Apocynin and Diphenyleneiodonium Induce Oxidative Stress and Modulate PI3K/Akt and MAPK/Erk Activity in Mouse Embryonic Stem Cells

Abstract

Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.

Figure 1

Relative expression of NOX1 (a), NOX2 (b), NOX3 (c), NOX4 (d), DUOX1 (e), and DUOX2 (f) mRNA in mES and selected tissues. Comparison of relative expression of individual NOXs and DUOXs mRNA within mES cells is also shown (g). Data are presented as mean + SEM from at least two independent experiments.

Figure 2

Effect of APO (a) and DPI (b) on mES proliferation after 48 h treatment based on total cellular protein mass. Data represent mean + SEM from four independent experiments. Statistical significance was determined by ANOVA post hoc Bonferroni's Multiple Comparison test, P < 0.05. The groups marked differently by symbol letters are statistically significantly different from each other.

Figure 3

ROS production in mES cells treated by 1 mM APO and 100 nM DPI computed from the automated time-lapse image acquisition of HE fluorescence for 120 minutes (a, b); selected single time point 60 minutes (c). HPLC determination of specific 2-OH-E(+) and nonspecific product E(+) of HE oxidation in the presence of 1 mM APO and 100 nM DPI (d, e). Data are presented as mean + SEM from four independent experiments. Statistical significance was determined by ANOVA post hoc Bonferroni's Multiple Comparison test, P < 0.05. The groups marked by an asterisk are statistically significantly different from control.

Figure 4

Effect of APO (2 mM) and DPI (100 nM) on the Stat3, Akt, and Erk phosphorylation in serum starved mES cells (SF) treated by drugs for 20 minutes followed by LIF (5 ng/mL) or FBS (15%) stimulation for 20 minutes. Total level of β-actin was used as a loading control. A typical representative western blot is shown.

Figure 5

Effect of APO (1, 2, 4 mM), DPI (0.1, 1, 100 μM), NAC (5, 10, 20 mM), and H₂O₂ (0.5, 1 mM) on the Akt and Erk phosphorylation in serum starved mES cells treated by drugs for 20 minutes followed by 15% FBS stimulation for 20 minutes (a) and cells treated for 1 hour in complete medium (b). Total level of β-actin was used as a loading control. A typical representative western blot is shown.

Figure 6

Effect of APO (1 mM) and DPI (10 nM) in absence and presence of antioxidant NAC (10 mM) on the Akt and Erk, phosphorylation and Nanog protein level in mES cells after 24 hours in complete medium. Total level of β-actin was used as a loading control. A typical representative western blot is shown.

Figure 7

Effect of APO and DPI on Wnt pathway activity determined by TOPflash assay and GSK3 phosphorylation on S9. Effect of the Wnt3a conditioned media or exogenous nondegradable β-catenin on the transcriptional activation of a reporter gene (a). Effect of LY (10 μM), APO (1 mM), and DPI (10 nM) on the spontaneous (b), Wnt3a conditioned media induced (c), and exogenous nondegradable β-catenin-induced transcriptional activation (d). Data represent mean + SEM, from at least four independent experiments. Statistical significance was determined by ANOVA post hoc Bonferroni's Multiple Comparison test (^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001). Effect of LY, APO, and DPI on GSK3β (S9) phosphorylation (e). Total level of β-actin was used as a loading control. A typical representative western blot is shown.