CRIF1 Deficiency Induces p66shc-Mediated Oxidative Stress and Endothelial Activation

Abstract

Mitochondrial dysfunction has been implicated in the pathophysiology of various cardiovascular diseases. CRIF1 is a protein present in the mitochondria associated with large mitoribosomal subunits, and CRIF1 knockdown induces mitochondrial dysfunction and promotes ROS production. p66shc is a redox enzyme implicated in mitochondrial ROS generation and translation of oxidative signals and, therefore, is a key factor for oxidative stress in endothelial cells. In this study, we investigated whether mitochondrial dysfunction induced by CRIF1 knockdown induces p66shc stimulation and plays any role in mitochondrial dysfunction-induced endothelial activation. Knockdown of CRIF1 decreased the expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, III and IV, leading to increased mitochondrial ROS (mtROS) and hyperpolarization of the mitochondrial membrane potential. Knockdown of CRIF1 also stimulated phosphorylation of p66shc and increased cytosolic ROS in endothelial cells. Furthermore, the expression of vascular cell adhesion molecule-1 and endoplasmic reticulum stress proteins were increased upon CRIF1 knockdown in endothelial cells. However, p66shc knockdown blunted the alteration in mitochondrial dynamics and ROS production in CRIF1 knockdown endothelial cells. In addition, p66shc knockdown reduced the CRIF1 knockdown-induced increases in adhesion between monocytes and endothelial cells. Taken together, these results suggest that CRIF1 knockdown partially induces endothelial activation via increased ROS production and phosphorylation of p66shc.

CR6-interacting factor 1 deficiency reduces endothelial nitric oxide synthase activity by inhibiting biosynthesis of tetrahydrobiopterin

“…Furthermore, some studies showed that BH4 is susceptible to oxidation by reactive radicals, and increased ROS is associated with a deficiency in total biopterin and BH4 in mitochondria 37 . We have previously shown that CRIF1 deletion reduced the synthesis of OXPHOS and triggered severe ROS production in endothelial cells 17,18 . To further reveal whether CRIF1 knockdown induced ROS or whether CRIF1 itself impeded BH4 synthesis, we used the MitoTEMPO to remove mitochondrial ROS or free-radical scavenger N-acetyl-L-cysteine (NAC) to eliminate both cytosolic and mitochondrial ROS, and then detected changes in the levels of BH4 and NO.…”

“…Therefore, a lack of CRIF1 is a major factor underlying misfolded mitochondrial OXPOS subunits. This deficiency leads to a production of excessive mitochondrial ROS in vascular endothelial cells which stimulates endothelial dysfunction 18 . Furthermore, CRIF1-deficiency-induced mitochondrial dysfunction stimulates impaired vascular function via the inactivation of eNOS and decreased NO production 19 .…”

Differential contribution of the mitochondrial translation pathway to the survival of diffuse large B-cell lymphoma subsets

“…34 However, our observations are consistent with studies in other cell types that reported increased mitochondrial superoxide content following knockdown of mitochondrial translation factors and examination of MitoSOX Red and DHR123 probe intensities, which specifically measure mitochondrial ROS. 29,45 The basis of the difference between tigecycline's effect on ROS content in DLBCL and AML cells is unclear. It is also possible that tigecycline may invoke cell context-specific mechanisms that contribute to its toxicity.…”

<p>p66shc siRNA Nanoparticles Ameliorate Chondrocytic Mitochondrial Dysfunction in Osteoarthritis</p>

“…Recombinant adenovirus encoding shRNA targeting p66shc was generated using the AdEasy system, as described previously. 17 Primary chondrocytes were infected with adenovirus at a multiplicity of infection (MOI) of 100 in 6-well culture plates. After 48 h, mitochondrial ROS levels were determined by mitoSOX staining (Molecular probes, M36008).…”

“…13 p66shc has been associated with oxidative stress in kidney, cardiovascular, and lung disease. [16][17][18][19] However, the role played by p66shc during articular cartilage degeneration has not been investigated although chondrocytes exhibit reduced mitochondrial membrane potential. 20 Meanwhile, siRNA-loaded nanoparticles (NPs) for drug delivery in various diseases has been in the spotlight recently.…”