Phytosphingosine Derivatives Ameliorate Skin Inflammation by Inhibiting NF-κB and JAK/STAT Signaling in Keratincoytes and Mice

      Phytosphingosine is abundant in plants and fungi and is found in mammalian epidermis, including the stratum corneum. Phytosphingosine and its derivatives N-acetyl phytosphingosine and tetraacetyl phytosphingosine are part of the natural defense system of the body. However, these molecules exhibit strong toxicities at high concentrations. We synthesized phytosphingosine derivatives, mYG-II-6 ((Z)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid) and fYG-II-6 ((E)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid), to increase efficacy and decrease toxicity, and the biological activities of the derivatives in the inflammatory response were examined. Both YG-II-6 compounds effectively suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced inflammatory skin damage and inflammatory response in a mouse model. In addition, topical application of fYG-II-6 suppressed ear swelling and psoriasiform dermatitis in the ears of IL-23-injected mice. Anti-inflammatory and antipsoriatic activities of the phytosphingosine derivatives inhibited NF-κB, JAK/signal transducer and activator of transcription (JAK/STAT), and mitogen-activated protein kinase (MAPK) signaling. Finally, the YG-II-6 compounds induced programmed cell death in keratinocytes and mouse skin and were less toxic than phytosphingosine. Our study demonstrated that the phytosphingosine-derived YG-II-6 compounds have much stronger biological potencies than the lead compounds. The YG-II-6 compounds ameliorated inflammatory skin damage. Thus, YG-II-6 compounds are potential topical agents for treating chronic inflammatory skin diseases, such as psoriasis.

      Abbreviations

      fYG-II-6
      (E)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid
      MAPK
      mitogen-activated protein kinase
      mYG-II-6
      (Z)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid
      PBMC
      peripheral blood mononuclear cell
      STAT
      signal transducer and activator of transcription
      TPA
      12-O-tetradecanoylphorbol-13-acetate

      INTRODUCTION

      The skin is the outermost organ of the human body and serves as a primary wall-like barrier against environmental substances, such as viruses, bacteria, UV radiation, toxic agents, injury, and mechanical insults (
      • Chuong C.M.
      • Nickoloff B.J.
      • Elias P.M.
      • et al.
      What is the ‘true’ function of skin?.
      ;
      • Tobin D.J.
      Biochemistry of human skin—our brain on the outside.
      ). The outermost viable layer of the skin is the epidermis, which has a highly active lipid-synthesizing tissue consisting of several distinct layers. The stratum corneum is the outer layer of the epidermis and is composed of corneocytes embedded in lamellar lipid regions. The stratum corneum also contains extracellular lipid membranes that are primarily composed of cholesterol, free fatty acids, and ceramides with small amounts of cholesterol esters and cholesterol sulfates (
      • Coderch L.
      • López O.
      • de la Maza A.
      • et al.
      Ceramides and skin function.
      ). Most of these lipids are derived from the lamellar bodies and synthesized in the keratinocytes of the upper stratum spinosum and stratum granulosum. Lipid synthesis is very active in the epidermis. The function of the skin as a protective barrier is regulated by synthesis of these lipids (
      • Lee S.H.
      • Jeong S.K.
      • Ahn S.K.
      An update of the defensive barrier function of skin.
      ;
      • Feingold K.R.
      The outer frontier: the importance of lipid metabolism in the skin.
      ). Lipids are organized into broad lipid bilayers in the intercorneocyte spaces. Mammalian skin lipid bilayers play essential roles in preventing water loss and maintaining moisture, maintaining a pliable skin barrier, and forming a permeability barrier from the environment (
      • Pappas A.
      Epidermal surface lipids.
      ).
      Phytosphingosine and its derivatives are known to prevent loss of moisture from the skin, regulate epidermal cell growth, differentiation, and apoptosis, and possess bactericidal and anti-inflammatory activities (
      • Kim H.J.
      • Kim H.J.
      • Kim S.H.
      • et al.
      Tetraacetyl phytosphingosine-induced caspase activation and apoptosis occur through G2 arrest in human keratinocyte HaCaT cells.
      • Kim H.J.
      • Shin W.
      • Park C.S.
      • et al.
      Differential regulation of cyclooxygenase-2 expression by phytosphingosine derivatives, NAPS and TAPS, and its role in the NAPS or TAPS-mediated apoptosis.
      • Kim H.J.
      • Kang S.Y.
      • Kim S.J.
      • et al.
      Potentiation of UVB-induced apoptosis by novel phytosphingosine derivative, tetraacetyl phytosphingosine in HaCaT cell and mouse skin.
      • Kim S.
      • Hong I.
      • Hwang J.S.
      • et al.
      Phytosphingosine stimulates the differentiation of human keratinocytes and inhibits TPA-induced inflammatory epidermal hyperplasia in hairless mouse skin.
      ;
      • Pavicic T.
      • Wollenweber U.
      • Farwick M.
      • et al.
      Anti-microbial and -inflammatory activity and efficacy of phytosphingosine: an in vitro and in vivo study addressing acne vulgaris.
      ;
      • Fischer C.L.
      • Drake D.R.
      • Dawson D.V.
      • et al.
      Antibacterial activity of sphingoid bases and fatty acids against Gram-positive and Gram-negative bacteria.
      ). Thus, phytosphingosine and its derivatives are important for maintaining healthy skin. The antibacterial and anti-inflammatory activities of phytosphingosine were clearly demonstrated in a clinical study in which acne patients were treated with a variety of products such as skin and hair conditioning agents that are currently used to treat acne (
      • Pavicic T.
      • Wollenweber U.
      • Farwick M.
      • et al.
      Anti-microbial and -inflammatory activity and efficacy of phytosphingosine: an in vitro and in vivo study addressing acne vulgaris.
      ). Therefore, phytosphingosine and its derivatives are associated with the natural defense system of the body. Although phytosphingosine and its derivatives possess beneficial biological activities, these compounds cannot be used at high concentrations because of toxicities (
      • Farwick M.
      • Watson R.E.
      • Rawlings A.V.
      • et al.
      Salicyloyl-phytosphingosine: a novel agent for the repair of photoaged skin.
      ;
      • Snel M.
      • Sleddering M.A.
      • Pijl H.
      • et al.
      The effect of dietary phytosphingosine on cholesterol levels and insulin sensitivity in subjects with the metabolic syndrome.
      ;
      • Song C.K.
      • Lee J.H.
      • Jahn A.
      • et al.
      In vitro and in vivo evaluation of N,N,N-trimethylphytosphingosine-iodide (TMP) in liposomes for the treatment of angiogenesis and metastasis.
      ).
      In this study, we synthesized the phytosphingosine derivatives, mYG-II-6 ((Z)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid) and fYG-II-6 ((E)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid), by combining phytosphingosine with either maleic anhydride or (E)-4-ethoxy-4-oxobut-2-enoic acid to reduce the toxicity and increase the biological activity of phytosphingosine. We examined the anti-inflammatory activities of these compounds in 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced human keratinocytes or cytokine-induced human peripheral blood mononuclear cells (PBMCs), mouse T cells, rat pre-T-lymphoma cells, and in cutaneous inflammatory models in mice.

      RESULTS

       Spectral analysis

      We synthesized two YG-II-6 compounds, mYG-II-6 and fYG-II-6, from phytosphingosine and maleic anhydride or (E)-4-ethoxy-4-oxobut-2-enoic acid (Figure 1a), respectively, as described in the Materials and Methods section. The proton nuclear magnetic resonance (1H-NMR) data for mYG-II-6 were 1H-NMR (DMSO-d6, 400 MHz) δ 15.74 (s, 1H), 9.23 (br s, 1H), 6.52 (br s, 1H), 6.24 (d, J=12.7 Hz, 1H), 4.81 (d, J=6.0 Hz, 1H), 4.61 (s, 1H), 4.46 (d, J=6.3 Hz, 1H), 4.13-4.05 (m, 1H), 3.71-3.63 (m, 1H), 3.56-3.49 (m, 1H), 1.56-1.36 (m, 2H), 1.32-1.14 (m, 24H), and 0.87-0.82 (m, 3H). The 1H-NMR data for fYG-II-6 were 1H-NMR (DMSO-d6, 400 MHz) δ 12.75 (br s, 1H), 8.28 (d, J=8.8 Hz, 1H), 7.05 (d, J=15.8 Hz, 1H), 6.50 (d, J=15.4 Hz, 1H), 4.66 (d, J=5.6 Hz, 1H), 4.54 (dd, J=5.08, 5.16 Hz, 1H), 4.05-3.88 (m, 1H), 3.63-3.60 (m, 1H), 3.54-3.48 (m, 1H), 3.41-3.37 (m, 2H), 3.26-3.23 (m, 1H), 1.53-1.41 (m, 3H), 1.23 (s, 24H), and 0.85 (t, J=6.2 Hz, 3H).
      Figure thumbnail gr1
      Figure 1YG-II-6 compounds inhibit TPA-induced NF-κB signaling. (a) YG-II-6 compounds were synthesized from phytosphingosine and either fumaric acid ethyl ester or maleic anhydride. (b) HaCaT cells were preincubated with compound for 1 hour and stimulated with TPA (100 nM). Western blot analysis was performed. (c) NF-κB luciferase activity was measured in TPA-stimulated HaCaT cells. Results are shown as the mean± SEM. #P<0.001 versus empty vector-transfected group, **P<0.001 versus NF-κB reporter-transfected group. (d) NF-κB nuclear translocation was examined in TPA-stimulated HaCaT cells. (e) Western blot analysis was performed on the skin of C57BL/6 mice as described in the Materials and Methods. DMAP, 4-dimethylaminopyridine; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; FA, fumaric acid ethyl ester; fYG-II-6, (E)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MA, maleic anhydride; mYG-II-6, (Z)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid; PS, phytosphingosine; TPA, 12-O-tetradecanoylphorbol-13-acetate.

       YG-II-6 compounds inhibit NF-κB signaling

      We first examined NF-κB signaling because phytosphingosine exhibits antiscratching, antimicrobial, and anti-inflammatory activities in histamine- or TPA-induced mouse skin and human skin (
      • Kim S.
      • Hong I.
      • Hwang J.S.
      • et al.
      Phytosphingosine stimulates the differentiation of human keratinocytes and inhibits TPA-induced inflammatory epidermal hyperplasia in hairless mouse skin.
      ;
      • Pavicic T.
      • Wollenweber U.
      • Farwick M.
      • et al.
      Anti-microbial and -inflammatory activity and efficacy of phytosphingosine: an in vitro and in vivo study addressing acne vulgaris.
      ;
      • Ryu K.R.
      • Lee B.
      • Lee I.A.
      • et al.
      Anti-scratching behavioral effects of N-stearoyl-phytosphingosine and 4-hydroxysphinganine in mice.
      ). Both YG-II-6 compounds effectively inhibited phosphorylation of IκBα and NF-κB p65 and degradation of IκBα in TPA-stimulated human keratinocytes (Figure 1b). The YG-II-6 compounds also strongly inhibited NF-κB reporter activity and nuclear translocation of NF-κB p65 in TPA-stimulated keratinocytes (Figure 1c and d). In addition, we observed that both YG-II-6 compounds significantly inhibited phosphorylation of IκBα and NF-κB p65 in mouse skin treated with a double application of TPA. In contrast, the lead compounds, including phytosphingosine, maleic anhydride, and fumaric acid, exhibited weak or no inhibitory effects on NF-κB signaling in TPA-induced keratinocytes and mouse skin (Figure 1e).

