María Monsalve

Direct coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1.

Transcription and mRNA processing are coupled events in vivo, but the mechanisms that coordinate these processes are largely unknown. PGC-1 is a transcriptional coactivator that plays a major role in the regulation of adaptive thermogenesis. PGC-1 also has certain motifs characteristic of splicing factors. We demonstrate here that mutations in the serine- and arginine-rich domain and RNA recognition motif of PGC-1 interfere with the ability of PGC-1 to induce mRNAs of target genes. These mutations also disrupt the ability of PGC-1 to co-localize and associate with RNA processing factors. PGC-1 can alter the processing of an mRNA, but only when it is loaded onto the promoter of the gene. These data demonstrate the coordinated regulation of RNA transcription and processing through PGC-1.

Mutual Dependence of Foxo3a and PGC-1α in the Induction of Oxidative Stress Genes

Oxidative stress is a hallmark of metabolism-related diseases and a risk factor for atherosclerosis. FoxO factors have been shown to play a key role in vascular endothelial development and homeostasis. Foxo3a can protect quiescent cells from oxidative stress through the regulation of detoxification genes such as sod2 and catalase. Here we show that Foxo3a is a direct transcriptional regulator of a group of oxidative stress protection genes in vascular endothelial cells. Importantly, Foxo3a activity requires the transcriptional co-activator PGC-1α, because it is severely curtailed in PGC-1α-deficient endothelial cells. Foxo3a and PGC-1α appear to interact directly, as shown by co-immunoprecipitation and in vitro interaction assays, and are recruited to the same promoter regions. The notion that Foxo3a and PGC-1α interact directly to regulate oxidative stress protection genes in the vascular endothelium is supported by the observation that PGC-1α transcriptional activity at the sod2 (manganese superoxide dismutase) promoter requires a functional FoxO site. We also demonstrate that Foxo3a is a direct transcriptional regulator of PGC-1α, suggesting that an auto-regulatory cycle regulates Foxo3a/PGC-1α control of the oxidative stress response.

Peroxisome Proliferator-activated Receptor α (PPARα) Induces PPARγ Coactivator 1α (PGC-1α) Gene Expression and Contributes to Thermogenic Activation of Brown Fat INVOLVEMENT OF PRDM16

Background: PPARα is a distinctive marker of the brown-versus-white fat phenotype. Results: PPARα induces PGC-1α gene transcription in brown adipocytes through mechanisms involving PRDM16. Conclusion: PPARα regulates brown fat thermogenesis via induction of PGC-1α and PRDM16 gene expression. Significance: Activation of PGC-1α by PPARα provides a molecular mechanism for concerted induction of thermogenic genes (UCP1, mitochondrial genes, and lipid oxidation genes) in brown fat. Peroxisome proliferator activated receptor α (PPARα) is a distinctive marker of the brown fat phenotype that has been proposed to coordinate the transcriptional activation of genes for lipid oxidation and for thermogenic uncoupling protein 1 in brown adipose tissue. Here, we investigated the involvement of PPARα in the transcriptional control of the PPARγ coactivator (PGC)-1α gene. Treatment with PPARα agonists induced PGC-1α mRNA expression in brown fat in vivo and in primary brown adipocytes. This enhancement of PGC-1α transcription was mediated by PPARα binding to a PPAR-responsive element in the distal PGC-1α gene promoter. PGC-1α gene expression was decreased in PPARα-null brown fat, both under basal conditions and in response to thermogenic activation. Moreover, PPARα- and cAMP-mediated pathways interacted to control PGC-1α transcription. PRDM16 (PRD1-BF1-RIZ1 homologous domain-containing 16) promoted PPARα induction of PGC-1α gene transcription, especially under conditions in which protein kinase A pathways were activated. This enhancement was associated with the interaction of PRDM16 with the PGC-1α promoter at the PPARα-binding site. In addition, PPARα promoted the expression of the PRDM16 gene in brown adipocytes, and activation of PPARα in human white adipocytes led to the appearance of a brown adipocyte pattern of gene expression, including induction of PGC-1α and PRDM16. Collectively, these results suggest that PPARα acts as a key component of brown fat thermogenesis by coordinately regulating lipid catabolism and thermogenic gene expression via induction of PGC-1α and PRDM16.

