Interleukin-6-Type Cytokines Upregulate Expression of Multidrug Resistance-Associated Proteins in NHEK and Dermal Fibroblasts
2005; Elsevier BV; Volume: 124; Issue: 1 Linguagem: Inglês
10.1111/j.0022-202x.2004.23499.x
ISSN1523-1747
AutoresAlexandra Dreuw, Heike M. Hermanns, Ruth Heise, Sylvia Joussen, Felipe Rodríguez, Yvonne Marquardt, Frank K. Jugert, Hans F. Merk, Peter C. Heinrich, Jens Malte Baron,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoNormal human epidermal keratinocytes (NHEK) and dermal fibroblasts express a cell-specific pattern of efflux transport proteins. Since regulatory mechanisms for these transporters in cells of the human skin were unknown, we analyzed the influence of inflammatory cytokines on the expression of multidrug resistance-associated proteins (MRP1, 3, 4, 5). Using real-time PCR, RT-PCR, cDNA microarray, immunostaining and efflux assays we demonstrated that stimulation of NHEK and primary human dermal fibroblasts with interleukin-6 (IL-6), in combination with its soluble α-receptor, or oncostatin M (OSM) for 24–72 h resulted in an upregulation of MRP expression and activity. Both cytokines induced a strong activation of signal transducer and activator of transcription (STAT)1 and STAT3 as well as the mitogen-activated protein kinase (MAPK) Erk1/2. OSM additionally activated proteinkinase B strongly. Using the MAPK/extracellular signal-regulated kinase kinase 1-specific inhibitor U0126 we could exclude a stimulatory effect of MAPK on MRP gene expression. Inhibition of the phosphatidylinositol 3-kinase, however, indicated that this pathway might be involved of OSM-mediated upregulation of MRP4 in dermal fibroblasts. Several inflammatory skin diseases show an enhanced expression of IL-6-type cytokines. Correspondingly, upregulation of MRP expression was found in lesional skin taken from patients with psoriasis and lichen planus. Normal human epidermal keratinocytes (NHEK) and dermal fibroblasts express a cell-specific pattern of efflux transport proteins. Since regulatory mechanisms for these transporters in cells of the human skin were unknown, we analyzed the influence of inflammatory cytokines on the expression of multidrug resistance-associated proteins (MRP1, 3, 4, 5). Using real-time PCR, RT-PCR, cDNA microarray, immunostaining and efflux assays we demonstrated that stimulation of NHEK and primary human dermal fibroblasts with interleukin-6 (IL-6), in combination with its soluble α-receptor, or oncostatin M (OSM) for 24–72 h resulted in an upregulation of MRP expression and activity. Both cytokines induced a strong activation of signal transducer and activator of transcription (STAT)1 and STAT3 as well as the mitogen-activated protein kinase (MAPK) Erk1/2. OSM additionally activated proteinkinase B strongly. Using the MAPK/extracellular signal-regulated kinase kinase 1-specific inhibitor U0126 we could exclude a stimulatory effect of MAPK on MRP gene expression. Inhibition of the phosphatidylinositol 3-kinase, however, indicated that this pathway might be involved of OSM-mediated upregulation of MRP4 in dermal fibroblasts. Several inflammatory skin diseases show an enhanced expression of IL-6-type cytokines. Correspondingly, upregulation of MRP expression was found in lesional skin taken from patients with psoriasis and lichen planus. calcein-acetoxymethylester ethylenediaminetetraacetic acid interleukin-6 Janus kinase mitogen-activated protein kinase multidrug resistance MAPK/extracellular signal-regulated kinase kinase multidrug resistance-associated protein normal human epidermal keratinocytes oncostatin M P-glycoprotein phosphaditylinositol 3-kinase protein kinase B retinoic acid reverse transcription PCR soluble human IL-6 receptor signal transducer and activator of transcription The subfamily C of the adenosine triphosphate-binding cassette transporter superfamily consists of 13 members, including the multidrug resistance-associated protein (MRP) family (Dean et al., 2001Dean M. Rzhetsky A. Allikmets R. The human ATP-binding cassette (ABC) transporter superfamily.Genome Res. 2001; 11: 1156-1166Crossref PubMed Scopus (1384) Google Scholar; Yabuuchi et al., 2002Yabuuchi H. Takayanagi S. Yoshinaga K. Taniguchi N. Aburatani H. Ishikawa T. ABCC13, an unusual truncated ABC transporter, is highly expressed in fetal human liver.Biochem Biophys Res Commun. 