1. Interleukin-6-Type Cytokines Upregulate Expression of Multidrug Resistance-Associated Proteins in NHEK and Dermal Fibroblasts 
Alexandra Dreuw, Heike M. Hermanns, Ruth Heise, Sylvia Joussen, Felipe Rodríguez, Yvonne Marquardt, Frank Jugert, Hans F. Merk, Peter C. Heinrich, Jens M. Baron 
Journal of Investigative Dermatology 
Volume 124, Issue 1, Pages (January 2005)
DOI: /j X x
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Real-time PCR analysis of multidrug resistance-associated proteins (MRP) expression in dermal fibroblasts in response to interleukin-6 (IL-6)/soluble human IL-6 receptor (sIL-6R) or oncostatin M (OSM). Primary human dermal fibroblasts were stimulated with 20 ng per mL IL-6 and 1 μg per mL sIL-6R or 20 ng per mL OSM for 48 h. Total RNA was isolated and untreated fibroblasts were used as control. The relative MRP mRNA levels of the subtypes 1, 3, 4, and 5 were normalized to β-actin.
Journal of Investigative Dermatology  , 28-37DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Real-time PCR, reverse transcription PCR (RT-PCR), and cDNA microarray analysis of multidrug resistance-associated proteins (MRP) expression in proliferating normal human epidermal keratinocytes (NHEK) after incubation with interleukin-6 (IL-6)/soluble human IL-6 receptor (sIL-6R), oncostatin M (OSM), or other inflammatory cytokines. (a) TaqMan real-time PCR analyses of MRP3 expression in proliferating NEHK. Primary keratinocytes were stimulated with 20 ng per mL IL-6 and 1 μg per mL sIL-6R or 20 ng per mL OSM for 48 h. Total RNA was isolated and untreated keratinocytes were used as control. The relative MRP3 RNA level was normalized to β-actin. (b) RT-PCR analysis of MRP1 and MRP3 expression in keratinocytes after stimulation for 48 h with IL-6 (20 ng per mL)/sIL-6R (1 μg per mL) or OSM (20 ng per mL). PCR products of MRP1, MRP3, and β-actin as internal standard were separated on agarose gels and stained with ethidium bromide. (c) RT-PCR analysis of MRP1, MRP3, and β-actin in proliferating NHEK after incubation with different cytokines for 24 h. Lane 1, unstimulated keratinocytes; lane 2, IL-6 (20 ng per mL)/sIL-6R (1 μg per mL); lane 3, IL-1β (2 ng per mL), lane 4: TNFα (1 ng per mL), lane 5: TGFβ (1 ng per mL); lane 6: DNA-Marker pBR322 HaeIII Digest. (d) Analysis of differentially expressed genes in NHEK using microarray analysis. Human keratinocytes were incubated with 20 ng per mL IL-6 and 1 μg per mL sIL-6R for 24 h; untreated NHEK were used as control. Generation of 33P-labeled cDNA probes was achieved by reverse transcription of 10 μg total RNA isolated from IL-6/sIL-6R-treated and control keratinocytes. Radiolabeled probes were applied to the GeneFilters microarrays (ID1001, Research Genetics) for hybridization.
Journal of Investigative Dermatology  , 28-37DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Coculture of dermal fibroblasts and normal human epidermal keratinocytes (NHEK) using the transwell system (TW) (left panel) in contrast to NHEK monoculture (MC) (right panel).Lane 1, control; lane 2, incubation with 20 ng per mL interleukin-6 (IL-6) and 1 μg per mL soluble human IL-6 receptor (sIL-6R) for 24 h; lane 3, incubation with 2 ng per mL IL-1β; lane 4, DNA-Marker pBR322 HaeIII Digest.
Journal of Investigative Dermatology  , 28-37DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Signaling pathways initiated by IL-6 and OSM in dermal fibroblasts and their impact on the enhanced transcription of MRP. (a) Analysis of signaling pathways initiated in dermal fibroblasts in response to oncostatin M (OSM) (left panel) or interleukin-6 (IL-6)/soluble human IL-6 receptor (sIL-6R) (right panel). Human dermal fibroblasts were stimulated with 20 ng per mL OSM or 20 ng per mL IL-6 and 1 μg per mL sIL-6R for the indicated time points. Total cell lysates were separated by SDS-PAGE. Western blots were developed with antisera specific for the indicated proteins. (b) Effect of inhibition of Erk1/2 on multidrug resistance-associated proteins (MRP) transcription. Real-time PCR analysis of MRP1, 3, 4, and 5 expression in cytokine-stimulated dermal fibroblasts pretreated with the MEK-1 inhibitor U0126: Human dermal fibroblasts were stimulated for 48 h with 20 ng per mL OSM or pretreated for 45 min with 5 μM U0126 followed by cytokine stimulation. The inhibitor as well as the cytokine were refreshed once after 24 h incubation. Total RNA was isolated, and untreated fibroblasts were used as control. The relative MRP RNA levels of the different subtypes were normalized to β-actin. (c) Effect of inhibition of phosphaditylinositol 3-kinase (PI3 kinase) on MRP4 transcription. Real-time PCR analysis of MRP4 expression in OSM-stimulated dermal fibroblasts pre-treated with the PI3-kinase inhibitor LY294002: Human dermal fibroblasts were stimulated for 48 h with OSM or pre-treated for 45 min with 5 μM LY followed by cytokine stimulation. The inhibitor as well as the cytokine were refreshed once after 24 h incubation. Total RNA was isolated and untreated fibroblasts were used as control. The relative MRP4 mRNA level was normalized to 18S rRNA.
Journal of Investigative Dermatology  , 28-37DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Immunohistological staining of human skin specimen using a monoclonal antibody specific for the detection of multidrug resistance-associated proteins (MRP1). (a) Staining of normal human skin, (b) skin sample taken from a patient with lichen planus, (c) skin sample from a psoriasis plaque. → indicates enhanced expression of MRP1 in the plasma membrane of the epidermal keratinocytes.
Journal of Investigative Dermatology  , 28-37DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Measurement of multidrug resistance-associated proteins (MRP) activity. The net accumulation of calcein-acetoxymethylester (calcein-AM) (a, b), and all-trans retinoic acid (RA) (c) was determined either fluorimetrically (calcein-AM) or radiometrically (all-trans [20-methyl-3H] RA) in normal human epidermal keratinocytes (NHEK) (a, c) or dermal fibroblasts (b). Cells were pre-treated with 20 ng per mL IL-6 and 1 μg per mL sIL-6R for 72 h, and untreated cells served as control. (a, b) Accumulation of calcein-AM was allowed to proceed for 15, 20, 30, or 60 min and was performed in triplicate. (c) Efflux of all-trans RA was measured in NHEK pre-incubated with 20 ng per mL IL-6 and 1 μg per mL sIL-6R for 24 h. Experiments were performed in hexaplicate after incubation with 1.4 × 10-8 mol per liter all-trans [20-methyl-3H] RA for 60 min, and median values were used for data analysis.
Journal of Investigative Dermatology  , 28-37DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions