p66Shc Activation Research Articles

Exploring the Gut-Mitochondrial Axis: p66Shc Adapter Protein and Its Implications for Metabolic Disorders.

This review investigates the multifaceted role of the p66Shc adaptor protein and the gut microbiota in regulating mitochondrial function and oxidative stress, and their collective impact on the pathogenesis of chronic diseases. The study delves into the molecular mechanisms by which p66Shc influences cellular stress responses through Rac1 activation, Forkhead-type transcription factors inactivation, and mitochondria-mediated apoptosis, alongside modulatory effects of gut microbiota-derived metabolites and endotoxins. Employing an integrative approach, the review synthesizes findings from a broad array of studies, including molecular biology techniques and analyses of microbial metabolites' impacts on host cellular pathways. The results underscore a complex interplay between microbial metabolites, p66Shc activation, and mitochondrial dysfunction, highlighting the significance of the gut microbiome in influencing disease outcomes through oxidative stress pathways. Conclusively, the review posits that targeting the gut microbiota-p66Shc-mitochondrial axis could offer novel therapeutic strategies for mitigating the development and progression of metabolic diseases. This underscores the potential of dietary interventions and microbiota modulation in managing oxidative stress and inflammation, pivotal factors in chronic disease etiology.

Abstract 14657: p66Shc Mediates SUMO2-Induced Endothelial Dysfunction and ROS Production

Endothelial dysfunction is one of the earliest changes promoting vascular pathologies. Studies suggest that promoting sumoylation increases endothelial dysfunction and development of atherosclerotic plaque. We showed that overexpression of SUMO2 (one of the sumo isoforms) leads to impaired vasorelaxation. However, the molecular signaling is not well understood.P66Shc is an adaptor protein known to regulate vascular function. Studies in mice suggest that p66Shc impairs endothelial function via promoting oxidative stress. Mice with p66Shc knockout are protected against endothelial dysfunction due to hyperlipidemia, diabetes, and aging. Therefore, we asked if p66Shc mediates endothelial effects of SUMO2. We show that p66Shc is modified by SUMO2 and knockdown of p66Shc in endothelial cells inhibits SUMO2-induced reactive oxygen species production as well as inflammation. To identify the specific residue of p66Shc getting sumoylated, we generated recombinant p66Shc and SUMO2ylated it with a commercially available kit. Using mass spectroscopy, we identified that lysine-81 in CH2 domain of p66Shc is specifically modified by SUMO2. We also show that SUMO2-modificaiton of p66ShcK81 promotes oxidative activation (serine-36 phosphorylation) and mitochondrial translocation of p66Shc which are essential for ROS production. P66Shc non-sumoylatable at lysine 81 (p66ShcK81R) is resistant to SUMO2-induced serine-36 phosphorylation and mitochondrial translocation. To examine physiological significance of p66ShcK81-SUMO2, we generated a mouse model having knock in of p66ShcK81R. Using aortic rings from p66ShcK81R knock in and wild type mice, we show that overexpression of SUMO2 significantly impairs endothelium dependent relaxation in wild type mice aortic rings while aortic rings from p66ShcK81R knock are protected against it. Together, we demonstrate that SUMO2-p66Shc is a novel molecular signaling regulating vascular endothelial function.

Sirtuin 2 deficiency aggravates ageing-induced vascular remodelling in humans and mice.

The mechanisms underlying ageing-induced vascular remodelling remain unclear. This study investigates the role and underlying mechanisms of the cytoplasmic deacetylase sirtuin 2 (SIRT2) in ageing-induced vascular remodelling. Transcriptome and quantitative real-time PCR data were used to analyse sirtuin expression. Young and old wild-type and Sirt2 knockout mice were used to explore vascular function and pathological remodelling. RNA-seq, histochemical staining, and biochemical assays were used to evaluate the effects of Sirt2 knockout on the vascular transcriptome and pathological remodelling and explore the underlying biochemical mechanisms. Among the sirtuins, SIRT2 had the highest levels in human and mouse aortas. Sirtuin 2 activity was reduced in aged aortas, and loss of SIRT2 accelerated vascular ageing. In old mice, SIRT2 deficiency aggravated ageing-induced arterial stiffness and constriction-relaxation dysfunction, accompanied by aortic remodelling (thickened vascular medial layers, breakage of elastin fibres, collagen deposition, and inflammation). Transcriptome and biochemical analyses revealed that the ageing-controlling protein p66Shc and metabolism of mitochondrial reactive oxygen species (mROS) contributed to SIRT2 function in vascular ageing. Sirtuin 2 repressed p66Shc activation and mROS production by deacetylating p66Shc at lysine 81. Elimination of reactive oxygen species by MnTBAP repressed the SIRT2 deficiency-mediated aggravation of vascular remodelling and dysfunction in angiotensin II-challenged and aged mice. The SIRT2 coexpression module in aortas was reduced with ageing across species and was a significant predictor of age-related aortic diseases in humans. The deacetylase SIRT2 is a response to ageing that delays vascular ageing, and the cytoplasm-mitochondria axis (SIRT2-p66Shc-mROS) is important for vascular ageing. Therefore, SIRT2 may serve as a potential therapeutic target for vascular rejuvenation.

Abstract 214: Sumo2 Regulates Oxidative Function Of Adaptor Protein P66shc

SUMOylation is a highly dynamic post-translational modification which regulates protein function and/or stability. It involves conjugation of SUMOs ( S mall U biquitin-Like Mo difier s ) to the lysine residue/s of target proteins mediated via a highly coordinated enzymatic mechanism. Studies have shown that promoting SUMOylation (SUMO2/3) causes vascular endothelial dysfunction and accelerates atherosclerosis. We reported that SUMO2 overexpression in endothelial cells promotes oxidative stress and impairs endothelial function. However, the mechanism is not well understood. Adaptor protein p66Shc is a master regulator of oxidative stress and endothelial function. Genetic deletion of p66Shc protects mice against endothelial dysfunction due to metabolic dysregulation. Prior studies have shown that lysine modification of p66Shc regulates its oxidative function. As SUMOylation primarily occurs on lysine residue, we examined if p66Shc is a target of SUMO2. In this study, we show that p66Shc is a direct target of SUMO2. Using endothelial cells and HEK-293 cells, we show that overexpression of SUMO2 leads to p66Shc SUMO2ylation which is further increased in presence of E2-ligase Ubc9 and reduced by deSUMOylating enzyme SENP1. SUMO2ylation of p66Shc increases oxidative activation (serine-36 phosphorylation) of p66Shc which is essential for reactive oxygen production by p66Shc. Using mass spectroscopic study, we identified that lysine-81 is the only lysine present in CH2 domain which is getting SUMO2ylated. Rendering p66ShcK81 non-sumoylatable (p66ShcK81R) prevented the SUMO2ylation of p66Shc. In endothelial cells, overexpression of p66ShcK81R prevented the SUMO2-induced increase in serine-36 phosphorylation and mitochondrial translocation of p66Shc, which are essential for oxidative function of p66Shc. Collectively, we present a novel molecular mechanism via which SUMO2 regulates the oxidative function of p66Shc and identify a unique post-translational regulation of p66Shc in endothelial cells.

Aberrant activation of p53/p66Shc-mInsc axis increases asymmetric divisions and attenuates proliferation of aged mammary stem cells.

Aging is accompanied by the progressive decline in tissue regenerative capacity and functions of resident stem cells (SCs). Underlying mechanisms, however, remain unclear. Here we show that, during chronological aging, self-renewing mitoses of mammary SCs (MaSCs) are preferentially asymmetric and that their progeny divides less frequently, leading to decreased number of MaSCs and reduced regenerative potential. Underlying mechanisms are investigated in the p66Shc-/- mouse, which exhibits several features of delayed aging, including reduced involution of the mammary gland (MG). p66Shc is a mitochondrial redox sensor that activates a specific p53 transcriptional program, in which the aging-associated p44 isoform of p53 plays a pivotal role. We report here that aged p66Shc-/- MaSCs show increased symmetric divisions, increased proliferation and increased regenerative potential, to an extent reminiscent of young wild-type (WT) MaSCs. Mechanistically, we demonstrate that p66Shc, together with p53: (i) accumulates in the aged MG, (ii) sustains expression of the cellpolarity determinant mInscuteable and, concomitantly, (iii) down-regulates critical cellcycle genes (e.g.,: Cdk1 and Cyclin A). Accordingly, overexpression of p53/p44 increases asymmetric divisions and decreases proliferation of young WT MaSCs in a p66Shc-dependent manner and overexpression of mInsc restores WT-like levels of asymmetric divisions in aged p66Shc-/- MaSCs. Notably, deletion of p66Shc has negligible effects in young MaSCs and MG development. These results demonstrate that MG aging is due to aberrant activation of p66Shc, which induces p53/p44 signaling, leading to failure of symmetric divisions, decreased proliferation and reduced regenerative potential of MaSCs.

Retracted: PKCδ Promotes High Glucose Induced Renal Tubular Oxidative Damage via Regulating Activation and Translocation of p66Shc.

[This retracts the article DOI: 10.1155/2014/746531.].

Exendin-4 inhibits high glucose-induced oxidative stress in retinal pigment epithelial cells by modulating the expression and activation of p66Shc

Purpose Activation of p66Sch, an adaptor protein, is associated with oxidative stress and apoptosis and has been implicated in the pathogenesis of diabetes-induced retinal pigment epithelial cell damage and diabetic retinopathy. Exendin-4 is a glucagon-like protein that protects against diabetic retinopathy, but the mechanism of action is not well understood. This study aimed to investigate whether Exendin-4 could protect against high glucose-induced oxidative stress and apoptosis in the adult human retinal pigment epithelial-19 cell line by modulating levels and activation of p66Shc and to study the underlying mechanisms. Materials and Methods Adult human retinal pigment epithelial-19 cells were cultured under low (5 µM) or high glucose (100 µM) conditions in the presence or absence of Exendin-4 and with or without pre-incubation with Exendin-9-39, a glucagon-like peptide-1 receptor antagonist. Results In a dose-dependent manner, Exendin-4 inhibited high glucose-induced cell death and decreased levels of reactive oxygen species, lactate dehydrogenase release, and single single-stranded DNA. At the most effective concentration (100 µM), Exendin-4 reduced mitochondrial levels of phospho-p66Shc (Ser36), cytoplasmic levels of cleaved caspase-3 and cytochrome-c, and NADPH oxidase levels in high glucose-treated cells. It also increased levels of glutathione and magnesium superoxide dismutase and protein levels of magnesium superoxide dismutase but downregulated total protein levels of protein kinase-β and p66Shc and inhibited c-Jun N-terminal kinase phosphorylation in both low- and high glucose-treated cells. All these Exendin-4 effects, however, were inhibited by Exendin-9-39. Conclusions Exendin-4 protects against high glucose-induced adult human retinal pigment epithelial-19 cell damage by increasing antioxidants, downregulating NADPH, and inhibiting mitochondria-mediated apoptosis, effects that are associated with the inhibition of c-Jun N-terminal kinase and downregulation of protein kinase-β and p66Shc.

Curcumin reverses diabetic nephropathy in streptozotocin-induced diabetes in rats by inhibition of PKCβ/p66Shc axis and activation of FOXO-3a

This study investigated if the nephroprotective effect of Curcumin in streptozotocin-induced type 1 diabetes mellitus (DM) in rats involves downregulation/inhibition of p66Shc and examined the underlying mechanisms. Rats were divided into 4 groups (n = 12/group) as control, control + Curcumin (100 mg/kg), T1DM, and T1DM + Curcumin. Curcumin was administered orally to control or diabetic rats for 12 weeks daily. As compared to diabetic rats, Curcumin didn't affect either plasma glucose or insulin levels but significantly reduced serum levels of urea, blood urea nitrogen, and creatinine, and concurrently reduced albumin/protein urea and increased creatinine clearance. It also prevented the damage in renal tubules and mitochondria, mesangial cell expansion, the thickness of the basement membrane. Mechanistically, Curcumin reduced mRNA and protein levels of collagen I/III and transforming growth factor- β-1 (TGF-β1), reduced inflammatory cytokines levels, improved markers of mitochondrial function, and suppressed the release of cytochrome-c and the activation of caspase-3. In the kidneys of both control and diabetic rats, Curcumin reduced the levels of reactive oxygen species (ROS), increased mRNA levels of manganese superoxide dismutase (MnSOD) and gamma-glutamyl ligase, increased glutathione (GSH) and protein levels of Bcl-2 and MnSOD, and increased the nuclear levels of nuclear factor2 (Nrf2) and FOXO-3a. Besides, Curcumin reduced the nuclear activity of the nuclear factor-kappa B (NF-κB), downregulated protein kinase CβII (PKCβII), NADPH oxidase, and p66Shc, and decreased the activation of p66Shc. In conclusion, Curcumin prevents kidney damage in diabetic rats by activating Nrf2, inhibiting Nf-κB, suppressing NADPH oxidase, and downregulating/inhibiting PKCβII/p66Shc axis.

Cadmium Chloride Induces Memory Deficits and Hippocampal Damage by Activating the JNK/p66Shc/NADPH Oxidase Axis.

