Sulforaphane-Induced Klf9/Prdx6 Axis Acts as a Molecular Switch to Control Redox Signaling and Determines Fate of Cells

Abstract

Sulforaphane (SFN), an activator of transcription factor Nrf2 (NFE2-related factor), modulates antioxidant defense by Nrf2-mediated regulation of antioxidant genes like Peroxiredoxin 6 (Prdx6) and affects cellular homeostasis. We previously observed that dose levels of SFN are crucial in determining life or death of lens epithelial cells (LECs). Herein, we demonstrated that higher doses of SFN (>6 μM) activated death signaling by overstimulation of Nrf2/ARE (antioxidant response element)-mediated Kruppel-like factor (Klf9) repression of Prdx6 expression, which increased reactive oxygen species (ROS) load and cell death. Mechanistically, Klf9 bound to its repressive Klf9 binding elements (RKBE; 5-CA/GCCC-3) in the Prdx6 promoter, and repressed Prdx6 transcription. Under the condition of higher dose of SFN, excessive Nrf2 abundance caused death signaling by enforcing Klf9 activation through ARE (5-RTGAYnnnGC-3) in Klf9 promoter that suppress antioxidant genes such as Prdx6 via a Klf9-dependent fashion. Klf9-depletion showed that Klf9 independently caused ROS reduction and subsequent cell survival, demonstrating that Klf9 upregulation caused cell death. Our work revealed the molecular mechanism of dose-dependent altered activity of SFN in LECs, and demonstrated that SFN activity was linked to levels of Nrf2/Klf9/Prdx6 axis. We proposed that in the development of therapeutic interventions for aging/oxidative disorders, combinations of Klf9-ShRNA and Nrf2 inducers may prove to be a promising strategy.

Regulation of Nrf2 by Mitochondrial Reactive Oxygen Species in Physiology and Pathology

“…Klf9-mediated oxidative stress was evaluated in vivo using bleomycin-induced pulmonary fibrosis model mice, and Klf9-KO mice exhibited a decrease in 8-OHdG levels and fibrosis [88]. Chhunchha et al also reported an adverse effect of Klf9 in human lens epithelial cells treated with high-dose sulforaphane [89]. Low-dose sulforaphane induces peroxiredoxin-6 (Prdx6) gene expression via Nrf2 and ARE in its regulatory region; however, high-dose sulforaphane suppresses Prdx6 expression via Klf9 and the repressive Klf9 binding element (RKBE).…”

“…Low-dose sulforaphane induces peroxiredoxin-6 (Prdx6) gene expression via Nrf2 and ARE in its regulatory region; however, high-dose sulforaphane suppresses Prdx6 expression via Klf9 and the repressive Klf9 binding element (RKBE). These reports implicate Klf9 as a factor determining cell fate in response to the magnitude of oxidative stress, and Klf9 suppression/inhibition is a possible strategy to alleviate the side effects of Nrf2-activating interventions [89] (Figure 4).…”

“…Nrf2 induces the expression of cytoprotective genes, such as HO-1 and NQO1, in response to a wide range of oxidative stresses[88]. High-dose H2O2 or sulforaphane induces Klf9 expression, which represses Txnrd2 gene expression as well as that of other Nrf2-target genes, such as Prdx6, and induces cytotoxic ROS production[88,89]. The line graphs represent the oxidative stress level (x-axis) and transcription (y-axis) relationship.…”

Flavonoids Activation of the Transcription Factor Nrf2 as a Hypothesis Approach for the Prevention and Modulation of SARS-CoV-2 Infection Severity

“…However, under conditions with an excessive level of ROS, the continuous activation of Nrf2 causes its accumulation in the nucleus and subsequent binding to the Kruppel-like factor 9 (Klf9) promoter. The upregulation of Klf9 transcription, which is a novel regulator of intracellular ROS, could then lead to increased levels of ROS and subsequent cell death [41,42]. Therefore, according to the studies, an excessive amount of oxidative stress is needed to activate the Klf9 transcription, which will be unlikely with a viral infection since the virus needs to keep oxidative stress at an optimal level to maintain viral metabolism without causing host cell death [43].…”

Potential Protective and Therapeutic Roles of the Nrf2 Pathway in Ocular Diseases: An Update

“…Therefore, we have summarized some studies on the protection of LECs under stress through Nrf2 pathways. The use of Nrf2 activators (such as SFN pretreatment [85]) or the overexpression of Nrf2 [86] can reduce DNA fracture; upregulate Nrf2, NQO1, HO-1, etc. [87]; and protect LECs from OS damage.…”