       YG-II-6 compounds inhibit JAK/STAT signaling

      Phytosphingosine is a class of sphingolipids that regulate the T cell–mediated immune response (
      • Walsh K.B.
      • Teijaro J.R.
      • Wilker P.R.
      • et al.
      Suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus.
      ;
      • Paget C.
      • Chow M.T.
      • Duret H.
      • et al.
      Role of γδ T cells in α-galactosylceramide-mediated immunity.
      ;
      • Zygmunt B.M.
      • Weissmann S.F.
      • Guzman C.A.
      NKT cell stimulation with α-galactosylceramide results in a block of Th17 differentiation after intranasal immunization in mice.
      ). The cytokine-mediated JAK/STAT signaling pathway also regulates the immune response (
      • Shuai K.
      • Liu B.
      Regulation of JAK-STAT signalling in the immune system.
      ). Thus, we examined whether the YG-II-6 compounds could regulate JAK/STAT signaling. Both YG-II-6 compounds inhibited phosphorylation of the JAK and STAT proteins in TPA- or IFN-γ-stimulated human keratinocytes (Figure 2a). In addition, both compounds effectively inhibited cytokine-induced phosphorylation of STAT1, STAT3, and STAT5 in mouse T cells (Figure 2b and c) and human PBMCs (Figure 2d and e).
      Figure thumbnail gr2
      Figure 2YG-II-6 compounds inhibit JAK/STAT signaling. (a) HaCaT cells were stimulated with either TPA (400 nM) for 3 hours or IFN-γ (200 U ml−1) for 10 minutes followed by treatment with YG-II-6 compound for 1 hour. (b–e) Mouse T cells (b c) or human PBMCs (d e) were stimulated with IL-6 (10 ng ml−1), IFN-γ (200 U ml−1), or IL-2 (100 ng ml−1) for 10 minutes followed by treatment with YG-II-6 compound for 1 hour, respectively. (f) Rat pre-T-lymphoma Nb2 cells were starved for 16 hours in the presence of DMSO or each compound and subsequently induced with either prolactin (PRL; 100 ng ml−1) or IL-2 (100 ng ml−1) for 10 minutes. Whole-cell extracts were subjected to western blot analysis with antibodies specific for the indicated molecules. fYG-II-6, (E)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mYG-II-6, (Z)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid; PBMC, peripheral blood mononuclear cell; STAT, signal transducer and activator of transcription; TPA, 12-O-tetradecanoylphorbol-13-acetate.
      Next, we assessed whether the YG-II-6 compounds inhibited JAK2- or JAK-3-dependent STAT5 phosphorylation by stimulating rat pre-T-lymphoma Nb2 cells with prolactin or IL-2. Levels of phosphorylated JAK2/STAT5 and JAK3/STAT5 were dramatically increased in response to prolactin or IL-2, whereas the YG-II-6 compounds decreased phosphorylated JAK2/STAT5 and JAK3/STAT5 (Figure 2f). Thus, in addition to inhibiting NF-κB signaling, the YG-II-6 compounds inhibited JAK/STAT signaling more effectively than the lead compounds.

       YG-II-6 compounds suppress cutaneous inflammation

      Inhibition of NF-κB and JAK/STAT signaling suggests that the YG-II-6 compounds may be effective therapies for inflammatory and immune disorders. We examined acute irritant contact dermatitis by applying TPA to the skin of mice to determine if YG-II-6 compounds exhibit anti-inflammatory activity in vivo. Increased skin thickness is the first hallmark of skin irritation and local inflammation and is due to increased vascular permeability, edema and swelling within the dermis, and proliferation of epidermal keratinocytes (
      • De Vry C.G.
      • Valdez M.
      • Lazarov M.
      • et al.
      Topical application of a novel immunomodulatory peptide, RDP58, reduces skin inflammation in the phorbol ester-induced dermatitis model.
      ;
      • Kalyan Kumar G.
      • Dhamotharan R.
      • Kulkarni N.M.
      • et al.
      Embelin reduces cutaneous TNF-α level and ameliorates skin edema in acute and chronic model of skin inflammation in mice.
      ). Epicutaneous TPA application resulted in a marked increase in inflammatory parameters, leading to increased skin thickness, edema and swelling, vascular permeability, and epidermal cell proliferation. Topical application of the YG-II-6 compounds effectively decreased TPA-induced inflammatory parameters, whereas the lead compounds exhibited weak to no inhibitory effects on skin inflammation (Figure 3a–c). Similarly, a patch test showed increased inflammatory parameters in the skin of mice upon application of TPA, including increased skin thickness, vascular permeability, and cell proliferation. In contrast, these parameters were effectively suppressed by fYG-II-6 application (Figure 3d).
      Figure thumbnail gr3
      Figure 3YG-II-6 compounds suppress TPA-induced cutaneous inflammation in mouse skin. (a) TPA was applied to the dorsal skin of C57BL/6 mice (n=6), and skin biopsies were stained with hematoxylin and eosin (H&E). Scale bar=100 μm. (b c) Epidermal thickness (b) and edema formation (c) were measured in the skin of mice. Results are shown as the mean±SEM. #P<0.001 versus DMSO-treated group, and *P<0.05 and **P<0.005 versus TPA-treated group. (d) TPA was topically administered to samples by transdermal patch for 24 hours, and skin biopsies were stained with proliferating cell nuclear antigen (PCNA) or H&E. Scale Bar=60 μm. FA, fumaric acid ethyl ester; fYG-II-6, (E)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid; MA, maleic anhydride; mYG-II-6, (Z)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid; PS, phytosphingosine; TPA, 12-O-tetradecanoylphorbol-13-acetate.
      Because topical application mainly affects the epidermis, we also examined mRNA levels of proinflammatory mediators in TPA-induced human keratinocytes. Similar to the histological results, the mRNA levels of proinflammatory mediators, including IL-1α, IL-6, IL-8, tumor necrosis factor—α, and cyclooxygenase-2, were increased by TPA application. However, these levels were effectively suppressed by the YG-II-6 compounds. In contrast, the YG-II-6 compounds increased the mRNA level of the anti-inflammatory cytokine IL-10. The anti-inflammatory activities of the YG-II-6 compounds were more effective than those of the lead compounds (Supplementary Figure S1a online).

       The fYG-II-6 compound ameliorates psoriasiform dermatitis

      In addition to the TPA-induced acute inflammatory model, we assessed a chronic inflammatory model by intradermal injection of IL-23 into the ears of mice (
      • Zheng Y.
      • Danilenko D.M.
      • Valdez P.
      • et al.
      Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis.
      ;
      • Rizzo H.L.
      • Kagami S.
      • Phillips K.G.
      • et al.
      IL-23-mediated psoriasis-like epidermal hyperplasia is dependent on IL-17A.
      ;
      • Maddur M.S.
      • Miossec P.
      • Kaveri S.V.
      • et al.
      Th17 cells: biology, pathogenesis of autoimmune and inflammatory diseases, and therapeutic strategies.
      ;
      • Kim B.H.
      • Na K.M.
      • Oh I.
      • et al.
      Kurarinone regulates immune responses through regulation of the JAK/STAT and TCR-mediated signaling pathways.
      ;
      • Lee Y.S.
      • Cheon I.S.
      • Kim B.H.
      • et al.
      Loss of extracellular superoxide dismutase induces severe IL-23-mediated skin inflammation in mice.
      ). Intradermal IL-23 injection into mouse ear skin induced psoriasiform dermatitis that leads to ear thickness, edematous swelling, vascular permeability, and epidermal cell proliferation. Topical application of fYG-II-6 suppressed these inflammatory responses (Figure 4a and b).
      Figure thumbnail gr4
      Figure 4YG-II-6 ((E)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid) compound ameliorates IL-23-induced psoriasiform dermatitis in mouse ears. (a) IL-23 was intradermally injected into the ears of C57BL/6 mice (n=3) every other day for 14 days followed by topical application of fYG-II-6. Mice were killed 24 hours after final injection, and the ears were stained with hematoxylin and eosin. Bar=100 μm. (b) Ear swelling was measured before the mouse was killed. (c) RNA was isolated from the ears of mice, and quantitative real-time reverse-transcriptase–PCR (qRT–PCR) was performed. Results are shown as the mean±SEM. #P<0.001 versus phosphate-buffered saline (PBS)–injected group, and *P<0.05 and **P<0.001 versus IL-23-injected group.
      Chemokine expression levels are controlled by proinflammatory mononuclear cells that are recruited to the inflammation site. fYG-II-6 effectively suppressed the mRNA levels of chemokines and T helper type 17 cell cytokines, including CXCL1, CCL17, CCL20, CCL27, IL-17A, and IL-22, that are highly expressed in psoriasis (
      • Harper E.G.
      • Guo C.
      • Rizzo H.
      • et al.
      Th17 cytokines stimulate CCL20 expression in keratinocytes in vitro and in vivo: implications for psoriasis pathogenesis.
      ;
      • Mabuchi T.
      • Takekoshi T.
      • Hwang S.T.
      Epidermal CCR6+ γδ T cells are major producers of IL-22 and IL-17 in a murine model of psoriasiform dermatitis.
      ;
      • Maeda S.
      • Hayami Y.
      • Naniwa T.
      • et al.
      The Th17/IL-23 axis and natural immunity in psoriatic arthritis.
      ). In addition, the mRNA levels of proinflammatory mediators, such as IL-1α, IL-1β, IL-6, IFN-γ, and tumor necrosis factor-α, were also suppressed by fYG-II-6 (Figure 4c). Similar to results obtained with TPA stimulation, the mRNA levels of IL-10 were increased by fYG-II-6 in IL-23-injected mouse ears (Figure 4c). IL-22 can also induce psoriasiform dermatitis in a murine model and HaCaT cells that express the IL-22 receptor (
      • Mabuchi T.
      • Chang T.W.
      • Quinter S.
      • et al.
      Chemokine receptors in the pathogenesis and therapy of psoriasis.
      ). The mRNA levels of chemokines were not highly increased in IL-22-stimulated HaCaT cells in comparison with IL-23-injected mouse ears. However, fYG-II-6 significantly suppressed mRNA expression levels of CXCL1, CXCL10, CCL17, CCL20, and CCL27 (Supplementary Figure S1b online). These results suggest that YG-II-6 compounds may be able to protect against cutaneous inflammation resulting from acute irritant contact dermatitis or chronic inflammatory diseases.

       YG-II-6 compounds inhibit MAP kinases signaling

      Mitogen-activated protein kinases (MAPKs) play key roles in inflammatory signaling in mammalian cells (
      • Johansen C.
      • Kragballe K.
      • Westergaard M.
      • et al.
      The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin.
      ;
      • Wang S.
      • Uchi H.
      • Hayashida S.
      • et al.
      Differential expression of phosphorylated extracellular signal-regulated kinase 1/2, phosphorylated p38 mitogen-activated protein kinase and nuclear factor-kappaB p105/p50 in chronic inflammatory skin diseases.
      ). Generally, the extracellular signal–regulated kinase pathway is involved in cell proliferation, differentiation, and survival, whereas the p38 and JNK pathways are associated with apoptosis, inflammation, and responses to environmental stress. In addition, extracellular signal–regulated kinase and p38 signaling regulate NF-κB signaling in various cell types (
      • Matsumoto M.
      • Sudo T.
      • Saito T.
      • et al.
      Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis mediated by receptor activator of NF-kappa B ligand (RANKL).
      ). We observed that the YG-II-6 compounds effectively inhibited phosphorylation of MAPKs, including extracellular signal–regulated kinase 1/2, p38, and JNK in TPA-stimulated keratinocytes (Figure 5). These results suggest that YG-II-6 compounds may regulate keratinocyte proliferation, survival, apoptosis, and inflammation.
      Figure thumbnail gr5
      Figure 5YG-II-6 compounds inhibit TPA-induced MAPK signaling. HaCaT cells were pretreated with compounds and then stimulated with TPA for 15 minutes. Western blot analysis was performed with primary antibodies against the target molecules. ERK1/2, extracellular signal–regulated kinase 1/2; FA, fumaric acid ethyl ester; fYG-II-6, (E)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid; MA, maleic anhydride; MAPK, mitogen-activated protein kinase; mYG-II-6, (Z)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid; PS, phytosphingosine; TPA, 12-O-tetradecanoylphorbol-13-acetate.

       YG-II-6 compounds induce apoptosis

      Because NF-κB, JAK/STAT, and MAPK signaling regulate cell survival, we examined whether YG-II-6 compounds could regulate cell proliferation and induce apoptosis. In previous reports, phytosphingosine and its derivatives were cytotoxic and induced apoptosis in keratinocytes and mouse skin (
      • Kim H.J.
      • Kim H.J.
      • Kim S.H.
      • et al.
      Tetraacetyl phytosphingosine-induced caspase activation and apoptosis occur through G2 arrest in human keratinocyte HaCaT cells.
      • Kim H.J.
      • Shin W.
      • Park C.S.
      • et al.
      Differential regulation of cyclooxygenase-2 expression by phytosphingosine derivatives, NAPS and TAPS, and its role in the NAPS or TAPS-mediated apoptosis.
      • Kim H.J.
      • Kang S.Y.
      • Kim S.J.
      • et al.
      Potentiation of UVB-induced apoptosis by novel phytosphingosine derivative, tetraacetyl phytosphingosine in HaCaT cell and mouse skin.
      ). Phytosphingosine was more cytotoxic than the YG-II-6 compounds and exhibited strong cytotoxic activity at concentrations between 5 and 10 μM. However, maleic anhydride and fumaric acid did not exhibit cytotoxic activities at concentrations as high as 2 mM (Figure 6a).
      Figure thumbnail gr6
      Figure 6YG-II-6 compounds induce apoptosis in cells and mouse skin. (a) HaCaT cells were incubated in the presence of DMSO or compound for 24 hours, and cell viability was measured with WST-1 reagent. Results are shown as the mean±SEM. **P<0.001 versus DMSO-treated group. (b) Western blot analysis was performed on lysates from HaCaT cells incubated with TPA in the presence of DMSO or compound for 24 hours. (c d) Primary human keratinocytes (c) or HaCaT cells (d) were incubated with the above mentioned, and TUNEL assay was performed. (e) Dorsal skins of C57BL/6 mice were prepared as mentioned in , and apoptotic cell death was visualized by FITC-stained TUNEL assay. Nuclei were counterstained with DAPI. Bcl-2, B-cell lymphoma 2; DAPI, 4',6-diamidino-2-phenylindole; FA, fumaric acid ethyl ester; fYG-II-6, (E)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MA, maleic anhydride; mYG-II-6, (Z)-4-oxo-4-(((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)amino)but-2-enoic acid; PARP, poly (ADP-ribose) polymerase; PS, phytosphingosine; TPA, 12-O-tetradecanoylphorbol-13-acetate.
      Western blots showed that the YG-II-6 compounds increased expression of the proapoptotic proteins Bax and Bad and induced fragmentation of the hallmark apoptotic proteins, poly (ADP-ribose) polymerase and caspase 3. In contrast, expression levels of cell cycle regulatory proteins cyclin D1 and E, antiapoptotic protein B-cell lymphoma 2, and cyclin-dependent kinases p21Cip/WAF1 and p27Kip1 were decreased by YG-II-6 compounds in TPA-stimulated keratinocytes (Figure 6b).
      Consistent results were observed by TUNEL assay in both keratinocytes and mouse skin. The YG-II-6 compounds increased the numbers of apoptotic cells in TPA-induced primary and immortalized human keratinocytes (Figure 6c and d) and in the skin of mice (Figure 6e). In comparison, phytosphingosine weakly induced apoptosis, and maleic anhydride and fumaric acid did not induce apoptosis in cells or mouse skin (Figure 6).

