The aim of the present study was to investigate the effect of receptor-interacting protein 4 (RIP4) on keratinocyte proliferation and its role in the pathogenesis of psoriasis vulgaris. The expression of RIP4 and Ki-67 in fixed sections from 30 patients with psoriasis vulgaris and 30 gender- and age-matched healthy controls was detected by two-step immunohistochemistry, prior to the correlation being examined with Pearson's analysis. Reverse transcription-semi-quantitative polymerase chain reaction and western blot analyses were carried out to detect the mRNA and protein expression of RIP4 in an immortalized human keratinocyte line, HaCaT, stimulated by different concentrations of interleukin-17 (IL-17), in order to analyze the change in RIP4 expression following IL-17 stimulation. The cell proliferation rate was measured using the cell counting kit-8 assay simultaneously. RIP4 was mainly present in the cytoplasm of the keratinocytes. Compared with its expression in the healthy control skin, RIP4 exhibited a significant upregulation in the psoriatic lesions (P<0.05). Pearson's correlation analysis revealed a positive correlation between the expression level of RIP4 and the proliferation index. Both RIP4 mRNA and protein levels were significantly increased following IL-17 stimulation. Exposure to IL-17 additionally increased the proliferation rate of the HaCaT cells. In conclusion, RIP4 may play a role in the pathogenesis of psoriasis vulgaris as a potential target of IL-17.
Psoriasis vulgaris is a common chronic inflammatory skin disease marked by the hyperproliferation of keratinocytes and infiltration of inflammatory cells. Although its pathogenesis has not been not fully elucidated, the condition is generally recognized as a polygenic disease resulting from a dysregulated interplay between hereditary and environmental factors, which gives rise to the hyperproliferative epidermis and altered differentiation through an immune-mediated common pathway. Immune dysfunction plays a vital role in the pathogenic progression of psoriasis vulgaris (
Receptor-interacting protein 4 (RIP4), as a novel member of the RIP kinase family, activates mitogen-activated protein kinases (MAPKs) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) during the proliferation, differentiation, apoptosis, inflammation and immune response of keratinocytes (
Thirty psoriatic biopsies were obtained from 15 male and 15 female patients (mean age, 27.96±8.64 years; range, 19–52 years) who had been diagnosed with psoriasis vulgaris in Qilu Hospital (Jinan, China) and who had not received any local or systemic treatment since the latest attack of the disease. Non-lesion biopsies, acquired from 30 age- and gender-matched healthy individuals, served as controls. All participants were negative for systemic disease and gave their informed consent prior to biopsy. The study received approval from the Ethics Committee of Qilu Hospital (no. QL2009019). The specimens were fixed in 10% neutral formaldehyde immediately subsequent to their collection.
Paraffin-embedded 10% neutral formaldehyde-fixed tissues were sectioned continuously at 3 µm, deparaffinized with xylene and rehydrated in a graded alcohol series. For antigen retrieval, the deparaffinized sections in 1 mM EDTA buffer (pH=9.0) were heated at 120°C for 5 min and cooled slowly to room temperature. In order to prevent nonspecific background staining, the slices were blocked with goat serum following the quenching of endogenous peroxidase activity with 3% H2O2 solution. Polyclonal rabbit anti-human RIP4 antibody (cat. no. ZA-0502; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), at a dilution of 1:100, and a ready-to-use anti-Ki-67 monoclonal antibody (ZSGB-Bio, Beijing, China) were applied to the sections at 37°C for 1 h, prior to the use of the PV-6000 polymer detection system (ZSGB-Bio) according to the manufacturer's instructions. 3,3′-Diaminobenzidine (ZSGB-Bio) was applied to visualize the antibody under a microscope, following which the sections were counterstained with Harris's hematoxylin. The slices were mounted with neural gum and images were captured using the ToupCam microscope camera system (Hangzhou ToupTek Photonics Co., Ltd., Hangzhou, China).
Cells were considered to be RIP4- and Ki-67-positive when yellow to brown granules appeared in the cytoplasm or nucleus of the keratinocytes, respectively. The proliferation index was calculated as a percentage by dividing the number of Ki-67-positive cells by the total number of epidermal cells (basal and suprabasal). Five high-power fields (x400 magnification) were counted in each sample.
Immortalized human keratinocyte HaCaT cells were obtained commercially from the American Type Culture Collection (Manassas, VA, USA) and frozen in our lab. The HaCaT cells were thawed using a routine method and grown in Dulbecco's modified Eagle's medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% (vol/vol) fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. For the subcultures, the cells were disaggregated with 0.25% trypsin/0.02% EDTA (1:1) solution and split at a ratio of 1:2 every 2 to 3 days, prior to inoculation into 12-well plates at a concentration of 1×105 cells/ml. HaCaT cells at 60–70% confluence were stimulated with IL-17 (50, 70 and 90 ng/ml; ProSpec-Tany TechnoGene, Ltd., Rehovot, Israel) for RNA isolation or western blotting. The control group cells were processed in parallel using Dulbecco's modified Eagle's medium without IL-17.
The CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was adopted to evaluate cell viability. Cells (5×103/ml) in the logarithmic phase were inoculated in 96-well plates with 100 µl normal growth medium containing 10% fetal calf serum. After 24 h, the medium was changed to serum-free medium. When the cell reached ~70% confluence, various doses of IL-17 (50, 70 and 90 ng/ml) in serum-free medium were added. A total of 10 µl CCK-8 was added to each well after 12, 24, 36 and 48 h incubation with IL-17, prior to further incubation for 2 h at 37°C. A microplate reader (Varioskan™ Flash; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was applied to measure the optical density (OD) of each well at 450 nm. The cell viability (percentage of control) is expressed as the percentage of (ODtest-ODblank)/(ODcontrol-ODblank).
Total RNA was isolated from the cells 48 h after stimulation with IL-17 using RNAiso Plus [Takara Biotechnology (Dalian) Co., Ltd., Dalian, China] in accordance with the manufacturer's instructions and quantified spectrophotometrically at 260 nm. A total of 1 µg RNA was converted in cDNA using oligo (dT) primers and then amplified with a Power RT kit (BioTeke, Beijing, China). The PCR was performed in a total reaction volume of 50 µl: 25 µl 2X PCR Mix (BioTeke), 2.5 µl forward and reverse primer, 10 µl templates and 10 µl sterile H2O. The mixture underwent predenaturation for 5 min at 95°C and was then subjected to 35 cycles of 95°C for 1 min, 60°C for 45 sec and 72°C for 45 sec, and finally 72°C for 10 min. The specific primer sets (
HaCaT cells were dissolved in lysis buffer (cell lysis buffer for western and immunoprecipitation; Beyotime Institute of Biotechnology, Haimen, China) containing 1 mM phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology). The mixture was centrifuged for 5 min at 12,000 × g and the bicinchoninic acid (BCA) assay method (Enhanced BCA Protein Assay kit; Beyotime Institute of Biotechnology) was utilized to quantify the total protein in the supernatant. A total of 30 µg protein from the cell lysate was solubilized in loading buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% acrylamide gel, prior to being electrotransferred onto polyvinylidene difluoride (PVDF) membranes. Following blocking with 5% skimmed milk at room temperature for 1 h, the PVDF membranes were incubated overnight at 4°C with polyclonal rabbit anti-human RIP4 antibody at a dilution of 1:400 (sc-83320; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China) or with mouse anti-human anti-β-actin antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:1,000. The membranes were then thoroughly washed with 0.01 M Tris-buffered saline containing 0.1% Tween 20 and incubated with horseradish peroxidase-labeled goat-anti-rabbit or goat-anti-mouse immunoglobulin G (heavy- and light-chain) antibody (EarthOx Life Sciences, Millbrae, CA, USA) at a dilution of 1:1,000 at room temperature for 1 h. A standard enhanced chemiluminescence (Shanghai Sangon Biotechnology Co., Ltd.) reaction was performed according to the manufacturer's instructions, and densitometric analysis of the band intensity was conducted using a chemiluminescence imaging analysis system (Tanon 5000; Tanon Science & Technology Co., Ltd.). Relative expression was measured by dividing the gray value of RIP4 by that of β-actin. High gray values represented low expression.
All statistical analysis was carried out using the SPSS 17.0 software package (SPSS, Inc., Chicago, IL, USA), and data are presented as the mean ± standard error of the mean. The Student's t-test was performed for the comparison of the results, and the correlation between RIP4 expression and the proliferation index was examined using Pearson's correlation analysis.