2002; 299: 410-417Crossref PubMed Scopus (51) Google Scholar). These proteins function as organic anion/conjugate transporters and participate in hepatic detoxification, drug distribution, and multidrug resistance (MDR) of tumor cells (Flens et al., 1996Flens M.J. Zaman J.R. van der Valk P. et al.Tissue distribution of the multidrug resistance protein.Am J Pathol. 1996; 148: 1237-1247PubMed Google Scholar; König et al., 1999König J. Nies A.T. Cui Y. Leier I. Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: Localization, substrate specificity, and MRP2-mediated drug resistance.Biochim Biophys Acta. 1999; 1461: 377-394Crossref PubMed Scopus (673) Google Scholar; Kruh and Belinsky, 2003Kruh G.D. Belinsky M.G. The MRP family of drug efflux pumps.Oncogene. 2003; 22: 7537-7552Crossref PubMed Scopus (533) Google Scholar). We were recently able to demonstrate that normal human epidermal keratinocytes (NHEK) and dermal fibroblasts express several metabolically active and transport-associated enzymes such as organic anion transporting polypeptide-B, -D, and -E (Schiffer et al., 2003Schiffer R. Neis M. Holler D. et al.Active influx transport is mediated by members of the organic anion transporting polypeptide family in human epidermal keratinocytes.J Invest Dermatol. 2003; 120: 285-291Crossref PubMed Scopus (75) Google Scholar) as well as MRP1 and MRP3–7 (Baron et al., 2001Baron J.M. Höller D. Schiffer R. Frankenberg S. Neis M. Merk H.F. Jugert F. Expression of multiple cytochrome P450 enzymes and MDR-associated transport proteins in human skin keratinocytes.J Invest Dermatol. 2001; 116: 541-548Crossref PubMed Scopus (200) Google Scholar) and are therefore capable of active uptake, metabolism, and efflux of large organic molecules. These are exported either directly, or in the case of many natural drugs, conjugated to acidic ligands, such as glutathione (GSH), glucuronate, or sulfate. Interestingly, it has recently become evident that MRP1, 2, and 3 can also augment resistance to unmodified organic drugs that are not conjugated to acidic ligands by transporting them together with free GSH (Rappa et al., 1997Rappa G. Lorico A. Flavell R.A. Sartorelli A.C. Evidence that the multidrug resistance protein (MRP) functions as a co-transporter of glutathione and natural product toxins.Cancer Res. 1997; 57: 5232-5237PubMed Google Scholar; Loe et al., 1998Loe D.W. Deeley R.G. Cole S.P. Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): Evidence for cotransport with reduced glutathione.Cancer Res. 1998; 58: 5130-5136PubMed Google Scholar). Although overexpression of MRP family members seems closely associated with the clinical outcome of various malignancies, their physiological functions are still poorly defined. Abrogation of MRP expression in mice, however, led not only to hypersensitivity to anticancer drugs but also to an impairment in the inflammatory response. This defect was attributed to a decreased secretion of leukotriene C4 (Wijnholds et al., 1997Wijnholds J. Evers R. van Leusden M.R. et al.Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein.Nat Med. 1997; 3: 1275-1279Crossref PubMed Scopus (388) Google Scholar). Meanwhile, several members of the MRP family have been shown to not only transport leukotrienes (König et al., 1999König J. Nies A.T. Cui Y. Leier I. Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: Localization, substrate specificity, and MRP2-mediated drug resistance.Biochim Biophys Acta. 1999; 1461: 377-394Crossref PubMed Scopus (673) Google Scholar) but also prostaglandins (Reid et al., 2003Reid G. Wielinga P. Zelcer N. et al.The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs.Proc Natl Acad Sci USA. 2003; 100: 9244-9249Crossref PubMed Scopus (393) Google Scholar). First evidence for a putative role of interleukin-6 (IL-6) in the regulation of MRP1 expression in HepG2 cells was described byLee and Piquette-Miller, 2001Lee G. Piquette-Miller M. Influence of IL-6 on MDR and MRP-mediated multidrug resistance in human hepatoma cells.Can J Physiol Pharmacol. 2001; 79: 876-884Crossref PubMed Scopus (76) Google Scholar. The family of IL-6-type cytokines, which comprises to date nine members, is involved in pro- as well as anti-inflammatory processes. They are major players in hematopoiesis, as well as in acute phase and immune responses of the organism and characterized by the use of a common signal transducing receptor chain glycoprotein 130 (Heinrich et al., 1998Heinrich P.C. Behrmann I. Müller-Newen G. Schaper F. Graeve L. Interleukin-6-type cytokine signaling through the gp130/Jak/STAT pathway.Biochem J. 1998; 334: 297-314Crossref PubMed Scopus (1657) Google Scholar; Heinrich et al., 2003Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Müller-Newen G. Schaper F. Principles of interleukin (IL)-6-type cytokine signaling and its regulation.Biochem J. 2003; 374: 1-20Crossref PubMed Scopus (2332) Google Scholar). This cytokine receptor lacks intrinsic kinase activity, but is constitutively associated with tyrosine kinases belonging to the Janus kinase (Jak) family. After ligand binding to their receptors, these kinases become activated and initiate downstream signaling events, which ultimately result in the phosphorylation of signal transducer and activator of transcription (STAT) factors and Ser/Thr-kinases of the MAP-kinase family (Erk1/2, p38, and JNK). Although STAT transcription factors appear to be direct targets of the Jak, the activation of the MAP-kinase cascade seems more complex and involves adapter molecules like Shc (Hermanns et al., 2000Hermanns H.M. Radtke S. Schaper F. Heinrich P.C. Behrmann I. Non-redundant signal transduction of interleukin-6-type cytokines. The adapter protein Shc is specifically recruited to rhe oncostatin M receptor.J Biol Chem. 2000; 275: 40742-40748Crossref PubMed Scopus (59) Google Scholar) or the tyrosine phosphatase SHP-2 (Schiemann et al., 1997Schiemann W.P. Bartoe J.L. Nathanson N.M. Box 3-independent signaling mechanisms are involved in leukemia inhibitory factor receptor alpha- and gp130-mediated stimulation of mitogen-activated protein kinase. Evidence for participation of multiple signaling pathways which converge at Ras.J Biol Chem. 1997; 272: 16631-16636Crossref PubMed Scopus (75) Google Scholar) as well as guanine nucleotide exchange factors such as Son of Sevenless. In the case of Erk1/2, the signaling route seems to depend on Ras, Raf-1, and the MAP-kinase kinase MEK-1 (MEK, MAPK/extracellular signal-regulated kinase kinase). Enhanced expression of some members of this cytokine family (IL-6, oncostatin M (OSM), leukemia inhibitory factor (LIF)) occurs in a number of inflammatory skin diseases (Grossman et al., 1989Grossman R.M. Krueger J. Yourish D. et al.Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes.Proc Natl Acad Sci USA. 1989; 86: 6367-6371Crossref PubMed Scopus (683) Google Scholar; Bonifati et al., 1998Bonifati C. Mussi A. D'Auria L. Carducci M. Trento E. Cordiali-Fei P. Ameglio F. Spontaneous release of leukemia inhibitory factor and oncostatin-M is increased in supernatants of short-term organ cultures from lesional psoriatic skin.Arch Dermatol Res. 1998; 290: 9-13Crossref PubMed Scopus (27) Google Scholar; Fayyazi et al., 1999Fayyazi A. Schweyer S. Soruri A. et al.T lymphocytes and altered keratinocytes express interferon-gamma and interleukin 6 in lichen planus.Arch Dermatol Res. 1999; 291: 485-490Crossref PubMed Scopus (49) Google Scholar). Since inflammation is not only regulated by pro- and anti-inflammatory cytokines, but many additional mediators like leukotrienes or prostaglandins, we became interested in the transcriptional regulation of transporters known to be involved in the secretion of these molecules. In this study, the inducibility of enhanced gene expression of MRP isoforms was characterized not only in primary NHEK and fibroblasts but also in human skin samples from patients with inflammatory skin disorders. Primary human dermal fibroblasts (Figure 1) and proliferating normal epidermal keratinocytes (Figure 2) isolated from foreskin were treated with IL-6/soluble human IL-6 receptor (sIL-6R) or OSM for 48 h. Expression of the subtypes 1, 3, 4, and 5 of the MRP family was studied by real-time PCR (TaqMan) or semiquantitative reverse transcription PCR (RT-PCR). As shown in Figure 1, mRNA levels for all four family members were significantly increased in OSM-treated dermal fibroblasts of three individual donors. Particularly, MRP3 and MRP4 were upregulated more than 2-fold. IL-6/sIL-6R had a reproducible stimulatory effect on the transcription of MRP3, whereas effects on MRP1, MRP4, and MRP5 were only marginal.Figure 2Real-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.View Large Image Figure ViewerDownload (PPT) Primary NHEK, on the other hand responded much stronger to IL-6/sIL-6R than to OSM. Although MRP3 mRNA levels demonstrated a 150% increase after incubation with IL-6/sIL6R for 48 h, the mRNA levels remained rather unchanged in response to OSM (Figure 2a). MRP1 mRNA levels had to be analyzed in RT-PCR experiments using established MRP1-specific primers (Kool et al., 1997Kool M. de Haas M. Scheffer G.L. et al.Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines.Cancer Res. 1997; 57: 3537-3547PubMed Google Scholar; Baron et al., 2001Baron J.M. Höller D. Schiffer R. Frankenberg S. Neis M. Merk H.F. Jugert F. Expression of multiple cytochrome P450 enzymes and MDR-associated transport proteins in human skin keratinocytes.J Invest Dermatol. 2001; 116: 541-548Crossref PubMed Scopus (200) Google Scholar) since the Assay-on-Demand primers used in dermal fibroblasts turned out to be unsuitable for the keratinocytes. Similar amounts of template mRNA and the same cycle no. (35) were used for each RT-PCR reaction using primer pairs for MRP1 and MRP3. As shown in Figure 2b, keratinocytes revealed a constitutive expression of MRP1. Stimulation for 48 h with IL-6, in combination with its soluble α receptor (sIL-6R), however, also enhanced the expression of this transport protein. For MRP3, an equivalent upregulation was observed as seen before in the real-time PCR (Figure 2a). Other pro- or anti-inflammatory cytokines such as IL-1β, tumor necrosis factor α (TNFα), or transforming growth factor (TGFβ) had no significant effect on mRNA expression of MRP in NHEK (Figure 2c). Additionally, we could detect higher amounts of MRP1 transcripts using a cDNA microarray focusing on genes expressed in human skin (Dermarray, ID1001). Incubation of NHEK with IL-6/sIL-6R for 24 h confirmed the RT-PCR data and revealed a 1.74-fold upregulation of MRP1 expression (Figure 2d). Expression of MRP was measured by RT-PCR in NHEK cocultured with dermal fibroblasts using the transwell system (TW) or in an NHEK from a monoculture (MC) (Figure 3). Incubation of cells with IL-6/sIL-6R for 24 h revealed the upregulation of MRP1 and -3 in both experimental setups, i.e. keratinocytes of the keratinocyte–fibroblast coculture and NHEK monoculture. Stimulation with IL-1β for 24 h, however, showed the upregulation of MRP1 and -3 only in those keratinocytes cocultured with fibroblasts in a TW (Figure 3), but not in keratinocytes taken from the NHEK monoculture. Both IL-6/sIL-6R as well as OSM are strong activators of the Jak/STAT and the mitogen-activated protein kinase (MAPK) pathway (Heinrich et al., 1998Heinrich P.C. Behrmann I. Müller-Newen G. Schaper F. Graeve L. Interleukin-6-type cytokine signaling through the gp130/Jak/STAT pathway.Biochem J. 1998; 334: 297-314Crossref PubMed Scopus (1657) Google Scholar, Heinrich et al., 2003Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Müller-Newen G. Schaper