Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1–/– phenotype. Here we demonstrate that cells derived from Bmi1–/– mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1–/– mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.

Previous studies have determined that mice with a homozygous deletion in the adapter protein p66shc have an extended life span and that cells derived from these mice exhibit lower levels of reactive oxygen species. Here we demonstrate that a fraction of p66 shc localizes to the mitochondria and that p66 shc؊/؊ fibroblasts have altered mitochondrial energetics. In particular, despite similar cytochrome content, under basal conditions, the oxygen consumption of spontaneously immortalized p66shc؊/؊ mouse embryonic fibroblasts were lower than similarly maintained wild type cells. shc may extend life span by repartitioning metabolic energy conversion away from oxidative and toward glycolytic pathways.Generation of ATP in the mitochondria represents the most efficient pathway to meet the energetic needs of a cell. This process, however, requires the consumption of molecular oxygen with a corresponding production of reactive oxygen species (ROS).2 Generation of ROS appears to be one of the central mechanisms that contribute to aging in a wide range of organisms (1-3). In contrast, under aerobic conditions, energy generation can also be achieved through glycolytic pathways present in the cytosol. These cytosolic pathways are inherently less efficient but do not produce ROS. Each cell employs a different relative balance between these two major energetic pathways, although relatively little is known about how this partition is established or maintained.In lower organisms, such as Caenorhabditis elegans and Drosophila, a number of longevity-associated genes have been isolated. One prominent and well characterized aging pathway regulates the activity of the transcription factor DAF-16, a member of the Forkhead family of transcriptional regulators. Evidence suggests that DAF-16 is involved in responding to numerous environmental stresses (4). A rise in intracellular ROS is one particular stress that may be relevant to life span determination, and in this regard, it is of interest that both DAF-16 and its closest mammalian ortholog Foxo3a appear to regulate a number of cellular antioxidant proteins (5-9).In addition to the DAF-16 pathway, RNAi screens performed in C. elegans has identified a number of other putative longevity genes (10, 11). Interestingly, functional characterization of these longevity-associated genes have determined that a number of them appear to be important for mitochondrial function. Similarly, direct knockdown of components of the electron transport chain has also been shown to extend the life span of the worm (12). Analysis of these long lived mitochondrial mutants, as well as in depth energetic analysis of the previously characterized DAF-16-related mutants, has led to the proposal that many life span-extending mutants in C. elegans slow aging by decreasing mitochondrial metabolism (13). This hypothesis suggests that a shift away from trichloroacetic acid-based mitochondrial metabolism might extend life by a reduction in oxidative stress. Nonetheless, it should be mentioned that the relationship bet...

Glioblastoma is a severe type of primary brain tumor, and its highly invasive character is considered to be a major therapeutic obstacle. Phospholipase D (PLD) isozyme is overexpressed in various human tumor tissues and involved in tumorigenesis. However, the molecular mechanisms by which PLD enhances glioma invasion are unknown. In this study, we demonstrate that the increased expression of PLD and its enzymatic activity in the glioma stimulate the secretion and expression of matrix metalloproteinase (MMP)-2 and induce the invasiveness of glioma cells. The upregulation of MMP-2 induced by phosphatidic acid (PA), the product of PLD, was mediated by protein kinase C (PKC), protein kinase A (PKA), nuclear factor-kappaB (NF-kappaB) and Sp1 and it enhanced glioma cell invasion. PA activated PKC and PKA and induced the nuclear translocation and transactivation of NF-kappaB. PA also increased the binding of NF-kappaB and Sp1 to the MMP-2 promoter. Mutation of the NF-kappaB- or Sp1-binding sites significantly attenuated MMP-2 promoter activity. This is the first report to show that NF-kappaB and Sp1 are essential transcriptional factors linking PLD to MMP-2 upregulation, providing evidence that PLD contributes to glioma progression by enhancing MMP-2 expression and tumor cell invasion via PKC/PKA/NF-kappaB/Sp1-mediated signaling pathways.

Phospholipase D (PLD) has been suggested to play an important role in a variety of cellular functions. PLD activity has been shown to be significantly elevated in many tumours and transformed cells, suggesting the possibility that PLD might be involved in tumorigenesis. In this study, we have established stable cell lines overexpressing PLD1 and PLD2 from fibroblast cells. These cells, but not control cells, showed altered growth properties and anchorage-independent growth in soft agar. Both PLD1 and PLD2 also induced an up-regulation of the activity of matrix metalloprotease-9 as detected by zymograms. Furthermore, both PLD1 and PLD2 transformants, but not vector-transfectants, induced undifferentiated sarcoma when transplanted into nude mice. Both PLD1- and PLD2-mediated cell cycle distributions in stable cell lines revealed an increased fraction of cells in the S phase compared with control cells. Interestingly, the level of cyclin D3 protein, known as an activator of G(1) to S phase transition in the cell cycle, was aberrantly high in cells overexpressing PLD1 and PLD2 compared with control cells. These results suggest that overexpression of PLD isozymes may play an important role in neoplastic transformation.

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The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-␥1 has two putative PH domains, an NH 2 -terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-␥1 with translational elongation factor (EF)-1␣, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-␥1 followed by peptide sequence analysis, we identified EF-1␣ as a binding partner of a split PH domain of PLC-␥1. Analysis by site-directed mutagenesis of the PH domain revealed that the ␤2-sheet of a split PH domain is critical for the interaction with EF-1␣. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP 2 ). So the PH domain of PLC-␥1 binds to both EF-1␣ and PIP 2 . The binding affinity of EF-1␣ to the GST⅐PH domain fusion protein increased in the presence of PIP 2 , although PIP 2 does not bind to EF-1␣ directly. This suggests that EF-1␣ may control the binding affinity between the PH domain and PIP 2 . PLC-␥1 is substantially activated in the presence of EF-1␣ with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-␥1 (Y509A/F510A) that lost its binding affinity to EF-1␣ shows basal level activity. Taken together, our data show that EF-1␣ plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-␥1 activity via a split PH domain. Regulation of phosphoinositide metabolism by PLC-␥11 is important for cell proliferation, differentiation, and migration.Many extracellular signals stimulate the hydrolysis of PIP 2 by the activation of PLC-␥1, which produces inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. Both second messengers regulate the release of Ca 2ϩ from intracellular stores and activate protein kinase C, respectively (1, 2). On the roles of PLC-␥1 in cell growth and differentiation, recent findings demonstrate that overexpression of PLC-␥1 induces malignant transformation in nude mice (3), and targeted deletion of PLC-␥1 results in embryonic lethality in mice (4).For protein-protein interactions, PLC-␥1 has two Src homology (SH) 2 domains to bind to tyrosine-phosphorylated proteins including several growth factor receptors (5-8). Although a large variety of proteins are identified as interacting counterparts of the SH2-SH2-SH3 domain of PLC-␥1, the activation mechanism of PLC-␥1 remains obscure.The PH domain is a 120-amino acid residue stretch that has been identified in over 100 proteins (9 -12). The PH domain binds with high specificity and affinity to phosphoinositides including PIP, PIP 2 , and IP 3 (13-15). The PH domain of signaling molecules is involved in targeted translocation of molecules to cell membranes (13,16,17). Also, the PH domain mediates protein-protein interaction as well ...

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