ENOS Regulation of Nrf2 and BNIP3 in Primary Murine Hepatocytes

Abstract

It is well known that endothelial nitric oxide synthase (eNOS)‐derived nitric oxide (NO) stimulates mitochondrial biogenesis in a cGMP‐PGC1α dependent manner in various tissues. However, the importance of eNOS in regulating mitophagy, the clearance of poorly functioning mitochondria, in hepatocytes remains unknown. We hypothesized that eNOS induces mitochondrial turnover through nuclear factor‐E2‐related factor‐2 (Nrf2) mediated BCL2/E1B 19 kDa protein‐interacting protein 3 (BNIP3) induction of mitophagy. To examine the relationship between eNOS, Nrf2, and BNIP3 in primary hepatocytes, we isolated primary hepatocytes from C57/BL6 wild‐type (WT) mice followed by siRNA treatment [Scramble control (Scr), eNOS, or Nfr2] for six hours. Growth media was applied for 12–18 hours after siRNA treatment, then 24 hours of either control media or 500 μM FFA (250 μM oleate + 250 μM palmitate); the 500 μM FFA condition also included one of the following treatments for 16 hours: 1 μM carbonyl cyanide 3‐chlorophenylhydrazone (CCCP, to depolarize mitochondria and induce mitophagy)/10 μM cholorquine (CQ, lysosomal inhibitor), 30 nM CDDO‐Methyl Ester (CDDO‐Me, NRF2 activator), or 50 μM diethylenetriamine (DETA) NONOate/CCCP/CQ. Silencing of eNOS in primary hepatocytes (50% reduction vs Scramble control, p<0.01) knocked down Nrf2 mRNA expression by ~40% and Nrf2 activation (NQO1 mRNA; target gene of Nrf2) and BNIP3 mRNA expression by ~40% following FFA challenge (p<0.05 vs Scr control). In contrast, treating primary hepatocytes with the NO donor DETA NONOate induced Nrf2 activation (NQO1 mRNA) and BNIP3 mRNA by 50% and 300%, respectively (p<0.05 vs Scr control). In support of Nrf2 regulating BNIP3, we provide data, that to our knowledge, is the first evidence that the direct knockdown of Nrf2 in primary hepatocytes by an siRNA approach (−80% knockdown of Nrf2 vs Scr control, p<0.001) not only reduces Nrf2 activity (−80% knockdown of NQO1 mRNA), but also blunts induction of mitophagy flux (BNIP3 induction with CCCP/CQ reduced 50% vs Scr) in primary hepatocytes. In addition, Nrf2 activation with CDDO‐Me treatment (3.5‐fold increase in NQO1 mRNA) induced a ~2‐fold increase in BNIP3 accumulation (w/CCCP+CQ), an effect completely ablated with Nrf2 siRNA. These preliminary observations point to a regulatory role of hepatocyte‐specific eNOS in Nrf2 and BNIP3, and a strong rationale for the further examination of the role of Nrf2 in the regulation of BNIP3 and mitophagy.Support or Funding InformationFunding: This work was supported by grants from the Missouri Foundation for Veterans Medical Research, the Richard Wallace Faculty Incentive, Department of Medicine Research Council, and a Veterans Affairs VHA‐CDA2 Grant (IK2BX001299).