The Human NADPH Oxidase, Nox4, Regulates Cytoskeletal Organization in Two Cancer Cell Lines, HepG2 and SH-SY5Y

Abstract

NADPH oxidases of human cells are not only functional in defense against invading microorganisms and for oxidative reactions needed for specialized biosynthetic pathways but also during the past few years have been established as signaling modules. It has been shown that human Nox4 is expressed in most somatic cell types and produces hydrogen peroxide, which signals to remodel the actin cytoskeleton. This correlates well with the function of Yno1, the only NADPH oxidase of yeast cells. Using two established tumor cell lines, which are derived from hepatic and neuroblastoma tumors, respectively, we are showing here that in both tumor models Nox4 is expressed in the ER (like the yeast NADPH oxidase), where according to published literature, it produces hydrogen peroxide. Reducing this biochemical activity by downregulating Nox4 transcription leads to loss of F-actin stress fibers. This phenotype is reversible by adding hydrogen peroxide to the cells. The effect of the Nox4 silencer RNA is specific for this gene as it does not influence the expression of Nox2. In the case of the SH-SY5Y neuronal cell line, Nox4 inhibition leads to loss of cell mobility as measured in scratch assays. We propose that inhibition of Nox4 (which is known to be strongly expressed in many tumors) could be studied as a new target for cancer treatment, in particular for inhibition of metastasis.

Redox Regulation of the Actin Cytoskeleton in Cell Migration and Adhesion: On the Way to a Spatiotemporal View

“…In this regard, an early study showed that blockade of NOXes in endothelial cell lines prevented Gactin incorporation into growing F-actin suggesting that ROS production by NOXes is critical for F-actin assembly (Moldovan et al, 2000). Similarly, blockade of NOXes downmodulated actin stress fiber formation and migration of tumor cell lines providing evidence for a positive role of ROS for localized actin polymerization and dynamics (Auer et al, 2017;Tamborindeguy et al, 2018). However, to the best of our knowledge, even though localized ROS production by NOXes during cell migration was elucidated (Kaplan et al, 2011;Tamborindeguy et al, 2018), there is no literature showing localized oxidation of actin on cysteine residues during physiological processes such as cell migration.…”

The defense and signaling role of NADPH oxidases in eukaryotic cells

“…Sequence comparisons of Yno1 with all seven human Nox enzymes show that Nox4 displays by far the largest similarity in sequence with Yno1 [ 93 ]. In both established tumor cell lines that the authors (HepG2 and SH-SY5Y), the enzyme, Nox4, was strongly expressed as compared with non-tumorous material from the same organ, and located in the ER, like the yeast enzyme.…”

Nox4 mediates RANKL-induced ER-phagy and Osteoclastogenesis via activating ROS/PERK/eIF-2α/ATF4 Pathway

“…Nox4 is composed of conserved transmembrane domains, FAD-and NADPH-binding domains in the Cterminal region, and two heme groups [43,44]. ER-localized Nox4 has been found to promote the proliferation, migration, differentiation and survival of cells [45,46]. The activity of ER-localized Nox4 in the regulation of cellular processes may be dependent upon its ability to produce H 2 O 2 , which can be a stable and diffusible signaling molecule, through its E-loop portion [47].…”