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1 KISEP Rhinology Korean J Otolaryngol 2006;49: 사람정상코점막상피세포에서겔형성점소 (Gel-Forming Mucin) 분비에대한 Prostaglandin E 2 의효과 김진국 1 김창훈 2,3 김규보 2 장현종 1 김현직 2 윤주헌 2,3,4 Effect of Prostaglandin E 2 on Gel-forming Mucin Secretion in Normal Human Nasal Epithelial ells Jin Kook Kim, MD 1, hang Hoon Kim, MD 2,3, Kyubo Kim, MD 2, Hyun Jong Jang, MD 1, Hyun Jik Kim, MD 2 and Joo Heon Yoon, MD 2,3,4 1 Department of Otorhinolaryngology-Head and Neck Surgery, KonKuk University ollege of Medicine, Seoul and 2 Department of Otorhinolaryngology 3 The Airway Mucin Institute 4 Brain Korea 21 Project for Medical Sciences, Yonsei University ollege of Medicine, Seoul, Korea ABSTRAT Background and Objectives:Recently, Prostaglandin E 2 ( ) was found to induce MU5A production via an agonist of E-prostanoid (EP2/EP4), but not EP1/EP3, in normal human airway epithelium. However, the receptor that mediates MU5A has not been determined. This study was aimed to investigate the MU5A mucin gene and mucin secretion by and its receptors in cultured normal human nasal epithelial cells. Materials and Method:After treatment with and/or antagonist, gel-forming mucin mrna expression was determined by reverse transcription-polymerase chain reaction. Total mucin and MU5A mucin levels were measured using an immuno-dot blotting assay. Results: increased the MU5A gene expressions and MU5A mucin, but not the expressions of other gel-forming mucin genes. An EP2 receptor antagonist (AH6809) did not suppress the -induced MU5A gene expression or MU5A mucin. However, an EP4 receptor antagonist (AH23848) significantly suppressed the level of -induced MU5A gene expression and MU5A mucin. onclusion:these findings indicate that induces MU5A gene expression and mucin secretion via EP4 receptor in cultured normal human nasal epithelial cells. (Korean J Otolaryngol 2006;49:307-12) KEY WORDS:Prostaglandin E 2 Mucin Secretion Prostaglandin receptor. 307

2 로유도된점소유전자발현과분비 Table 1. Designed primers used for PR amplification MU5A MU5B MU6 MU7 EP2 EP4 2M Name Sequence Annealing temperature ycle Size F- GA AA TA TT TG GG TG R- GA T AT TT T GT GTT F- AT A GAG AT GT A A R- TA A TGG TT GTG TG TA F- TA T AT A AA AA R- GGA GAA GAA GGA AAA AGA G F- A A TA ATT TT R- TA TTG T A AT GT F- GA GA GG AA GTG GT T R- GA GA GG GG TG GTA GA F-TG G AAG GAG AG AAG GAG A R-GA GGT GG GAG AAT GAG GAA GGA F- TG G TA TT T TTT TG G R- GT TA ATG TT GA T A TTA A bp bp bp bp bp bp bp 308 Korean J Otolaryngol 2006;49:307-12

김진국외 - - t EP2 EP4 2M 2 7 14 21 Days after confluence Fig. 1. EP2 gene expression increased as a function of mucociliary differentiation.

3 김진국외 - - t EP2 EP4 2M Days after confluence Fig. 1. EP2 gene expression increased as a function of mucociliary differentiation. However, EP4 gene expression was not altered as a function of differentiation M AH M AH M MU5A MU5B MU6 MU7 2M Relative intensities AH6809 AH23848 Fig. 2. Effects of prostaglandin E 2 on gel-forming mucin gene expressions and inhibitory effects of receptor antagonists. increased only MU5A gene expression, but not the expression other gelforming mucin genes. An EP2 receptor antagonist AH6809 did not suppress -induced MU5A gene expression. However, an EP4 receptor AH23848 antagonist significantly suppressed MU5A gene expression. 309

로유도된점소유전자발현과분비 Fold increase of total mucin 0.3 2.5 1.5 0.1 0.5 0.0 p0.05 p0.

01 AH6809 AH23848 Fig. 3. Effect of on total mucin and MU5A mucinsecretion.

An EP4 antagonist AH23848 significantly suppressed both total mucin and MU5A mucin secretion.

4 로유도된점소유전자발현과분비 Fold increase of total mucin p0.05 p0.01 AH6809 AH23848 Fold increase of MU5A mucin p0.05 p0.01 AH6809 AH23848 Fig. 3. Effect of on total mucin and MU5A mucinsecretion. increased total mucin secretion and promoted a 4-fold increase of MU5A mucin secretion. An EP4 antagonist AH23848 significantly suppressed both total mucin and MU5A mucin secretion. EP2 EP4 2M Fig. 4. Detection of EP E prostanoid mrna expression in cultured NHNE cells. did not affect EP2 or EP4 gene expression. 310 Korean J Otolaryngol 2006;49:307-12

5 김진국외 REFERENES 1) Thornton DJ, Gray T, Nettesheim P, Howard M, Koo JA, Sheehan JK. haracterization of mucins from cultured normal human tracheobronchial epithelial cells. Am J Physiol Lung ell Mol Physiol 2000;278: L ) Lu W, Illehoj EP, Kim K. Effect of dexamethasone on Muc5ac mucin production by promary airway goblet cells. Am J Physiol Lung ell Mol Physiol 2005;288:L ) Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: Structures, properties, and functions. Physiol Rev 1999;79: ) Gray T, Nettesheim P, Loftin, Koo JS, Bonner J, Peddada S, et al. Interleukin-1β induced mucin production in human airway epithelium is mediated by cyclooxygenase-2, prostaglandin E2 receptors, cyclic AMP-protein kinase A signaling. Mol Phamacol 2004;66: ) Song KS, Lee WJ, hung K, Koo JS, Yang EJ, hoi JY, et al. Interleukin-1β and tumor necrosis factor-α induce MU5A overexpression through a mechanism involving ERK/p38 mitogen-activated protein kinase-msk1-reb activation in human airway epithelial cells. J Biol hem 2003;278: ) Yoon JH, Kim KS, Kim HU, Linton JA, Lee JG. Effects of TNF-α and IL-1β on mucin, lysozyme, IL-6 and IL-8 in passage-2 normal human nasal epithelial cells. Acta Otolarygol 1999;119: ) Yoon JH, Kim KS, Kim SS, Lee JG, Park IY. Secretory differentiation of serially passaged normal human nasal epithelial cells by retinoic acid: Expression of mucin and lysozyme. Ann Otol Rhinol Laryngol 2000;109: ) Yoon JH, Gray T, Guzman K, Koo JS, Nettesheim P. Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix. Am J Respir ell Mol Biol 1997;16:

6 로유도된점소유전자발현과분비 9) Yoon JH, Kim KS, Kim SS, Lee JG, Park IY. Secretory differention of serially passaged normal human nasal epithelial cells by retinoic acid: Expression of mucin and lysozyme. Ann Otol Rhinol Laryngol 2000; 109: ) Fedyk ER, Phipps RP. Prostaglandin E2 receptors of the EP2 and EP4 subtypes regulate activation and differentiation of mouse B lymphocytes to IgE-secreting cells. Proc Natl Acad Sci USA 1996;93: ) Kim YD, Kwon EJ, Park DW, Song SY, Yoon SK, Baek SH. Interleukin-1induces MU2 and MU5A synthesis through cyclooxygenase-2 in NI-H292 cells. Mol Pharmacol 2002;62: ) ho KN, hoi JY, Kim H, Baek SJ, hung K, Moon UY, et al. Prostaglandin E2 induces MU8 gene expression via a mechanism involving ERK MAPK/RSK1/cAMP response element binding protein activation in human airway epithelial cells. J Biol hem 2005;280: ) Hill EM, Eling T, Nettesheim P. hanges in expression of 15-lipoxygenase and prostaglandin-h synthase during differentiation of human tracheobrochial epithelial cells. Am J Respir ell Mol Biol 1998;18: ) Gray T, oakley R, Hirsh A, Thornton D, Kirkham S, Koo JS, et al. Regulation of MU5A mucin secretion and airway surface liquid metabolism by IL-1β in human bronchial epithelia. Am J Physiol Lung ell Mol Physiol 2004;286:L ) Borchers MT, arty MP, Leikauf GD. Regulation of human airway mucins by acrolein and inflammatory mediators. Am J Physiol 1999; 276:L ) Takahashi S, Takeuchi K, Okabe S. EP4 receptor mediation of prostaglandin E2-stimulated mucus secretion by rabbit gastric epithelial cells. Biochem Pharmacol 1999;58: ) Belley A, hadee K. Prostaglandin E2-stimulates rat and human colonic mucin exocytosis via the EP4 receptor. Gastroenterology 1999;117: ) Regan JW. EP2 and EP4 prostanoid receptor signaling. Life Sci 2003; 74: Korean J Otolaryngol 2006;49:307-12

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Microsoft Word doc KISEP Otology Korean J Otolaryngol 2006;49:263-8 사람중이점막상피세포에서 Uridine-5 Triphosphate 에의한칼슘이동과점액분비에미치는카페인의효과 최재영 김성헌 정상호 손은진 박헌이 신중욱 윤주헌 Effect of Caffeine on UTP-induced Ca 2+ Mobilization and Mucin Secretion