Downregulation Of p66Shc Research Articles

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.

Low P66shc with High SerpinB3 Levels Favors Necroptosis and Better Survival in Hepatocellular Carcinoma.

Simple SummaryCell proliferation and escape from apoptosis are important pathological features of hepatocellular carcinoma, one of the tumors with the highest mortality rate worldwide. The aim of the study was to evaluate the expression of the pro-apoptotic p66shc and the anti-apoptotic SerpinB3 molecules in relation to clinical outcome in patients with hepatocellular carcinoma and to evaluate their effect on cell fate and tumor growth. In patients with hepatocellular carcinoma the best survival was observed in the subgroup with p66shc levels below median values and SerpinB3 levels above median values. Mice p66shc knockout showed high levels of SerpinB3, while in hepatoma cells overexpressing SerpinB3, p66shc expression was trivial. Hepatoma cells overexpressing SerpinB3 were more prone to die after oxidizing treatments. These cells injected in nude mice developed tumors five times smaller than those from controls. Tumors originating from hepatoma cells overexpressing SerpinB3 showed typical features of necroptosis. In conclusion, in patients affected by hepatocellular carcinoma, the pattern characterized by p66shc downregulation and elevated SerpinB3 levels was associated with markedly better survival. This pattern favored necroptosis in experimental high-stress conditions.Cell proliferation and escape from apoptosis are important pathological features of hepatocellular carcinoma (HCC), one of the tumors with the highest mortality rate worldwide. The aim of the study was to evaluate the expression of the pro-apoptotic p66shc and the anti-apoptotic SerpinB3 in HCCs in relation to clinical outcome, cell fate and tumor growth. p66shc and SerpinB3 were evaluated in 67 HCC specimens and the results were correlated with overall survival. Proliferation and cell death markers were analyzed in hepatoma cells overexpressing SerpinB3, under different stress conditions. p66shc−/− mice and xenograft models were also used to assess the effects of p66shc and SerpinB3 on tumor growth. In patients with HCC, the best survival was observed in the subgroup with p66shc levels below median values and SerpinB3 levels above median values. Mice p66shc−/− showed high levels of SerpinB3, while in HepG2 cells overexpressing SerpinB3, p66shc expression was trivial. HepG2 overexpressing SerpinB3 cells were more prone to die after oxidizing treatments, such as diamide or high concentration H2O2. These cells injected in nude mice developed tumors five times smaller than those from control HepG2 cells. Tumors originating from HepG2 overexpressing SerpinB3 cells showed decreased activated Caspase-8, with concomitant increase of RIP3K and decreased levels of cleaved RIP3K, typical features of necroptosis. In conclusion, in patients affected by HCC, the pattern characterized by p66shc downregulation and elevated SerpinB3 levels was associated with markedly better survival. This pattern favored necroptosis in experimental high-stress conditions.

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-β1 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.

P66shc siRNA Nanoparticles Ameliorate Chondrocytic Mitochondrial Dysfunction in Osteoarthritis.

BackgroundOsteoarthritis (OA) is the most common type of joint disease associated with cartilage breakdown. However, the role played by mitochondrial dysfunction in OA remains inadequately understood. Therefore, we investigated the role played by p66shc during oxidative damage and mitochondrial dysfunction in OA and the effects of p66shc downregulation on OA progression.MethodsMonosodium iodoacetate (MIA), which is commonly used to generate OA animal models, inhibits glycolysis and biosynthetic processes in chondrocytes, eventually causing cell death. To observe the effects of MIA and poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles, histological analysis, immunohistochemistry, micro-CT, mechanical paw withdrawal thresholds, quantitative PCR, and measurement of oxygen consumption rate and extracellular acidification rate were conducted.Resultsp-p66shc was highly expressed in cartilage from OA patients and rats with MIA-induced OA. MIA caused mitochondrial dysfunction and reactive oxygen species (ROS) production, and the inhibition of p66shc phosphorylation attenuated MIA-induced ROS production in human chondrocytes. Inhibition of p66shc by PLGA-based nanoparticles-delivered siRNA ameliorated pain behavior, cartilage damage, and inflammatory cytokine production in the knee joints of MIA-induced OA rats.Conclusionp66shc is involved in cartilage degeneration in OA. By delivering p66shc-siRNA-loaded nanoparticles into the knee joints with OA, mitochondrial dysfunction-induced cartilage damage can be significantly decreased. Thus, p66shc siRNA PLGA nanoparticles may be a promising option for the treatment of OA.

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.

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.

Curcumin ameliorates alveolar epithelial injury in a rat model of chronic obstructive pulmonary disease

AimsTo investigate the effects of curcumin on alveolar epithelial injury in a rat model of chronic obstructive pulmonary disease (COPD) and its potential mechanism. Main methodsThe rat COPD model was established by cigarette smoke exposure combined with intratracheal administration of lipopolysaccharide. Thirty-eight male Sprague-Dawley rats were randomly divided into four groups: control, COPD model, COPD with curcumin and COPD with solvent groups. Neutrophil and macrophage infiltration in bronchoalveolar lavage fluid (BALF) was evaluated, and the levels of IL-6, IL-8 and TNF-α in BALF and serum were determined by ELISA. Histopathological examination and TUNEL staining were used to assess the alveolar epithelial injury. The protein expression of p66Shc and p-p66Shc in the lung tissues was determined by immunohistochemistry and western blot. Key findingsCurcumin significantly decreased the numbers of total cells, neutrophils and macrophages in BALF from COPD rats. In addition, the levels of IL-6, IL-8 and TNF-α in BALF and serum of COPD rats were significantly decreased after treatment with curcumin. Moreover, curcumin ameliorated emphysema and ultrastructural damage of alveolar epithelial cells in COPD rats. The apoptosis index of alveolar epithelial cells in the COPD with curcumin group was significantly lower than that in the COPD model group. Furthermore, the protein expression of p66Shc and p-p66Shc in alveolar epithelia was significantly decreased in the COPD with curcumin group compared with COPD model group. SignificanceCurcumin attenuates alveolar epithelial injury in COPD rats, which may be partially due to the down-regulation of p66Shc.

Ergothioneine oxidation in the protection against high-glucose induced endothelial senescence: Involvement of SIRT1 and SIRT6

Ergothioneine (Egt), the betaine of 2-mercapto-L-histidine, is a dietary antioxidant protecting against many diseases, including cardiovascular disease (CVD), through a redox mechanism different from alkylthiols. Here, experiments were designed to evaluate the mechanisms underlying the beneficial effect of Egt against hyperglycaemia-induced senescence in endothelial cells. To this end, cells were incubated with increasing concentrations of Egt (0.01–1.00mM) for 12h followed by incubation for 48h with high-glucose (25mM). Cell evaluation indicated that viability was not affected by mM concentrations of Egt and that the high-glucose cytotoxicity was prevented with the highest efficacy at 0.5mM Egt. The cytoprotective effect of Egt was paralleled by reduced ROS production, cell senescence, and, interestingly, the formation of hercynine (EH), a betaine we recently found to be produced during the Egt oxidation pathway. Notably, the Egt beneficial effect was exerted through the upregulation of sirtuin 1 (SIRT1) and sirtuin 6 (SIRT6) expression and the downregulation of p66Shc and NF-κB. SIRT1 activity inhibition and SIRT6 gene silencing by small interfering RNA abolished the protective effect of Egt against the high-glucose-induced endothelial senescence. These data provide the first evidence of the Egt ability to interfere with endothelial senescence linked to hyperglycaemia through the regulation of SIRT1 and SIRT6 signaling, thus further strengthening the already assessed role of these two histone deacetylases in type 2 diabetes.

Small molecule compounds alleviate anisomycin-induced oxidative stress injury in SH-SY5Y cells via downregulation of p66shc and Aβ1-42 expression.

Oxidative stress and ageing are important factors contributing to the pathogenesis of Alzheimer's disease (AD), which is associated with neuronal damage and β-amyloid (Aβ) deposition. The p66shc adaptor protein is important for the regulation of oxidative stress and ageing. In the present study, SH-SY5Y human neuroblastoma cells were treated with anisomycin in order to establish a cell model of oxidative stress-induced neuronal damage. The results from quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and western blotting demonstrated that anisomycin was able to stimulate the secretion of Aβ1-42 from SH-SY5Y cells and upregulate the expression levels of p66shc, which was associated with concomitant damage to SH-SY5Y cells. In addition, the protective effects of various small molecule compounds with antioxidant properties against neuronal damage were evaluated. Notably, treatment of SH-SY5Y cells with gallic acid was associated with significant downregulation of p66shc protein expression levels, reduced anisomycin-induced secretion of Aβ1-42, and increased superoxide dismutase activity and acetylcholine secretion levels. The results of the present study suggested that anisomycin is able to promote oxidative neuronal damage by inducing the secretion of Aβ1-42 from neurons and increasing the neuronal expression of p66shc, and this damage may be attenuated by treatment with gallic acid. Therefore, gallic acid and similar small molecule compounds may be considered for the alleviation of neuronal oxidative stress injury in patients with AD.

The mitochondrial reactive oxygen species regulator p66Shc controls PDGF-induced signaling and migration through protein tyrosine phosphatase oxidation

Growth factor receptors induce a transient increase in reactive oxygen species (ROS) levels upon receptor binding to promote signaling through oxidation of protein tyrosine phosphatases (PTPs). Most studies have focused on NADPH oxidases as the dominant source of ROS to induce PTP oxidation. A potential additional regulator of growth factor-induced PTP oxidation is p66Shc, which stimulates mitochondrial ROS production. This study explores the contribution of p66Shc-induced ROS to PTP oxidation and growth factor receptor-induced signaling and migration through analyses of p66Shc-KO fibroblasts and cells with siRNA-mediated p66Shc downregulation. Analyses of PDGFβR phosphorylation in two independent cell systems demonstrated a decrease in PDGFβR phosphorylation after p66Shc deletion or downregulation, which occurred in a partially site-selective and antioxidant-sensitive manner. Deletion of p66Shc also reduced PDGF-induced activation of downstream signaling of Erk, Akt, PLCγ-1, and FAK. Importantly, reduced levels of p66Shc led to decreased oxidation of DEP1, PTP1B, and SHP2 after PDGF stimulation. The cell biological relevance of these findings was indicated by demonstration of a significantly reduced migratory response in PDGF-stimulated p66Shc-KO fibroblasts, consistent with reduced PDGFβR-Y1021 and PLCγ-1 phosphorylation. Downregulation of p66Shc also reduced EGFR phosphorylation and signaling, indicating that the positive role of p66Shc in receptor tyrosine kinase signaling is potentially general. Moreover, downregulation of the mitochondrial hydrogen peroxide scavenger peroxiredoxin 3 increased PDGFβR phosphorylation, showing that mitochondrial ROS in general promote PDGFβR signaling. This study thus identifies a previously unrecognized role for p66Shc in the regulation of PTP oxidation controlling growth factor-induced signaling and migration. In more general terms, the study indicates a regulatory role for mitochondrial-derived ROS in the control of PTP oxidation influencing growth factor signaling.

Salvianolic acid A preconditioning confers protection against concanavalin A-induced liver injury through SIRT1-mediated repression of p66shc in mice

Salvianolic acid A (SalA) is a phenolic carboxylic acid derivative extracted from Salvia miltiorrhiza. It has many biological and pharmaceutical activities. The purpose of this study was to investigate the effect of SalA on concanavalin A (ConA)-induced acute hepatic injury in Kunming mice and to explore the role of SIRT1 in such an effect. The results showed that in vivo pretreatment with SalA significantly reduced ConA-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and decreased levels of the hepatotoxic cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Moreover, the SalA pretreatment ameliorated the increases in NF-κB and in cleaved caspase-3 caused by ConA exposure. Whereas, the pretreatment completely reversed expression of the B-cell lymphoma-extra large (Bcl-xL). More importantly, the SalA pretreatment significantly increased the expression of SIRT1, a NAD+-dependent deacetylase, which was known to attenuate acute hypoxia damage and metabolic liver diseases. In our study, the increase in SIRT1 was closely associated with down-regulation of the p66 isoform (p66shc) of growth factor adapter Shc at both protein and mRNA levels. In HepG2 cell culture, SalA pretreatment increased SIRT1 expression in a time and dose-dependent manner and such an increase was abrogated by siRNA knockdown of SIRT1. Additionally, inhibition of SIRT1 significantly reversed the decreased expression of p66shc, and attenuated SalA-induced p66shc down-regulation. Collectively, the present study indicated that SalA may be a potent activator of SIRT and that SalA can alleviate ConA-induced hepatitis through SIRT1-mediated repression of the p66shc pathway.

Aberrant expression of the transcriptional factor Twist1 promotes invasiveness in ALK-positive anaplastic large cell lymphoma

The transcriptional factor Twist1 has been shown to play a key role in regulating epithelial mesenchymal transition, invasiveness and migratory properties in solid tumors. We found that Twist1 is aberrantly expressed in ALK-positive anaplastic large cell lymphoma (ALK+ALCL), a type of T-cell lymphoid malignancy. Using RT-PCR and Western blots, Twist1 was detectable in all 3 ALK+ALCL cell lines examined but absent in normal T-cells. By immunohistochemistry, Twist1 was detectable in all 10 cases of ALK+ALCL examined; benign lymphoid tissues were consistently negative. Twist1 expression in ALK+ALCL cells can be attributed to the NPM-ALK/STAT3 signaling axis, the key oncogenic driving force in this tumor type. Twist1 is biologically important in ALK+ALCL cells, as Twist1 knockdown resulted in a significant decrease in their invasiveness in an in-vitro assay. Further investigation revealed that this increase in invasiveness is linked to the activation of AKT and down-regulation of p66Shc, two signaling proteins known to be involved in NPM-ALK-mediated oncogenesis. Lastly, knockdown of Twist1 sensitizes ALK+ALCL cells to the growth inhibitory effect of PF-2341066 (Crizotinib®), an ALK inhibitor being used in clinical trials. In conclusion, Twist1 expression, owing to the abnormal NPM-ALK/STAT3 signaling, contributes to its invasiveness and decreased sensitivity to PF-2341066 in ALK+ALCL.

Genistein protects against UVB-induced senescence-like characteristics in human dermal fibroblast by p66Shc down-regulation

Genistein, as an active compound of dietary antioxidants, has shown considerable promise as an effective agent against aging process. However, the effect of genistein on skin photoaging and the associated mechanism remain unclear. To delineate the effect of genistein on UVB-induced senescence in human dermal fibroblasts (HDFs) with emphasis on the mechanism of oxidative pathway regulated by p66Shc involved in the events. HDFs were induced to premature senescence by repetitive subcytotoxic doses of UVB irradiation. Cellular apoptosis and DNA cell cycle were analyzed using flow cytometry. Intracellular levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by ELISA. Mutation levels of two large deletions of mitochondrial DNA, 4977bp and 3895bp deletion, were determined by quantitative PCR. Western blot was applied to detect the expression and activation of p66Shc (the 66-kilodalton isoform of the growth factor adapter Shc) and FKHRL1 (a forkhead protein that is intimately linked with intracellular oxidation). Strong activity of senescence-associated beta-galactosidase (SA-beta-gal), high percent of cell apoptosis as well as cell cycle arrest in G0/G1 phase, and increased intracellular oxidative stress were observed in HDFs irradiated by UVB. Genistein exerted dramatically protective effects on HDFs in a dose-dependent manner. Elevated copy numbers of large deletions in mitochondrial DNA were also inhibited by genistein. Down-regulation of total and phosphorylated p66Shc on Ser36, as well as FKHRL1 and its phosphorylation on Thr32, were observed after genistein treatment. The results indicate that genistein protects UVB-induced senescence-like characteristics in HDFs via maintenance of antioxidant enzyme activities and modulation of mitochondrial oxidative stress through down-regulation of a p66Shc-dependent signaling pathway, which may provide potential prevention against skin aging and even photoaging.

P66Shc Links α 1 -Adrenergic Receptors to a Reactive Oxygen Species-Dependent AKT-FOXO3A Phosphorylation Pathway in Cardiomyocytes

p66Shc is an adapter protein that is induced by hypertrophic stimuli and has been implicated as a major regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. This study implicates p66Shc in an alpha(1)-adrenergtic receptor (alpha(1)-AR) pathway that requires the cooperative effects of protein kinase (PK)Cepsilon and PKCdelta and leads to AKT-FOXO3a phosphorylation in cardiomyocytes. alpha(1)-ARs promote p66Shc-YY(239/240) phosphorylation via a ROS-dependent mechanism that is localized to caveolae and requires epidermal growth factor receptor (EGFR) and PKCepsilon activity. alpha(1)-ARs also increase p66Shc-S(36) phosphorylation via an EGFR transactivation pathway involving PKCdelta. p66Shc links alpha(1)-ARs to an AKT signaling pathway that selectively phosphorylates/inactivates FOXO transcription factors and downregulates the ROS-scavenging protein manganese superoxide dismutase (MnSOD); the alpha(1)-AR-p66Shc-dependent pathway involving AKT does not regulate GSK3. Additional studies show that RNA interference-mediated downregulation of endogenous p66Shc leads to the derepression of FOXO3a-regulated genes such as MnSOD, p27Kip1, and BIM-1. p66Shc downregulation also increases proliferating cell nuclear antigen expression and induces cardiomyocyte hypertrophy, suggesting that p66Shc exerts an antihypertrophic action in neonatal cardiomyocytes. The novel alpha(1)-AR- and ROS-dependent pathway involving p66Shc identified in this study is likely to contribute to cardiomyocyte remodeling and the evolution of heart failure.

P66shc regulates endothelial NO production and endothelium-dependent vasorelaxation: implications for age-associated vascular dysfunction

The p66shc adaptor protein mediates age-associated oxidative stress. We examined the role of p66shc in endothelial nitric oxide synthase (eNOS) signaling. Overexpression of p66shc inhibited eNOS-dependent NO production. RNAi-mediated down-regulation of endogenous p66shc led to activation of the proto-oncogene ras, and Akt kinase, with a corresponding increase in phosphorylation of eNOS at S1177 (S1179 on bovine eNOS). In rat aortic rings, down-regulation of p66shc suppressed the vasoconstrictor response to phenyephrine that was abrogated by treatment with the NOS inhibitor l-NAME, and enhanced vasodilation induced by sub-maximal doses of acetylcholine. These findings highlight a pivotal role for p66shc in inhibiting endothelial NO production, and endothelium-dependent vasorelaxation, that may provide important mechanistic information about endothelial dysfunction seen with aging.

218 SELECTIVE REDUCTION OF p66shc mRNA IN BOVINE OOCYTES/EMBRYOS BY RNA INTERFERENCE

High embryo loss occurs during the first week of in vitro bovine embryo development with a high percentage of embryo death and arrest. These early stages of development are regulated by stored maternal mRNAs that accumulate in the oocyte cytoplasm during its growth phase. In our in vitro production (IVP) system approximately 13.5% of embryos arrest at the 2–4-cell stage, displaying no characteristics of apoptosis. We hypothesized that these arrested embryos enter an oxidative stress-induced senescence-like state. We previously showed that elevated levels of reactive oxygen species and increased levels of the stress-adaptor protein p66shc were associated with this early embryonic arrest. The aim of this study was to selectively down-regulate p66shc mRNA levels in bovine oocytes, using post-transcriptional gene silencing by RNA interference (RNAi), to study the effects of p66shc mRNA “knock-down” on early arrest of IVP bovine embryos. Approximately 12,000 short hairpin (sh) RNAi molecules specific for p66shc were microinjected into bovine germinal vesicle (GV) oocytes. Experiments consisted of a control group undergoing IVF alone, and two groups microinjected with and without p66shc shRNAi molecules. Quantification of p66shc mRNA by real-time PCR was used to assure down-regulation of p66shc mRNA in 2-cell embryos collected at 35 hours post-insemination (hpi). The amount of p66shc mRNA detected in control IVF, vehicle-injected, and p66shc RNAi-injected groups was 0.92 ± 0.02 × 10-5 pg, 1.2 ± 0.20 × 10-5 pg, and 0.46 ± 0.06 × 10-5 pg, respectively. This statistically significant (P < 0.001) reduction in p66shc mRNA levels by 54% upon p66shc shRNAi microinjection was selective for p66shc mRNA, as both histone H2a and p53 mRNA levels were not altered. Percentage of 2–4-cell arrest was evaluated at Day 8 post-insemination and related to p66shc mRNA down-regulation. While there were no significant differences in the percentage of 2–4-cell arrested embryos between the control (13.3 ± 0.8%) and vehicle-microinjected (10.8 ± 0.7%) embryos, there was a significant decrease (P < 0.001) in the incidence of arrest in p66shc shRNAi-microinjected embryos (0.9 ± 0.9%). Quantification by real time PCR in blastocysts from the three groups showed no significant differences in p66shc mRNA levels (P = 0.314) among control IVF, vehicle, or p66shc hairpin RNAi microinjected at 0.59 ± 0.1 × 10-5 pg, 0.57 ± 0.2 × 10-5 pg, and 0.51 ± 0.2 × 10-5 pg, respectively. This suggests that p66shc down-regulation by RNAi might be temporary. Thus, using this novel approach of RNAi and microinjection of oocytes at the GV stage, we were able to selectively down-regulate the expression of p66shc mRNA and correlate this down-regulation with a significant decrease in 2–4 cell arrest. These data reinforce our hypothesis that p66shc is involved in a stress-induced pathway that executes a senescent-like embryonic arrest at the 2–4-cell stage, thereby postulating the possible role of p66shc as a new molecular marker for developmental competence. Statistics performed using ANOVA with Fisher LSD test for multiple comparisons. This work was funded by NSERC, CIHR, OGS, and OMAFRA.