Regulation of reactive-oxygen-species generation in fibroblasts by Rac 1

Abstract

In a variety of non-phagocytic cell types, there is a marked increase in intracellular levels of reactive oxygen species (ROS), including superoxide and H2O2, after ligand stimulation. We demonstrate that in NIH 3T3 cells transient expression of constitutively activated forms of the small GTP-binding proteins Ras or Rac1 leads to a significant increase in intracellular ROS. An increase in intracellular ROS is also demonstrated after growth factor [platelet-derived growth factor (PDGF) or epidermal growth factor (EGF)] or cytokine [tumour necrosis factor-alpha (TNF-alpha) or interleukin (IL)-1 beta] stimulation of NIH 3T3 cells. Expression of a dominant negative allele of Rac1 inhibits the rise in ROS seen after Ras expression or after stimulation by either growth factors or cytokines. These results provide the first demonstration of the pathway by which ligand stimulation of ROS occurs in non-phagocytic cells and suggest that the family of Ras-related small GTP-binding proteins may function as regulators of the intracellular redox state.

Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: Studies on TGF-β1 regulation

“…Our group has contributed to the knowledge of this regulation by demonstrating that H 2 O 2 induces mesangial cell contraction (6), stimulates cell proliferation (8) To decreased intracellular ROS concentration, CAT was added to the extracellular media of the cells. Extracellular CAT decreases H 2 O 2 in cell supernatants, with the subsequent transfer of this ROS from the intra to the extracellular media, but also penetrates into the cells (24,34). Our experiments confirm that intracellular CAT activity increased about three-fold after addition of 320 U/ml CAT to the cell supernatants for 16 h. Moreover, they confirmed that intracellular ROS concentration decreased about 40% with this same CAT concentration, measured by fluorescence of DCFHDA.…”

“…As previously reported (24,34), the intracellular CAT activity significantly increased (Fig. 1A) and a dose-dependent reduction of intracellular ROS concentration, measured by flow cytometry with fluorescent dye DCFHDA, was observed (Fig.…”

Role of redox potential and reactive oxygen species in stress signaling

“…Of interest is the independent role implicated for p85, the regulatory subunit of PI3K, in p53-dependent apoptotic response to oxidative stress (Yin et al, 1998). Another GTP binding protein, Rac1, has been associated with the generation of ROS and activation of NF-kB (Sundaresan et al, 1996;Sulciner et al, 1996). ROS activation of Rac1 has also been implicated in the alteration of cell shape via transactivation of interleukin1a and of collagenase-1, both of which a ect cellular cytoskeleton reorganization associated with wound healing, in¯ammation and malignancy (Kheradmand et al, 1998).…”

Reactive oxygen species-mediated regulation of the Na+–H+ exchanger 1 gene expression connects intracellular redox status with cells' sensitivity to death triggers

“…However, during the 1990s it became increasingly evident that nonphagocytic cells could also produce low levels of O 2 KÀ through mechanisms similar to those in phagocytes. In particular, Sudaresan et al 20 showed the production of O 2 KÀ upon growth factor-mediated activation of the small GTPase Rac1 in fibroblasts that could be inhibited by the expression of RacN17, the dominantnegative allele of Rac1. Moreover, Irani et al 21 To support further a role for Rac1 activation in the induction of NHE-1 promoter activity, we used mouse muscle L6 cells (L6 1.1 Kb), 23 mouse renal fibroblast MCT cells (MCT 1.1 kb) and NIH3T31A8 cells stably transfected with the 1.1-kb proximal fragment of the mouse NHE1 promoter/enhancer inserted 5 0 to a luciferase gene.…”