Concomitant Nrf2- and ATF4-Activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes

Abstract

: Carnosic acid (CA) is a phytochemical found in some dietary herbs, such as Rosmarinus officinalis L., and possesses antioxidative and anti-microbial properties. We previously demonstrated that CA functions as an activator of nuclear factor, erythroid 2 (NF-E2)-related factor 2 (Nrf2), an oxidative stress-responsive transcription factor in human and rodent cells. CA enhances the expression of nerve growth factor (NGF) and antioxidant genes, such as HO-1 in an Nrf2-dependent manner in U373MG human astrocytoma cells. However, CA also induces NGF gene expression in an Nrf2-independent manner, since 50 μM of CA administration showed striking NGF gene induction compared with the classical Nrf2 inducer tert-butylhydroquinone (tBHQ) in U373MG cells. By comparative transcriptome analysis, we found that CA activates activating transcription factor 4 (ATF4) in addition to Nrf2 at high doses. CA activated ATF4 in phospho-eIF2α- and heme-regulated inhibitor kinase (HRI)-dependent manners, indicating that CA activates ATF4 through the integrated stress response (ISR) pathway. Furthermore, CA activated Nrf2 and ATF4 cooperatively enhanced the expression of NGF and many antioxidant genes while acting independently to certain client genes. Taken together, these results represent a novel mechanism of CA-mediated gene regulation evoked by Nrf2 and ATF4 cooperation.

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

“…In addition, we recently identified several antioxidant and cytoprotective genes including nerve growth facts (NGF) are cooperatively regulated by both Nrf2 and ATF4 when both are activated by the phytochemical carnosic acid in the human glioma U373MG cells [124]. Interestingly, genes involved in glutathione synthesis are regulated to various extent in either an Nrf2 and ATF4-dependent or in a both Nrf2-and ATF4-dependent manner ( Figure 6) ( [124] and our unpublished observation). Furthermore, genes involved in NADPH synthesis in the mitochondria such as SHMT2 and MTHFD2 are regulated by ATF4 [29] and those in the cytosol (i.e.…”

“…We have also reported that proteasome inhibitors activate both Nrf2 and ATF4 and cooperatively induce the expression of the cystine transporter xCT in bladder cancer cells via Nrf2 binding to ARE and ATF4 binding to amino acid response elements (AAREs), respectively [123]. In addition, we recently identified several antioxidant and cytoprotective genes including nerve growth facts (NGF) are cooperatively regulated by both Nrf2 and ATF4 when both are activated by the phytochemical carnosic acid in the human glioma U373MG cells [124]. Interestingly, genes involved in glutathione synthesis are regulated to various extent in either an Nrf2 and ATF4-dependent or in a both Nrf2-and ATF4-dependent manner ( Figure 6) ( [124] and our unpublished observation).…”

“…Carnosic acid activates both Nrf2 and ATF4 and induces antioxidant genes and genes involved in amino acid synthesis/transport and GSH synthesis. The contributions of Nrf2 and ATF4 to the gene activation were analyzed by knockdown of Nrf2 or ATF4 and shown with color characters ( [124] and our unpublished observation). Induction of cystine transporter xCT and GSH synthetic enzymes (GCLC, GCLM, GSS, and GSR) was dependent on both Nrf2 and ATF4 (genes indicated with blue).…”

Metabolic features of cancer cells in NRF2 addiction status

“…This is because the sustained activation of PI3K-AKT pathway inhibits GSK3, which phosphorylates NRF2 and allows its recognition and ubiquitination by βTrCP-CUL1 ubiquitin E3 ligase for degradation. As for another cooperative factor, ATF4 activation has a synergistic effect with NRF2 activation on xCT expression (Ye et al 2014;Mimura et al 2019). Consequently, cystine uptake coupled with glutamate excretion and glutathione synthesis are enhanced (Fig.…”

Evaluation of Antioxidant Activity of Spice-Derived Phytochemicals Using Zebrafish

“…It is likely that capsaicin indirectly activates some cellular antioxidant mechanisms, but it may not function well in zebrafish larvae. Carnosic acid has been reported to exhibit antioxidant activity through the Nrf2 pathway in various cells and mice [31,52]. However, in zebrafish larvae, the toxicity of carnosic acid was so strong that we could not analyze it at concentrations ≥5 µM, and even at 1 µM, the numbers of surviving larvae were significantly decreased due to cotoxicity when they were further treated with H 2 O 2 .…”