Expression of Cholesterol Sulfotransferase (SULT2B1b) in Human Skin and Primary Cultures of Human Epidermal Keratinocytes Yuko Higashi, Hirotoshi Fuda,

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Expression of Cholesterol Sulfotransferase (SULT2B1b) in Human Skin and Primary Cultures of Human Epidermal Keratinocytes Yuko Higashi, Hirotoshi Fuda, Hidekatsu Yanai, Young Lee, Tomoko Fukushige, Tamotsu Kanzaki, Charles A. Strott Journal of Investigative Dermatology Volume 122, Issue 5, Pages 1207-1213 (May 2004) DOI: 10.1111/j.0022-202X.2004.22416.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of SULT2B1b in normal human skin and epidermal keratinocytes. (A) Reverse transcription-PCR analysis of SULT2A1 (lanes 1,5), SULT2B1a (lanes 2,6), SULT2B1b (lanes 3, 7) and β-actin (lanes 4, 8) of human skin (lanes 1–4) and NHEK after incubation with 1.2 mM Ca2+ for 9 days (lanes 5–8). (B) SULT2A1 and SULT2B1 isoforms were analyzed by immunoblotting. Extracts (30 μg) of normal human epidermis (lanes 2, 4) and NHEK that had been grown in 1.2 mM Ca2+ for 9 d (lanes 3, 5) were gel loaded. Positive controls were 15 ng of recombinant SULT2A1 (lane 1) and 70 ng each of SULT2B1a and SULT2B1b (lane 6). Journal of Investigative Dermatology 2004 122, 1207-1213DOI: (10.1111/j.0022-202X.2004.22416.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Ca2+-induction of SULT2B1b mRNA and protein expression in cultured normal human epidermal keratinocytes. Cells were treated with 0.05, 0.2, or 1.2 mM Ca2+. At the indicated times, cell extracts and total RNA were prepared and analyzed by immunoblotting and real-time PCR. The inset shows expression of SULT2B1b protein after loading of 30 μg of cell extract. Real-time PCR values represent the mean±SEM, n=6. *p<0.005 vs 0.05 mM Ca2+-maintained cells using Scheffe's F test; §,time-dependent significance (Kruskal–Wallis ANOVA, p<0.001) and p<0.005 versus cells at day 0 by Scheffe's F test. Journal of Investigative Dermatology 2004 122, 1207-1213DOI: (10.1111/j.0022-202X.2004.22416.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunohistochemical detection of SULT2B1b and barrier proteins in normal human skin. Tissues were stained with antibody to SULT2B1 (A), filaggrin (B) or involucrin (C). Scale bar=10 μm. Journal of Investigative Dermatology 2004 122, 1207-1213DOI: (10.1111/j.0022-202X.2004.22416.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunohistochemical detection of SULT2B1b in disorders of human skin. Tissues were stained with antibody to SULT2B1 in recessive X-linked ichthyosis (A), lichen planus (B), and psoriasis vulgaris (C). Scale bar=40 μm. Journal of Investigative Dermatology 2004 122, 1207-1213DOI: (10.1111/j.0022-202X.2004.22416.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Cholesterol sulfotransferase activity in cultures of normal human epidermal keratinocytes. Cells were treated with either 0.05 or 1.2 mM Ca2+. At the designated times, cells were detached, cytosol prepared and assayed for enzymatic activity. Control indicates activity in the absence of cytosol protein. Column heights represent mean values (n=4); error bars are indicated. *p<0.05 vs 0.05 mM Ca2+-maintained cells using Scheffe's F test. §,Time-dependent significance (Kruskal–Wallis ANOVA, p<0.005) and p<0.005 versus cells at day 3 by Scheffe's F test. Journal of Investigative Dermatology 2004 122, 1207-1213DOI: (10.1111/j.0022-202X.2004.22416.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Subcellular localization of cholesterol sulfate in culture of normal human epidermal keratinocytes. Cells were cultured for 9 d in medium containing either 0.05 or 1.2 mM Ca2+ and 20 μCi per mL Na235SO4. Membrane and cytosol fractions were prepared and assayed for cholesterol sulfate. The results are representative of two independent experiments. Journal of Investigative Dermatology 2004 122, 1207-1213DOI: (10.1111/j.0022-202X.2004.22416.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions