Elucidation of the respective roles of itaconate and nitric oxide in the nuclear accumulation and activation of Nrf2 in activated macrophages

Abstract

Abstract Nrf2 is a transcription factor that, under basal conditions, is produced in the cytosol and is marked for degradation by its repressor Keap1. Oxidative stress causes Keap1 to release Nrf2, and it accumulates in the nucleus to upregulate certain antioxidant gene targets. Itaconate, the metabolic product of IRG1, has been suggested to play a role in alkylating Keap1, leading to the release of Nrf2. However, there is much ambiguity and even controversy surrounding this role of itaconate. Our study therefore seeks to definitively elucidate the role of itaconate in the regulation and activation of Nrf2. Here we utilize expression analysis and targeted metabolomics in bone marrow-derived macrophages isolated from varying murine backgrounds (Acod1−/−, Nos2−/−, and double knockouts). Despite previously published data suggesting a role for itaconate in Nrf2 activity, our data demonstrate a significant role of Nos2, but not itaconate, in Nrf2 activation, as Acod1−/− mice show little change in the activity of Nrf2 transcription targets, while Nos2−/− mice show severe impairment of Nrf2 gene target activity. For Nrf2 to properly upregulate its gene targets, it must be released from Keap1, phosphorylated, and then allowed to translocate to the nucleus; all three must occur. This may help explain why some studies have found itaconate to be important in Keap1’s regulation of Nrf2, while others demonstrate that this same itaconate mechanism produces comparatively little Nrf2 activity.