Impact of the epithelial hypoxia-inducible factor 2 alpha/fetal liver kinase-1 system on murine lung development
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In the present study, we hypothesized that the HIF 2 alpha/flk-1 system has a unique role in the pulmonary epithelial system, with further impact on lung development. This hypothesis was based on data by Compernolle et al. showing a severe respiratory distress syndrome upon global HIF 2 alpha deletion which could be overcome by the application of VEGF (Compernolle et al., 2002). However, key functions of the HIF 2 alpha/flk-1 system have been demonstrated to be restricted to the vascular system, to regulate physiological and pathological blood vessel formation. In contrast, reports by Brown et al. and Raoul et al. suggest a direct influence of the flk-1/VEGF system on pulmonary epithelial cell function (Brown et al., 2001; Raoul et al., 2004). Since flk-1 has been shown to be regulated by HIF 2 alpha (Elvert et al., 2003) it represents the functional target molecule of the HIF 2 alpha/flk-1 system. Accordingly, we characterized flk-1 expression from ED12.5 at daily intervals throughout lung development until postnatal stages. We found flk-1 expression, as previously described, in the early primitive vascular network. At the end of the pseudoglandular stage, at ED16.5 we found widespread epithelial expression of flk-1 which persisted until postnatal stages. Furthermore, we analyzed flk-1 expression and functional integrity in isolated adult (Ahlbrecht et al., 2008). To test our hypothesis of a direct functional impact of the HIF 2 alpha/flk-1 system on the pulmonary epithelial system, we analyzed isolated AEC for HIF 2 alpha expression, and found strong expression of HIF 2 alpha at the protein level. Thus, we demonstrated that the HIF 2 alpha/flk-1 system is not restricted to the endothelial system, and is present in the pulmonary alveolar epithelial system. To analyze the functional impact of this newly characterized pulmonary epithelial system, we generated an inducible in vivo pulmonary epithelial HIF 2 alpha deletion combining the SPCrtTA and Cre-loxP system. We succeeded in generating the epithelial HIF 2 alpha deletion which could be demonstrated at the protein level. But, in contrast to our hypothesis that the epithelial HIF 2 alpha/flk-1 system might have functional impact on lung development, epithelial HIF 2 alpha deletion did not lead to a disorder or phenotype. Even though flk-1 was strongly downregulated, no changes were observed regarding lung structure or AEC morphology and SPC expression respectively. This was unexpected. Thus we postulate that the epithelial HIF 2 alpha system works within a complex network. Upon HIF 2 alpha deletion, a compensatory system takes over the HIF 2 alpha-related functions to rescue the deletion to protect the process of lung development. This is highly speculative and further investigations are needed to characterize the different factors involved which were not addressed in the present study. Taken together our study demonstrates: Characterization of flk-1 expression at daily intervals throughout lung development with epithelial expression of flk-1 rising from ED16.5 and persisting at postnatal stages (Ahlbrecht et al., 2008). Furthermore HIF 2 alpha and flk-1 expression could be demonstrated in isolated AEC. Thus, we were able to demonstrate the existence of the epithelial HIF 2 alpha/flk-1 system. Analysis of the direct epithelial function of the newly characterized pulmonary epithelial system could be addressed by successful generation of an pulmonary epithelial-restricted HIF 2 alpha in vivo deletion using the SPCrtTA-tetO-Cre system. Surprisingly, characterization of the phenotype of pulmonary epithelial HIF 2 alpha knock-out mice did not show any disorders or phenotype, or changes in lung structure and epithelial cell development. These data may be basis for future studies elucidating the network of cooperating factors regulating pulmonary epithelial development.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Am J Respir Cell Mol Biol 39 (2008), S. 163-170.