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

Background and purpose:  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.

Experimental approach:  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.

Key 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‐κB (NF‐κB). ET‐1 promoted binding of AP‐1 c‐Jun subunit and NF‐κB p65 subunit to the MCP‐1 promoter. Blocking the inhibitor of κB 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 and implications:  ET‐1 regulated MCP‐1 transcriptionally, via NF‐κB 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.

DOI: 10.1111/j.1476-5381.2009.00143.x

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