Cyclophilin A Is Required for Angiotensin II–Induced p47phox Translocation to Caveolae in Vascular Smooth Muscle Cells

Abstract

Objective Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl prolyl cis-trans isomerase (PPIase) activity, scaffold function and, significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane. Approach and Results Overexpression of CyPA in CyPA deficient VSMC (CyPA−/−VSMC) significantly increased AngII-stimulated ROS production. NADPH oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox overexpressing CyPA−/−VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA−/−VSMC compared to WT-VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting for p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA−/−VSMC. Mechanistically, inhibition of p47phox phosphorylation and PX domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-PPIase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC suggesting that PPIase activity was required for their interaction. Conclusions These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton which enhances the translocation of the p47phox to the caveolae.

Extracellular Cyclophilin A, Especially Acetylated, Causes Pulmonary Hypertension by Stimulating Endothelial Apoptosis, Redox Stress, and Inflammation

“…11 Our lab has identified CypA as a secreted oxidative stress induced factor that also participates in the generation of oxidative stress through autocrine and paracrine mechanisms. 12–15 CypA may also play a role in endothelial stress signaling in which low concentrations protect EC from apoptosis while high concentrations promote apoptosis 16 . In large blood vessels, VSMC are thought to be the primary source of secreted CypA.…”

RhoA/Rho-Kinase in the Cardiovascular System

“…Indeed, CyPA may be a key mediator of Rho-kinase that generates a vicious cycle of ROS augmentation, affecting EC, VSMC, and inflammatory cells (Figure 4). 143 Importantly, CyPA plays a crucial role in the translocation of Nox enzymes, such as p47phox, 154 contributing to VSMC proliferation and vascular diseases. 117 Because ROS production by Nox enzymes activates other oxidase systems, CyPA and Nox enzymes amplify ROS formation in a synergistic manner, leading to augmentation of oxidative stress.…”

Cyclophilin A in Cardiovascular Homeostasis and Diseases

“…In addition, intracellular CyPA has a variety of other functions, including intracellular trafficking, signal transduction, and transcriptional regulation (Zhu et al 2007). Importantly, intracellular CyPA plays a crucial role in the translocation of Nox enzymes such as p47phox (Soe et al 2013), which are known to contribute to VSMC proliferation (Lassegue et al 2012). Since ROS production by Nox enzymes activates other oxidase systems, CyPA and Nox enzymes amplify ROS formation in a synergistic manner, leading to increased oxidative stress.…”