Active Influx Transport is Mediated by Members of the Organic Anion Transporting Polypeptide Family in Human Epidermal Keratinocytes  Ruth Schiffer, Mark Neis, Daniela Höller, Felipe Rodríguez, Andreas Geier, Carsten Gartung, Frank Lammert, Alexandra Dreuw, Gabriele Zwadlo-Klarwasser, Hans Merk, Frank Jugert, Jens M. Baron  Journal of Investigative Dermatology  Volume 120, Issue 2, Pages 285-291 (February 2003) DOI: 10.1046/j.1523-1747.2003.12031.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 RT-PCR analysis of proliferating keratinocytes derived from the human epidermis using primers specific for various OATPs and β-actin. RT-PCR analysis of OATP-A, OATP-B, OATP-C, OATP-D, OATP-E and β-actin expression. Lane 1, donor 1; lane 2, donor 1, induction with dexamethasone (DEX); lane 3, donor 1, induction with TNF-α; lane 4, human adult liver; lane 5, DNA marker pBR322 Hae III Digest. Induction of cells was performed with dexamethasone (10–7 M) or TNF-α (100 U per ml) for 24 h. Journal of Investigative Dermatology 2003 120, 285-291DOI: (10.1046/j.1523-1747.2003.12031.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Northern blot analysis of OATP mRNA expression in keratinocytes derived from human epidermis of different donors, in HepG2 cells and in human adult liver. Twenty micrograms total RNA were subjected to northern blot analysis for OATP-D (a,b) and OATP-E (c). Equal amounts of RNA are indicated by the 18 S rRNA. Journal of Investigative Dermatology 2003 120, 285-291DOI: (10.1046/j.1523-1747.2003.12031.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Analysis of OATP-D expression in different human tissues studied by northern blot analysis. A human multiple tissue northern blot containing 20 μg of total RNA per lane was hybridized using an α-32P-cDNA fragment of OAPT-D as probe. β-actin was used as loading control. Journal of Investigative Dermatology 2003 120, 285-291DOI: (10.1046/j.1523-1747.2003.12031.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunoblots with antibodies against OATP. The expression of OATP-A and OATP-B was detected via 7.5% SDS polyacrylamide gel electrophoresis using specific antibodies. Generally, 100 μg of protein are loaded per lane. The protein bands were detected on an autoradiography film using enhanced chemiluminescence. Lanes 1, 2, donors 1 and 2; lane 3, human adult liver tissue. Journal of Investigative Dermatology 2003 120, 285-291DOI: (10.1046/j.1523-1747.2003.12031.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunohistochemistry and immunofluorescence. Immunohistochemistry: formalin-fixed and paraffin-embedded skin specimen stained with a polyclonal antibody specific for OATP-B (a), and isotype control (b). Immunofluorescence: frozen skin specimen stained with a polyclonal antibody specific for OATP-B (c), and isotype control (d). ⋄, Positive stained keratinocytes located in all layers of the epidermis. (b), (d) Isotypic controls; no positive staining detectable. Journal of Investigative Dermatology 2003 120, 285-291DOI: (10.1046/j.1523-1747.2003.12031.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 The inhibitory effect of taurocholate on the uptake of 17β-D-glucuronide [estradiol-6,7–3H(N)] and estrone sulfate [6,7–3H(N)] by OATP and its cytotoxicity in primary human keratinocytes and dermal fibroblasts. (a) Uptake of 17β-D-glucuronide [estradiol-6,7–3H(N)] was measured in quadruplicate in NHEKs (donor 1) for 60 min in the presence of taurocholate (1 and 5 mmol per l) and in the absence of the inhibitor. (b) Uptake of estrone sulfate [6,7–3H(N)] was measured in quadruplicate in NHEKs (donor 1) for 60 min in the presence of taurocholate (1 and 5 mmol per l) and in the absence of the inhibitor. (c) Uptake of 17β-D-glucuronide [estradiol-6,7–3H(N)] was measured in quadruplicate in dermal fibroblasts (donor 1) for 60 min in the presence of taurocholate (1 and 5 mmol per l) and in the absence of the inhibitor. (d) For the cytotoxicity assays NHEKs were incubated with taurocholate (1 and 5 mmol per l) for 2 h. The amount of cell death was then assessed by the release of ATP calculated by use of an ATP standard. Results of the cytotoxicity assay are displayed as median values. Journal of Investigative Dermatology 2003 120, 285-291DOI: (10.1046/j.1523-1747.2003.12031.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions