Ruth Heise

Differential expression of influx and efflux transport proteins in human antigen presenting cells

Abstract:  Human macrophages (MΦ) express cytochrome P450 enzymes verifying their capacity to metabolize a variety of endogenous and exogenous substances. Here we analysed the mRNA and protein expression of transport proteins involved in the uptake or export of drugs, hormones and arachidonic acid metabolites in dendritic cells (DC) and MΦ compared to their precursors – blood monocytes – using cDNA microarray, RT‐PCR, Western‐blot and immunostaining techniques. The transport proteins studied included members of the solute carrier organic anion transporter family (SLCO) and the multidrug resistance associated proteins (MRP) 1–6 belonging to the ATP‐binding cassette subfamily C (ABCC). We found that only mRNA for SLCO‐2B1, ‐3A1, and ‐4A1 were present in monocytes, MΦ and DC. Most interestingly the expression of SLCO‐2B1 was markedly enhanced in MΦ as compared to monocytes and DC. The presence of mRNA for ABCC1, 3, 4, 5 and 6 in all three cell types was demonstrated. On protein level ABCC1/MRP1 which has been identified as leukotriene C4 transporter was found to be the most abundant transporter in MΦ and DC. Blocking the ABCC1/MRP1 activity with the specific inhibitor MK571 resulted in a phenotypic change in DC but not in MΦ. Our data show that human blood monocytes and monocyte derived MΦ as well as DC express a specific profile of transporters involved in uptake and export of exogenous molecules like allergens or drugs, but also of endogenous substances in particular of inflammatory lipid mediators like leukotrienes and prostaglandins.

Cutaneous Metabolic Activation of Carvoxime, a Self-Activating, Skin-Sensitizing Prohapten

Bioactivation of low molecular weight compounds in the skin can cause contact sensitization. We have previously shown that the alpha, beta-R-unsaturated oxime R-carvoxime [1, (R)-2-methyl-5-isopropenylcyclohex-2-enone oxime] is bioactivated to two diastereomeric highly reactive and strongly sensitizing alpha, beta-epoxy oxime metabolites. To investigate if this metabolic activation is catalyzed by the major cytochrome P450 (P450) enzymes found in human skin, incubations of 1 with a skinlike P450 cocktail in the presence of glutathione were carried out. We identified three glutathione conjugates in the incubation mixture arising from two diasteomeric alpha, beta-epoxy oxime metabolites of 1, thus showing that the metabolic activation of 1 is P450-mediated. A P450 identification study using the individual P450 enzymes present in the skinlike P450 cocktail showed the involvement of P450 1A1 and 1B1 and also to some extent 2B6. P450 1B1 metabolism of 1 was found to be stereoselective as glutathione conjugates from only one of the alpha, beta-epoxyoxime metabolites were identified (metabolite 2). Additionally, 1 was found to be an inducer of P450 1B1 (but not 1A1) in human monocyte-derived dendritic cells (moDCs) and to some extent in normal human epidermal keratinocytes. A further transcriptional gene expression change observed in moDCs was a 44-fold upregulation of IL-8, a marker often used for assessment of sensitizing potential of contact allergens. The autoinduction of P450 1B1 by 1 may be a key event in the development of contact allergy to 1 and may also explain why only metabolite 2, and not 3, was found to elicit an allergic response in mice sensitized to 1. Our data show that the alpha, beta-unsaturated oxime 1 is bioactivated by human cutaneous P450, thus forming highly allergenic metabolites, and has the potential to induce its own bioactivation pathway, particularly in antigen-presenting cells.

Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts.

Abstract:  Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 μg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 μg/ml of pantothenate compared with untreated cells using the GeneChip® Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)‐6, IL‐8, Id1, HMOX‐1, HspB7, CYP1B1 and MARCH‐II. Regulation of these genes was subsequently verified by quantitative real‐time polymerase chain reaction analysis. Induction of HMOX‐1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts.

Dexpanthenol modulates gene expression in skin wound healing in vivo.

Topical application of dexpanthenol is widely used in clinical practice for the improvement of wound healing. Previous in vitro experiments identified a stimulatory effect of pantothenate on migration, proliferation and gene regulation in cultured human dermal fibroblasts. To correlate these in vitro findings with the more complex in vivo situation of wound healing, a clinical trial was performed in which the dexpanthenol-induced gene expression profile in punch biopsies of previously injured and dexpanthenol-treated skin in comparison to placebo-treated skin was analyzed at the molecular level by Affymetrix® GeneChip analysis. Upregulation of IL-6, IL-1β, CYP1B1, CXCL1, CCL18 and KAP 4–2 gene expression and downregulation of psorasin mRNA and protein expression were identified in samples treated topically with dexpanthenol. This in vivo study might provide new insight into the molecular mechanisms responsible for the effect of dexpanthenol in wound healing and shows strong correlations to previous in vitro data using cultured dermal fibroblasts.

Concepts in molecular dermatotoxicology.

Abstract:  In the recent years, molecular research has successfully elucidated some of the major mechanisms through which environmental noxae damage human skin. From this knowledge, novel concepts for skin protection have been developed. Here, we provide a brief overview of some of the most exciting and intriguing concepts in molecular dermatotoxicology.

High-Resolution Transcriptional Profiling of Chemical-Stimulated Dendritic Cells Identifies Immunogenic Contact Allergens, but Not Prohaptens

Allergic contact dermatitis is a complex syndrome and knowledge about the in vitro detection of small-molecular-weight compounds, particularly prohaptens, is limited. Therefore, we investigated chemical-induced gene expression changes in human antigen-presenting cells upon stimulation with immunogenic contact allergens, prohaptens and irritants. Monocyte-derived dendritic cells (moDCs) and THP-1 cells were stimulated with the prohapten cinnamic alcohol (CAlc), the hapten cinnamic aldehyde (CAld), an irritant and an obligatory sensitizer in vitro. Whole-genome screening and consecutive PCR analysis of differential gene expression in moDCs stimulated with either CAld or the obligatory sensitizer revealed coregulation of 11 marker genes which were related to immunological reactions (IL-8, CD1e, CD200R1, PLA2G5, TNFRSF11A), oxidative or metabolic stress responses (AKR1C3, SLC7A11, GCLM) or other processes (DPYLS3, TFPI, TRIM16). In contrast, the prohapten CAlc and the irritant did not change marker gene expression. In THP-1 cells, CAld and the positive control elicited similar expression changes in only 4 of the previously identified genes (IL-8, TRIM16, CD200R1, GCLM). In conclusion, we provide important insights into the pathophysiological basis of allergic contact dermatitis, identify marker genes suitable for skin hazard assessment and demonstrate that contact-allergenic prohaptens escape in in vitro detection if their skin metabolism is not taken into account.

Active transport of contact allergens in human monocyte-derived dendritic cells is mediated by multidrug resistance related proteins

The multidrug resistance related proteins (MRPs) function as efflux transporters of a variety of large organic anions or their conjugates. In recent studies we demonstrated that antigen-presenting cells express a specific pattern of MRPs. MRP-mediated efflux activity of human monocyte-derived dendritic cells (moDCs) was analyzed using an in vitro transport assay. The efflux transport of radiolabeled contact allergens was inhibited using the specific MRP inhibitor indomethacin. Treatment with indomethacin increased intracellular concentration of [³H] eugenol and [³H] isoeugenol in moDCs. In addition by using MRP1 expressing inside-out membrane vesicles we revealed that the transport of eugenol is mediated by MRP1. Human DCs were employed to assess the sensitizing potential of contact allergens and alters their cytokine gene expression profile. Hence, to survey the functionality of indomethacin after stimulation with contact allergens IL-8 and TRIM16 regulation was measured by a DC-based in vitro assay. Incubation with isoeugenol after pre-treatment with indomethacin leads to increased IL-8 and TRIM16 gene expression. These results strongly support the functional role of MRPs in the active efflux of contact allergens also in antigen-presenting cells like moDCs, a novel mechanism which could possibly play a role in the pathogenesis of contact allergy.

Downregulation of STRA6 Expression in Epidermal Keratinocytes Leads to Hyperproliferation-Associated Differentiation in Both In Vitro and In Vivo Skin Models

Retinoids are known to affect skin cell proliferation and differentiation and are key molecules that target retinoid and retinoic acid receptors (RXRs and RARs), leading to physiological and pharmacologic effects. Our aim was to elucidate the role of the retinol-binding protein receptor STRA6, mediating cellular uptake of retinol, on skin structure and function. Our results indicate that STRA6 is constitutively expressed in human epidermal keratinocytes and dermal fibroblasts and is regulated via RAR/RXR-mediated pathways. HaCaT (Human adult low Calcium high Temperature) cells with stable STRA6 knockdown (STRA6KD) showed increased proliferation. Consistently, human organotypic 3D skin models using stable STRA6KD HaCaT cells showed a significantly thicker epidermis and enhanced expression of activation, differentiation, and proliferation markers. The effects were reversible after treatment with free retinol. Human skin reconstitution employing STRA6KD HaCaT cells leads to massive epithelial thickening under in vivo conditions in SCID mice. We propose that STRA6KD could lead to cellular vitamin A deficiency in keratinocytes. Consequently, STRA6 has a role for regulating retinoid homeostasis and in helping to program signaling that drives proliferation and differentiation of human skin cells. By its influence on hyperproliferation-associated differentiation, STRA6 could also have a role in skin regeneration and could be a target for pharmacological approaches to improve wound healing.

Active transport of contact allergens and steroid hormones in epidermal keratinocytes is mediated by multidrug resistance related proteins.

TO THE EDITOR The phenomenon of multidrug resistance (MDR) is defined as the ability of a cell to show resistance to a wide variety of structurally and functionally unrelated molecules and is, in large part, related to membrane efflux transporters because its principal mechanism is the active transport of substrates out of the cell (Higgins, 2007). Multidrug transporters have broad specificity for a wide range of chemically unrelated agents (Higgins, 2007). One superfamily of active membrane transporters is the family of ATP-binding cassette (ABC) transport proteins that use the energy of ATP hydrolysis to transport large organic molecules either directly or, in case of many natural substrates, conjugated to acidic ligands, such as glutathione, glucuronate, or sulfate out of the cell (König et al., 1999). Although most ABC transporters were discovered as drug transporters, they frequently transport a wide range of physiological substrates, including peptides, steroids, and inflammatory lipid mediators (Robbiani et al., 2000; Borges-Walmsley et al., 2003; Reid et al., 2003). Therefore, it is further understood that these efflux pumps have, in addition to conferring resistance of tumor cells to various chemotherapeutic drugs, crucial physiological roles (Piddock, 2006). Several studies have been carried out to investigate the expression, regulation, and specific substrates of ABC efflux transport proteins, such as P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRP, gene name: ATP binding cassette C transporters, ABCC) (Flens et al., 1996; Ishikawa et al., 2000). Earlier it has been shown that cells of the skin express a specific pattern of several efflux proteins (Sleeman et al., 2000; Baron et al., 2001; Colone et al., 2008). Gene expression analysis of normal human epidermal keratinocytes (NHEK) showed constitutive expression of MRP1 (ABCC1) as well as MRP 3–7 (ABCC3–7), but was negative for MDR1 and MRP2 (ABCC2) (Baron et al., 2001). With regard to the regulatory mechanisms of these transport proteins in skin cells, it was shown that the expression of different MRP family members can be significantly enhanced by IL-6-type cytokines. Furthermore, upregulation of MRP expression was found in lesional skin samples collected from patients with inflammatory skin disorders such as psoriasis and lichen planus (Dreuw et al., 2005). In this study, we provide evidence for the functional MRP-mediated efflux activity of NHEKs using an in vitro transport assay. Therefore, the efflux transport of various radiolabeled compounds, including eugenol (4-allyl-2methoxyphenol), isoeugenol, estradiol 17beta-D-glucuronid (E217bG), oestrone3-sulfate, cyclosporine, and dexpanthenol, was inhibited by the specific MRP inhibitors, such as indomethacin (Figure 1) (Draper et al., 1997; Zhou et al., 2008) or MK571 (Figure 2) (Skazik et al., 2008; Zhou et al., 2008), and the intracellular accumulation of these substrates was determined after 15, 30, and 60 minutes. Cells not treated with the inhibitor served as a control. Treatment with indomethacin at a concentration of 1 mM decreased the efflux transport of [H]E217bG up to 6.5-fold and of [H]oestrone-3sulfate up to 3.6-fold in NHEKs in comparison with control transport measured in the absence of the inhibitor (Figure 1a and b). Treatment of NHEKs with MK571 decreased the efflux of [H]E217bG in a concentrationdependent manner at a concentration of 25 mM up to 4.1-fold and at a concentration of 100mM up to 5.1-fold (Figure 2a). MK571 strongly reduced the efflux of [H]oestrone-3-sulfate as well in a concentration-dependent manner, as the cells accumulated this steroid hormone up to 3.2-fold using 25 mM of MK571 and up to 6.1-fold using 100mM of the inhibitor (Figure 2b) in comparison with control transport that is measured in the absence of the inhibitor. Indomethacin blocked the efflux transport of [H]eugenol up to 1.6-fold compared with control cells without indomethacin treatment (Figure 1c). Treatment of cells with indomethacin increased intracellular concentration of [H]isoeugenol only slightly up to 1.1-fold (Figure 1d). Treatment of NHEKs with MK571 at concentrations of 100mM and 25 mM decreased efflux transport of [H]eugenol and [H]isoeugenol, respectively, to 1.6-fold after 60 minutes of incubation (Figure 2c and d). Time course experiments show stronger effects of both inhibitors on the accumulation of E217bG, oestrone-3-sulfate, eugenol, and isoeugenol (Figures 1a–d and 2a–d) in the cell after 60 minutes compared with 15 or 30 minutes. This adds to the suggestion that these effects are due to the inhibition of efflux transport and not due to influx transport. Treatment of cells with indomethacin or MK571

Adjuvant interferon alfa treatment for patients with malignant melanoma stimulates transporter proteins associated with antigen processing and proteasome activator 28

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