P214 : Increased expression of stem cell factor in keratinocytes via direct PAR-2 activation: possible involvement of PAR-2 in melanogenesis and melanocyte growth

( Hyo Jung Sohn ),( Dae Suk Kim ),( He Min Lee ),( Ji Young Kim ),( Sang Ho Oh ) 대한피부과학회 2013 대한피부과학회 학술발표대회집 Vol.65 No.2

Background: The role of protease-activated receptor-2 (PAR-2) in skin pigmentation is well-known for its involvement in melanosome transfer from melanocytes to keratinocytes. Objectives: We aimed to further study the function of PAR-2 in skin pigmentation. Methods: We examined whether stem cell factor (SCF), which is a crucial paracrine factor from keratinocytes in melanogenesis, could be modulated via direct activation of PAR-2 expressed on keratinocytes. Results: The activation of PAR-2 with kallikrein 5 and PAR-2-activating peptide increased SCF expression at the protein and mRNA levels. Furthermore, increased expression of SCF was confirmed to be mediated by direct PAR-2 activation through experiments using a PAR-2 antagonist and PAR-2 siRNA. PAR-2 activation increased MITF expression in melanocyte-keratinocyte coculture experiments. Conclusion: Our data indicate that PAR-2 may be involved in melanogenesisor the growth of melanocytes through the induction of SCF expression in keratinocytes.

Role of Protease Activated Receptor 2 (PAR2) in Aspergillus Protease Allergen Induces Th2 Related Airway Inflammatory Response

Hak Sun Yu(유학선) 한국생명과학회 2010 생명과학회지 Vol.20 No.4

대부분의 알려진 알러젠들은 단백분해효소의 성격을 가지고 있고 이는 알레르기 반응에서 Th2 면역 반응을 일으키는 데 중요한 역할을 하는 것으로 알려져 있다. 이러한 단백분해효소들과 반응하는 것으로 알려진 protease activated receptor (PAR)는 4가지 종류가 있으며, 이 중 PAR2의 경우 알레르기 질환과 많은 상관관계를 보여 많은 연구가 되고 있다. 본 연구는 Aspergillus protease 알러젠에 의한 초기 및 만성 Th2 면역반응에서 PAR2의 역할을 규명하기 위해 Aspergillus protease 알러젠으로 정상쥐와 PAR2 유전자 결핍쥐 모두 Th2 반응을 유도한 후 면역세포의 침윤 정도 및 Th2 관련 cytokine 및 chemokine 유전자들의 발현 정도를 비교하였다. 그 결과 Aspergillus protease 알러젠으로 비강내로 1회 처리했을 경우 중성구의 침윤이 두드러지는데, 이때 PAR2 결핍 마우스는 이러한 면역세포의 침윤이 유의적으로 감소하였다. 또한, 이와 관련된 IL-25, TSLP, Eotaxin 유전자들의 발현 역시 PAR2 결핍 마우스에 현저히 감소하였다. 한편, Aspergillus protease 알러젠으로 비강내로 6회 처리했을 경우 중성구 대신 호산구의 침윤이 두드러지지만 PAR2 결핍 마우스에서 그 정도가 유의적으로 낮았다. OVA 특이 IgE와 IgGl 농도 역시 현저하게 PAR2 결핍 마우스에서 낮았고, CCL21의 발현이 PAR2 결핍마우스 MEF cells에서 현저히 감소하였다. Th2 초기 면역반응에서 가장 중요한 IL-25의 발현에 MAKP p38 pathway가 관여한다는 것을 이번 연구에서 알 수 있다. 본 연구를 통해 Aspergillus protease 알러젠으로 유도된 알러지성기관지 염증 반응에서 초기 반응뿐만 아니라 만성반응에서도 PAR2가 중요한 것을 알 수 있다. Most allergens have protease activities, suggesting that proteases may be a key link between Th2-type immune reactions in allergic responses. Protease activated receptor (PAR) 2 is activated via the proteolytic cleavage of its N-terminal domain by proteinases. To know the role of PAR2 in Aspergillus protease allergen activated Th2 immune responses in airway epithelial cells, we investigated and compared immune cell recruitment and level of chemokines and cytokines between PAR2 knock out (KO) mice and wild type (WT) mice. There were evident immune cell infiltrations into the bronchial alveolar lavage fluid (BALF) of WT mice, but the infiltrations in PAR2 KO mice were significantly lowered than those of WT mice. The IL-25, TSLP, and eotaxin gene expressions were profoundly increased after Aspergillus protease, but their expression was significantly lowered in PAR2 KO mice in this study. Compared to PAR2 KO mice, OVA specific IgE concentrations in serum of WT mice were quite increased; moreover, the IgE level of PAR2 KO mice was lower than in WT mice. The IL-25 expression by Aspergillus protease stimulation was significantly reduced by p38 specific inhibitor treatment. In this study, we determined that Th2 response was initiated with IL-25 and TSLP mRNA up-regulation in lung epithelial cells via PAR2 after Aspergillus protease allergen treatment.

비용에서 Protease-activated Receptor 2의 발현:면역조직화학적 연구

이승균,박용수,전은주,조진희,박용진,강준명,김명원 대한이비인후과학회 2005 대한이비인후과학회지 두경부외과학 Vol.48 No.4

Background and Objectives:Protease-activated receptor 2 (PAR-2) has been known to play an important role in modulatinghomeostasis and inflammation in the airways. But only a little is known about the localization of PAR-2 in nasal mucosa andrelationship between PAR-2 and nasal mucosal inflammation. In this study, we investigated the localization of PAR-2 in nasalpolyp and turbinate mucosa and examined the relationship between the PAR-2 expression and eosinophil infiltration. Materialsand Method:Nasal polyp tissues were obtained from 20 nasal polyposis patients and inferior turbinate tissues were taken from12 nasal septal deviation patients as control. PAR-2 expression was assessed by immunohistochemical staining. We comparedPAR-2 immunoreactivity between nasal polyp and turbinate mucosa. Infiltrated eosinophils were counted ands its correlationwith the intensity of PAR-2 immunoreactivity in nasal polyp was determined. Also, we evaluated the PAR-2 expression in thepresence of allergic rhinitis. Results:PAR-2 immunoreactivity was detected in the nasal mucosal epithelium, endothelial cells,vascular myocytes, submucosal glands, fibroblasts and inflammatory cells. PAR-2 expression was similar between nasal polypand turbinate mucosa, but inflammatory cells of nasal polyp showed stronger immunoreactivity than those of turbinate. Immunoreactivityin inflammatory cells showed correlation with eosinophil infiltration in nasal polyp. There were no differences inthe PAR-2 expression between allergic rhinitis patients and non-allergic rhinitis patients. Conclusion:Increased expression ofPAR-2 in inflammatory cells of nasal polyp and its correlation with eosinophil infiltration raise the possibility that PAR-2 mayplay a role in pathophysiology of nasal polyposis.

Effects of Par2-Antagonist on Inflammatory Signals and Tight Junction Expression in Protease Activated Canine Primary Epithelial Keratinocytes

( Ha-jung Kim ),( Se Kyoo Jeong ),( Soo-jong Hong ),( Kim Ahrens ),( Rosanna Marsella ) 한국피부장벽학회 2016 한국피부장벽학회지 Vol.18 No.2

Atopic dermatitis (AD) is a common allergic eczematous skin disorder in humans, the incidence of which is increasing worldwide. Protease activated receptor 2 (PAR2) is a mediator of innate immunity expressed on keratinocytes that can induce Th2-related inflammation in AD. Using a validated canine model of spontaneously occurring AD, we previously assessed the expression of PAR2 and thymic stromal lymphopoietin (TSLP) and reported that the expression pattern of skin biopsy samples differed in normal and atopic dogs. Pilot studies in our canine atopic model have also shown decrease tight junction protein expression such as and Zonula Occludens-1 (ZO-1). This study investigated whether PAR2 mediates the expression of TSLP and ZO-1 in canine primary epithelial keratinocytes (CPEKs) by assessing the effects of a PAR2-antagonist (PAR2-ant) on PAR2, TSLP, and ZO-1 immunofluorescence. CPEKs were cultured with serine protease over time with or without PAR2-antagonist and the expressions were quantitated by real-time PCR and fluorescence intensity in CPEKs. Protease significantly increased the expressions of PAR2 and TSLP, but decreased the expression of ZO-1 at each specific incubation time. PAR2-antagonist blocked those effects on each mediator and tight junction. Therefore, blockage of PAR2 can suppress the trypsin-activated initiation of inflammatory signals and the disturbance of tight junction protein CPEK.

Increased Expression of Cathelicidin by Direct Activation of Protease-Activated Receptor 2: Possible Implications on the Pathogenesis of Rosacea

김지영,오상호,김윤지,임범진,손효정,신동윤 연세대학교의과대학 2014 Yonsei medical journal Vol.55 No.6

Purpose: Recent findings of increased cathelicidin protein and its proteolytic fragmentsin rosacea suggest a pathogenic role for cathelicidin in this disease. The relationshipbetween cathelicidin and protease-activated receptor 2 (PAR-2) is thereforeof interest, as PAR-2, expressed principally in keratinocytes, regulates pro-inflammatory cytokine expression in the skin. The purpose of this study was to determine the relationship between expression of PAR-2 and cathelicidin in rosaceaand to test the effect of direct PAR-2 activation on cathelicidin expression in keratinocytes. Materials and Methods: Samples from 40 patients with clinicopathologicdiagnosis of rosacea and facial skin tissue samples from 20 patients with no specific findings or milium without inflammation were retrieved. Intensitiesof immunohistochemical staining for PAR-2 and cathelicidin were compared between normal and rosacea-affected skin tissues. Additionally, correlations betweenPAR-2 and cathelicidin staining intensities within rosacea patients were analyzed. In cultured keratinocytes, changes in PAR-2, cathelicidin, and vascular endothelialgrowth factor (VEGF) mRNA and protein were analyzed after treatment with PAR-2 activating peptide (AP). Results: Cathelicidin expression was significantlyhigher in rosacea skin tissues than in normal tissues (p<0.001), while PAR-2 expression was not significantly higher in rosacea tissues than in normal skin tissues. A positive correlation between PAR-2 and cathelicidin within rosacea samples was observed (R=0.330, p=0.037). After treatment of PAR-2 AP, both mRNA and protein levels for PAR-2, cathelicidin, and VEGF significantly increased in culturedkeratinocytes, compared with PAR-2 control peptide treatment. Conclusion: PAR-2 may participate in the pathogenesis of rosacea through activation of cathelicidinLL-37, a mediator of innate immune responses in the skin.

인간 신장암 Caki세포에서 Par-4에 의한 MMP-2 활성 저해를 통한 세포 이동 조절

우선민(Seon Min Woo),권택규(Taeg Kyu Kwon) 한국생명과학회 2016 생명과학회지 Vol.26 No.5

Par-4는 다양한 세포사멸 자극에 세포 사멸을 조절하고, 종양 억제기능을 가지고 있다. 그러나, Par-4에 의한 암세포의 이동 및 침윤에 대한 연구는 수행되지 않았다. 본 연구에서 Par-4단백질의 과발현이 인간 신장암 Caki세포에서 MMP-2의 활성화를 억제하지만 MMP-9 활성에는 영향을 주지 않았다. Par-4에 의한 MMP-2의 활성 억제는 leucine zipper domain이 결실된 Par-4 에서는 확인되지 않았다. Par-4 siRNA를 이용한 knock-down 실험에서 PMA 처리 시 세포이동 및 침윤 증가함을 확인하였다. Par-4의 과발현과 knock-dwon에서 MMP-2 mRNA 발현의 변화를 확인 할 수 없었다. 이 점은 Par-4 매개의 MMP-2 활성 억제는 전사 후 조절을 통하여 야기됨을 추측 할 수 있다. The prostate-apoptosis-response-gene-4 (Par-4) protein has been identified as an effector of cell death in response to various apoptotic stimuli in prostate cancer cells. We found that overexpression of Par-4 by stable transfection inhibits cell migration and invasion in Caki cells. The expression of various matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. In this study, we investigated whether ectopic expression of Par-4 modulates MMP-2 expression and activity in human renal carcinoma Caki cells. We found that overexpression of Par-4 markedly inhibited MMP-2 activity, but not MMP-9 activity. However, loss of the leucine zipper domain of Par-4 (Par-4 ΔLZ#1 and #2) did not inhibit MMP-2 activity. Further, knock-down of Par-4 with the corresponding siRNA resulted in increased invasion and metastasis of renal carcinoma Caki cells. Interestingly, overexpression or knock-down of Par-4 did not affect the expression levels of MMP-2 mRNA. Taken together, our findings suggest that Par-4 may inhibit MMP-2 activity through its post-transcriptional regulation in renal carcinoma Caki cells.

The induction of the collagen capsule synthesis by Trichinella spiralis is closely related to protease-activated receptor 2

Park, M.K.,Cho, M.K.,Kang, S.A.,Kim, B.Y.,Yu, H.S. Elsevier Scientific Pub. Co 2016 Veterinary parasitology Vol.230 No.-

<P>The muscle-stage larvae of the parasite Trichinella spiralis have the ability to survive within host muscle tissue by virtue of the formation a nurse cell-parasite complex, which is surrounded by collagen. The formation of the complex is initiated by excretory-secretory (ES) proteins produced by the parasite. To determine the mechanisms underlying collagen capsule formation, we investigated the expression levels of several types of collagen genes and TGF-beta I signaling-related genes (Smad2 and Smad3) in muscle cells. Synthesis of type I, IV, and VI collagen, which are major constituents of the collagen capsule, significantly increased during T. spiralis infection. In addition, we found that expression of the protease-activated receptor 2 (PAR2) gene was significantly increased during this period. Expression levels of the collagen genes and TGF-beta 1, Smad2, and Smad3 were induced by ES proteins and a PAR2 agonist, whereas their enhanced expression levels were reduced by a PAR2 antagonist and serine protease inhibitors. To evaluate the involvement of PAR2 during T. spiralis infection in vivo, we infected wild-type and PAR2 knockout (KO) mice with T. spiralis. Expression levels of type I, IV, and VI collagen genes and TGF-beta I signaling-related genes (Smad2 and Smad3) were also decreased in the PAR2 KO mice. Phosphorylation of Smad2/3, which was increased by T. spiralis infection, was significantly diminished in the PAR2 KO mice. In conclusion, ES proteins containing serine protease most likely activate collagen synthesis via PAR2 and TGF-beta I signaling, and this event could influence collagen capsule formation. (C) 2016 Elsevier B.V. All rights reserved.</P>

Protease-Activated Receptor-2 (PAR-2) 억제제 Pal-KTTKS 펩타이드 탐색 및 이를 함유한 국소도포제에 의한 아토피피부염의 임상적 호전

이윤희 ( Yoon Hee Lee ),김민정 ( Min Jung Kim ),공인덕 ( In Duck Kong ),류종석 ( Jong Sung Ryu ),장민열 ( Min Yeol Jang ),이천구 ( Cheon Gu Lee ),최응호 ( Eung Ho Choi ) 대한피부과학회 2010 대한피부과학회지 Vol.48 No.11

Background: Serine protease promotes desquamatation of the stratum corneum and this is controlled by serine protease inhibitors (SPI). After disruption of the skin barrier, signals for barrier recovery are started with the activation of cytokines and a migration of calcium ions. On the other hand, the protease-activated receptor-2 (PAR-2) pathway is initiated as a negative signal. As the pH of the stratum corneum become neutral, activated serine protease and PAR-2 inhibit the secretion of lamellar bodies and the formation of the lamellar structure. Objective: We wanted to screen noble synthetic peptides and identify the efficacy of a selected peptide, Palmitic acid-Lysine Threonine Threonine Lysine Serine (Pal-KTTKS), on PAR-2 in vitro and in vivo, and a clinical study was performed. Methods: In vitro: Changes of the intracellular calcium ion concentration were measured in cultured HaCaT cells by fluorescence imaging according to treatment with sample peptides and trypsin. In vivo animal study: The efficacy of 2% Pal-KTTKS cream as a selected noble peptide was evaluated in an oxazolone-induced atopic dermatitis animal model. Clinical study: A total of twenty three atopic dermatitis patients applied 2.5% Pal-KTTKS peptide-containing cream on the one side of their extremities and pseudo-ceramide containing moisturizer on the other side of the extremities as a control twice a day for 4 weeks. Clinical improvement was evaluated by the Eczema Area Severity Index (EASI) score, a subject questionnaire and comparison of photographs. Results: Suppression of the intracellular calcium concentration via PAR-2 inhibition was noted in the Pal-KTTKS peptide treated cultured HaCaT cells. In the oxazolone-induced atopic dermatitis hairless mice model, 2% Pal-KTTKS peptide containing lotion was more effective than vehicle lotion only. In the atopic dermatitis patients, the sites treated with 2.5% Pal-KTTKS peptide-containing cream showed better improvement for the EASI score, the subject questionnaire and the clinical photographs as compared to that of the control sites. There were no remarkable side effects related to the treatment. Conclusion: A PAR-2 inhibitor-containing topical agent would be an effective and safe modality for treating atopic dermatitis. (Korean J Dermatol 2010;48(11):966∼974)

Mechanism of Lipid Accumulation through PAR2 Signaling in Diabetic Male Mice

김대현,김예라,방은진,하수경,노상균,김병무,정성호,정희진,이지영,정해영 대한내분비학회 2021 Endocrinology and metabolism Vol.36 No.1

Background: Protease-activated protein-2 (PAR2) has been reported to regulate hepatic insulin resistance condition in type 2 diabetes mice. However, the mechanism of lipid metabolism through PAR2 in obesity mice have not yet been examined. In liver, Forkhead box O1 (FoxO1) activity induces peroxisome proliferator-activated receptor γ (PPARγ), leading to accumulation of lipids and hyperlipidemia. Hyperlipidemia significantly influence hepatic steatoses, but the mechanisms underlying PAR2 signaling are complex and have not yet been elucidated. Methods: To examine the modulatory action of FoxO1 and its altered interaction with PPARγ, we utilized db/db mice and PAR2-knockout (KO) mice administered with high-fat diet (HFD). Results: Here, we demonstrated that PAR2 was overexpressed and regulated downstream gene expressions in db/db but not in db+ mice. The interaction between PAR2/β-arrestin and Akt was also greater in db/db mice. The Akt inhibition increased FoxO1 activity and subsequently PPARγ gene in the livers that led to hepatic lipid accumulation. Our data showed that FoxO1 was negatively controlled by Akt signaling and consequently, the activity of a major lipogenesis-associated transcription factors such as PPARγ increased, leading to hepatic lipid accumulation through the PAR2 pathway under hyperglycemic conditions in mice. Furthermore, the association between PPARγ and FoxO1 was increased in hepatic steatosis condition in db/db mice. However, HFD-fed PAR2-KO mice showed suppressed FoxO1-induced hepatic lipid accumulation compared with HFD-fed control groups. Conclusion: Collectively, our results provide evidence that the interaction of FoxO1 with PPARγ promotes hepatic steatosis in mice. This might be due to defects in PAR2/β-arrestin-mediated Akt signaling in diabetic and HFD-fed mice.

Inhibitory effect of FSLLRY-NH2 on inflammatory responses induced by hydrogen peroxide in HepG2 cells

이연주,김수진,권경완,이원모,임위준,손의동 대한약학회 2017 Archives of Pharmacal Research Vol.40 No.7

Proteinase activated receptor 2 (PAR2), which is localized in the GI tract, the respiratory system, and the kidney tubules is a G protein-coupled receptor associated with inflammation, metabolism, and disease. The aim of this study was to explore the role of PAR2 in hydrogen peroxide (H2O2)-induced HepG2 cells by using FSLLRYNH2 a PAR2 antagonist. H2O2 treatment resulted in induction of PAR2 in esophageal, gastric, and liver cells, with the most robust response being in HepG2 cells. Furthermore, this effect was dose-dependent in HepG2 cells. Treatment with H2O2 at concentrations above 400 lM for 24 h also reduced HepG2 cell viability. H2O2 treatment increased both the protein and mRNA levels of IL-1b, IL- 8, and TNF-a, as well as those of SAPK/JNK. The increased levels of these pro-inflammatory genes and SAPK/JNK induced by H2O2 were attenuated in a dosedependent manner when cells were co-treated with H2O2 and FSLLRY-NH2. In summary, the PAR2 antagonist peptide, FSLLRY-NH2, reduces the level of the pro-inflammatory genes IL-8, IL-1b, and TNF-a induced by H2O2, through the SAPK/JNK pathways in HepG2 cells. These data suggest that a PAR2 antagonist could be an anti-inflammatory agent in HepG2 cells.