The Effects of Macrophage Migration Inhibitory Factor on Antioxidant Responses During Cigarette Smoke Exposure

COPD;Macrophage migration inhibitory factor;MIF;cigarette smoke;endothelial cell;Nrf2;antioxidants;CAND1;oxidative stress;DJ-1;ROS;UBC12;Neddylation;CRL3;Environmental Health & Engineering

Aladdin, MeenaHassoun, Paul M. ;

Johns Hopkins University

关键词: COPD;    Macrophage migration inhibitory factor;    MIF;    cigarette smoke;    endothelial cell;    Nrf2;    antioxidants;    CAND1;    oxidative stress;    DJ-1;    ROS;    UBC12;    Neddylation;    CRL3;    Environmental Health & Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/58640/ALADDIN-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States. Cigarette smoke (CS) is the leading cause of emphysema, a component of COPD, which is characterized by airway inflammation and destruction of alveolar sacs and gas exchange impairment. The pathogenesis of emphysema remains to be elucidated, but CS-induced endothelial cell (EC) apoptosis is an established mechanism that is both necessary and sufficient to cause emphysematous remodeling. Fallica et al have previously shown that expression of the cytokine macrophage migration inhibitory factor (MIF) is altered in patients with COPD and is significantly attenuated in advanced stage COPD. Preclinical data reveals that chronic CS exposure antagonizes MIF gene expression in vivo. Furthermore, MIF deficient mice have increased ROS production, DNA damage and exacerbated emphysematous tissue remodeling. Damico et al have also previously shown that EC apoptosis is enhanced in the absence MIF in the context of CS. Taken together, these provide evidence for MIF’s cytoprotective role in the context of CS-induced oxidative stress.MIF has also been shown to antagonize oxidant-mediated injury in other models, which may be linked to its capacity to impact the antioxidant regulator and transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2). The goal of this thesis is to elucidate the mechanisms by which MIF is impacting antioxidant responses during CS-induced oxidative stress. Our results reported here demonstrate that MIF is a necessary mediator of Nrf2-dependent transcription in vivo in a model of CS-induced oxidative injury and emphysematous remodeling. We then demonstrated in primary EC in vitro that exogenous MIF was sufficient to drive antioxidant response element (ARE)-driven transcriptional activity and stabilize Nrf2 protein via a proteasomal-dependent mechanism. There are two recognized regulators of Nrf2 stability. The Kelch-like ECH-associated protein 1 (KEAP1)-Cullin3 (CUL3) ligase complex dynamically regulates Nrf2 by targeting it for proteasomal-mediated degradation under basal conditions. In contrast, DJ-1 has been demonstrated to directly and indirectly promote Nrf2 stability in select models. Given that MIF capacity to interfer with Nrf2’s proteasomal-mediated degradation, we sought to understand MIF’s effects on the members and regulators of the KEAP1-CUL3 ligase complex, as well as DJ-1.The CUL3 component of the KEAP1-CUL3 complex is regulated by two established mechanisms 1) post-translational modification (PTM) of CUL3 and 2) via inhibitory protein-protein interactions with CAND1. The recognized PTM of CUL3 involves the cyclic addition and removal of a small ubiquitin-like molecule, NEDD8, through a process known as neddylation and deneddylation, respectively. We found that chronic CS exposure in vivo was associated with increased CUL3 neddylation in the lung. This observation is lost in MIF deficient animals exposed to CS, implicating MIF as a regulator of CUL3 PTM in the context of CS-induced oxidative injury. To test the hypothesis that the observed CUL3 neddylation status was sufficient to alter Nrf2 activity, we altered the PTM of CUL3 by ectopic expression of the neddylase, UBC12. This was sufficient to enhance CUL3 neddylation and, importantly, drive Nrf2 expression and ARE activity, supporting a model in which MIF stabilizes Nrf2 in the context of CS in vivo via alterations in CUL3 neddylation status. MIF however, was neither necessary nor sufficient to alter CUL3 neddylation in vitro, suggesting that the mechanism was indirect or contextual. We then investigated the effects of MIF on the known CUL3 inhibitor, CAND1 and determined that MIF is a novel positive regulator of CAND1 expression. Further, over expression of CAND1 was sufficient to stabilized Nrf2 in ECs in vitro.In addition to negative Nrf2 regulators, we also explored MIF’s effects on the DJ-1. DJ-1 is a redox-sensitive molecule that is important in mediating antioxidant responses and antagonizing ROS in neuronal cells. We tested the hypothesis that MIF-mediated Nrf2 stability via a DJ-1-dependent mechanism. Despite reports of DJ-1 regulating Nrf2 in pulmonary epthelial cells (EpiCs) in vitro, we found that DJ-1 was not sufficient for Nrf2 stabilization in EC in vitro, suggesting that the molecular mechanisms of Nrf2 stabilization may differ between pulmonary EC and EpiCs. In conclusion, we provide evidence that MIF’s ability to enhance Nrf2 stability as a novel positive regulator of CAND1, and inhibitor of the KEAP1- CUL3 ligase complex.