PerspectiveCell Signaling

H2O2, a Necessary Evil for Cell Signaling

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Science  30 Jun 2006:
Vol. 312, Issue 5782, pp. 1882-1883
DOI: 10.1126/science.1130481

For many years, hydrogen peroxide (H2O2) was viewed as the inevitable but unwanted by-product of an aerobic existence. Given the damage inflicted by H2O2, it was assumed that the faster the elimination of this toxic waste, the better for the cell. However, as highlighted in recent forums (1, 2), we now know that mammalian cells produce H2O2 to mediate diverse physiological responses such as cell proliferation, differentiation, and migration (3, 4). This has led to implications of cellular “redox” signaling in regulating normal processes and disease progression, including angiogenesis, oxidative stress and aging, and cancer. This changing view of H2O2 has partly evolved from a clearer understanding of redox chemistry as it affects biology—that is, cellular signaling that is linked to reductive-oxidative-based mechanisms. As the components and mechanisms involved in performing cellular redox chemistry become better defined, new areas of research are emerging as to how the cells spatially and temporally channel H2O2 into specific signaling pathways to achieve desired cellular outcomes.

H2O2 production has been studied most extensively in neutrophils. These immune cells defend a host against infections by engulfing and killing foreign microorganisms. The system relies on Nox [the NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase complex], which generates millimolar quantities of H2O2 within the safe confines of an organelle [phagosome (see the figure)] for the purpose of microbial killing. In the classical phagocyte paradigm, stimulation of neutrophils by invading microoganisms leads to assembly at the plasma membrane of an active Nox complex, which comprises a catalytic subunit—the integral membrane protein gp91 Phox—and regulatory proteins including the small guanosine triphosphatase Rac. This complex releases the reactive oxygen species superoxide (the free radical anion O2) into the phagosome, and superoxide dismutation yields another reactive oxygen species, H2O2.

We do know that in nonphagocytic cells, H2O2 affects numerous intracellular signaling pathways. Nonphagocytic cells express gp91 Phox and its homologs (5), and these proteins are the major source of H2O2 in cells stimulated with various growth factors and cytokines including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, tumor necrosis factor-α (TNFα), and interleukin-1 (IL-1) (3, 4). However, the coupling of receptor activation to Nox activation in nonphagocytic cells still remains poorly understood.

H2O2 production, protection, and signaling actions.

Activation of various cell surface receptors activates Nox situated either in the plasma membrane or in the membrane of organelles such as endosomes to produce H2O2. To function as an intracellular signaling molecule, H2O2 must be imported into the cytosol. Cytosolic H2O2 enhances protein tyrosine phosphorylation by inactivating protein tyrosine phosphatases while activating protein tyrosine kinases. Transient protection of the H2O2 signal from abundant cytosolic peroxiredoxin appears to result from the reversible inactivation of these enzymes through either hyperoxidation or phosphorylation.


We are also trying to understand the mechanisms by which H2O2 can modify the activity of key signaling proteins. Biological redox reactions catalyzed by H2O2 typically involve the oxidation of cysteine residues on proteins, which may affect protein function. Phosphorylation of tyrosine residues in proteins is governed by the opposing activities of protein tyrosine phosphatases and protein tyrosine kinases. The protein tyrosine phosphatase family features a common Cys-X-X-X-X-X-Arg active-site motif (where X = any amino acid). As a result of the invariant arginine, the conserved catalytic cysteine possesses a low PKa (where Ka is the acid dissociation constant) and exists as a thiolate anion with enhanced susceptibility to oxidation by H2O2. Oxidation of the essential cysteine abolishes phosphatase activity and can be reversed by cellular thiols. Reversible inactivation of several different protein tyrosine phosphatases has been demonstrated in relevant cell types stimulated with PDGF, EGF, insulin, extracellular matrix molecules, and B cell receptor ligands (3, 6). Oxidative inactivation of these phosphatases and increased tyrosine phosphorylation of target proteins were found to be dependent on H2O2 production. Moreover, in TNF-α-stimulated cells, the resulting H2O2 that is generated inactivates mitogen-activated protein kinase phosphatases. This in turn results in sustained activation of c-Jun N-terminal kinase, a subfamily of the mitogen-activated protein kinases that elicits specific cellular responses.

H2O2 also appears to promote tyrosine phosphorylation by activating protein tyrosine kinases. For example, upon cell attachment to extracellular matrix and associated generation of H2O2, the tyrosine kinase Src becomes oxidized at two cysteine residues and thus becomes activated (7). Moreover, antioxidant treatment of cells that express an oncogenic form of Src (v-Src), or mutation of the oxidation-sensitive cysteine residues of v-Src, reduces the potency of v-Src to transform cells. This redox-dependent activation of Src occurs alongside dephosphorylation of a carboxyl-terminal tyrosine, a modification that is needed to activate Src.

For H2O2 to serve as a signal—through modification of signaling proteins—its concentration must increase rapidly above a certain threshold. How can this occur in the presence of antioxidant enzymes such as catalase, glutathione perioxidase, and peroxiredoxin? Whereas catalase is confined to the peroxisome, several peroxiredoxin isoforms are abundant in the cytosol. Therefore, H2O2 must be protected from destruction by peroxiredoxin in selected contexts. Indeed, multiple protective mechanisms of this type are being uncovered. During catalysis of H2O2 reduction, the active-site residue, Cys-SH, of peroxiredoxin occasionally reacts with two molecules of H2O2, and thus becomes hyperoxidized to Cys-SOOH. Consequently, peroxiredoxins are inactivated (8). This inactivation, which can be reversed by sulfiredoxin, an adenosine triphosphate-dependent enzyme, may represent a built-in mechanism to prevent damping of the H2O2 signal. Prokaryotes do not express sulfiredoxin and their peroxiredoxins are resistant to hyperoxidation. Thus, this regulatory mode appears unique to eukaryotes. Peroxiredoxins are also reversibly inactivated upon phosphorylation by cyclin B-dependent kinase during mitosis (9).

Given the toxicity of H2O2, spatial and temporal regulatory strategies must exist to ensure that Nox activation occurs only where needed and that the H2O2 signal is terminated in a timely fashion. Recent work on cells stimulated with TNF-α. suggests that Nox proteins are assembled in specific subcellular compartments within membranes such as lipid rafts (10). Localized Nox assembly also occurs at focal complexes, points of contact between a moving cell and the extracellular matrix, in response to migratory stimuli (11). The relevant oxidation targets that are presumably enriched in these microenvironments remain to be identified.

Despite the increasingly sophisticated molecular descriptions of H2O2 action, disturbingly little is understood about how H2O2 is actually delivered to the cytosol. The classical neutrophil studies demonstrate that Nox releases H2O2 into the phagosome, which is topologically equivalent to the extracellular space. How, then, does H2O2 modulate intracellular signaling? In one scenario, Nox situated at the plasma membrane releases H2O2 into the extracellular space as an autocrine factor to be imported into the cell. Alternatively, Nox proteins assembled at organelle membranes discharge H2O2 into the lumenal space. For example, binding of IL-1 to its receptor in the plasma membrane triggers Rac-mediated Nox association with the IL-1 receptor and endocytosis (internalization) of the receptor complex (12). This results in superoxide production and conversion into H2O2 in the lumen of the endosome. In addition, Nox isoforms and their regulatory subunits have been detected in other cell organelles including the endoplasmic reticulum and nucleus.

Regardless of whether the Nox complex is activated at the cell surface or within an organelle, the resultant H2O2 must traverse the lipid bilayer to access the cytosol, where most if not all of its target proteins exist. Although H2O2 is believed to diffuse freely across membranes, recent studies indicate that some membranes are poorly permeable to H2O2. Instead, H2O2 transport might be regulated by changes in membrane lipid composition or by aquaporins (13), which are diffusion-facilitating channel proteins for noncharged solutes such as water.

The current picture of H2O2 -based redox regulation of signaling processes is rapidly expanding beyond those issues focused on here. The development of a sensitive and specific probe for H2O2 that allows quantitative and dynamic assessment in live cells, conspicuously lacking in studies to date, will be a great boon for the study of this misunderstood and maligned molecule.

References and Notes

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