Involvement of Phospholipase D 1 and 2 in the subcellular localization and activity of formyl-peptide-receptors in the human colonic cell line HT29.
Epithelial cells of the alimentary tract play a central role in the mucosal host defence against pathogens and in the recognition of agonists that interact with mucosal surfaces. In particular, the formyl peptide receptor (FPR) family and their three human subtypes: FPR, formyl-peptide-receptor-like-1 (FPRL1) and FPRL2, are involved in the host defence against pathogens that mediate epithelial responses thus upregulating inflammation. To elucidate the mechanisms by which FPR function, we examined the influence of phospholipase D (PLD) 1 and 2 on the activity and signal transduction of human enterocytes cell line HT29. PLD is a key enzyme involved in secretion, endocytosis and receptor signalling. We inhibited PLD1 and 2 by small interference RNA (siRNA) and determined the activity of formyl peptide receptors using Western blotting and cAMP level measurements. We then analyzed the distribution of formyl peptide receptors FPR, FPRL1 and FPRL2 compared to a control. In this study, we demonstrated that the depletion of PLD1 and 2 resulted in a marked reduction of formyl peptide receptor activity due to inhibited extracellular-signal regulated kinases 1/2 (ERK1/2), phosphorylation and cAMP level reduction. In addition, we observed an intracellular accumulation of FPR, FPRL1 and FPRL2 as a result of receptor recycling inhibition using fluorescence microscopy. The constitutive internalization rate was unaffected. Our results support the importance of PLD1 and 2 in formyl peptide receptor function and the role of endocytosis, receptor recycling and reactivation for receptor activity.
Brandenburg
Lars-Ove
LO
Department of Anatomy and Cell Biology, RWTH Aachen University, Wendlingweg 2, Aachen, Germany. lbrandenburg@ukaachen.de
Seyferth
Svenja
S
Wruck
Christoph Jan
CJ
Koch
Thomas
T
Rosenstiel
Philip
P
Lucius
Ralph
R
Pufe
Thomas
T
D019794:2,3-Diphosphoglycerate / Q000378:metabolism; D000704:Analysis of Variance; D003106:Colon / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007150:Immunohistochemistry; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D044042:Receptors, Formyl Peptide / Q000037:antagonists & inhibitors / Q000378:metabolism*; D044067:Receptors, Lipoxin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
C469986:FPR1 protein, human; C105101:FPR2 protein, human; D034741:RNA, Small Interfering; D044042:Receptors, Formyl Peptide; D044067:Receptors, Lipoxin; D019794:2,3-Diphosphoglycerate; D000242:Cyclic AMP; D048049:Extracellular Signal-Regulated MAP Kinases; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1