Role of Taurine Accumulation in Keratinocyte Hydration

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Role of Taurine Accumulation in Keratinocyte Hydration Wilfried Siefken, Stefanie Carstensen, Gunja Springmann, Klaus-Peter Wittern, Horst Wenck, Franz Stäb, Gerhard Sauermann, Volker Schreiner, Thomas Doering Journal of Investigative Dermatology Volume 121, Issue 2, Pages 354-361 (August 2003) DOI: 10.1046/j.1523-1747.2003.12366.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of TAUT in normal human skin. Human foreskin was prepared from three different donors and separated into dermis and epidermis. Proteins were extracted and separated by SDS-PAGE, and TAUT was detected by western blot analysis using a rabbit antirat TAUT antiserum. 20 μg of total protein were loaded per lane. Note that TAUT is strongly expressed in the epidermis but not in the dermis. Journal of Investigative Dermatology 2003 121, 354-361DOI: (10.1046/j.1523-1747.2003.12366.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Localization of TAUT in normal human skin. Full-thickness skin was prepared from the abdomen and paraffin-embedded thin sections were stained with a rabbit antirat TAUT antiserum. A representative phase contrast microscope image (A) and the respective fluorescence image (B) are shown. Bars: 25 μm. Journal of Investigative Dermatology 2003 121, 354-361DOI: (10.1046/j.1523-1747.2003.12366.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Regional and age-related variations in human epidermal TAUT expression. Skin samples were prepared from 17 donors. Dermis and epidermis were separated and TAUT levels were quantified by measuring chemiluminescence intensity on western blots stained with a rabbit antirat TAUT antiserum. Fetal palm skin samples were prepared after 14, 16, 20, 22, and 25 wk of gestation. Foreskin epidermis was obtained from donors between 1 and 15 y of age and abdominal skin from donors between 55 and 70 y of age. Data represent the mean±SD (fetal palm epidermis, n=5; foreskin epidermis, n=9; abdominal epidermis, n=3). Journal of Investigative Dermatology 2003 121, 354-361DOI: (10.1046/j.1523-1747.2003.12366.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Characteristics of keratinocyte taurine accumulation. NHEK were cultured for 72 h in medium containing 1.1 mM Ca2+ ions and taurine at concentrations indicated. Cellular taurine levels were quantified by HPLC analysis. (A) The concentration-dependent uptake of taurine measured under isoosmotic culture conditions (i.e., 320 mOsM). (B) The effect of increased extracellular osmolarity on taurine uptake. NHEK were cultivated for 72 h at 320 mOsM or 400 mOsM adjusted using NaCl. Data represent the mean±SD (n=6). *p<0.05. Journal of Investigative Dermatology 2003 121, 354-361DOI: (10.1046/j.1523-1747.2003.12366.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Modulation of osmotically driven taurine uptake by various osmolytes and taurine analogs. NHEK were cultured for 72 h in medium at 400 mOsM containing 1.1 mM Ca2+ ions, 150 μM taurine, and osmolytes (i.e., β-alanine, betaine, inositol, sorbitol) or taurine analogs (i.e., GES, homotaurine, N-methyl-taurine, hypotaurine). Cellular taurine levels were quantified by HPLC analysis. Data represent the mean±SD (n=6). *p<0.05. Journal of Investigative Dermatology 2003 121, 354-361DOI: (10.1046/j.1523-1747.2003.12366.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Osmotic regulation of TAUT mRNA levels. NHEK were grown to confluency in medium containing 0.1 mM Ca2+ ions. The cells were then incubated in hyperosmotic culture medium at 400 mOsM adjusted with NaCl for the indicated periods of time in the presence of either 0.1 mM Ca2+ ions (open bars) or 1.1 mM Ca2+ ions (closed bars). The cells were harvested and TAUT mRNA levels were quantified using real-time RT-PCR. The TAUT mRNA levels were normalized to the respective levels of 18S rRNA, which were quantified in parallel. Data represent the mean±SD (n=3). Journal of Investigative Dermatology 2003 121, 354-361DOI: (10.1046/j.1523-1747.2003.12366.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 TAUT and involucrin mRNA levels in keratinocytes cultivated at the air–liquid interface. Reconstructed human epidermis (EPI-201 from MatTek) was cultivated at the air–liquid interface for the periods indicated. The tissues were harvested and the levels of TAUT mRNA (closed bars) and involucrin mRNA (open bars) were quantified using real-time RT-PCR. The mRNA levels were normalized to the respective levels of 18S rRNA and to the mRNA level detected after 9 d of air-exposed culture. Data represent the mean±SD (n=3). Journal of Investigative Dermatology 2003 121, 354-361DOI: (10.1046/j.1523-1747.2003.12366.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Inhibition of osmotically induced and UV-induced apoptosis by taurine. NHEK were cultured for 48 h in the presence of 0.1 mM Ca2+ ions and 0 or 150 μM taurine. The cells were then subjected to either osmotic stress (i.e., 500 mOsM adjusted with NaCl) for 6 h or UV stress. UV irradiation was performed with an Oriel solar UV simulator equipped with a WG 320 filter at a UVB dosage of 120 mJ per cm2. UV-irradiated NHEK were cultivated for an additional 24 h prior to detection of apoptotic and necrotic cells using the Roche Annexin-V-Fluos-Staining assay. Data represent the mean±SD (n=3). Journal of Investigative Dermatology 2003 121, 354-361DOI: (10.1046/j.1523-1747.2003.12366.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions