The Influence of Birch Bark Triterpenes on. Keratinocytes and Fibroblasts from Diabetic and

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1 The Influence of Birch Bark Triterpenes on Keratinocytes and Fibroblasts from Diabetic and Nondiabetic Donors 4 5 Tina Wardecki,#, Philipp Werner,#, Maria Thomas, Markus F. Templin, Gudula Schmidt, Johanna M. Brandner, Irmgard Merfort, * Pharmaceutical Biology and Biotechnology, Albert-Ludwigs-University Freiburg, Freiburg, Germany Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, and University of Tübingen, Tübingen, Germany Institute of Natural and Medical Sciences at the University of Tübingen, Reutlingen, Germany Institute for Experimental and Clinical Pharmacology and Toxicology, Albert-Ludwigs- University Freiburg, Freiburg, Germany Department of Dermatology and Venerology, University Hospital Hamburg-Eppendorf, Hamburg, Germany # These authors contributed equally to this work. 18

2 2 19 ASSOCIATED CONTENT 20 Supporting information Fig. S1 Birch bark extract (TE) and betulin influence mrna expression of various genes in primary human keratinocytes (NHK-nd and NHK-d). The mrna expression in primary human keratinocytes derived from nondiabetic young and adult (NHK-nd) and diabetic adult (NHK-d) donors was measured after treatment with TE (1 or 5 µg/ml) or betulin (0.87 µg/ml = 1.96 µm or 4.35 µg/ml = 9.81 µm) for 6 h with the Fluidigm s Biomark high throughput qpcr chip platform. The results are expressed as fold change (= fold) in relation to the solvent control (DMSO 0.1%). Results from independent experiments (2-4) of two different donors per group are given and altogether 5-7 independent experiments were performed. mrna of the adhesion molecules ICAM-1 and VCAM-1 was measured by standard qrt-pcr from three independent experiments with cells of one donor. ± SD was calculated from the average of these experiments, respectively. The figure lists the genes where no gene was influenced statistically significant. Oneway ANOVA followed by Bonferroni s post-test was used for statistical analysis. The colors

3 indicate the extent of change in mrna expression in relation to the control. Blue: fold change < 0.3; light blue: fold change ; light red: fold change 1.3-4; red: fold change > 4. Abbreviations: NT: not tested; bdl: below detection limit Fig. S2 Birch bark extract (TE) and betulin influence mrna expression of various genes in fibroblasts (HDF). The mrna expression in primary human fibroblasts derived from a nondiabetic (HDF-nd) and a diabetic (HDF-d) donor was measured after treatment with TE (1 or 5 µg/ml) or betulin (0.87 µg/ml = 1.96 µm or 4.35 µg/ml = 9.81 µm) for 6 h with the Fluidigm s Biomark high throughput qpcr chip platform. The results are expressed as fold change in relation to the solvent control (DMSO 0.1%). Values represent means of four independent experiments ± SD. For each group (HDF-d, HDF-nd young and adult) cells from one donor were used. The figure lists the genes where no gene was influenced statistically significant. One-way ANOVA followed by Bonferroni s post-test was used for statistical analysis. The colors indicate the extent of change in mrna expression in relation to the control. Blue: fold change < 0.3; light blue: fold change ; light red: fold change 1.3-4; red: fold change > 4.

4 Fig. S3 Differences in baseline mrna expression in keratinocytes (NHK) and fibroblasts (HDF) isolated from nondiabetic (nd) and diabetic (d) as well as young and old donors. The baseline mrna expression of 48 different genes in primary human keratinocytes derived from nondiabetic and diabetic donors (A) as well as young and old donors (B) was compared. Results from independent experiments (2-4) with cells from two different donors per group are given. Differences in baseline expression of human dermal fibroblasts isolated from a nondiabetic and a diabetic (C) as well as a young and an old donor (D) were analyzed. Significant differences are listed. The results are expressed as normalized and averaged 2 -ΔCT values that were put in relation to each other. Statistical analysis was determined using Student s t-test. * p < 0.05; ** p < 0.01; *** p <

5 Fig. S4 Influence of betulin on the production of different mediators important for wound healing in human fibroblasts and keratinocytes. Keratinocytes (A) and fibroblasts (B) from nondiabetic and diabetic origin were treated with betulin (0.87 µg/ml = 1.96 µm) or DMSO (0.1%) for 24 h. Protein levels of the mediators were determined in the supernatant using a bead-based suspension array technology (xmap, Luminex Corp., Austin, TX, USA). Values represent means of three independent experiments + SD. # One value was below the detection limit and was substituted by the value for the detection limit. Data are not significant, but often show a trend.

6 Fig. S5 Shape change of the actin cytoskeleton of fibroblasts. Fluorescence microscopy pictures of the actin cytoskeleton of fibroblasts from nondiabetic donors (HDF-nd) (A) and from diabetic donors (HDF-d) (B) after treatment with triterpenes betulinic acid or oleanolic acid for 2 h. Fibroblasts with additional treatment of p38-mapk inhibitor LN 950 are shown in (C) for HDFnd and in (D) for HDF-d. The inhibitor was added 30 minutes before treatment with the test compounds. Representative pictures are shown.

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8 Fig. S6 Shape change of the actin cytoskeleton of keratinocytes. Fluorescence microscopy pictures of the actin cytoskeleton of a young nondiabetic donor (NHK-nd) (A) and an adult nondiabetic donor (NHK-nd) (B) after treatment with the positive control CNF1 (150 ng/ml), TE (5.1 ng/ml), betulin (10 nm) or lupeol (10 nm) for 2 h. For inhibition of p38-mapk, inhibitor LN 950 (100 nm) was used 30 min before adding the test compounds. Representative pictures are shown. 82

9 S1 Protocol. This section contains all experimental details which are not shown in the experimental section of the paper Isolation and cultivation of the primary human keratinocytes and fibroblasts The keratinocytes NHK and NHK were derived from foreskin that was obtained following medical circumcisions. Briefly, the foreskin explant was washed with PBS containing 8% Antibiotic-Antimycotic and 1% Ciprobay 400 mg solution for infusion and the dermis was removed gently. The skin was cut into small pieces and incubated with trypsin/edta (0.05%/0.02%) for 1 h. The pieces were transferred into PBS containing 10% FCS and 1% Ciprobay and the epidermis was scraped with a scalpel to separate the normal human keratinocytes (NHKs). The cell suspension was centrifuged at 4 C and the cells were cultivated in Keratinocyte SFM supplemented with human recombinant EGF (rhegf), bovine pituitary extract (BPE) and penicillin-streptomycin 1%. Information on the isolation of the other NHKs and HDFs is provided in Pollok et al Primary keratinocytes (NHK-nd and NHK-d) and fibroblasts (HDF-nd and HDF-d) were grown in monolayer cultures. The cells were kept in a humidified environment with 5% CO2 at 37 C. NHK were cultivated in Keratinocyte SFM supplemented with BPE, rhegf and penicillinstreptomycin 1%. When NHK were starved medium without recombinant EGF and bovine pituitary extract (BPE) was used. HDF were cultured in DMEM (high glucose, 4.5 g/l) supplemented with 10% FCS and 1% penicillin-streptomycin. The glucose content in the medium of NHK was 5.5 mm, that one of HDF 25 mm. The cells were cultivated as mentioned above and split when they reached a confluency of 80%. Therefore, cells were washed with PBS, brought in

10 suspension with trypsin/edta 0.05%/0.02% and were centrifuged at 1200 rpm and 4 C for 5 min. The cells were resuspended in the corresponding cell culture media and seeded into tissue culture flasks or wells at the desired concentrations. NHK were maximally used up to passage 5 and HDF up to passage 8. For RNA analysis or Luminex assay, the cells were serum starved by using medium without FCS or supplements 30 h prior to cell harvest qrt-pcr RNA was amplified in a total reaction volume of 25 µl consisting of 6 µl of cdna (corresponding to 60 ng of total RNA input), 2x conc. KAPA PROBE FAST Master Mix, 50 nm primer and 100 nm probe for the reference gene and 300 nm primers and 100 nm probe for the gene of interest. The 18S rrna was used as reference. For relative quantification ΔCT values of treated cells were referred to untreated control cells resulting in a ΔΔCT value. The fold change was calculated as 2 - ΔΔCT Staining of the actin cytoskeleton The actin cytoskeleton was analyzed by fluorescence microscopy after staining with rhodamine phalloidin. For this purpose cells per sample were seeded on 10 mm glass coverslips in 24 well plates. For fibroblasts, the coverslips were coated with type I collagen for 2 h before plating. Keratinocytes were starved for 2 d prior to the experiment to reduce basal stimulation of the cells. After treatment with TE, the triterpenes or the positive control CNF1 (cytotoxic necrotizing factor 1 of Escherichia coli) for 2 h, cells were fixed with 400 µl of 3.7% formaldehyde and

11 permeabilized with 0.1% triton-x 100 for 10 min. Subsequently, the cells were washed in PBS, stained for 1 h with 1 µl rhodamine phalloidin (0.13 Units) in 24 µl PBS, and then washed again in PBS. Thereafter coverslips were fixed with 3.5 µl Prolong Gold Antifade reagent on microscope slides. The following day pictures were taken with an Axiovert fluorescence microscope (Zeiss)

12 fibroblasts keratinocytes Table A: Origin of the used human primary keratinocytes and fibroblasts Diabetic Classification Internal Name Age Gender Biopsy site status NHK nondiabetic <15 male no DM foreskin (FR) young (NHKnd) <15 male no DM foreskin NHK (FR) NHK nondiabetic 50 male no DM inguinal (HF) adult (NHK-nd) K80 (HH) 89 unknown no DM unknown diabetic adult (NHK-d) nondiabetic young (HDFnd) nondiabetic adult (HDF-nd) diabetic adult (HDF-d) Abbreviations: K787 (HH) 45 male DM type 2 unknown K705 (HH) unknown unknown DM type 2 unknown K758 (HH) 96 unknown DM type 2 unknown F223 (HH) <15 male no DM foreskin F188 (HH) <15 male no DM foreskin F-NHF (HF) 40 female no DM inguinal F31 (HH) 37 unknown no DM unknown F306 (HH) 42 unknown DM type 2 unknown HH = University Hospital Hamburg-Eppendorf; FR = keratinocytes from foreskins obtained from the Department of Dermatology, Medical Center, University of Freiburg, Prof. Dr. L. Bruckner-Tuderman; HF = a gift from the Department of Dermatology, Medical Center, University of Freiburg, Prof. Dr. L. Bruckner-Tuderman; NHK-nd: keratinocytes derived from nondiabetic donors; NHK-d: keratinocytes derived from diabetic donors; HDF-nd: fibroblasts from nondiabetic donors; HDF-d: fibroblasts from diabetic donors. 140

13 Table B. Genes analyzed on the Fluidigm s Biomark high throughput qpcr chip platform and their respective AB Assay Number Gene Gene name Protein AB Assay Number CCL2 chemokine (C-C motif) ligand 2 MCP-1 Hs _m1 ACTB actin, beta Actin-β Hs _g1 AQP3 aquaporin 3 (Gill blood group) Aquaporin 3 Hs _g1 CCL4 chemokine (C-C motif) ligand 4 MIP-1β Hs _m1 CCL5 chemokine (C-C motif) ligand 5 RANTES/CCL5 Hs _m1 CCR3 chemokine (C-C Motif) receptor 3 CCR3 Hs _s1 CLDN1 claudin 1 Claudin 1 Hs _m1 CLDN4 claudin 4 Claudin 4 Hs _s1 COL1A1 collagen, type I, alpha 1 Collagen 1 Hs _m1 COL5A2 collagen, type V, alpha 2 Collagen 5 Hs _m1 CRP C-reactive protein, pentraxin-related CRP Hs _g1 CSF3 colony stimulating factor 3 (Granulocyte) G-CSF Hs _g1 CXCL10 chemokine (C-X-C motif) receptor 10 IP-10 Hs _g1 DEFB103B defensin, beta 103B Beta-Defensin 3 Hs _g1 ELN elastin Elastin Hs _m1 FBN1 fibrillin 1 Fibrillin 1 Hs _m1 FGF2 fibroblast growth factor 2 (basic) FGF2 Hs _m1 FN1 fibronectin 1 Fibronectin Hs _m1 FOS FBJ murine osteosarcoma viral oncogene homolog c-fos Hs _s1 GAPDH glyceraldehyde-3-phosphate dehydrogenase GAPDH Hs _g1 GDF15 growth differentiation factor 15 GDF15 Hs _m1 GJA1 gap junction protein, alpha 1, 43 kda Connexin 43 Hs _s1 GJB2 gap junction protein, beta 2, 26 kda Connexin 26 Hs _s1 IFNG interferon, gamma IFNγ Hs _m1 IGF1 insulin-like growth factor 1 IGF-1 Hs _m1 IL10 interleukin 10 IL-10 Hs _m1 IL1B interleukin 1, beta IL-1β Hs _m1 IL1R1 interleukin 1 receptor, type I IL1R1 Hs _m1 IL2 interleukin 2 IL-2 Hs _m1 IL6 interleukin 6 IL-6 Hs _m1 IL8 interleukin 8 IL-8 Hs _m1 JUN jun proto-oncogene c-jun Hs _s1 MMP1 matrix metallopeptidase 1 (interstitial collagenase) MMP-1 Hs _m1 MMP2 matrix metallopeptidase 2 MMP-2 Hs _m1 MMP9 matrix metallopeptidase 9 MMP-9 Hs _m1 NFE2L2 nuclear factor, erythroid 2-like 2 Nrf2 Hs _g1 NFKBIA nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha IκBα Hs _g1

