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B Repressing Factor1
* Department of Thoracic Medicine, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan;
Department of Medicine, Chang Gung University College of Medicine, Taoyuan, Taiwan;
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan;
Airways Disease Section, National Heart and Lung Institute, Imperial College London, United Kingdom; and
¶ School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan
NF-
B repressing factor (NRF), a nuclear inhibitor of NF-B, is constitutively expressed and is implicated in the basal silencing of specific NF-B targeting genes, including IFN-β, 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-B in primary human ASM (hASM), leading to induction of TGF-β1. In this study, we describe that, instead of inducing the NF-B 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-B 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.
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1 This work was supported by grants from the National Science Council of Taiwan (NSC 93-2314-B-182-043, NSC 94-2314-B-182-007, and NSC 95-2314-B-182-001).
2 Address correspondence and reprint requests to Dr. Han-Pin Kuo, Department of Thoracic Medicine, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 199 Tun-Hwa North Road, Taipei, Taiwan; E-mail address: q8828{at}ms11.hinet.net; or Dr. Chien-Huang Lin, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan; E-mail address: chlin{at}tmu.edu.tw
3 Abbreviations used in this paper: ASM, airway smooth muscle; ChIP, chromatin immunoprecipitation; hASM, human ASM; IP-DNA, immunoprecipitated DNA; LTR, long terminal repeat; NE, neutrophil-derived elastase; NRF, NF-B repressing factor; PMN, polymorphonuclear cell; qPCR, quantitative real-time PCR; RNA Pol 2, RNA polymerase II; SFM, serum-free medium; si, small interfering; 3'-UTR, 3'-untranslated region.
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