The Regulatory Mechanism for NF-Kappa B Induced NF-Kappa B Repression Factor (NRF)

NF-κB is well known as a pro-inflammatory transcription factor which mediates the induction of most pro-inflammatory mediators and has attracted interest as a target for the treatment of inflammatory diseases. The consequence of NF-κB activation is cell and context dependent and sometimes Janus-like. This is in part due to involvement of variable subunits which have distinct and non-overlapping functions. NF-κB repressing factor (NRF) has been implicated in repressing basal expression of several NF-κB mediated gene expression, including IL-8, iNOS, interferon-β and IP-10, but conversely in the induction of IL-8 by IL-1β in HeLa cells. In our preliminary study, we have demonstrated that neutrophil-derived elastase (NE) specifically inhibits the expression of the proinflammatory chemokine IL-8/CXCL8 in primary human airway smooth muscle (hASM) cells through up-regulation of NRF. NRF is induced by NF-κB activation and is subsequently recruited to the native IL-8/CXCL8 promoter leading to removal of RNA polymerase II from the promoter. Knockdown of NRF by SiRNA prevents NE-induced suppression of IL-8 expression. In contrast, NE stimulates IL-8/CXCL8 expression in lung epithelial cells without a change in NRF expression. A similar effect is also seen in IL-1β-stimulated primary hASM cells. Since the expression of NRF can be differentially induced by NF-κB activation by different stimuli or in different cellular contexts, understanding the precise regulatory mechanisms may not only advance our knowledge into the role of NRF as an endogenous modulation of NF-κB mediated responses which may be applied to human inflammatory diseases, but also provide a new therapeutic direction for developing specific NF-κB inhibitors. This 3-year project is designed to investigate 1) NF-κB family subunit activation implicated in NRF up-regulation by TransAM assay analysis of differential subunit activation between NE and IL-1β stimulation or between A549 cells and hASM cells, or by ChIP assay to determine the specific subunit binding to NRF promoter site; 2) The upstream of NF-κB family subunit activation pathway (classical IKK1 or non-canonical IKK2 pathway) involved in differential stimuli (such as NE, LPS vs IL-1β) in regulation of NRF expression by treatment with specific inhibitors, transfection with SiRNA targeting IKK1 or IKK2; 3) The implication of histone acetylation in NRF-mediated repression of NF-κB mediated genes by ChIP assay for histone H3, H4 acetylation, RNA polymerase or HDAC recruitment, or by treatment with specific inhibitors; 4) Basal and up-regulated NRF responsive genes in pro-inflammatory cells, such as monocytes (THP-1 cells) by over-expression of NRF in A549 cells, hASM cells with or without stimulation with NE, IL-1β, TNF-α or LPS and microaaray assay analysis targeting NF-κB responsive genes. This project may explore a novel regulatory mechanism for NF-κB mediated responses.

Project ID:PC9902-0609
External Project ID:NSC97-2314-B182-024-MY3