Nitric oxide regulates mitochondrial oxidative stress protection via the transcriptional coactivator PGC-1α

Nitric oxide (NO) has both prooxidant and antioxidant activities in the endothelium; however, the molecular mechanisms involved are still a matter of controversy. PGC‐1α [peroxisome proliferators‐activated receptor (PPAR) γ coactivator 1‐α] induces the expression of several members of the mitochondrial reactive oxygen species (ROS) detoxification system. Here, we show that NO regulates this system through the modulation of PGC‐1α expression. Short‐term (<12 h) treatment of endothelial cells with NO donors down‐regulates PGC‐1α expression, whereas long‐term (>24 h) treatment up‐regulates it. Treatment with the NOS inhibitor L‐NAME has the opposite effect. Down‐regulation of PGC‐1α by NO is mediated by protein kinase G (PKG). It is blocked by the soluble guanylate cyclase (sGC) inhibitor ODQ and the PKG inhibitor KT5823, and mimicked by the cGMP analog 8‐Br‐cGMP. Changes in PGC‐1α expression are in all cases paralleled by corresponding variations in the mitochondrial ROS detoxification system. Cells that transiently overexpress PGC‐1α from the cytomeglovirus (CMV) promoter respond poorly to NO donors. Analysis of tissues from eNOS−/− mice showed reduced levels of PGC‐1α and the mitochondrial ROS detoxification system. These data suggest that NO can regulate the mitochondrial ROS detoxification system both positively and negatively through PGC‐1α. —Borniquel, S., Valle, I., Cadenas, S., Lamas, S., and Monsalve, M. Nitric oxide regulates mitochondrial oxidative stress protection via the transcriptional coactivator PGC‐1α. FASEB J. 20, E1216–E1227 (2006)

Redox regulation of FoxO transcription factors

Transcription factors of the forkhead box, class O (FoxO) family are important regulators of the cellular stress response and promote the cellular antioxidant defense. On one hand, FoxOs stimulate the transcription of genes coding for antioxidant proteins located in different subcellular compartments, such as in mitochondria (i.e. superoxide dismutase-2, peroxiredoxins 3 and 5) and peroxisomes (catalase), as well as for antioxidant proteins found extracellularly in plasma (e.g., selenoprotein P and ceruloplasmin). On the other hand, reactive oxygen species (ROS) as well as other stressful stimuli that elicit the formation of ROS, may modulate FoxO activity at multiple levels, including posttranslational modifications of FoxOs (such as phosphorylation and acetylation), interaction with coregulators, alterations in FoxO subcellular localization, protein synthesis and stability. Moreover, transcriptional and posttranscriptional control of the expression of genes coding for FoxOs is sensitive to ROS. Here, we review these aspects of FoxO biology focusing on redox regulation of FoxO signaling, and with emphasis on the interplay between ROS and FoxOs under various physiological and pathophysiological conditions. Of particular interest are the dual role played by FoxOs in cancer development and their key role in whole body nutrient homeostasis, modulating metabolic adaptations and/or disturbances in response to low vs. high nutrient intake. Examples discussed here include calorie restriction and starvation as well as adipogenesis, obesity and type 2 diabetes.

SirT1 Regulation of Antioxidant Genes Is Dependent on the Formation of a FoxO3a/PGC-1α Complex

UNLABELLED SirT1 is a class III histone deacetylase that has been implicated in metabolic and reactive oxygen species control. In the vasculature it has been shown to decrease endothelial superoxide production, prevent endothelial dysfunction and atherosclerosis. However, the mechanisms that mediate SirT1 antioxidant functions remain to be characterized. The transcription factor FoxO3a and the transcriptional coactivator peroxisome proliferator activated receptor γ-coactivator 1α (PGC-1α) have been shown to induce the expression of antioxidant genes and to be deacetylated by SirT1. AIMS Here we investigated SirT1 regulation of antioxidant genes and the roles played by FoxO3a and PGC-1α in this regulation. RESULTS We found that SirT1 regulates the expression of several antioxidant genes in bovine aortic endothelial cells, including Mn superoxide dismutase (MnSOD), catalase, peroxiredoxins 3 and 5 (Prx3, Prx5), thioredoxin 2 (Trx2), thioredoxin reductase 2 (TR2), and uncoupling protein 2 (UCP-2) and can be localized in the regulatory regions of these genes. We also found that knockdown of either FoxO3a or PGC-1α prevented the induction of antioxidant genes by SirT1 over-expression. Furthermore, SirT1 increased the formation of a FoxO3a/PGC-1α complex as determined by co-immunoprecipitation (IP) assays, concomitantly reducing H2O2-dependent FoxO3a and PGC-1α acetylation. Data showing that FoxO3a knockdown increases PGC-1α acetylation levels and vice versa, suggest that SirT1 activity on FoxO3a and PGC-1α may be dependent of the formation of a FoxO3a/PGC-1α complex. INNOVATION A unifying mechanism for SirT1 activities is suggested. CONCLUSION We show that SirT1 regulation of antioxidant genes in vascular endothelial cells depends on the formation of a FoxO3a/PGC-1α complex.

