p66Shc Expression Research Articles

Circ-PRKCB acts as a ceRNA to regulate p66Shc-mediated oxidative stress in intestinal ischemia/reperfusion.

Background: Oxidative stress has emerged as an essential factor in the pathogenesis of intestinal ischemia/reperfusion (I/R) injury. The adaptor protein p66Shc is a key regulator of reactive oxygen species (ROS) generation and a mediator of I/R damage in the intestine, but the upstream mechanisms that directly regulate p66Shc expression during intestinal I/R remain largely unknown. Recent studies have suggested that noncoding RNAs, such as circular RNAs (circRNAs), are important players in physiological and pathological processes based on their versatile regulatory roles in gene expression. The aim of this study was to elucidate the contribution of p66Shc to oxidative damage in intestinal I/R and to investigate the regulation of p66Shc by circRNA sponges.Methods: Intestinal I/R was induced in mice via superior mesenteric artery (SMA) occlusion. A miR-339-5p agomir or circ-protein kinase C beta (PRKCB) siRNA was injected intravenously before I/R challenge. In addition, Caco-2 cells were subjected to hypoxia/reoxygenation (H/R) in vitro to simulate an in vivo I/R model.Results: In vitro, p66Shc deficiency significantly reduced H/R-induced ROS overproduction by attenuating mitochondrial superoxide anion (O2-) levels, suppressing NADPH oxidase activity and enhancing antioxidant enzyme expression. Moreover, miR-339-5p was identified to directly regulate p66Shc expression in the intestine. Furthermore, we found that a circRNA transcribed from the PRKCB gene, named circ-PRKCB, acted as an endogenous miR-339-5p sponge to regulate p66Shc expression. circ-PRKCB silencing or miR-339-5p overexpression significantly downregulated p66Shc expression and attenuated oxidative stress levels and I/R injury in vivo and in vitro. Notably, the increased circ-PRKCB levels and decreased miR-339-5p levels associated with murine intestinal I/R were consistent with those in patients with intestinal infarction.Conclusions: Our findings reveal a crucial role for the circ-PRKCB/miR-339-5p/p66Shc signaling pathway in regulating oxidative stress in the I/R intestine. This pathway may be a potential therapeutic target for intestinal I/R injury.

Berberine Ameliorates Doxorubicin-Induced Cardiotoxicity via a SIRT1/p66Shc-Mediated Pathway.

Doxorubicin- (DOX-) induced cardiotoxicity is associated with oxidative stress and cardiomyocyte apoptosis. The adaptor protein p66Shc regulates the cellular redox status and determines cell susceptibility to apoptosis. This study is aimed at investigating the involvement of sirtuin 1- (SIRT1-) mediated p66Shc inhibition in DOX-induced redox signalling and exploring the possible protective mechanisms of berberine (Ber) against DOX-triggered cardiac injury in rats and a cultured H9c2 cell line. Our results showed that the Ber pretreatment markedly increased CAT, SOD, and GSH-PX activities, decreased the levels of MDA, and improved the electrocardiogram and histopathological changes in the myocardium in DOX-treated rats (in vivo). Furthermore, Ber significantly ameliorated the DOX-induced oxidative insult and mitochondrial damage by adjusting the levels of intracellular ROS, ΔΨm, and [Ca2+]m in H9c2 cells (in vitro). Importantly, the Ber pretreatment increased SIRT1 expression following DOX exposure but downregulated p66Shc. Consistent with the results demonstrating the SIRT1-mediated inhibition of p66Shc expression, the Ber pretreatment inhibited DOX-triggered cardiomyocyte apoptosis and mitochondrial dysfunction. After exposing H9c2 cells to DOX, the increased SIRT1 expression induced by Ber was abrogated by a SIRT1-specific inhibitor (EX527) or the use of siRNA against SIRT1. Accordingly, SIRT1 inhibition significantly abrogated the suppression of p66Shc expression and protection of Ber against DOX-induced oxidative stress and apoptosis. These results suggest that Ber protects the heart from DOX injury through SIRT1-mediated p66Shc suppression, offering a novel mechanism responsible for the protection of Ber against DOX-induced cardiomyopathy.

