Different Inhibition of Nrf2 by Two Keap1 Isoforms α and β to Shape Malignant Behaviour of Human Hepatocellular Carcinoma

Abstract

Nrf2 (nuclear factor E2-related factor 2, encoded by Nfe2l2) acts as a master transcriptional regulator in mediating antioxidant, detoxification, and cytoprotective responses against oxidative, electrophilic, and metabolic stress, but also plays a crucial role in cancer metabolism and multiple oncogenic pathways, whereas the redox sensor Keap1 functions as a predominant inhibitor of Nrf2 and, hence, changes in its expression abundance directly affect the Nrf2 stability and transcriptional activity. However, nuanced functional isoforms of Keap1 α and β have rarely been identified to date. Herein, we have established four distinct cell models stably expressing Keap1−/−, Keap1β(Keap1Δ1–31), Keap1-Restored, and Keap1α-Restored aiming to gain a better understanding of similarities and differences of two Keap1 isoforms between their distinct regulatory profiles. Our experimental evidence revealed that although Keap1 and its isoforms are still localized in the cytoplasmic compartments, they elicited differential inhibitory effects on Nrf2 and its target HO-1. Furthermore, transcriptome sequencing unraveled that they possess similar but different functions. Such functions were further determined by multiple experiments in vivo (i.e., subcutaneous tumour formation in nude mice) and in vitro (e.g., cell cloning, infection, migration, wound healing, cell cycle, apoptosis, CAT enzymatic activity, and intracellular GSH levels). Of note, the results obtained from tumourigenesis experiments in xenograft model mice were verified based on the prominent changes in the PTEN signaling to the PI3K-AKT-mTOR pathways, in addition to substantially aberrant expression patterns of those typical genes involved in the EMT (epithelial–mesenchymal transition), cell cycle, and apoptosis.

A novel crosstalk between Nrf2 and Smad2/3 bridged by two nuanced Keap1 isoforms

“…Distinct genotypic cell lines Keap1 +/+ , Keap1 −/− , Keap1β( Keap1△ 1-31 ), Keap1-restored and Keap1α-restored , that had been confirmed by Western blotting (Fig. 3a) and others described previously 24 , were subjected to transcriptome sequencing so as to identify differentially regulated gene expression profiles. As shown in a Venn diagram (Fig.…”

“…2h), as reported previously in the cytoplasm of COS-1 cells 31 . Further subcellular fractionation of distinct genotypic cell lines Keap1 +/+ , Keap1 −/− and Keap1β( Keap1△ 1-31 ), along with two stably Keap1- or Keap1α -expressing cell lines (all derived from HepG2 cells 24 ) unravelled that Keap1, Keap1α and Keap1β were recovered primarily in the cytosolic and mitochondrial factions, and only modestly in nuclear fractions (Figs. 2i & S1c).…”

“…In the present study, we have discovered an unusual interaction of Keap1 with Smad2/3 by parsing transcriptome sequencing, protein profiling, and co-immunoprecipitation data. Further examinations validated that Smad2/3 enable a physical interaction with Keap1, including its full-length Keap1α and Keap1β (i.e., Keap1 △1-31 ) 24 , by both EDGETSD and DLG motifs located in the linker region of Smad2 or Smad3, so that they are segregated in the cytoplasmic compartments, but with their protein stability being enhanced by co-expression of Keap1 or its isoforms. Keap1α and Keap1β were also further demonstrated to monitor the TGF-β1-Smad2/3 signalling transduction of the integral signal from the cell surface to the nucleus.…”

“…), Keap1-Restored and Keap1α-Restored cell lines Both Keap1 (sgRNA-Keap1: 5'-TATGAGCCAGAGCGGGATG-3') and Keap1α (sgRNA-Keap1α: 5'-AGGCCTAGCGGGGCTGGG GC-3') genomes had been created via CRISPR/Cas9 and identified 24 . Then, each of those Cas9 plasmids for Keap1 and Keap1α were introduced into HepG2 cells to construct Keap1 −/− and Keap1β(Keap1 △1-31 ) cell lines (Fig.…”

“…The intrinsic developmental signalling cascades are predominantly induced by growth factors (including cytokines such as TGF- 11 ), morphogens (e.g., WNT 12 ) mitogens and survival factors (e.g., Hedgehog 13 ). However, only considerably less attention has been attracted on such a putative as-yet-unidentified interplay between the TGF--Smad and Keap1-Nrf2 signalling pathways, even though a large number of studies have indeed elucidated that the transforming growth factor-physical interaction with Keap1, including its full-length Keap1α and Keap1β (i.e., Keap1 △1-31 ) 24 , by both EDGETSD and DLG motifs located in the linker region of Smad2 or Smad3, so that they are segregated in the cytoplasmic compartments, but with their protein stability being enhanced by co-expression of Keap1 or its isoforms. Keap1α and Keap1β were also further demonstrated to monitor the TGF-β1-Smad2/3 signalling transduction of the integral signal from the cell surface to the nucleus.…”