated once with 4OHT. Dorsal skins of K14-Raf:ER mice were treated once with 1
mg 4OHT and at the indicated times (in hours), they were harvested and subjected to immunohistochemistry using a anti-phospho-Erk1/2 antibody. (B) The expression of phospho-Erk1/2 in psoriatic epidermis. Lesional skins of patients with psoriasis vulgaris (n=3) and normal skins from healthy volunteers (n=3) were subjected to immunohistochemistry using the anti-phospho-Erk1/2 antibody. Bars: 20μm.
As shown in Fig. 2B, the expression of phospho-Erk1/2 was also evident in psoriatic epidermis, where the nuclei of epidermal cells were clearly positive for phospho-Erk1/2. In contrast, phospho-Erk1/2 expression was observed only faintly in the epidermis of healthy controls.
3.3. Production of cytokines and chemokines by K14-Raf:ER keratinocytes
The expression of cytokines and chemokines produced by keratinocytes cultured from newborn K14-Raf:ER transgenic mice was examined. When the cells were incubated with 0, 10 or 100nM 4OHT for 24h, cellular phospho-Erk1/2 levels were increased in a dose-dependent manner (Fig. S1), and VEGF, IL-18, G-CSF and CXCL1 (KC) in the medium were significantly increased (Fig. 3). In contrast, the production of Th2 chemokines, CCL5 (RANTES) and CCL11 (Eotaxin), was not induced by the treatment with 4OHT (Fig. 3). In keratinocytes derived from wild-type mice, those cytokines and chemokines were not altered by the 4OHT treatment (data not shown).
Fig. 3.
The production of cytokines and chemokines by keratinocytes cultured from K14-Raf:ER mouse skin and then induced by 4OHT. Keratinocytes from newborn K14-Raf:ER mice were incubated with 0, 10 or 100
nM 4OHT for 24h, after which the concentration of each cytokine or chemokine noted was measured. Data are shown as means±standard deviation of triplicate experiments. Significance of differences was assessed using One-way analysis of variance (ANOVA) followed by Scheffe's test. *p<0.05, **p<0.01 (vs 0nM). ND: not detected.
3.4. Induction of cytokines and chemokines in K14-Raf:ER mouse skin
We next examined the expression of cytokines and chemokines in K14-Raf:ER mouse skin sampled before and 8h after a single treatment with 1mg 4OHT. As shown in Fig. 4, IL-15, IL-18, LIF, M-CSF, VEGF, CXCL1 (KC), CXCL2 (MIP-2), CCL2 (MCP-1) and CCL4 (MIP-1β) were significantly increased in the skin by the 4OHT treatment. In contrast, the expression of the Th2 cytokine IL-5 and the Th2 chemokines CCL5 (RANTES) and CCL11 (Eotaxin) was not altered. Those cytokines and chemokines were not altered in wild-type mice treated with 4OHT or in K14-Raf:ER mice treated only with ethanol, a vehicle of 4OHT (data not shown).
Fig. 4.
Rapid expression of cytokines and chemokines in K14-Raf:ER skin treated once with 4OHT. Dorsal skins of K14-Raf:ER mice were treated once with 1
mg 4OHT and the concentrations of cytokines and chemokines in skin homogenates prepared before or 8h after treatment were measured. Data are shown as means±standard deviation of triplicate experiments. Significance of differences was assessed using the unpaired Student's t-test. *p<0.05 (vs 0h). There were no significant differences in IL-5, CCL5 (RANTES) and CCL11 (Eotaxin).
3.5. The differentiation potential of CD4-positive regional lymph node cells of K14-Raf:ER mice
Seven days after a single topical treatment of the dorsal skin of K14-Raf:ER mice with 4OHT, the immobilized anti-CD3 antibody-stimulated cytokine production by CD4-positive cells isolated from inguinal and axillary lymph nodes of those transgenic mice were examined. As shown in Fig. 5, the production of IFN-γ and IL-17 by the CD4-positive cells was increased at 24 and 48h after the stimulation with anti-CD3 antibody, whereas IL-4 was unaltered.
Fig. 5.