CRIF1 deficiency induced mitophagy via p66shc-regulated ROS in endothelial cells

Inhibition of mitochondrial protein CR6 interacting factor 1 (CRIF1) disturbs mitochondrial function, depolarizes membrane potential, and increases reactive oxygen species (ROS) levels in endothelial cells. Impaired mitochondrial function accompanied by oxidative damage is a major contributor to the initiation of mitophagy. We hypothesized that CRIF1 deficiency-induced harmful effects may promote mitophagy, and explored the mechanism underlying this effect in human umbilical vein endothelial cells (HUVECs). Our results showed that CRIF1 downregulation not only induced the mitophagy-related markers LC3 (LC3-II/Ⅰ), PTEN-induced putative kinase 1 (PINK1) and parkin, but also stimulated redox enzyme p66shc expression. Scavenging mitochondrial ROS markedly blunted the CRIF1 deficiency-induced increase in p66shc expression. In addition, knockdown of p66shc inhibited the CRIF1 deletion-triggered mitochondrial ROS increase, membrane potential depolarization, and mitochondrial fusion. The restoration of mitochondrial dysfunction by p66shc downregulation also decreased CRIF1 deficiency-induced mitophagy, by elevating the levels of LC3-II/Ⅰ, PINK1 and parkin. These findings suggest that CRIF1 deficiency induces mitophagy via p66shc-regulated ROS in endothelial cells.

Perinatal protein restriction with postnatal catch-up growth leads to elevated p66Shc and mitochondrial dysfunction in the adult rat liver.

Epidemiological data suggest an inverse relationship between birth weight and long-term metabolic deficits, which is exacerbated by postnatal catch-up growth. We have previously demonstrated that rat offspring subject to maternal protein restriction (MPR) followed by catch-up growth exhibit impaired hepatic function and ER stress. Given that mitochondrial dysfunction is associated with various metabolic pathologies, we hypothesized that altered expression of p66Shc, a gatekeeper of oxidative stress and mitochondrial function, contributes to the hepatic defects observed in MPR offspring. To test this hypothesis, pregnant Wistar rats were fed a control (20% protein) diet or an isocaloric low protein (8%; LP) diet throughout gestation. Offspring born to control dams received a control diet in postnatal life, while MPR offspring remained on a LP diet (LP1) or received a control diet post weaning (LP2) or at birth (LP3). At four months, LP2 offspring exhibited increased protein abundance of both p66Shc and the cis-trans isomerase PIN1. This was further associated with aberrant markers of oxidative stress (i.e. elevated 4-HNE, SOD1 and SOD2, decreased catalase) and aerobic metabolism (i.e., increased phospho-PDH and LDHa, decreased complex II, citrate synthase and TFAM). We further demonstrated that tunicamycin-induced ER stress in HepG2 cells led to increased p66Shc protein abundance, suggesting that ER stress may underlie the programmed effects observed in vivo. In summary, because these defects are exclusive to adult LP2 offspring, it is possible that a low protein diet during perinatal life, a period of liver plasticity, followed by catch-up growth is detrimental to long-term mitochondrial function.

MiR-124 attenuates doxorubicin-induced cardiac injury via inhibiting p66Shc-mediated oxidative stress

Previous studies showed that miR-124 had a protective role by reducing oxidant stress and preventing cell apoptosis and autophagy. However, its role in doxorubicin-induced cardiomyopathy was less known. In our study, we confirmed increased ROS and decreased expression of miR-124 in doxorubicin-treated heart tissues and primary cardiomyocytes. The oxidative stress and cell apoptosis were alleviated by overexpressing miR-124, characterized by decreased activity of MDA and increased activity of SOD. While inhibiting miR-124 generated opposed effects. Mechanistically, our bioinformatic prediction and luciferase assay confirmed that miR-124 inhibited the expression of p66Shc, a proapoptotic signaling pathway. Our results suggested that miR-124 was hopeful to become a therapeutic target in doxorubicin-related cardiomyopathy.

P66Shc regulates podocyte autophagy in high glucose environment through the Notch-PTEN-PI3K/Akt/mTOR pathway.

