Hypoxia-inducible transcription factor 1alpha (HIF-1alpha) is a key player in the response to hypoxia. Additionally, HIF-1alpha responds to growth factors and hormones which can act via protein kinase B (Akt). However, HIF-1alpha is not a direct substrate for this kinase. Therefore, we investigated whether the protein kinase B target glycogen synthase kinase 3 (GSK-3) may have an impact on HIF-1alpha. We found that the inhibition or depletion of GSK-3 induced HIF-1alpha whereas the overexpression of GSK-3beta reduced HIF-1alpha. These effects were mediated via three amino acid residues in the oxygen-dependent degradation domain of HIF-1alpha. In addition, mutation analyses and experiments with von Hippel-Lindau (VHL)-defective cells indicated that GSK-3 mediates HIF-1alpha degradation in a VHL-independent manner. In line with these observations, the inhibition of the proteasome reversed the GSK-3 effects, indicating that GSK-3 may target HIF-1alpha to the proteasome by phosphorylation. Thus, the direct regulation of HIF-1alpha stability by GSK-3 may influence physiological processes or pathophysiological situations such as metabolic diseases or tumors.
The molecular mechanisms regulating sympathetic innervation of the heart during embryogenesis and its importance for cardiac development and function remain to be fully elucidated. We generated mice in which conditional knockout (CKO) of the Hif1a gene encoding the transcription factor hypoxia-inducible factor 1α (HIF-1α) is mediated by an Islet1-Cre transgene expressed in the cardiac outflow tract, right ventricle and atrium, pharyngeal mesoderm, peripheral neurons, and hindlimbs. These Hif1aCKO mice demonstrate significantly decreased perinatal survival and impaired left ventricular function. The absence of HIF-1α impaired the survival and proliferation of preganglionic and postganglionic neurons of the sympathetic system, respectively. These defects resulted in hypoplasia of the sympathetic ganglion chain and decreased sympathetic innervation of the Hif1aCKO heart, which was associated with decreased cardiac contractility. The number of chromaffin cells in the adrenal medulla was also decreased, indicating a broad dependence on HIF-1α for development of the sympathetic nervous system.
Summary Plasmid DNA was encapsulated in liposomes to protect from degradation and to support the incorporation into sperm cells. Transgenic spermatozoa carry the foreign DNA to the target cell when fertilizing the egg. Several plasmid constructs were transferred into rabbits, cattle and chicken. The plasmid apparently can persist when provided with a murine amplification promoting sequence. The transgeneity of sperm cells as well as fetuses or offspring was demonstrated by dot blot, Southern blot analysis and PCR. Expression of the transgene was shown as enzymatic activities of the reporter genes SEAP or β‐galactosidase and by gelelectrophoretic characterization of the expressed gene products. The transfer efficiency and the persistence of the gene constructs is significantly increased due to the use of sperm cells as gene carriers mediated by liposomes and the amplification element. Zusammenfassung Gentransfer mittels Liposomen und Spermien Plasmid‐DNA wurde in Liposomen eingeschlossen, um sie vor enzymatischem Abbau zu schützen und ihre Inkorporation in die Spermienzelle zu ermöglichen. Bei der Befruchtung bringt das Spermium die Fremd‐DNA in die Eizelle. Es wurden verschiedene Genkonstrukte in Kaninchen, Rind und Huhn übertragen. Das Plasmid kann offensichtlich persistieren, wenn es eine murine amplifikationsfördernde Sequenz enthält. Die Transgenität von Spermien, Feten und Nachkommen wurde mit Dot Blot, Southern Blot und PCR nachgewiesen, die Expression der Reportergene SEAP und β‐Galaktosidase gelelektrophoretisch bzw. enzymatisch charakterisiert. Der Einsatz von Spermien und Liposomen zur Genübertragung sowie die Verwendung einer ‘amplification promoting sequence' erhöhen die Transfereffizienz und die Persistenz der Genkonstrukte beträchtlich.
Here, we report that activation of different types of tissue macrophages, including microglia, by lipopolysaccharide (LPS) or GM-CSF stimulation correlates with the quantitative redistribution of NADPH oxidase (cyt b(558)) from the plasma membrane to an intracellular stimulus-responsive storage compartment. Cryo-immunogold labeling of gp91(phox) and CeCl(3) cytochemistry showed the presence of gp91(phox) and oxidant production in numerous small (<100 nm) vesicles. Cell homogenization and sucrose gradient centrifugation in combination with transferrin-HRP/DAB ablation showed that more than half of cyt b(558) is present in fractions devoid of endosomal markers, which is supported by morphological evidence to show that the cyt b(558)-containing compartment is distinct from endosomes or biosynthetic organelles. Streptolysin-O-mediated guanosine 5'-3-O-(thio)triphosphate loading of Ra2 microglia caused exocytosis of a major complement of cyt b(558) under conditions where lysosomes or endosomes were not mobilized. We establish phagocytic particles and soluble mediators ATP, TNFα, and CD40L as physiological inducers of cyt b(558) exocytosis to the cell surface, and by shRNA knockdown, we identify Rab27A/B as positive or negative regulators of vesicular mobilization to the phagosome or the cell surface, respectively. Exocytosis was followed by clathrin-dependent internalization of cyt b(558), which could be blocked by a dominant negative mutant of the clathrin-coated pit-associated protein Eps15. Re-internalized cyt b(558) did not reach lysosomes but associated with recycling endosomes and undefined vesicular elements. In conclusion, cyt b(558) depends on clathrin for internalization, and in activated macrophages NADPH oxidase occupies a Rab27A/B-regulated secretory compartment, which allows rapid agonist-induced redistribution of superoxide production in the cell.
The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.
Zielsetzung: Bei Adipositas kommt es zu einer vermehrten Einwanderung von Immunzellen, wie z.B. Monozyten und T-Zellen in das Fettgewebe. Bei diesem Infiltrationsprozess spielt das mikrovaskuläre Endothel als Barriere zwischen Fettgewebe und zirkulierenden Immunzellen im Blut eine aktive Rolle. Mit der Fettgewebsexpansion kommt es dort lokal zu einem hypoxischen Milieu (Befunde aus Mausmodellen) und zur Synthese von pro-inflammatorischen Molekülen. Ziel des Projektes ist die Aufklärung der zellulären Interaktionen und molekularen Mechanismen zwischen Prä/Adipozyten und Endothelzellen unter verschiedenen physiologischen und pathophysiologischen Bedingungen sowie die Identifizierung von Faktoren in diesem Wechselspiel.
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