Cathelicidin LL-37 Induces Semaphorin 3A Expression in Human Epidermal Keratinocytes: Implications for Possible Application to Pruritus

Abstract

atopic dermatitis normal human epidermal keratinocyte P2X7 receptor semaphorin 3A 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, 2014Tominaga M. Takamori K. Itch and nerve fibers with special reference to atopic dermatitis: therapeutic implications.J Dermatol. 2014; 41: 205-212Crossref PubMed Scopus (141) Google Scholar). 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., 2008Tominaga M. Ogawa H. Takamori K. Decreased production of semaphorin 3A in the lesional skin of atopic dermatitis.Br J Dermatol. 2008; 158: 842-844Crossref PubMed Scopus (86) Google Scholar). Sema3A replacement in AD model NC/Nga mice normalized hyperinnervation, resulting in the suppression of itching (Yamaguchi et al., 2008Yamaguchi J. Nakamura F. Aihara M. et al.Semaphorin3A alleviates skin lesions and scratching behavior in NC/Nga mice, an atopic dermatitis model.J Invest Dermatol. 2008; 128: 2842-2849Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar; Negi et al., 2012Negi O. Tominaga M. Tengara S. et al.Topically applied semaphorin 3A ointment inhibits scratching behavior and improves skin inflammation in NC/Nga mice with atopic dermatitis.J Dermatol Sci. 2012; 66: 37-43Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To defend against pathogens, human skin contains antimicrobial peptides, including cathelicidins and β-defensins, which are induced by inflammation (Schauber and Gallo, 2008Schauber J. Gallo R.L. Antimicrobial peptides and the skin immune defense system.J Allergy Clin Immunol. 2008; 122: 261-266Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar; Niyonsaba et al., 2009Niyonsaba F. Nagaoka I. Ogawa H. et al.Multifunctional antimicrobial proteins and peptides: natural activators of immune systems.Curr Pharm Des. 2009; 15: 2393-2413Crossref PubMed Scopus (75) Google Scholar). The induction of these peptides, such as cathelicidin LL-37 and human β-defensins, produced by epidermal keratinocytes is impaired in lesional skin in patients with AD, explaining their frequent infections (Ong et al., 2002Ong P.Y. Ohtake T. Btandt C. et al.Endogenous antimicrobial peptides and skin infections in atopic dermatitis.N Engl J Med. 2002; 347: 1151-1160Crossref PubMed Scopus (1638) Google Scholar; de Jongh et al., 2005de Jongh G.J. Zeeuwen P.L. Kucharekova M. et al.High expression levels of keratinocyte antimicrobial proteins in psoriasis compared with atopic dermatitis.J Invest Dermatol. 2005; 125: 1163-1173Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). 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. 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 three passages. LL-37 (L1LGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES37) was synthesized by the solid phase method on a peptide synthesizer (model PSSM-8; Shimadzu, Kyoto, Japan). NHEKs were incubated with human β-defensin-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 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 human β-defensins had little effect (Figure 1a). This LL-37-induced Sema3A expression was dose- and time dependent (Figure 1b). Furthermore, Sema3A protein levels measured using ELISA kits (USCN Life Science, Wuhan, China) were markedly higher in the supernatants of NHEKs incubated with 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., 2001Niyonsaba F. Someya A. Hirata M. et al.Evaluation of the effects of peptide antibiotics human β-defensins-1/-2 and LL-37 on histamine release and prostaglandin D2 production from mast cells.Eur J Immunol. 2001; 31: 1066-1075Crossref PubMed Scopus (289) Google Scholar). 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). Activation of either mitogen-activated protein kinase or phosphatidylinositol 3 kinase was found to be involved in the LL-37 signaling pathway (Niyonsaba et al., 2010Niyonsaba F. Ushio H. Hara M. et al.Antimicrobial peptides human β-defensins and cathelicidin LL-37 induce the secretion of a pruritogenic cytokine IL-31 by human mast cells.J Immunol. 2010; 184: 3526-3534Crossref PubMed Scopus (211) Google Scholar). We therefore assessed the effects of mitogen-activated protein kinase and phosphatidylinositol 3 kinase inhibitors on Sema3A induction in cultured NHEKs. LL-37-induced Sema3A expression was inhibited by pretreatment with the extracellular signal-regulated kinase 1/2 inhibitor PD98059 (Cell Signaling Technology, Beverly, MA), but not the phosphatidylinositol 3 kinase inhibitor wortmannin (Sigma-Aldrich), the p38 inhibitor SB203580 (Sigma-Aldrich), and the c-Jun N-terminal kinase inhibitor SP600125 (Calbiochem, Darmstadt, Germany; Figure 2b). We next examined whether formyl peptide receptor-like 1 and P2X7 receptor (P2X7R), both candidate LL-37 receptors (Niyonsaba et al., 2009Niyonsaba F. Nagaoka I. Ogawa H. et al.Multifunctional