The differentiation capacity of regional lymph node CD4-positive cells in K14-Raf:ER mice treated once with 4OHT. Seven days after a single treatment with 1
mg 4OHT, immobilized CD3-stimulated production of IFN-γ, IL-4 or IL-17 by CD4-positive cells from lymph nodes was measured. Experiments were repeated 3 times and representative data are shown.
3.6. The effect of an anti-Gr-1 antibody on the skin of K14-Raf:ER mice
Gr-1 is a marker of peripheral neutrophils and the infiltration of Gr-1-positive cells was examined in K14-Raf:ER mouse skin after a single topical treatment with 1mg 4OHT. As shown in Fig. 6E, Gr-1-positive cells accumulated in the stratum corneum and epidermis as well as in the dermis after treatment with 4OHT. Several CD3-positive cells and a few CD8-positive cells were seen in the skin infiltrates (data not shown). When 2.5mg anti-Gr-1 antibody was injected intraperitoneally 1 day before the 4OHT treatment and that injection was repeated every 2 days, the Gr-1-positive cells were almost completely depleted from the skin (Fig. 6F) and the expression of I-A, MHC class II, was reduced at 7 days after treatment with 4OHT (Fig. 6H and I). Furthermore, the epidermal hyperplasia and hyperkeratosis evoked by the single topical treatment with 4OHT was markedly improved by the injections with anti-Gr-1 antibodies (Fig. 6A–C).
Fig. 6.
The effect of an anti-Gr-1 antibody on epidermal hyperplasia and inflammation of K14-Raf:ER skin treated once with 4OHT. PBS (A, B, D, E, G, H) or anti-Gr-1 antibody (C, F, I) was administered intraperitoneally to K14-Raf:ER mice 1 day before topical treatment with 4OHT (B, C, E, F, H, I) or with vehicle alone (A, D, G) and every 2 days thereafter. Seven days after treatment with 4OHT, skins were processed and subjected to hematoxylin and eosin staining (A–C), to immunofluorescence for Gr-1 (D–F), or for I-A (G–I). Solid lines indicate the contours of the skin surface; dotted lines denote the basement membrane. Bars: 400
μm in A–C; 200μm in D–I.
4. Discussion
In K14-Raf:ER mice, the Raf effector pathway in the epidermis can be transiently induced by a topical single application of 4OHT. The up-regulation of phospho-Erk1/2 downstream of Raf indicates that the Raf pathway is activated as early as 8h after the treatment with 4OHT, followed by epidermal thickening and elongation of rete ridges in response to that stimulation. Those pathologic changes are transient and subside within 4 weeks. In contrast with K14-Raf:ER or K14-ER:Ras mice treated every day with 4OHT [3], no papillomatous skin hyperplasia was observed during the transient Raf expression in K14-Raf:ER mice. Therefore, this transgenic model with a single application of 4OHT is suitable to elucidate the non-tumorigenic cutaneous responses to Raf expression in the epidermis.
Besides epidermal hyperplasia and hyperkeratosis, the induced epidermal Raf expression in K14-Raf:ER mouse skin causes vascular dilatation, cutaneous inflammatory infiltrates (including neutrophils) into the epidermis, and increased expression of MHC class II (I-A) molecules (which are possibly induced by the infiltrating immune cells). Those pathological characteristics resemble those seen in psoriasis, including epidermal hyperplasia and hyperkeratosis with cutaneous inflammatory infiltrates, neutrophil-dominant infiltrates and formation of microabscesses in the epidermis, and capillary dilatation of the papillary dermis. The up-regulation of Ras in psoriatic lesional epidermis is suggestive of the activation of the Raf effector pathway in that tissue [8]. Indeed, the expression of phospho-Erk1/2 in the psoriatic lesional epidermis implies that the Raf downstream effector pathway is involved in the pathological findings in psoriasis.
Interestingly, the Raf expression in keratinocytes derived from K14-Raf:ER skin induces the production of CXCL1 (KC), which is strongly chemotactic for neutrophils as is human CXCL8/IL-8. Cytokines IL-18 and VEGF are also increased in keratinocytes by the Raf expression. In addition to those cytokines and chemokines, the transient Raf expression in the skin of K14-Raf:ER mice rapidly induces IL-18 and CCL2 (MCP-1), which have been suggested to contribute to the pathogenesis of psoriasis [21], [22], [23]. In contrast, IL-5, CCL5 (RANTES) and CCL11 (Eotaxin), which are abundantly expressed in atopic dermatitis skin [24], are not induced by transient Raf expression in keratinocytes or in skins from K14-Raf:ER mice. TNF-α, which participates in the development of psoriatic lesions, is not induced by the Raf expression and hence the up-regulation of TNF-α is possibly independent of the Raf effector pathway in the epidermis. IL-15 is anti-apoptotic for keratinocytes [25], and therefore it might contribute to epidermal hyperplasia in mice and in psoriatic epidermis. VEGF, a cytokine that promotes cutaneous angiogenesis, is up-regulated in psoriatic epidermis [26], and the epidermal expression of VEGF causes the development of psoriasiform dermatitis in mouse skin [27]. The psoriasis-like pathological changes in the skin induced by the Raf expression might be in part due to the expression of VEGF.
Both Th1 and Th17 cells have been shown to participate in the immuno-pathogenesis of psoriasis [28], [29]. Interestingly, in K14-Raf:ER mice with the induced epidermal Raf expression, CD4-positive cells in the regional lymph nodes have the capacity to produce IFN-γ and IL-17, but not IL-4. Thus, the helper T cell subsets in K14-Raf:ER mice with transient epidermal Raf expression are compatible with those predominant in psoriasis. The Th17-predominance of K14-Raf:ER mice might be related to the induction of VEGF and epidermal hyperplasia because transgenic mice that express epidermis-specific VEGF also reveal Th17-like responses in the phorbol ester-induced hyperplastic skins [13]. However, the precise immunological mechanisms of the Th17 responses in those mice have not yet been uncovered.
In the present study, the activation of Raf in the epidermis possibly increases the production of CXCL1 (KC) which recruits neutrophils to infiltrate the epidermis. Administration of anti-Gr-1 antibodies depletes the Gr-1-positive infiltrates including neutrophils and reduces the cutaneous expression of MHC class II (I-A). Along with reducing the inflammation, the anti-Gr-1 antibody improves the epidermal thickening and hyperkeratosis. Hence, the inflammatory reactions elicited by the Raf expression in the skin are at least in part involved in the acceleration of the epidermal hyperplasia and the hyperkeratosis, but further studies including the effect of Erk inhibitors will be necessary to elucidate the mechanisms of Raf-induced epidermal changes and skin inflammation.
Our results reveal that the stimulation of epidermal growth via the activation of the Raf effector pathway amplifies the cutaneous inflammation and increases inflammatory infiltrates, which accelerates the epidermal thickening via the induction of a set of chemokines and cytokines. The K14-Raf:ER transgenic mouse with transient epidermal Raf expression is a novel model of psoriasis that mimics epidermal hyperplasia and neutrophil-dominant cutaneous inflammation.
Acknowledgements
The authors would like to thank Prof. Paul A. Khavari, Program in Epithelial Biology, Stanford University School of Medicine, for providing the K14-Raf:ER mice. The authors thank Dr. Tatsuya Tsuda and Mrs. Hiroe Konishi, Department of Dermatology, and the members of the Joint-Use Research Facilities, Hyogo College of Medicine, for their technical assistance. This work was supported in part by JSPS KAKENHI [20591359, 21591476], by a High-Tech Research Center Grant, by a grant from the Ministry of Health, Labour and Welfare, Health and Labour Sciences Research Grants for Research on intractable diseases.
Appendix A. Supplementary data
Fig. S1.
The expression of phospho-Erk1/2 by 4OHT in K14-Raf:ER keratinocytes. Keratinocytes from newborn K14-Raf:ER mice and from wild-type mice were incubated with 0, 10 or 100
nM 4OHT for 24h, then cellular protein extracts were subjected to western blotting using a phospho-ERK1/2 antibody.
