Urocortin induced expression of COX-2 and ICAM-1 via corticotrophin-releasing factor type 2 receptor in rat aortic endothelial cells.
OBJECTIVE
Our previous study showed that urocortin (Ucn1) exacerbates the hypercoagulable state and vasculitis in a rat model of sodium laurate-induced thromboangiitis obliterans. Furthermore, the inflammatory molecules COX-2 and ICAM-1 may participate in this effect. In the present study, the effects of Ucn1 on COX-2 and ICAM-1 expression in lipopolysaccharide (LPS)-induced rat aortic endothelial cells (RAECs) were investigated and the mechanisms involved explored.


METHODS
RAECs were isolated from adult male Wistar rats, and identified at the first passage. Experiments were performed on cells, from primary culture, at passages 5-8. The expression of COX-2 and ICAM-1 at both mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blot analysis. Levels of PGE(2) and soluble ICAM-1 (sICAM-1) in culture medium were measured by enzyme-linked immunosorbent assay. Furthermore, the phosphorylation status of p38MAPK, ERK1/2, JNK, Akt and NF-kappaB was analysed by Western blot; nuclear translocation of NF-kappaB was observed by immunofluorescence.


RESULTS
Ucn1 augmented LPS-induced expression of COX-2 and ICAM-1 in RAECs in a time- and concentration-dependent manner. Ucn1 increased PGE(2) and sICAM-1 levels. These effects were abolished by the CRF(2) receptor antagonist, antisauvagine-30, but not by the CRF(1) receptor antagonist, NBI-27914. Moreover, Ucn2 activated p38MAPK and augmented NF-kappaB nuclear translocation and phosphorylation, whereas ERK1/2, JNK and Akt pathways were not involved in this process.


CONCLUSIONS
These findings suggest that Ucn1 exerts pro-inflammatory effects by augmenting LPS-induced expression of COX-2 and ICAM-1 in RAECs via CRF(2) receptors and the activation of p38MAPK and NF-kappaB.
Zhang
Rongjian
R
Department of Pharmacology, Nanjing Medical University, Nanjing, China.
Xu
Youhua
Y
Fu
Hong
H
Wang
Juejin
J
Jin
Lai
L
Li
Shengnan
S
D021581:Active Transport, Cell Nucleus; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000096:biosynthesis*; D015232:Dinoprostone / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D004789:Enzyme Activation; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018019:Receptors, Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors; D054832:Urocortins / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000099052:CRF Receptor, Type 1
C471210:2-methyl-4-(N-propyl-N-cycloproanemethylamino)-5-chloro-6-(2,4,6-trichloranilino)pyrimidine; D000814:Aniline Compounds; C101531:CRF receptor type 2; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010446:Peptide Fragments; D011743:Pyrimidines; D018019:Receptors, Corticotropin-Releasing Hormone; D054832:Urocortins; C116607:antisauvagine 30; D018799:Intercellular Adhesion Molecule-1; D000099052:CRF Receptor, Type 1; D051546:Cyclooxygenase 2; C489691:Ptgs2 protein, rat; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone
Acta Histochem. 2002;104(2):177-84
12086338
J Immunol. 2002 Jun 1;168(11):5860-6
12023390
Nat Rev Immunol. 2008 Nov;8(11):837-48
18927578
Am J Physiol Lung Cell Mol Physiol. 2001 Mar;280(3):L450-7
11159028
Br J Pharmacol. 1993 Dec;110(4):1282-4
8306065
FASEB J. 1998 Sep;12(12):1063-73
9737710
J Pharmacol Exp Ther. 1999 Mar;288(3):1349-56
10027877
J Biol Chem. 2002 Apr 5;277(14):12280-7
11790788
Endocrinology. 2004 Feb;145(2):890-900
14592950
Peptides. 2004 Oct;25(10):1745-9
15476941
J Immunol. 2004 Jun 1;172(11):7015-23
15153523
Cell Physiol Biochem. 2008;21(1-3):173-82
18209484
Toxicol Lett. 2006 Sep 10;165(3):212-20
16777358
J Immunol. 1995 Oct 1;155(7):3538-45
7561050
In Vitro Cell Dev Biol Anim. 2001 Jan;37(1):50-4
11249206
Endocrinology. 2006 Sep;147(9):4454-62
16777977
Hypertension. 2000 Jan;35(1 Pt 1):68-75
10642277
Nature. 1995 Nov 16;378(6554):287-92
7477349
Ann N Y Acad Sci. 1999 Jun 22;876:1-11; discussion 11-3
10415589
J Neurosci. 2003 Dec 10;23(36):11436-43
14673008
Mol Pharmacol. 2001 Dec;60(6):1439-48
11723253
Proc Natl Acad Sci U S A. 2002 May 28;99(11):7734-9
12032352
Biochem Biophys Res Commun. 2008 Jul 4;371(3):345-9
18267110
Atherosclerosis. 2009 Apr;203(2):360-70
18640679
Mol Pharmacol. 1999 Mar;55(3):575-83
10051543
Endocrinology. 1998 Jan;139(1):403-13
9421440
Curr Pharm Des. 2006;12(27):3497-508
17017943
Peptides. 2009 Feb;30(2):365-72
19026699
Am J Physiol. 1997 Jul;273(1 Pt 1):L217-26
9252559
J Endocrinol. 2006 Apr;189(1):167-78
16614391
Mol Endocrinol. 2008 Mar;22(3):689-706
18048641
J Immunol. 2003 May 1;170(9):4767-75
12707358
J Immunol. 2000 Sep 1;165(5):2719-28
10946303
Peptides. 2001 May;22(5):809-20
11337095
J Invest Dermatol. 1989 Aug;93(2 Suppl):33S-38S
2666521
J Clin Endocrinol Metab. 2004 Nov;89(11):5352-61
15531481
J Mol Endocrinol. 2000 Aug;25(1):43-52
10915217
J Leukoc Biol. 2000 Jun;67(6):885-93
10857863
Haematologica. 2000 Sep;85(9):967-72
10980636
Annu Rev Biochem. 1990;59:129-70
1695830
Ann Rheum Dis. 2001 Nov;60 Suppl 3:iii6-12
11890657
Trends Pharmacol Sci. 2003 May;24(5):245-52
12767724
Semin Thromb Hemost. 2000;26(5):571-87
11129414
Gut. 2007 Sep;56(9):1210-7
17412781
Endocrinology. 1995 Dec;136(12):5745-50
7588332
J Cardiovasc Pharmacol. 2006;47 Suppl 1:S26-36
16785826
Vascul Pharmacol. 2006 Apr;44(4):238-46
16488667
J Clin Endocrinol Metab. 1998 Feb;83(2):708-11
9467598
Cytokine. 2005 Jul 21;31(2):127-34
15919214
Front Neuroendocrinol. 2007 Apr;28(1):1-27
17083971
Neuropeptides. 1998 Apr;32(2):167-71
9639256
J Clin Endocrinol Metab. 2001 Sep;86(9):4344-52
11549672
Eur J Immunol. 2003 Mar;33(3):597-605
12616480
Kidney Int. 2000 Oct;58(4):1664-76
11012900
Trends Endocrinol Metab. 1999 Nov;10(9):359-368
10511695
J Cardiovasc Pharmacol. 2008 Jan;51(1):32-7
18209566
J Cell Physiol. 2007 Mar;210(3):774-83
17117478
Immunol Lett. 2008 Oct 30;120(1-2):57-64
18675302
J Biol Chem. 2003 Mar 14;278(11):9944-52
12645577
Endocrinology. 2008 Jun;149(6):2866-76
18325997
Infect Immun. 2002 Nov;70(11):6068-74
12379683
Br J Pharmacol. 2008 Mar;153 Suppl 2:S1-209
18347570
J Neurosci. 1998 Mar 1;18(5):1633-41
9464988
Lab Invest. 1987 Jul;57(1):94-105
3298852
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3939-44
18308934