Autophagy has been found to be involved in podocyte injury, which is a key factor in the progression of diabetic kidney disease (DKD). p66Shc is an important protein adaptor that regulates production of reactive oxygen species (ROS) and induction of apoptosis, and is a novel biomarker for oxidative damage of renal tubules. Our preliminary studies showed that p66Shc expression in podocytes of DKD patients is increased, while autophagic flux and podocyte number is decreased in DKD patients. The mechanism by which p66Shc may regulate podocyte autophagy and injury remains unknown. The present study aimed to investigate the molecular function of p66Shc under high glucose condition and its possible therapeutic utility in DKD. We histologically evaluated kidney injury in a streptozocin (STZ)-induced mouse model of diabetes using HE, PAS, PASM, and Masson staining and assessed glomerular structure by transmission electron microscopy. The apoptosis rate of high glucose-treated podocytes was assessed by TUNEL and Annexin V/PI staining. Markers of podocyte autophagy were measured by immunofluorescence and western blotting. DHE/ET fluorescence quantification was used for ROS detection and quantification. Urine creatinine, serum creatinine, urinary microalbumin, and p66Shc expression were significantly increased in STZ-induced diabetic mice. Cultured MPC5 podocytes subjected to high glucose showed reduced viability, and p66Shc overexpression further accelerated apoptosis. p66Shc knockdown enhanced HG-induced autophagy, while p66Shc overexpression reduced the expression of PTEN and increased the expression of mTOR and phospho-mTOR. LC3 protein expression was higher in cells with p66Shc knockdown, indicating that activation of p66Shc inhibits podocyte autophagy. DAPT, an inhibitor of the Notch pathway, downregulated the expression of p66Shc. These findings indicate that p66Shc inhibits podocyte autophagy and induces apoptosis through the Notch -PTEN-PI3K/Akt/ mTOR signaling pathway in high glucose environment, providing novel evidence for its potential role in DKD treatment.

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

Downregulation of p66Shc can reduce oxidative stress and apoptosis in oxidative stress model of marginal cells of stria vascularis in Sprague Dawley rats.

Backgroundp66Shc, a Src homologue and collagen homologue (Shc) adaptor protein, mediates oxidative stress signaling. The p66Shc-null mice have increased lifespan and enhanced resistance to oxidative stress. Studies have also indicated its potential role in inner ear aging, which can lead to deafness.ObjectiveThe aim of this study was to determine the effects of p66Shc down-regulation on the marginal cells (MCs) of the inner ear stria vascularis.MethodsPrimary MCs were isolated from neonatal rats and treated with glucose oxidase to induce oxidative stress. The cells were transduced with adenovirus expressing siRNA, and the knockdown was verified by Western blotting. The reactive oxygen species (ROS) levels and apoptosis were analyzed using the DCFH-DA probe and Annexin-V/7-AAD staining respectively. The ultrastructure of the differentially-treated cells was examined by transmission electron microscopy (TEM).Results: The in vitro oxidative stress model was established successfully in rat MCs. Knockdown of p66Shc alleviated the high ROS levels and apoptosis in the glucose oxidase-treated cells. In addition, glucose oxidase significantly increased the number of peroxisomes in the MCs, which was decreased by p66Shc inhibition.ConclusionOxidative stress increases p66Shc levels in the marginal cells of the inner ear, which aggravates ROS production and cellular injury. Blocking p66Shc expression can effectively reduce oxidative stress and protect the MCs.

P66Shc promotes HCC progression in the tumor microenvironment via STAT3 signaling

The development of hepatocellular carcinoma (HCC) is strongly associated with chronic inflammation. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species. However, the importance and biologic functions of p66Shc in HCC are unclear. The clinical significance of p66Shc was assessed in a large cohort of patients with HCC. High Shc1 expression was closely correlated with poor clinical outcomes and early recurrence of HCC. p66Shc expression was also determined in HCC samples and cell lines and found to be increased. Moreover, knockdown of p66Shc significantly inhibited cell proliferation, motility in vitro and tumor growth in vivo and could attenuate the proliferation, and motility of cells stimulated by activated macrophage conditioned media. Mechanically, p66Shc knockdown inhibited phosphorylation of STAT3 on serine 727 in vitro and in vivo. Our results show that high p66Shc expression in HCC predicts a worse prognosis for survival. Furthermore, p66Shc may serve as a novel candidate target for HCC therapy.

Protocatechuic Acid-Mediated miR-219a-5p Activation Inhibits the p66shc Oxidant Pathway to Alleviate Alcoholic Liver Injury.

