Allergy Asthma Immunol Res.  2013 Jul;5(4):224-231. 10.4168/aair.2013.5.4.224.

Effect of Prostaglandin E2 on Vascular Endothelial Growth Factor Production in Nasal Polyp Fibroblasts

• Han DY
• Cho JS
• Moon YM
• Lee HR
• Lee HM
• Lee BD
• Baek BJ

• KMID: 2260316
• DOI: http://doi.org/10.4168/aair.2013.5.4.224

Abstract

PURPOSE
Angiogenesis is involved in the pathogenesis of chronic rhinosinusitis with nasal polyps. We aimed to investigate the effects of prostaglandin E2 (PGE2) on vascular endothelial growth factor (VEGF) production, the role of E-prostanoid (EP) 4 receptors, and the signal transduction pathway mediating VEGF production in nasal polyp-derived fibroblasts (NPDFs).
METHODS
Eight primary NPDF cultures were established from nasal polyps, which were incubated with or without PGE2. Reverse transcription-polymerase chain reaction amplification of EP receptors (EP1, EP2, EP3, and EP4) and immunofluorescence staining for VEGF production were performed. VEGF production via the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K) pathways was evaluated by enzyme-linked immunosorbent assay.
RESULTS
All EP receptors were expressed in NPDFs. PGE2 significantly increased VEGF production concentration- and time dependently, and VEGF production was regulated by an EP4 receptor. Activation of intracellular cAMP regulated VEGF production. VEGF production was decreased by PKA and PI3K inhibitors via intracellular cAMP.
CONCLUSIONS
PGE2 stimulates VEGF production via the EP4 receptor in NPDFs. These results indicate that PGE2-induced VEGF production is mediated, at least partially, through cAMP-dependent signaling pathways. Therapies targeting the EP4 receptor may be effective in inhibiting the development of nasal polyps.

Keyword

Nasal polyp; fibroblast; prostaglandin E2; vascular endothelial growth factor; E-prostanoid receptor; cyclic adenosine monophosphate

MeSH Terms

Adenosine Monophosphate
Cyclic AMP-Dependent Protein Kinases
Dinoprostone
Fibroblasts
Fluorescent Antibody Technique
Nasal Polyps
Negotiating
Phosphatidylinositol 3-Kinase
Signal Transduction
Vascular Endothelial Growth Factor A
Adenosine Monophosphate
Cyclic AMP-Dependent Protein Kinases
Dinoprostone
Phosphatidylinositol 3-Kinase
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 Analysis of EP receptor mRNA expression using reverse transcriptase-polymerase chain reaction in NPDFs. EP, E-prostanoid; GAPDH, glyceraldehyde phosphate dehydrogenase; NPDFs, nasal polyp-derived fibroblasts.

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  Fig. 2 Effect of PGE2 on VEGF production in NPDFs as determined by RT-PCR (A, C) and ELISA (B, D). (A, B) RT-PCR and ELISA showed that the relative ratio of VEGF/GADPH mRNA and VEGF protein were increased in a dose-dependent manner. (C, D) The results of RT-PCR and ELISA showed that the relative ratio of VEGF/GADPH mRNA and VEGF protein were increased in a time-dependent manner. Monolayer cultures were stimulated with PGE2 (10 mM), and the medium was harvested at varying time points followed by determination of VEGF levels by ELISA. The means±SE of data from six experiments are shown. *P<0.05 and †P<0.01 vs. control. VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde phosphate dehydrogenase; PGE2, prostaglandin E2; NPDF, nasal polyp derived fibroblast; RT-PCR, reverse transcription-polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; SE, standard error.

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  Fig. 3 (A) Effect of EP receptor agonists on VEGF production by NPDFs. CAY10580 increased VEGF production significantly; however, no other EP receptor agonist showed an increase in VEGF production. (B) The effect of EP receptor antagonists on VEGF production. AH23848 decreased VEGF production markedly, but no other EP receptor antagonist elicited a decrease in VEGF production. (C) The effect of PGE2 combined with an EP4 receptor agonist or antagonist on VEGF production by immunofluorescence staining. Strong immunoreactivity for VEGF was observed in NPDF cells treated with PGE2 and CAY10580 by confocal laser scanning microscopy. NPDFs treated with PGE2 and EP4 receptor agonists showed blue-stained nuclei with a greenish cytoplasm (representative of four independent experiments; original magnification, ×200). The means±SE of data from six experiments are shown. *P<0.05 vs. control and †P<0.05 vs. PGE2. PGE2, prostaglandin E2; EP, E-prostanoid; Sulprostone, EP1/3 receptor agonist; Butaprost, EP2 receptor agonist; CAY10580, EP4 receptor agonist; SC51322, EP1 receptor antagonist; AH6809, EP2 receptor antagonist; L-798106, EP3 receptor antagonist; AH23848, EP4 receptor antagonist; VEGF, vascular endothelial growth factor; NPDFs, nasal polyp-derived fibroblasts; SE, standard error.

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  Fig. 4 (A) The effect of PGE2 on cAMP release. PGE2 stimulated cAMP release in a dose-dependent manner. (B) The effect of forskolin on VEGF production. Forskolin increased VEGF production in a dose-dependent manner. *P<0.05 and †P<0.01 vs. the control. (C) The effect of cAMP on VEGF production via the EP4 receptor. CAY10580 increased cAMP release. When AH 23848 was added before the treatment of PGE2, cAMP level was significantly decreased. The means±SE of data from six experiments are shown. *P<0.05 vs. control and †P<0.05 vs. PGE2. cAMP, cyclic adenosine monophosphate; PGE2, prostaglandin E2; VEGF, vascular endothelial growth factor; EP, E-prostanoid; CAY10580, EP4 receptor agonist; AH23848, EP4 receptor antagonist; Forskolin, cAMP activator; SE, standard error.

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  Fig. 5 (A) The effect of PKA and PI3K inhibitors on VEGF production in NPDFs. KT5720 and LY294002 significantly decreased VEGF production. (B) The effect of PKA and PI3K inhibitors on cAMP release. PGE2 with KT5720 elicited a decrease in cAMP release, whereas PGE2 with LY294002 did not. The means±SE of data from six experiments are shown. *P<0.05 vs. control and †P<0.05 vs. PGE2. VEGF, vascular endothelial growth factor; cAMP, cyclic adenosine monophosphate; PGE2, prostaglandin E2; PKA, protein kinase A; PI3K, phosphatidylinositol 3-kinase; KT5720, PKA inhibitor; LY294002, PI3K inhibitor; Forskolin, cAMP activator; VEGF, vascular endothelial growth factor; NPDFs, nasal polyp-derived fibroblasts; SE, standard error.

Reference

1. Coste A, Brugel L, Maître B, Boussat S, Papon JF, Wingerstmann L, Peynègre R, Escudier E. Inflammatory cells as well as epithelial cells in nasal polyps express vascular endothelial growth factor. Eur Respir J. 2000; 15:367–372.

2. Biteman B, Hassan IR, Walker E, Leedom AJ, Dunn M, Seta F, Laniado-Schwartzman M, Gronert K. Interdependence of lipoxin A4 and heme-oxygenase in counter-regulating inflammation during corneal wound healing. FASEB J. 2007; 21:2257–2266.

3. Tilley SL, Coffman TM, Koller BH. Mixed messages: modulation of inflammation and immune responses by prostaglandins and thromboxanes. J Clin Invest. 2001; 108:15–23.

4. Cho KN, Choi JY, Kim CH, Baek SJ, Chung KC, Moon UY, Kim KS, Lee WJ, Koo JS, Yoon JH. Prostaglandin E2 induces MUC8 gene expression via a mechanism involving ERK MAPK/RSK1/cAMP response element binding protein activation in human airway epithelial cells. J Biol Chem. 2005; 280:6676–6681.

5. Yoshimura T, Yoshikawa M, Otori N, Haruna S, Moriyama H. Correlation between the prostaglandin D(2)/E(2) ratio in nasal polyps and the recalcitrant pathophysiology of chronic rhinosinusitis associated with bronchial asthma. Allergol Int. 2008; 57:429–436.

6. Peacock CD, Misso NL, Watkins DN, Thompson PJ. PGE 2 and dibutyryl cyclic adenosine monophosphate prolong eosinophil survival in vitro. J Allergy Clin Immunol. 1999; 104:153–162.

7. Ben-Av P, Crofford LJ, Wilder RL, Hla T. Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS Lett. 1995; 372:83–87.

8. Sugimoto Y, Narumiya S. Prostaglandin E receptors. J Biol Chem. 2007; 282:11613–11617.

9. Breyer RM, Bagdassarian CK, Myers SA, Breyer MD. Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol. 2001; 41:661–690.

10. Hatazawa R, Tanigami M, Izumi N, Kamei K, Tanaka A, Takeuchi K. Prostaglandin E2 stimulates VEGF expression in primary rat gastric fibroblasts through EP4 receptors. Inflammopharmacology. 2007; 15:214–217.

11. Inoue H, Takamori M, Shimoyama Y, Ishibashi H, Yamamoto S, Koshihara Y. Regulation by PGE2 of the production of interleukin-6, macrophage colony stimulating factor, and vascular endothelial growth factor in human synovial fibroblasts. Br J Pharmacol. 2002; 136:287–295.

12. Cheng T, Cao W, Wen R, Steinberg RH, LaVail MM. Prostaglandin E2 induces vascular endothelial growth factor and basic fibroblast growth factor mRNA expression in cultured rat Muller cells. Invest Ophthalmol Vis Sci. 1998; 39:581–591.

13. Bradbury D, Clarke D, Seedhouse C, Corbett L, Stocks J, Knox A. Vascular endothelial growth factor induction by prostaglandin E2 in human airway smooth muscle cells is mediated by E prostanoid EP2/EP4 receptors and SP-1 transcription factor binding sites. J Biol Chem. 2005; 280:29993–30000.

14. Sales KJ, Maudsley S, Jabbour HN. Elevated prostaglandin EP2 receptor in endometrial adenocarcinoma cells promotes vascular endothelial growth factor expression via cyclic 3',5'-adenosine monophosphate-mediated transactivation of the epidermal growth factor receptor and extracellular signal-regulated kinase 1/2 signaling pathways. Mol Endocrinol. 2004; 18:1533–1545.

15. Fujino H, Salvi S, Regan JW. Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. Mol Pharmacol. 2005; 68:251–259.

16. Bateman ND, Fahy C, Woolford TJ. Nasal polyps: still more questions than answers. J Laryngol Otol. 2003; 117:1–9.

17. Pawankar R. Nasal polyposis: an update: editorial review. Curr Opin Allergy Clin Immunol. 2003; 3:1–6.

18. Wang QP, Escudier E, Roudot-Thoraval F, Abd-Al Samad I, Peynegre R, Coste A. Myofibroblast accumulation induced by transforming growth factor-beta is involved in the pathogenesis of nasal polyps. Laryngoscope. 1997; 107:926–931.

19. Harris SG, Padilla J, Koumas L, Ray D, Phipps RP. Prostaglandins as modulators of immunity. Trends Immunol. 2002; 23:144–150.

20. Samuelsson B, Morgenstern R, Jakobsson PJ. Membrane prostaglandin E synthase-1: a novel therapeutic target. Pharmacol Rev. 2007; 59:207–224.

21. Sheng H, Shao J, Kirkland SC, Isakson P, Coffey RJ, Morrow J, Beauchamp RD, DuBois RN. Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2. J Clin Invest. 1997; 99:2254–2259.

22. Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev. 1999; 79:1193–1226.

23. Kohyama T, Ertl RF, Valenti V, Spurzem J, Kawamoto M, Nakamura Y, Veys T, Allegra L, Romberger D, Rennard SI. Prostaglandin E(2) inhibits fibroblast chemotaxis. Am J Physiol Lung Cell Mol Physiol. 2001; 281:L1257–L1263.

24. Arosh JA, Banu SK, Chapdelaine P, Emond V, Kim JJ, MacLaren LA, Fortier MA. Molecular cloning and characterization of bovine prostaglandin E2 receptors EP2 and EP4: expression and regulation in endometrium and myometrium during the estrous cycle and early pregnancy. Endocrinology. 2003; 144:3076–3091.

25. Fujino H, West KA, Regan JW. Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. J Biol Chem. 2002; 277:2614–2619.