Normalization of Epidermal Calcium Distribution Profile in Reconstructed Human Epidermis Is Related to Improvement of Terminal Differentiation and Stratum.

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Normalization of Epidermal Calcium Distribution Profile in Reconstructed Human Epidermis Is Related to Improvement of Terminal Differentiation and Stratum Corneum Barrier Formation Jana Vičanová, Esther Boelsma, A. Mieke Mommaas, Johanna A. Kempenaar, Bo Forslind, Jan Pallon, Torbjörn Egelrud, Henk K. Koerten, Maria Ponec Journal of Investigative Dermatology Volume 111, Issue 1, Pages 97-106 (July 1998) DOI: 10.1046/j.1523-1747.1998.00251.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Ca2+ distribution gradient in native human skin reaches the highest levels in the uppermost granular cells. The Ca2+ was localized by the oxalate-pyroanimonate precipitation method. On the transmission electron micrographs, the Ca2+-containing precipitates appear as electron dense granules or aggregates. (a) The Ca2+-containing deposits (arrows) were localized within the cytoplasm of granular cells. The SC contains small quantities of Ca2+ deposits in the lowermost layers; scale bar, 0.5 μm. (b) In one of the six specimens analyzed, Ca2+-containing precipitates were found higher in the SC; scale bar, 0.4 μm. (c) Occasionally, labeled desmosomes (arrowheads) were found in the inner SC; scale bar, 0.3 μm. Journal of Investigative Dermatology 1998 111, 97-106DOI: (10.1046/j.1523-1747.1998.00251.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Incomplete terminal differentiation of keratinocytes grown in serum-containing medium is associated with accumulation of Ca2+ within corneocytes throughout the entire SC. (a) The thick and highly compact SC contains lipid droplets (asterisks) and fragments of cytoplasmic organelles (white arrow). A low number of keratohyalin granules (black arrow) is present in the SG. Nu, nucleus; scale bar, 8 μm. (b) Ca2+-containing precipitates present in the upper SG and SC are localized mainly in the intracellular matrix (arrows). Small arrowheads mark keratohyalin granules; scale bar, 2 μm. (c) In the SC, Ca2+ deposits are associated within corneocytes, extracellularly to desmosomes (white arrowheads), but not with lipid droplets (asterisk). Immature desmosomes form long connections (black arrowheads); scale bar, 0.5 μm. Journal of Investigative Dermatology 1998 111, 97-106DOI: (10.1046/j.1523-1747.1998.00251.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 SC structure and Ca2+ distribution profile in RE generated in serum-free medium are similar to those in the native skin. (a) The number of interconnected keratohyalin granules (arrowheads) and the normal SC ultrastructure suggests an improved terminal differentiation. Nu, nucleus; scale bar, 3 μm. (b) Ca2+-containing precipitates (arrows) are abundantly present in the upper SG. The SC contains little precipitate in the inner part (unstained section); scale bar, 1 μm. (c) Cells of 2–3 uppermost layers of the SC formed during the first days of culture are incompletely differentiated and contain Ca2+ deposits (arrows); scale bar, 0.4 μm. Journal of Investigative Dermatology 1998 111, 97-106DOI: (10.1046/j.1523-1747.1998.00251.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Deregulation of epidermal differentiation by retinoic acid results in the re-appearance of calcium-containing precipitates within the SC. (a) The SC consists of incompletely differentiated cells; asterisks, lipid droplets; scale bar, 3 μm. (b) The Ca2+-containing precipitates were localized in the cytoplasm of granular cells (arrow). In the SC, Ca2+ deposits were found mainly associated with fragments of organelles (black arrowheads) and with desmosomes (white arrowhead); scale bar, 0.8 μm. (c) Ca2+-labeled membrane structures (black arrowheads) and Ca2+-containing precipitates present free in the intracellular matrix (arrow) of SC cells; scale bar, 0.4 μm. Journal of Investigative Dermatology 1998 111, 97-106DOI: (10.1046/j.1523-1747.1998.00251.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Proton probe analysis demonstrates a comparable calcium concentration in both native epidermis and RE. The distribution curves of calcium and phosphorus over skin cross-section (0–100 μm from the skin surface) were obtained using the proton microprobe (particle induced X-ray emission). The presence of phosphorus in the epidermis indicates the site of epidermal strata containing nucleated cells. (a) The calcium concentration reaches a maximum in the SG in native epidermis. (b) The calcium distribution profile in RE generated in serum-free medium is similar to that in native epidermis. Journal of Investigative Dermatology 1998 111, 97-106DOI: (10.1046/j.1523-1747.1998.00251.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 RE contains high levels of pro-SCCE, but a relatively small fraction of the total SCCE is in the activated form. (a) Zymogram in 10% polyacryl-amide gel with 1% casein. Extracts (1–10 μl) derived from RE cultured in serum-free medium (RE) and plantar SC (SC) were applied. Arrowhead indicates SCCE. (b) Immunoblot after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions with SCCE-specific antibodies, 10 μl of each extract was applied. Molecular mass markers are to the right. (c) Magnification of the immunoblot. 1, Glycosylated pro-SCCE; 2, mixture of unglycosylated pro-SCCE and glyco-sylated active SCCE; 3, unglyco-sylated active SCCE. Journal of Investigative Dermatology 1998 111, 97-106DOI: (10.1046/j.1523-1747.1998.00251.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Normalization of penetration pathways is suggestive for improved barrier function in RE generated in serum-free medium. Fluorescent images of diffusion pathways across the SC in native epidermis (a–c) and RE (d–f) as visualized by confocal laser scanning microscopy 30 min after the application of 10 mM NR in dimethyl sulfoxide. The images were obtained by optical sectioning at 10 (a, d), 20 (b, e), and 30 μm (c, f), proceeding downwards from the outer surface. Scale bar, 10 μm. Note that the difference in the thickness of the SC obtained by transmission electron microscopy and confocal laser scanning microscopy can be most probably ascribed to the state of hydration of the tissue specimen. Journal of Investigative Dermatology 1998 111, 97-106DOI: (10.1046/j.1523-1747.1998.00251.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions