Abstract
The purinergic nucleotide ATP is released from stressed cells and is implicated in vascular inflammation. Glucocorticoids are essential to stress responses and are used therapeutically, yet little information is available that describes the effects of glucocorticoids on ATP-induced inflammation. In a human microvascular endothelial cell line, extracellular ATP-induced interleukin (IL)-6 secretion in a dose- and time-dependent manner. When cells were pretreated with dexamethasone, a prototypic glucocorticoid, ATP-induced IL-6 production was enhanced in a time- and dose-dependent manner. Mifepristone, a glucocorticoid receptor antagonist, blocked these effects. ATP-induced IL-6 release was significantly inhibited by a phospholipase C inhibitor [1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)] (63.2 ± 3%, p < 0.001) and abolished by a p38 mitogen-activated protein kinase inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580)] (88 ± 1%, p < 0.001). Cells treated with dexamethasone induced mRNA expression of the purinergic P2Y2 receptor (P2Y2R) 1.8- ± 0.1-fold and, when stimulated with ATP, enhanced Ca2+ release and augmented IL-6 mRNA expression. Silencing of the P2Y2R by its small interfering RNA decreased ATP-induced IL-6 production by 81 ± 1% (p < 0.001). Dexamethasone enhanced the transcription rate of P2Y2R mRNA and induced a dose-related increase in the activity of the P2Y2R promoter. Furthermore, dexamethasone-enhanced ATP induction of adhesion molecule transcription and augmented the release of IL-8. Dexamethasone leads to an unanticipated enhancement of endothelial inflammatory mediator production by extracellular ATP via a P2Y2R-dependent mechanism. These data define a novel positive feedback loop of glucocorticoids and ATP-induced endothelial inflammation.
Footnotes
This work was supported by the Intramural Research Program of the National Institutes of Health Clinical Center and the National Institute of Diabetes and Digestive and Kidney Diseases.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.171975.
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ABBREVIATIONS:
- P2
- purinergic receptor 2
- RU486
- 11β-(4-dimethylamino)phenyl-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one, mifepristone
- U73122
- 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione
- Gö6976
- 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole
- PD 98059
- 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
- SB 203580
- 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole
- SP 600125
- 1,9-pyrazoloanthrone, anthrapyrazolone
- LY294002
- 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride
- DMSO
- dimethyl sulfoxide
- LPS
- lipopolysaccharide
- IL
- interleukin
- TNFα
- tumor necrosis factor α
- IL-1ra
- IL-1 receptor antagonist
- MCP-1
- monocyte chemotactic factor
- PCR
- polymerase chain reaction
- RT-PCR
- reverse transcription PCR
- siRNA
- small interfering RNA
- PLC
- phospholipase C
- MAPK
- mitogen-associated protein kinase
- PKC
- protein kinase C
- ERK
- extracellular signal-regulated kinase
- [Ca2+]i
- intracellular calcium
- JNK
- c-Jun N-terminal kinase
- TNFAIP3
- TNFα-induced protein 3
- VCAM-1
- vascular cell adhesion molecule-1
- ICAM-1
- intracellular adhesion molecule-1
- SELE
- E- selectin
- DEX
- dexamethasone.
- Received June 24, 2010.
- Accepted September 2, 2010.
- U.S. Government work not protected by U.S. copyright
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