Nox4-generated superoxide drives angiotensin II-induced neural stem cell proliferation

Abstract

Reactive oxygen species (ROS) have been reported to affect neural stem cell self-renewal and therefore may be important for normal development and may influence neurodegenerative processes when ROS activity is elevated. To determine if increasing production of superoxide, via activation of NADPH oxidase (Nox), increases neural stem cell proliferation, 100 nM angiotensin II (Ang II) - a strong stimulator of Nox - was applied to cultures of a murine neural stem cell line, C17.2. Twelve hours following a single treatment with Ang II, there was a doubling of the number of neural stem cells. This increase in neural stem cell numbers was preceded by a gradual elevation of superoxide levels (detected by dihydroethidium fluorescence) from the steady state at 0, 5, and 30 min and gradually increasing from 1 h to the maximum at 12 h, and returning to baseline at 24 h. Ang II-dependent proliferation was blocked by the antioxidant N-acetyl-L-cysteine. Confocal microscopy revealed the presence of two sources of intracellular ROS in C17.2 cells: (i) mitochondrial and (ii) extramitochondrial; the latter indicative of the involvement of one or more specific isoforms of Nox. Of the Nox family, mRNA expression for one member, Nox4, is abundant in neural stem cell cultures, and Ang II treatment resulted in elevation of the relative levels of Nox4 protein. SiRNA targeting of Nox4 mRNA reduced both the constitutive and Ang II-induced Nox4 protein levels and attenuated Ang II-driven increases in superoxide levels and stem cell proliferation. Our findings are consistent with our hypothesis that Ang II-induced proliferation of neural stem cells occurs via Nox4-generated superoxide, suggesting that an Ang II/Nox4 axis is an important regulator of neural stem cell self-renewal and as such may fine-tune normal, stress- or disease-modifying neurogenesis.

Fig. 1

Ang II increases stem cell proliferation. (a, b) Bright-field photomicrographs of C17.2 stem cell cultures grown for 12 h in the absence (a) or presence (b) of 100 nM Ang II. (b) At 12 h post-treatment there was an obvious increase in the numbers of cells. (c) the BrdU Cell Proliferation ELISA Kit, which detects newly synthesized DNA, confirmed a significant elevation in cell proliferation (BrdU incorporation) in response to Ang II resulting in higher cell numbers 24 and 48 h after a single treatment with Ang II. (d) The CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay, which measures mitochondrial function, detected increases in mitochondrial activity with Ang II treatment. * represents p<0.001 (c) and p<0.05 (d) compared to control cultures. (e) Hemocytometric cell counts at 48 h validated the observation of an Ang II-driven proliferative response. In addition, an antioxidant, N-acetyl cysteine (NAC) prevented Ang II-induced proliferation. * represents p<0.05 compared to control, NAC, and Ang II + NAC cultures. Data are from three independent experiments with n=6 wells per experiment.

Fig. 2

Ang II drives superoxide generation in a time-dependent manner. The increase in superoxide was assessed by (a) microscopic and (b) microplate DHE assay of fluorescent product of DHE oxidation. * represents p<0.05 compared to 0, 5, 30 min, and 24 h. # represents p<0.05 compared to 0, 5, 30 min, and 1 and 24 h. Data are from three independent experiments with n=6 wells per experiment.

Fig. 3

Superoxide originates inside and outside of mitochondria. When treated with Ang II, neural stem cells generate superoxide (DHE, red) in mitochondria identified here by MitoTracker Green as well as at sites, which are extramitochondrial, and that likely represent membrane-associated Nox as a source of superoxide. As illustrated here, fluorescence in the nucleus represents DHE oxidized to ethidium bromide (a fluorescent nucleic acid stain), which when generated intracellularly intercalates into DNA.

Fig. 4

Superoxide production, generated in response to Ang II, is reduced by inhibitors of NADPH oxidase such as phenylarsine oxide (PAO; 0.6 μM) and apocynin (10 μM). Cultures were pretreated with either PAO or apocynin for six hours prior to one-hour treatment with Ang II. * represents p<0.05 compared to control and PAO-treated cultures. # represents p<0.05 compared to Ang II-treated cultures. Data are from three independent experiments with n=6 wells per experiment.

Fig. 5

Nox4 mRNA is the most abundant Nox isoform mRNA in C17.2 cells (a). Murine aortic smooth muscle cell line (MOVAS) was used as a positive control for Nox4 mRNA expression. Ang II treatment increases Nox4 synthesis as illustrated by (b) Western immunoblot and quantified densitometrically and analyzed using NIH image J (c). * represents p<0.05 compared to control cultures, which were not treated with Ang II. Data are from three independent experiments with n=6 wells per experiment.

Fig. 6

Ang II-driven proliferation of C17.2 neural stem cells requires Nox4-generated superoxide. Cells were grown sequentially in complete medium with 15% serum (48 h), in medium with 0.1% serum (24 h), transfected with siRNAs (24 h), and allowed to recover for 48 h while being treated for 12 or 48 h with Ang II. The effect of media only (control), transfection reagent (TKO), scrambled non-targeting siRNA (SCR siRNA), or Nox4 siRNA on: (a) expression of Nox4 in C17.2 neural stem cells illustrated by Western analysis; (b) quantification of Nox4 expression; (c) superoxide levels; and (d) proliferation. The effect of media only on C17.2 neural stem cells versus Ang II treatment with transfection reagent (TKO), scrambled non-targeting siRNA (SCR siRNA), or Nox4 siRNA in the presence of Ang II on: (e) Nox4 expression in C17.2 neural stem cells illustrated by Western analysis; (f) quantification of Nox4 expression; (g) superoxide levels; and (h) proliferation. † represents p<0.01 compared to all other culture conditions; * represents p<0.01 compared with cultures not treated with Ang II; # represents p<0.01 compared with all other cultures that were treated with Ang II. Data are from three independent experiments with n=6 wells per experiment.

Fig. 7

A conceptualization of Angiotensin II signaling in Nox4-dependent self-renewal of neural stem cells. Angiotensin II binding to its most abundant receptor, AT1 receptor, is known to stimulate signaling cascades leading to activation of various isoforms of Nox in a tissue- and cell-type dependent manner [68;69]. Here, we show that in C17.2 stem cells, Ang II leads to increased synthesis and activation of Nox4 and thereby enhancement of superoxide generation toward increased stem cell proliferation. Inhibiting the activity of Nox4 via Nox4 siRNA targeting, or pharmacologically with inhibitors of Nox (Apocynin and PAO), as well as a general antioxidant, NAC, prevented Ang II from increasing production of superoxide. The siRNA targeting Nox4 abolished the following responses to Ang II treatment: i) increase in Nox4 protein levels, ii) increase in superoxide production, and iii) increase in stem cell numbers.