In healthy skin, RIP4 was found predominantly in the basal layer of the epidermis; by contrast, the expression in the psoriatic lesions was concentrated in the basal and spinous layers, in the cytoplasm and nuclei of the cells. Analysis of the OD showed the mean OD (MOD) of RIP4 in the healthy controls to be 0.11±0.03, compared with 0.30±0.05 in the psoriasis vulgaris lesions. This revealed a statistically significant upregulation in the psoriatic lesions (t=7.71, P<0.01) (
In the healthy control skin, Ki-67-positive cells only appeared sporadically in the basal layer. By contrast, Ki-67-positive cells in the psoriatic specimens were distributed in not only the basal layer but also the middle and lower parts of the rete ridges of the lesional epidermis. The proliferation index of the psoriatic lesions was 20.01±7.90%, while that of the healthy control skin was 10.67±4.92%; this suggested that the Ki-67 expression in the psoriatic lesions was significantly higher than that in the healthy control skin (t=4.535, P<0.05) (
To further investigate the role of RIP4 in psoriasis, an immortalized line of human epidermal keratinocytes, HaCaT, which has been extensively adopted as an
RT-semi-quantitative PCR and western blot analyses were applied to study the change in RIP4 mRNA and protein expression, respectively, following exposure to IL-17. The RIP4 mRNA and protein were isolated 48 h after the stimulation of the cells with 50, 70 and 90 ng/ml IL-17 and subjected to RT-semi-quantitative PCR or western blot analyses with β-actin as a housekeeping control. Compared with the control group, it was found that IL-17 dose-dependently induced a significant upregulation of RIP4 expression at both the mRNA (t50=5.091, P50=0.036; t70=7.022, P70=0.020; t90=18.621, P90=0.003) and protein (t50=9.076, P50=0.012; t70=6.600, P70=0.022; t90=6.650, P90=0.022) levels (
Psoriasis vulgaris is a common chronic skin disease in which a thicker epidermis, excessive keratinocyte proliferation and parakeratosis, accompanied by extensive inflammatory cell infiltration, can be observed (
The RIP kinase family comprises a group of specific serine/threonine kinases that are crucial mediators of multiple signal transduction processes, which lead to the activation of NF-κB and MAPKs (
RIP4 activates NF-κB by inducing inhibitor of κB phosphorylation, which requires an active kinase domain. Similar to NF-κB activation, RIP4-mediated JNK signal initiation depends on the presence of the kinase domain (
IL-17 is one of the most important inflammatory factors in psoriasis. In the present study, the elevated proliferation rate of HaCaT cells and the higher levels of RIP4 expression were shown to be enhanced by stimulation with an increasing concentration of IL-17. Furthermore, the RIP4 protein level, which was shown to be positively correlated with the proliferation index, was notably upregulated in the psoriatic lesions compared with the healthy control skin. It could thus be inferred that RIP4 is a possible downstream molecule of IL-17.
Interacting with IL-17R on the surface of keratinocytes, IL-17 produces proinflammatory and proliferation-regulatory effects by means of activating NF-κB and the JNK signaling pathway, in which RIP4 is directly involved (
This study was supported by the 2010 National Science Foundation (grant no. 81071291).
Receptor-interacting protein 4 expression in (A) psoriatic lesions and (B) healthy controls. Magnification, x200.
Difference in RIP4 expression between the psoriatic lesions (group 1) and healthy controls (group 2) (t=7.71, P<0.01). Data are presented as the mean ± standard error of the mean. RIP4, receptor-interacting protein 4; MOD, mean optical density.
Difference in Ki-67 expression (used to calculate PI) between the psoriatic lesions (group 1) and healthy controls (group 2) (t=4.535, P<0.05). Data are presented as the mean ± standard error of the mean. PI, proliferation index.
Correlation of RIP4 expression and the PI (r=0.69, P<0.01). RIP4, receptor-interacting protein 4; MOD, mean optical density; PI, proliferation index.
Proliferation index of HaCaT cells following stimulation with different concentration of IL-17. Data are presented as the mean ± standard error of the mean. IL-17, interleukin-17.
Reverse transcription-semi-quantitative polymerase chain reaction analysis of RIP4 mRNA following IL-17 stimulation. RIP4, receptor-interacting protein 4; IL-17, interleukin-17.
Difference in receptor-interacting protein 4 mRNA levels following IL-17 stimulation: t50=5.091, P50=0.036; t70=7.022, P70=0.020; t90=18.621, P90=0.003. Data are presented as the mean ± standard error of the mean. IL-17, interleukin-17.
Western blot analysis of RIP4 protein following IL-17 stimulation. RIP4, receptor-interacting protein 4; IL-17, interleukin-17.
Difference in receptor-interacting protein 4 protein levels following IL-17 stimulation: t50=9.076, P50=0.012; t70=6.600, P70=0.022; t90=6.650, P90=0.022. Data are presented as the mean ± standard error of the mean. IL-17, interleukin-17.
Primer sequences.
Gene | Primer sequence |
---|---|
RIP4 | Upstream: 5′-TGTTAGGTGATTTGGGATAGG-3′ |
Downstream: 5′-AAAGGCACAATGAGGCATA-3′ | |
β-actin | Upstream: 5′-CATTTGCTGCATGGGTTA-3′ |
Downstream: 5′-TCCTACGGCTTGGACTTT-3′ | |
RIP4, receptor-interacting protein 4. |