Reactive oxygen species (ROS) were seen as destructive molecules, but recently, they have been shown also to act as second messengers in varying intracellular signaling pathways. This review concentrates on hydrogen peroxide (H2O2), as it is a more stable ROS, and delineates its role as a survival molecule. In the first part, the production of H2O2 through the NADPH oxidase (Nox) family is investigated. Through careful examination of Nox proteins and their regulation, it is determined how they respond to stress and how this can be prosurvival rather than prodeath. The pathways on which H2O2 acts to enable its prosurvival function are then examined in greater detail. The main survival pathways are kinase driven, and oxidation of cysteines in the active sites of various phosphatases can thus regulate those survival pathways. Regulation of transcription factors such as p53, NF-kappaB, and AP-1 also are reviewed. Finally, prodeath proteins such as caspases could be directly inhibited through their cysteine residues. A better understanding of the prosurvival role of H2O2 in cells, from the why and how it is generated to the various molecules it can affect, will allow more precise targeting of therapeutics to this pathway.

Reactive oxygen species have been implicated in processes involving cellular damage and subsequent cell death, especially in organs such as the eye that are constantly exposed to excitatory signals. However, recent studies have shown that oxidant species can also act as intracellular signalling molecules promoting cell survival, but little is known about this mechanism in the retina. The present study demonstrates for the first time that hydrogen peroxide (H 2 O 2 ) is generated rapidly and acts as a prosurvival signal in response to a variety of apoptotic stimuli in retina-derived 661W cells and in the retinal ganglion cell line RGC-5. Focussing on 661Ws and serum deprivation, we systematically investigated pro-survival and pro-death pathways and discovered that the rapid and transient burst of H 2 O 2 activates the AKT survival pathway. Activation of the apoptotic machinery takes place following the decline of H 2 O 2 to basal levels. To substantiate this proposed pro-survival role of H 2 O 2 , we inhibited the oxidant burst, which exacerbated cell death. Conversely, maintenance of the oxidant signal using exogenous H 2 O 2 enhanced cell survival. Overall, the results presented in this study provide evidence for a novel role of H 2 O 2 in mediating survival of retinal cells in response to apoptotic stimuli.

The interaction of soluble forms of the human cationindependent insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-IIR) with IGFs and mannosylated ligands was analyzed in real time. IGF-IIR proteins containing domains 1-15, 10 -13, 11-13, or 11-12 were combined with rat CD4 domains 3 and 4. Following transient expression in 293T cells, secreted protein was immobilized onto biosensor chips. ␤-Glucuronidase and latent transforming growth factor-␤1 bound only to domains 1-15. IGF-II bound to all constructs except a control, which contained a point mutation in domain 11. The affinity of domains 1-15, 10 -13, 11-13, and 11-12 to IGF-II were 14, 120, 100, and 450 nM, respectively. Our data suggest that domain 13 acts as an enhancer of IGF-II affinity by slowing the rate of dissociation, but additional enhancement by domains other than 10 -13 also occurs. As the receptor functions to transport ligands from either the trans-Golgi network or extracellular space to the endosomes, the interaction of IGF-IIR extracellular domains with IGF-II was analyzed over a pH range of 5.0 -7.4. The constructs behaved differently in response to pH and in recovery after low pH exposure, suggesting that pH stability of the extracellular domains depends on domains other than 10 -13.

Reactive oxygen species (ROS) have traditionally been viewed as a toxic group of molecules; however, recent publications have shown that these molecules, including H2O2, can also strongly promote cell survival. Even though the retina has a large capacity to produce ROS, little is known about its non‐mitochondrial sources of these molecules, in particular the expression and function of NADPH oxidase (Nox) proteins which are involved in the direct generation of superoxide and indirectly H2O2. This study demonstrated that 661W cells, a retina‐derived cell line, and mouse retinal explants express Nox2, Nox4 and certain of their well‐established regulators. The roles of Nox2 and Nox4 in producing pro‐survival H2O2 were determined using 661W cells and some of the controlling factors were identified. To ascertain if this phenomenon could have physiological relevance, the novel technique of time‐lapse imaging of dichlorofluorescein fluorescence (generated upon H2O2 production) in retinal explants was established and it showed that explants also produce a burst of H2O2. The increase in H2O2 production was partly blocked by an inhibitor of Nox proteins. Overall, this study demonstrates a pro‐survival role of Nox2 and Nox4 in retina‐derived cells, elucidates some of the regulatory mechanisms and reveals that a similar phenomenon exists in retinal tissue as a whole.

Cell repulsion responses to Eph receptor activation are linked to rapid actin cytoskeletal reorganizations, which in turn are partially mediated by Rho-ROCK (Rho kinase) signalling, driving actomyosin contractility. In the present study, we show that Rho alone is not sufficient for this repulsion response. Rather, Cdc42 (cell division cycle 42) and its effector MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) are also critical for ephrinB-induced cell retraction. Stimulation of endothelial cells with ephrinB2 triggers rapid, but transient, cell retraction. We show that, although membrane retraction is fully blocked by blebbistatin (a myosin-II ATPase inhibitor), it is only partially blocked by inhibiting Rho-ROCK signalling, suggesting that there is ROCK-independent signalling to actomyosin contractility downstream of EphBs. We find that a combination of either Cdc42 or MRCK inhibition with ROCK inhibition completely abolishes the repulsion response. Additionally, endocytosis of ephrin-Eph complexes is not required for initial cell retraction, but is essential for subsequent Rac-mediated re-spreading of cells. Our data reveal a complex interplay of Rho, Rac and Cdc42 in the process of EphB-mediated cell retraction-recovery responses.