Beta-tryptase regulates IL-8 expression in airway smooth muscle cells by a PAR-2-independent mechanism.
Mast cells are central in the development of several allergic diseases and contain a number of pre-formed mediators. beta-tryptase, the most abundant mast cell product, is increasingly recognized as a key inflammatory mediator, as it causes the release of cytokines, particularly the chemokine IL-8, from both inflammatory and structural cells. The molecular mechanisms, however, remain largely unknown. In this study we sought to investigate whether beta-tryptase could induce IL-8 expression in human airway smooth muscle (ASM) cells and to explore the molecular mechanisms involved. We found that purified human beta-tryptase stimulated IL-8 production in a time- and concentration-dependent manner, which was inhibited by protease inhibitors and mimicked by recombinant human beta-tryptase, but not by the protease-activated receptor-2 (PAR-2) agonist SLIGKV-NH(2), consistent with the low-level expression of PAR-2 protein in these cells. beta-tryptase also up-regulated IL-8 mRNA expression, as analyzed by RT-PCR and real-time PCR, which was abolished by the transcription inhibitor actinomycin D. Reporter gene assay showed that beta-tryptase-induced IL-8 transcription was mediated by the transcription factors activator protein-1, CCAAT/enhancer binding protein, and NF-kappaB, and chromatin immunoprecipitation assay demonstrated that beta-tryptase induced in vivo binding of these transcription factors to the IL-8 gene promoter. Furthermore, beta-tryptase stabilized IL-8 mRNA, suggesting additional post-transcriptional regulation. Collectively these findings show that beta-tryptase up-regulates IL-8 expression in ASM cells through a PAR-2-independent proteolytic mechanism and coordinated transcriptional and post-transcriptional regulation, which may be of particular importance in understanding the role and the mechanisms of action of beta-tryptase in regulating chemokine expression in mast cell-related disorders.
Mullan
Charlotte S
CS
Division of Respiratory Medicine, Clinical Sciences Building, City Hospital, University of Nottingham, Hucknall Road, Nottingham NG5 1PB, UK.
Riley
Michael
M
Clarke
Deborah
D
Tatler
Amanda
A
Sutcliffe
Amy
A
Knox
Alan J
AJ
Pang
Linhua
L
D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D006868:Hydrolysis; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D044464:Receptor, PAR-2 / Q000819:agonists / Q000378:metabolism / Q000502:physiology*; D014132:Trachea / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D053802:Tryptases / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology
D016209:Interleukin-8; D044464:Receptor, PAR-2; D053802:Tryptases