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Skin Barrier Recovery by Protease-Activated Receptor-2 Antagonist Lobaric Acid

Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-NH2 and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-NH2-induced PAR2 activation resulting in decreased mobilization of intracellular Ca2+ in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-NH2 and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-α) and IFN-γ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-NH2-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-NH2 downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-NH2 in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis.


INTRODUCTION
Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with cutaneous hyperreactivity to environmental triggers. AD is the product of interaction between susceptibility genes, the environment, defective skin barrier function, and immunologic responses. AD is characterized by dry skin and increased transepidermal water loss (TEWL). Impairment of the skin barrier function in AD leads to increased antigen absorption contributing to cutaneous hyperreactivity (Leung et al., 2004;Lee et al., 2010).
Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases (SPs). Cleavage of PAR2 exposes the tethered ligand neoepitope that binds intramolecularly within the receptor to stimulate signal transduction via coupled G proteins. PAR2-mediated signal transduction is also experimentally stimulated by hexapeptide agonist that are homologous to the tethered ligand sequence. The irreversible nature of PAR2 proteolysis means that downstream signal transduction is tightly regulated (Bohm et al., 1996;Oldham and Hamm, 2008). In skin, PAR2 is associated with regulation of keratinocyte proliferation, differentiation, epidermal barrier recovery and inflammation (Derian et al., 1997;Macfarlane and Plevin, 2003;Frateschi et al., 2011).
The epidermis has protective and defensive functions that are mediated by its unique differentiation end product, the stratum corneum. The stratum corneum comprises a unique, two-compartment system: protein-enriched corneocyte "bricks" embedded in a lipid-enriched, extracellular matrix "mortar" (Elias, 1983;Elias, 2005;Elias and Choi, 2005;Elias and Choi, 2008). Both compartments are required to form an effective permeability barrier (Madison, 2003). Inhibition of serine protease-mediated signaling stimulates barrier recovery responses by both differentiating keratinocytes and the LB secretory machinery (Hachem et al., 2006). For example, recent studies demonstrated that proteolytically active allergens, house dust mites and cockroaches activated PAR2 and delayed barrier recovery by inhibiting LB secretion (Jeong et al., 2008).
Lobaric acid, a phenolic compound isolated from lichen Stereocaulon aplinum, has been reported to show antitumor and antimitotic effects (Gissurarson et al., 1997, Morita et al., 2009. However, there is no report showing how lobaric acid affects skin barrier function.
This study investigated, the effects of lobaric acid on PAR2 activation, the inflammation response, keratinocyte differentiation, and permeability barrier recovery after acute barrier abrogation in mouse skin.
Normal human epidermal keratinocytes (NHEKs) of neonatal origin were from Invitrogen. NHEKs were used until passage 5. Cells were cultured in EpiLife medium with 60 µM calcium and human keratinocyte growth supplement (Invitrogen), 100 units/ml penicillin and 100 mg/ml streptomycin and incubated at 37ºC in a humidified atmosphere of 5% CO2 and 95% air.

Measurement of intracellular Ca 2+
HaCaT cells were seeded in 96-well plates (black wells and clear bottoms, Greiner Bio-one, Germany) at 4×10 4 cells/ well. After 24 hours, medium was changed to 2 µM Fluo-4, AM (Invitrogen), 0.02% Pluronic F-127 (Invitrogen) and 2.5 mM Probenecid (Sigma) in Hank's balanced salt solution (HBSS, Sigma) containing 20 mM HEPES (Sigma) for 30 minutes at 37ºC, protected from light. Cells were stabilized for 15 minutes at room temperature during shaking. Cells were pretreated with lobaric acid before PAR2 activation. After 30 minutes, 20 µM PAR2-activating peptide SLIGKV-NH2 or 2 unit/ml trypsin (Sigma) was added and intracellular Ca 2+ was simultaneously measured using Flexstation 3 (Molecular Devices, Sunnyvale, CA, USA), 485 nm excitation and 525 nm emission at room temperature. Data were analyzed with SoftMax Pro (Molecular Devices), and mobilization of intracellular Ca 2+ was calculated as the minimum value subtracted from the maximum value.

Measurement of secreted IL-8
HaCaT cells were seeded in 96-well plates as above. After 24 hours, cells were starved in DMEM without FBS for 12 hours and pretreated with lobaric acid for 30 minutes before adding 100 µM SLIGKV-NH2 and culturing for 24 hours. Supernatants were harvested and analyzed with Human CXCL8/ IL-8 DuoSet kits (R&D Systems, Minneapolis, MN, USA). Absorbance was measured at 450 nm and corrected at 540 nm using auto microplate reader (infinite M200, TECAN, Männedorf, Switzerland).

TARC measurement
HaCaT cells were seeded in 96-well plates at 3×10 4 cells/ well. After 24 hours, cells were starved using DMEM without FBS for 12 hours. The inflammatory response was induced by treating with both INF-γ and TNF-α (East Brunswick) at 10 ng/ml for 24 hours. Lobaric acid was added for 72 hours. Dexamethasone (Sigma), the positive control was also treated with 100 µg/ml. Supernatants were harvested and analyzed with Quantikine Human CCL17/TARC immunoassay kits (R&D Systems). Absorbance was measured and corrected as above.

Animal studies
Female hairless mice (SKH-1), aged 7-12 weeks, were from HOSHINO laboratory animals (Shizuoka, Japan) and fed mouse diet and water ad libitum. All animal procedures were approved by the Institutional Animal Care and Use Committees of Gyeonggi Bio Center and performed in accordance with their guidelines. Procedures were as in Hachem et al. (2006). Before acute barrier disruption by sequential cellophane tape stripping, mice were treated with a topical application of lobaric acid (0.2 µM) in propylene glycol (7:3 vol/vol) or vehicle alone. After 2 hours, tape-stripping was performed (TEWL rate ≥50 g/m 2 h; normal ≤8.5 g/m 2 h) and samples were applied again. Permeability barrier function was assessed as After pretreatment with lobaric acid, 100 µM SLIGKV-NH 2 was added to HaCaT keratinocytes for 24 hours and immunoassays were performed (A). INF-γ and TNF-α were added to HaCaT keratinocytes followed by lobaric acid or dexamethasone. Supernatants were used in immunoassays (B). ERK phosphorylation in NHEKs induced by 100 µM SLIGKV-NH 2 was blocked by lobaric acid. NHEKs were stimulated for indicated times with 100 µM SLIGKV-NH 2 and stimulation was blocked by pretreatment with 2 µM lobaric acid (C). Whole cell lysates were used to determine ERK phosphorylation (p-ERK) and total-ERK (t-ERK) by Western blots. Data are mean ± SEM, n=2. *p<0.05, **p<0.01, ***p<0.005, compared with controls. TEWL with a tewameter (TM300/Courage+Khazaka electronic GmbH, Köln, Germany), before treatment and immediately after treatment and 3 and 24 hours after acute barrier disruption.

Statistical analysis
Data are presented as means ± standard error of the mean (SEM). All statistical analysis was done using SPSS statistical software package Version 17.0, considering below p<0.05 as statistically significant.
www.biomolther.org was used as positive control and also significantly decreased TARC. To further assess the pathway of blocking inflammatory response, we investigated whether ERK1/2 activation by SLIGKV-NH2 could be influenced by lobaric acid. Treatment with 100 µM SLIGKV-NH2 induced phosphorylation of ERK1/2 (p-ERK), starting from 4 min after treatment and declining at 12 min. When NHEKs were pretreated with 2 µM lobaric acid for 2 hours, SLIGKV-NH2-induced upregulation of p-ERK was inhibited (Fig. 3C).
In HaCaT keratinocytes, treatment with SLIGKV-NH2 (100 µM) decreased involucrin expression that was induced by calcium treatment for 72 hours. The SLIGKV-NH2 effect was strongest at 0.4 mM calcium, so 0.4 mM was used for experiments (Fig. 3A). SLIGKV-NH2 decreased involucrin expression and this effect was antagonized by lobaric acid treatment (Fig. 3B). PTX (0.5 µg/ml), or a G-protein inhibitor that was used as a positive control also increased expression of involucrin. PTX-B (0.5 µg/ml), which was used as a negative control to PTX, decreased involucrin expression. Treatment with 2.8 mM calcium was used as a positive control to induce involucrin expression. Treatment of SLIGKV-NH2 with PTX reversed the effect of SLIGKV-NH2 alone and high concentrations of calcium (2.8 mM) induced normal keratinocyte differentiation.