F. Principles of interleukin (IL)-6-type cytokine signaling and its regulation.Biochem J. 2003; 374: 1-20Crossref PubMed Scopus (2332) Google Scholar). Numerous studies have identified the Jak-mediated phosphorylation, dimerization, and nuclear translocation of STAT3 as a key component for transcription of a majority of IL-6-type cytokine-stimulated genes. But previous studies on regulatory mechanisms driving MDR genes have suggested a significant contribution of the MAPK pathway (Sukhai and Piquette-Miller, 2000Sukhai M. Piquette-Miller M. Regulation of the multidrug resistance genes by stress signals.J Pharm Pharmaceut Sci. 2000; 3: 268-280PubMed Google Scholar). The increase in mRNA levels of the various MRP in primary dermal fibroblasts is a long-term effect visible after 24–48 h incubation with either OSM or IL-6/sIL-6R. We therefore first investigated the time kinetics of the different signaling pathways known to be activated by IL-6-type cytokines (Heinrich et al., 2003Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Müller-Newen G. Schaper F. Principles of interleukin (IL)-6-type cytokine signaling and its regulation.Biochem J. 2003; 374: 1-20Crossref PubMed Scopus (2332) Google Scholar). As expected, STAT3 as well as STAT1 are strongly tyrosine phosphorylated in response to OSM as well as to IL-6/sIL-6R. Interestingly, after the initial peak between 10 min and 1 h, an enhanced level of tyrosine phosphorylated STAT remained detectable at least up to 24 h (Figure 4a, first and third panel). The effect was more pronounced for STAT3 than for STAT1. In contrast, the increased phosphorylation status of the MAPK Erk1/2 and p38 as well as of PKB/Akt was only transient with the same initial peak between 10 min and 1 h, but then a rapid return to basal levels (Figure 4a, fifth, seventh, and ninth panel). In contrast to IL-6, OSM turned out to be a much more potent activator of p38 and PKB/Akt (Figure 4a). In order to analyze whether the activation of MAPK is important to modify MRP gene transcription, dermal fibroblasts were pre-incubated with the MEK-1 inhibitor (U0126) 45 min before stimulation with OSM for 48 h. The inhibitor and the cytokine were refreshed once during the incubation period. Real-time PCR analysis revealed that pre-treatment of dermal fibroblasts with 5 μM of U0126 had no inhibitory effect on the enhanced transcription of MRP1, MRP3, or MRP4 under the influence of OSM (Figure 4b). Western blot studies verified that U0126 was suppressing the phosphorylation of Erk1/2, as expected, but also the phosphorylation of the stress-activated kinase p38. This unspecific activity of the inhibitor might be because of the long incubation time of 48 h. Tyrosine phosphorylation of STAT3, however, as well as serine phosphorylation of PKB/Akt were not influenced (data not shown). We then examined whether inhibition of the phosphaditylinositol 3-kinase (PI3-kinase) pathway by the specific inhibitor LY294002 altered OSM-induced MRP expression. Interestingly, the significant increase in MRP4 expression mediated through 48 h stimulation with OSM was strongly impaired in dermal fibroblasts treated with OSM in the presence of LY294002 (Figure 4c). Unfortunately, MRP1 and MRP3 mRNA could not be evaluated, since LY294002 itself already had a strong stimulatory effect on transcription of these two transporters (data not shown). LY294002 remained specific even after 48 h incubation and inhibited only the phosphorylation of PKB/Akt, but not of Erk1/2, p38, or STAT3 (not shown). The immunohistochemical staining of skin samples clearly demonstrated the expression of MRP1 in the cell membrane of epidermal keratinocytes (Figure 5; MRP1 expression indicated by arrow). In contrast to normal human skin (Figure 5a), expression of MRP1 was more pronounced in skin samples of patients with lichen planus (Figure 5b) and especially in psoriasis vulgaris (Figure 5c). To further corroborate the efficient translation of the increased mRNA for the various MRP, the efflux activity of human keratinocytes as well as dermal fibroblasts was determined fluorimetrically using the calcein-acetoxymethylester (calcein-AM) efflux assay (Figure 6a, b. This chemical diffuses passively into cells, remains non-fluorescent in its ester form, and can efficiently be exported by MRP. Cytoplasmic and mitochondrial esterases within the cells convert calcein-AM into a hydrophilic fluorescent dye (calcein) that is trapped inside the cytoplasm. Therefore, a low level of fluorescence indicates a low intracellular retention of calcein, reflecting a high functional activity of efflux transport proteins. Untreated NHEK and fibroblasts were compared with cells incubated with IL-6 and sIL-6R for 72 h. After 3 d of incubation with the cytokine, accumulation of calcein-AM was allowed to proceed for the indicated times. Cells were washed and the remaining incorporated fluorescence was measured. In accordance with the PCR data (Figure 1 and Figure 2), enhanced expression of MRP transporters in the plasma membrane of NHEK as well as in dermal fibroblasts correlated with a decrease in fluorescence after IL-6/sIL-6R treatment (Figure 6a, b). A novel transport assay for the detection of MRP-mediated efflux activity in NHEK was established using all-trans [20-methyl-3H] retinoic acid (RA) as a substrate. As seen with calcein-AM, treatment of cells with IL-6 and sIL-6R (Figure 6c) increased the active efflux transport of all-trans [20-methyl-3H] RA in NHEK by 25% in comparison with control transport measured in the absence of the retinoid. Dysregulated expression of transport proteins like the MRP has since many years been associated with increased resistance of various cancer cells to chemotherapeutic drugs (König et al., 1999König J. Nies A.T. Cui Y. Leier I. Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: Localization, substrate specificity, and MRP2-mediated drug resistance.Biochim Biophys Acta. 1999; 1461: 377-394Crossref PubMed Scopus (673) Google Scholar; Kruh and Belinsky, 2003Kruh G.D. Belinsky M.G. The MRP family of drug efflux pumps.Oncogene. 2003; 22: 7537-7552Crossref PubMed Scopus (533) Google Scholar). In recent years, it has become clear that MRP are not only involved in exporting xenobiotica, but also seem to be involved in the export of lipid mediators like leukotrienes or prostaglandins (König et al., 1999König J. Nies A.T. Cui Y. Leier I. Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: Localization, substrate specificity, and MRP2-mediated drug resistance.Biochim Biophys Acta. 1999; 1461: 377-394Crossref PubMed Scopus (673) Google Scholar; Reid et al., 2003Reid G. Wielinga P. Zelcer N. et al.The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs.Proc Natl Acad Sci USA. 2003; 100: 9244-9249Crossref PubMed Scopus (393) Google Scholar). Therefore, they seem to be target genes of potential interest when studying the inflammatory response. Indeed, first hints that cytokines involved in coordinating the body's inflammatory reaction as well as bacterial products might be involved in the regulation of MRP came from studies in the liver (Lee and Piquette-Miller, 2001Lee G. Piquette-Miller M. Influence of IL-6 on MDR and MRP-mediated multidrug resistance in human hepatoma cells.Can J Physiol Pharmacol. 2001; 79: 876-884Crossref PubMed Scopus (76) Google Scholar; Hartmann et al., 2002Hartmann G. Cheung A.K. Piquette-Miller M. Inflammatory cytokines, but not bile acids, regulate expression of murine hepatic anion transporters in endotoxemia.J Pharmacol Exp Ther. 2002; 303: 273-281Crossref PubMed Scopus (179) Google Scholar), but nothing was known about the regulation of these transporters in human skin cells. The presence of MRP1 and MRP3–7 in human skin has been verified in earlier studies (Baron et al., 2001Baron J.M. Höller D. Schiffer R. Frankenberg S. Neis M. Merk H.F. Jugert F. Expression of multiple cytochrome P450 enzymes and MDR-associated transport proteins in human skin keratinocytes.J Invest Dermatol. 