Abstract
Generation of reactive oxygen species (ROS) by Ras oncogene-induced NADPH oxidase (Nox) 1 is required for Ras transformation phenotypes including anchorage-independent growth, morphological transformation, and tumorigenesity, but the signaling mechanism downstream of Nox1 remains elusive. Rho is known to be a critical regulator of actin stress fiber formation. Nonetheless, Rho was reported to no longer couple to loss of actin stress fibers in Ras-transformed Swiss3T3 cells despite the elevation of Rho activity. In this study, however, we demonstrate that Rho is inactivated in K-Ras-transformed normal rat kidney cells, and that abrogation of Nox1-generated ROS by Nox1 small interference RNAs or diphenyleneiodonium restores Rho activation, suggesting that Nox1-generated oxidants mediate down-regulation of the Rho activity. This down-regulation involves oxidative inactivation of the low molecular weight protein-tyrosine phosphatase by Nox1-generated ROS and a subsequent elevation in the tyrosine-phosphorylated active form of p190RhoGAP, the direct target of the phosphatase. Furthermore, the decreased Rho activity leads to disruption of both actin stress fibers and focal adhesions in Ras-transformed cells. As for Rac1, Rac1 also appears to participate in the down-regulation of Rho via Nox1. Our discovery defines a mediating role of Nox1-redox signaling for Ras oncogene-induced actin cytoskeletal changes.
Highlights
To act as signaling molecules in various physiological processes because of their regulated production in response to ligands, the existence of catabolic metabolism to terminate their signaling, and redox-dependent reversible modification of target proteins [1, 2]
As the results make it very unlikely that the observed effects of siRNAs are the results of an unusual cell clone or off-target effect from the siRNAs used, we mostly focus on KNRKsiNox1 cells in the subsequent study
Our previous study demonstrated that generation of superoxide is augmented in Kirstein-Ras-transformed NRK (KNRK) cells compared with NRK cells due to constitutive induction of Nox1 expression by Ras oncogenes via the RafMEK-MAPK pathway [8]
Summary
A distinct mode of intracellular ROS-mediated signaling could be expected for Ras transformation compared with cell spreading Another issue is contribution of Rho family GTPases to loss of both stress fibers and cell adhesions characteristic of Rastransformed cells. We demonstrate that K-Ras transformation reduces the level of active RhoGTP, and that depletion of Nox1-generated ROS reverts the reduction of the RhoGTP amount, suggesting the mediating role of Nox1-generated ROS in down-regulation of Rho. Nox1-generated ROS suppress the LMWPTP-p190RhoGAP pathway and thereby inactivate Rho, leading to loss of stress fiber formation and focal adhesions. Our discovery, unlike the model described by Sahai et al [20], reveals a new mechanism involving the Nox1-redox signaling-mediated down-regulation of Rho for altered stress fibers and focal adhesions in Ras-transformed cells
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