Age associated low mitochondrial biogenesis may be explained by lack of response of PGC-1α to exercise training

Low mitochondriogenesis is critical to explain loss of muscle function in aging and in the development of frailty. The aim of this work was to explain the mechanism by which mitochondriogenesis is decreased in aging and to determine to which extent it may be prevented by exercise training. We used aged rats and compared them with peroxisome proliferator-activated receptor-γ coactivator-1α deleted mice (PGC-1α KO). PGC-1α KO mice showed a significant decrease in the mitochondriogenic pathway in muscle. In aged rats, we found a loss of exercise-induced expression of PGC-1α, nuclear respiratory factor-1 (NRF-1), and of cytochrome C. Thus muscle mitochondriogenesis, which is activated by exercise training in young animals, is not in aged or PGC-1α KO ones. Other stimuli to increase PGC-1α synthesis apart from exercise training, namely cold induction or thyroid hormone treatment, were effective in young rats but not in aged ones. To sum up, the low mitochondrial biogenesis associated with aging may be due to the lack of response of PGC-1α to different stimuli. Aged rats behave as PGC-1α KO mice. Results reported here highlight the role of PGC-1α in the loss of mitochondriogenesis associated with aging and point to this important transcriptional coactivator as a target for pharmacological interventions to prevent age-associated sarcopenia.

The complex biology of FOXO

FOXO transcription factors control proliferation, apoptosis, differentiation and metabolic processes. Loss of FOXO function has been identified in several human cancers, and results in increased cellular survival and a predisposition to neoplasia, especially in epithelial cancer. FOXO factors are therefore bona fide tumor suppressors, and their potential use as therapeutic targets in cancer has been a matter of debate. Importantly, FOXO factors can also positively regulate cell survival through the activation of several detoxification genes, complicating its putative therapeutic potential. Targeting of FOXO factors has also been proposed for the treatment of metabolic dysfunctions such as diabetes mellitus, immunological disorders and neurodegeneration, as well as for the prevention of aging by maintaining the hematopoyetic stem cells niche. But again, data has accumulated that cautions against the potential use of the FOXO activators in these settings. Therefore, greater understanding of the regulation of FOXO target specificity is still needed to boost its use as a therapeutic target. The four members of the FOXO family (FOXO1, FOXO3A, FOXO4 and FOXO6) have distinct but overlapping cellular functions, although they seem to bind a common set of DNA sites. This fact together with the observation that FOXOs are only partially dependent on their DNA binding activity to regulate their target genes highlights the fact that the interaction of the FOXOs with other transcription factors is crucial for the FOXO-mediated transcriptional programs. In this review, we provide an overview of recent progress in the understanding of the modulation of FOXO activity and target specificity by transcription factors and coactivators.

Transcription Activation or Repression by Phage Φ29 Protein p4 Depends on the Strength of the RNA Polymerase–Promoter Interactions

Phage psi 29 protein p4 activates the late A3 promoter and represses the early A2c promoter, in both cases by binding upstream from RNA polymerase (RNAP) and interacting with the C-terminal domain of the RNAP alpha subunit. To investigate how this interaction leads to activation at PA3 and to repression at PA2c, mutant promoters were constructed. We show that the position of protein p4 relative to that of RNAP, which is different at each promoter, does not dictate the outcome of the interaction. Rather, in the absence of a-35 consensus box for sigma A-RNAP activation was observed, while in its presence repression occurred. The results support the view that stabilization of RNAP at the promoter over a threshold level leads to repression.

Mitochondrial dysfunction in human pathologies.

The integrity of mitochondrial function is fundamental to cell life. The cell demands for mitochondria and their complex integration into cell biology, extends far beyond the provision of ATP. It follows that disturbances of mitochondrial function lead to disruption of cell function, expressed as disease or even death. Mitochondria are major producers of free radical species and also possibly of nitric oxide, and are, at the same time, major targets for oxidative damage. In this review we consider recent developments in our knowledge of how the mitochondrial production of reactive oxygen species (ROS) plays a critical role in several major human pathologies. We will also consider recent advances in our understanding of the molecular mechanisms involved in mitochondrial ROS detoxification.