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 · 3.87 Impact Factor
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    • "The aquaporins that have been identified demonstrate tissue-specific localization and membranes that express aquaporins are more water permeable than membranes lacking aquaporins (King and Agre 1996; Saadoun et al. 2005; Yang et al. 2001). AQP1 is highly expressed in microvascular endothelia and has been shown to accelerate cytosolic hypoxia and to regulate hypoxiainducible genes (Echevarria et al. 2007). Although mice lacking AQP1 show decreased pulmonary vascular permeability and are not able to create a hypertonic renal medullary interstitium to regulate urine concentration, no clinical symptoms have yet been identified as a consequence of AQP1 deficiency (King and Agre 1996; Ma et al. 1998; Umenishi and Schrier 2003; Verkman 2008). "
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    ABSTRACT: VACM-1, a cul5 gene product, when overexpressed in vitro, has an antiproliferative effect. In vivo, VACM-1/cul5 is present in tissues involved in the regulation of water balance. Neither proteins targeted for VACM-1/cul5-specific degradation nor factors that may regulate its expression in those tissues have been studied. To identify genes that may be misregulated by VACM-1 cDNA, we performed microarray analysis. Our results indicate that in cos-1 cells transfected with VACM-1 cDNA, mRNA levels for several genes, including AQP1, were decreased when compared to the control group. Our results also indicate that in cos-1 cells transfected with VACM-1 cDNA, endogenous AQP1 protein was decreased about 6-fold when compared to the controls. To test the hypothesis that VACM-1/cul5 may be regulated by conditions that compromise water homeostasis in vivo, we determined if 24 h of water deprivation affects VACM-1/cul5 levels or the effect of VACM-1/cul5 on AQP1. VACM-1 mRNA and protein levels were significantly higher in rat mesenteric arteries, skeletal muscle and the heart ventricle but not in the heart atrium from 24-h water-deprived rats when compared to the controls. Interestingly, 24 h of water deprivation increased modification of VACM-1 by an ubiquitin-like protein, Nedd8, essential for cullin-dependent E3 ligase activity. Although water deprivation did not significantly change AQP1 levels in the mesenteric arteries, AQP1 protein concentrations were inversely correlated with the ratio of the VACM-1 to Nedd8-modified VACM-1. These results suggest that VACM-1/cul5 may regulate endothelial AQP1 concentration both in vivo and in vitro.
    Cell and Tissue Research 05/2012; 349(2):527-39. DOI:10.1007/s00441-012-1419-3 · 3.33 Impact Factor
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    • "However, some studies suggest that oxygen diffusion through pure phospholipid membranes may be some orders of magnitude slower than expected, mainly due to the high microviscosity of bilayers [9]. As a matter of fact, an active line of research is trying to identify potential protein channels that could facilitate transport of gases through cell membranes [10] [11]. "
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    ABSTRACT: During respiration, it is accepted that oxygen diffuses passively from the lung alveolar spaces through the respiratory epithelium until reaching the pulmonary capillaries, where blood is oxygenated. It is also widely assumed that pulmonary surfactant, a lipid-protein complex secreted into alveolar spaces, has a main surface active function, essential to stabilize the air-liquid interface, reducing in this way the work of breathing. The results of the present work show that capillary water layers containing enough density of pulmonary surfactant membranes transport oxygen much faster than a pure water phase or a water layer saturated with purely lipidic membranes. Membranes reconstituted from whole pulmonary surfactant organic extract, containing all the lipids plus the hydrophobic surfactant proteins, permit also very rapid oxygen diffusion, substantially faster than achieved by membranes prepared from the surfactant lipid fraction depleted of proteins. A model is proposed suggesting that protein-promoted membrane networks formed by pulmonary surfactant might have important properties to facilitate oxygenation through the thin water layer covering the respiratory surface.
    Biochimica et Biophysica Acta 03/2010; 1798(6):1281-4. DOI:10.1016/j.bbamem.2010.03.008 · 4.66 Impact Factor
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