Vitamin D Enhances ALA-Induced Protoporphyrin IX Production and Photodynamic Cell Death in 3-D Organotypic Cultures of Keratinocytes  Nobuyuki Sato, Brian W. Moore, Samantha Keevey, Judy A. Drazba, Tayyaba Hasan, Edward V. Maytin  Journal of Investigative Dermatology  Volume 127, Issue 4, Pages 925-934 (April 2007) DOI: 10.1038/sj.jid.5700595 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Effects of Vit D and RA upon cell growth, differentiation, and PpIX production in keratinocyte monolayer cultures. (a) Drop cultures were grown for 3 days in the presence of either Vit D (VD) or RA on 60mm dishes at the molar concentration indicated, stained with hematoxylin, and photographed at low magnification on a light box. Bar=1cm. (b) Graph of the surface areas of drop cultures in two experiments using Vit D and two experiments using RA. Data are mean±SD of 5–10 cultures per condition (arbitrary units (a.u.)). Vitamin concentration on the abscissa is plotted on a logarithmic scale. (c–e) Phase-contrast microscope images of hematoxylin-stained drop cultures, taken at a location midway between the edge and the center of each culture. Arrows, examples of stratifying islands. (f) Graph of the relative change in the number of stratifying (cornified) islands at different concentrations of Vit D or RA. Data are the mean±SD of areas, traced out on 6–10 digital images. (g–j) Confocal fluorescence micrographs of living cell monolayers incubated for 4hours in the presence of ALA (1mM), taken under conditions that specifically detect PpIX (see Materials and Methods). (k) Phase-contrast image of the same field as in panel j, to demonstrate bright PpIX signals corresponding to stratifying islands (arrows) within the cultures. Bar=200μm. P-values of Student's t-test, difference from control: *P<0.01; **P<0.005; ***P<0.0005. Journal of Investigative Dermatology 2007 127, 925-934DOI: (10.1038/sj.jid.5700595) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Vit D effects upon markers of proliferation and differentiation in 3-D organotypic keratinocyte cultures. Cells were either untreated (−VD) or treated (+VD) with Vit D at 0.1nM. (a, b) H&E stains, showing significant thickening of the stratum corneum with Vit D. Vertical brackets, stratum corneum. (c, d) Stained with antisera to PCNA; no change with Vit D is observed. (e, f) Stained with antisera to keratin 10 (K10); an increase in expression is observed with Vit D. (g, h) Stained with antisera to loricrin; a large increase in signal intensity is seen with Vit D. Dashed line, epidermal–dermal junction. Dotted line, top of stratum corneum. Bar=25μm. Journal of Investigative Dermatology 2007 127, 925-934DOI: (10.1038/sj.jid.5700595) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Vit D effects upon proliferation and differentiation in organotypic keratinocyte cultures; semiquantitative image analysis. Multiple histological sections, stained as in Figure 2, were subjected to digital image capture and measurements of integrated intensity (pixel-by-pixel) as described in Materials and Methods. Graphs summarize the following: (a) proliferative index, that is, percentage of cells that stain positively with an anti-PCNA antibody. No significant change is seen with Vit D (VD). Mean±SD, six images per condition. (b) Relative stratum corneum thickness as a function of Vit D concentration. Significant increases are seen with Vit D. Mean±SD, data pooled from two experiments, two cultures/experiment, six images per culture. (c) K10 expression (control versus Vit D at 0.1μM). Mean±SD, data pooled from two independent cultures per condition, eight images per culture. (d) Loricrin expression (control versus Vit D at 0.1μM). Mean±SD, data pooled from two independent cultures per condition, eight images per culture. P-values of Student's t-test, difference from control: *P<0.05; **P<0.005; ***P<0.001. Journal of Investigative Dermatology 2007 127, 925-934DOI: (10.1038/sj.jid.5700595) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effects of Vit D upon PpIX levels in the organotypic keratinocyte cultures. (a–e) Fluorescent confocal images of REKs incubated 4 days in the presence of various Vit D doses, ALA 0.1mM added in the final 2hours, and frozen sections analyzed by confocal microscopy. PpIX signal resulting from excitation at 635nm, with emission at 670–700nm, is shown in gray scale. Dashed line, epidermal–dermal junction. Dotted line, top of stratum corneum. Bar=50μm. (f) Dose–response curve of PpIX levels (arbitrary fluorescence intensity units) as a function of Vit D concentration. For all datapoints, the cultures were incubated for 4 days in the presence of Vit D at the indicated concentration, given ALA (1mM) for 4hours, then harvested and PpIX levels measured by confocal fluorescence microscopy of frozen sections. Data are pooled from three independent experiments, normalized to the control (no Vit D) levels of PpIX, mean±SEM. P-values of Student's t-test, difference from control: *P<0.05; **P<0.01; ***P<0.005. Solid circle, no ALA. Journal of Investigative Dermatology 2007 127, 925-934DOI: (10.1038/sj.jid.5700595) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Vit D preconditioning enhances photodynamic killing in organotypic cultures exposed to ALA and visible light. REK lift cultures were cultured for 4 days in the (a, c, e, g, i, k) absence or (b, d, f, h, j, l) presence of 0.1μM Vit D. After addition of ALA (1mM) for 4hours, the cultures were exposed to 635nm visible light at the doses indicated, then returned to the incubator for 24hours before fixation and paraffin embedding. Apoptotic cells (arrows), areas of liquefactive degeneration (boxes), and areas of acantholytic blistering (asterisks) are seen. (k, l) Cultures exposed to 82mJ/cm2 of light in the absence of ALA. Bar=50μm. Journal of Investigative Dermatology 2007 127, 925-934DOI: (10.1038/sj.jid.5700595) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of activated Casp3 in organotypic keratinocyte cultures, following ALA treatment and exposure to light. Cultures were preconditioned in the (a, c, e) absence or (b, d, f) presence of Vit D for 4 days, then exposed to visible light at the doses indicated and incubated a further 24hours before fixation and paraffin embedding. Histological sections were stained with an antiserum that specifically recognizes activated Casp3 (bright signal). Despite a clear positive correlation between irradiation dose and the number of Casp3-positive cells, there is no apparent difference between Vit D-preconditioned and unconditioned cultures. Bar=50μm. Journal of Investigative Dermatology 2007 127, 925-934DOI: (10.1038/sj.jid.5700595) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions