Study on the Regulation of Txnip in Nicotine-Induced Inflammatory C-Reactive Protein Expression in Macrophages and Its Pathological Effects

In Taiwan, cardiovascular diseases remain the top two ranks of ten leading causes of death. Atherosclerosis is recognized as the major cause of cardiovascular disease. Oxides and pro-inflammatory substances can activate NF-κB- and AP-1-mediated pro-inflammatory cytokine, cell adhesion molecule, and C-reactive protein (CRP) expression, resulting in atherosclerosis. CRP is not only identified as a highly correlated-risk factor with an incidence of disease, but also a direct participator in the formation of atherosclerosis. Nicotine, a causative agent for atherosclerosis, is reported to induce NF-κB-mediated CRP, TNF-α, and inducible nitric oxide synthase (iNOS) expression in macrophages. However, the underlying mechanisms of nicotine-induced NF-κB activation and further inflammation remain unclear. Thioredoxin-interacting protein (Txnip) is originally described as a vitamin D3-up-regulated protein, which can block the antioxidative function of thioredoxin (Trx). Our preliminary results show that the expression of Txnip is suppressed in nicotine-treated macrophages. Txnip deficiency is identified to prompt excessive nitric oxide (NO) induction via the highly activation of NF-κB. Therefore, the involvement of Txnip in nicotine-mediated NF-κB activation and subsequent inflammation is hypothesized. In this project, we plan to clarify the molecular mechanisms of Txnip in nicotine-mediated CRP or inflammatory mediator induction in macrophages, and develop potential drugs against CRP-mediated atherosclerosis. Specific Aim 1 is to investigate the mechanisms involved in NADPH oxidase activation, NF-κB activation, CRP expression, and the inflammatory mediator induction in nicotine-treated macrophages. Previous reports showed that nicotine induces CRP production in an acetylcholine receptor dependent ERK1/2/p38 MAPK-NF-κB signal pathway. Since NADPH oxidase is involved in cigarette smoking extract-induced NF-κB activation, we will thus investigate the involvements of β-arrestin, c-Src, NADPH oxidase, and ROS production in nicotine-induced NF-κB activation. Moreover, we will also determine the production of CRP, TNF-α and NO to demonstrate that nicotine induces NF-κB-mediated CRP, TNF-α and NO induction through regulating β-arrestin/c-Src/NADPH oxidase activation. Specific Aim 2 is to clarify the involvement of Txnip in regulating nicotine-induced NF-κB activation. Our preliminary results showed that nicotine effectively suppresses Txnip expression in Raw264.7 cells. The decreased Txnip expression is associated with the activation of JNK in response to oxidative stress. Accordingly, we will further verify the regulation of NADPH oxidase, MAPKs, and Itch E3 ligase in nicotine-mediated Txnip suppression. The reduction of Txnip in nicotine-induced NF-κB activation and CRP, TNF-α and NO production will also be investigated. In this aim, we plan to demonstrate that nicotine induces NF-κB-mediated CRP, TNF-α and NO induction via NADPH oxidase-dependent Txnip suppression. Specific Aim 3 is to discover the potential drugs to negatively modulate nicotine-mediated CRP and cytokine induction, and further apply to the treatment of atherosclerosis-associated foam cell formation. Base on the findings from aim 1 and 2, the inhibitors, (clomipramine, PP1, DPI and NAC), anti-hypercholesterolemia drugs (simvastatin, pravastatin, lovastatin, fluvastatin, and atorvastatin), and anti-inflammatory drugs (dextromethorphan) will be used to verify their regulations in nicotine-induced macrophage differentiation and further foam cell formation. Taken together, this project will help to clarify the molecular mechanisms of nicotine-induced inflammation in macrophages, and provide potential therapeutic strategies in the future.