Biomedical subjects

M Celeste Simon

Publications and source records attributed to M Celeste Simon.

At least 19 recordsLinked to original sources

PubMed2007

Hypoxia-inducible factors: central regulators of the tumor phenotype.

Low oxygen levels are a defining characteristic of solid tumors, and responses to hypoxia contribute substantially to the malignant phenotype. Hypoxia-induced gene transcription promotes characteristic tumor behaviors, including angiogenesis, invasion, metastasis, de-differentiation and enhanced glycolytic metabolism. These effects are mediated, at least in part, by targets of the hypoxia-inducible factors (HIFs). The HIFs function as heterodimers comprising an oxygen-labile alpha-subunit and a stable beta-subunit also referred to as ARNT. HIF-1alpha and HIF-2alpha stimulate the expression of overlapping as well as unique transcriptional targets, and their induction can have distinct biological effects. New targets and novel mechanisms of dysregulation place the HIFs in an ever more central role in tumor biology and have led to development of pharmacological inhibitors of their activity.

Cell Hypoxia↗

PubMed2006

The transcriptional repressor GFI-1 antagonizes PU.1 activity through protein-protein interaction.

Mice lacking the zinc finger transcriptional repressor protein GFI-1 are neutropenic. These mice generate abnormal immature myeloid cells exhibiting characteristics of both macrophages and granulocytes. Furthermore, Gfi-1(-/-) mice are highly susceptible to bacterial infection. Interestingly, Gfi-1(-/-) myeloid cells overexpress target genes of the PU.1 transcription factor such as the macrophage colony-stimulating factor receptor and PU.1 itself. We therefore determined whether GFI-1 modulates the transcriptional activity of PU.1. Our data demonstrate that GFI-1 physically interacts with PU.1, repressing PU.1-dependent transcription. This repression is functionally significant, as GFI-1 blocked PU.1-induced macrophage differentiation of a multipotential hematopoietic progenitor cell line. Retroviral expression of GFI-1 in primary murine hematopoietic progenitors increased granulocyte differentiation at the expense of macrophage differentiation. We interbred Gfi-1(+/-) and PU.1(+/-) mice and observed that heterozygosity at the PU.1 locus partially rescued the Gfi-1(-/-) mixed myeloid lineage phenotype, but failed to restore granulocyte differentiation. Our data demonstrate that GFI-1 represses PU.1 activity and that lack of this repression in Gfi-1(-/-) myeloid cells contributes to the observed mixed lineage phenotype.

Animals↗

PubMed2006

Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro.

Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-alpha) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O(2))-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O(2) deprivation (hypoxia). Using a fusion protein containing the human HIF-1alpha O(2)-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1alpha during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1alpha accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O(2) depletion.

Animals↗

PubMed2006

PML inhibits HIF-1alpha translation and neoangiogenesis through repression of mTOR.

Loss of the promyelocytic leukaemia (PML) tumour suppressor has been observed in several human cancers. The tumour-suppressive function of PML has been attributed to its ability to induce growth arrest, cellular senescence and apoptosis. Here we identify PML as a critical inhibitor of neoangiogenesis (the formation of new blood vessels) in vivo, in both ischaemic and neoplastic conditions, through the control of protein translation. We demonstrate that in hypoxic conditions PML acts as a negative regulator of the synthesis rate of hypoxia-inducible factor 1alpha (HIF-1alpha) by repressing mammalian target of rapamycin (mTOR). PML physically interacts with mTOR and negatively regulates its association with the small GTPase Rheb by favouring mTOR nuclear accumulation. Notably, Pml-/- cells and tumours display higher sensitivity both in vitro and in vivo to growth inhibition by rapamycin, and lack of PML inversely correlates with phosphorylation of ribosomal protein S6 and tumour angiogenesis in mouse and human tumours. Thus, our findings identify PML as a novel suppressor of mTOR and neoangiogenesis.

Animals↗

PubMed2006

Ribosomal stress couples the unfolded protein response to p53-dependent cell cycle arrest.

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2alpha leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK(-/-) murine embryonic fibroblasts have an attenuated G(1)/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

Cell Cycle↗

PubMed2006

HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth.

The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic "knock-in" strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1alpha with HIF-2alpha results in expanded expression of HIF-2alpha-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2alpha, but not HIF-1alpha, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2alpha KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2alpha severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2alpha-specific target gene and indicate that HIF-2alpha can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2alpha's tumor promoting activity.

Alleles↗

PubMed2006

Hypoxia, HIF and the placenta.

Early in mammalian development the placenta, a highly vascularized organ, develops to facilitate exchange of oxygen (O2), nutrients and waste between mother and offspring. This process is intricately regulated by O2 tension and the hypoxic (low O2) uterine environment. Consequently, the placenta provides an excellent model for understanding the relationship between hypoxia (low O2 tension), organogenesis (organ development)and angiogenesis (blood vessel development). Herein we describe recent research on Hypoxia Inducible Factor (HIF), a heterodimeric transcription factor regulated by hypoxia that is crucial for proper placental development. Complete disruption of HIF signaling through loss of the HIFbeta (ARNT) or HIF1alpha and HIF2alpha subunits results in improper placental development, characterized by a diminished spongiotrophoblast layer and insufficient chorio/allantoic fusion. Experiments using placental stem cells (TS cells) derived from Hif1alpha-/- Hif2alpha-/- (Hifalpha-/-) and Arnt-/- mice indicate that there is increased expression of the labyrinthine specific transcription factors GCM and TFEB and a deficiency in the spongiotrophoblast transcription factor Mash2. Furthermore Hifalpha-/- and Arnt-/- TS cells subjected to differentiating conditions tend to adopt a labyrinthine like syncytial fate, and do not form giant cells or spongiotrophoblasts. These observations demonstrate a crucial role for HIF in the formation of the spongiotrophoblast that is probably regulated by Mash2, and suggest a complex interaction between hypoxia, HIF and Mash2 in the formation of the spongiotrophoblast.

Animals↗

PubMed2006

cGMP-dependent protein kinase phosphorylates p21-activated kinase (Pak) 1, inhibiting Pak/Nck binding and stimulating Pak/vasodilator-stimulated phosphoprotein association.

Endothelial cells are normally non-motile and quiescent; however, endothelial cells will become permeable and invade and proliferate to form new blood vessels (angiogenesis) in response to wounding, cancer, diabetic retinopathy, age-related macular degeneration, or rheumatoid arthritis. p21-activated kinase (Pak), an effector for the Rho GTPases Rac and Cdc42, is required for angiogenesis and regulates endothelial cell permeability and motility. Although Pak is primarily activated by Rac and Cdc42, there are additional proteins that regulate Pak activity and localization, including three AGC protein kinase family members, Akt-1, PDK-1, and cAMP-dependent protein kinase. We describe phosphorylation and regulation of Pak localization by a fourth AGC kinase family member, cGMP-dependent protein kinase (PKG). Using in vitro mapping, a phosphospecific antibody, co-transfection assays, and untransfected bovine aortic endothelial cells we determined that PKG phosphorylates Pak at serine 21. Phosphorylation was accompanied by changes in proteins associated with Pak. The adaptor protein Nck was released, whereas a novel complex with vasodilator-stimulated phosphoprotein was stimulated. Furthermore Ser-21 phosphorylation of Pak appears to be important for regulation of cell morphology. In both human umbilical vein endothelial cells and HeLa cells, activation of PKG in the presence of Pak stimulated tail retraction and cell polarization. However, in cells expressing S21A mutant Pak1, PKG activation or treatment with a peptide that blocks Nck/Pak binding caused aberrant cell morphology, blocked cell retraction, and mislocalized Pak, producing uropod (tail-like) structures. These data suggest that PKG regulates Pak and that the interaction plays a role in tail retraction.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Hypoxia-induced energy stress regulates mRNA translation and cell growth.

Oxygen (O2) deprivation, or hypoxia, has profound effects on cell metabolism and growth. Cells can adapt to low O2 in part through activation of hypoxia-inducible factor (HIF). We report here that hypoxia inhibits mRNA translation by suppressing multiple key regulators, including eIF2alpha, eEF2, and the mammalian target of rapamycin (mTOR) effectors 4EBP1, p70S6K, and rpS6, independent of HIF. Hypoxia results in energy starvation and activation of the AMPK/TSC2/Rheb/mTOR pathway. Hypoxic AMP-activated protein kinase (AMPK) activation also leads to eEF2 inhibition. Moreover, hypoxic effects on cellular bioenergetics and mTOR inhibition increase over time. Mutation of the TSC2 tumor suppressor gene confers a growth advantage to cells by repressing hypoxic mTOR inhibition and hypoxia-induced G1 arrest. Together, eIF2alpha, eEF2, and mTOR inhibition represent important HIF-independent mechanisms of energy conservation that promote survival under low O2 conditions.

AMP-Activated Protein Kinases↗

PubMed2006

Mitochondrial reactive oxygen species are required for hypoxic HIF alpha stabilization.

Multicellular organisms initiate adaptive responses when oxygen (O2) availability decreases. The underlying mechanisms of O2 sensing remain unclear. Mitochondria have been implicated in many hypoxia-inducible factor (HIF) -dependent and -independent hypoxic responses. However, the role of mitochondria in mammalian cellular O2 sensing has remained controversial, particularly regarding the use pharmacologic agents to effect hypoxic HIF alpha stabilization, which has produced conflicting data in the literature. Using murine embryonic cells lacking cytochrome c, we show that mitochondrial reactive O2 species (ROS) are essential for O2 sensing and subsequent HIF alpha stabilization at 1.5% O2. In the absence of this signal, HIF alpha subunits continue to be hydroxylated and degraded via the proteasome. Importantly, exogenous treatment with H2O2 and severe O2 deprivation is sufficient to stabilize HIF alpha even in the absence of functional mitochondrial. These results demonstrate that mitochondria function as O2 sensors and signal hypoxic HIF alpha stabilization by releasing ROS to the cytoplasm. The cytochrome c mutant embryonic cells provide a unique reagent to further dissect the role of mitochondria in O2 mediated-intracellular events.

Animals↗

PubMed2006

Coming up for air: HIF-1 and mitochondrial oxygen consumption.

Hypoxic cells induce glycolytic enzymes; this HIF-1-mediated metabolic adaptation increases glucose flux to pyruvate and produces glycolytic ATP. Two papers in this issue of Cell Metabolism (Kim et al., 2006; Papandreou et al., 2006) demonstrate that HIF-1 also influences mitochondrial function, suppressing both the TCA cycle and respiration by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 regulation in hypoxic cells promotes cell survival.

Animals↗

PubMed2006

HIF-dependent hematopoietic factors regulate the development of the embryonic vasculature.

Hypoxia inducible factors (HIFs) regulate adaptive responses to changes in oxygen (O(2)) tension during embryogenesis, tissue ischemia, and tumorigenesis. Because HIF-deficient embryos exhibit a number of developmental defects, the precise role of HIF in early vascular morphogenesis has been uncertain. Using para-aortic splanchnopleural (P-Sp) explant cultures, we show that deletion of the HIF-beta subunit (ARNT) results in defective hematopoiesis and the inhibition of both vasculogenesis and angiogenesis. These defects are rescued upon the addition of wild-type Sca-1(+) hematopoietic cells or recombinant VEGF. Arnt(-/-) embryos exhibit reduced levels of VEGF protein and increased numbers of apoptotic hematopoietic cells. These results suggest that HIF coordinates early endothelial cell emergence and vessel development by promoting hematopoietic cell survival and paracrine growth factor production.

Animals↗

PubMed2006

Regulation of angiogenesis by hypoxia and hypoxia-inducible factors.

Maintenance of oxygen homeostasis is critical for the survival of multicellular organs. As a result, both invertebrates and vertebrates have developed highly specialized mechanisms to sense changes in oxygen levels and to mount adequate cellular and systemic responses to these changes. Hypoxia, or low oxygen tension, occurs in physiological situations such as during embryonic development, as well as in pathological conditions such as ischemia, wound healing, and cancer. A primary effector of the adaptive response to hypoxia in mammals is the hypoxia-inducible factor (HIF) family of transcription regulators. These proteins activate the expression of a broad range of genes that mediate many of the responses to decreased oxygen concentration, including enhanced glucose uptake, increased red blood cell production, and the formation of new blood vessels via angiogenesis. This latter process is dynamic and results in the establishment of a mature vascular system that is indispensable for proper delivery of oxygen and nutrients to all cells in both normal tissue and hypoxic regions. Angiogenesis is essential for normal development and neoplastic disease as tumors must develop mechanisms to stimulate vascularization to meet increasing metabolic demands. The link between hypoxia and the regulation of angiogenesis is an area of intense research and the molecular details of this connection are still being elaborated. This chapter will provide an overview of current knowledge and highlight new insights into the importance of HIF and hypoxia in angiogenesis in both physiological and pathophysiological conditions.

Animals↗

PubMed2006

Hypoxia-inducible factors, hypoxia, and tumor angiogenesis.

PURPOSE OF REVIEW: The transcription factor hypoxia-inducible factor is activated by low oxygen to promote the expression of target genes that allow cellular, tissue, and organismal adaptation to low oxygen. Hypoxia-inducible factor is activated not only by hypoxia but also as a consequence of genetic mutations in a variety of tumors. This review summarizes recent studies on hypoxia-inducible factor-alpha functions in development and tumorigenesis. RECENT FINDINGS: Deficiency of the tumor suppressor von Hippel-Lindau leads to constitutively active hypoxia-inducible factor and hypoxia-inducible factor target gene expression. Other genetic lesions, however, e.g. in JunD, can also result in elevated hypoxia-inducible factor levels. The specific functions of hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha during development and tumor growth remain incompletely understood. Whereas hypoxia-inducible factor-2alpha seems to be the critical hypoxia factor in renal cell carcinoma, hypoxia-inducible factor-1alpha plays a significant role in the growth of tumors in other tissues. Loss of von Hippel-Lindau is not sufficient for neoplastic transformation, suggesting that hypoxia-inducible factor does not act alone to cause tumors. SUMMARY: It will be important to further characterize the specific roles of hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha during tumorigenesis to design therapies targeting the relevant isoform for specific diseases. It is also necessary to investigate the effect of reducing hypoxia-inducible factor levels on other angiogenic factors.

Animals↗

PubMed2006

pVHL function is essential for endothelial extracellular matrix deposition.

The tumor suppressor von Hippel-Lindau protein (pVHL) is critical for cellular molecular oxygen sensing, acting to target degradation of the hypoxia-inducible factor alpha transcription factor subunits under normoxic conditions. We have found that independent of its function in regulating hypoxic response, the VHL gene plays a critical role in embryonic endothelium development through regulation of vascular extracellular matrix assembly. We created mice lacking the VHL gene in endothelial cells; these conditional null mice died at the same stage as homozygous VHL-null mice, with similar vascular developmental defects. These included defective vasculogenesis in the placental labyrinth, a collapsed endocardium, and impaired vessel network patterning. The defects in embryonic vascularization were correlated with a diminished vascular fibronectin deposition in vivo and defective endothelial extracellular fibronectin assembly in vitro. We found that the impaired migration and adhesion of VHL-null endothelial cells can be partially rescued by the addition of back exogenous fibronectin, which indicates that pVHL regulation of fibronectin deposition plays an important functional role in vascular patterning and maintenance of vascular integrity.

Animals↗

PubMed2006

Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells.

Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factors (HIFs), HIF-1alpha and HIF-2alpha. The HIF-1alpha and HIF-2alpha subunits are structurally similar in their DNA binding and dimerization domains but differ in their transactivation domains, implying they may have unique target genes and require distinct transcriptional cofactors. Our previous results demonstrated that HIF-1alpha and HIF-2alpha regulate distinct target genes. Here, we report that HIF-2alpha is not transcriptionally active in embryonic stem (ES) cells, as well as possible inhibition by a HIF-2alpha-specific transcriptional repressor. Using DNA microarray analysis of hypoxia-inducible genes in wild-type (WT), Hif-1alpha(-)(/)(-), and Hif-2alpha(-)(/)(-) ES cells, we show that HIF-1alpha induces a large number of both confirmed and novel hypoxia-inducible genes, while HIF-2alpha does not activate any of its previously described targets. We further demonstrate that inhibition of HIF-2alpha function occurs at the level of transcription cofactor recruitment to endogenous target gene promoters. Overexpression of WT and, notably, a DNA-binding-defective HIF-2alpha mutant restores endogenous HIF-2alpha protein activity, suggesting that ES cells express a HIF-2alpha-specific corepressor that can be titrated by overexpressed HIF-2alpha protein. HIF-2alpha repression may explain why patients with mutations in the VHL tumor suppressor gene display cancerous lesions in specific tissue types.

Animals↗

PubMed2005

Mxi1 is induced by hypoxia in a HIF-1-dependent manner and protects cells from c-Myc-induced apoptosis.

HIF-1, a hypoxia inducible transcription factor, plays a pivotal role in the cellular response to hypoxia by activating genes involved in glucose metabolism, vascular remodeling, and erythropoiesis. We identified Mxi1, a c-Myc antagonist, as a novel target gene induced in hypoxia. Mxi1 was not induced in cells deficient in ARNT (HIF-1beta), suggesting that Mxi1 is a transcriptional target of the HIF-1 complex. Notably, c-Myc protein levels decreased during hypoxia but were stabilized by a proteasome inhibitor. Analysis of downstream transcriptional targets of c-Myc during hypoxia revealed that genes regulated by c-Myc, such as ornithine decarboxylase (ODC), were downregulated during hypoxia. In contrast, genes that are regulated by c-Myc and HIF-1, such as LDH-A, were upregulated. Mxi1 protects against c-Myc-dependent sensitization to hypoxia-induced apoptosis. The results suggest a coordinated mechanism for opposing c-Myc signaling during hypoxia that is mediated by a reduction in c-Myc levels, the induction of Mxi1, and a dominant effect of HIF-1 transcriptional activity.

Animals↗

PubMed2005

Hypoxia-inducible factor-dependent histone deacetylase activity determines stem cell fate in the placenta.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of HIFalpha and the arylhydrocarbon receptor nuclear translocator (ARNT/HIF1beta). Previously, we have reported that ARNT function is required for murine placental development. Here, we used cultured trophoblast stem (TS) cells to investigate the molecular basis of this requirement. In vitro, wild-type TS cell differentiation is largely restricted to spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS cells differentiated into chorionic trophoblasts and syncytiotrophoblasts, as demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the HIV receptor CXCR4. During this process, a region of the differentiating Arnt-null TS cells underwent granzyme B-mediated apoptosis, suggesting a role for this pathway in murine syncytiotrophoblast turnover. Surprisingly, HIF1alpha and HIF2alpha were induced during TS cell differentiation in 20% O2; additionally, pVHL levels were modulated during the same time period. These results suggest that oxygen-independent HIF functions are crucial to this differentiation process. As histone deacetylase (HDAC) activity has been linked to HIF-dependent gene expression, we investigated whether ARNT deficiency affects this epigenetic regulator. Interestingly, Arnt-null TS cells had reduced HDAC activity, increased global histone acetylation, and altered class II HDAC subcellular localization. In wild-type TS cells, inhibition of HDAC activity recapitulated the Arnt-null phenotype, suggesting that crosstalk between the HIFs and the HDACs is required for normal trophoblast differentiation. Thus, the HIFs play important roles in modulating the developmental plasticity of stem cells by integrating physiological, transcriptional and epigenetic inputs.

Animals↗