to 65 years old, including 5 males and 5 females. Patients did not receive any treatment 4

Transcription

1 MATERIALS AND METHODS Human Skin Tissues For immunofluorescence analysis, the skin samples of psoriatic lesions were obtained from 10 adult outpatients at Jiangsu Province Hospital diagnosed with vulgaris psoriasis, aged 18 to 65 years old, including 5 males and 5 females. Patients did not receive any treatment 4 weeks before the biopsy. The normal skin tissues were obtained from 10 age- and sex-matched healthy adults undergoing plastic surgery. For cell purification, total protein extraction for co-immunoprecipitation, or double fluorescent staining, skin samples were obtained from 5 healthy adults undergoing plastic surgery. The study was approved by the Ethical Committee of Jiangsu Province Hospital, China. Biopsies were used after the subjects provided their informed consent. Primary Human Keratinocytes Culture Human skin samples were kept on ice in PBS and treated within 2 hours after collection. The skin was pinned, the very thin epidermis layers were detached using a surgical knife. The epidermis tissues were cut into strips of 2 2 mm, and put into a dish containing 5 ml of α-mem medium (Gibco-Invitrogen, Carlsbad, USA) supplemented with 10% FCS, 2 mg/ml Collagenase P (Roche, Mannheim, German), and 1% penicillin / streptomycin (Gibco-Invitrogen). These were incubated for 4 hours at 37. The suspension of digested tissues was then filtered through a 70-mm cell strainer (BD, Franklin Lakes, USA). The cell suspension was washed twice in PBS and centrifuged. Cells were seeded into cell culture flasks containing EpiLife serum-free keratinocyte medium containing growth supplement (5% HKGS) and 1% penicillin / streptomycin (Gibco-Invitrogen). Cells at passage 3 were 1

2 identified as keratinocytes using immunohistochemistry analysis with anti-cytokeratin antibody (abcam, UK). HaCaT Cells Culture The human keratinocyte cell line HaCaT cells obtained from American Type Culture Collection were cultured in Dulbecco s Modified Eagle Medium (DMEM, Gibco-Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum and antibiotics (Invitrogen). Cell Treatment in vitro The primary keratinocytes at passage 4 or HaCaT cells were pretreated with SIRT1 activator SRT1720 (1 μm), or resveratrol (25 μm) for 4 hours, or SIRT1 inhibitor EX527 (50 μm) for 6 hours (Selleck Chemical, Houston, USA) at 60% confluence, before stimulated with recombinant human IL-6 (50 ng/ml), or IL-22 (50 ng/ml) (PeproTech, Rocky Hill, USA). HaCaT cells were transfected with SIRT1 sirna or over-expression plasmid, or STAT3 sirna, and incubated for 48 hours before cytokines stimulation. The cultured HaCat cells were transfected with sirnas and GV141-SIRT1 plasmids by a lipofection technique using Lipofectamine 2000 (Invitrogen) for 48 hours before the stimulation with cytokines. Short interfering sirna to knock down SIRT1 was synthesized by Genechem, Shanghai, China. The target sequence of SIRT1-siRNA is GGCTTGATGGTAATCAGTA, and the scrambled sirna sequence is GGATTAGGCGTAGCTATTA. To obtain the recombinant SIRT1 over-expression plasmid, the full-length cdna of human SIRT1 (NM_012238) gene was PCR-amplified using the following primers: 5 -ACGGGCCCTCTAGACTCGAGATGGCGGACGAGGCGGCCCTC-3 and 5 -AGTCACTTAAGCTTGGTACCGATGATTTGTTTGATGGATAGTTCATG-3, after 2

3 digestion with restriction enzymes XhoI and KpnI, SIRT1 gene were cloned into the Mammalian Expression Vector GV141 (GENECHEM). The final construct, named GV141-SIRT1, containing a CMV promoter and the neomycin resistance gene as a selection marker, was confirmed by DNA sequencing. GV141 plasmid was used as a negative control. An antisense ologonucleotide of STAT3 (HSS186131, Invitrogen, Carlsbad, USA) was used to silence STAT3. Stealth RNAi negative control was ordered from Invitrogen ( ). STAT3, PY-STAT3, Ac-STAT3 were detected by Western blot after 30-minute stimulation of cytokines. Cyclin D was detected after 24-hour stimulation, and Keratin 17 was detected after 48-hour stimulation. The values for the western analysis were normalized to GAPDH. Values were confirmed from at least three independent experiments. Then the relative quantitation in each group was expressed as fold induction of its value vs. that of the PBS group, and expressed as Mean±SEM. Cell Proliferation Assay HaCaT cells ( ) were seeded in 96-well plates, and were treated with SIRT1 regulators for 4 to 6 hours, the day after. SIRT1 silence or overexpression of HaCaT cells was also administrated by transfected with SIRT1 sirna or plasmid. Then, cell proliferation assay was performed following 24 hours stimulation of cytokines. The number of viable HaCaT cells in proliferation was determined using a commercially available kit (CellTiter 96 AQ One Solution Cell Proliferation Assay, Promega, Madison, USA). The CellTiter 96 AQ ueous One Solution Reagent contains a novel tetrazolium compound [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H -tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). 3

4 The MTS tetrazolium compound is bioreduced by cells into a colored formazan product that is soluble in tissue culture medium. This conversion is presumably accomplished by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. Assay was performed by adding a small amount of the reagent directly to culture wells, incubating for 4 hours and then recording the absorbance at 490nm. The quantity of formazan product as measured by the absorbance at 490nm is directly proportional to the number of living cells in culture. The absorbance of the untreated group was given as 1, than the intensity of cell proliferation in each group was expressed as fold induction of its absorbance vs. that of the untreated group (Mean±SEM). There were 3 replications in each group, and the experiment was repeated for 3 times. Animals and Treatment Female BALB/c mice were purchased from Model Animal Research Center of Nanjing University. Mice at 8 to 11 weeks of age were used. For induction, mice received a daily dose of 62.5 mg of Aldara cream (5% IMQ) (3M Pharmaceuticals, St. Paul, USA) on their shaved back for six consecutive days. The control group was treated similarly with a control Vaseline cream (van der Fits et al., 2009). To investigate the effect of SIRT1, mice were randomly divided into 5 groups (5 mice per group): resveratrol (50 mg per kg body weight, i.p.)+ Aldara group, EX527 (10 mg per kg body weight, i.p.)+ Aldara group, DMSO (1% DMSO, i.p.) + Aldara group as a mock control, Aldara group, and control group treated with Vaseline cream. The resveratrol and EX527 were administered every other day for 5 times starting at four days before the IMQ application. The severity of inflammation was scored based on the clinical Psoriasis Area and Severity Index (PASI) (van der Fits et al., 2009). 4

5 Paraffin sections of the mouse skins (n=5) were blocked with 5% bovine serum albumin, and relevant protein expression were analyzed by immunofluorescence and immunohistochemistry. The extracts of lesion skin epithelia in each group (n=5) were measured for the relevant protein expression by western analysis. The values were normalized to GAPDH. The relative quantitation in each group was expressed as fold induction of its value vs. that of control group. For co-immunoprecipitation of STAT3 and SIRT1, the extracts of lesion skin epithelia in Aldara group (n=5) and those of normal skin epithelia in control group (n=5) was analyzed. All animal studies were conducted with prior approval from the Nanjing University Animal Care and Use Committee and in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institute of Health (NIH). Antibodies The primary antibodies for immunofluorescence and immunohistochemistry analysis were Phospho-STAT3 (Tyr705) mab (Cell Signaling Technology, USA), SIRT1 mab, Ki67 mab, Keratin 17 mab, and CD4 mab (Abcam, UK). The secondary antibodies used for immunofluorescence were Alexa Fluor 488-labeled or 594-labeled donkey anti rabbit IgG (Invitrogen). The primary antibodies for Western Blot or co-immunoprecipitation included: STAT3 mab, Phospho-STAT3 (Tyr705) mab, Acetyl-STAT3 (Lys685) pab (Cell Signaling Technology), SIRT1 mab, Keratin 17 mab, Cyclin D (Abcam), and GAPDH pab (Bioworld Technology, Co., Ltd., Nanjing, P.R. China). The secondary antibody was goat anti-rabbit IgG (H & L)-HRP (Bioworld Technology). 5

