Neutrophil elastase represses IL-8/CXCL8 synthesis in human airway smooth muscle cells through induction of NF-kappa B repressing factor.
NF-kappaB repressing factor (NRF), a nuclear inhibitor of NF-kappaB, is constitutively expressed and is implicated in the basal silencing of specific NF-kappaB targeting genes, including IFN-beta, IL-8/CXCL8, and iNOS. Little is known about the regulation of NRF and its role in response to stimuli. Airway smooth muscle (ASM) is a rich source of inflammatory mediators that may regulate the development and progression of airway inflammation. We have previously reported that NE activates NF-kappaB in primary human ASM (hASM), leading to induction of TGF-beta1. In this study, we describe that, instead of inducing the NF-kappaB response gene IL-8/CXCL8, NE suppressed IL-8/CXCL8 release and mRNA expression in hASM cells. Transcriptional blockade studies using actinomycin D revealed a similar degradation rate of IL-8/CXCL8 mRNA in the presence or absence of NE, suggesting an involvement at the transcription level. Mechanistically, the NE repressive effect was mediated by inducing NRF, as shown by RT-PCR and Western blotting, which was subsequently recruited to the native IL-8/CXCL8 promoter leading to removal of RNA polymerase II from the promoter, as demonstrated by chromatin immunoprecipitation assays. Knockdown of NRF by small interfering RNA prevented NE-induced suppression of IL-8/CXCL8 expression. In contrast, NE did not induce NRF expression in A549 and Beas-2B cells, where NE only stimulates NF-kappaB activation and IL-8/CXCL8 induction. Forced expression of NRF in A549 cells by an NRF expression plasmid suppressed IL-8/CXCL8 expression. Hence, we describe a novel negative regulatory mechanism of NE-induced NRF, which is restricted to hASM and mediates the suppression of IL-8/CXCL8 expression.
Ho
Shu-Chuan
SC
Department of Thoracic Medicine, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan.
Lee
Kang-Yun
KY
Chan
Yao-Fei
YF
Kuo
Lu-Wei
LW
Ito
Kazuhiro
K
Adcock
Ian M
IM
Chen
Bing-Chang
BC
Sheu
Joen-Rong
JR
Lin
Chien-Huang
CH
Kuo
Han-Pin
HP
D001980:Bronchi / Q000166:cytology / Q000201:enzymology* / Q000276:immunology* / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D015536:Down-Regulation / Q000235:genetics / Q000276:immunology; D006801:Humans; D016209:Interleukin-8 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D019272:Leukocyte Elastase / Q000502:physiology*; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology* / Q000276:immunology* / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000276:immunology; D034622:RNA Interference / Q000276:immunology; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors; D015854:Up-Regulation / Q000235:genetics / Q000276:immunology*
D016209:Interleukin-8; C111027:NKRF protein, human; D012097:Repressor Proteins; D051996:Transcription Factor RelA; D019272:Leukocyte Elastase