Jong-Kyung Youm

Mite and Cockroach Allergens Activate Protease- Activated Receptor 2 and Delay Epidermal Permeability Barrier Recovery

Protease-activated receptor-2 (PAR-2) is known to be involved in epidermal permeability barrier function homeostasis. PAR-2 activation occurs after barrier disruption and further activation of PAR-2 by activating peptide significantly delays barrier recovery rate. Cockroach and house dust mite allergens, both known to be associated with the development of asthma, allergic rhinitis, and atopic dermatitis, have protease activity, which can activate PAR-2. In this study, we investigated the effects of both allergens on the epidermal barrier function as well as on the epidermal calcium gradient. Both allergens, when topically applied on the barrier-disrupted site, increased protease activities in the epidermis and delayed barrier recovery and lamellar body secretion in murine skin. The topical application of PAR-2-specific antagonist or protease inhibitors normalized the barrier recovery. Cockroach allergens induced intracellular calcium oscillations in cultured human keratinocytes through PAR-2-involved pathway, which was confirmed by desensitization protocol. Abnormal calcium ion distribution after barrier disruption was also observed in allergens-applied skin. These results suggest that allergens with protease activity can influence the epidermal permeability barrier homeostasis through PAR-2 activation and consequent modulation of the calcium ions in skin.

Inhibition of atopic dermatitis-like skin lesions by topical application of a novel ceramide derivative, K6PC-9p, in NC/Nga mice.

Abstract:u2002 Atopic dermatitis (AD) is a chronic inflammatory skin disease that commonly begins in childhood. K6PC‐9p (N‐(Ethyl dihydrogenphosphate)‐2‐hexyl‐3‐oxo‐decanamide) is a synthetic ceramide derivative of PC‐9S (N‐Ethanol‐2‐mirystyl‐3‐oxo‐staramide), which was known to be effective in atopic patients. In this study, we examined the effect of topical application of K6PC‐9p on skin inflammation and AD‐like skin lesions in mouse models. K6PC‐9p dose‐dependently inhibited phorbol ester‐induced increase in ear thickness in BALB/c mice. Moreover, topical application of K6PC‐9p suppressed dust mite extract‐induced AD‐like skin lesions in NC/Nga mice. Histopathological analysis revealed that both ear swelling and leucocyte infiltration were suppressed by K6PC‐9p treatment. K6PC‐9p also suppressed IL‐4 and TNF‐α expression in the ears and mast cell infiltration into the ears in NC/Nga mice. Further study demonstrated that K6PC‐9p inhibited ConA‐induced IL‐4 secretion and LPS‐induced macrophage activation. Taken together, our results showed that topical application of K6PC‐9p exerts beneficial effects in animal model of skin inflammation and AD, suggesting that K6PC‐9p might be a promising topical agent for the treatment of inflammatory skin diseases.

K6PC-5, a Direct Activator of Sphingosine Kinase 1, Promotes Epidermal Differentiation Through Intracellular Ca2+ Signaling

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SphK) is the enzyme directly responsible for production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. Here we synthesized a previously unidentified SphK activator, K6PC-5, and have studied its effects on intracellular Ca(2+) signaling in HaCaT cells and epidermal differentiation in murine skin. K6PC-5, a hydrophobic compound chemically named N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide, activated SphK (obtained from C57BL/6 murine blood and F9-12 cell lysates) in a dose-dependent manner. K6PC-5 induced both intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in HaCaT cells and Ca(2+) mobilization in hairless mouse epidermis. Both dimethylsphingosine (DMS) and dihydroxysphingosine (DHS), SphK inhibitors, and transfection of SphK1-siRNA blocked K6PC-5-induced increases in [Ca(2+)](i). The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry, but independent of the classical phospholipase C-mediated pathway. In addition, K6PC-5 enhanced the expression of involucrin and filaggrin, specific differentiation-associated marker proteins in HaCaT cells, whereas transfection of SphK1-siRNA blocked the increase of involucrin. Topical K6PC-5 also enhanced the expression of involucrin, loricrin, filaggrin, and keratin 5 in intact murine epidermis. Finally, topical K6PC-5 inhibited epidermal hyperplasia by exerting antiproliferative effects on keratinocytes in murine epidermis. These results suggest that K6PC-5 acts to regulate both differentiation and proliferation of keratinocytes via [Ca(2+)](i) responses through S1P production. Thus, regulation of S1P levels may represent a novel approach for treatment of skin disorders characterized by abnormal differentiation and proliferation, such as atopic dermatitis and psoriasis.

K6PC-5, a sphingosine kinase activator, induces anti-aging effects in intrinsically aged skin through intracellular Ca2+ signaling.

BACKGROUNDnSphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SK) is the enzyme directly responsible for the production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. To date, there are no reports to demonstrate a chemically induced, direct activation of SK.nnnOBJECTIVEnHere we have studied the effects of K6PC-5 as a newly synthesized SK activator on fibroblast proliferation in both human fibroblasts and aged mouse skin. To demonstrate that K6PC-5 has S1P-mediated action mechanism in fibroblasts, we have measured SK-dependent intracellular Ca(2+) signaling.nnnMETHODSnFibroblasts were cultured primarily from human foreskin and were used to study the effect of K6PC-5 and S1P on intracellular Ca(2+) signaling and fibroblast proliferation. Changes in intracellular Ca(2+) were detected by fluorescence with fura-2/AM. To study skin anti-aging effects of K6PC-5, we used intrinsically aged hairless mice (56 weeks old).nnnRESULTSnK6PC-5 promoted fibroblast proliferation and procollagen production in human fibroblasts significantly. K6PC-5 induced intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in human fibroblasts. Both dimethylsphingosine and dihydroxysphingosine, SK inhibitors, and the transfection of SK1-siRNA blocked the K6PC-5-induced increases in [Ca(2+)](i), an effect independent of the classical PLC/IP(3)-mediated pathway. The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry. Topical application of K6PC-5 for 2 weeks to intrinsically aged hairless mice enhanced fibroblast proliferation, collagen production, and eventually increased dermal thickness (10%). K6PC-5 also promoted specific epidermal differentiation marker proteins, including involucrin, loricrin, filaggrin, and keratin 5, without any alterations on epidermal barrier function.nnnCONCLUSIONnThese results suggest that K6PC-5 acts to regulate fibroblast proliferation through intracellular S1P production, and can further promote keratinocyte differentiation. We anticipate that the regulation of S1P levels may represent a novel approach for the treatment of skin disorders, including skin aging.

Novel synthetic ceramide derivatives increase intracellular calcium levels and promote epidermal keratinocyte differentiation

Ceramide is an important constituent of stratum corneum lipids, which act as both physical barriers and signal modulators. We synthesized several ceramide derivatives and investigated their effects on keratinocyte differentiation. RT-PCR and Western blotting showed that the novel synthetic ceramide derivatives K6PC-4 (N-ethanol-2-hexyl-3-hydroxy-decanamide), K6PC-5, (N-ethanol-3-oxo-2-tetradecyl/hexadecyl-octadecanamide/eicosanamide)and K6PC-9 [N-(1,3-dihydroxypropyl)-2-hexyl-3-oxo-decanamide] markedly increased keratin 1 and involucrin expression in normal human epidermal keratinocytes cultured in vitro. These ceramide derivatives elicited a rapid transient increase in intracellular calcium levels, which were measured using laser scanning confocal microscopy. In addition, K6PC-4, K6PC-5, and K6PC-9 stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In a reconstituted epidermis model, K6PC-4, K6PC-5, and K6PC-9 significantly increased keratin 1 expression in the suprabasal layer. These results indicate that these novel synthetic ceramide derivatives have the potential to promote keratinocyte differentiation, suggesting that the lipid molecules are applicable for treating skin diseases involving abnormal keratinocyte differentiation.

K6PC-5, a novel sphingosine kinase activator, improves long-term ultraviolet light-exposed aged murine skin.

Abstract:u2002 Sphingosine‐1‐phosphate (S1P), which is formed by phosphorylation of sphingosine through a process catalysed by sphingosine kinase (SK), is a multifunctional mediator of a variety of cellular responses including proliferation, differentiation, motility, and survival. K6PC‐5, which was recently synthesized as a novel SK activator, is expected to increase S1P levels. Indeed studies have already demonstrated that K6PC‐5 exhibits anti‐aging effects on intrinsic aged murine skin by increasing fibroblasts, collagen synthesis, dermal thickness, and epidermal differentiation. However, photoaging and intrinsic aging have highly different clinical and histopathological properties. In this study, we developed a photoaged murine model by exposing mice that were 56u2003weeks old to ultraviolet (UV)B and UVA radiation for 8u2003weeks. We then investigated whether K6PC‐5, as an SK activator, had anti‐aging effects on photoaged murine skin in addition to its effects on intrinsic aged murine skin and determined the mechanism. K6PC‐5 increased dermal collagen density in photoaged skin through increases in fibroblasts and collagen production. Photoaged murine skin treated with K6PC‐5 showed an increase in stratum corneum (SC) integrity with increased corneodesmosome density and an improvement in barrier recovery rate. Matrix metalloproteinase 13 remained unchanged. These results indicate that topical application of K6PC‐5 improves photoaged skin by improving skin barrier and increasing fibroblast count and function. In conclusion, K6PC‐5, as an S1P activator, improves long‐term UV‐exposed aged skin as well as intrinsic aged skin.

Anti-ageing effects of a new synthetic sphingolipid (K6EAA-L12) on aged murine skin.

Abstract:u2002 Recently, we reported on the anti‐ageing effects of K6PC‐5. This compound induced keratinocyte differentiation and fibroblast proliferation by increasing sphingosine‐1 phosphate synthesis. We performed this study to confirm the anti‐ageing effects of new synthetic products (the K6EAA series) derived from K6PC‐5 through an amino group induction. Cellular responses such as differentiation, proliferation and calcium mobilization were investigated using cultured human keratinocytes and fibroblasts. Also, we measured the expressions of collagen mRNA and protein using real time RT‐PCR and ELISA, respectively. The K6EAA‐L12 product, selected by in vitro screening, was evaluated for anti‐ageing effects on intrinsically and extrinsically (photo) aged models of hairless mice. In the intrinsically aged murine skin, K6EAA‐L12 showed anti‐ageing effects by activating collagen synthesis, eventually causing dermal thickening. Also, in the photo‐aged skin, the dermal collagen density and dermal thickness were increased. In photo‐aged murine skin, K6EAA‐L12 increased stratum corneum integrity by increasing corneodesmosome density and improved the barrier recovery rate. However, there were no changes in the expressions of epidermal differentiation maker proteins. In conclusion, topical K6EAA‐L12, a new synthetic K6PC‐5 derivative, improves intrinsically and extrinsically (photo) aged skin by increasing the collagen density and improving the skin barrier function.

Abietic acid inhibits UVB-induced MMP-1 expression in human dermal fibroblast cells through PPARα/γ dual activation

Peroxisome proliferator‐activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand‐activated transcription factors and consist of three isotypes: PPARα, PPARβ/δ and PPARγ. PPARs are expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, these receptors are highly studied in dermato‐endocrine research, and their ligands are targets for the treatment of various skin disorders, such as photoageing and chronological ageing of skin. Intensive studies have revealed that PPARα/γ functions in photoageing and age‐related inflammation by regulating matrix metalloproteinases (MMPs) via nuclear factor‐kappa B (NF‐κB) and activator protein‐1 (AP‐1). However, the detailed mechanism of PPARα/γs role in photoageing has not yet been elucidated. In this study, we confirmed that abietic acid (AA) is a PPARα/γ dual ligand and significantly decreased UVB‐induced MMP‐1 expression by downregulating UVB‐induced MAPK signalling and downstream transcription factors, subsequently reducing IκBα degradation and blocking NF‐κB p65 nuclear translocation in Hs68 human dermal fibroblast cells. Treatment of cells with AA and GW6471 or bisphenol A diglycidyl ether (BADGE), PPARα or PPARγ antagonists, respectively, reversed the effect on UVB‐induced MMP‐1 expression and inflammatory signalling pathway activation. Taken together, our data suggest that AA acts as a PPARα/γ dual activator to inhibit UVB‐induced MMP‐1 expression and age‐related inflammation by suppressing NF‐κB and the MAPK/AP‐1 pathway and can be a useful agent for improving skin photoageing.

Polyphenol‐rich apple extract inhibits dexamethasone‐induced sebaceous lipids production by regulating SREBP1 expression

Sebum production and excretion is a primary function of the sebaceous glands, but abnormally increased sebum production is a major cause of acne vulgaris. To identify a new candidate that regulates sebum production, we investigated the possible inhibitory effects of apple polyphenols (APP) purified from unripe apples on primary cultured human sebocytes and in patients with acne vulgaris. Dexamethasone (Dex) increased lipid synthesis and expression of the sterol response element‐binding protein 1 (SREBP 1) and its target enzymes, acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS), in the sebocytes. However, APP inhibited Dex‐induced lipid production and expression of SREBP‐1, ACC and FAS. APP also inhibited the increase in the expression and activation of glucocorticoid receptor in the sebocytes. Taken together, these results suggest that APP may be useful to regulate sebum production and may alleviate sebum‐involved skin disease, such as acne vulgaris.