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Background and Purpose: The TP53 induced glycolysis and apoptosis regulator (TIGAR) functions to lower fructose-2,6-bisphosphate (Fru-2,6-P(2)) levels in cells, consequently decreasing glycolysis and leading to the scavenging of reactive oxygen species (ROS), which correlate with a higher resistance to cell death. The decrease in intracellular ROS levels in response to TIGAR may also play a role in the ability of p53 to protect from the accumulation of genomic lesions. Given these good prospects of TIGAR for metabolic regulation and p53-response modulation, we analyzed the effects of TIGAR knockdown in U87MG and T98G glioblastoma-derived cell lines., Methods/results: After TIGAR-knockdown in glioblastoma cell lines, different metabolic parameters were assayed, showing an increase in Fru-2,6-P(2), lactate and ROS levels, with a concomitant decrease in reduced glutathione (GSH) levels. In addition, cell growth was inhibited without evidence of apoptotic or autophagic cell death. In contrast, a clear senescent phenotype was observed. We also found that TIGAR protein levels were increased shortly after irradiation. In addition, avoiding radiotherapy-triggered TIGAR induction by gene silencing resulted in the loss of capacity of glioblastoma cells to form colonies in culture and the delay of DNA repair mechanisms, based in γ-H2AX foci, leading cells to undergo morphological changes compatible with a senescent phenotype. Thus, the results obtained raised the possibility to consider TIGAR as a therapeutic target to increase radiotherapy effects., Conclusion: TIGAR abrogation provides a novel adjunctive therapeutic strategy against glial tumors by increasing radiation-induced cell impairment, thus allowing the use of lower radiotherapeutic doses., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Activation of p38 MAPK has been shown to be relevant for a number of bone morphogenetic protein (BMP) physiological effects. We report here the involvement of noncanonical phosphorylated mothers against decapentaplegic (Smad) signaling in the transcriptional induction of Cox2 (Ptgs2) by BMP-2 in mesenchymal cells and organotypic calvarial cultures. We demonstrate that different regulatory elements are required for regulation of Cox2 expression by BMP-2: Runt-related transcription factor-2 and cAMP response element sites are essential, whereas a GC-rich Smad binding element is important for full responsiveness. Efficient transcriptional activation requires cooperation between transcription factors because mutation of any element results in a strong decrease of BMP-2 responsiveness. BMP-2 activation of p38 leads to increased recruitment of activating transcription factor-2, Runx2, Smad, and coactivators such as p300 at the responsive sites in the Cox2 proximal promoter. We demonstrate, by either pharmacological or genetic analysis, that maximal BMP-2 effects on Cox2 and JunB expression require the function of p38 and its downstream effector mitogen/stress-activated kinase 1. Altogether our results strongly suggest that cooperative effects between canonical and noncanonical BMP signaling allow the fine-tuning of BMP transcriptional responses on specific target genes.

Trypanosoma cruzi infection produces an intense inflammatory response in diverse tissues including the heart. The inflammatory reaction is critical for the control of the parasites' proliferation and evolution of Chagas disease. 15-Deoxy-Δ(12,14) prostaglandin J(2) (15dPGJ2) can repress the inflammatory response in many experimental models. However, the precise role of peroxisome proliferator-activated receptor γ (PPARγ) ligands in T. cruzi infection or in Chagas disease is poorly understood. This work reports the first evidence that 15dPGJ2 treatment increases the number of intracellular parasites as shown by fluorescence microscopy and it is also able to inhibit the expression and activity of different inflammatory enzymes such as inducible nitric oxide synthase (NOS-2), matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), as well as pro-inflammatory cytokine (TNF-α and IL-6) mRNA expression in neonatal mouse cardiomyocytes after T. cruzi infection. Transfection of cardiomyocytes with small interfering RNA (siRNA) induces silencing of PPARγ and impairs the effects of 15dPGJ2 on the modulation of pro-inflammatory enzymes. Moreover, transfection restores the ability of these cells to control the intracellular growth of T. cruzi. We also found that PPARγ-independent pathways are involved, since 15dPGJ2 also exerts its effect through extracellular signal-regulated kinases-mitogen-activated protein kinase (Erk-MAPK) and nuclear factor-κB (NF-κB). The use of specific pharmacological inhibitors confirmed these findings. Our data point out that 15dPGJ2 is a potent modulator of the inflammatory process and regulator of parasites growth through PPARγ-dependent and independent (Erk-MAPK- and NF-κB) pathways in T. cruzi infected neonatal cardiac cells., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

The activity of mammalian target of rapamycin (mTOR) complexes regulates essential cellular processes, such as growth, proliferation, or survival. Nutrients such as amino acids are important regulators of mTOR complex 1 (mTORC1) activation, thus affecting cell growth, protein synthesis, and autophagy. Here, we show that amino acids may also activate mTOR complex 2 (mTORC2). This activation is mediated by the activity of class I PI3K and of Akt. Amino acids induced a rapid phosphorylation of Akt at Thr-308 and Ser-473. Whereas both phosphorylations were dependent on the presence of mTOR, only Akt phosphorylation at Ser-473 was dependent on the presence of rictor, a specific component of mTORC2. Kinase assays confirmed mTORC2 activation by amino acids. This signaling was functional, as demonstrated by the phosphorylation of Akt substrate FOXO3a. Interestingly, using different starvation conditions, amino acids can selectively activate mTORC1 or mTORC2. These findings identify a new signaling pathway used by amino acids underscoring the crucial importance of these nutrients in cell metabolism and offering new mechanistic insights.

Recent reports point out the importance of the complex GK-GKRP in controlling glucose and lipid homeostasis. Several GK mutations affect GKRP binding, resulting in permanent activation of the enzyme. We hypothesize that hepatic overexpression of a mutated form of GK, GK(A456V), described in a patient with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) and could provide a model to study the consequences of GK-GKRP deregulation in vivo. GK(A456V) was overexpressed in the liver of streptozotocin diabetic mice. Metabolite profiling in serum and liver extracts, together with changes in key components of glucose and lipid homeostasis, were analyzed and compared to GK wild-type transfected livers. Cell compartmentalization of the mutant but not the wild-type GK was clearly affected in vivo, demonstrating impaired GKRP regulation. GK(A456V) overexpression markedly reduced blood glucose in the absence of dyslipidemia, in contrast to wild-type GK-overexpressing mice. Evidence in glucose utilization did not correlate with increased glycogen nor lactate levels in the liver. PEPCK mRNA was not affected, whereas the mRNA for the catalytic subunit of glucose-6-phosphatase was upregulated ~4 folds in the liver of GK(A456V)-treated animals, suggesting that glucose cycling was stimulated. Our results provide new insights into the complex GK regulatory network and validate liver-specific GK activation as a strategy for diabetes therapy.

Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.

Aim: To examine the relevance of hypoxia inducible factor (HIF-1) and nitric oxide (NO) on the preservation of fatty liver against cold ischemia-reperfusion injury (IRI)., Methods: We used an isolated perfused rat liver model and we evaluated HIF-1alpha in steatotic and non-steatotic livers preserved for 24 h at 4 degrees C in University of Wisconsin and IGL-1 solutions, and then subjected to 2 h of normothermic reperfusion. After normoxic reperfusion, liver enzymes, bile production, bromosulfophthalein clearance, as well as HIF-1alpha and NO [endothelial NO synthase (eNOS) activity and nitrites/nitrates] were also measured. Other factors associated with the higher susceptibility of steatotic livers to IRI, such as mitochondrial damage and vascular resistance were evaluated., Results: A significant increase in HIF-1alpha was found in steatotic and non-steatotic livers preserved in IGL-1 after cold storage. Livers preserved in IGL-1 showed a significant attenuation of liver injury and improvement in liver function parameters. These benefits were enhanced by the addition of trimetazidine (an anti-ischemic drug), which induces NO and eNOS activation, to IGL-1 solution. In normoxic reperfusion, the presence of NO favors HIF-1alpha accumulation, promoting also the activation of other cytoprotective genes, such as heme-oxygenase-1., Conclusion: We found evidence for the role of the HIF-1alpha/NO system in fatty liver preservation, especially when IGL-1 solution is used.

The ribosomal protein S6 kinase 1 (S6K1) is emerging as a common downstream target of signalling by hormones and nutrients such as insulin and amino acids. Here, we have investigated how amino acids signal through the S6K1 pathway. First, we found that a commercial anti-phospho-Thr389-S6K1 antibody detects an 80-90 kDa protein that is rapidly phosphorylated in response to amino acids. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI-3 kinase inhibitors, and knockdown experiments showed that this protein was not S6K1. Looking for candidate targets of this phosphorylation, we found that amino acids stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. In turn, these phosphorylations required the activity of either p38 or ERK MAP kinases, which could compensate for each other. Moreover, we show that these MAP kinases are also needed for the amino acid-induced phosphorylation of S6K1 at Thr421/Ser424, as well as for that of S6K1 substrate, the S6 ribosomal protein. Consistent with these results, concomitant inhibition of p38 and ERK pathways also antagonised the well-known effects of amino acids on the process of autophagy. Altogether, these findings demonstrate a previously unknown role for MAP kinases in amino acid signalling.

As described by Warburg more than 50 years ago, tumour cells maintain a high glycolytic rate even in conditions of adequate oxygen supply. However, most of tumours are subjected to hypoxic conditions due to the abnormal vasculature that supply them with oxygen and nutrients. Thus, glycolysis is essential for tumour survival and spread. A key step in controlling glycolytic rate is the conversion of fructose-6-P to fructose-1,6-P(2) by 6-phosphofructo-1-kinase (PFK-1). The activity of PFK-1 is allosterically controlled by fructose-2,6-P(2), the product of the enzymatic activity of a dual kinase/phosphatase family of enzymes (PFKFB1-4) that are increased in a significant number of tumour types. In turn, these enzymes are induced by hypoxia through the activation of the HIF-1 complex (hypoxia-inducible complex-1), a transcriptional activator that controls the expression of most of hypoxia-regulated genes. HIF-1 complex is overexpressed in a variety of tumours and its expression appears to correlate with poor prognosis and responses to chemo or radiotherapy. Thus, targeting PFKFB enzymes, either directly or through inhibition of HIF-1, appears as a promising approach for the treatment of certain tumours.

This study investigates how the addition of trimetazidine (TMZ) and aminoimidazole-4-carboxamide ribonucleoside (AICAR) to University of Wisconsin (UW) solution protects steatotic livers. Steatotic and nonsteatotic livers were preserved for 24 hours at 4 degrees C in UW and UW with TMZ and AICAR (separately or in combination) and then perfused ex vivo for 2 hours at 37 degrees C. Adenosine monophosphate-activated protein kinase (AMPK) or nitric oxide (NO) synthesis inhibition in livers preserved in UW with TMZ was also investigated. Hepatic injury and function (transaminases, bile production, and sulfobromophthalein clearance) and factors potentially involved in the susceptibility of steatotic livers to ischemia-reperfusion (I/R), including vascular resistance, mitochondrial damage, adenosine triphosphate depletion, and oxidative stress were evaluated. AMPK, NO synthase (NOS), nitrate, and nitrite levels were also determined. The addition of TMZ and AICAR (separately or in combination) to UW reduced hepatic injury, improved functionality, and protected against the mechanisms responsible for the vulnerability of steatotic livers to I/R. Like AICAR, TMZ increased AMPK, constitutive NOS, and nitrates and nitrites, and conversely, AMPK or NO synthesis inhibition abolished the benefits of TMZ. In conclusion, TMZ, by means of AMPK, increased NO, thus protecting steatotic livers against their vulnerability to I/R injury. TMZ and AICAR may constitute new additives to UW solution in steatotic liver preservation, whereas a combination of both seems unnecessary., ((c) 2007 AASLD.)

Hepatic ischemia-reperfusion injury associated with liver transplantation is an as yet unresolved problem in clinical practice. Preconditioning protects the liver against the deleterious effects of ischemia, although the mechanism underlying this preconditioning is still unclear. To profile gene expression patterns involved in hepatic ischemic preconditioning, we analyzed the changes in gene expression in rat livers by DNA microarray analysis. Approximately 116 genes were found to have altered gene expression after 8 hours of cold ischemia. Moreover, the expression of 218 genes was modified by classic preconditioning followed by the same ischemia process. Given the importance of the effects of ischemic preconditioning (IP) in minimizing the liver damage induced by sustained ischemia before reperfusion, this study analyzed the putative genes involved in the beneficial role of IP in liver grafts undergoing cold ischemia before its implantation in the recipient (IP+I). Great differences were found in the gene expression pattern of ischemic preconditioning + long cold ischemia (IP+I) group when compared with the long cold ischemia alone condition (I), which could explain the protective regulatory mechanisms that take place after preconditioning. Twenty-six genes that were downregulated in cold ischemia were found upregulated after preconditioning preceding a long cold ischemia period. These would be genes activated or maintained by preconditioning. Heat shock protein genes and 3-hydroxy-3-methylglutaryl-coenzyme A reductase are among the most markedly induced transcripts., ((c) 2006 AASLD)

Background/aims: Hepatic steatosis is a risk factor for transplantation. We examined the role of AMP-activated protein kinase (AMPK) and nitric oxide (NO) in the benefits of preconditioning in steatotic liver transplantation., Methods: Steatotic liver transplantation with or without preconditioning was induced in Zucker rats. The activities of AMPK and NO synthase (NOS) were measured and altered pharmacologically., Results: Preconditioning or AMPK activation with aminoimidazole-4-carboxamide ribonucleoside (AICAR) increased AMPK and constitutive NOS activities and protected against lipid peroxidation, nitrotyrosine formation and hepatic injury in both grafts. Inhibition of AMPK activity removed the benefits of preconditioning. NO synthesis inhibition abolished the benefits of preconditioning or AICAR. Therefore, preconditioning or AICAR, through AMPK activation, may induce NO synthesis, thus protecting against hepatic injury in both steatotic and non-steatotic liver transplantation. In non-steatotic grafts, NO donors simulated the benefits of preconditioning. However, in steatotic grafts, NO supplementation was ineffective., Conclusions: These results indicate (a) a potential relationship between AMPK and NO in the benefits of preconditioning in steatotic liver transplantation, (b) AICAR as a new phamacological strategy in steatotic liver transplantation and (c) a differential effect of NO supplementation in both grafts.