Elucidating the role of caveolin-1 in postnatal lung

Abstract

Caveolin-1 (cav-1), an integral membrane protein is the main structural component of 50-100 nm omega-shaped invaginations of the plasma membrane called caveolae. The Caveolins, a gene family comprised of Caveolins -1, -2, and -3, are expressed in numerous cell types, and have been shown to regulate a variety of cellular processes including signal transduction, cholesterol homeostasis, and endocytosis. The cav-1-null mouse is known to develop a fibrotic lung phenotype with endothelial and interstitial hyperproliferation. The aim of this study is to characterize postnatal alveolar development in the cav-1-null mouse, and to characterize the expression and function of cav-1 in the alveolar epithelium. Morphometric analysis of lung sections from the cav-1-null mouse revealed that both alveolar diameter and septal thinning are significantly reduced in the null as compared to wild type. Septal outgrowth is developmentally delayed, and is decreased both in number and in length at key developmental timepoints. Markedly increased numbers of type II alveolar epithelial cells are associated with each alveolus, as observed by immunohistochemical staining of surfactant proteins and ultrastructural analysis by transmission electron microscopy. The ultrastructure of tight junctions of epithelial membranes is also altered, as is the morphology of the membranes of type I cells. Hypercellularity observed in the interstitial compartment is comprised immature blast-like endothelial cell precursors. Molecular analysis by immunofluorescence, immunohistochemistry, and Western blot revealed that the two isoforms of cav-1 show non-overlapping patterns of expression in the lungs, with cav-1beta localizing to the alveolar epithelium, while the more prevalent alpha-isoform is highly expressed in the endothelium. Interestingly, the expression of glucocorticoid receptor (GR) protein, a key mediator of alveolar development, is upregulated in the cav-1-null mouse throughout postnatal lung development, with the highest expression localizing to type II cells. Furthermore, immunoprecipitation from lung lysates showed that the GR interacts selectively with cav-1beta. The novel interaction of cav-1beta with GR is intriguing, as a unique function for the beta-isoform, which lacks the N-terminal 31 amino acids of the longer alpha-isoform, has yet to be demonstrated in any cell type. The potential biological consequences of this interaction were assessed utilizing isolated primary type II cells: a novel nuclear translocation assay demonstrated that the sub-cellular localization of GR was not altered in the absence of cav-1 - cytoplasmic GR translocated to the nucleus following the administration of the GR-agonist dexamethasone in cells isolated from both wild-type and cav-1-null mice. Despite the observation that cav-1 is upregulated as type II cells differentiate in vivo and in vitro, the spontaneous differentiation of cav-1-null primary cells into type I-like cells in culture appears to progress normally, as assessed by morphology and the expression of type I-cell specific molecular markers. As it is known that cav-1 can function a mediator of signal transduction, the activity of GR-mediated target genes was characterized. Western blot analysis of lung lysates utilizing phospho-specific antibodies demonstrated that the MAP Kinase pathway is resistant to downregulation in cav-1-null mice following the in vivo administration of dexamethasone. Utilizing quantitative real-time RT-PCR, we assessed the transcriptional activity of lung-specific genes and primary GR target genes in vivo throughout differentiation and in response to treatment with dexamethasone, demonstrating that GR-mediated signaling is dysregulated in vivo, with constitutive activity of GR target genes. Intriguingly, this dysregulation is limited to type II AEC-specific genes, while signaling in other cell types remains intact. The results of these experiments suggest that cav-1 may serve as a modulator of GR activity and function. As cav-1 has been shown to play a role in human lung cancer, fibrotic lung diseases, and perinatal lung development, future work exploring the mechanism and biological implications of the GR/cav-1beta interaction could yield important insight into the developmental biology and pathobiology of the lung.