Lung development is a continuous process involving epithelio-mesenchymal interactions. Laminin, a basement membrane-related glycoprotein, plays a critical role in this process. Preliminary evidence indicates that both epithelium and mesenchyme produce laminin, and that the biological activity of this glycoprotein in the mouse embryonic lung is mediated at least by two different molecular domains. The goal of these investigations will be to precisely define laminin sites active during murine lung development and to establish structure-function correlations. The effects of a series of antibodies to specific laminin epitopes, synthetic peptides, including sequences from the two molecular domains of interest, and laminin fragments will be used for functional studies. Cell-cell and cell-basement membrane interactions will be perturbed by microinjecting nanomolar quantities of these probes into the extracellular space of the epithelial and mesenchymal compartments of embryonic lung explants developing in vitro. The effects of these interventions on morphogenesis, branching activity (at branching and non-branching points), cell phenotype, proliferation and basement membrane formation will be determined. These studies will establish whether the effects of laminin in the lung are general or cell/site- specific and will define molecular sites biologically active in lung organogenesis. The role of laminin active sites in modulating primary cell behaviors essential for morphogenesis will be established by using synthetic peptides and antibodies to promote or block epithelial and mesenchymal cell adhesion, motility and proliferation. The role of laminin in morphogenic events requiring cell-cell recognition will be elucidated next by determining molecular domains involved in cell sorting and polarization as it takes place in organotypic cultures of embryonic lung cells. Elucidation of laminin involvement in adhesion, motility, proliferation, sorting and polarization of epithelial and mesenchymal cells will provide the cellular basis to further interpret the results observed at the organ culture level. Mechanistic conclusions will be drawn on how epithelium, mesenchyme and laminin interact during normal lung development. Eventually, this investigation will define possible reciprocal interactions between epithelium and mesenchyme in the control of laminin expression by studying the effects of transient exposure of one cell type to the other. A combination of SDS-PAGE, Northern blot analysis, and mRNA in situ hybridization will be employed to detect changes in laminin expression. This integrative approach will provide new information important for the understanding of normal lung development. It may also contribute to the elucidation of pathological conditions that result or lead to defective lung development, such as lung hypoplasia, bronchopulmonary sequestration, bronchopulmonary dysplasia, as well as other abnormal conditions in which the extracellular matrix is involved.
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| Badri, Kameswara Rao; Yue, Ming; Carretero, Oscar A et al. (2013) Blood pressure homeostasis is maintained by a P311-TGF-? axis. J Clin Invest 123:4502-12 |
