Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

Abstract

Non-alcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA) and hepatocyte lipoapoptosis. This lipoapoptosis requires increased JNK phosphorylation and activation of the pro-apoptotic BH3-only proteins Bim and PUMA. Kelch-like ECH-associated protein (Keap)-1 is a BTB/Kelch protein that can regulate the expression of Bcl-2 protein and control apoptotic cell death. Yet, the role of Keap1 in hepatocyte lipotoxicity is unclear. Here we demonstrate that Keap1 protein was rapidly degraded in hepatocytes, through autophagy in a p62-dependent manner, in response to the toxic saturated FFA palmitate, but not following incubation with the non-toxic FFA oleic acid. Stable knockdown of Keap1 expression, using shRNA technology, in hepatocarcinoma cell lines induced spontaneous cell toxicity that was associated with JNK1-dependent upregulation of Bim and PUMA protein levels. Also, Keap1 knockdown further sensitized hepatocytes to lipoapoptosis by palmitate. Likewise, primary hepatocytes isolated from liver-specific Keap1 À / À mice displayed higher Bim and PUMA protein levels and demonstrated increased sensitivity to palmitate-induced apoptosis than wild-type mouse hepatocytes. Finally, stable knockdown of Bim or PUMA expression prevented cell toxicity induced by loss of Keap1. These results implicate p62-dependent autophagic degradation of Keap1 by palmitate as a mechanism contributing to hepatocyte lipoapoptosis.

p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB

“…However, the interaction of DmKeap1 with DmAtg8a is interesting, both because DmKeap1 appears to recognize a different binding surface in DmAtg8a than Ref(2)P and other LIR-containing proteins and because the interaction might have an important role for DmAtg8a-mediated autophagic degradation of DmKeap1 during Drosophila development. Recent studies show mammalian Keap1 to be degraded by autophagy under nutritional starvation and oxidative stress in a p62-dependent manner (34,76), whereas degradation of mammalian Keap1 by basal autophagy has not been clearly demonstrated. Consistent with this, we observed no degradation of DmKeap1 by basal autophagy in Drosophila, but DmKeap1 was degraded under programmed autophagy during Drosophila development.…”

Transcription Factor NRF2 as a Therapeutic Target for Chronic Diseases: A Systems Medicine Approach

“…2C) (Rada et al, 2011(Rada et al, , 2012. Moreover, KEAP1 interacts with other proteins that also contain the high-affinity binding motif ETGE (Hast et al, 2013), such as Bcl-2 and IKKb (Kim et al, 2010a;Cazanave et al, 2014). Therefore, some results obtained from KEAP1-deficient cells may not necessarily be related to NRF2 activation.…”

“…Table 3 compiles recent patents addressing these small molecules. Although these compounds are very promising, it is still needed to demonstrate that they are selective for the KEAP1/NRF2 interaction, because KEAP1 also targets at least Bcl2 and IKK (Kim et al, 2010a;Hast et al, 2013;Cazanave et al, 2014).…”

KEAP the balance between life and death

“…3 Therefore, in our recent study we evaluated the role of KEAP1 in hepatocyte lipotoxicity. 4 In both primary and transformed liver cells, we observed that sFFA-induced toxicity was associated with degradation of KEAP1, in part through autophagy in a sequestrosome (SQSTM)1/p62-dependent manner. We also identified that loss of Keap1 induced spontaneous liver cell death and further increased hepatocyte susceptibility to a lipotoxic insult.…”

“…This effect was associated with increased activation of c-Jun NH2 terminal kinase (JNK) in an NRF2-independent manner and upregulation of protein levels of Bcl-2-interacting mediator (BIM) and p53 upregulated modulator of apoptosis (PUMA), 2 proapoptotic BH3-only proteins that induce hepatocyte cell death. 4 Autophagy is often observed in dying cells as it is activated by oxidative stress. Although it is mostly considered a prosurvival pathway it can also contribute to cell death, especially if the process relates to lysosomal dysfunction and the activation of caspase-8, which are both implicated in lipotoxicity.…”