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Role of Epac1 in mediating anti-proliferative effects of prostanoid EP2 receptors and cAMP in human lung fibroblasts

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Abstract

In lung fibroblasts, proliferation is inhibited by activation of EP2 prostanoid receptors which are known to couple to adenylyl cyclase. Beside the classic target of cAMP, protein kinase A (PKA), alternative cAMP effectors have been identified, among them Epac (exchange protein activated by cAMP). The present study aimed to illuminate transduction pathways mediating the anti-proliferative effects of EP2 receptors in lung fibroblasts. Proliferative activity of human lung fibroblasts was determined by measuring [3H]-thymidine incorporation. The selective EP2 receptor agonist butaprost inhibited [3H]-thymidine incorporation by 75%, an effect mimicked by forskolin, the phosphodiesterase inhibitor IBMX, the stable cAMP analogues dibutyryl-cAMP and bromo-cAMP, as well as by the Epac selective cAMP analogues 8-pCPT-2′-O-Me-cAMP and Sp-8-pCPT-2′-O-Me-cAMPS, whereas the PKA selective agonist 6-Bnz-cAMP was inactive. The PKA inhibitor Rp-8-Br-cAMPS inhibited butaprost-induced phosphorylation of CREB (cAMP response element-binding protein), but did not affect butaprost-induced inhibition of [3H]-thymidine incorporation. Partial knockdown of Epac1 by specific siRNA transfection resulted in a marked attenuation of the inhibitory potency of butaprost, whereas transfection of Epac2 siRNA or non-silencing siRNA did not affect the effectiveness of butaprost to inhibit [3H]-thymidine incorporation. In conclusion, Epac1 rather than the classic cAMP effector PKA is a crucial element in the signal transduction pathway mediating anti-proliferative effects of EP2 receptor activation.

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References

  • Abramovitz M, Adam M, Boie Y, Carrière M, Denis D, Godbout C, Lamontagne S, Rochette C, Sawyer N, Tremblay NM, Belley M, Gallant M, Dufresne C, Gareau Y, Ruel R, Juteau H, Labelle M, Ouimet N, Metters KM (2000) The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim Biophys Acta 1483:285–293

    PubMed  CAS  Google Scholar 

  • Boie Y, Stocco R, Sawyer N, Slipetz DM, Ungrin MD, Neuschäfer-Rube F, Püschel GP, Metters KM, Abramovitz M (1997) Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes. Eur J Pharmacol 340:227–241

    Article  PubMed  CAS  Google Scholar 

  • Bos JL (2006) Epac proteins: multi-purpose cAMP targets. Trends Biochem Sci 31:680–686

    Article  PubMed  CAS  Google Scholar 

  • Charbeneau RP, Peters-Golden M (2005) Eicosanoids: mediators and therapeutic targets in fibrotic lung disease. Clin Sci 108:479–491

    Article  PubMed  CAS  Google Scholar 

  • Davies SP, Reddy H, Caivano M, Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351:95–105

    Article  PubMed  CAS  Google Scholar 

  • de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL (1998) Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396:474–477

    Article  PubMed  Google Scholar 

  • Dremier S, Kopperud R, Doskeland SO, Dumont JE, Maenhaut C (2003) Search for new cyclic AMP-binding proteins. FEBS Lett 546:103–107

    Article  PubMed  CAS  Google Scholar 

  • Dunkern TR, Feurstein D, Rossi GA, Sabatini F, Hatzelmann A (2007) Inhibition of TGF-beta induced lung fibroblast to myofibroblast conversion by phosphodiesterase inhibiting drugs and activators of soluble guanylyl cyclase. Eur J Pharmacol 572:12–22

    Article  PubMed  CAS  Google Scholar 

  • Ferro A (2006) beta-adrenoceptors and potassium channels. Naunyn Schmiedebergs Arch Pharmacol 373:183–185

    Article  PubMed  CAS  Google Scholar 

  • Freitag A, Reimann A, Wessler I, Racké K (1996) Effects of bacterial lipopolysaccharides (LPS) and tumour necrosis factor-alpha (TNF alpha) on rat tracheal epithelial cells in culture: morphology, proliferation and induction of nitric oxide (NO) synthase. Pulm Pharmacol 9:149–156

    Article  PubMed  CAS  Google Scholar 

  • Furchgott RF (1972) The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory. In: Blaschko H, Muscholl E (eds) Handbook experimental pharmacology, vol 33. Springer, Berlin Heidelberg New York, pp 283–335

