Transcriptional regulation of monocyte chemotactic protein-1 release by endothelin-1 in human airway smooth muscle cells involves NF-kappaB and AP-1.
OBJECTIVE
Endothelin-1 (ET-1) is implicated in airway inflammation in asthma, but the mechanisms of its effects are poorly understood. We studied the effect of ET-1 on expression of the chemokine, monocyte chemotactic protein-1 (MCP-1), in primary cultures of human airway smooth muscle cells.


METHODS
MCP-1 release was measured by elisa. Pharmacological antagonists/inhibitors, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to study ET receptors and kinase cascades. Transcriptional regulation was studied by real-time RT-PCR, transient transfection studies and chromatin immunoprecipitation assay. Major findings were confirmed in cells from three donors and mechanistic studies in cells from one donor.


RESULTS
ET-1 increased MCP-1 release through an ET(A) and ET(B) receptor-dependent mechanism. ET-1 increased MCP-1 mRNA levels but not mRNA stability suggesting it was acting transcriptionally. ET-1 increased the activity of an MCP-1 promoter-reporter construct. Serial deletions of the MCP-1 promoter mapped ET-1 effects to a region between -213 and -128 base pairs upstream of the translation start codon, containing consensus sequences for activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB). ET-1 promoted binding of AP-1 c-Jun subunit and NF-kappaB p65 subunit to the MCP-1 promoter. Blocking the inhibitor of kappaB kinase-2 with 2-[(aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA-1) decreased ET-1-stimulated MCP-1 production. p38 and p44/p42 mitogen-activated protein kinases were involved in upstream signalling.


CONCLUSIONS
ET-1 regulated MCP-1 transcriptionally, via NF-kappaB and AP-1. The upstream signalling involved ET(A), ET(B) receptors, p38 and p44/p42 mitogen-activated protein kinases. These may be targets for novel asthma therapies.
Sutcliffe
Amy M
AM
Nottingham Respiratory Biomedical Research Unit, University of Nottingham, City Hospital, Nottingham NG5 1PB, UK.
Clarke
Deborah L
DL
Bradbury
Dawn A
DA
Corbett
Lisa M
LM
Patel
Jamie A
JA
Knox
Alan J
AJ
D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism*; D019332:Endothelin-1 / Q000494:pharmacology / Q000502:physiology*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3 / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D016328:NF-kappa B / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D044022:Receptor, Endothelin A / Q000502:physiology; D044023:Receptor, Endothelin B / Q000502:physiology; D012137:Respiratory System / Q000166:cytology*; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000502:physiology*; D014158:Transcription, Genetic; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D019332:Endothelin-1; D016328:NF-kappa B; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D018808:Transcription Factor AP-1; D019869:Phosphatidylinositol 3-Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases