p66Shc-generated oxidative signal promotes fat accumulation

Reactive oxygen species (ROS) and insulin signaling in the adipose tissue are critical determinants of aging and age-associated diseases. It is not clear, however, if they represent independent factors or they are mechanistically linked. We investigated the effects of ROS on insulin signaling using as model system the p66(Shc)-null mice. p66(Shc) is a redox enzyme that generates mitochondrial ROS and promotes aging in mammals. We report that insulin activates the redox enzyme activity of p66(Shc) specifically in adipocytes and that p66(Shc)-generated ROS regulate insulin signaling through multiple mechanisms, including AKT phosphorylation, Foxo localization, and regulation of selected insulin target genes. Deletion of p66(Shc) resulted in increased mitochondrial uncoupling and reduced triglyceride accumulation in adipocytes and in vivo increased metabolic rate and decreased fat mass and resistance to diet-induced obesity. In addition, p66(Shc-/-) mice showed impaired thermo-insulation. These findings demonstrate that p66(Shc)-generated ROS regulate the effect of insulin on the energetic metabolism in mice and suggest that intracellular oxidative stress might accelerate aging by favoring fat deposition and fat-related disorders.     

p66(Shc) gene has a pro-apoptotic role in human cell lines and it is activated by a p53-independent pathway

p66(Shc) protein has been proposed to be an indispensable factor for p53-dependent, mitochondria-mediated apoptosis in mice. Here, we show that p66(Shc) plays a pro-apoptotic role also in cell lines of human origin such as SaOs-2 and HeLa, where p53 is either absent or inactivated, thus, suggesting that p66(Shc) pro-apoptotic role is independent from the presence of a functional form of p53. The active form of p66(Shc) is phosphorylated in Serine 36. We confirm the importance of Serine 36 phosphorylation for p66(Shc) pro-apoptotic role, and our results suggest that the kinase involved in this process is activated independently from p53.     

p66Shc is involved in promoting HIF-1alpha accumulation and cell death in hypoxic T cells

Hypoxia results in adaptationally appropriate alterations of gene expression through the activation of hypoxia-inducible factor (HIF)-1 to overcome any shortage of oxygen. Peripheral blood mononuclear cells may be exposed to low oxygen tensions for different times as they migrate between blood and various tissues. We and others have previously shown that T-cell adaptation to hypoxia is characterized by a modulation of cytokine expression and an inhibition of T-cell activation. We have recently demonstrated that the adaptor protein p66Shc negatively regulates T-cell activation and survival. We here show that hypoxia enhances HIF-1alpha accumulation and vascular endothelial growth factor production in T cells. Hypoxic T cells expressed high levels of p21(WAF1/CIP1), of the pro-apoptotic molecules BNIP3, a classic HIF target gene, and BAX, as well as low levels of the anti-apoptotic molecule BCLxl, associated with an induction of cell death. We found out that hypoxic T cells expressed p66Shc. Furthermore, using T-cell transfectants expressing p66Shc, as well as T cells derived from mice p66Shc-/-, we defined a role of p66Shc in T-cell responses to hypoxia. Of interest, hypoxic p66Shc-positive transfectants expressed higher level of HIF-1alpha than negative controls. Thus, p66Shc may play an important role in downstream hypoxic signaling, involving HIF-1alpha protein accumulation and cell death in T lymphocytes.     

The Shc locus regulates insulin signaling and adiposity in mammals

Longevity of a p66Shc knockout strain (ShcP) was previously attributed to increased stress resistance and altered mitochondria. Microarrays of ShcP tissues indicated alterations in insulin signaling. Consistent with this observation, ShcP mice were more insulin sensitive and glucose tolerant at organismal and tissue levels, as was a novel p66Shc knockout (ShcL). Increasing and decreasing Shc expression in cell lines decreased and increased insulin sensitivity, respectively - consistent with p66Shc's function as a repressor of insulin signaling. However, differences between the two p66Shc knockout strains were also observed. ShcL mice were fatter and susceptible to fatty diets, and their fat was more insulin sensitive than controls. On the other hand, ShcP mice were leaner and resisted fatty diets, and their adipose was less insulin sensitive than controls. ShcL and ShcP strains are both highly inbred on the C57Bl/6 background, so we investigated gene expression at the Shc locus, which encodes three isoforms, p66, p52, and p46. Isoform p66 is absent in both strains; thus, the remaining difference to which to attribute the 'lean' phenotype is expression of the other two isoforms. ShcL mice have a precise deletion of p66Shc and normal expression of p52 and p46Shc isoforms in all tissues; thus, a simple deletion of p66Shc results in a 'fat' phenotype. However, ShcP mice in addition to p66Shc deletion have a fourfold increase in p46Shc expression in white fat. Thus, p46Shc overexpression in fat, rather than p66Shc deletion, is the likely cause of decreased adiposity and reduced insulin sensitivity in the fat of ShcP mice, which has implications for the longevity of the strain.     

P66Shc mediates increased platelet activation and aggregation in hypercholesterolemia

Background and hypothesis

Hypercholesterolemia leads to a prothrombotic phenotype. Platelet hyperactivity associated with hypercholesterolemia has been attributed, in part, to oxidative stress. P66Shc is a well-known determinant of cellular and organismal oxidative stress. However, its role in platelet biology is not known. We hypothesized that p66Shc mediates platelet hyperactivation and hyperaggregation in hypercholesterolemia.

Methods and results

P66Shc was expressed in both human and mouse platelets, as determined by qRT-PCR and immunoblotting. Mouse platelet p66Shc expression was upregulated by hypercholesterolemia induced by high-fat diet feeding. Compared to wild-type mice, high-fat diet-induced p66Shc expression in platelets was suppressed in transgenic mice expressing a short hairpin RNA targeting p66Shc (p66ShcRNAi). High-fat diet feeding of wild-type mice amplified surface P-selectin expression on platelets stimulated by the thrombin receptor agonist protease-activated receptor-4 (PAR4), and increased aggregation of platelets induced by thrombin. These exaggerated platelet responses induced by high-fat diet feeding were significantly blunted in p66ShcRNAi mice. Finally, thrombin-stimulated platelet reactive oxygen species were suppressed in p66ShcRNAi mice.

Conclusions

Hypercholesterolemia stimulates p66Shc expression in platelets, promoting platelet oxidative stress, hyperreactivity and hyperaggregation via p66Shc.     

Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells

p66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. However, a role of p66Shc in VEGF signaling and physiological responses in ECs is unknown. Here we show that VEGF promotes p66Shc phosphorylation at Ser36 through the JNK/ERK or PKC pathway as well as Rac1 binding to a nonphosphorylated form of p66Shc in ECs. Depletion of endogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role in VEGFR2 phosphorylation in caveolae/lipid rafts as well as downstream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube formation. These studies uncover a novel role of p66Shc as a positive regulator for ROS-dependent VEGFR2 signaling linked to angiogenesis in ECs and suggest p66Shc as a potential therapeutic target for various angiogenesis-dependent diseases.     

p66SHC promotes apoptosis and antagonizes mitogenic signaling in T cells

Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc(-/-) T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.     

p66Shc is a negative regulator of FcεRI-dependent signaling in mast cells

Aggregation of FcεRI on mast cells activates signaling pathways, resulting in degranulation and cytokine release. Release of mast cell-derived inflammatory mediators is tightly regulated by the interplay of positive and negative signals largely orchestrated by adapter proteins. Among these, the Shc family adapter p52Shc, which couples immunoreceptors to Ras activation, positively regulates FcεRI-dependent signaling. Conversely, p66Shc was shown to uncouple the TCR for the Ras-MAPK pathway and prime T cells to undergo apoptotic death. Loss of p66Shc in mice results in breaking of immunologic tolerance and development of lupus-like autoimmune disease, which includes alopecia among its pathological manifestations. The presence of numerous activated mast cells in alopecic skin areas suggests a role for this adapter in mast cells. In this study, we addressed the involvement of p66Shc in FcεRI-dependent mast cell activation. We showed that p66Shc is expressed in mast cells and that mast cells from p66Shc(-/-) mice exhibit enhanced responses following Ag stimulation of FcεRI. Furthermore, using RBL-2H3 cell transfectants, we showed that aggregation of FcεRI resulted in the recruitment of a p66Shc-SHIP1 complex to linker for activation of T cells. Collectively, our data identified p66Shc as a negative regulator of mast cell activation.     

Expression in T-cells of the proapoptotic protein p66SHC is controlled by promoter demethylation

p66Shc plays a key role in oxidative stress-induced apoptosis. p66Shc gene expression is tissue-specific and controlled by promoter methylation. In T-cells p66Shc expression is induced by a variety of apoptotic stimuli. We have addressed the mechanisms regulating p66Shc expression in T-cells. We show that the increase in p66Shc protein following stimulation with a Ca2+ ionophore results from enhanced gene expression, which is primarily dependent on DNA replication-independent promoter demethylation. Our data underline the role of CpG methylation in the control of p66Shc gene expression and provide evidence that Ca2+ signaling may lead to epigenetic modifications in nondividing cells.     

A cryptic targeting signal induces isoform-specific localization of p46Shc to mitochondria

The human Src homology and collagen (Shc) gene encodes three protein isoforms of 46, 52, and 66 kDa that belong to a family of molecular adapters involved in several signal transduction pathways. Recently, the 66-kDa isoform has been shown to play a central role in controlling reactive oxygen species metabolism and life span in mammals. Despite the large amount of information available on the biology and biochemistry of Shc proteins, very little is known regarding the regulation of their subcellular localization. Here we demonstrate the specific and selective localization of p46Shc to the mitochondrial matrix. Through deletion mapping experiments, we show that targeting of p46Shc to mitochondria is mediated by its first 32 amino acids, which behave as a bona fide mitochondrial targeting sequence. We further demonstrate that the N-terminal location of the signal peptide is critical for its function. This accounts for the observation that p52Shc and p66Shc, containing the same sequence but more internally located, display a remarkably different subcellular localization. These findings indicate that p46Shc may exert a non-redundant biological function in signal transduction pathways involving mitochondria.     

Deletion of p66Shc in mice increases the frequency of size‐change mutations in the lacZ transgene

Upon oxidative challenge the genome accumulates adducts and breaks that activate the DNA damage response to repair, arrest, or eliminate the damaged cell. Thus, reactive oxygen species (ROS) generated by endogenous oxygen metabolism are thought to affect mutation frequency. However, few studies determined the mutation frequency when oxidative stress is reduced. To test whether in vivo spontaneous mutation frequency is altered in mice with reduced oxidative stress and cell death rate, we crossed p66Shc knockout (p66KO) mice, characterized by reduced intracellular concentration of ROS and by impaired apoptosis, with a transgenic line harboring multiple copies of the lacZ mutation reporter gene as part of a plasmid that can be recovered from organs into Escherichia coli to measure mutation rate. Liver and small intestine from 2‐ to 24‐month‐old, lacZ (p66Shc+/+) and lacZp66KO mice, were investigated revealing no difference in overall mutation frequency but a significant increase in the frequency of size‐change mutations in the intestine of lacZp66KO mice. This difference was further increased upon irradiation of mice with X‐ray. In addition, we found that knocking down cyclophilin D, a gene that facilitates mitochondrial apoptosis acting downstream of p66Shc, increased the size‐change mutation frequency in small intestine. Size‐change mutations also accumulated in death‐resistant embryonic fibroblasts from lacZp66KO mice treated with H₂O₂. These results indicate that p66Shc plays a role in the accumulation of DNA rearrangements and suggest that p66Shc functions to clear damaged cells rather than affect DNA metabolism.     

Age-related changes in levels of p66Shc and serine 36-phosphorylated p66Shc in organs and mouse tissues

Mammalian life span can be controlled by p66Shc protein through regulation of cellular response to oxidative stress. We investigated age-related changes in the amount of p66Shc and its Ser36-phosphorylated form in various mouse organs and tissues and correlated it with the level of antioxidant enzymes. Comparing to the newborn, in adult 6-month-old mice, the level of p66Shc was increased particularly in liver, lungs, skin and diaphragm. In older animals the level of p66Shc decreased while signaling pathway responsible for Ser36 phosphorylation of p66Shc protein seemed to be continually enhanced. The amount of p66Shc phosphorylated at Ser36, significantly increased with age, resulted in higher free radical production and, in consequence accumulation of damages caused by free radicals. The increased amount of Ser36-phosphorylated p66Shc in livers of 12- and 23-month-old mice was correlated with the decreased level of antioxidant enzymes. Moreover, we found that p66Shc is a resident of mitochondria- and plasma membrane-associated membranes and that its level there depends on the age of animal.     

The Influence of Shc Proteins and Aging on Whole Body Energy Expenditure and Substrate Utilization in Mice

While it has been proposed that Shc family of adaptor proteins may influence aging by regulating insulin signaling and energy metabolism, the overall impact of Shc proteins on whole body energy metabolism has yet to be elucidated. Thus, the purpose of this study was to determine the influence of Shc proteins and aging on whole body energy metabolism in a mouse model under ambient conditions (22°C) and acute cold exposure (12°C for 24 hours). Using indirect respiration calorimetry, we investigated the impact of Shc proteins and aging on EE and substrate utilization (RQ) in p66 Shc−/− (ShcKO) and wild-type (WT) mice. Calorimetry measurements were completed in 3, 15, and 27 mo mice at 22°C and 12°C. At both temperatures and when analyzed across all age groups, ShcKO mice demonstrated lower 24 h total EE values than that of WT mice when EE data was expressed as either kJ per mouse, or adjusted by body weight or crude organ mass (ORGAN) (P≤0.01 for all). The ShcKO mice also had higher (P<0.05) fed state RQ values than WT animals at 22°C, consistent with an increase in glucose utilization. However, Shc proteins did not influence age-related changes in energy expenditure or RQ. Age had a significant impact on EE at 22°C, regardless of how EE data was expressed (P<0.05), demonstrating a pattern of increase in EE from age 3 to 15 mo, followed by a decrease in EE at 27 mo. These results indicate a decline in whole body EE with advanced age in mice, independent of changes in body weight (BW) or fat free mass (FFM). The results of this study indicate that both Shc proteins and aging should be considered as factors that influence energy expenditure in mice.