Interplay between Nuclear Factor Erythroid 2–Related Factor 2 and Amphiregulin during Mechanical Ventilation

Abstract

Mechanical ventilation (MV) elicits complex and clinically relevant cellular responses in the lungs. The current study was designed to define the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), a major regulator of the cellular antioxidant defense system, in the pulmonary response to MV. Nrf2 activity was quantified in ventilated isolated perfused mouse lungs (IPL). Regulation of amphiregulin (AREG) was investigated in BEAS-2B cells with inactivated Nrf2 or Keap1, the inhibitor of Nrf2, using a luciferase vector with AREG promoter. AREG-dependent Nrf2 activity was examined in BEAS-2B cells, murine precision-cut lung slices (PCLS), and IPL. Finally, Nrf2 knockout and wild-type mice were ventilated to investigate the interplay between Nrf2 and AREG during MV in vivo. Lung functions and inflammatory parameters were measured. Nrf2 was activated in a ventilation-dependent manner. The knockdown of Nrf2 and Keap1 via short hairpin RNA in BEAS-2B cells and an EMSA with lung tissue revealed that AREG is regulated by Nrf2. Conversely, AREG application induced a significant Nrf2 activation in BEAS-2B cells, PCLS, and IPL. The signal transduction of ventilation-induced Nrf2 activation was shown to be p38 MAP kinase-dependent. In vivo ventilation experiments indicated that AREG is regulated by Nrf2 during MV. We conclude that Areg expression is regulated by Nrf2. During high-pressure ventilation, Nrf2 becomes activated and induces AREG, leading to a positive feedback loop between Nrf2 and AREG, which involves the p38 MAPK and results in the expression of cytoprotective genes.

Reference Gene Selection for Gene Expression Analyses in Mouse Models of Acute Lung Injury

“…Il-6 and Il-8 levels correlate with tidal volume and mortality in human ARDS, as already shown over 20 years ago [32][33][34]. They are equally important in experimental ALI, and belong to the standard readout parameters in most ALI studies [6,8,[35][36][37]. While Il-6 is a cytokine that elicits inflammation and, moreover, cytokine storm [24], the chemokine Il-8 is co-responsible for neutrophil recruitment, a hallmark of ARDS/ALI pathogenesis [38].…”

Nrf2 Ameliorates DDC-Induced Sclerosing Cholangitis and Biliary Fibrosis and Improves the Regenerative Capacity of the Liver

“…Nrf2 binds to the "antioxidant response element" (ARE) and induces the expression of target genes coding for antioxidative enzymes and proteins. In addition, Nrf2 regulates the expression of genes that influence tissue regeneration such as Notch, amphiregulin, VEGF, and IL-6 (Kobayashi et al, 2016;Reiss et al, 2014;Serhan and Savill, 2005;Wakabayashi et al, 2010a;Wruck et al, 2011). Inducers of Nrf2 signaling such as isothiocyanates, flavonoids, or kavalactones are shown to be protective against various diseases (Brandenburg et al, 2010;Fragoulis et al, 2012Fragoulis et al, , 2017Wruck et al, 2007Wruck et al, , 2008.…”

Association of Nrf2 with airway pathogenesis: lessons learned from genetic mouse models

“…Conventional MV with high tidal volumes and low positive end expiratory pressure causes excessive alveolar distention, resulting in biotrauma characterized by lung edema and inflammation in patients (Ware et al 2006). In response to experimentally applied MV, greater levels of lung permeability, inflammation, and disturbed redox balance and impaired gas exchange were found in Nrf2 −/− mice compared to Nrf2 +/+ mice (ICR or B6) (Papaiahgari et al 2007; Reiss et al 2014). Supplementation of Nrf2 −/− mice (ICR) with NAC, a precursor for glutathione biosynthesis, significantly attenuated ventilator-induced lung injury, indicating a role for Nrf2-mediated thiol homeostasis in protection against ventilator-induced lung injury (Papaiahgari et al 2007).…”

“…In addition, hyperoxia-inducible NADPH oxidase 4 ( Nox4 ) contained functional AREs (−438_−458, −619_−636), and was also regulated by Nrf2 in mouse lungs for endothelial cell function and signaling (Pendyala et al 2011). Moreover, MV caused Nrf2-dependent induction of amphiregulin ( Areg ), a ligand for epidermal growth factor (EGF) receptor (Papaiahgari et al 2007), and it had a functional ARE (−760) for Nrf2 biding (Reiss et al 2014). Interestingly, Pparg or Areg are not only Nrf2 downstream targets but their protein products also mutually activate Nrf2 (Cho et al 2010; Reiss et al 2014).…”

“…Moreover, MV caused Nrf2-dependent induction of amphiregulin ( Areg ), a ligand for epidermal growth factor (EGF) receptor (Papaiahgari et al 2007), and it had a functional ARE (−760) for Nrf2 biding (Reiss et al 2014). Interestingly, Pparg or Areg are not only Nrf2 downstream targets but their protein products also mutually activate Nrf2 (Cho et al 2010; Reiss et al 2014).…”