Abstract
Vascular endothelial cells (ECs) are important for maintaining vascular homeostasis. Dysfunction of ECs contributes to cardiovascular diseases, including atherosclerosis, and can impair the healing process during vascular injury. An important mediator of EC response to stress is the GTPase Rac1. Rac1 responds to extracellular signals and is involved in cytoskeletal rearrangement, reactive oxygen species generation and cell cycle progression. Rac1 interacts with effector proteins to elicit EC spreading and formation of cell-to-cell junctions. Rac1 activity has recently been shown to be modulated by glutathiolation or S-nitrosation via an active site cysteine residue. However, it is not known whether other redox signaling compounds can modulate Rac1 activity. An important redox signaling mediator is the electrophilic lipid, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2). This compound is a downstream product of cyclooxygenase and forms covalent adducts with specific cysteine residues, and induces cellular signaling in a pleiotropic manner. In this study, we demonstrate that a biotin-tagged analog of 15d-PGJ2 (bt-15d-PGJ2) forms an adduct with Rac1 in vitro at the C157 residue, and an additional adduct was detected on the tryptic peptide associated with C178. Rac1 modification in addition to modulation of Rac1 activity by bt-15d-PGJ2 was observed in cultured ECs. In addition, decreased EC migration and cell spreading were observed in response to the electrophile. These results demonstrate for the first time that Rac1 is a target for 15d-PGJ2 in ECs, and suggest that Rac1 modification by electrophiles such as 15d-PGJ2 may alter redox signaling and EC function.
Highlights
Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1) is a small GTP hydrolyzing (GTPase) protein in the Ras superfamily
When Recombinant Rac1 (rRac1) was treated with equimolar amounts of bt-15d-PGJ2, a biotin signal was detected by western blot at the expected molecular weight of rRac1 (21 kDa), whereas no signal was detected in the untreated rRac1 (Fig. 1B)
It is becoming increasingly clear that electrophilic lipids have additional advantages as signaling mediators since they form covalent adducts allowing the effect of adduction to persist and accumulate over time
Summary
Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1) is a small GTP hydrolyzing (GTPase) protein in the Ras superfamily. Rac is ubiquitously expressed in many cell types, and regulation is both dynamic and contextual. Previous studies have described Rac dysregulation as a contributing factor in an array of different pathologies including cardiovascular disease and cancer [1,2,3,4]. Dysregulation of Rac in endothelial cells (ECs) may be important in disease initiation and progression during cardiovascular disease. Many studies have shown that Rac functions downstream of many cell surface receptors and is a major pathway by which ECs migrate and align in the direction of flow [5]. The mechanisms by which Rac can be dysregulated by vascular oxidative stress, and contribute to vascular injury, are not clear
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