6 Statistics Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). The significance of the differences between cell proliferation intensity values was determined by Wilcoxon s signed rank test. The Mann-Whitney test was used for calculating the significant differences of the psoriasis severity scores, the ratio of the PY-STAT3 positive cells, or Ki-67 positive cells, and the relative mrna level of Keratin 17 between the groups. P-values < 0.05 represent significant differences. Supplementary Figure Figure S1. The keratinocytes in psoriatic lesion skin were undergoing hyperproliferation and abnormal differentiation. Ki-67 (red) and Keratin 17 (green) immunofluorescence stainings were performed in the biopsies of lesion skin from psoriatic patients and normal skin from healthy individuals (n=10). 6

7 Figure S2. The lesion skin of the mice treated with Aldara+DMSO (the vehicle control) showed no detectable differences on the pathologic features, when compared with the littermates treated with Aldara alone. (a) The psoriasiform phenotype was induced by Aldara. (b). The PASI score (means ± SD) indicated disease severity induced by Aldara. (c) Parakeratosis (arrows), acanthosis (asterisks), inflammatory infiltrates (black arrowheads) were revealed in H&E-stained mouse lesion skins ( 100). 7

8 Figure S3. SIRT1 counteracts psoriasiform pathology in Aldara induced mouse skin. (a) Immunohistochemistry for Keratin 17 (stained in brown) was performed on sections of formalin-fixed back skin biopsies from mice (magnification 100). (b) qpcr analysis of Keratin 17 expression of the mouse back in the four groups. Relative quantification is performed according to the ΔΔCT method, and results are expressed as fold of 2-ΔΔCT of the treated groups vs. the control group. *P < (c) Bars represent the ratio of Ki-67 + cells against total cells per microscopic field ( 200). *P < (d) Bars represent the ratio of PY-STAT3 + cells against total cells per microscopic field ( 200). *P < (e) SIRT1 was detected in complexes immunoprecipitated with anti-stat3 antibody. 8

9 Figure S4. SIRT1 counter-regulated cytokines induced STAT3 activation and STAT3-linked phenotypes of HaCaTs. (a) HaCaTs were stimulated with IL-22 for 30 minutes,following a pretreatment of SIRT1 regulators. (b) SIRT1 was detected in complexes immunoprecipitated with anti-stat3 antibody from the extracts of HaCaTs. (c, d) HaCaTs were treatment of SIRT1 regulators (c) or transfected with SIRT1 sirna or over-expression plasmid (d), before a 24 hours stimulation of IL-22. Cell proliferation was measured by MTS assay. *P < 0.05, Wilcoxon s signed rank test. (e) HaCaT cells were transfected with STAT3 sirna. PY-STAT3 (30 minutes treatment with IL-6) and Keratin 17 (48 hours treatment with IL-6) were analyzed by Western blot. The values for the western analysis were normalized to GAPDH. Then the relative quantitation in each group was expressed as fold induction of its value vs. that of PBS group. 9

10 Figure S5. Cultured primary human keratinocytes. (a) The morphology of cultured primary human keratinocytes was observed under phase - contrast microscope (magnification 100). (b) Cells at passage 3 were identified using immunohistochemistry analysis with anti-cytokeratin antibody. The cytoplasm is stained in brown (magnification 400). 10