Adenovirus has re-emerged as a key pathogen linked to epidemic acute respiratory illness. Fatal adenoviral infection produces the acute respiratory distress syndrome (ARDS) with prominent injury to alveolar epithelia. Alveolar type II epithelia produce surfactant, an essential surface-active mixture deficient in ARDS and highly enriched with dipalmitoylphosphatidylcholine (DPPC). Newly synthesized surfactant in type II epithelial cells is packaged into a storage form and secreted into the alveolus by a well-established apical route. To date, there is limited information on the molecular mechanisms whereby viruses might interfere with this model of surfactant trafficking. The ATP-binding cassette transporter 1, ABCA1, has emerged as a key protein that directs basolateral trafficking of lipids to suitable acceptor proteins within the circulation. This revised competing renewal expands on recent advances in our laboratory showing that i) wild-type adenovirus decreases surfactant DPPC levels in vivo and in vitro, ii) alveolar epithelia export primarily nonsurfactant phosphatidylcholine (PC) basolaterally, iii) this latter process is mediated by ABCA1, and iv) that adenovirus stimulates basolateral surfactant export by increasing ABCA1 activity, protein, and mRNA. These observations led to our overall hypothesis that ABCA1 is a virally-regulated molecular sensor that modulates alveolar epithelial surfactant PC content and composition by increasing basolateral phospholipid efflux. Thus, this proposal will investigate for the first time a novel, basolateral exit route for surfactant and its regulation by adenovirus. We will determine if ABCA1 is an adenovirally-regulated basolateral export pump that controls surfactant PC content and composition (Aim 1) and determine if adenovirus decreases surfactant PC by transcriptional activation of the ABCA1 gene (Aim 2).
In Aim 1, we will modulate ABCA1 activity using complementary strategies (ABCA1 dominant-negative, apical ABCA1 targeting, and siRNA approaches) to counteract adenoviral effects on surfactant trafficking.
In Aim 2, we will perform deletional and mutational analysis to identify the adenovirally-regulated c/s-acting elements within the 5'flanking region of the ABCA1 gene. Our hypothesis will be tested by in vivo administration of adenovirus with analysis conducted in primary type II alveolar epithelial cells. These studies will be supplemented with ABCA1 knockout mice, cells from Tangier Disease patients (lacking a functional ABCA1), and an adenoviral-sensitive type II (MLE-12) cell line. The unique contributions of this proposal impacting the field of lung injury include 1) delineation of a novel exit pathway for surfactant, catalyzed in part by ABCA1, that impacts the type II cell lipophenotype, 2) studies linking adenoviral-signaling with surfactant trafficking, and 3) investigation of ABCA1 gene transcription which represents a new effector mechanism whereby viruses might deplete cells of surfactant in the setting of acute lung injury.
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