Zoltan Arany

Activation of Hypoxia-inducible Transcription Factor Depends Primarily upon Redox-sensitive Stabilization of Its α Subunit

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is critical for hypoxic induction of a number of physiologically important genes. We present evidence that regulation of HIF-1 activity is primarily determined by the stability of the HIF-1α protein. Both HIF-1α and HIF-1β mRNAs were constitutively expressed in HeLa and Hep3B cells with no significant induction by hypoxia. However, the HIF-1α protein was barely detectable in normoxic cells, even when HIF-1α was overexpressed, but was highly induced in hypoxic cells, whereas HIF-1β protein levels remained constant, regardless of pO2. Hypoxia-induced HIF-1 binding as well as the HIF-1α protein were rapidly and drastically decreased in vivo following an abrupt increase to normal oxygen tension. Moreover, short pre-exposure of cells to hydrogen peroxide selectively prevented hypoxia-induced HIF-1 binding via blocking accumulation of HIF-1α protein, whereas treatment of hypoxic cell extracts with H2O2 had no effect on HIF-1 binding. These observations suggest that an intact redox-dependent signaling pathway is required for destablization of the HIF-1α protein. In hypoxic cell extracts, HIF-1 DNA binding was reversibly abolished by sulfhydryl oxidation. Furthermore, the addition of reduced thioredoxin to cell extracts enhanced HIF-1 DNA binding. Consistent with these results, overexpression of thioredoxin and Ref-1 significantly potentiated hypoxia-induced expression of a reporter construct containing the wild-type HIF-1 binding site. These experiments indicate that activation of HIF-1 involves redox-dependent stabilization of HIF-1α protein.

HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1|[agr]|

Ischaemia of the heart, brain and limbs is a leading cause of morbidity and mortality worldwide. Hypoxia stimulates the secretion of vascular endothelial growth factor (VEGF) and other angiogenic factors, leading to neovascularization and protection against ischaemic injury. Here we show that the transcriptional coactivator PGC-1α (peroxisome-proliferator-activated receptor-γ coactivator-1α), a potent metabolic sensor and regulator, is induced by a lack of nutrients and oxygen, and PGC-1α powerfully regulates VEGF expression and angiogenesis in cultured muscle cells and skeletal muscle in vivo. PGC-1α-/- mice show a striking failure to reconstitute blood flow in a normal manner to the limb after an ischaemic insult, whereas transgenic expression of PGC-1α in skeletal muscle is protective. Surprisingly, the induction of VEGF by PGC-1α does not involve the canonical hypoxia response pathway and hypoxia inducible factor (HIF). Instead, PGC-1α coactivates the orphan nuclear receptor ERR-α (oestrogen-related receptor-α) on conserved binding sites found in the promoter and in a cluster within the first intron of the VEGF gene. Thus, PGC-1α and ERR-α, major regulators of mitochondrial function in response to exercise and other stimuli, also control a novel angiogenic pathway that delivers needed oxygen and substrates. PGC-1α may provide a novel therapeutic target for treating ischaemic diseases.

PGC-1α protects skeletal muscle from atrophy by suppressing FoxO3 action and atrophy-specific gene transcription

Maintaining muscle size and fiber composition requires contractile activity. Increased activity stimulates expression of the transcriptional coactivator PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α), which promotes fiber-type switching from glycolytic toward more oxidative fibers. In response to disuse or denervation, but also in fasting and many systemic diseases, muscles undergo marked atrophy through a common set of transcriptional changes. FoxO family transcription factors play a critical role in this loss of cell protein, and when activated, FoxO3 causes expression of the atrophy-related ubiquitin ligases atrogin-1 and MuRF-1 and profound loss of muscle mass. To understand how exercise might retard muscle atrophy, we investigated the possible interplay between PGC-1α and the FoxO family in regulation of muscle size. Rodent muscles showed a large decrease in PGC-1α mRNA during atrophy induced by denervation as well as by cancer cachexia, diabetes, and renal failure. Furthermore, in transgenic mice overexpressing PGC-1α, denervation and fasting caused a much smaller decrease in muscle fiber diameter and a smaller induction of atrogin-1 and MuRF-1 than in control mice. Increased expression of PGC-1α also increased mRNA for several genes involved in energy metabolism whose expression decreases during atrophy. Transfection of PGC-1α into adult fibers reduced the capacity of FoxO3 to cause fiber atrophy and to bind to and transcribe from the atrogin-1 promoter. Thus, the high levels of PGC-1α in dark and exercising muscles can explain their resistance to atrophy, and the rapid fall in PGC-1α during atrophy should enhance the FoxO-dependent loss of muscle mass.

Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinase

Cyclin A-kinase, an enzyme required for coordinating S phase progression, forms stable in vivo complexes with E2F-1, a growth-promoting transcription factor, which binds to the retinoblastoma gene product and is involved in the timely activation of genes whose products contribute to G1 exit and S phase traversal. Complex formation results in a negative biochemical effect of cyclin A-kinase: the shut-off of E2F-1-dependent DNA binding function in S/G2. Thus, specific and timely cell cycle-dependent interactions of E2F-1 with proteins that inhibit its function (i.e., RB during G1 and cyclin A-kinase during S/G2) may contribute to the periodicity of expression of certain E2F-1-responsive genes at the G1/S transition.

Transcriptional control of preadipocyte determination by Zfp423

The worldwide epidemic of obesity has increased the urgency to develop a deeper understanding of physiological systems related to energy balance and energy storage, including the mechanisms controlling the development of fat cells (adipocytes). The differentiation of committed preadipocytes to adipocytes is controlled by PPARγ and several other transcription factors, but the molecular basis for preadipocyte determination is not understood. Using a new method for the quantitative analysis of transcriptional components, we identified the zinc-finger protein Zfp423 as a factor enriched in preadipose versus non-preadipose fibroblasts. Ectopic expression of Zfp423 in non-adipogenic NIH 3T3 fibroblasts robustly activates expression of Pparg in undifferentiated cells and permits cells to undergo adipocyte differentiation under permissive conditions. Short hairpin RNA (shRNA)-mediated reduction of Zfp423 expression in 3T3-L1 cells blunts preadipocyte Pparg expression and diminishes the ability of these cells to differentiate. Furthermore, both brown and white adipocyte differentiation is markedly impaired in Zfp423-deficient mouse embryos. Zfp423 regulates Pparg expression, in part, through amplification of the BMP signalling pathway, an effect dependent on the SMAD-binding capacity of Zfp423. This study identifies Zfp423 as a transcriptional regulator of preadipocyte determination.

The transcriptional coactivator PGC-1α mediates exercise-induced angiogenesis in skeletal muscle

Peripheral arterial disease (PAD) affects 5 million people in the US and is the primary cause of limb amputations. Exercise remains the single best intervention for PAD, in part thought to be mediated by increases in capillary density. How exercise triggers angiogenesis is not known. PPARγ coactivator (PGC)-1α is a potent transcriptional co-activator that regulates oxidative metabolism in a variety of tissues. We show here that PGC-1α mediates exercise-induced angiogenesis. Voluntary exercise induced robust angiogenesis in mouse skeletal muscle. Mice lacking PGC-1α in skeletal muscle failed to increase capillary density in response to exercise. Exercise strongly induced expression of PGC-1α from an alternate promoter. The induction of PGC-1α depended on β-adrenergic signaling. β-adrenergic stimulation also induced a broad program of angiogenic factors, including vascular endothelial growth factor (VEGF). This induction required PGC-1α. The orphan nuclear receptor ERRα mediated the induction of VEGF by PGC-1α, and mice lacking ERRα also failed to increase vascular density after exercise. These data demonstrate that β-adrenergic stimulation of a PGC-1α/ERRα/VEGF axis mediates exercise-induced angiogenesis in skeletal muscle.

Cardiac angiogenic imbalance leads to peripartum cardiomyopathy

Peripartum cardiomyopathy (PPCM) is an often fatal disease that affects pregnant women who are near delivery, and it occurs more frequently in women with pre-eclampsia and/or multiple gestation. The aetiology of PPCM, and why it is associated with pre-eclampsia, remain unknown. Here we show that PPCM is associated with a systemic angiogenic imbalance, accentuated by pre-eclampsia. Mice that lack cardiac PGC-1α, a powerful regulator of angiogenesis, develop profound PPCM. Importantly, the PPCM is entirely rescued by pro-angiogenic therapies. In humans, the placenta in late gestation secretes VEGF inhibitors like soluble FLT1 (sFLT1), and this is accentuated by multiple gestation and pre-eclampsia. This anti-angiogenic environment is accompanied by subclinical cardiac dysfunction, the extent of which correlates with circulating levels of sFLT1. Exogenous sFLT1 alone caused diastolic dysfunction in wild-type mice, and profound systolic dysfunction in mice lacking cardiac PGC-1α. Finally, plasma samples from women with PPCM contained abnormally high levels of sFLT1. These data indicate that PPCM is mainly a vascular disease, caused by excess anti-angiogenic signalling in the peripartum period. The data also explain how late pregnancy poses a threat to cardiac homeostasis, and why pre-eclampsia and multiple gestation are important risk factors for the development of PPCM.

Association of p300 and CBP with simian virus 40 large T antigen.

p300 and the CREB-binding protein CBP are two large nuclear phosphoproteins that are structurally highly related. Both function, in part, as transcriptional adapters and are targeted by the adenovirus E1A oncoprotein. We show here that p300 and CBP interact with another transforming protein, the simian virus 40 large T antigen (T). This interaction depends on the integrity of a region of T which is critical for its transforming and mitogenic properties and includes its LXCXE Rb-binding motif. T interferes with normal p300 and CBP function on at least two different levels. The presence of T alters the phosphorylation states of both proteins and inhibits their transcriptional activities on certain promoters. Although E1A and T show little sequence similarity, they interact with the same domain of p300 and CBP, suggesting that this region exhibits considerable flexibility in accommodating diverse protein ligands.

Metabolic Signatures of Exercise in Human Plasma

Measurement by mass spectrometry of 200 blood metabolites reveals that individuals who are more fit respond more effectively to exercise, as shown by larger exercise-induced increase in glycerol. What Happens When You Run the Boston Marathon? We used to call it toil; now, we call it exercise. The human body has evolved to perform physical labor, and modern sedentary lifestyles are at odds with this evolutionary mandate. This disconnect makes it all the more imperative that we understand the physiology of how the body converts fuel to work. Lewis and colleagues have moved us toward that goal by comprehensively surveying blood metabolites in people of varying fitness levels before and during exercise. Through the use of a high-sensitivity mass spectrometry method, they have characterized these exercise-induced metabolic changes in unprecedented detail. The authors measured 200 blood metabolites in groups of people before, during, and after exercise on a treadmill. They found that the elevated glycolysis, lipolysis, and amino acid catabolism that occur in skeletal muscle cells during use are reflected in a rise in marker metabolites of these processes in blood. Also appearing in the blood after exercise were niacinamide, which enhances insulin release and improves glycemic control, and allantoin, an indicator of oxidative stress. Even when other variables were controlled for, the people who were more fit—as measured by their maximum oxygen use—exhibited more lipolysis during exercise (98% increase) than did the less fit (48% increase) participants or those who developed heart ischemia upon exertion (18% increase). Even more striking was the increase in lipolysis (1128%) in runners after they finished the Boston Marathon, a 26.2-mile run through the winding roads of Boston and its environs. From these data, the authors could not tell whether the more well-conditioned individuals were fitter because their metabolism used fat more effectively or whether, once attaining fitness, these able-bodied metabolic systems were better at burning fat. A mechanistic clue is provided by a final experiment in which the authors show that a combination of six of the metabolites elevated by exercise reflects an increase in glucose utilization and lipid metabolism in skeletal muscle cells, whereas none of the individual elevated molecules signal this effect. Thus, a cost of our sedentary lives may be to deoptimize the operation of the complicated system that is human metabolism. Sorting out how this backsliding occurs and how to restore the vigor of our metabolism will be facilitated by the findings and tools reported here. Exercise provides numerous salutary effects, but our understanding of how these occur is limited. To gain a clearer picture of exercise-induced metabolic responses, we have developed comprehensive plasma metabolite signatures by using mass spectrometry to measure >200 metabolites before and after exercise. We identified plasma indicators of glycogenolysis (glucose-6-phosphate), tricarboxylic acid cycle span 2 expansion (succinate, malate, and fumarate), and lipolysis (glycerol), as well as modulators of insulin sensitivity (niacinamide) and fatty acid oxidation (pantothenic acid). Metabolites that were highly correlated with fitness parameters were found in subjects undergoing acute exercise testing and marathon running and in 302 subjects from a longitudinal cohort study. Exercise-induced increases in glycerol were strongly related to fitness levels in normal individuals and were attenuated in subjects with myocardial ischemia. A combination of metabolites that increased in plasma in response to exercise (glycerol, niacinamide, glucose-6-phosphate, pantothenate, and succinate) up-regulated the expression of nur77, a transcriptional regulator of glucose utilization and lipid metabolism genes in skeletal muscle in vitro. Plasma metabolic profiles obtained during exercise provide signatures of exercise performance and cardiovascular disease susceptibility, in addition to highlighting molecular pathways that may modulate the salutary effects of exercise.

The transcription factor E2F-1 is a downstream target of RB action.

Reintroduction of RB into SAOS2 (RB-/-) cells causes a G1 arrest and characteristic cellular swelling. Coexpression of the cellular transcription factor E2F-1 could overcome these effects. The ability of E2F-1 to bind to RB was neither necessary nor sufficient for this effect, and S-phase entry was not accompanied by RB hyperphosphorylation under these conditions. Furthermore, E2F-1 could overcome the actions of a nonphosphorylatable but otherwise intact RB mutant. These data, together with the fact that RB binds to E2F-1 in vivo, suggest that E2F-1 is a downstream target of RB action. Mutational analysis showed that the ability of E2F-1 to bind to DNA was necessary and sufficient to block the formation of large cells by RB, whereas the ability to induce S-phase entry required a functional transactivation domain as well. Thus, the induction of a G1 arrest and the formation of large cells by RB in these cells can be genetically dissociated. Furthermore, the ability of the E2F-1 DNA-binding domain alone to block one manifestation of RB action is consistent with the notion that RB-E2F complexes actively repress transcription upon binding to certain E2F-responsive promoters. In keeping with this view, we show here that coproduction of an E2F1 mutant capable of binding to DNA, yet unable to transactivate, is sufficient to block RB-mediated transcriptional repression.