Transcriptional regulation of monocyte chemotactic protein‐1 release by endothelin‐1 in human airway smooth muscle cells involves NF‐κB and AP‐1

Abstract

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. 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. 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. 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.