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.
Nrf2 plays a crucial role in the management of oxidative and electrophilic stress. Nrf2 acts as a master regulator of oxidative or metabolic stress responses and is involved in cancer cell metabolism and multiple oncogenic pathways. Keap1 is the main inhibitor of Nrf2, and its high or low expression also causes changes in Nrf2. The function of Keap1 isoforms has rarely been reported. Here, to gain a better understanding of their similarities and differences in distinct regulatory profiles, four distinct cell models stably expressing Keap1-/-, Keap1[beta]; (Keap1[Delta];1-31), Keap1-Restored or Keap1[alpha];-Restored have been established. Immunofluorescence localization and nucleocytoplasmic separation experiments found that Keap1 and its isoforms were localized in the cytoplasm and had inhibitory effects on Nrf2 and HO-1. Further transcriptome analysis revealed that they had similar and different functions. Their functions were further explored in vivo (subcutaneous tumour formation in nude mice) and in vitro (cell cloning, infection, migration, wound healing, CAT, GSH, cell cycle and apoptosis). Next, the results of tumorigenesis experiments in nude mice were verified based on changes in the PTEN and PI3K-mTOR pathways. Finally, some typical genes of EMT, cell cycle and apoptosis were detected, and the experimental results were verified by in vitro experiments.
The Keap1-Nrf2 signalling to transcriptionally regulate antioxidant response element (ARE)-driven target genes has been accepted as key redox-sensitive pathway governing a vast variety of cellular stresses during healthy survival and disease development. Herein, we identified two nuanced isoforms α and β of Keap1, arising from its first and another in-frame translation starting codons, respectively. Those common and specific genes monitored by Keap1α and/or Keap1β were unravelled by transcriptomic sequencing of indicated experimental cell lines. Amongst them, an unusual interaction of Keap1 with Smad2/3 was discovered by parsing transcriptome sequencing, protein profiling, and immunoprecipitation data. Further examinations validated that Smad2/3 enable physical interaction with Keap1, as well as its isoforms α and β, by both EDGETSD and DLG motifs in the linker regions between their MH1 and MH2 domains, such that the stability of Smad2/3 and its transcriptional activity are enhanced with the prolonged half-lives and signalling responsiveness from the cytoplasmic to nuclear compartments. Such activation of Smad2/3 by Keap1, Keap1α or Keap1β was contributable to a competitively inhibitory effect of Nrf2. Overall, this discovery presents a novel functional bridge crossing the Keap1-Nrf2 and the TGF-β1-Smad2/3 signalling pathways in healthy growth and development.
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