Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling

Abstract

Hydrogen peroxide (H 2 O 2 ) produced by cell-surface NADPH Oxidase (Nox) enzymes is emerging as an important signaling molecule for growth, differentiation, and migration processes. However, how cells spatially regulate H 2 O 2 to achieve physiological redox signaling over nonspecific oxidative stress pathways is insufficiently understood. Here we report that the water channel Aquaporin-3 (AQP3) can facilitate the uptake of H 2 O 2 into mammalian cells and mediate downstream intracellular signaling. Molecular imaging with Peroxy Yellow 1 Methyl-Ester (PY1-ME), a new chemoselective fluorescent indicator for H 2 O 2 , directly demonstrates that aquaporin isoforms AQP3 and AQP8, but not AQP1, can promote uptake of H 2 O 2 specifically through membranes in mammalian cells. Moreover, we show that intracellular H 2 O 2 accumulation can be modulated up or down based on endogenous AQP3 expression, which in turn can influence downstream cell signaling cascades. Finally, we establish that AQP3 is required for Noxderived H 2 O 2 signaling upon growth factor stimulation. Taken together, our findings demonstrate that the downstream intracellular effects of H 2 O 2 can be regulated across biological barriers, a discovery that has broad implications for the controlled use of this potentially toxic small molecule for beneficial physiological functions.growth factor signaling | redox biology | reactive oxygen species | fluorescent sensor | membrane regulation H ydrogen peroxide (H 2 O 2 ) is garnering increased attention as a molecule involved not only in immune response and oxidative stress, but also as a physiological effector in essential cellular processes (1-5). Seminal contributions have elucidated ligand stimulants (6-10) and enzymatic sources (11-13) for cellular H 2 O 2 production as well as putative downstream targets (14-24), but principles of how this reactive oxygen species (ROS) is spatially and temporally regulated to promote redox signaling over oxidative stress pathways remain insufficiently understood. Because many of the signaling functions of H 2 O 2 rely on its generation by nonphagocytic forms of NADPH (Nox) proteins on the extracellular side of cell membranes, understanding how cells funnel H 2 O 2 toward beneficial pathways in these locales is of significant interest. Despite its reactive nature, H 2 O 2 has been long thought to be freely diffusible across biological membranes in a manner akin to the related canonical small-molecule signal nitric oxide (NO) (25). More recent studies implicate the role of AQP water channels, a class of membrane-spanning proteins that facilitate the diffusion of water and other substrates with varying specificity (26-28), in mediating H 2 O 2 passage across the plasma membrane of reconstituted yeast (29) and plant (30) cells. However, given that the experiments described in these reports utilized nonspecific chemical reagents for determination of the redox signal that was passed into the cell, direct evidence that aquaporins influence the cellular uptake of H 2 O 2 in a na...

A respiratory chain controlled signal transduction cascade in the mitochondrial intermembrane space mediates hydrogen peroxide signaling

“…Relevant other sources of extracellular and intracellular H 2 O 2 include environmental exposure to ozone, UV light, and ionizing radiation (1,2). Among the most conserved defense mechanisms against the oxidizing effects of H 2 O 2 are detoxifying enzymes, such as catalase, as well as aquaporins that control the influx of H 2 O 2 into the cell (3)(4)(5)(6)(7). These mechanisms are highly efficient and adaptable, and provide an explanation for the great variation in susceptibility to H 2 O 2 among cell types despite the generally low intracellular concentrations across bacteria, plants, and mammals (3,8,9).…”

Aquaporins facilitate hydrogen peroxide entry into guard cells to mediate ABA- and pathogen-triggered stomatal closure

“…Altogether, these findings are reminiscent of results obtained in animal cells. A pioneering work using HyPer unraveled the role of AQP3 in H 2 O 2 transport and epidermal growth factor (EGF) signaling (31). This function was recently extended to NF-κB signaling in keratinocytes (32) or in response to environmental stresses in colonic epithelia (33).…”

Role of aquaporins in cell proliferation: What else beyond water permeability?

“…We 13 and others 17,[83][84][85] have shown that both AQP1 and AQP3 mediate uptake of H 2 O 2 in cells, postulating that transport of H 2 O 2 into mammalian systems by AQPs might interfere with intracellular signaling, amplifying cascades that depend on ROS, or increase the phosphorylation status of a cell (AKT/protein kinase B) and thus favor proliferation cascades.…”

“…12,[38][39][40] In addition, several metabolically important small neutral solutes and ions have been identified as AQP substrates, namely hydrogen peroxide (H 2 O 2 ). 13,17,41,42 carbon dioxide, 43,44 nitric oxide, 45,46 ammonia, 47,48 metalloids such as arsenite and antimonite, 49-51 selenite, 52 boric acid, 53 and silicic acid. 51 AQP6 with low intrinsic water permeability may function primarily as an anion transporter.…”

“…Reviewing the literature, we find that this association has been suggested mainly in relation to a role for these proteins in cell volume changes, 3,5,8 or in the case of a subgroup of AQPs, called aquaglyceroporins, 11,12 to their permeability to solutes such as glycerol and hydrogen peroxide. [13][14][15][16][17][18] Additionally, various studies have demonstrated that AQP expression is modified over the course of the cell cycle [19][20][21][22][23] and, more recently, we have shown that overexpression of AQP1 and AQP3 alters the levels of essential checkpoint proteins (cyclins) relevant for cell cycle progression, in addition to modifying the levels of transcription factors, and cytokines. 13,24 In this review, we have sought to gather the most relevant evidence associating AQPs with cell proliferation and we will discuss whether the canonical function of water permeation of these proteins would be always essential for this process or the role in proliferation might be independent of this function.…”