Abstract
Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O2 transport. It has been reported that this protein contributes to gas permeation (CO2, NO and O2) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5′ proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl2) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.
Highlights
During the last decade new functions, locations, and regulatory pathways have been described for AQP1
Induction of vascular endothelial growth factor (Vegf), a well-established hypoxia-responsive gene, was analyzed as control. No induction of this gene was detected in lung tissue, it was observed that Vegf was induced faster than Aqp1 in brain tissue, indicating that expression kinetics of the two genes are different in this organ (Supplementary Figure S1A)
A functional correlation between hypoxic induction of AQP1 and increases in water permeability is indicated here for the first time, and the involvement of the hypoxia-inducible transcription factor hypoxia-inducible transcription factors (HIF)-1a in this phenomenon is analyzed in detail
Summary
During the last decade new functions, locations, and regulatory pathways have been described for AQP1. Expression of AQP1 is known to be associated with inflammatory and neoplastic processes and has been detected in response to mechanical injury in numerous cells and tissues, including astrocytes, pneumocytes, lymphocytes, and several types of tumor cell [7,8,9,10,11,12], in which its presence had not previously suspected. Chronic hypoxia induces the expression of numerous genes by activation of hypoxia-inducible transcription factors (HIF), mainly HIF-1a and HIF-2a [15,16]. In the cerebellum of rats subjected to hypoxia, increments in the mRNA and protein levels of vascular endothelial growth factor (VEGF) and aquaporin-4 (AQP4) were found to be closely associated to an increase in HIF-1a expression. Inhibition of HIF1a by 2-methoxyestradiol reduced the up-regulated levels of both these AQPs [18]
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