Molecular mechanisms regulating lung development

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Betaglycan is a cell-surface proteoglycan that binds to many cytokines and growth factors. Although altering betaglycan expression is thought to affect lung development, the mechanism(s) involved remain(s) unknown. Our previous studies showed that betaglycan binds TGF receptors and enhances TGF-dependent gene transcription. However, our recent results suggest that siRNA targeting betaglycan increases the expression of the hepatocyte growth factor (HGF) receptor, c-Met, as well as the metalloproteinases MMP1 and MMP14. Based on these observations, we hypothesize that betaglycan inhibits lung branching morphogenesis by suppressing receptor tyrosine kinase (c-Met) and/or MMP activities. We will test this hypothesis in the following three aims. Aim 1. Assess how betaglycan influences HGF/c-Met signalling in lung cells. Our preliminary results indicate that siRNA targeting betaglycan increases the expression of the HGF receptor, c-Met. To test if betaglycan influences HGF signaling in cultured lung cells, we will incubate control or betaglycan siRNA cells with HGF and carry out western blotting using phospho-specific antibodies to assess pathway activation. We will also assess gene expression via qPCR, followed by western blotting to assess protein levels. Identification of the signaling pathways involved in betaglycan modulation of HGF signaling will be assessed using specific pharmacological inhibitors. Aim 2. Assess the role of betaglycan on MMP activity of lung cells. We will assess the activities of MMP1 and MMP14 in lung cells, as siRNA targeting betaglycan increases the expression of these two MMP family members. Briefly, MMP1 production, secretion and activity will be assessed from cultured media using sensitive ELISA kits. SDS-PAGE zymography will be used to detect MMP-1 and membrane-tethered MMP14 activity in culture media and/or cellular lysates. The effects of pharmacological MMP inhibition will then be assessed in mouse lung explant assays. Aim 3. Characterize the role of betaglycan in mouse lung branching morphogenesis. We plan to carry out ex vivo cultures of E11.5-12.5 mouse lungs, and branching morphology will be assessed by brightfield microscopy. We will analyze lungs from WT, betaglycan (Tbr3-/-) whole body null and conditional lung null mice, and assess cell migration, tissue remodeling and specific signaling pathways using IHC. The relative roles of c-Met and MMP involvement will be analyzed using ligands and/or pharmacological inhibitors described in aims 1 and 2. Significance Our previous work established that betaglycan influences TGF-dependent transcription. However, the specific regulation of betaglycan in growth factor and MMP-dependent regulation of lung development remains unclear. Further research will elucidate the mechanism(s) of how this proteoglycan influences lung morphogenesis, thus expanding our understanding of the fundamentals of lung development.