    Google Scholar 

  • Gjertsen BT, Mellgren G, Otten A, Maronde E, Genieser HG, Jastorff B, Vintermyr OK, McKnight GS, Døskeland SO (1995) Novel (Rp)-cAMPS analogs as tools for inhibition of cAMP-kinase in cell culture. Basal cAMP-kinase activity modulates interleukin-1 beta action. J Biol Chem 27020599-20607

  • Haag S, Matthiesen S, Juergens UR, Racké K (2008a) Muscarinic receptors mediate stimulation of collagen synthesis in human lung fibroblasts. Eur Respir J doi:10.1183/09031936.00129307

  • Haag S, Warnken M, Juergens KU, Racké K (2008b) Anti-proliferative effects of prostanoid EP2 receptors and cAMP in human lung fibroblasts are mediated via Epac1. Naunyn-Schmiedeberg’s Arch Pharmacol 377(Suppl 1):14

    Google Scholar 

  • Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, Cherniack RM, Rogers RM, Sciurba FC, Coxson HO, Pare PD (2004) The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 350:2645–2653

    Article  PubMed  CAS  Google Scholar 

  • Holz GG, Chepurny OG, Schwede F (2008) Epac-selective cAMP analogs: New tools with which to evaluate the signal transduction properties of cAMP-regulated guanine nucleotide exchange factors. Cell Signal 20:10–20

    Article  PubMed  CAS  Google Scholar 

  • Huang S, Wettlaufer SH, Hogaboam C, Aronoff DM, Peters-Golden M (2007) Prostaglandin E(2) inhibits collagen expression and proliferation in patient-derived normal lung fibroblasts via E prostanoid 2 receptor and cAMP signaling. Am J Physiol Lung Cell Mol Physiol 292:L405–L413

    Article  PubMed  CAS  Google Scholar 

  • Huang S, Scott H, Wettlaufer SH, Peters-Golden M (2008) Prostaglandin E2 inhibits specific lung fibroblast functions via selective actions of PKA and Eapc-1. Am J Respir Cell Mol Biol doi:10.1165/rcmb.2008–0080OC

  • Jacobs JP, Jones CM, Baille JP (1970) Characteristics of a human diploid cell designated MRC-5. Nature 227:168–170

    Article  PubMed  CAS  Google Scholar 

  • Jeffery PK (2004) Remodeling and inflammation of bronchi in asthma and chronic obstructive pulmonary disease. ProcAm Thorac Soc 1:176–183

    Article  CAS  Google Scholar 

  • Kassel KM, Wyatt TA, Panettieri RA Jr, Toews ML (2008) Inhibition of human airway smooth muscle cell proliferation by ß2-adrenergic receptors and cAMP is PKA-independent: Evidence for EPAC involvement. Am J Physiol Lung Cell Mol Physiol 294:L131–L138

    Article  PubMed  CAS  Google Scholar 

  • Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, Graybiel AM (1998) A family of cAMP-binding proteins that directly activate Rap1. Science 282:2275–2279

    Article  PubMed  CAS  Google Scholar 

  • Kiriyama M, Ushikubi F, Kobayashi T, Hirata M, Sugimoto Y, Narumiya S (1997) Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br J Pharmacol 122:217–224

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  • Kolodsick JE, Peters-Golden M, Larios J, Toews GB, Thannickal VJ, Moore BB (2003) Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation. Am J Respir Cell Mol Biol 29:537–544

    Article  PubMed  CAS  Google Scholar 

  • Kooistra MR, Corada M, Dejana E, Bos JL (2005) Epac1 regulates integrity of endothelial cell junctions through VE-cadherin. FEBS Lett 579:4966–4972

    Article  PubMed  CAS  Google Scholar 

  • Liu X, Ostrom RS, Insel PA (2004) cAMP-elevating agents and adenylyl cyclase overexpression promote an antifibrotic phenotype in pulmonary fibroblasts. Am J Physiol Cell Physiol 286:C1089–C1099

    Article  PubMed  CAS  Google Scholar 

  • Matthiesen S, Bahulayan A, Kempkens S, Fuhrmann R, Stichnote C, Haag S, Juergens UR, Racké K (2006) Muscarinic receptors mediate stimulation of human lung fibroblast proliferation. Am J Respir Cell Mol Biol 35:621–627

    Article  PubMed  CAS  Google Scholar 

  • Matthiesen S, Bahulayan A, Holz O, Racké K (2007) MAPK pathway mediates muscarinic receptor-induced human lung fibroblast proliferation. Life Sci 80:2259–2262

    Article  PubMed  CAS  Google Scholar 

  • Molfino NA, Jeffery PK (2007) Chronic obstructive pulmonary disease: histopathology, inflammation and potential therapies. Pulm Pharmacol Ther 20:462–472

    Article  PubMed  CAS  Google Scholar 

  • Peterkofsky B, Diegelmann R (1997) Use of a mixture of proteinase-free collagenases for the specific assay of radioactive collagen in the presence of other proteins. Biochemistry 10:988–994

    Article  Google Scholar 

  • Racké K, Haag S, Matthiesen S (2007) Role of EPAC in transmission of ß-adrenoceptor and EP2-receptor mediated antiproliferative effects in human lung fibroblasts. Abstract, Eur Respir Soc Meeting 2007. Available at: http://www.ersnet.org/learning_resources_player/abstract_print_07/main_frameset.htm

  • Racké K, Haag S, Bahulayan A, Warnken M (2008) Pulmonary fibroblasts, an emerging target for anti-obstructive drugs. Naunyn-Schmiedeberg’s Arch. Pharmacol doi:10.1007/s00210–008–0264–0

  • Regan JW (2003) EP2 and EP4 prostanoid receptor signaling. Life Sci 74:143–153

    Article  PubMed  CAS  Google Scholar 

  • Roscioni SS, Elzinga CR, Schmidt M (2008) Epac: effectors and biological functions. Naunyn-Schmiedeberg’s Arch Pharmacol 377:345–357

    Article  CAS  Google Scholar 

  • Selman M, King TE, Pardo A (2001) Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 134:136–151

    PubMed  CAS  Google Scholar 

  • Shaywitz AJ, Greenberg ME (1999) CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu Rev Biochem 68:821–861

    Article  PubMed  CAS  Google Scholar 

  • Skålhegg BS, Taskén K (2000) Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front Biosci 5:D678–D693

    Article  PubMed  Google Scholar 

  • Stork PJ, Schmitt JM (2002) Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol 12:258–266

    Article  PubMed  CAS  Google Scholar 

  • Tallarida RJ, Murray RB (1988) Manual of pharmacological calculations with computer programs. Springer, Berlin

    Google Scholar 

  • White ES, Atrasz RG, Dickie EG, Aronoff DM, Stambolic V, Mak TW, Moore BB, Peters-Golden M (2005) Prostaglandin E(2) inhibits fibroblast migration by E-prostanoid 2 receptor-mediated increase in PTEN activity. Am J Respir Cell Mol Biol 32:135–141

    Article  PubMed  CAS  Google Scholar 

  • Wilson RJ, Rhodes SA, Wood RL, Shield VJ, Noel LS, Gray DW, Giles H (2004) Functional pharmacology of human prostanoid EP2 and EP4 receptors. Eur J Pharmacol 501:49–58

    Article  PubMed  CAS  Google Scholar 

  • Woodward DF, Pepperl DJ, Burkey TH, Regan JW (1995) 6-Isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a human EP2 receptor antagonist. Biochem Pharmacol 50:1731–1733

    Article  PubMed  CAS  Google Scholar 

  • Yokoyama U, Patel HH, Lai NC, Aroonsakool N, Roth DM, Insel PA (2008) The cyclic AMP effector Epac integrates pro- and anti-fibrotic signals. Proc Natl Acad Sci USA 105:6386–6391

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (Ra-400/12-2) and BONFOR Medical Faculty University of Bonn. This paper contains part of the Dr. rer. nat. thesis of S.H.

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Authors and Affiliations

  1. Institute of Pharmacology and Toxicology, University of Bonn, Reuterstraße 2b, 53113, Bonn, Germany

    S. Haag, M. Warnken & K. Racké

  2. Department of Pulmonary Diseases, Medical Polyclinic, University Hospital Bonn, Bonn, Germany

    U. R. Juergens

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  1. S. Haag
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  2. M. Warnken
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  3. U. R. Juergens
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  4. K. Racké
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Correspondence to K. Racké.

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Haag, S., Warnken, M., Juergens, U.R. et al. Role of Epac1 in mediating anti-proliferative effects of prostanoid EP2 receptors and cAMP in human lung fibroblasts. Naunyn-Schmied Arch Pharmacol 378, 617–630 (2008). https://doi.org/10.1007/s00210-008-0334-3

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