      DISCUSSION

      Phytosphingosine is essential for healthy skin and helps to regulate lipid production in the epidermal stratum corneum. However, imbalances in the lipid constituents of the stratum corneum leads to dry skin because of loss of moisture and can cause skin diseases, such as psoriasis, atopic dermatitis, eczema, ichthyosis, severe xerosis, and acne (
      • Coderch L.
      • López O.
      • de la Maza A.
      • et al.
      Ceramides and skin function.
      ;
      • Lee S.H.
      • Jeong S.K.
      • Ahn S.K.
      An update of the defensive barrier function of skin.
      ;
      • Jungersted J.M.
      • Hellgren L.I.
      • Jemec G.B.
      • et al.
      Lipids and skin barrier function - a clinical perspective.
      ). The in vitro and in vivo studies showed that phytosphingosine and its derivatives exhibited biological activities, such as proliferation and differentiation of keratinocytes, and provided skin care for acne because of anti-inflammatory and antimicrobial activities (
      • Pavicic T.
      • Wollenweber U.
      • Farwick M.
      • et al.
      Anti-microbial and -inflammatory activity and efficacy of phytosphingosine: an in vitro and in vivo study addressing acne vulgaris.
      ;
      • Paragh G.
      • Schling P.
      • Ugocsai P.
      • et al.
      Novel sphingolipid derivatives promote keratinocyte differentiation.
      ). Thus, phytosphingosine and its derivatives are potential cosmetic or medicinal ingredients. In fact, fumaric acid esters, including dimethyl fumarate, were developed for the treatment of psoriasis by the German Chemist Schweckendiek in 1959 and are known to have immunomodulatory, anti-inflammatory, antiproliferative, and apoptotic actions (
      • Seidel P.
      • Goulet S.
      • Hostettler K.
      • et al.
      DMF inhibits PDGF-BB induced airway smooth muscle cell proliferation through induction of heme-oxygenase-1.
      ;
      • Wilms H.
      • Sievers J.
      • Rickert U.
      • et al.
      Dimethylfumarate inhibits microglial and astrocytic inflammation by suppressing the synthesis of nitric oxide, IL-1beta, TNF-alpha and IL-6 in an in-vitro model of brain inflammation.
      ;
      • Albrecht P.
      • Bouchachia I.
      • Goebels N.
      • et al.
      Effects of dimethyl fumarate on neuroprotection and immunomodulation.
      ). Therefore, fumaric acid esters and salts are potential chemicals or drugs for treating adults with moderate to severe plaque psoriasis (
      • Ghoreschi K.
      • Brück J.
      • Kellerer C.
      • et al.
      Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells.
      ;
      • Heelan K.
      • Markham T.
      Fumaric acid esters as a suitable first-line treatment for severe psoriasis: an Irish experience.
      ) and granulomatous skin diseases, such as granuloma annulare and cutaneous sarcoidosis (
      • Kreuter A.
      • Gambichler T.
      • Altmeyer P.
      • et al.
      Treatment of disseminated granuloma annulare with fumaric acid esters.
      ;
      • Breuer K.
      • Gutzmer R.
      • Völker B.
      • et al.
      Therapy of noninfectious granulomatous skin diseases with fumaric acid esters.
      ). Fumaric acid esters and salts are also currently being investigated as treatments for relapsing–remitting multiple sclerosis (
      • Moharregh-Khiabani D.
      • Linker R.A.
      • Gold R.
      • et al.
      Fumaric acid and its esters: an emerging treatment for multiple sclerosis.
      ;
      • Ghoreschi K.
      • Brück J.
      • Kellerer C.
      • et al.
      Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells.
      ).
      Although phytosphingosine and its derivatives have biological activities, these compounds exhibit strong toxicities at high concentrations. In addition, fumaric acid esters and their salts have adverse effects, including diarrhea, abdominal pain, facial flushing, renal damage, appetite, tiredness, leucopenia, lymphopenia, transient eosinophilia, and spontaneous persistent erythema. We synthesized the phytosphingosine derivatives, mYG-II-6 and mYG-II-6, to reduce toxicity and increase efficacy. The biological and pharmacological activities of these derivatives were examined in TPA-induced human keratinocytes and animal models of skin inflammation induced by TPA or IL-23 stimulation.
      Keratinocytes are major cells in the epidermis of the skin and play important roles in protecting the body from environmental stimuli and skin inflammation (
      • Pivarcis A.
      • Nagy I.
      • Kemeny L.
      Innate immunity in the skin: how keratinocytes fight against pathogens.
      ). We found that YG-II-6 compounds were less cytotoxic and induced more apoptotic cell death compared with phytosphingosine in human keratinocytes and mouse skin by regulating proapoptotic proteins, cell cycle regulators, and antiapoptotic proteins. These data suggest that YG-II-6 compounds can regulate physiological processes that promote tissue development in the skin with reduced cytotoxicity.
      YG-II-6 compounds inhibited the NF-κB, JAK/STAT, and MAPK signaling in TPA- or cytokine-induced human keratinocytes, human PBMCs, mouse T cells, and rat pre-T-lymphoma cells. NF-κB and STAT proteins are transcription factors, and MAPKs are serine/threonine-specific protein kinases that play important roles in immune and inflammatory responses. However, aberrantly activated these signaling cascades are important therapeutic targets in inflammatory diseases and cancer (
      • Mudter J.
      • Neurath M.F.
      The role of signal transducers and activators of transcription in T inflammatory bowel diseases.
      ;
      • Shuai K.
      • Liu B.
      Regulation of JAK-STAT signalling in the immune system.
      ;
      • Brown K.D.
      • Claudio E.
      • Siebenlist U.
      The roles of the classical and alternative nuclear factor-kappaB pathways: potential implications for autoimmunity and rheumatoid arthritis.
      ;
      • Sarkar F.H.
      • Li Y.
      • Wang Z.
      • et al.
      NF-kappaB signaling pathway and its therapeutic implications in human diseases.
      ;
      • Lawrence T.
      The nuclear factor NF-kappaB pathway in inflammation.
      ;
      • Sikorski K.
      • Czerwoniec A.
      • Bujnicki J.M.
      • et al.
      STAT1 as a novel therapeutical target in pro-atherogenic signal integration of IFNγ, TLR4 and IL-6 in vascular disease.
      ). Phytosphingosine and its derivatives inhibit NF-κB and MAPK signaling (
      • Kim H.J.
      • Shin W.
      • Park C.S.
      • et al.
      Differential regulation of cyclooxygenase-2 expression by phytosphingosine derivatives, NAPS and TAPS, and its role in the NAPS or TAPS-mediated apoptosis.
