Abstract
The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5), we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70%) decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels.
Highlights
Aquaporins are a family of membrane water channels that are required for the transport of water through many secretory and absorptive epithelia [1,2,3]
Since it is well known that the alveolar epithelium in the lung is a key anatomical site for both gas exchange and fluid transport, we considered the possibility that oxygen tension regulates the expression of Aquaporin 5 (AQP5), and tested this hypothesis by examining the effect of hypoxic stress on AQP5 expression in lungs of mice exposed to hypoxia and in the mouse lung epithelial cell line MLE12
To investigate whether hypoxia affects AQP5 expression, MLE12 cells were exposed to 1% O2 for 24 h in a hypoxic chamber, and total RNA or protein extracts were prepared after addition of a chaotropic agent that prevented reoxygenation
Summary
Aquaporins are a family of membrane water channels that are required for the transport of water through many secretory and absorptive epithelia [1,2,3]. Aquaporin 5 (AQP5), a member of the AQP family is highly expressed in the mammalian lung, brain, salivary glands, and lachrymal glands. In the lung, it is expressed on the apical surface of both type I and type II alveolar epithelial cells [4,5]. Hypoxic stress occurs in many physiologic and pathologic conditions, such as decrease in alveolar oxygen tension during ascent to high altitude, or as a consequence of hypoventilation related to central nervous disorders, obstructive airway disease, or acute lung injury [8,9]. Previous studies have shown that hypoxia and Co++ affect the expression of a number of genes that play a central role in remodeling the lung in response to hypoxic stress, including up-regulation of the transcriptional activator hypoxiainducible factor (HIF-1a) [10,11,12], sometimes considered a ‘‘master regulator’’ of adaptive responses to hypoxia
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