This study investigated whether the mechanism underlying the neurotoxic effects of cadmium chloride (CdCl2) in rats involves p66Shc. This study comprised an initial in vivo experiment followed by an in vitro experiment. For the in vivo experiment, male rats were orally administered saline (vehicle) or CdCl2 (0.05 mg/kg) for 30 days. Thereafter, spatial and retention memory of rats were tested and their hippocampi were used for biochemical and molecular analyses. For the in vitro experiment, control or p66Shc-deficient hippocampal cells were treated with CdCl2 (25 µM) in the presence or absence of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. Cadmium chloride impaired the spatial learning and retention memory of rats; depleted levels of glutathione and manganese superoxide dismutase; increased reactive oxygen species (ROS), tumor necrosis factor α, and interleukin 6; and induced nuclear factor kappa B activation. Cadmium chloride also decreased the number of pyramidal cells in the CA1 region and induced severe damage to the mitochondria and endoplasmic reticulum of cells in the hippocampi of rats. Moreover, CdCl2 increased the total unphosphorylated p66Shc, phosphorylated (Ser36) p66Shc, phosphorylated JNK, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, cytochrome c, and cleaved caspase-3. A dose-response increase in cell death, ROS, DNA damage, p66Shc, and NADPH oxidase was also observed in cultured hippocampal cells treated with CdCl2. Of note, all of these biochemical changes were attenuated by silencing p66Shc or inhibiting JNK with SP600125. In conclusion, CdCl2 induces hippocampal ROS generation and apoptosis by promoting the JNK-mediated activation of p66Shc.

Rigosertib-Activated JNK1/2 Eliminate Tumor Cells through p66Shc Activation.

Rigosertib, via reactive oxygen species (ROS), stimulates cJun N-terminal kinases 1/2 (JNK1/2), which inactivate RAS/RAF signaling and thereby inhibit growth and survival of tumor cells. JNK1/2 are not only regulated by ROS—they in turn can also control ROS production. The prooxidant and cell death function of p66Shc requires phosphorylation by JNK1/2. Here, we provide evidence that establishes p66Shc, an oxidoreductase, as a JNK1/2 effector downstream of Rigosertib-induced ROS production, DNA damage, and cell death. This may provide a common pathway for suppression of tumor cell growth by Rigosertib.

Exendin-4 protects the hearts of rats from ischaemia/reperfusion injury by boosting antioxidant levels and inhibition of JNK/p66 Shc/NADPH axis.

Exendin-4, a glucagon-like peptide-1 receptor agonist, was shown to protect against cardiac ischaemia/reperfusion (I/R) injury by suppressing oxidative stress. p66 Shc, a pro-oxidant and an apoptotic protein, is activated in the infarcted left ventricles (LVs) after induction of I/R. This study investigated if the cardiac protective effect of Exendin-4 against I/R injury in rats involves inhibition of p66 Shc and to determine the underlying mechanisms behind this. Adult male rats (n=12/group) were divided into four groups as a sham, a sham+Exendin-4, an I/R, and an I/R+Exendin-4. Exendin-4 was administered to rats 7days before the induction of I/R. Ischaemia was induced by ligating the left anterior descending (LAD) coronary artery for 40minutes followed by reperfusion for 10minutes. The infarct myocardium was used for further analysis. Exendin-4 significantly reduced infarct area (by 62%), preserved LV function and lowered serum levels of LDH and CK-MB in I/R-induced rats. Also, it significantly reduced LV levels of ROS and MDA and protein levels of cytochrome-c and cleaved caspase-3 but significantly increased levels of glutathione (GSH) and manganese superoxide dismutase (MnSOD) in LVs of I/R rats indicating antioxidant and anti-apoptotic effects. Furthermore, it inhibited JNK and p66 Shc activation and downregulated protein levels of p66 Shc and NADPH oxidase with no effect on protein levels/activity of p53 and PKCβII. Of note, Exendin-4 also increased GSH and MnSOD in LVs of control rats. In conclusion, Exendin-4 cardioprotective effect in I/R hearts is mediated mainly by antioxidant effect and inhibition of JNK/P66 Shc/NADPH oxidase.

Resolvin D1 Prevents the Impairment in the Retention Memory and Hippocampal Damage in Rats Fed a Corn Oil-Based High Fat Diet by Upregulation of Nrf2 and Downregulation and Inactivation of p66Shc.

This study investigated the effect of a high-fat diet rich in corn oil (CO-HFD) on the memory retention and hippocampal oxidative stress, inflammation, and apoptosis in rats, and examined if the underlying mechanisms involve modulating Resolvin D1 (RvD1) levels and activation of p66Shc. Also, we tested if co-administration of RvD1 could prevent these neural adverse effects induced by CO-HFD. Adult male Wistar rats were divided into 4 groups (n = 18/each) as control fed standard diet (STD) (3.82kcal/g), STD + RvD1 (0.2µg/Kg, i.p/twice/week), CO-HFD (5.4kcal/g), and CO-HFD + RvD1. All treatments were conducted for 8weeks. With normal fasting glucose levels, CO-HFD induced hyperlipidemia, hyperinsulinemia, increased HOMA-IRI and reduced the rats' memory retention. In parallel, CO-HFD increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), cytoplasmic cytochrome-c, and cleaved caspase-3 and significantly decreased levels of glutathione (GSH), Bcl-2, and manganese superoxide dismutase (MnSOD) in rats' hippocampi. Besides, CO-HFD significantly reduced hippocampal levels of docosahexaenoic acid (DHA) and RvD1, as well as total protein levels of Nrf2 and significantly increased nuclear protein levels of p-NF-κB. Concomitantly, CO-HFD increased hippocampal protein levels of p-JNK, p53, p66Shc, p-p66Shc, and NADPH oxidase. However, without altering plasma and serum levels of glucose, insulin, and lipids, co-administration of RvD1 to CO-HFD completely reversed all these events. It also resulted in similar effects in the STD fed-rats. In conclusion, CO-HFD impairs memory function and induces hippocampal damage by reducing levels of RvD1 and activation of JNK/p53/p66Shc/NADPH oxidase, effects that are prevented by co-administration of RvD1.

Racial Differences in p66shc Activity in Activated Endothelial Cells

BackgroundOxidative stress plays a pivotal role in the development of endothelial dysfunction and the pathophysiology of atherosclerosis. P66shc is an adaptor protein that acts as a redox‐sensitive enzyme involved in mitochondrial reactive oxygen species (ROS) production. Upon endothelial cell activation, protein kinase C‐β2 (PKC‐β2) mediates Ser36 phosphorylation of p66shc, thereby allowing p66shc translocation from the cytosol to the mitochondria. Furthermore, we have shown that African American (AA) endothelial cells display higher levels of inflammation and oxidative stress markers compared to Caucasian American (CA) endothelial cells. Therefore, the purpose of this study was to investigate racial differences in p66shc activity in response to tumor necrosis factor‐α (TNF‐α)‐induced endothelial cell activation.MethodsHuman umbilical vein endothelial cells (HUVECs) from three AA donors and three CA donors were cultured and incubated with TNF‐α (10ng/ml, 4hr) to assess racial differences in p66shc, PKC‐β2 and endothelial nitric oxide synthase (eNOS) protein levels and bioactivity.ResultsEnzymatically active eNOS (p‐eNOSS1177) was significantly lower in AA HUVECs under both basal and TNF‐α incubation conditions (p= 0.01, Fig. 1A). Additionally, p‐Ser36 p66shc/total p66shc was significantly lower in AA HUVECs compared to CA HUVECs (p= 0.04, Fig. 3C). However, there was no group or treatment effect on any on the other variables measured.Conclusionp66shc represents an important molecular pathway that mediates ROS production and plays an integral role in the development of cardiovascular disease. According to our results, racial differences in endothelial function cannot be explained by p66shc. However, future research should focus on assessing cytosolic vs. mitochondrial p66shc levels in AA endothelial cells compared to CA.eNOS protein expression and bioactivity in CA and AA HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐eNOS. B) Densiometric quantification and representative Western blot of eNOS. C) p‐eNOS bioactivity. (* p= .01 vs. CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 1p66shc protein expression and bioactivity in CA and AA HUVECs after 4 hours of incubation with TNF‐α (10ng/ml).A) Densiometric quantification and representative Western blot of p‐p66shc. B) Densiometric quantification and representative Western blot of p66shc. C) p‐p66shc bioactivity. (* p= .04 vs. CA HUVECs, n= 4 independent experiments. Data are shown as mean ± SEM).Figure 2

P2250P66Shc mediates the influence of Sirt-1 on age and obesity related endothelial function

