scholarly journals Embryonal Carcinoma P19 Cells Produce Erythropoietin Constitutively But Express Lactate Dehydrogenase in an Oxygen-Dependent Manner

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1185-1195 ◽  
Author(s):  
Taiho Kambe ◽  
Junko Tada ◽  
Mariko Chikuma ◽  
Seiji Masuda ◽  
Masaya Nagao ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Binding Site ◽  
Embryonal Carcinoma ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Gene Promoter ◽  
Dependent Manner ◽  
P19 Cells ◽  
Independent Manner ◽  
Hep3b Cells

Abstract Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3′ enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

scholarly journals Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

2012 ◽  
Vol 198 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Yanmei Qi ◽  
Xiaoxiang Tian ◽  
Jie Liu ◽  
Yaling Han ◽  
Alan M. Graham ◽  
...  
Keyword(s):  
Protein A ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Epithelial Morphogenesis ◽  
Dependent Manner ◽  
Interacting Protein ◽  
The Core ◽  
Adenovirus E1b ◽  
Induced Apoptosis

Apoptosis is an essential step in cavitation during embryonic epithelial morphogenesis, but its mechanisms are largely unknown. In this paper, we used embryonic stem cell–differentiated embryoid bodies (EBs) as a model and found that Bnip3 (Bcl-2/adenovirus E1B 19-kD interacting protein), a BH3-only proapoptotic protein, was highly up-regulated during cavitation in a hypoxia-dependent manner. Short hairpin RNA silencing of Bnip3 inhibited apoptosis of the core cells and delayed cavitation. We show that the Bnip3 up-regulation was mediated mainly by hypoxia-inducible factor (HIF)–2. Ablation of HIF-2α or HIF-1β, the common β subunit of HIF-1 and -2, suppressed Bnip3 up-regulation and inhibited apoptosis and cavitation. We further show that apoptosis-inducing factor (AIF) cooperated with Bnip3 to promote lumen clearance. Bnip3 silencing in AIF-null EBs nearly blocked apoptosis and cavitation. Moreover, AIF also regulated Bnip3 expression through mitochondrial production of reactive oxygen species and consequent HIF-2α stabilization. These results uncover a mechanism of cavitation through hypoxia-induced apoptosis of the core cells mediated by HIFs, Bnip3, and AIF.

scholarly journals A dynamic balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and retinoic acid receptor:retinoid X receptor heterodimers regulates Oct-3/4 expression in embryonal carcinoma cells.

1995 ◽  
Vol 15 (2) ◽  
pp. 1034-1048 ◽  
Author(s):  
E Ben-Shushan ◽  
H Sharir ◽  
E Pikarsky ◽  
Y Bergman
Keyword(s):  
Transcription Factors ◽  
Retinoic Acid ◽  
Binding Site ◽  
Transcriptional Repression ◽  
Embryonal Carcinoma ◽  
Dependent Manner ◽  
Orphan Receptors ◽  
Positive Regulators ◽  
Ec Cells ◽  
Kinetics Of

The Oct-3/4 transcription factor is a member of the POU family of transcription factors and, as such, probably plays a crucial role in mammalian embryogenesis and differentiation. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Similarly, Oct-3/4 is expressed in embryonal carcinoma (EC) cells and is repressed in retinoic acid (RA)-differentiated EC cells. Previously we have shown that the Oct-3/4 promoter harbors an RA-responsive element, RAREoct, which functions in EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for negative regulators. Our present results demonstrate that in P19 and RA-treated P19 cells, the orphan receptors ARP-1/COUP-TFII and EAR-3/COUP-TFI repress Oct-3/4 promoter activity through the RAREoct site in a dose-dependent manner. While the N-terminal region of the ARP-1/COUP-TFII receptor is dispensable for this repression, the C-terminal domain harbors the silencing region. Interestingly, three different RA receptor:retinoid X receptor (RAR:RXR) heterodimers, RAR alpha:RXR alpha, RAR beta:RXR alpha, and RAR beta:RXR beta, specifically bind and activate Oct-3/4 promoter through the RAREoct site in a ligand-dependent manner. We have shown that antagonism between ARP-1/COUP-TFII or EAR-3/COUP-TFI and the RAR:RXR heterodimers and their intracellular balance modulate Oct-3/4 expression. Oct-3/4 transcriptional repression by the orphan receptors can be overcome by increasing amounts of RAR:RXR heterodimers. Conversely, activation of Oct-3/4 promoter by RAR:RXR heterodimers was completely abolished by EAR-3/COUP-TFI and by ARP-1/COUP-TFII. The orphan receptors bind the RAREoct site with a much higher affinity than the RAR:RXR heterodimers. This high binding affinity provides ARP-1/COUP-TFII and EAR-3/COUP-TFI with the ability to compete with and even displace RAR:RXR from the RAREoct site and subsequently to actively silence the Oct-3/4 promoter. We have shown that RA treatment of EC cells results in up-regulation of ARP-1/COUP-TFII and EAR-3/COUP-TFI expression. Most interestingly, in RA-treated EC cells, the kinetics of Oct-3/4 repression inversely correlates with the kinetics of ARP-1/COUP-TFII and EAR-3/COUP-TFI activation. These findings are in accordance with the suggestion that these orphan receptors participate in controlling a network of transcription factors, among which Oct-3/4 is included, which may establish the pattern of normal gene expression during development.

scholarly journals Estrogen receptor α (ESR1) over-expression mediated apoptosis in Hep3B cells by binding with SP1 proteins

2013 ◽  
Vol 51 (1) ◽  
pp. 203-212 ◽  
Author(s):  
Chuan-Chou Tu ◽  
V Bharath Kumar ◽  
Cecilia Hsuan Day ◽  
Wei-Wen Kuo ◽  
Su-Peng Yeh ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Gene Promoter ◽  
Estrogen Receptor Α ◽  
Dependent Manner ◽  
Estradiol Treatment ◽  
Binding Interaction ◽  
Normal Human Liver ◽  
Hep3b Cells ◽  
Factor Α ◽  
Active Caspase

Previous studies have reported that estrogen receptors (ERs) are expressed in normal human liver, chronic hepatitis, and benign hepatic tumor tissues. However, decreased expression of ERs can be observed in hepatocellular carcinoma (HCC) and the role of ERs in HCC is not fully understood. Thus, the present study aimed to investigate the molecular mechanism induced by the overexpression of ERα (ERα (ESR1)) in Hep3B cells. We first detected the induction of apoptosis in ER-negative Hep3B cells using DNA fragmentation assay and flow cytometry. We found that ERα and ERα plus 17β-estradiol treatment increased apoptosis in Hep3B cells. Additionally, western blotting showed increased expression of active caspase 3 and tumor necrosis factor α (TNFα (TNF)) inERα-transfected cells. To further understand the importance of SP1-binding sites in theTNFαpromoter, ERα-negative Hep3B cells were co-transfected withERαand a wild-type TNFα plasmid orTNFαwith deleted SP1 regions. Deletion of both distant and primal SP1 sites abolished the activity of ERα, and similar results were observed by blocking the expression of SP1 protein using mithramycin (MA). This result indicates that SP1 protein is essential for ERα-activatedTNFαpromoter activity. Co-immunoprecipitation assay further confirmed the binding interaction between ERα and SP1 in a ligand-dependent manner. In general, we demonstrate that the overexpression of ERα mediates apoptosis in ERα-negative Hep3B cells by the binding of ERα to SP1 protein. Additionally, this ERα–SP1 complex binds to the proximal and distal sites of theTNFαgene promoter and further induces the expression of active caspase 3 in a ligand-dependent manner.

scholarly journals Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

2007 ◽  
Vol 12 (3) ◽  
Author(s):  
Grzegorz Terlecki ◽  
Elżbieta Czapińska ◽  
Katarzyna Hotowy
Keyword(s):  
Skeletal Muscle ◽  
Lactate Dehydrogenase ◽  
Binding Site ◽  
Adenine Nucleotides ◽  
Low Ph ◽  
Nicotinamide Adenine ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cofactor Binding ◽  
Acidic Phospholipids

AbstractLactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids. The contribution of the ATP/ADP binding site to this process can also be considered. In the case of pig skeletal muscle LDH, neither the cofactor binding site nor the subunit interfacing areas seem to be involved in the interaction.

scholarly journals Hypoxia Downregulates MAPK/ERK but Not STAT3 Signaling in ROS-Dependent and HIF-1-Independent Manners in Mouse Embryonic Stem Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-16 ◽  
Author(s):  
Jan Kučera ◽  
Julie Netušilová ◽  
Stanislava Sladeček ◽  
Martina Lánová ◽  
Ondřej Vašíček ◽  
...  
Keyword(s):  
Cell Fate ◽  
Intracellular Signaling ◽  
Es Cells ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Erk Signaling ◽  
Independent Manner ◽  
Hypoxic Response ◽  
Stat3 Phosphorylation ◽  
Phosphorylation Pattern

Hypoxia is involved in the regulation of stem cell fate, and hypoxia-inducible factor 1 (HIF-1) is the master regulator of hypoxic response. Here, we focus on the effect of hypoxia on intracellular signaling pathways responsible for mouse embryonic stem (ES) cell maintenance. We employed wild-type and HIF-1α-deficient ES cells to investigate hypoxic response in the ERK, Akt, and STAT3 pathways. Cultivation in 1% O2 for 24 h resulted in the strong dephosphorylation of ERK and its upstream kinases and to a lesser extent of Akt in an HIF-1-independent manner, while STAT3 phosphorylation remained unaffected. Downregulation of ERK could not be mimicked either by pharmacologically induced hypoxia or by the overexpression. Dual-specificity phosphatases (DUSP) 1, 5, and 6 are hypoxia-sensitive MAPK-specific phosphatases involved in ERK downregulation, and protein phosphatase 2A (PP2A) regulates both ERK and Akt. However, combining multiple approaches, we revealed the limited significance of DUSPs and PP2A in the hypoxia-mediated attenuation of ERK signaling. Interestingly, we observed a decreased reactive oxygen species (ROS) level in hypoxia and a similar phosphorylation pattern for ERK when the cells were supplemented with glutathione. Therefore, we suggest a potential role for the ROS-dependent attenuation of ERK signaling in hypoxia, without the involvement of HIF-1.

Lysine demethylase KDM6B regulates HIF-1α mediated systemic and cellular responses to intermittent hypoxia

2021 ◽  
Author(s):  
Jayasri Nanduri ◽  
Ning Wang ◽  
Benjamin L Wang ◽  
Nanduri R. Prabhakar
Keyword(s):  
Transcriptional Activation ◽  
Intermittent Hypoxia ◽  
Hypoxia Inducible Factor ◽  
Plasma Catecholamines ◽  
Gene Promoter ◽  
Independent Manner ◽  
Obstructive Sleep ◽  
Induced Hypertension ◽  
Responsive Element ◽  
Lysine Demethylase

Intermittent hypoxia (IH) is a hallmark manifestation of Obstructive Sleep Apnea (OSA). Rodents treated with IH exhibit hypertension. Hypoxia-inducible factor (HIF)-1-dependent transcriptional activation of NADPH oxidases (Nox) and the resulting increase in reactive oxygen species (ROS) levels is a major molecular mechanism underlying IH/OSA-induced hypertension. Jumanji C (JmjC)-containing histone lysine demethylases (JmjC-KDMs) are coactivators of HIF-1-dependent transcriptional activation. In the present study, we tested the hypothesis that JmjC-KDMs are required for IH-evoked HIF-1 transcriptional activation of Nox4 and the ensuing hypertension. Studies were performed on pheochromocytoma (PC)12 cells and rats. IH increased KDM6B protein and enzyme activity in PC12 cells in a HIF-1-independent manner as evidenced by unaltered KDM6B activation by IH in HIF-1α shRNA treated cells. Cells treated with IH showed increased HIF-1-dependent Nox4 transcription as indicated by increased HIF-1α binding to hypoxia responsive element (HRE) sequence of the Nox4 gene promoter demonstrated by chromatin immunoprecipitation (ChiP) assay. Pharmacological blockade of KDM6B with GSKJ4, a specific KDM6 inhibitor, or genetic silencing of KDM6B with shRNA abolished IH-induced Nox4 transcriptional activation by blocking HIF-1α binding to the promoter of the NOX4 gene. Treating IH exposed rats with GSKJ4 showed: a) absence of KDM6B activation and HIF-1-dependent Nox4 transcription in the adrenal medullae, as well as b) absence of elevated plasma catecholamines and hypertension. Collectively, these findings indicate that KDM6B functions as a coactivator of HIF-1-mediated Nox4 transactivation and demonstrate a hitherto uncharacterized role for KDM's in IH-induced hypertension by HIF-1.

scholarly journals The ldhA Gene Encoding Fermentative L-Lactate Dehydrogenase in Corynebacterium glutamicum Is Positively Regulated by the Global Regulator GlxR

2021 ◽  
Vol 9 (3) ◽  
pp. 550
Author(s):  
Koichi Toyoda ◽  
Masayuki Inui
Keyword(s):  
Lactate Dehydrogenase ◽  
Binding Site ◽  
Corynebacterium Glutamicum ◽  
Lactate Production ◽  
Exponential Phase ◽  
Aerobic Bacterium ◽  
Global Regulator ◽  
Gene Encoding ◽  
Fermentation Products ◽  
Ldha Gene

Bacterial metabolism shifts from aerobic respiration to fermentation at the transition from exponential to stationary growth phases in response to limited oxygen availability. Corynebacterium glutamicum, a Gram-positive, facultative aerobic bacterium used for industrial amino acid production, excretes L-lactate, acetate, and succinate as fermentation products. The ldhA gene encoding L-lactate dehydrogenase is solely responsible for L-lactate production. Its expression is repressed at the exponential phase and prominently induced at the transition phase. ldhA is transcriptionally repressed by the sugar-phosphate-responsive regulator SugR and L-lactate-responsive regulator LldR. Although ldhA expression is derepressed even at the exponential phase in the sugR and lldR double deletion mutant, a further increase in its expression is still observed at the stationary phase, implicating the action of additional transcription regulators. In this study, involvement of the cAMP receptor protein-type global regulator GlxR in the regulation of ldhA expression was investigated. The GlxR-binding site found in the ldhA promoter was modified to inhibit or enhance binding of GlxR. The ldhA promoter activity and expression of ldhA were altered in proportion to the binding affinity of GlxR. Similarly, L-lactate production was also affected by the binding site modification. Thus, GlxR was demonstrated to act as a transcriptional activator of ldhA.

scholarly journals Advanced maternal age perturbs mouse embryo development and alters the phenotype of derived embryonic stem cells

2021 ◽  
pp. 1-11
Author(s):  
Pooja Khurana ◽  
Neil R. Smyth ◽  
Bhavwanti Sheth ◽  
Miguel A. Velazquez ◽  
Judith J. Eckert ◽  
...  
Keyword(s):  
Maternal Age ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Advanced Maternal Age ◽  
Preimplantation Embryos ◽  
Dependent Manner ◽  
Ki 67 ◽  
Altered Expression ◽  
Developmental Potential ◽  
Developmental Mechanisms

Abstract Advanced maternal age (AMA) is known to reduce fertility, increases aneuploidy in oocytes and early embryos and leads to adverse developmental consequences which may associate with offspring lifetime health risks. However, investigating underlying effects of AMA on embryo developmental potential is confounded by the inherent senescence present in maternal body systems further affecting reproductive success. Here, we describe a new model for the analysis of early developmental mechanisms underlying AMA by the derivation and characterisation of mouse embryonic stem cell (mESC-like) lines from naturally conceived embryos. Young (7–8 weeks) and Old (7–8 months) C57BL/6 female mice were mated with young males. Preimplantation embryos from Old dams displayed developmental retardation in blastocyst morphogenesis. mESC lines established from these blastocysts using conventional techniques revealed differences in genetic, cellular and molecular criteria conserved over several passages in the standardised medium. mESCs from embryos from AMA dams displayed increased incidence of aneuploidy following Giemsa karyotyping compared with those from Young dams. Moreover, AMA caused an altered pattern of expression of pluripotency markers (Sox2, OCT4) in mESCs. AMA further diminished mESC survival and proliferation and reduced the expression of cell proliferation marker, Ki-67. These changes coincided with altered expression of the epigenetic marker, Dnmt3a and other developmental regulators in a sex-dependent manner. Collectively, our data demonstrate the feasibility to utilise mESCs to reveal developmental mechanisms underlying AMA in the absence of maternal senescence and with reduced animal use.

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