High glucose-induced p66Shc mitochondrial translocation regulates autophagy initiation and autophagosome formation in syncytiotrophoblast and extravillous trophoblast.

BACKGROUND: p66Shc, as a redox enzyme, regulates reactive oxygen species (ROS) production in mitochondria and autophagy. However, the mechanisms by which p66Shc affects autophagosome formation are not fully understood. METHODS: p66Shc expression and its location in the trophoblast cells were detected in vivo and in vitro. Small hairpin RNAs or CRISPR/Cas9, RNA sequencing, and confocal laser scanning microscope were used to clarify p66Shc's role in regulating autophagic flux and STING activation. In addition, p66Shc affects mitochondrial-associated endoplasmic reticulum membranes (MAMs) formation were observed by transmission electron microscopy (TEM). Mitochondrial function was evaluated by detected cytoplastic mitochondrial DNA (mtDNA) and mitochondrial membrane potential (MMP). RESULTS: High glucose induces the expression and mitochondrial translocation of p66Shc, which promotes MAMs formation and stimulates PINK1-PRKN-mediated mitophagy. Moreover, mitochondrial localized p66Shc reduces MMP and triggers cytosolic mtDNA release, thus activates cGAS/STING signaling and ultimately leads to enhanced autophagy and cellular senescence. Specially, we found p66Shc is required for the interaction between STING and LC3II, as well as between STING and ATG5, thereby regulates cGAS/STING-mediated autophagy. We also identified hundreds of genes associated several biological processes including aging are co-regulated by p66Shc and ATG5, deletion either of which results in diminished cellular senescence. CONCLUSION: p66Shc is not only implicated in the initiation of autophagy by promoting MAMs formation, but also helps stabilizing active autophagic flux by activating cGAS/STING pathway in trophoblast.

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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.

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.

The longevity protein p66Shc is required for neonatal heart regeneration.

The longevity protein p66Shc is essential for the senescence signaling that is involved in heart regeneration and remodeling. However, the exact role of p66Shc in heart regeneration is unknown. In this study, we found that p66Shc deficiency decreased neonatal mouse cardiomyocyte (CM) proliferation and impeded neonatal heart regeneration after apical resection injury. RNA sequencing and functional verification demonstrated that p66Shc regulated CM proliferation by activating ß-catenin signaling. These findings reveal the critical role of p66Shc in neonatal heart regeneration and provide new insights into senescence signaling in heart regeneration.

P66shc in the spinal cord is an important contributor in complete Freund's adjuvant induced inflammatory pain in mice.

PURPOSE: The aim of this study is to investigate whether p66shc is involved in inflammatory pain and the potential molecular mechanisms of p66shc in inflammatory pain. METHODS: Inflammatory pain model was established by complete Freund's adjuvant (CFA) injection. Paw withdrawal latency (PWL) and paw withdrawal frequency (PWF) was recorded. The expression of spinal p66shc were determined by immunohistochemical staining, immunofluorescence staining. P66shc knockdown was performed by an adeno-associated virus (AAV) vector infusion. NLRP3 inflammasome complexes were determined by Western blot. DHE staining was used to evaluate reactive oxygen species (ROS) generation. RESULTS: P66Shc expression was progressively elevated in spinal cord of inflammatory pain mice, and p66Shc knockdown in vivo significantly attenuated CFA injection triggers hyperalgesia. Furthermore, knockdown of p66Shc significantly inhibited ROS production and NOD-like receptor protein 3 (NLRP3) inflammasome activation, which were reversed by a ROS donor (t-BOOH). However, post-treatment with nigericin, a agonist of NLRP3, reversed AAV-shP66shc analgesic effect. CONCLUSION: Spinal p66shc may facilitate the development of inflammatory pain by promoting the activation of NLRP3 inflammasome through ROS.

P66Shc is increased in peripheral blood mononuclear cells of the patients with obstructive sleep apnea.

Objective: Obstructive sleep apnea (OSA) is characterized by nocturnal intermittent hypoxemia and linked to oxidative stress. Evidence demonstrated that p66Shc plays a key role in regulating oxidative stress. This study aimed to investigate the expression of p66Shc in peripheral blood mononuclear cells (PBMCs) of patients with OSA and the association with polysomnographic parameters. Methods: Fifty-four OSA subjects and 19 no OSA controls were enrolled in this study. All the subjects underwent standard polysomnography. P66Shc mRNA and protein levels in the PBMCs were detected by quantitative real-time polymerase chain reaction and western blotting. Plasma 3-nitrotyrosine (3-NT), oxidized low density lipoprotein (oxLDL), and advanced oxidation protein products (AOPP) were measured by ELISA method. Results: P66Shc mRNA and protein levels in PBMCs were significantly higher in OSA patients than in controls. P66Shc mRNA was positively correlated with plasma 3-NT, oxLDL, AOPP, hypopnea index (AHI), oxygen desaturation index (ODI), percentage of total sleep time with oxygen saturation (SaO2) below 90% (CT90), epworth sleepiness scale (ESS) and lymphocytes; negatively correlated with lowest SaO2 (LSaO2) and mean SaO2 (MSaO2). Further multivariate linear regression analysis showed that p66Shc mRNA levels were independently associated with AHI, MSaO2 and CT90. Conclusions: Oxidative stress regulator p66Shc may play a role in the pathophysiology of OSA and might serve as a potential biomarker for this disease.

SIRT6 reduces the symptoms of premature ovarian failure and alleviates oxidative stress and apoptosis in granulosa cells by degrading p66SHC via H3K9AC.

CONTEXT: Substantial evidence suggests that ovarian oxidative stress can result in severe ovarian dysfunction. OBJECTIVE: The purpose of this article is to investigate the potential of SIRT6 in alleviating premature ovarian failure (POF) by inhibiting oxidative stress. METHODS: To mimic POF, mice were administered daily subcutaneous injections of d-galactose. The levels of E2, FSH, LH, AMH, and progesterone in serum were measured, along with changes in follicles and SIRT6 levels. Mice were treated with the SIRT6 agonist MDL-800, SIRT6 levels, follicles, and aforementioned hormones were reassessed. The effects of MDL-800 on oxidative stress and apoptosis were subsequently identified. Primary granulosa cells were isolated from mice, and the effects of H2O2 and MDL-800 on cell viability, oxidative stress, SIRT6 level, and apoptosis were evaluated. In addition, the regulation of SIRT6 on H3K9AC/p66SHC was verified by examining changes in protein levels, promoter activity, and the reversal effects of p66SHC overexpression. RESULTS: MDL-800 mitigated hormone fluctuations, reduced follicle depletion in ovarian tissue, and attenuated oxidative stress and apoptosis in mice. In vitro experiments demonstrated that MDL-800 enhanced the resilience of primary granulosa cells against H2O2, as evidenced by increased cell viability and reduced oxidative stress and apoptosis. Furthermore, SIRT6 was found to decrease H3K9AC and p66SHC levels, as well as attenuate p66SHC promoter activity. The protective effects of MDL-800 on cells were reversed upon p66SHC overexpression. CONCLUSION: In summary, this study highlights that activation of SIRT6 can alleviate POF and reduce oxidative stress by degrading H3K9AC and suppressing p66Shc levels in granulosa cells.

The p66Shc Redox Protein and the Emerging Complications of Diabetes.

Diabetes mellitus is a chronic metabolic disease, the prevalence of which is constantly increasing worldwide. It is often burdened by disabling comorbidities that reduce the quality and expectancy of life of the affected individuals. The traditional complications of diabetes are generally described as macrovascular complications (e.g., coronary heart disease, peripheral arterial disease, and stroke), and microvascular complications (e.g., diabetic kidney disease, retinopathy, and neuropathy). Recently, due to advances in diabetes management and the increased life expectancy of diabetic patients, a strong correlation between diabetes and other pathological conditions (such as liver diseases, cancer, neurodegenerative diseases, cognitive impairments, and sleep disorders) has emerged. Therefore, these comorbidities have been proposed as emerging complications of diabetes. P66Shc is a redox protein that plays a role in oxidative stress, apoptosis, glucose metabolism, and cellular aging. It can be regulated by various stressful stimuli typical of the diabetic milieu and is involved in various types of organ and tissue damage under diabetic conditions. Although its role in the pathogenesis of diabetes remains controversial, there is strong evidence regarding the involvement of p66Shc in the traditional complications of diabetes. In this review, we will summarize the evidence supporting the role of p66Shc in the pathogenesis of diabetes and its complications, focusing for the first time on the emerging complications of diabetes.

p66Shc signaling does not contribute to tubular damage induced by renal ischemia-reperfusion injury in rat.

Renal ischemia-reperfusion (IR) injury is one of the major causes of acute kidney injury and represents a significant risk factor for renal transplantation. The level of renal damage is influenced by the ischemic duration and is caused by excessive amounts of produced reactive oxygen species (ROS). Adaptor protein p66Shc is known to regulate cellular and organ's sensitivity to oxidative stress and to contribute significantly to mitochondrial production of hydrogen peroxide in stress conditions. Studies carried out in cultured renal cells suggest that p66Shc-mediated mitochondrial dysfunction and ROS production are responsible for renal ischemic injury. We used our genetically modified rats, which either lack p66Shc expression, or express p66Shc variant, which constitutively generates increased quantities of hydrogen peroxide, to evaluate potential contribution of p66Shc signaling to renal damage in ischemia reperfusion rat model. Analysis of outer medulla tubule damage revealed the lack of contribution of either p66Shc expression or its constitutive signaling to IR injury in rat model.

Inhibition of p66Shc attenuates retinal ischemia-reperfusion injury-induced damage by activating the akt pathway.

Retinal ganglion cell (RGC) death is the direct cause of several optic neuropathies. Several studies have reported that the loss of p66Shc ameliorates neuronal injury and vascular abnormalities in ischemia-reperfusion (I/R) injury. However, whether p66Shc is involved in the loss of RGC remains unclear. Therefore, this study aimed to investigate the function of p66Shc due to retinal ischemia in mice. The retinal I/R model was constructed after an intravitreal injection of recombinant adeno-associated viruses (rAAV-EGFP or rAAV-p66Shc-EGFP) for 4 weeks. The expression of p66Shc was detected by western blotting, quantitative real-time polymerase chain reaction, and immunofluorescence staining. The survival of RGCs was determined using immunofluorescence staining. Retinal function was analyzed based on electroretinogram (ERG) findings. Retinal cell apoptosis was detected by TdT-mediated dUTP nick-end labeling staining. The protein expressions of Akt, phospho-Akt, Bax, and PARP were analyzed by western blotting. After rAAVs were successfully transfected, enhanced green fluorescent protein was expressed in all retinal cell layers, and the level of p66Shc after I/R injury was successfully reduced. We found that inhibition of p66Shc expression remarkably decreased the death of RGCs and prevented the loss of ERG a- and b-wave amplitudes caused by retinal ischemia. Mechanistically, downregulation of p66Shc resulted in reduced Bax, whereas increased phospho-Akt and PARP. Taken together, our study revealed that p66Shc acts through the Akt pathway to protect RGCs from retinal I/R injury-induced apoptosis and retinal dysfunction, making p66Shc a possible therapeutic target for glaucoma treatment.

Genistein suppresses ox-LDL-elicited oxidative stress and senescence in HUVECs through the SIRT1-p66shc-Foxo3a pathways.

The anti-senescence function of genistein is related to inhibiting oxidative stress, however, the mechanism has not been clarified. The present study aimed to explore the effects of genistein on oxidized low-density lipoprotein (ox-LDL)-induced endothelial senescence and the role of the sirtuin-1 (SIRT1)-66-kDa Src homology 2 domain-containing protein (p66Shc)-forkhead box protein O3 (Foxo3a) pathways in the process. In this paper, human umbilical vein endothelial cells were pretreated with 1000 nM genistein for 30 min and then incubated with 50 mg/L ox-LDL for another 12 h; meanwhile, the functions of adenovirus-mediated overexpression of p66shc and small interfering RNA-mediated silencing of SIRT1 were investigated. Results showed that genistein pretreatment alleviated ox-LDL-induced mitochondrial reactive oxygen species, the levels of oxidatively modified DNA (8-OHdG) and pai-1, and the activity of SA-ß-gal, which was associated with mitigating p66shc. Further studies indicated the inhibitory effect of genistein on p66shc was correlated with suppressing the acetylation and phosphorylation of p66shc, and ameliorating its mitochondrial translocation by activating SIRT1. Moreover, the inactivated p66shc could enhance the activity of Foxo3a via restraining the phosphorylation and triggering nucleus accumulation. The study demonstrates genistein could prevent ox-LDL-induced mitochondrial oxidative stress and senescence through the SIRT1-p66shc-Foxo3a pathways.

Modulation of p66Shc impairs cerebrovascular myogenic tone in low renin but not low nitric oxide models of systemic hypertension.

Cerebral blood flow and perfusion are tightly maintained through autoregulation despite changes in transmural pressure. Oxidative stress impairs cerebral blood flow, precipitating cerebrovascular events. Phosphorylation of the adaptor protein p66Shc increases mitochondrial-derived oxidative stress. The effect of p66Shc gain or loss of function in nonhypertensive rats is unclear. We hypothesized that p66Shc gain of function would impair autoregulation of cerebral microcirculation under physiological and pathological conditions. Three previously established transgenic [salt-sensitive (SS) background] p66Shc rats were used, p66-Del/SS (express p66Shc with a nine-amino acid deletion), p66Shc-knockout (KO)/SS (frameshift premature termination codon), and p66Shc signaling and knock-in substitution of Ser36Ala (p66Shc-S36A)/SS (substitution of Ser36Ala). The p66Shc-Del were also bred on Sprague-Dawley (SD) backgrounds (p66-Del/SD), and a subset was exposed to a hypertensive stimulus [NG-nitro-l-arginine methyl ester (l-NAME)] for 4 wk. Active and passive diameters to increasing transmural pressure were measured and myogenic tone was calculated in all groups (SS and SD). Myogenic responses to increasing pressure were impaired in p66Shc-Del/SS rats relative to wild-type (WT)/SS and knock-in substitution of Ser36Ala (S36A; P < 0.05). p66-Del/SD rats did not demonstrate changes in active/passive diameters or myogenic tone relative to WT/SD but did demonstrate attenuated passive diameter responses to higher transmural pressure relative to p66-Del/SS. Four weeks of a hypertensive stimulus (l-NAME) did not alter active or passive diameter responses to increasing transmural pressure (P = 0.86-0.99), but increased myogenic responses relative to p66-Del/SD (P < 0.05). Collectively, we demonstrate the functional impact of p66Shc within the cerebral circulation and demonstrate that the genetic background of p66Shc rats largely drives changes in cerebrovascular function.NEW & NOTEWORTHY We demonstrate that the modulation of p66Shc signaling impairs cerebral artery myogenic tone in a low renin model of hypertension. This impairment is dependent upon the genetic background, as modulated p66Shc signaling in Sprague-Dawley rats does not impair cerebral artery myogenic tone.

Epidermal growth factor receptor and integrins meet redox signaling through P66shc and Rac1.

This review examines the concerted role of Epidermal Growth Factor Receptor (EGFR) and integrins in regulating Reactive oxygen species (ROS) production through different signaling pathways. ROS as such are not always deleterious to the cells but they also act as signaling molecules, that regulates numerous indespensible physiological fuctions of life. Many adaptor proteins, particularly Shc and Grb2, are involved in mediating the downstream signaling pathways stimulated by EGFR and integrins. Integrin-induced activation of EGFR and subsequent tyrosine phosphorylation of a class of acceptor sites on EGFR leads to alignment and tyrosine phosphorylation of Shc, PLCγ, the p85 subunit of PI-3 K, and Cbl, followed by activation of the downstream targets Erk and Akt/PKB. Functional interactions between these receptors result in the activation of Rac1 via these adaptor proteins, thereby leading to Reactive Oxygen Species. Both GF and integrin activation can produce oxidants independently, however synergistically there is increased ROS generation, suggesting a mutual cooperation between integrins and GFRs for redox signalling. The ROS produced further promotes feed-forward stimulation of redox signaling events such as MAPK activation and gene expression. This relationship has not been reviewed previously. The literature presented here can have multiple implications, ranging from looking at synergistic effects of integrin and EGFR mediated signaling mechanisms of different proteins to possible therapeutic interventions operated by these two receptors. Furthermore, such mutual redox regulation of crosstalk between EGFR and integrins not only add to the established models of pathological oxidative stress, but also can impart new avenues and opportunities for targeted antioxidant based therapeutics.

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

AIMS: 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. METHODS AND RESULTS: 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). CONCLUSION: 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. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT02796976 (https://clinicaltrials.gov/ct2/show/NCT02796976).

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-ß 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-ß and p66Shc.

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.

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.

Suppression of p66Shc prevents hyperandrogenism-induced ovarian oxidative stress and fibrosis.

BACKGROUND: Rats with hyperandrogen-induced polycystic ovary syndrome (PCOS) have been shown to develop ovarian oxidative stress (OS) and fibrosis. The Sirt1 agonist, resveratrol, can reduce OS through inhibiting p66Shc in other models of OS. METHODS: We created a rat PCOS model with increased OS levels following treatment with one of the two androgens, dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT). The PCOS related features were determined by measurement of malondialdehyde (MDA) and superoxide dismutase (SOD) levels or by examining the reactive oxygen species (ROS) levels using the DCF-DA probe. The potential mechanisms by which p66Shc/Sirt1 mediates ovarian fibrosis were explored by western blotting, quantitative reverse transcription-PCR, immunofluorescence staining, and immunohistochemistry. RESULTS: Hyperandrogen dramatically augmented OS and activation of fibrotic factors in the ovary. Our data demonstrated that treatment with resveratrol enhanced Sirt1 and decreased ovarian OS as well as inhibited phosphorylation of p66Shc both in vivo and in vitro. The treatment suppressed fibrotic factor activation and improved ovarian morphology. Lentivirus- or siRNA-mediated p66Shc knockdown resulted in a dramatic enhancement of Sirt1 expression, down-regulation of ROS and suppression of fibrotic factors in granulosa cells. Moreover, p66Shc overexpression markedly increased the expression of fibrotic factors. Additionally, silencing Sirt1 induced a dramatic increase in p66Shc and enhanced activation of fibrotic factors. CONCLUSIONS: p66Shc may be a direct target of Sirt1 for inducing ROS and thus promoting fibrosis. Further exploration of the mechanisms of p66Shc in both fibrosis and OS may provide novel therapeutic strategies that will facilitate the improvement in PCOS symptoms and reproductive functions.

P66Shc Deletion Ameliorates Oxidative Stress and Cardiac Dysfunction in Pressure Overload-Induced Heart Failure.

OBJECTIVE: p66Shc is a redox enzyme that plays an important role in the response of oxidative stress and the p53-dependent apoptosis. The expression level of p66Shc has a negative correlation with the resistance of oxidative stress in vivo and in vitro. We aim to demonstrate the function of p66Shc in pressure overload-induced heart failure. METHODS AND RESULTS: The pressure overload-induced heart failure was induced in mice by transverse aortic constriction (TAC). Cardiac dysfunction was shown by transthoracic echocardiography. Western blot was used to check the protein levels of phosphodiesterase type 5 (PDE5) and ventricular oxidative stress markers. Superoxide dismutase (SOD) mimetic M40401 and PDE5 inhibitor sildenafil were used in the treatment of mice. The deletion of p66Shc attenuated cardiac dysfunction and oxidative stress in pressure overload-induced heart failure. p66Shc deletion also decreased the expression of ventricular oxidative stress markers and enhanced PKG signaling by promoting the expression of PDE5. M40401 and sildenafil attenuated the TAC-induced cardiac dysfunction and oxidative stress in p66Shc overexpression mice. CONCLUSIONS: Our findings suggest that p66Shc participates in the regulation of cardiac dysfunction and oxidative stress in TAC-derived pressure overload-induced heart failure in mice, and SOD and PDE5 are molecules downstream of p66Shc in this regulatory process.

LncRNA Mical2/miR-203a-3p sponge participates in epithelial-mesenchymal transition by targeting p66Shc in liver fibrosis.

Epithelial-mesenchymal transition (EMT) is regulated by reactive oxygen species (ROS) in liver fibrosis. p66Shc is a redox enzyme, but its role of EMT is unclear in liver fibrosis. Long noncoding RNAs (lncRNAs) have been implicated as important regulators in numerous physiological and pathological processes and generally acting as a microRNA (miRNA) sponge to regulate gene expression. The aim of the current study was to evaluate the contribution of p66Shc to EMT in liver fibrosis and the regulation of p66Shc by lncRNA sponge. In vivo, p66Shc silencing prevented carbon tetrachloride (CCl4)-induced EMT as evidenced by the upregulation of E-cadherin, downregulation of Vimentin and N-cadherin, and inhibition of oxidative stress and extracellular matrix (ECM) components. Moreover, in vitro, TGF-ß1 significantly enhanced ECM components, as well as the development of the EMT phenotype. These effects were abrogated by p66Shc downregulation and aggravated by p66Shc overexpression. Mechanistically, p66Shc contributed to EMT via mediating ROS, as evidenced by p66Shc downregulation inhibiting EMT under exogenous hydrogen peroxide (H2O2) stimulation. Furthermore, we found that molecule interacting with CasL2 (Mical2) lncRNA functioned as an endogenous miR-203a-3p sponge to regulate p66Shc expression. Both Mical2 silencing and miR-203a-3p agomiR treatment downregulated p66Shc expression, thus suppressing EMT in vivo and in vitro. Notably, the increased p66Shc and Mical2 levels and decreased miR-203a-3p levels in murine fibrosis were consistent with those in patients with liver fibrosis. In sum, our study reveals that p66Shc is critical for liver fibrosis and that Mical2, miR-203a-3p and p66Shc compose a novel regulatory pathway in liver fibrosis.