Metabolic changes in Japanese medaka (Oryzias latipes) during embryogenesis and hypoxia as determined by in vivo 31P NMR

In vivo (31)P nuclear magnetic resonance spectroscopy (NMR) was used to determine phosphometabolite changes in medaka (Oryzias latipes) during embryogenesis and hypoxia. NMR data were acquired using a flow-through NMR tube perfusion system designed to both deliver oxygenated water to embryos and accommodate a hypoxic challenge. Measurements of embryogenesis at 12- and 24-h intervals throughout 8 days of development (n = 3 per time point, 900 embryos per replicate) and during acute hypoxia (n = 6, 900 embryos at Iwamatsu stage 37 per replicate) were performed via NMR, and replicate samples (n = 4, 250 embryos each) were flash frozen for HPLC analysis. The hypoxic challenge experiment consisted of data acquisition with recirculating water (pre-hypoxic control period; 1 h), without recirculating water (hypoxic challenge; 1 h), then again with recirculating water (recovery period; 1.3 h). Concentrations of ATP, phosphocreatine (PCr), orthophosphate (P(i)), phosphomonoesters (PME), phosphodiesters (PDE), and intracellular pH (pH(i)) were determined by NMR, and ATP, ADP, AMP, GTP, GDP, and PCr were also determined via HPLC. During embryogenesis, [ATP] and [PCr] as determined by HPLC increased from 1-day post fertilization (DPF) levels of 0.93+/-0.08 and 2.48+/-0.21 micromol/mg (dry tissue), respectively, to 7.24+/-0.77 and 15.66+/-1.08 micromol/mg, respectively, by day 8. [ATP] and [PCr] measured by both NMR and HPLC fluctuated over 1-3 DPF, then increased significantly (p<0.05) over 3-8 DPF, while [PME] and [PDE] decreased (p<0.05) throughout embryogenesis. NMR and HPLC measurements revealed 1-3, 4-5, and 6-8 DPF as periods of embryogenesis significantly different from each other (p<0.05), and representing important transitions in metabolism and growth. During hypoxic challenge, [ATP] and [PCr] declined (p<0.05), [PME] and [PDE] decreased slightly, and [P(i)] increased (p<0.05). All phosphometabolites returned to pre-hypoxia concentrations during recovery. The pH(i) decreased (p<0.05) from 7.10+/-0.03 to 6.94+/-0.03 as a result of hypoxia, and failed to return to pre-hypoxic levels within the 1.3-h recovery phase. Results demonstrate the utility of in vivo (31)P NMR to detect significant alterations in phosphorylated nucleotides and phosphometabolites at specific developmental stages during medaka development and that late-stage medaka utilize PCr to generate ATP under hypoxic conditions.     

Effects of hypoxia on the proliferation and differentiation of NSCs

Oxygen is vital to nearly all forms of life on Earth via its role in energy homeostasis and other cell functions. Until recently, the effects of oxygen on the proliferation and differentiation of neural stem cells (NSCs) have been largely ignored. Some studies have been carried out on the basis of the fact that NSCs exists within a “physiological hypoxic” environment at 1 to 5% O₂ in both embryonic and adult brains. The results showed that hypoxia could promote the growth of NSCs and maintain its survival in vitro. In vivo studies also showed that ischemia/hypoxia increased the number of endogenous NSCs in the subventricular zone and dentate gyrus. In addition, hypoxia could influence the differentiation of NSCs. More neurons, especially more doparminergic neurons, were produced under hypoxic condition. The effects of hypoxia on the other kind of stem cell were briefly introduced as additional evidence. The mechanism of these responses might be primarily involved in the hypoxic inducible factor-1 (HIF-1) signal pathway. The present review summarizes recent works on the role of hypoxia in the proliferation and differentiation of NSCs both in vitro and in vivo, and the mechanism involved in HIF-1 signaling pathway behind this response was also discussed.     

PNUTS (phosphatase nuclear targeting subunit) inhibits retinoblastoma-directed PP1 activity

Protein phosphatase type 1 catalytic subunit (PP1c) is a serine/threonine phosphatase involved in the dephosphorylation of many proteins in eukaryotic cells. It associates with several known targeting or regulatory subunits that directly regulate PP1c activity toward specific substrates. The recently identified Phosphatase Nuclear Targeting Subunit (PNUTS) binds to PP1c and inhibits PP1 activity toward phosphorylase a. One of the substrates of PP1c has been shown to be the cell cycle regulatory protein, Retinoblastoma (pRb). In this study, we show that PNUTS dissociates from PP1c under mildly hypoxic cell growth conditions that lead to an increase of PP1c activity toward pRb. We developed an assay that measures pRb-directed PP1c activity and show that a GST-PNUTS fusion protein inhibits phosphatase activity toward pRb when using PP1c from cell lysates, GST-PP1c, or purified PP1c. These studies suggest that PNUTS is involved in the regulation of PP1c activity toward pRb.     