Lobaric acid accelerated permeability barrier recovery after mouse skin barrier disruption
To investigate whether lobaric acid aided in recovery of skin barrier functions in vivo, we assessed barrier recovery kinetics in hairless mice. Mice were treated with 0.2 µM of lobaric acid or vehicle (70:30 propylene glycol: ethanol) 2 hours before tape stripping. Acute barrier disruption was induced by sequential tape stripping and TEWL levels measured. TEWL levels were elevated more than six fold compared to the normal conditions and samples were reapplied to acutely disrupted skin. Lobaric acid significantly accelerated the kinetics of barrier recovery at 3 hours following tape-stripping. Skin treated with either lobaric acid or vehicle had recovered by over 80% after 24 hours (Fig. 4).

DISCUSSION
AD is a chronic inflammatory skin disease characterized by eczema, pruritus, and cutaneous hyperreactivity to environmental factors that are innocuous to normal nonatopic individuals (Briot et al., 2009). Children with poorly controlled AD have a poorer quality of life, and AD results in financial, time, and psychologic burdens (Spergel and Paller, 2003). AD has a complex etiology that results from interactions between environment and several susceptibility genes involved in skin barrier function and systemic and local immunological responses (Cork et al., 2006). AD and PAR2 are closely related. AD-like lesions caused by PAR2 are seen in a serine protease inhibitor knockout mouse model (Briot et al., 2009). In the absence of PAR2, mice have a rescued inflammation response and altered skin barrier permeability (Frateschi et al., 2011). Thus, PAR2 correlates with the inflammatory response and barrier permeability and homeostasis. The proliferation and differentiation of keratinocytes in the epidermis also controls barrier permeability and homeostasis.
The aim of this study was to find new PAR2 antagonists that interfered with PAR2 activation and might halt AD symptoms. We found that lobaric acid decreased intracellular Ca 2+ mobilization induced by SLIGKV-NH2 and trypsin in HaCaT keratinocytes (Fig. 1). Our results indirectly indicated that PAR2 activation was specifically inhibited by lobaric acid. We also showed that lobaric acid inhibited PAR2-induced inflammation and keratinocyte differentiation in vitro. PAR2 activation increases secretion of IL-8 via ERK activation (Hou et al., 1998;Guo et al., 2011). Lobaric acid blocked ERK activation and decreased secretion of IL-8 level significantly induced by SLIGKV-NH2 (Fig. 2). TARC was also downregulated significantly by lobaric acid in HaCaT keratinocytes (Fig. 2B).
A number of autocrine and paracrine factors drive keratinocyte differentiation and calcium is crucial in this process (Su et al., 1994). The epidermis has a calcium gradient with the lowest concentrations in the basal layer and the highest in the granulosum layer, where critical proteins for barrier function are produced (Yuspa et al., 1989). PAR2 activation regulates terminal differentiation of keratinocytes; however, the effect of tion. Acute barrier disruption in hairless mice was by repeated cellophane tape stripping followed by application of lobaric acid (0.2 mM) and vehicle. Lobaric acid before and after tape stripping significantly improved barrier recovery compared to vehicle-treated skin. TEWL was measured at baseline, immediately after, and 3 and 24 h after acute barrier disruption. Results are mean ± SEM, n=9. *p<0.05 compared with control.
To determine the effects of lobaric acid on skin barrier recovery, we assessed permeability barrier homeostasis in mice. SP activity increased after acute perturbation of the permeability barrier and inhibition of SP activation. Topical serine protease inhibitors accelerate barrier recovery in rodents and in humans (Hachem et al., 2006). A tryptic SP signals downstream responses through PAR2 so we measured barrier recovery kinetics of skin after topical lobaric acid application compared to application of vehicle only. Topical treatment with lobaric acid significantly accelerated barrier recovery compared to the control (Fig. 4).
In summary, blocking a PAR2-mediated signal pathway with lobaric acid rescued inflammation and promoted skin barrier recovery. Our results suggested that lobaric acid was a PAR2 antagonist and might be a potential therapeutic agent for AD. Further studies needed include measuring the effects of lobaric acid on AD using NC/Nga mice, a strain that spontaneously develops AD.