Abstract Background Sirtuins are histone deacetylase downregulated in conditions of excessive calory intake, with important roles in controlling cellular ageing pathways. Among their different isoforms, Sirt-1 is the most studied and experimental models suggest it could contribute to the downregulation of the ageing protein p66Shc in conditions of reduced energy intake, ultimately resulting in a reduced mitochondrial free radical production (mtROS). Purpose We assessed the combined influence of ageing and obesity (a condition of excessive calory intake) on the expression and functional activity of Sirt-1 in humans small resistance arteries, as well as whether the effects of Sirt-1 on endothelial function might depend from the regulation of p66Shc activity and its influence on mtROS. Methods Small resistance arteries were isolated from subcutaneous tissue of obese subjects and healthy controls undergoing bariatric surgery of inguinal hernia repair, respectively. The obese and control groups were further divided into young (<40 years old) and old (>40 years old) subjects. Arteries were mounted on a pressurised myograph and endothelial function was assessed using dose-response curves to acetylcholine before and after incubation with SRT1720 (selective agonist of Sirt-1), L-NAME (eNOS inhibitor), MitoTEMPO (mtROS scavenger) and gp91ds-tat (NADPH oxidase inhibitor), alone or in combination. The expression of Sirt-1 and p66Shc in the vascular wall of obese and control subjects was assessed by qPCR while the ChIP assay was used to assess the binding of Sirt-1 to the promoter region of p66Shc. Results Endothelial function was lower in older than younger and in obese than control groups (p<0.001). Incubation with SRT1720 did not affect endothelial function in young controls, slightly improved endothelial function in young obese (p<0.01) and induced a substantial improvement in endothelial function in both the old control and old obese groups (p<0.001). Pre-incubation with L-NAME suggested that the effects of Sirt-1 were dependent from an improved vascular NO availability. Incubation with gp91ds-tat improved endothelial function in both obese groups and the old control group (p<0.01), but to a lesser extent than SRT1720. Conversely, incubation with MitoTEMPO induced the same improvement of endothelial function than SRT1720 in all study groups and, when vessels were preincubated with MitoTEMPO, the addition of Sirt-1 did not produce further improvement of endothelial function. The expression of Sirt-1 and p66Shc was increased in the vessels from obese subjects compared to the non-obese group (p<0.001), while the ChIP assay revealed a lower binding of Sirt-1 to the promoter region of p66Shc in obese than controls subjects (p<0.001). Conclusions Our results suggest that Sirt-1 has an important role in the obesity and age-related endothelial dysfunction, throughout epigenetic regulation of p66Shc and, thus, controlling the production of mtROS. Acknowledgement/Funding Ministero dell'istruzione, dell'università e della ricerca

P66SHC KNOCK-OUT PREVENTS ALDOSTERONE INDUCED VASCULAR REMODELING AND DYSFUNCTION

Objective: Recent evidences suggested that aldosterone may induce the activation of p66shc in vascular smooth muscle cells. This in vivo study sought to investigate whether aldosterone may induce functional and structural alterations in part through p66shc-dependent manner. Design and method: P66shc−/− mice (14 weeks, 6 per group) and age-matched wild type (WT) control mice were treated or not with aldosterone (300 μg /kg/ day) for 4 weeks. Systolic blood pressure (SBP) was measured by tail-cuff method. Endothelium-dependent and independent relaxations were assessed by concentration-response curves to acetylcholine (1 nM to 100 μM) ± L-NAME (100 μM) and sodium nitroprusside (SNP) (10 nM to 1 mM) respectively, in mesenteric arteries pre-contracted with norepinephrine (10 μM). Media-to-lumen ratio (M/L) and cross sectional area (CSA) were evaluated on pressurized preparations. Results: SBP was similar in WT and p66shc−/− mice before treatment and was significantly increased after 4 weeks of aldosterone treatment in both WT (192.3 ± 4.522 vs 103.3 ± 2.996; p < 0.05) and p66shc−/− mice (176.8 ± 5.313 vs 105.3 ± 1.542; p < 0.05). However the SBP increase was significantly blunted in p66shc−/− mice treated with aldosterone as compared to WT treated mice (176.8 ± 5.313 vs 192.3 ± 4.522, p < 0.05). Acetylcholine-induced vasodilation was similar in WT and p66shc−/− mice and was significantly reduced only in WT after aldosterone treatment. Acetylcholine induced relaxation was blunted by L-NAME in all the groups although at less extent in WT. This suggests that aldosterone may induce endothelial dysfunction only in this group. Endothelium-independent vasodilation was similarly preserved in all the groups. M/L ratio was similar in both WT and p66shc−/− before treatment and was significantly increased by aldosterone only in WT group (p < 0.001). CSA was similar in all the groups. Conclusions: Despite BP increase, aldosterone induced endothelial dysfunction and vascular remodelling only in WT but not in p66shc−/− mice. Thus, p66shc may be involved in the mechanisms of endothelial dysfunction and vascular remodelling induced by aldosterone in the cardiovascular system.

Klotho inhibits PKCα/p66SHC-mediated podocyte injury in diabetic nephropathy

Diabetic nephropathy (DN) is a progressive disease, the main pathogeny of which is podocyte injury. As a calcium-dependent serine/threonine protein kinase involved in podocyte injury, protein kinase C isoform α (PKCα) was reported to regulate the phosphorylation of p66SHC. However, the role of PKCα/p66SHC in DN remains unknown. Klotho, an anti-aging protein with critical roles in protecting kidney, is expressed predominantly in the kidney and secreted in the blood. Nonetheless, the mechanism underlying amelioration of podocyte injury by Klotho in DN remains unclear. Our data showed that Klotho was decreased in STZ-treated mice and was further declined in diabetic KL ± mice. As expected, Klotho deficiency aggravated diabetes-induced proteinuria and podocyte injury, accompanied by the activation of PKCα and p66SHC. In contrast, overexpression of Klotho partially ameliorated PKCα/p66SHC-mediated podocyte injury and proteinuria. In addition, in vitro experiments showed that activation of PKCα and subsequently increased intracellular reactive oxygen species (ROS) was involved in podocytic apoptosis induced by high glucose (HG), which could be partially reversed by Klotho. Hence, we conclude that Klotho might inhibit PKCα/p66SHC-mediated podocyte injury in diabetic nephropathy.

CD36 dependent redoxosomes promotes ceramide-mediated pancreatic β-cell failure via p66Shc activation

Altered metabolism is implicated in the pathogenesis of beta-cell failure in type 2 diabetes (T2D). Plasma and tissue levels of ceramide species play positive roles in inflammatory and oxidative stress responses in T2D. However, oxidative targets and mechanisms underlying ceramide signaling are unclear. We investigated the role of CD36-dependent redoxosome (redox-active endosome), a membrane-based signaling agent, in ceramide-induced beta-cell dysfunction and failure. Exposure of beta cells to C2-ceramide (N-acetyl-sphingosine) induced a CD36-dependent non-receptor tyrosine kinase Src-mediated redoxosome (Vav2-Rac1-NOX) formation. Activated Rac1-GTP-NADPH oxidase complex induced c-Jun-N-terminal kinase (JNK) activation and nuclear factor (NF)-kB transcription, which was associated with thioredoxin-interacting protein (TXNIP) upregulation and thioredoxin activity suppression. Upregulated JNK expression induced p66Shc serine36 phosphorylation and peroxiredoxin-3 hyperoxidation, causing beta-cell apoptosis via mitochondrial dysfunction. CD36 inhibition by sulfo-N-succinimidyl oleate (SSO) or CD36 siRNA blocked C2-ceramide-induced redoxosome activation, thereby decreasing JNK-dependent p66Shc serine36 phosphorylation. CD36 inhibition downregulated TXNIP expression and promoted thioredoxin activity via enhanced thioredoxin reductase activity, which prevented peroxiredoxin-3 oxidation. CD36 inhibition potentiated glucose-stimulated insulin secretion and prevented beta-cell apoptosis. Our results reveal a new role of CD36 during early molecular events that lead to Src-mediated redoxosome activation, which contributes to ceramide-induced pancreatic beta-cell dysfunction and failure.

P66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

A key pathological feature of Alzheimer’s disease (AD) is the accumulation of the neurotoxic amyloid beta (Aβ) peptide within the brains of affected individuals. Previous studies have shown that neuronal cells selected for resistance to Aβ toxicity display a metabolic shift from mitochondrial-dependent oxidative phosphorylation (OXPHOS) to aerobic glycolysis to meet their energy needs. The Src homology/collagen (Shc) adaptor protein p66Shc is a key regulator of mitochondrial function, ROS production and aging. Moreover, increased expression and activation of p66Shc promotes a shift in the cellular metabolic state from aerobic glycolysis to OXPHOS in cancer cells. Here we evaluated the hypothesis that activation of p66Shc in CNS cells promotes both increased OXPHOS and enhanced sensitivity to Aβ toxicity. The effect of altered p66Shc expression on metabolic activity was assessed in rodent HT22 and B12 cell lines of neuronal and glial origin respectively. Overexpression of p66Shc repressed glycolytic enzyme expression and increased both mitochondrial electron transport chain activity and ROS levels in HT22 cells. The opposite effect was observed when endogenous p66Shc expression was knocked down in B12 cells. Moreover, p66Shc activation in both cell lines increased their sensitivity to Aβ toxicity. Our findings indicate that expression and activation of p66Shc renders CNS cells more sensitive to Aβ toxicity by promoting mitochondrial OXPHOS and ROS production while repressing aerobic glycolysis. Thus, p66Shc may represent a potential therapeutically relevant target for the treatment of AD.