Systemic hypoxia alters gene expression levels of structural proteins and growth factors in knee joint cartilage

We investigated the effects of short- (8- and 24-h) and long-term (3 weeks) exposure to systemic normobaric hypoxia (13%) on the gene expression level of structural proteins and growth factors in knee joint cartilage of rabbits. Collagen type Ia2, II, and Va1, TGF-beta1, and b-FGF were upregulated after short-term hypoxia in both menisci, but not in articular cartilage. In contrast, long-term hypoxia downregulated gene expression level of collagens, aggrecan, and growth factors in articular cartilage and meniscal fibrocartilage. Interestingly, gene expression levels of non-collagenous proteins biglycan, decorin, and versican were not affected by short-term or by long-term hypoxia in knee joint cartilage. The present study suggests that changes in oxygen level differentially affect gene expression levels of growth factors, collagens, and non-collagenous proteins in normal knee joint cartilage in rabbits.     

Structural requirements for O2 sensing by the human tandem-P domain channel, hTREK1

TREK1 is a member of the tandem-P domain K+ channel family which is expressed almost exclusively in the nervous system. It is modulated by a number of important factors including arachidonic acid and cell swelling. Since both factors are associated with brain ischemia, it has been suggested that activation of TREK1 may confer neuroprotection. However, it has been reported that the stably expressed human homologue of TREK1 is inhibited by hypoxia, calling into question its neuroprotective role in ischemia. Here, using transient transfection of HEK 293 cells with several hTREK1 mutations and whole-cell patch-clamp, we show that: hypoxic inhibition: (a) requires the C-terminal domain of the channel; (b) does not involve redox modulation of the C-terminal domain cysteine residues C365 and C399; and (c) is critically dependent on the glutamate residue at position 306. These data suggest strongly that neuroprotection is unlikely to be provided by this channel in low O2 environments and continue to cast a shadow of doubt over the precise role that TREK may have during hypoxic episodes.     

Regulation of MAP kinase-dependent apoptotic pathway: implication of reactive oxygen and nitrogen species

Mitogen-activated protein (MAP) kinase signaling cascades are multi-functional signaling networks that influence cell growth, differentiation, apoptosis, and cellular responses to stress. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase that triggers apoptogenic kinase cascade leading to the phosphorylation/activation of c-Jun N-terminal kinases and p38-MAP kinase, which are responsible for inducing apoptotic cell death. This pathway plays a pivotal role in transduction of signals from different apoptotic stimuli. In the present review, we summarized the recent evidence concerning MAP kinase-dependent apoptotic pathway and its regulation in the mammalian cells and organism in vivo. We have shown that the key messengers of regulation of this pathway are the reactive oxygen and nitrogen species. The role of protein oxidation and S-nitrosation in induction of apoptotic cell death via ASK1 is discussed. Also we have outlined other recently discovered signal transduction processes involved in the regulation of ASK1 activity and downstream pathway.     

Hepatocyte growth factor enhances CXCR4 expression favoring breast cancer cell invasiveness

Microenvironmental factors affect different aspects of tumor cell biology, including cell survival, invasion, and metastasis. Here, we report that hepatocyte growth factor and hypoxia may contribute to breast carcinoma cell invasiveness by inducing the chemokine receptor CXCR4. Hepatocyte growth factor enhanced CXCR4 mRNA and protein expression exclusively in MCF-7 (low invasive) carcinoma cells, while in response to hypoxia, CXCR4 induction was observed in both MCF-7 and MDA-MB 231 (highly invasive) carcinoma cells. The receptor induction had a functional role in cancer cells, as demonstrated by the fact that hepatocyte growth factor pretreatment promoted MCF-7 cell migration toward the CXCR4-specific ligand CXCL12. Extracellular signal-regulated protein kinase 1/2 (ERK1/2) and phosphoinositide-3-kinase (PI3K) transduction pathways seemed to be differently implicated in the early induction of CXCR4 by hepatocyte growth factor or hypoxia in the two breast carcinoma cells examined.