References
- . Ras proteins in the control of the cell cycle and cell differentiation. Cell Mol Life Sci. 2000;57:1613–1636
- . Understanding Ras: ‘it ain’t over ‘til it's over’. Trends Cell Biol. 2000;10:147–154
- . Inducible activation of Ras and Raf in adult epidermis. Cancer Res. 2003;63:319–323
- . Ras/Erk MAPK signaling in epidermal homeostasis and neoplasia. Cell Cycle. 2007;6:2928–2931
- . Mek1/2 gene dosage determines tissue response to oncogenic Ras signaling in the skin. Oncogene. 2009;28:1485–1495
- . An association between the risk of cancer and mutations in the HRAS1 minisatellite locus. N Engl J Med. 1993;329:517–523
- . Ras gene mutation and amplification in human nonmelanoma skin cancers. Mol Carcinog. 1991;4:196–202
- . Increased activation of Ras in psoriatic lesions. Skin Pharmacol Appl Skin Physiol. 1999;12:90–97
- . The pathogenesis of psoriasis: immunological facts and speculations. Immunol Today. 1999;20:40–46
- . Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis. Cell. 1995;83:957–968
- . Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder. EMBO J. 2004;23:1770–1781
- Stat3 links activated keratinocytes and immunocytes required for development of psoriasis in a novel transgenic mouse model. Nat Med. 2005;11:43–49
- . TPA induction leads to a Th17-like response in transgenic K14/VEGF mice: a novel in vivo screening model of psoriasis. Int Immunol. 2008;20:1097–1106
- Resolution of psoriasis following allogeneic bone marrow transplantation for chronic myelogenous leukemia: case report and review of the literature. Am J Hematol. 2002;71:41–44
- . Potential of tumor necrosis factor inhibitors in psoriasis and psoriatic arthritis. Arch Dermatol. 2004;140:218–225
- A human interleukin-12/23 monoclonal antibody for the treatment of psoriasis. N Engl J Med. 2007;356:580–592
- . The cytokine network in lesional and lesion-free psoriatic skin is characterized by a T-helper type 1 cell-mediated response. J Invest Dermatol. 1993;101:701–705
- . Pathophysiology of psoriasis: recent advances on IL-23 and Th17 cytokines. Curr Rheumatol Rep. 2007;9:461–467
- Increase in TNF-alpha and inducible nitric oxide synthase-expressing dendritic cells in psoriasis and reduction with efalizumab (anti-CD11a). Proc Natl Acad Sci USA. 2005;102:19057–19062
- Contribution of IL-18 to atopic-dermatitis-like skin inflammation induced by Staphylococcus aureus product in mice. Proc Natl Acad Sci USA. 2006;103:8816–8821
- . Pathogenesis and therapy of psoriasis. Nature. 2007;445:866–873
- . A functional promoter polymorphism in monocyte chemoattractant protein-1 is associated with psoriasis. Int J Immunogenet. 2008;35:45–49
- . Interleukin-18 levels in the plasma of psoriatic patients correlate with the extent of skin lesions and the PASI score. Acta Derm Venereol. 2003;83:262–265
- . Tacrolimus decreases the expression of eotaxin, CCR3, RANTES and interleukin-5 in atopic dermatitis. Br J Dermatol. 2005;152:1173–1181
- Inhibition of keratinocyte apoptosis by IL-15: a new parameter in the pathogenesis of psoriasis?. J Immunol. 2000;165:2240–2250
- Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis. J Exp Med. 1994;180:1141–1146
- . Transgenic delivery of VEGF to mouse skin leads to an inflammatory condition resembling human psoriasis. Blood. 2003;102:161–168
- . Th17 cells in human disease. Immunol Rev. 2008;223:87–113
- Psoriasis vulgaris lesions contain discrete populations of Th1 and Th17 T cells. J Invest Dermatol. 2008;128:1207–1211
PII: S0923-1811(10)00127-1
doi:10.1016/j.jdermsci.2010.01.006
© 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Inc. All rights reserved.
Refers to article:
- Introduction to Erratum , 12 July 2010
- Neutrophil-dominant psoriasis-like skin inflammation induced by epidermal-specific expression of Raf in mice , 12 July 2010
