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. 2013 Nov 1;19(13):1447-51.
doi: 10.1089/ars.2013.5330. Epub 2013 Sep 18.

Tyrosine Kinase Signal Modulation: A Matter of H2O2 Membrane Permeability?

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Free PMC article

Tyrosine Kinase Signal Modulation: A Matter of H2O2 Membrane Permeability?

Milena Bertolotti et al. Antioxid Redox Signal. .
Free PMC article

Abstract

Abstract H2O2 produced by extracellular NADPH oxidases regulates tyrosine kinase signaling inhibiting phosphatases. How does it cross the membrane to reach its cytosolic targets? Silencing aquaporin-8 (AQP8), but not AQP3 or AQP4, inhibited H2O2 entry into HeLa cells. Re-expression of AQP8 with silencing-resistant vectors rescued H2O2 transport, whereas a C173A-AQP8 mutant failed to do so. Lowering AQP8 levels affected H2O2 entry into the endoplasmic reticulum, but not into mitochondria. AQP8 silencing also inhibited the H2O2 spikes and phosphorylation of downstream proteins induced by epidermal growth factor. These observations lead to the hypothesis that H2O2 does not freely diffuse across the plasma membrane and AQP8 and other H2O2 transporters are potential targets for manipulating key signaling pathways in cancer and degenerative diseases.

Figures

FIG. 1.
FIG. 1.
Entry of exogenous H2O2 into HeLa cells requires AQP8. (A) Time course response of wt cytosolic HyPer (red line) or a redox-insensitive mutant (C199S, blue line) to 50 μM H2O2. (B) Kinetics of H2O2-dependent HyPerCyto activation in HeLa cells silenced for AQP8 expression (8i), and then transfected with silencing-resistant constructs driving the expression of wt (8i+wt) or mutant AQP8 (8i+C173A, see Methods for details). Graphs in A and B show mean fold change±SEM of the 488/405 nm ratio measured by confocal laser scanning. (C) The HyPer 488/405 nm ratio is not influenced by the expression levels of the probe. Transiently transfected HeLa cells were analyzed by confocal laser scanning. Despite cells being varied in the extent of probe expression, the ratio remained rather constant, as demonstrated by the linear correlation of 405 and 488 nm light excitation (r=0.92). (A.U. stands for Arbitrary Units). (D) H2O2-dependent activation of HyPerCyto was analyzed in HeLa cells treated with specific silencing oligos for AQP8, AQP4, or AQP3 by Typhoon scanning (see Methods). Data are expressed as fold increase in the 488 nm fluorescence±SEM, relative to cells that were not treated with H2O2. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
FIG. 2.
FIG. 2.
H2O2 transport into the endoplasmic reticulum (ER) is facilitated by AQP8. HeLa cells expressing HyPerERLum (A) or HyperMito (B) were silenced with AQP8-specific (blue line) or irrelevant (red line) oligos before semipermeabilization with 40 μg/ml digitonin (7) to bypass the plasma membrane and allow access to ER and mitochondria. The kinetics of H2O2 entry into the two organelles were measured by confocal laser scanning. Graphs show mean fluorescence±SEM. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
FIG. 3.
FIG. 3.
Epidermal growth factor (EGF) induces transport of H2O2 through AQP8. (A) Time course analysis of H2O2 uptake by HeLa cells expressing HyPerCyto by confocal laser scanning after treatment with 10 μg/ml EGF. Color code as in Figure 2; (B). Western blot analysis with the indicated antibodies showing changes in phosphorylation of total cell tyrosines on whole HeLa cell extracts that were either silenced for AQP8 (8i) or treated with catalase before treatment with EGF. (C) The intensity of protein phosphotyrosine signals in the blots shown in B were quantified by densitometry and normalized to Tubulin. Bars show average band intensities relative to untreated control cells±SEM. (D) A schematic view of H2O2 generation and transport during EGF signaling. NOX and DuOX enzymes on the cell surface are activated to produce H2O2. As demonstrated by the inhibitory effects of catalase, extracellularly generated H2O2 is important for efficient amplification of signaling and enters the cell through AQP8, as indicated by silencing (B, C). The residual activity in silenced cells could reflect incomplete AQP8 downregulation, the presence of additional H2O2 transporters in the plasma membrane and/or its generation in mitochondria. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

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