Alcohol abuse has become common worldwide and has been recognized as a major cause of chronic alcoholic liver disease (ALD). ALD encompasses a complex process that includes a broad scope of hepatic lesions, ranging from steatosis to cirrhosis. In particular, reactive oxygen species (ROS) are mainly involved. Numerous studies have shown that p66shc plays a significant role in ALD. Protocatechuic acid (PCA), a dihydroxybenzoic acid that is naturally found in green tea, vegetables, and fruits, has efficient free radical scavenging effects. In this study, we aimed to assess the protective effect of PCA on ALD and to evaluate the microRNA- (miRNA-) p66shc-mediated reduction of ROS formation in ALD. Our results demonstrated that PCA treatment significantly decreased p66shc expression and downstream ROS formation in ALD. miR-219a-5p, which was identified by bioinformatics and experimental analysis, was enhanced by PCA and subsequently suppressed p66shc expression. Importantly, p66shc played an essential role in the protection of PCA-stimulated miR-219a-5p overexpression. Overall, these findings show that PCA-stimulated miR-219a-5p expression mitigates ALD by reducing p66shc-mediated ROS formation. This study may contribute to the development of therapeutic interventions for ALD.

N160 of Aiolos Determines its DNA-Binding Activity.

Aiolos is a transcription factor of the Ikaros family. It is expressed in lymphocytes and plays a vital role in the development of lymphatic system. Recently, Aiolos has been found to be ectopically expressed in lung cancer cells and promotes cancer metastasis through repression of p66Shc expression. Blocking DNA-binding activity of Aiolos may benefit cancer patients by preventing further metastasis. However, it is not clear which sequence is essential for its DNA binding. In this study, we found that N160 is a key amino acid to determine the DNA-binding activity of Aiolos. Mutation of N160A resulted in loss of peripheral heterochromatin localization and dissociation with its target gene. Accordingly, Aiolos with N160A could not change the expression of its target genes. Thus, we identified the amino acid (N160) that is essential for Aiolos binding to DNA. Anat Rec, 302:2014-2019, 2019. © 2019 American Association for Anatomy.

The p52 isoform of SHC1 is a key driver of breast cancer initiation

BackgroundSHC1 proteins (also called SHCA) exist in three functionally distinct isoforms (p46SHC, p52SHC, and p66SHC) that serve as intracellular adaptors for several key signaling pathways in breast cancer. Despite the broad evidence implicating SHC1 gene products as a central mediator of breast cancer, testing the isoform-specific roles of SHC1 proteins have been inaccessible due to the lack of isoform-specific inhibitors or gene knockout models.MethodsHere, we addressed this issue by generating the first isoform-specific gene knockout models for p52SHC and p66SHC, using germline gene editing in the salt-sensitive rat strain. Compared with the wild-type (WT) rats, we found that genetic ablation of the p52SHC isoform significantly attenuated mammary tumor formation, whereas the p66SHC knockout had no effect. Rats were dosed with 7,12-dimethylbenz(a)anthracene (DMBA) by oral gavage to induce mammary tumors, and progression of tumor development was followed for 15 weeks. At 15 weeks, tumors were excised and analyzed by RNA-seq to determine differences between tumors lacking p66SHC or p52SHC.ResultsCompared with the wild-type (WT) rats, we found that genetic ablation of the p52SHC isoform significantly attenuated mammary tumor formation, whereas the p66SHC knockout had no effect. These data, combined with p52SHC being the predominant isoform that is upregulated in human and rat tumors, provide the first evidence that p52SHC is the oncogenic isoform of Shc1 gene products in breast cancer. Compared with WT tumors, 893 differentially expressed (DE; FDR < 0.05) genes were detected in p52SHC KO tumors compared with only 18 DE genes in the p66SHC KO tumors, further highlighting that p52SHC is the relevant SHC1 isoform in breast cancer. Finally, gene network analysis revealed that p52SHC KO disrupted multiple key pathways that have been previously implicated in breast cancer initiation and progression, including ESR1 and mTORC2/RICTOR.ConclusionCollectively, these data demonstrate the p52SHC isoform is the key driver of DMBA-induced breast cancer while the expression of p66SHC and p46SHC are not enough to compensate.

Involvement of microRNA-34a in Age-Related Susceptibility to Oxidative Stress in ARPE-19 Cells by Targeting the Silent Mating Type Information Regulation 2 Homolog 1/p66shc Pathway: Implications for Age-Related Macular Degeneration.

The aging retinal pigment epithelium and oxidative stress, mediated by reactive oxygen species (ROS) accumulation, have been implicated in the mechanisms of age-related macular degeneration (AMD). The expression level of the adapter protein p66shc, a key protein that regulates cellular oxidative stress, is relatively low under normal conditions because of the effects of silent mating type information regulation 2 homolog 1 (SIRT1) on the binding of fully deacetylated histone H3’ to the p66shc promoter region, thus inhibiting p66shc transcription and expression. The equilibrium between SIRT1 and p66shc is disrupted in the presence of various stresses, including AMD. As a major target gene, SIRT1 is regulated by microRNA-34a (miR-34a), and overexpression of miR-34a results in significant inhibition of post-transcriptional expression of SIRT1. Furthermore, our recent studies demonstrated that miR-34a is significantly upregulated, accompanied by reduced tolerance to oxidative stress in hydrogen peroxide-induced prematurely senescent ARPE-19 cells. Moreover, the expression of SIRT1 is decreased, whereas that of p66shc is increased in these cells. Accordingly, miR-34a may play a key role in age-related susceptibility to oxidative stress in ARPE-19 cells by targeting the SIRT1/p66shc pathway, leading to AMD. In this review article, we discuss the functions of miR-34a in modulating the SIRT1/p66shc pathway in age-related conditions, including AMD.

Inhibition of S-Adenosylhomocysteine Hydrolase Induces Endothelial Dysfunction via Epigenetic Regulation of p66shc-Mediated Oxidative Stress Pathway.

Elevated levels of S-adenosylhomocysteine (SAH), the precursor of homocysteine, are positively associated with the risk of cardiovascular disease and with the development and progression of atherosclerosis. However, the role of SAH in endothelial dysfunction is unclear. Apolipoprotein E-deficient ( apoE-/-) mice received dietary supplementation with the SAH hydrolase (SAHH) inhibitor adenosine dialdehyde or were intravenously injected with a retrovirus expressing SAHH shRNA. These 2 approaches, along with the heterozygous SAHH gene knockout ( SAHH+/-) mouse model, were used to elevate plasma SAH levels and to examine the role of SAH in aortic endothelial dysfunction. The relationship between plasma SAH levels and endothelial dysfunction was also investigated in human patients with coronary artery disease and healthy control subjects. Plasma SAH levels were increased in SAHH+/- mice and in apoE-/- mice after dietary administration of adenosine dialdehyde or intravenous injection with SAHH shRNA. SAHH+/- mice or apoE-/- mice with SAHH inhibition showed impaired endothelium-dependent vascular relaxation and decreased nitric oxide bioavailability after treatment with acetylcholine; this was completely abolished by the administration of the endothelial nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester. Furthermore, SAHH inhibition induced production of reactive oxygen species and p66shc expression in the mouse aorta and human aortic endothelial cells. Antioxidants and p66shc siRNA prevented SAHH inhibition-induced generation of reactive oxygen species and attenuated the impaired endothelial vasomotor responses in high-SAH mice. Moreover, inhibition of SAHH induced hypomethylation in the p66shc gene promoter and inhibited expression of DNA methyltransferase 1. Overexpression of DNA methyltransferase 1, induced by transduction of an adenovirus, was sufficient to abrogate SAHH inhibition-induced upregulation of p66shc expression. Finally, plasma SAH levels were inversely associated with flow-mediated dilation and hypomethylation of the p66shc gene promoter and positively associated with oxidative stress levels in patients with coronary artery disease and healthy control subjects. Our findings indicate that inhibition of SAHH results in elevated plasma SAH levels and induces endothelial dysfunction via epigenetic upregulation of the p66shc-mediated oxidative stress pathway. Our study provides novel molecular insight into mechanisms of SAH-associated endothelial injury that may contribute to the development of atherosclerosis. URL: https://www.clinicaltrials.gov . Unique identifier: NCT03345927.

MiR-412-5p targets Xpo1 to regulate angiogenesis in hemorrhoid tissue

Hemorrhoid is a common and recurrent proctological disease, which is often accompanied by angiogenesis and edema. MicroRNAs in the DLK1-DIO3 imprinted clusters are involved in the development and pathogenesis of mammalian hemorrhoids. Results of the present study indicated multiple, differential expression of DLK1-DIO3 imprinted cluster microRNA between hemorrhoid and normal tissues, where miR-412-5p expression in hemorrhoid tissue was significantly decreased. Fluorescein reporter assays showed that miR-412-5p silenced Xpo1 mRNA expression by targeting its 3′-UTR. Overexpression of miR-412-5p in human umbilical vein endothelial cells (HUVECs) indicated that proliferation, migration and formation of vascular structures in HUVECs were inhibited in vitro. In addition, overexpression of miR-412-5p significantly inhibited Xpo1 expression and promoted upregulation of the p53 protein and its retention in the nucleus. Simultaneously, expression of p66SHC and p16 proteins was activated. In summary, downregulation of endogenous miR-412-5p expression in hemorrhoid vascular endothelial cells leads to high expression of the target gene Xpo1 and translocation of the p53 protein out of the nucleus, rendering it unable to activate p66SHC and p16. This ultimately weakens regulation of the vascular endothelial cell cycle, thereby accelerating the division of hemorrhoid vascular endothelial cells, leading to angiogenesis.

High-intensity interval training modulates retinal microvascular phenotype and DNA methylation of p66Shc gene: a randomized controlled trial (EXAMIN AGE)

Impairments of retinal vessel diameter are associated with major adverse cardiovascular (CV) events. Promoter DNA methylation is a repressor of the mitochondrial adaptor p66Shc gene transcription, a key driver of ageing-induced reactive oxygen species. The study aimed to investigate whether high-intensity interval training (HIIT) affects retinal microvascular phenotype as well as p66Shc expression and oxidative stress in ageing subjects with increased CV risk from the EXAMIN AGE cohort. Eighty-four sedentary subjects (mean age 59.4 ± 7.0 years) with ≥2 CV risk factors were randomized into either a 12-week HIIT or standard physical activity recommendations. Retinal arteriolar and venular diameters were measured by use of a retinal vessel analyser. As a marker of oxidative stress plasma 3-nitrotyrosine (3-NT) level was determined by ELISA. Gene expression of p66Shc and DNA methylation were assessed in mononuclear cells by RT-qPCR and methylated-DNA capture (MethylMiner Enrichment Kit) coupled with qPCR, respectively. High-intensity interval training reduced body mass index, fat mass, low-density lipoprotein and increased muscle mass, as well as maximal oxygen uptake (VO2max). Moreover, HIIT restored microvascular phenotype by inducing retinal arteriolar widening (pre: 175 ± 14 µm vs. post: 181 ± 13 µm, P = 0.001) and venular narrowing (pre: 222 ± 14 µm vs. post: 220 ± 14 µm, P = 0.007). After HIIT, restoration of p66Shc promoter methylation (P = 0.034) reduced p66Shc gene expression (P = 0.037) and, in turn, blunted 3-NT plasma levels (P = 0.002). High-intensity interval training rescues microvascular dysfunction in ageing subjects at increased CV risk. Exercise-induced reprogramming of DNA methylation of p66Shc gene may represent a putative mechanistic link whereby exercise protects against age-related oxidative stress. ClinicalTrials.gov: NCT02796976 (https://clinicaltrials.gov/ct2/show/NCT02796976).

P66Shc deficiency in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia enhances leukemogenesis by altering the chemokine receptor landscape.

The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eμ-TCL1/p66Shc−/− mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eμ-TCL1 mice. Eμ-TCL1/p66Shc−/− mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells’ level of p66Shc expression. p66Shc expression declined with disease progression in Eμ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.

P66Shc Contributes to Liver Fibrosis through the Regulation of Mitochondrial Reactive Oxygen Species.

Background: p66Shc is a redox enzyme that mediates mitochondrial reactive oxygen species (ROS) generation. p66Shc inhibition confers protection against liver injury, however, its functional contribution to liver fibrosis remains unclear. The aim of this study is to explore the involvement of p66Shc in liver fibrosis and underlying mechanism of p66Shc by focusing on mitochondrial ROS.Methods: p66Shc-silenced mice were injected with carbon tetrachloride (CCl4). Primary hepatic stellate cells (HSCs) were performed with p66Shc silencing or overexpression prior to TGF-β1 stimulation.Results: p66Shc expression was progressively elevated in mice with CCl4-induced liver fibrosis, and p66Shc silencing in vivo significantly attenuated fibrosis development, reducing liver damage, oxidative stress and HSC activation, indicated by the decreased α-SMA, CTGF and TIMP1 levels. Furthermore, in primary HSCs, p66Shc-mediated mitochondrial ROS production played a vital role in mitochondrial morphology and cellular metabolism. Knockdown of p66Shc significantly inhibited mitochondrial ROS production and NOD-like receptor protein 3 (NLRP3) inflammasome activation, which were closely associated with HSC activation, indicated by the decreased α-SMA, CTGF and TIMP1 levels. However, p66Shc overexpression exerted the opposite effects, which were suppressed by a specific mitochondrial ROS scavenger (mito-TEMPO). More importantly, p66Shc expression was significantly increased in human with liver fibrosis, accompanied by NLRP3 inflammasome activation.Conclusions: p66Shc is a key regulator of liver fibrosis by mediating mitochondrial ROS production, which triggers NLRP3 inflammasome activation.

Targeting of miR-96-5p by Catalpol Ameliorates Oxidative Stress and Hepatic Steatosis in LDLr-/- Mice via p66shc/Cytochrome C Cascade

Background: Hepatic steatosis and oxidative stress are considered to sequential steps in the development of non-alcoholic fatty liver disease (NAFLD). We previously found the protective effects of catalpol, an iridoid glucoside extracted from the root of Romania glutinosa L. Here, the increase expression of p66shc was found in NAFLD models and catalpol could inhibit p66shc expression ameliorating NAFLD effectively. Then, we investigated p66shc-targeting miRNAs in regulating oxidative stress and hepatic steatosis, and the potential role of p66shc in NAFLD. Methods: An NAFLD animal model established by low density lipoprotein receptor knockout (LDLr-/-) mice fed with high fat diet and cellular model by palmitic acid -treated hepG2 cells were employed to investigate the role of catalpol on NAFLD and the underling mechanism miR-96-5p involve in. With histological analysis, western blot, qPCR and transfection assay, identified miR-96-5p as being strongly differentially expressed between NAFLD and normal individuals and catalpol can ameliorate NAFLD via increasing miR-96-5p expression. Findings: We found that p66Shc, a protein induced by oxidative stress was significantly increased in NAFLD. Results also show that high level of p66shc is correlated with oxidative stress and hepatic steatosis. Moreover, targeting miR-96-5p by catalpol ameliorates oxidative stress and hepatic steatosis in LDLr-/- mice via p66shc/cytochrome C cascade. Interpretation: These data identify a role for miR-96-5p in NAFLD through inhibiting oxidative stress and hepatic steatosis via p66shc/cytochrome C cascade and the role of catalpol involve in. Funding Statement: This work was supported by grants from the Chinese National Natural Science Foundation (No.81273508). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All experiments were approved by the Animal Care and Use Committee of Dalian Medical University, and the experimental procedures were performed in strict accordance with Legislation Regarding the Use and Care of Laboratory Animals of China.

Propofol attenuates high glucose-induced P66shc expression in human umbilical vein endothelial cells through Sirt1.

Perioperative hyperglycemia is a common metabolic disorder in clinic settings. Hyperglycemia leads to endothelial inflammation, endothelial cell apoptosis, and dysfunction, thus resulting in endothelial injury. Propofol (2,6-diisopropylphenol) is a widely used intravenous anesthetic in clinic settings. Our previous study indicated that propofol inhibits mitochondrial reactive oxygen species (ROS) production via down-regulation of phosphatase A2 (PP2A) expression, inhibition of Ser36-p66shc dephosphorylation and mitochondrial translocation, thus improving high glucose-induced endothelial injury. The expression of p66shc was inhibited by propofol in hyperglycemic human umbilical vein endothelial cells (HUVECs). However, the mechanism by which propofol inhibits p66shc expression in hyperglycemic HUVECs is still obscure. In the present study, we mainly examined how propofol inhibited high glucose-induced p66shc expression in HUVECs. Compared with 5 mM glucose treatment, high glucose increased p66shc expression and decreased sirt1 expression, which was inhibited by propofol treatment. Moreover, EX527 (a sirt1 inhibitor) reversed the effect of propofol against high glucose-induced p66shc expression. However, EX527 did not reverse the effects of propofol against high glucose-induced ROS accumulation, endothelial inflammation, and apoptosis. Furthermore, when cells were incubated with propofol, EX527, and FTY720 (a PP2A activator) simultaneously, the effects of propofol against high glucose-induced ROS accumulation, inflammation, and apoptosis were reversed. Our results suggested that propofol inhibited high glucose-induced p66shc expression via upregulation of sirt1 expression in hyperglycemic HUVECs. Moreover, propofol protects against high glucose-mediated ROS accumulation and endothelial injury via both inhibition of p66shc expression and dephosphorylation of Ser36-p66shc.