14 14 Gene Gene name Protein AB Assay Number 143 NOS2 nitric oxide synthase 2, inducible NOS2 Hs _m1 PDGFB platelet-derived growth factor beta polypeptide PDGFB Hs _m1 PTGS2 prostaglandin-endoperoxide synthase 2 Cox-2 Hs _m1 SOCS3 suppressor of cytokine signaling 3 SOCS-3 Hs _s1 SOD2 superoxide dismutase 2, mitochondrial SOD2 Hs _m1 TGFB1 transforming growth factor, beta TGF-β1 Hs _m1 TIMP1 TIMP metallopeptidase inhibitor 1 TIMP-1 Hs _m1 TIMP2 TIMP metallopeptidase inhibitor 2 TIMP-2 Hs _m1 TNF tumor necrosis factor TNFα Hs _g1 VEGFA vascular endothelial growth factor A VEGF-A Hs _m1 WDR26 WD repeat domain 26 MIP-2 Hs _m1

15 PCR with the LightCycler Gene Species Protein Sequence (5-3 ) Table C. Studied genes and the nucleotide sequence of the used primers and probes for qrt- RNA18S5 human 18S rrna Sense 5 CGGCTACCACATCCAAGG 3 Antisense 5 CGGGTCGGGAGTGGGT 3 Probe 5 -[HEX] TTGCGCGCCTGCTGCCT-[TAM] 3 VCAM-1 human VCAM-1 Sense 5 -GAGAGTGTCAAAGAAGGA-3 Antisense 5 -GCGCCATCTATAGATTTTAG-3 Probe 5 -[6-FAM]-CTGTGTCTCCTGTCTCCGCTT- [TAM]-3 ICAM-1 human ICAM-1 Sense 5 -GAGTGCCCAGGGAATATG-3 Antisense 5 -GAGGGAGTCCTCCAATAC-3 Probe 5 -[6-FAM]-ATGCCTTGTCCTCTTGTCCTGT- [TAM]-3

16 Table D. Proteins analyzed on a Luminex 100 Total System Classification Protein Annotation Protein name adhesion molecules soluble receptors chemokines pro-inflammatory mediators antiinflammatory mediators miscellaneous se-selectin 2 Soluble E-Selectin svcam-1 Soluble vascular adhesion molecule 1 sicam-1 Soluble intercellular adhesion molecule 1 srage 1.2 Soluble receptor for advanced glycation end products IL-6sR Interleukin-6 soluble receptor sgp130 Soluble gp130 stnf-r1 Soluble tumor necrosis factor receptor 1 stnf-r2 1 Soluble tumor necrosis factor receptor 2 GRO-α Chemokine (C-X-C motif) ligand 1 (CXCL1) IL-8 Interleukin-8 MCP-1 Monocyte chemotactic protein 1 MIP-1α 2 Macrophage inflammatory protein 1 alpha MIP-1β 1.2 Macrophage inflammatory protein 1 beta RANTES Regulated on activation, normal T cell expressed and secreted IFNγ 1.2 Interferon gamma IL-1β 1.2 Interleukin-1 beta IL Interleukin-2 IL-12p Interleukin-12p40 subunit IL-17A 1.2 Interleukin-17A MIF Macrophage migration inhibitory factor PLA2G7 1.2 Phospholipase A2 group VII scd40l 1.2 Soluble CD40 ligand TNFα 2 Tumor necrosis factors alpha IL Interleukin-4 IL Interleukin-10 IL-1RA Interleukin-1 receptor antagonist FGF Fibroblast growth factor 23 GM-CSF 2 Granulocyte-macrophage colony-stimulating factor Leptin 1.2 Leptin Osteocalcin 1.2 Osteocalcin Osteopontin 1.2 Osteopontin sfas Soluble Fas The proteins that were below the detection limit are marked with the numbers 1 (keratinocytes) and 2 (fibroblasts)

17 References S1 Protocol (1) Pollok, S.; Pfeiffer, A.-C.; Lobmann, R.; Wright, C. S.; Moll, I.; Martin, P. E. M.; Brandner, J. M. J. Cell. Mol. Med. 2011, 15 (4),