PO-217 Tumour suppressor function of the oxidoreductase p66Shc in BRAFV600E-transformed cells

IntroductionElevated levels of reactive oxygen species (ROS) frequently observed in tumours are part of the metabolic reprogramming, which is critical for cancer initiation and progression. However, cancer cells remain sensitive to treatments which further increase or decrease ROS. Tailored modulation of ROS levels thus may become a new strategy in cancer therapy. In contrast to other tumours, melanoma have low ROS levels and we have shown previously that RAF kinases are able to prevent excessive mitochondrial ROS production. To understand the underlying reasons for impaired ROS production in BRAFV600E transformed cells, we studied a possible link to the oxidoreductase p66Shc, a protein that is directly involved in the generation of mitochondrial ROS and frequently overexpressed in tumours.Material and methodsMitochondrial ROS production was analysed in fibroblasts singly transformed by mutant BRAFV600E, the melanoma cell lines A375 and M238 carrying the identical BRAF mutation, and the vemurafenib resistant variant M238R. Mitochondrial ROS production was analysed by microscopic imaging or FACS analysis of MitoTracker Red CM-H2XRos stained cells. Intracellular signalling was monitored through the use of phosphorylation-specific and control antibodies.Results and discussionsWe show that following treatment with the cell death inducing agent phenethyl isothiocyanate (PEITC) oncogenic BRAFV600E renders cells refractory to phosphorylation of S36 in p66Shc, which is essential for p66Shc activation, mitochondrial translocation and ROS production. Consistent with this observation, the activation of JNK1/2, the kinases phosphorylating S36, as demonstrated by us previously, was blunted, while other MAPKs were not affected. Use of a JNK1/2 inhibitor most efficiently prevented ROS production while inhibition of BRAF or MEK increased ROS levels, without affecting S36 phosphorylation. Melanoma cells, which had become resistant to the mutant BRAF-specific inhibitor vemurafenib, were impaired in S36 phosphorylation and ROS production after PEITC treatment. Moreover, they failed to increase ROS levels after MEK/BRAF inhibition. Finally, shRNA-mediated knockdown of p66Shc led to increased colony size of BRAFV600E transformed cells in soft agar. Taken together these data suggest that phoshpo-p66Shc functions as a tumour suppressor in melanoma.ConclusionOur findings demonstrate that interference with cytoplasmic signalling pathways regulating p66Shc activation may be therapeutically exploited to alter ROS levels in tumour cells.

The oxidoreductase p66Shc acts as tumor suppressor in BRAFV600E-transformed cells.

Metabolic reprogramming, as exemplified by the shift from oxidative phosphorylation to glycolysis, is a common feature of transformed cells. In many tumors, altered metabolism is also reflected in increased reactive oxygen species (ROS) levels, which contribute to proliferation and survival signaling. However, despite high ROS levels, cancer cells can be efficiently killed by further increasing ROS production. We have shown previously that both wild‐type and oncogenic CRAF and BRAF prevent excessive mitochondrial ROS production. Subsequently, it has been demonstrated that raising ROS levels in BRAFV600E‐transformed melanoma cells by inhibiting BRAF or MEK rendered them susceptible to cell death induction. To understand how oncogenic BRAF affects mitochondrial ROS production in melanoma, we studied the mitochondrial ROS‐producing oxidoreductase p66Shc, which is frequently overexpressed in tumors. Using NIH 3T3 BRAFV600E fibroblasts and the melanoma cell lines A375 and M238 carrying the same BRAF mutation, we show that under treatment with the ROS‐inducing agent phenethyl isothiocyanate (PEITC), oncogenic BRAF renders cells refractory to p66ShcS36 phosphorylation, which is essential for p66Shc activation and mitochondrial ROS production. Consistent with this, the activation of JNK1/2, which phosphorylate S36, was blunted, while other mitogen‐activated protein kinases were not affected. Inhibition of JNK1/2 efficiently prevented ROS production, while BRAF and MEK inhibitors increased ROS levels. Vemurafenib‐resistant M238R melanoma cells were impaired in S36 phosphorylation and ROS production following PEITC treatment. Moreover, they failed to increase ROS levels after MEK/BRAF inhibition. Finally, shRNA‐mediated knockdown of p66Shc led to increased growth of BRAFV600E‐transformed NIH 3T3 cells in soft agar assay. Taken together, these data suggest that phosphorylation‐activated p66Shc functions as a tumor suppressor in melanoma cells.