Aquaporin-8 (AQP8) allows the bidirectional transport of water and hydrogen peroxide across biological membranes. Depending on its concentration, H 2 O 2 exerts opposite roles, amplifying growth factor signaling in physiological conditions, but causing severe cell damage when in excess. Thus, H 2 O 2 permeability is likely to be tightly controlled in living cells. Aims: In this study, we investigated whether and how the transport of H 2 O 2 through plasma membrane AQP8 is regulated, particularly during cell stress. Results: We show that diverse cellular stress conditions, including heat, hypoxia, and ER stress, reversibly inhibit the permeability of AQP8 to H 2 O 2 and water. Preventing the accumulation of intracellular reactive oxygen species (ROS) during stress counteracts AQP8 blockade. Once inhibition is established, AQP8-dependent transport can be rescued by reducing agents. Neither H 2 O 2 nor water transport is impaired in stressed cells expressing a mutant AQP8, in which cysteine 53 had been replaced by serine. Cells expressing this mutant are more resistant to stress-, drug-, and radiation-induced growth arrest and death. Innovation and Conclusion: The control of AQP8-mediated H 2 O 2 transport provides a novel mechanism to regulate cell signaling and survival during stress. Antioxid.

H 2 O 2 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 H 2 O 2 entry into HeLa cells. Re-expression of AQP8 with silencing-resistant vectors rescued H 2 O 2 transport, whereas a C173A-AQP8 mutant failed to do so. Lowering AQP8 levels affected H 2 O 2 entry into the endoplasmic reticulum, but not into mitochondria. AQP8 silencing also inhibited the H 2 O 2 spikes and phosphorylation of downstream proteins induced by epidermal growth factor. These observations lead to the hypothesis that H 2 O 2 does not freely diffuse across the plasma membrane and AQP8 and other H 2 O 2 transporters are potential targets for manipulating key signaling pathways in cancer and degenerative diseases.

Limited amounts of reactive oxygen species are necessary for cell survival and signaling, but their excess causes oxidative stress. H(2)O(2) and other reactive oxygen species are formed as byproducts of several metabolic pathways, possibly including oxidative protein folding in the endoplasmic reticulum. B- to plasma-cell differentiation is characterized by a massive expansion of the endoplasmic reticulum, finalized to sustain abundant immunoglobulin (Ig) synthesis and secretion. The increased production of disulfide-rich Ig might cause oxidative stress that could serve signaling roles in the differentiation and lifespan control of antibody-secreting cells. Here we show that terminal B-cell differentiation entails redox stress, NF-E2-related factor-2 (Nrf2) activation, and reshaping of the antioxidant responses. However, plasma-cell differentiation was not dramatically impaired in peroxiredoxin (Prx)1-, 2-, 3-, and 4-, glutathione peroxidase 1-, and Nrf2-knockout splenocytes, suggesting redundancy and robustness in antioxidant systems. Endoplasmic reticulum (ER)-resident Prx4 increases dramatically during differentiation. In its absence, IgM secretion was not significantly affected, but more high-molecular-weight covalent complexes accumulated intracellularly. Our results suggest that the early intracellular production of H(2)O(2) facilitates B-cell proliferation and reveal a role for the Nrf2 pathway in the differentiation and function of IgM-secreting cells.

During their differentiation to antibody-secreting plasma cells, B lymphocytes undergo dramatic changes in metabolism, structure, and function. Here we show that this transition entails extensive intra- and extracellular redox changes. Lipopolysaccharide (LPS)-driven activation and differentiation of naïve murine B splenocytes is paralleled by increased production of reactive oxygen species (ROS) from different sources, followed by a strong antioxidant response. This response includes upregulation of thioredoxin and of the cystine transporter xCT, and increased production and extracellular release of nonprotein thiols, mainly glutathione (GSH) and cysteine. Although ROS levels are higher in late-differentiating B cells, an early oxidative step is likely required to start the differentiation program, because inhibition of NADPH oxidase-dependent early ROS production impairs B-cell activation and differentiation. Addition of reducing agents such as 2-ME results in increased IgM secretion per cell, suggesting that the antioxidant response not only is aimed at restoring the redox homeostasis but also plays a functional role. A highly reduced environment coincident with the presence of large ROS-producing cells is observed in histologic sections of spleens from immunized mice, indicating that the redox modifications observed in LPS-induced B-cell differentiation in vitro occur also in vivo during physiologic immune responses.