2001; 116: 541-548Crossref PubMed Scopus (200) Google Scholar). Here, we could demonstrate that in primary NHEK as well as dermal fibroblasts the inflammatory cytokines IL-6/sIL-6R and OSM are able to upregulate the expression of different MRP family members. Lesional psoriatic skin (Sehgal, 1990Sehgal P.B. Interleukin-6: Molecular pathophysiology.J Invest Dermatol. 1990; 94: 2S-6SAbstract Full Text PDF PubMed Google Scholar; Bonifati et al., 1998Bonifati C. Mussi A. D'Auria L. Carducci M. Trento E. Cordiali-Fei P. Ameglio F. Spontaneous release of leukemia inhibitory factor and oncostatin-M is increased in supernatants of short-term organ cultures from lesional psoriatic skin.Arch Dermatol Res. 1998; 290: 9-13Crossref PubMed Scopus (27) Google Scholar) or keratinocytes isolated from patients with lichen planus (Fayyazi et al., 1999Fayyazi A. Schweyer S. Soruri A. et al.T lymphocytes and altered keratinocytes express interferon-gamma and interleukin 6 in lichen planus.Arch Dermatol Res. 1999; 291: 485-490Crossref PubMed Scopus (49) Google Scholar) are known to display elevated levels of IL-6-type cytokines. The highest level of IL-6 expression was observed in the basal and suprabasal keratinocytes of the involved skin. The role of these IL-6-type cytokines in the pathogenesis of psoriatic lesions is completely unknown. However, an in vitro study has indicated that OSM can directly induce GM-CSF and IL-6 production by endothelial cells and the endothelium seems to be involve d early in the psoriatic process (Bonifati et al., 1998Bonifati C. Mussi A. D'Auria L. Carducci M. Trento E. Cordiali-Fei P. Ameglio F. Spontaneous release of leukemia inhibitory factor and oncostatin-M is increased in supernatants of short-term organ cultures from lesional psoriatic skin.Arch Dermatol Res. 1998; 290: 9-13Crossref PubMed Scopus (27) Google Scholar). In our experiments, specimens from normal human skin revealed a low basal expression and activity of MRP efflux proteins in cultured keratinocytes (Figure 2b, c and Figure 5a). This expression was enhanced in lesional skin of patients with lichen planus (Figure 5b) and psoriasis (Figure 5c), indicating both increased transcription and protein synthesis of the MRP transporters. This increase in MRP visible at the cell membrane was then analyzed in vitro using freshly isolated NHEK and dermal fibroblasts. Stimulation of NHEK with IL-6, in combination with its agonistically acting soluble α receptor (sIL-6R), led to a strong upregulation of MRP3, and to a lesser extent of MRP1 (Figure 2a, b, and d). Interestingly, in an inflammatory situation infiltrating leukocytes can indeed secrete a soluble form of IL-6Rα or induce shedding of membrane-bound IL-6R (Jones et al., 2001Jones S.A. Horiuchi S. Topley N. Yamamoto N. Fuller G.M. The soluble interleukin 6 receptor: Mechanisms of production and implications in disease.FASEB J. 2001; 15: 43-58Crossref PubMed Scopus (511) Google Scholar). Assessment of other cytokines such as IL-1, TNFα, and TGFβ expressed in inflammatory skin tissue revealed no effect on the expression of these proteins in NHEK (Figure 2c). By analyzing the keratinocyte–fibroblast interaction in a two-chamber cell cultivation systemBoxman et al., 1993Boxman I. Lowik C. Aarden L. Ponec M. Modulation of IL-6 production and IL-1 activity by keratinocyte-fibroblast interaction.J Invest Dermatol. 1993; 101: 316-324Abstract Full Text PDF PubMed Google Scholar, we were able to show that fibroblasts are also an important source of IL-6 in normal human skin. This expression can be significantly upregulated by IL-1, which is released from keratinocytes after skin injury. Our data indicate that stimulation of keratinocyte–fibroblast cocultures with IL-1 leads to an upregulation of IL-6 production in fibroblasts, which could subsequently induce upregulation of MRP expression in NHEK in a paracrine manner (Figure 3). Stimulation of a keratinocyte monoculture with IL-1 had no effect on the M
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