Se Kyoo Jeong

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.

An Update of the Defensive Barrier Function of Skin

Skin, as the outermost organ in the human body, continuously confronts the external environment and serves as a primary defense system. The protective functions of skin include UV-protection, anti-oxidant and antimicrobial functions. In addition to these protections, skin also acts as a sensory organ and the primary regulator of body temperature. Within these important functions, the epidermal permeability barrier, which controls the transcutaneous movement of water and other electrolytes, is probably the most important. This permeability barrier resides in the stratum corneum, a resilient layer composed of corneocytes and stratum corneum intercellular lipids. Since the first realization of the structural and biochemical diversities involved in the stratum corneum, a tremendous amount of work has been performed to elucidate its roles and functions in the skin, and in humans in general. The perturbation of the epidermal permeability barrier, previously speculated to be just a symptom involved in skin diseases, is currently considered to be a primary pathophysiologic factor for many skin diseases. In addition, much of the evidence provides support for the idea that various protective functions in the skin are closely related or even co-regulated. In this review, the recent achievements of skin researchers focusing on the functions of the epidermal permeability barrier and their importance in skin disease, such as atopic dermatitis and psoriasis, are introduced.

Protease and protease-activated receptor-2 signaling in the pathogenesis of atopic dermatitis.

Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD.

Protease-activated receptor-2 mediates the expression of inflammatory cytokines, antimicrobial peptides, and matrix metalloproteinases in keratinocytes in response to Propionibacterium acnes

Propionibacterium acnes (P. acnes) has been known to produce various exogenous proteases, however, their role in acne pathogenesis remains largely unknown. Proteases elicit cellular responses, at least in part, via proteinase-activated receptor-2 (PAR-2), which is known to mediate inflammation and immune response. In this study, we investigated whether proteases from P. acnes could activate PAR-2 on keratinocytes and induce pro-inflammatory cytokines, antimicrobial peptides (AMPs), and matrix metalloproteinases (MMPs) via PAR-2 signaling. We examined PAR-2 expression and protease activity in acne lesions using immunofluorescence staining and in situ zymography. The effect of the culture supernatant of P. acnes on Ca2+ signaling in immortalized keratinocytes (HaCaT) was measured using a fluorescence method. HaCaT cells were treated with P. acnes strain ATCC 6919 culture supernatant, with or without pretreatment with serine protease inhibitor or selective PAR-2 antagonist and the gene expression of pro-inflammatory cytokines, AMPs, and MMPs was detected using real-time reverse transcription-polymerase chain reaction. We found that the protease activity and PAR-2 expression were increased in acne lesions. The P. acnes culture supernatant induced calcium signaling in keratinocytes via PAR-2 and stimulated the mRNA expression of interleukin (IL)-1α, -8, tumor necrosis factor (TNF)-α, human beta defensin (hBD)-2, LL-37, MMP-1, -2, -3, -9, and -13 in keratinocytes, which was significantly inhibited by serine protease inhibitor as well as selective PAR-2 specific antagonist. These results indicate that PAR-2 plays an important role in the pathogenesis of acne by inducing inflammatory mediators in response to proteases secreted from P. acnes.

Protease Activity Enhances Production of Thymic Stromal Lymphopoietin and Basophil Accumulation in Flaky Tail Mice

Epidermal barrier abnormality due to filaggrin deficiency is an important predisposing factor in the development of atopic dermatitis (AD). In addition, the expression of thymic stromal lymphopoietin (TSLP) in keratinocytes (KCs), induced by barrier disruption, can promote type 2 helper T-cell polarization. Protease activity, including protease-activated receptor-2 (PAR-2), is also known to be involved in epidermal barrier function in AD. However, to our knowledge, the relationship between protease activity and filaggrin deficiency from the perspective of AD has not been elucidated. Flaky tail (Flg(ft)) mice, known to have a mutation in the filaggrin gene, were used to assess the role of protease in KCs in the steady state and the mite-induced AD-like skin inflammation model. In the steady state, the expression and activity levels of endogenous proteases, kallikreins 5, 7, and 14, in the skin and TSLP were higher in Flg(ft) than in control mice. In addition, activation of PAR-2 by its agonist induced the production of TSLP in KCs of Flg(ft) mice, which was abrogated by a newly developed PAR-2 antagonist. Application of the PAR-2 antagonist improved symptoms and basophil accumulation in Flg(ft) mice treated with mite extracts. These results suggest that possibly through the PAR-2 activation in KCs, filaggrin deficiency induces TSLP production and basophil accumulation, which play important roles in the establishment of AD.

