Development of Cytosolic Hypoxia and Hypoxia-inducible Factor Stabilization Are Facilitated by Aquaporin-1 Expression

Laboratorio de Investigaciones Biomédicas, Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío, Universidad de Sevilla, Sevilla, 41013 Spain.
Journal of Biological Chemistry (Impact Factor: 4.57). 11/2007; 282(41):30207-15. DOI: 10.1074/jbc.M702639200
Source: PubMed

ABSTRACT O(2) is essential for aerobic life, and the classic view is that it diffuses freely across the plasma membrane. However, measurements of O(2) permeability of lipid bilayers have indicated that it is much lower than previously thought, and therefore, the existence of membrane O(2) channels has been suggested. We hypothesized that, besides its role as a water channel, aquaporin-1 (AQP-1) could also work as an O(2) transporter, because this transmembrane protein appears to be CO(2)-permeable and is highly expressed in cells with rapid O(2) turnover (erythrocytes and microvessel endothelium). Here we show that in mammalian cells overexpressing AQP-1 and exposed to hypoxia, the loss of cytosolic O(2), as well as stabilization of the O(2)-dependent hypoxia-inducible transcription factor and expression of its target genes, is accelerated. In normoxic endothelial cells, knocking down AQP-1 produces induction of hypoxia-inducible genes. Moreover, lung AQP-1 is markedly up-regulated in animals exposed to hypoxia. These data suggest that AQP-1 has O(2) permeability and thus could facilitate O(2) diffusion across the cell membrane.

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    • "The resuspended pellet was left on ice 5 min, vortex, and then centrifuged at 16,000g for 15 min at 4°C, and extracted proteins remain in the supernatant. Protein concentration was analyzed with the Bradford method (BioRad Protein Assay, BioRad, Berkeley, CA) and kept at –20°C until Western blot assay (Echevarría et al., 2007). Afterwards, 20–40 mg of whole-cell extracts were resolved by SDS–PAGE (10%) for AQP3 and Cyclin A, B1, D1, and E. After electrophoresis, proteins were transferred into PVDF membranes (Hybond-P, Amersham Biosciences, Pittsburgh, PA) using a Novex apparatus (Novel Experimental Technology, San Diego, CA). "
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    ABSTRACT: AQP3 has been correlated with higher transport of glycerol, increment of ATP content, and larger proliferation capacity. Recently, we described the gold(III) complex Auphen as a very selective and potent inhibitor of AQP3's glycerol permeability (Pgly). Here we evaluated Auphen effect on the proliferation of various mammalian cell lines differing in AQP3 expression level: no expression (PC12), moderate (NIH/3T3) or high (A431) endogenous expression, cells stably expressing AQP3 (PC12-AQP3), and human HEK293T cells transiently transfected (HEK-AQP3) for AQP3 expression. Proliferation was evaluated in the absence or presence of Auphen (5 μM) by counting number of viable cells and analyzing 5-bromo-2′-deoxyuridine (BrdU) incorporation. Auphen reduced ≈50% the proliferation in A431 and PC12-AQP3, ≈15% in HEK-AQP3 and had no effect in wt-PC12 and NIH/3T3. Strong arrest in the S-G2/M phases of the cell cycle, supported by analysis of cyclins (A, B1, D1, E) levels, was observed in AQP3-expressing cells treated with Auphen. Flow-cytometry of propidium iodide incorporation and measurements of mitochondrial dehydrogenases activity confirmed absence of cytotoxic effect of the drug. Functional studies evidenced ≈50% inhibition of A431 Pgly by Auphen, showing that the compound's anti-proliferative effect correlates with its ability to inhibit AQP3 Pgly. Role of Cys-40 on AQP3 permeability blockage by Auphen was confirmed by analyzing the mutated protein (AQP3-Ser-40). Accordingly, cells transfected with mutated AQP3 gained resistance to the antiproliferative effect of Auphen. These results highlight an Auphen inhibitory effect on proliferation of cells expressing AQP3 and suggest a targeted therapeutic effect on carcinomas with large AQP3 expression. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 11/2014; 229(11). DOI:10.1002/jcp.24632 · 3.84 Impact Factor
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    • "This study demonstrated that pulmonary edema associated with oral gavage in the acute toxicity test was categorized as alveologenic edema exhibiting higher AQP-1 and -4 expression (Fig. 1M-W) than in the sub-chronic test (Table 2). Lung AQP-1 is markedly upregulated in animals exposed to hypoxia, suggesting that AQP-1 has O2 permeability and thus could facilitate O2 diffusion across the cell membrane37. AQP-4 mRNA expression is upregulated on the alveolar type II cell membrane to regulate the exchange of fluid between the alveolar space and alveolar epithelium barrier and play an important compensational role in pulmonary liquid clearance in the event of sodium transport damages in acute lung injury19,45. "
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    ABSTRACT: A one-time oral gavage can be enough to cause of alveologenic edema with higher expression of AQP-1 and -4 than that with repeated-dose oral gavage, which caused both profound perivascular edema and hydrostatic pressure edema, while AQP-5 was similarly expressed. The alteration of AQPs expression was probably related to alveolar fluid clearance across the alveolar and bronchiolar epithelium in different stages of lung injury. The results clarified the type of lung edema in acute and sub-chronic toxicity studies without treatment related effect of tested material. The pathogenesis of pulmonary edema due to oral gavage toxicological study is associated with the cellular immune response to the reflux materials. Mast cell and leukocyte accumulation may contribute to increase vascular permeability leading to permeability edema. The increase in alveolar septum epithelium, perivascular and peribronchial cuffing, accumulation alveolar lipid containing macrophage and medial hyperplasia of the pulmonary artery might have been caused to increase airway resistance, which resulted in hydrostatic pressure edema.
    Journal of Toxicologic Pathology 09/2013; 26(3):283-91. DOI:10.1293/tox.26.283 · 0.53 Impact Factor
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    • "Levels of AQP overexpression were determined by Northern blot analysis and expressed relative to wild type PC12. RNA analysis and functional characterization of these clones have been described in detail previously [43]. "
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    ABSTRACT: Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC(50) = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.
    PLoS ONE 05/2012; 7(5):e37435. DOI:10.1371/journal.pone.0037435 · 3.23 Impact Factor
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