Reactive oxygen species (ROS) regulation of RAS and vulva development in the nematode (roundworm), C. elegans

This topic for these GEITP pages is a good example of hysteresis (in biology, this is a non-linear dose-response curve, where low doses are ineffective, a higher dose is beneficial, and a higher-yet dose is toxic). Examples of gases involved in signal-transduction pathways include nitrous and nitric oxide (N2O and NO), hydrogen sulfide (H2S), carbon dioxide and monoxide (CO2 and CO), hydrogen peroxide (H2O2), and oxygen, ozone, singlet-oxygen and superoxide (O2, O3, O. and O2•-). Each of these is essential to life at some concentration in living cells, ineffective at lower doses, and of course toxic at higher doses. Singlet-oxygen, superoxide and hydrogen peroxide are examples of reactive oxygen species (ROS), which are critically important signaling molecules; ROS studies in the intact organisms are often hampered by their potential toxicity. In the roundworm Caenorhabditis elegans, development of the vulva is regulated by a signal transduction cascade — which includes LET-602ras (the worm homolog of mammalian RAS, an oncogene in the GTPase family), MPK-1 (the worm homolog of ERK1/2, mitogen-activated protein kinases), and LIN-1 (an ETS proto-oncogene transcription factor).

Using C. elegans vulva development as a model, authors [see attached article] show both mitochondrial and cytoplasmic ROS act on the worm homolog of RAS — by way of a redox-sensitive cysteine (at position 118 in the protein, i.e. C118). However, in contrast to what is observed in cultured mammalian cells, it was found that C118 is oxidized by hydrogen peroxide, rather than by superoxide or nitric oxide, and that its oxidation inhibits, rather than activates, this pathway. Furthermore, authors show that regulation of LET-602ras (by its oxidation) does not act through MPK-1 to affect vulva development. In addition, authors identified two additional ROS-signaling pathways that promote vulva development: elevated cytoplasmic superoxide promotes vulva formation independently of C118 of LET-602ras, and downstream of

LIN-1, whereas NADPH oxidases (BLI-3, ortholog of human dual oxidase-1; and DUOX-2, dual oxidase-2; these exhibit heme-binding activity and peroxidase activity), and their redox-sensitive activator CED-10rac act in yet another parallel pathway to promote vulval formation. CED-10rac is orthologous to human RAC1 and RAC2 (small GTPases), which participates in cell migration, embryonic morphogenesis (e.g. vulva development, and generation of neurons.

In detail, authors show that the pro-oxidant paraquat (PQ) (as well as isp-1, nuo-6 and sod-2 mutants, which increase mitochondrial ROS), inhibit the activity of LET-602ras on vulval development. In contrast, the anti-oxidant N-acetylcysteine (NAC) and loss of sod-1 (encoding superoxide dismutase) — both of which decrease cytoplasmic H2O2 — enhance the activity of LET-602ras. CRISPR replacement of C118 with the non-oxidizable serine (C118S) stimulated LET-602ras activity, whereas replacement of C118 with aspartate (C118D) — which mimics a strongly oxidized cysteine — inhibited LET-602ras; these data indicate that C118 is oxidized by cytoplasmic H2O2 generated from dismutation of mitochondrial and/or cytoplasmic superoxide, and that this oxidation inhibited LET-602ras. This finding contrasts with results in mammalian cells where it is mostly nitric oxide (not found in worms) that oxidizes C118 and activates RAS. Interestingly, PQ, NAC and the C118S mutation do not act on the phosphorylation of MPK-1, suggesting that oxidation of LET-602ras acts on an as yet uncharacterized MPK-1-independent pathway. Elevated cytoplasmic superoxide was found to promote vulva formation independently of the C118 in LET-602ras and downstream of LIN-1. Lastly, authors discovered a role for the NADPH oxidases (BLI-3 and DUOX-2) and their redox-sensitive activator CED-10rac in stimulating vulva development. Therefore, there are at least three genetically separable pathways — by which ROS regulates vulval development.

How is this study related to gene-environment interactions? This study shows that endogenous (intracellular) signals of gases/chemical agents are really no different from foreign environmental signals — in promoting genes that function in up- or down-regulation in signaling cascades. In this case, the signal transduction pathways are critically important to organ morphogenesis in the developing embryo. 😊


PLoS Genet Jun 2020; 16: e1008838

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