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.

Effects of α-melanocyte-stimulating hormone on calcium concentration in SZ95 sebocytes.

Abstract:u2002 Melanocortins have been implicated in human sebum secretion for a long time. However, the signalling pathways of α‐melanocyte‐stimulating hormone (α‐MSH) in human sebocytes expressing melanocortin receptors (MC‐Rs) are still poorly understood. Because calcium ions play a central role in MC‐R signalling, we investigated whether α‐MSH affects calcium signalling in the immortalized human sebocyte cell line SZ95. In addition, we investigated the impact of α‐MSH on MC‐1R expression and lipid synthesis in these cells. α‐MSH increased intracellular calcium levels. α‐MSH‐mediated calcium mobilization originated from intracellular calcium stores and was mediated by inositol triphosphate. Moreover, α‐MSH increased MC‐1R immunoreactivity and lipid synthesis in SZ95 sebocytes in the presence of testosterone. Our data demonstrate that α‐MSH in human sebocytes controls a key cellular signalling pathway, the calcium ion response, which may coordinate MC‐1R‐mediated sebum secretion.

Stimulation of epidermal calcium gradient loss and increase in TNF-alpha and IL-1alpha expressions by glycolic acid in murine epidermis.

Abstract:u2002 In a previous study, we reported that α‐hydroxy acids (AHA), such as glycolic acid and lactic acid, did not induce any significant changes in transepidermal water loss for normal murine skin. The ultrastructural observations, however, showed that the extent of lamellar body exocytosis significantly increased. Because AHA can theoretically decrease the calcium ion concentration by chelation, topical AHA may induce the loss of epidermal calcium gradient by lowering the calcium ion concentration in the granulocytes and, subsequently, induce lamellar body secretion. The aim of this study is to verify that glycolic acid could modulate the epidermal calcium gradient and increase lamellar body exocytosis. Seventy per cent of glycolic acid aqueous solution was applied to the normal skin of hairless mice and biochemical and morphological studies were performed. The loss of epidermal calcium gradient was observed in glycolic‐acid‐applied skin of hairless mice and subsequent barrier function recovery processes, such as an increase in lamellar body secretion, were observed. The extracellular glycolic acid was found to inhibit the change in intracellular calcium ion concentration in response to extracellular calcium ion concentration changes in the cultured mouse keratinocyte in vitro. The protein and mRNA expressions of tumour necrosis factor‐α and interleukin‐1α in the murine epidermis were significantly increased after glycolic acid application. An in vitro study using cultured keratinocytes suggested that glycolic acid could lower the calcium ion concentration, at least in part, through the chelating effects of the glycolic acid on the cationic ions.

Role of PKC-delta as a signal mediator in epidermal barrier homeostasis

The skin shows an important “epidermal permeability barrier homeostasis” in response to barrier disruption. Calcium ion (Ca2+), a major regulator in keratinocyte differentiation and proliferation, plays a crucial role in skin barrier homeostasis. Acute barrier disruption induces an immediate depletion of both extra- and intracellular calcium ions in the epidermis, especially in the upper granular layers, and results in the loss of normal epidermal calcium gradient. Currently, we hypothesize that the change in the intracellular calcium ion concentration triggers the barrier repair responses, such as lamellar body (LB) secretion and increased lipid synthesis in the epidermis. In this article, we suggest that PKC-delta is a signaling mediator for the changes in extracellular and intracellular calcium ion concentration.

Antibody to FcεRIα Suppresses Immunoglobulin E Binding to High-Affinity Receptor I in Allergic Inflammation

Purpose High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. Materials and Methods Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1atm1Knt Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. Results NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. Conclusion Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases.