Skin Retinoid Concentrations Are Modulated by CYP26AI Expression Restricted to Basal Keratinocytes in Normal Human Skin and Differentiated 3D Skin Models

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Skin Retinoid Concentrations Are Modulated by CYP26AI Expression Restricted to Basal Keratinocytes in Normal Human Skin and Differentiated 3D Skin Models Ruth Heise, Jörg Mey, Mark M. Neis, Yvonne Marquardt, Sylvia Joussen, Hagen Ott, Tonio Wiederholt, Peter Kurschat, Mosaad Megahed, David R. Bickers, Hans F. Merk, Jens M. Baron Journal of Investigative Dermatology Volume 126, Issue 11, Pages 2473-2480 (November 2006) DOI: 10.1038/sj.jid.5700432 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 RT-PCR of CYP26AI expression in normal human epidermal keratinocytes and primary human dermal fibroblasts in response to all-trans RA or 4-oxo-all-trans RA. For RT-PCR analysis, NHEK (a and b) and dermal fibroblasts (c) were stimulated with 10−6m all-trans RA (a and c) or 4-oxo-all-trans RA (b) dissolved in DMSO and untreated as well as DMSO-treated cells were used as control. Cells were harvested 24hours after induction. RT-PCR was performed using specific primers for CYP26AI (5′ to 3′: sense: AGG CAC TAA AGC AAT CTT CAA; antisense: CAT GGA AAT GGG TGA ATC TTG) and β-actin (5′ to 3′: sense: CGG AAC CGC TCA TTG CC; antisense: CGG AAC CGC TCA TTG CC). PCR products of CYP26AI and β-actin as internal standard were separated on agarose gels and stained with ethidium bromide. Journal of Investigative Dermatology 2006 126, 2473-2480DOI: (10.1038/sj.jid.5700432) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Western blot analysis of CYP26AI expression in normal human epidermal keratinocytes in response to all-trans RA using different solvents. For Western blot analysis, NHEK were stimulated with 10−6m all-trans RA dissolved in DMSO (a) or ethanol (b) and, DMSO- or ethanol-treated cells were used as control. Cells were harvested at the indicated time points and total cell lysates were prepared and separated by SDS-PAGE. Western blots were developed with a generated anti-peptide antibody against the N-terminus of CYP26AI. Blots were stripped and reprobed with a primary monoclonal anti-human β-actin antibody. CYP26AI relative immunoreactivity related to β-actin immunoreativity was defined by performing a densidometric analysis. Journal of Investigative Dermatology 2006 126, 2473-2480DOI: (10.1038/sj.jid.5700432) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Immunofluorescence examination of CYP26AI expression in keratinocyte monolayers. Keratinocytes were cultivated in chamber slides and stimulated with 10−6m all-trans RA for 24 and 48hours. DMSO-treated cells were used as control. Intracellular staining of CYP26AI was performed using a generated peptide antibody specific for the detection of the N-terminus of CYP26AI. Journal of Investigative Dermatology 2006 126, 2473-2480DOI: (10.1038/sj.jid.5700432) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunofluorescence examination of human skin specimens and 3D skin models using peptide antibody specific for the N-terminus of CYP26AI. (a) Staining of normal skin, (b) skin sample taken from a patient with psoriasis, (c) 3D skin model day 7, (d) 3D skin model day 21, (e) 3D skin model day 14, and (f) 3D skin model day 14 incubated for 24hours with 0.2625% liquor carbonis detergens. ⇒ Indicates expression of CYP26AI in keratinocytes of the stratum basale. Journal of Investigative Dermatology 2006 126, 2473-2480DOI: (10.1038/sj.jid.5700432) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunohistochemistry of CYP26 expression in normal skin. (a) Overview, (b) staining of the sebaceous gland, (c) the eccrine sweat gland, and (d) the epidermis. indicates an isotypic control, ⇒ indicates expression of CYP26AI. Bar=200μm. Journal of Investigative Dermatology 2006 126, 2473-2480DOI: (10.1038/sj.jid.5700432) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions