Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1

The polycomb protein Bmi-1 represses the INK4a locus, which encodes the tumor suppressors p16 and p14(ARF). Here we report that Bmi-1 is downregulated when WI-38 human fibroblasts undergo replicative senescence, but not quiescence, and extends replicative life span when overexpressed. Life span extension by Bmi-1 required the pRb, but not p53, tumor suppressor protein. Deletion analysis showed that the RING finger and helix-turn-helix domains of Bmi-1 were required for life span extension and suppression of p16. Furthermore, a RING finger deletion mutant exhibited dominant negative activity, inducing p16 and premature senescence. Interestingly, presenescent cultures of some, but not all, human fibroblasts contained growth-arrested cells expressing high levels of p16 and apparently arrested by a p53- and telomere-independent mechanism. Bmi-1 selectively extended the life span of these cultures. Low O(2) concentrations had no effect on p16 levels or life span extension by Bmi-1 but reduced expression of the p53 target, p21. We propose that some human fibroblast strains are more sensitive to stress-induced senescence and have both p16-dependent and p53/telomere-dependent pathways of senescence. Our data suggest that Bmi-1 extends the replicative life span of human fibroblasts by suppressing the p16-dependent senescence pathway.     

Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress

Increasing cellular glucose uptake is a fundamental concept in treatment of type 2 diabetes, whereas nutritive calorie restriction increases life expectancy. We show here that increased glucose availability decreases Caenorhabditis elegans life span, while impaired glucose metabolism extends life expectancy by inducing mitochondrial respiration. The histone deacetylase Sir2.1 is found here to be dispensable for this phenotype, whereas disruption of aak-2, a homolog of AMP-dependent kinase (AMPK), abolishes extension of life span due to impaired glycolysis. Reduced glucose availability promotes formation of reactive oxygen species (ROS), induces catalase activity, and increases oxidative stress resistance and survival rates, altogether providing direct evidence for a hitherto hypothetical concept named mitochondrial hormesis or "mitohormesis." Accordingly, treatment of nematodes with different antioxidants and vitamins prevents extension of life span. In summary, these data indicate that glucose restriction promotes mitochondrial metabolism, causing increased ROS formation and cumulating in hormetic extension of life span, questioning current treatments of type 2 diabetes as well as the widespread use of antioxidant supplements.     

The dihydrolipoamide acetyltransferase is a novel metabolic longevity factor and is required for calorie restriction-mediated life span extension

Calorie restriction (CR) extends life span in a wide variety of species. Recent studies suggest that an increase in mitochondrial metabolism mediates CR-induced life span extension. Here we present evidence that Lat1 (dihydrolipoamide acetyltransferase), the E2 component of the mitochondrial pyruvate dehydrogenase complex, is a novel metabolic longevity factor in the CR pathway. Deleting the LAT1 gene abolishes life span extension induced by CR. Overexpressing Lat1 extends life span, and this life span extension is not further increased by CR. Similar to CR, life span extension by Lat1 overexpression largely requires mitochondrial respiration, indicating that mitochondrial metabolism plays an important role in CR. Interestingly, Lat1 overexpression does not require the Sir2 family to extend life span, suggesting that Lat1 mediates a branch of the CR pathway that functions in parallel to the Sir2 family. Lat1 is also a limiting longevity factor in nondividing cells in that overexpressing Lat1 extends cell survival during prolonged culture at stationary phase. Our studies suggest that Lat1 overexpression extends life span by increasing metabolic fitness of the cell. CR may therefore also extend life span and ameliorate age-associated diseases by increasing metabolic fitness through regulating central metabolic enzymes.     

Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression

The relationships between mitochondrial respiration, reactive oxygen species (ROS), and life span are complex and remain controversial. Inhibition of the target of rapamycin (TOR) signaling pathway extends life span in several model organisms. We show here that deletion of the TOR1 gene extends chronological life span in Saccharomyces cerevisiae, primarily by increasing mitochondrial respiration via enhanced translation of mtDNA-encoded oxidative phosphorylation complex subunits. Unlike previously reported pathways regulating chronological life span, we demonstrate that deletion of TOR1 delays aging independently of the antioxidant gene SOD2. Furthermore, wild-type and tor1 null strains differ in life span only when respiration competent and grown in normoxia in the presence of glucose. We propose that inhibition of TOR signaling causes derepression of respiration during growth in glucose and that the subsequent increase in mitochondrial oxygen consumption limits intracellular oxygen and ROS-mediated damage during glycolytic growth, leading to lower cellular ROS and extension of chronological life span.     

Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly

The oxidative stress hypothesis of aging predicts that a reduction in the generation of mitochondrial reactive oxygen species (ROS) will decrease oxidative damage and extend life span. Increasing mitochondrial proton leak-dependent state 4 respiration by increasing mitochondrial uncoupling is an intervention postulated to decrease mitochondrial ROS production. When human UCP2 (hUCP2) is targeted to the mitochondria of adult fly neurons, we find an increase in state 4 respiration, a decrease in ROS production, a decrease in oxidative damage, heightened resistance to the free radical generator paraquat, and an extension in life span without compromising fertility or physical activity. Our results demonstrate that neuronal-specific expression of hUCP2 in adult flies decreases cellular oxidative damage and is sufficient to extend life span.     

