Ventilator-induced lung injury induces a positive signalling feedback loop between Nrf2 and amphiregulin

Aims and objectives: Mechanical ventilation (MV) is a life saving intervention, but also bears the risk of ventilator-induced lung injury (VILI) with pulmonary inflammation. The mechanisms underlying VILI are not yet fully understood. This study was designed to examine a possible interaction of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), a major regulator of the cellular antioxidant defence system, with the stretch-induced growth factor amphiregulin (AREG) during VILI. Methods: In silico approaches were used to analyze Areg promoter sequences for the Nrf2 binding site ARE. HeLa cells with inactivated Keap1, the inhibitor of Nrf2, were used to examine the regulation of AREG by Nrf2. Ventilation-dependent Nrf2 activation was quantified in isolated perfused mouse lungs (IPL). Further, activation of Nrf2 by AREG was examined in HeLA cells, murine precision cut lung slices (PCLS) and IPL. In vivo ventilation experiments of Nrf2-deficient mice, including analyses of functional and inflammatory pulmonary parameters, completed the study. Results: Promoter analyses revealed ARE sequences within the Areg promoter. Based on this, the regulation of AREG by Nf2 was shown by shRNA knockdown of Keap1. In turn, Nrf2 was activated in a ventilation-dependent manner as well as by exogenously administered AREG. The induction of Nrf2 by AREG was inhibited by p38 MAP kinase repression. Finally, the Nrf2-dependent regulation of AREG was validated in vivo in mechanically ventilated mice. Conclusions: We conclude that MV induces a positive feedback signalling loop between Nrf2 and AREG, which might play a protective role in VILI.