      ;
      • Park M.T.
      • Choi J.A.
      • Kim M.J.
      • et al.
      Suppression of extracellular signal-related kinase and activation of p38 MAPK are two critical events leading to caspase-8- and mitochondria-mediated cell death in phytosphingosine-treated human cancer cells.
      ;
      • Ryu K.R.
      • Lee B.
      • Lee I.A.
      • et al.
      Anti-scratching behavioral effects of N-stearoyl-phytosphingosine and 4-hydroxysphinganine in mice.
      ), whereas their effects on JAK/STAT signaling have not yet been reported. We observed that YG-II-6 compounds effectively inhibited the NF-κB and JAK/STAT signaling cascades in TPA- or cytokine-stimulated keratinocytes, human PBMCs, mouse T cells, and TPA-induced mouse skin. These inhibitory activities were much stronger than those of the lead compounds, phytosphingosine, maleic anhydride, and fumaric acid. The inhibitory activities of phytosphingosine and its derivatives on JAK/STAT signaling are unreported findings. Furthermore, the inhibitory activities of the derivatives were much stronger than those of the lead compounds in all of the cells that were tested.
      Application of these derivatives as biologically active medicines and cosmetic ingredients may be important for skin care, because phytosphingosine and its derivatives have recently been sold in cosmetic markets. Although phytosphingosine exhibited beneficial effects in cosmetic markets, topical application at high doses is limited because of high toxicity. Several researchers modified and developed phytosphingosine derivatives to reduce toxic effects, and they examined the biological activities of these compounds (
      • Kim H.J.
      • Kim H.J.
      • Kim S.H.
      • et al.
      Tetraacetyl phytosphingosine-induced caspase activation and apoptosis occur through G2 arrest in human keratinocyte HaCaT cells.
      • Kim H.J.
      • Shin W.
      • Park C.S.
      • et al.
      Differential regulation of cyclooxygenase-2 expression by phytosphingosine derivatives, NAPS and TAPS, and its role in the NAPS or TAPS-mediated apoptosis.
      • Kim H.J.
      • Kang S.Y.
      • Kim S.J.
      • et al.
      Potentiation of UVB-induced apoptosis by novel phytosphingosine derivative, tetraacetyl phytosphingosine in HaCaT cell and mouse skin.
      • Kim S.
      • Hong I.
      • Hwang J.S.
      • et al.
      Phytosphingosine stimulates the differentiation of human keratinocytes and inhibits TPA-induced inflammatory epidermal hyperplasia in hairless mouse skin.
      ;
      • Park M.T.
      • Choi J.A.
      • Kim M.J.
      • et al.
      Suppression of extracellular signal-related kinase and activation of p38 MAPK are two critical events leading to caspase-8- and mitochondria-mediated cell death in phytosphingosine-treated human cancer cells.
      ;
      • Pavicic T.
      • Wollenweber U.
      • Farwick M.
      • et al.
      Anti-microbial and -inflammatory activity and efficacy of phytosphingosine: an in vitro and in vivo study addressing acne vulgaris.
      ;
      • Ryu K.R.
      • Lee B.
      • Lee I.A.
      • et al.
      Anti-scratching behavioral effects of N-stearoyl-phytosphingosine and 4-hydroxysphinganine in mice.
      ;
      • Walsh K.B.
      • Teijaro J.R.
      • Wilker P.R.
      • et al.
      Suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus.
      ;
      • Paget C.
      • Chow M.T.
      • Duret H.
      • et al.
      Role of γδ T cells in α-galactosylceramide-mediated immunity.
      ;
      • Zygmunt B.M.
      • Weissmann S.F.
      • Guzman C.A.
      NKT cell stimulation with α-galactosylceramide results in a block of Th17 differentiation after intranasal immunization in mice.
      ). The biological and pharmacological activities of YG-II-6 compounds were tested in an animal model, in which skin inflammation was induced by topical application of TPA or intradermal injection of IL-23 in mouse skin. Topical application of TPA induces IL-1α-mediated acute and chronic inflammation and hyperplasia in mouse skin (
      • Lee W.Y.
      • Lockniskar M.F.
      • Fischer S.M.
      Interleukin-1 alpha mediates phorbol ester-induced inflammation and epidermal hyperplasia.
      ). In addition, intradermal IL-23 injection into the ears of mice induces chronic inflammation (
      • Zheng Y.
      • Danilenko D.M.
      • Valdez P.
      • et al.
      Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis.
      ;
      • Rizzo H.L.
      • Kagami S.
      • Phillips K.G.
      • et al.
      IL-23-mediated psoriasis-like epidermal hyperplasia is dependent on IL-17A.
      ;
      • Maddur M.S.
      • Miossec P.
      • Kaveri S.V.
      • et al.
      Th17 cells: biology, pathogenesis of autoimmune and inflammatory diseases, and therapeutic strategies.
      ;
      • Kim B.H.
      • Na K.M.
      • Oh I.
      • et al.
      Kurarinone regulates immune responses through regulation of the JAK/STAT and TCR-mediated signaling pathways.
      ;
      • Lee Y.S.
      • Cheon I.S.
      • Kim B.H.
      • et al.
      Loss of extracellular superoxide dismutase induces severe IL-23-mediated skin inflammation in mice.
      ). These stimuli induce inflammatory responses in the skin that increase dermal edematous swelling, erythema, leukocyte infiltration, epidermal hyperplasia, and proliferation of epidermal keratinocytes. YG-II-6 compounds significantly suppressed progression of inflammatory skin damage in the skin and ears of mice. In addition to suppressing skin damage, YG-II-6 compounds significantly suppressed the expression of proinflammatory mediators, such as enzymes, cytokines, and chemokines, whereas the expression of anti-inflammatory cytokine IL-10 was markedly increased.
      In conclusion, we determined biological and pharmacological activities of the phytosphingosine derivatives, mYG-II-6 and fYG-II-6. Both YG-II-6 compounds exhibited effective anti-inflammatory activities and therapeutic effects in TPA- or cytokine-stimulated keratinocytes, human PBMCs, mouse T cells, and in cutaneous inflammatory models. The potencies of these compounds were much stronger than those of the lead compounds, phytosphingosine, maleic anhydride, and fumaric acid. The biological activities of YG-II-6 compounds were associated with inhibition of the NF-κB, JAK/STAT, and MAPK signaling with induction of programmed cell death. Inhibition of JAK/STAT signaling by these phytosphingosine derivatives is an unreported finding. Our data suggest that YG-II-6 compounds are promising candidates for the treatment of acute and chronic inflammatory skin diseases and potential ingredients for cosmetic skin care agents.

      MATERIALS AND METHODS

       Isolation of human PBMCs and mouse T cells

      This study was conducted according to the Declaration of Helsinki Principles and was approved by the institutional review board (IRB) of The Catholic University of Korea (KC12TISI0433). We obtained human blood samples from 3 male healthy donors aged from 27 to 43 years with their written informed consent. The donors did not take any therapy and were not hospitalized for at least 6 months. PBMCs were isolated from heparinized blood samples by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences, Uppsala, Sweden). Cells collected from interface were washed with phosphate-buffered saline and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics. Mouse T cells were isolated from spleens and lymph nodes of C57BL/6 mice as described previously and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics (
      • Kim B.H.
      • Na K.M.
      • Oh I.
      • et al.
      Kurarinone regulates immune responses through regulation of the JAK/STAT and TCR-mediated signaling pathways.
      ).

       Cell culture

      The immortalized human keratinocyte HaCaT cells were purchased from CLS Cell Line Service (Eppelheim, Germany) and maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics. The human keratinocytes were isolated from the skin of healthy volunteers using a keratinocyte primary isolation kit (Cascade Biologics, Portland, OR) and cultured as described previously (
      • Kim Y.
      • Kim B.H.
      • Lee H.
      • et al.
      Regulation of skin inflammation and angiogenesis by EC-SOD via HIF-1α and NF-κB pathways.
      ). Rat pre-T lymphoma Nb2 cells (kindly provided by Dr Charles Clevenger, Northwestern University, Evanston, IL) were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 5 mM HEPES (pH 7.4), and antibiotics.

       Western blot analysis

      Protein levels were analyzed by western blot analysis with specific primary antibodies for the target molecules, followed by appropriate horseradish peroxidase–conjugated secondary antibodies (Invitrogen, Grand Island, NY).

       Quantitative real-time PCR

      Total RNA was isolated using TRIzol Reagent (Ambion, Carlsbad, CA). The complementary DNA synthesis and quantitative real time-PCR were performed as described previously (
      • Kim Y.
      • Kim B.H.
      • Lee H.
      • et al.
      Regulation of skin inflammation and angiogenesis by EC-SOD via HIF-1α and NF-κB pathways.
      ).

       Transfection and NF-κB reporter assay

      HaCaT cells were transfected with plasmids containing both NF-κB-luciferase construct and pRL-TK vector using Effectene Transfection Reagent (Qiagen, Hilden, Germany) and the luciferase activity was measured using a Dual-Luciferase Assay kit (Promega, Sunnyvale, CA) as described previously (
      • Kim Y.
      • Kim B.H.
      • Lee H.
      • et al.
      Regulation of skin inflammation and angiogenesis by EC-SOD via HIF-1α and NF-κB pathways.
      ).

       Cell viability assay and TUNEL staining

      Cells were seeded into 96-well plate at a density of 20,000 cells per well. After overnight, the cells were incubated with either compound or vehicle for 24 hours, and cell viability was determined using EZ-CyTox Enhanced Cell Viability Assay Kit (Daeil Lab Service, Seoul, Korea) according to the manufacturer’s protocols. For performing TUNEL assay, samples were stained using an In Situ Apoptosis Detection Kit (Clontech, Mountain View, CA) to detect in situ DNA fragmentation according to the manufacturer’s protocol, and FITC signals were visualized with a confocal Laser Scanning Microscopy (Carl Zeiss, Oberkochen, Germany).

       Histological and immunohistochemical analysis

      Skin samples were fixed with 4% paraformaldehyde in phosphate-buffered saline for 24 hours, washed with tap water, dehydrated with grade ethanol, and then embedded in paraffin. Then, 5 μm sections were mounted on glass slides, dewaxed, rehydrated with grade ethanol, and stained with hematoxylin and eosin. For immunohistochemical analysis, deparaffinized sections were removed endogenous peroxidase, blocked, and then incubated with proliferating cell nuclear antigen antibody. The sections were labeled with biotin–streptavidin–immunoperoxidase complex, developed with diaminobenzidine (Dako, Carpinteria, CA), and counterstained with 0.2% Mayer’s hematoxylin (Sigma-Aldrich, St Louis, MO). Analysis was carried out using an inverted fluorescence microscope (Carl Zeiss).

       TPA-induced cutaneous inflammation model

      The protocol for animal use was approved by The Catholic Research Institute of the Medical Science Committee on Institutional Animal Care and Use. Cutaneous inflammation was induced by double TPA treatment as reported previously (
      • Ha H.Y.
      • Kim Y.
      • Ryoo Z.Y.
      • et al.
      Inhibition of the TPA-induced cutaneous inflammation and hyperplasia by EC-SOD.
      ). The mice were killed 1 hour after the second TPA application and the dorsal skins of mouse were collected for further experiments. Another cutaneous inflammatory model was performed by patch test. Tape stripping was performed with a 3 mm Tegaderm Film (6 × 7 cm, 3M Health Care, Neuss, Germany). TPA was applied on the back of hairless mouse in the presence or absence of fYG-II-6 for 24 hours, and the skins of mice were collected for further experiments.

       IL-23-induced psoriasis-like model

      Phosphate-buffered saline or recombinant mouse IL-23 (500 ng per 10 μl, eBioscience, San Diego, CA) was intradermally injected into C57BL/6 mouse ears, followed by topical application of compounds every other day for 14 days as described previously (
      • Kim B.H.
      • Na K.M.
      • Oh I.
      • et al.
      Kurarinone regulates immune responses through regulation of the JAK/STAT and TCR-mediated signaling pathways.
      ;
      • Lee Y.S.
      • Cheon I.S.
      • Kim B.H.
      • et al.
      Loss of extracellular superoxide dismutase induces severe IL-23-mediated skin inflammation in mice.
      ). Ear thickness was measured 24 hours after final injection and the ears were collected for further experiments.

       Statistical analysis

      Results in figures were represented as mean±SEM. Statistical analysis of differences among groups was determined by Student’s t-test. Differences were considered statistically significant at a level of *P<0.05 and **P<0.001.

      Acknowledgments

      This study was supported by a grant from the BioGreen 21 Program (PJ007175) of the Rural Development Administration, Republic of Korea.

      Supplementary Material

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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