Regulation of yeast chronological life span by TORC1 via adaptive mitochondrial ROS signaling

Here we show that yeast strains with reduced target of rapamycin (TOR) signaling have greater overall mitochondrial electron transport chain activity during growth that is efficiently coupled to ATP production. This metabolic alteration increases mitochondrial membrane potential and reactive oxygen species (ROS) production, which we propose supplies an adaptive signal during growth that extends chronological life span (CLS). In strong support of this concept, uncoupling respiration during growth or increasing expression of mitochondrial manganese superoxide dismutase significantly curtails CLS extension in tor1Δ strains, and treatment of wild-type strains with either rapamycin (to inhibit TORC1) or menadione (to generate mitochondrial ROS) during growth is sufficient to extend CLS. Finally, extension of CLS by reduced TORC1/Sch9p-mitochondrial signaling occurs independently of Rim15p and is not a function of changes in media acidification/composition. Considering the conservation of TOR-pathway effects on life span, mitochondrial ROS signaling may be an important mechanism of longevity regulation in higher organisms.     

Relief of oxidative stress by ascorbic acid delays cellular senescence of normal human and Werner syndrome fibroblast cells

Primary human cells have a definite life span and enter into cellular senescence before ceasing cell growth. Oxidative stress produced by aerobic metabolism has been shown to accelerate cellular senescence. Here, we demonstrated that ascorbic acid, used as an antioxygenic reagent, delayed cellular senescence in a continuous culture of normal human embryonic cells, human adult skin fibroblast cells, and Werner syndrome (WS) cells. The results using human embryonic cells showed that treatment with ascorbic acid phospholic ester magnesium salt (APM) decreased the level of oxidative stress, and extended the replicative life span. The effect of APM to extend the replicative life span was also shown in normal human adult cells and WS cells. To understand the mechanism of extension of cellular life span, we determined the telomere lengths of human embryonic cells, both with and without APM treatment, and demonstrated that APM treatment reduced the rate of telomere shortening. The present results indicate that constitutive oxidative stress plays a role in determining the replicative life span and that suppression of oxidative stress by an antioxidative agent, APM, extends the replicative life span by reducing the rate of telomere shortening.     

FoxO3A promotes metabolic adaptation to hypoxia by antagonizing Myc function

Exposure of metazoan organisms to hypoxia engages a metabolic switch orchestrated by the hypoxia-inducible factor 1 (HIF-1). HIF-1 mediates induction of glycolysis and active repression of mitochondrial respiration that reduces oxygen consumption and inhibits the production of potentially harmful reactive oxygen species (ROS). Here, we show that FoxO3A is activated in hypoxia downstream of HIF-1 and mediates the hypoxic repression of a set of nuclear-encoded mitochondrial genes. FoxO3A is required for hypoxic suppression of mitochondrial mass, oxygen consumption, and ROS production and promotes cell survival in hypoxia. FoxO3A is recruited to the promoters of nuclear-encoded mitochondrial genes where it directly antagonizes c-Myc function via a mechanism that does not require binding to the consensus FoxO recognition element. Furthermore, we show that FoxO3A is activated in human hypoxic tumour tissue in vivo and that FoxO3A short-hairpin RNA (shRNA)-expressing xenograft tumours are decreased in size and metabolically changed. Our findings define a novel mechanism by which FoxO3A promotes metabolic adaptation and stress resistance in hypoxia.     

Proteasome modulates mitochondrial function during cellular senescence

Proteasome plays fundamental roles in the removal of oxidized proteins and in the normal degradation of short-lived proteins. Previously we have provided evidence that the impairment in proteasome observed during the replicative senescence of human fibroblasts has significant effects on MAPK signaling, proliferation, life span, senescent phenotype, and protein oxidative status. These studies have demonstrated that proteasome inhibition and replicative senescence caused accumulation of intracellular protein carbonyl content. In this study, we have investigated the mechanisms by which proteasome dysfunction modulates protein oxidation during cellular senescence. The results indicate that proteasome inhibition during replicative senescence has significant effects on intra- and extracellular ROS production in vitro. The data also show that ROS impaired the proteasome function, which is partially reversible by antioxidants. Increases in ROS after proteasome inhibition correlated with a significant negative effect on the activity of most mitochondrial electron transporters. We propose that failures in proteasome during cellular senescence lead to mitochondrial dysfunction, ROS production, and oxidative stress. Furthermore, it is likely that changes in proteasome dynamics could generate a prooxidative condition at the immediate extracellular microenvironment that could cause tissue injury during aging, in vivo.     

Inhibition of mitochondrial respiration by the anticancer agent 2-methoxyestradiol

2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have antiproliferative, antiangiogenic, and proapoptotic activity. Mechanistically, 2ME2 has been shown to downregulate hypoxia-inducible factor 1alpha (HIF1alpha) and to induce apoptosis in tumour cells by generating reactive oxygen species (ROS). In this study we report that 2ME2 inhibits mitochondrial respiration in both intact cells and submitochondrial particles, and that this effect is due to inhibition of complex I of the mitochondrial electron transport chain (ETC). The prevention by 2ME2 of hypoxia-induced stabilisation of HIF1alpha in HEK293 cells was found not to be due to an effect on HIF1alpha synthesis but rather to an effect on protein degradation. This is in agreement with our recent observation using other inhibitors of mitochondrial respiration which bring about rapid degradation of HIF1alpha in hypoxia due to increased availability of oxygen and reactivation of prolyl hydroxylases. The concentrations of 2ME2 that inhibited complex I also induced the generation of ROS. 2ME2 did not, however, cause generation of ROS in 143B rho(-) cells, which lack a functional mitochondrial ETC. We conclude that inhibition of mitochondrial respiration explains, at least in part, the effect of 2ME2 on hypoxia-dependent HIF1alpha stabilisation and cellular ROS production. Since these actions of 2ME2 occur at higher concentrations than those known to inhibit cell proliferation, it remains to be established whether they contribute to its therapeutic effect.     

Hypoxia‐inducible factors: coupling glucose metabolism and redox regulation with induction of the breast cancer stem cell phenotype

Reduced oxygen availability (hypoxia) leads to increased production of reactive oxygen species (ROS) by the electron transport chain. Here, I review recent work delineating mechanisms by which hypoxia‐inducible factor 1 (HIF‐1) mediates adaptive metabolic responses to hypoxia, including increased flux through the glycolytic pathway and decreased flux through the tricarboxylic acid cycle, in order to decrease mitochondrial ROS production. HIF‐1 also mediates increased flux through the serine synthesis pathway and mitochondrial one‐carbon (folate cycle) metabolism to increase mitochondrial antioxidant production (NADPH and glutathione). Dynamic maintenance of ROS homeostasis is required for induction of the breast cancer stem cell phenotype in response to hypoxia or cytotoxic chemotherapy. Consistently, inhibition of phosphoglycerate dehydrogenase, the first enzyme of the serine synthesis pathway, in breast cancer cells impairs tumor initiation, metastasis, and response to cytotoxic chemotherapy. I discuss how these findings have important implications for understanding the logic of the tumor microenvironment and for improving therapeutic responses in women with breast cancer.     

Inhibition of Hypoxia Inducible Factor 1α by siRNA‐Induced Apoptosis in Human Retinoblastoma Cells

Hypoxia, which activates the hypoxia inducible factor 1α (HIF‐1α), is an essential feature of retinoblastoma (RB) and contributes to poor prognosis and resistance to conventional therapy. In this study, the effect of HIF‐1α knockdown by small interfering RNA (siRNA) on cell proliferation, apoptosis, and apoptotic pathways of human Y‐79 RB cells was first investigated. Exposure to hypoxia induced the increased expression of HIF‐1α both in mRNA and protein levels. Then, knockdown of HIF‐1α by siRNA_HIF‐1α resulted in inhibition of cell proliferation and induced cell apoptosis in human Y‐79 RB cells under both normoxic and hypoxic conditions, with hypoxic conditions being more sensitive. Furthermore, knockdown of HIF‐1α could enhance hypoxia‐induced slight increase of Bax/Bcl‐2 ratio and activate caspase‐9 and caspase‐3. These results together indicated that suppression of HIF‐1α expression may be a promising strategy for the treatment of human RB in the future.     

Qiliqiangxin attenuates hypoxia‐induced injury in primary rat cardiac microvascular endothelial cells via promoting HIF‐1α‐dependent glycolysis

Protection of cardiac microvascular endothelial cells (CMECs) against hypoxia injury is an important therapeutic strategy for treating ischaemic cardiovascular disease. In this study, we investigated the effects of qiliqiangxin (QL) on primary rat CMECs exposed to hypoxia and the underlying mechanisms. Rat CMECs were successfully isolated and passaged to the second generation. CMECs that were pre‐treated with QL (0.5 mg/mL) and/or HIF‐1α siRNA were cultured in a three‐gas hypoxic incubator chamber (5% CO₂, 1% O₂, 94% N₂) for 12 hours. Firstly, we demonstrated that compared with hypoxia group, QL effectively promoted the proliferation while attenuated the apoptosis, improved mitochondrial function and reduced ROS generation in hypoxic CMECs in a HIF‐1α‐dependent manner. Meanwhile, QL also promoted angiogenesis of CMECs via HIF‐1α/VEGF signalling pathway. Moreover, QL improved glucose utilization and metabolism and increased ATP production by up‐regulating HIF‐1α and a series of glycolysis‐relevant enzymes, including glucose transport 1 (GLUT1), hexokinase 2 (HK2), 6‐phosphofructokinase 1 (PFK1), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Our findings indicate that QL can protect CMECs against hypoxia injury via promoting glycolysis in a HIF‐1α‐dependent manner. Lastly, the results suggested that QL‐dependent enhancement of HIF‐1α protein expression in hypoxic CMECs was associated with the regulation of AMPK/mTOR/HIF‐1α pathway, and we speculated that QL also improved HIF‐1α stabilization through down‐regulating prolyl hydroxylases 3 (PHD3) expression.