antimicrobial proteins and peptides: natural activators of immune systems.Curr Pharm Des. 2009; 15: 2393-2413Crossref PubMed Scopus (75) Google Scholar), are involved in Sema3A induction in cultured NHEKs. LL-37-induced Sema3A expression was partially inhibited by pretreatment with the P2X7R antagonist BBG (Tokyo Chemical Industry, Tokyo, Japan) but not by pretreatment with the formyl peptide receptor-like 1 antagonist WRW4 (ABGENT, San Diego, CA; Figure 2c). Taken together, these findings showed that the antimicrobial peptide LL-37 markedly upregulated the expression of Sema3A mRNA and protein in cultured human keratinocytes, whereas the four human β-defensins were inactive (Figure 1), indicating that LL-37 is an endogenous inducer of Sema3A expression in keratinocytes. Vitamin D3, which increases cathelicidin expression in keratinocytes (Schauber and Gallo, 2008Schauber J. Gallo R.L. Antimicrobial peptides and the skin immune defense system.J Allergy Clin Immunol. 2008; 122: 261-266Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), might therefore also drive the LL-37-Sema3A pathway. We found that certain Gi-coupled receptors are involved in Sema3A induction (Figure 2a). Although LL-37 has been shown to activate G protein–coupled receptors, such as formyl peptide receptor-like 1, P2X7R, and Mas-related G protein–coupled receptor X2 (MrgX2; Barlow et al., 2014Barlow P.G. Findlay E.G. Currie S.M. et al.Antiviral potential of cathelicidins.Future Microbiol. 2014; 9: 55-73Crossref PubMed Scopus (58) Google Scholar), LL-37 receptors are less well-characterized in human keratinocytes. We showed that LL-37-induced Sema3A expression was partly inhibited by a P2X7R antagonist, but not by a formyl peptide receptor-like 1 antagonist (Figure 2c), and that MrgX2 mRNA expression was undetectable in NHEKs (Umehara et al., unpublished observation). These findings suggest that, although other receptors may be involved in induction, LL-37 activation of P2X7R is at least partly involved in Sema3A induction in keratinocytes. We also found that LL-37-induced Sema3A expression was completely inhibited by PD98059 (Figure 2b), indicating that extracellular signal-regulated kinase 1/2 signaling may be required for Sema3A induction. In contrast, SP600125 increased rather than inhibiting Sema3A expression (Figure 2b), suggesting that c-Jun N-terminal kinase signaling is involved in the suppression of Sema3A expression in keratinocytes. Altered c-Jun N-terminal kinase signaling might reduce Sema3A production in AD. Taken together, these findings suggest that LL-37 may bind to certain Gi-coupled receptors, including P2X7R, activating the extracellular signal-regulated kinase 1/2 signaling pathway, and finally inducing Sema3A expression in human epidermal keratinocytes (Figure 2d). Although 70–90% of patients with psoriasis also have pruritus, they are rarely accompanied by intense itch as in AD (Yosipovitch et al., 2000Yosipovitch G. Goon A. Wee J. et al.The prevalence and clinical characteristics of pruritus among patients with extensive psoriasis.Br J Dermatol. 2000; 143: 969-973Crossref PubMed Scopus (344) Google Scholar; Reich and Szepietowski, 2007Reich A. Szepietowski J.C. Mediators of pruritus in psoriasis.Mediators Inflamm. 2007; 2007: 64727Crossref PubMed Scopus (58) Google Scholar). We previously showed that Sema3A production was not reduced in the epidermis of psoriatic patients with or without itch (Taneda et al., 2011Taneda K. Tominaga M. Negi O. et al.Evaluation of epidermal nerve density and opioid receptor levels in psoriatic itch.Br J Dermatol. 2011; 165: 277-284Crossref PubMed Scopus (82) Google Scholar). In contrast to AD, high levels of expression of several antimicrobial peptides including LL-37 were observed in psoriatic lesions (Ong et al., 2002Ong P.Y. Ohtake T. Btandt C. et al.Endogenous antimicrobial peptides and skin infections in atopic dermatitis.N Engl J Med. 2002; 347: 1151-1160Crossref PubMed Scopus (1638) Google Scholar; Nomura et al., 2003Nomura I. Goleva E. Howell M.D. et al.Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes.J Immunol. 2003; 171: 3262-3269Crossref PubMed Scopus (644) Google Scholar), suggesting that epidermal Sema3A expression is not reduced in these patients. These findings suggest that LL-37 may restore Sema3A production in keratinocytes of certain pathological conditions such as psoriasis and that the decreased epidermal Sema3A production in AD lesional skin may be partly caused by a breakdown in this recovery system. Thus, the topical application of LL-37, which not only has antimicrobial activity but also enhances Sema3A production, might be a useful therapeutic strategy for AD. The authors state no conflict of interest. This work was partly supported by KAKENHI (Grant number 26860898) and by a grant of Strategic Research Foundation Grant-aided Project for Private Universities from MEXT (Grant number S1311011).