Both Sphingosine Kinase 1 and 2 Coordinately Regulate Cathelicidin Antimicrobial Peptide Production during Keratinocyte Differentiation  Kyong-Oh Shin, Kun Pyo Kim, Yunhi Cho, Min-Kyung Kang, Young-Hee Kang, Yong-Moon Lee, Hiroko Ikushiro, Mami Yokota, Takato Yano, Sung Jay Choe, Eung Ho Choi, Chae Jin Lim, Keedon Park, Walter M. Holleran, Kyungho Park, Yoshikazu Uchida  Journal of Investigative Dermatology  Volume 139, Issue 2, Pages 492-494 (February 2019) DOI: 10.1016/j.jid.2018.08.015 Copyright © 2018 The Authors Terms and Conditions

Figure 1 CAMP expression is increased by the actions of KCs of SPHK1 and SPHK2 during KC differentiation. S1P (a, h) and DHS1P levels (h, n), CAMP mRNA (b, j, m), and protein (c, k) were assessed by LC-ESI-MS/MS, quantitative real-time reverse transcriptase–PCR and Western blot analysis, respectively. LC-MS/MS spectra and chromatogram of both S1P and DHS1P are illustrated in Supplementary Figures S3 and S4 online, respectively. Both intact and/or phosphorylated form of SPHK1 and SPHK2 in cells or human skin tissues were assessed by Western blot (d) or immunohistochemistry (g) analyses. SPHK1 (e) and SPHK2 (f) activities were determined by LC-ESI-MS/MS. KC were overexpressed with pSPHK1-Myc-wt, pSPHK2-HA-wt and pSPHK2-HA-dn (h, j–l). The intensity of CAMP protein from experiment of (k) was quantified and the integrated areas were normalized to the corresponding value of β-actin (l). KCs were incubated with ABC294640 (0.5–5 μM), SPHK2-specific inhibitor, for 24 hours (m). CAMP, cathelicidin antimicrobial peptide; dn, dominant negative; DHS1P, dihydrosphingosine-1-phosphate; EDK, early stage of differentiated keratinocyte; KC, keratinocyte; LC-ESI-MS/MS, liquid chromatography-electrospray ionization-tandem mass spectrometry; LDK, late stage of differentiated keratinocyte; UDK, undifferentiated keratinocyte. Scale bar = 10 μm. Journal of Investigative Dermatology 2019 139, 492-494DOI: (10.1016/j.jid.2018.08.015) Copyright © 2018 The Authors Terms and Conditions