Molecular mechanisms regulating lung development

β聚糖是一种细胞表面蛋白聚糖，可与多种细胞因子和生长因子结合。 尽管认为改变β聚糖表达会影响肺发育，但涉及的机制仍不清楚。 我们以前的研究表明，β聚糖结合TGF受体并增强TGF依赖性基因转录。然而，我们最近的研究结果表明，siRNA靶向betaglycan增加肝细胞生长因子（HGF）受体，c-Met，以及金属蛋白酶MMP 1和MMP 14的表达。基于这些观察结果，我们假设β聚糖通过抑制受体酪氨酸激酶（c-Met）和/或MMP活性来抑制肺分支形态发生。我们将在以下三个目标中检验这一假设。 目标1.评估betaglycan如何影响肺细胞中的HGF/c-Met信号传导。 我们的初步结果表明，靶向betaglycan的siRNA增加了HGF受体c-Met的表达。为了测试β聚糖是否影响培养的肺细胞中的HGF信号传导，我们将用HGF孵育对照或β聚糖siRNA细胞，并使用磷酸特异性抗体进行蛋白质印迹以评估途径活化。我们还将通过qPCR评估基因表达，然后通过蛋白质印迹法评估蛋白质水平。将使用特定药理学抑制剂评估参与HGF信号传导的β聚糖调节的信号传导途径的鉴定。 目标2.评估β聚糖对肺细胞MMP活性的作用。 我们将评估MMP 1和MMP 14在肺细胞中的活性，因为靶向β聚糖的siRNA增加了这两个MMP家族成员的表达。简言之，将使用灵敏的ELISA试剂盒从培养基评估MMP 1的产生、分泌和活性。将使用SDS-PAGE酶谱法检测培养基和/或细胞裂解物中的MMP-1和膜系MMP 14活性。然后将在小鼠肺外植体测定中评估药理学MMP抑制的作用。 目标3.表征β聚糖在小鼠肺分支形态发生中的作用。 我们计划进行E11.5-12.5小鼠肺的离体培养，并通过明视野显微镜评估分支形态。我们将分析来自WT、β聚糖（Tbr 3-/-）全身无效和条件性肺无效小鼠的肺，并使用IHC评估细胞迁移、组织重塑和特异性信号传导途径。将使用目的1和2中描述的配体和/或药理学抑制剂分析c-Met和MMP参与的相对作用。 意义 我们先前的工作确定β聚糖影响TGF依赖性转录。然而，β聚糖在生长因子和MMP依赖性肺发育调节中的特异性调节仍不清楚。进一步的研究将阐明这种蛋白多糖如何影响肺形态发生的机制，从而扩大我们对肺发育基本原理的理解。

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.