Accepted Article Preview: Published ahead of advance online publication www.jidonline.org Cathelicidin LL-37 Induces Semaphorin 3A Expression in
Human Epidermal Keratinocytes: Implications for Possible
Application to Pruritus
Yoshie Umehara, Yayoi Kamata, Mitsutoshi Tominaga,
Franc¸ois Niyonsaba, Hideoki Ogawa, Kenji Takamori
Cite this article as: Yoshie Umehara, Yayoi Kamata, Mitsutoshi Tominaga,
Franc¸ois Niyonsaba, Hideoki Ogawa, Kenji Takamori, Cathelicidin LL-37
Induces Semaphorin 3A Expression in Human Epidermal Keratinocytes:
Implications for Possible Application to Pruritus, Journal of Investigative
Dermatology accepted article preview 29 June 2015; doi: 10.1038/jid.2015.243.
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Accepted article preview online 29 June 2015 © 2015 The Society for Investigative Dermatology 1
Journal of Investigative Dermatology (Letters to the Editor)
Cathelicidin LL-37 induces semaphorin 3A expression in human epidermal keratinocytes: implications for possible application to pruritus
Yoshie Umehara 1 , Yayoi Kamata 1 , Mitsutoshi Tominaga 1 , François Niyonsaba 2,3 ,
Hideoki Ogawa 1,2 , Kenji Takamori 1,4* 1
Institute for Environmental and Gender Specific Medicine, Juntendo University
Graduate School of Medicine, 2-1-1 Tomioka, Urayasu, Chiba 279-0021, Japan 2
Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan 3
Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku,
Tokyo 113-8421, Japan 4
Department of Dermatology, Juntendo University Urayasu Hospital, 2-1-1 Tomioka,
Urayasu, Chiba 279-0021, Japan
Short title: LL-37 induces Sema3A expression (31 characters)
Abbreviations: AD, atopic dermatitis; ELISA, enzyme-linked immunosorbent assay;
ERK, extracellular signal-regulated kinase; FPRL1, formyl peptide receptor-like 1; hBD, human β-defensin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NHEK, normal human epidermal keratinocyte; PI3K, phosphatidylinositol 3 © 2015 The Society for Investigative Dermatology 2 kinase; P2X7R, P2X7 receptor; RPS18, ribosome protein S18; Sema3A, semaphorin 3A
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TO THE EDITOR
Higher density of epidermal nerve fibers has been associated with itch sensitization in the periphery, with nerve fiber density being higher in atopic dermatitis (AD) lesional skin than in normal skin (Tominaga and Takamori, 2014). Concomitantly, the epidermal levels of the nerve repulsion factor semaphorin 3A (Sema3A) are lower in AD patients and AD model NC/Nga mice than in controls (Tominaga et al., 2008). Sema3A replacement in AD model NC/Nga mice normalized hyperinnervation, resulting in the suppression of itching (Yamaguchi et al., 2008; Negi et al., 2012).
To defend against pathogens, human skin contains antimicrobial peptides, including cathelicidins and β-defensins, which are induced by inflammation (Schauber et al., 2008; Niyonsaba et al., 2009). The induction of these peptides, such as cathelicidin
LL-37 and human β-defensins (hBDs), produced by epidermal keratinocytes is impaired in lesional skin in patients with AD, explaining their frequent infections (Ong et al., 2002; de Jongh et al., 2005). However, the interrelationships among Sema3A and antimicrobial peptides are unclear. We therefore investigated the effects of antimicrobial peptides on Sema3A expression in cultured normal human epidermal keratinocytes (NHEKs), as well as the signaling pathways involved in LL-37-induced Sema3A expression. © 2015 The Society for Investigative Dermatology 4
NHEKs derived from adult epidermis (Lonza, Basel, Switzerland) were cultured in
KBM-Gold (Lonza), a serum-free medium containing a low concentration (0.15 mM) of calcium, at 37˚C with 5% CO2 and used within 3 passages. LL-37 (L 1
LGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES 37 ) was synthesized by the solid phase method on a peptide synthesizer (model PSSM-8; Shimadzu, Kyoto, Japan).
NHEKs were incubated with hBD-1-4 (Peptide Institute, Osaka, Japan) or LL-37, and the levels of Sema3A mRNA expression at different time points were assessed by quantitative real-time PCR using SYBR Premix Ex Taq (TaKaRa, Kyoto, Japan) and the primers 5’-ACCCAACTATCAATGGGTGCCTTA-3’ (forward) and 5’-AACACTGGATTGTACATGGCTGGA-3’ (reverse). As controls, ribosome protein
S18 (RPS18) mRNA was amplified using the primers 5’-TTTGCGAGTACTCAACACCAACATC-3’ (forward) and 5’-GAGCATATCTTCGGCCCACAC-3’ (reverse).
Incubation with LL-37 for 48 hours increased Sema3A expression, whereas the individual hBDs had little effect (Figure 1a). This LL-37-induced Sema3A expression was dose- and time-dependent (Figure 1b). Furthermore, Sema3A protein levels measured using enzyme-linked immunosorbent assay (ELISA) kits (USCN Life Science,
Wuhan, China), were markedly higher in the supernatants of NHEKs incubated with © 2015 The Society for Investigative Dermatology 5
LL-37 than in control supernatants (Figure 1c).
LL-37 has been shown to activate rat mast cells via G protein-coupled receptors (Niyonsaba et al., 2001). Therefore, the effects of cholera toxin (Wako Pure Chemical
Industries, Ltd. Osaka, Japan) and pertussis toxin (Sigma-Aldrich, St Louis, MO), inhibitors of the Gs and Gi subfamilies of G protein α subunit, respectively, on Sema3A induction in cultured NHEKs were analyzed. LL-37-induced Sema3A expression was completely inhibited by pretreatment with pertussis toxin, but not cholera toxin (Figure 2a).