Journal of Signal Transduction molecular analysis has revealed that Rac proteins function as a necessary switch for ROS generation and that the protein is recruited to the membrane following neutrophil activation where it can bind to both p67phox and gp91phox [20]. Many evidences suggest that certain aspects of neutrophil biology appear to be conserved in the ROS signaling of non- phagocytic cells. In particular, homologues of the NADPH oxidase were found in vascular endothelial cells and smooth muscle cells, as well as in other normal or transformed cells such as colon cancer or melanoma [21]. Several isoforms of the catalytic NOX2 protein were identiﬁed, including NOX1, NOX3, NOX4, and NOX5, and shown to localize in prox- imity of speciﬁc redox-sensitive molecular targets within dis- crete subcellular compartments, thereby facilitating the com- partmentalization of redox signaling [15]. In addition, the expression of a constitutively activated form of Rac1 was noted to increase the basal level of hydrogen peroxide in immortalized ﬁbroblasts [22] as well as in certain trans- formed cell lines [23], while the expression of a dominant negative form of Rac1 was shown to inhibit the production of ROS following addition of various ligands [22]. These data suggest that a Rac-regulated oxidase exists in a wide range of cell types and participates in normal signal transduction. It has been shown that NOX1 constitutively binds the RacGEF βPIX, and the interaction is caused by growth factor stimulation [24]. This and previous studies [25] also support a pathway where ligand addition results in the sequential activation of phosphatidylinositol 3-kinase (PI3K), which in turn generates lipid products that can activate GEFs through the PH (pleckstin homology) domain present within the exchange factors. Activation of the GEF leads to increased Rac activity that is presumed to directly stimulate NOX [14]. The hypothesis that ROS generation is regulated by Rac and the role of ROS as speciﬁc eﬀector molecules that act downstream of Rac is supported by several evidences. In a recent paper there has been shown a role of Rac-regulated ROS in the crosstalk between G-protein-coupled receptors (GPCRs) and the JAK/STAT pathway [26], while diﬀerent studies support a role of Rac1 as a crucial, common upstream mediator of ROS production in integrin-mediated outside-in signaling [11–13, 16, 27]. Several evidences have implicated ROS in the integration of signals from VEGF and Rac to regulate the integrity of the endothelial barrier [22, 28–32]. Further studies demon- strated that the VEGF-dependent phosphorylation of VE- cadherin and β-catenin are dependent on Rac and ROS and result in decreased junctional integrity and enhanced vascular permeability [33, 34]. In addition to NADPH oxidase, Rac1 has been demon- strated to act upstream of AA-metabolizing enzymes, such as PLA2 [35, 36], 5-LOX [13, 26, 27], and COX-2 [37], whereas many reports show that AA metabolism modulates NADPH oxidase and mitochondrial ROS production [16]. Another aspect of oxidant signaling derived from the initial observation that Rac proteins regulate ROS levels is the demonstration of redox-dependent crosstalk between diﬀerent small GTPase family members. ROS production is apparently an essential component in signaling cascades that mediate Rac1/p190RhoGAP-induced 3 downregulation of RhoA and concomitant formation of membrane ruﬄes and integrin-mediated cell spreading. The pathway linking generation of ROS to downregulation of Rho involves inhibition of the low-molecular-weight pro- tein tyrosine phosphatase (LMW-PTP) and a consequent increase in the activation by phosphorylation of the Rho inhibitor p190Rho-GAP [38]. It has been shown that ROS production causes p190RhoGAP translocation to the adherens junctions (AJs), where it binds p120ctn, and sub- sequently inhibits local Rho activity [39]. It thus plays a role in the stabilization of cell-cell contacts [34]. These ﬁndings suggest that Rac1 downregulates Rho and stress ﬁber formation in a redox-dependent manner and deﬁne a mechanism for the coupling of changes in cellular re- dox state to the control of actin cytoskeleton rearrangements by Rho GTPases. In addition to Rac, the production of ROS by nonphago- cytic cell types stimulated by growth factors or cytokines includes the participation of p21Ras [22]. Fibroblasts ex- pressing constitutively active mutants of both Rac and Ras produce high levels of ROS associated with a high rate of proliferation. In the same study experimental evidence was provided suggesting that Rac is positioned downstream to Ras. Similar overexpression of Ras in other cell types such as keratinocytes [40] and epithelial cells [41] also demonstrated an increase in basal ROS levels. The pathway by which Ras regulates the levels of ROS remains incompletely understood. It has been shown that in some cells it proceeds through a PI3K and Rac-dependent pathway [25] leading to the regulation of a NOX-dependent oxidase. In other cell types the source of Ras-induced ROS appears to be linked to the mitochondria [42]. Mitochondria have the highest levels of antioxidants in the cell and play an important role in the maintenance of cellular redox status, thereby acting as a ROS and redox sink and limiting NADPH oxidase activity. However, mitochon- dria are not only a target for ROS produced by NADPH oxidase but also a signiﬁcant source of ROS, which under certain conditions may stimulate NADPH oxidases. Many ﬁndings indicate the existence of a bidirectional signaling crosstalk between mitochondria and NADPH oxidase, where small GTPases can orchestrate a complex web of regulation for ROS production [43–45]. Indeed, in integrin signaling, the regulation of mitochon- dria by both Rac and RhoA appears to be related to their ability to alter intracellular ROS [12]. It has been shown that Nerve Growth Factor- (NGF-) induced diﬀerentiation of PC12 cells is mediated by signif- icant alteration of mitochondrial metabolism by reducing mitochondrial-produced ROS and stabilizing the electro- chemical gradient. This is accomplished by stimulation of mitochondrial manganese superoxide dismutase (MnSOD) via Ki-Ras and ERK1/2 [46]. Thus ROS produced by small GTPases could regulate mitochondrial properties, including the overall metabolic rate and the generation of mitochondrial oxidants with im- portant signaling functions within the cell [14].