Molecular mechanisms regulating lung development

Betaglycan是一种细胞表面蛋白多糖，可与多种细胞因子和生长因子结合。虽然改变β-聚糖的表达被认为会影响肺的发育，但其中涉及的机制(S)(S)仍不清楚。我们先前的研究表明，β-葡聚糖能结合转化生长因子受体，增强转化生长因子β依赖的基因转录。然而，我们最近的结果表明，针对β-聚糖的siRNA增加了肝细胞生长因子(HGF)受体c-Met以及金属蛋白酶MMP1和MMP14的表达。基于这些观察，我们假设Betaglycan通过抑制受体酪氨酸激酶(c-Met)和/或MMP的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。目的1.研究β-葡聚糖对肺细胞HGF/c-Met信号转导的影响。我们的初步结果表明，针对β-多糖的siRNA增加了HGF受体c-Met的表达。为了测试Betaglycan是否影响培养的肺细胞中的HGF信号，我们将对照或Betaglycan siRNA细胞与HGF孵育，并使用磷酸化特异性抗体进行蛋白质印迹，以评估途径激活。我们还将通过定量聚合酶链式反应(QPCR)评估基因表达，然后用蛋白质印迹法评估蛋白质水平。将使用特定的药理抑制剂来评估参与β-多聚糖调节HGF信号的信号通路。目的2.探讨β-葡聚糖对肺细胞基质金属蛋白酶活性的影响。我们将评估MMP1和MMP14在肺细胞中的活性，因为针对β-聚糖的siRNA增加了这两个MMP家族成员的表达。简而言之，将使用敏感的酶联免疫吸附试验试剂盒从培养上清液中评估MMP1的产生、分泌和活性。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法检测培养上清液和/或细胞裂解物中的基质金属蛋白酶-1和膜系带基质金属蛋白酶14的活性。然后将在小鼠肺外植体试验中评估药物对基质金属蛋白酶的抑制作用。目的3.研究β-多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小鼠肺的体外培养，分支形态将通过Brightfield显微镜进行评估。我们将分析WT，Betaglycan(Tbr3-/-)全身缺失和条件性肺缺失小鼠的肺，并使用IHC评估细胞迁移、组织重塑和特定的信号通路。将使用目标1和2中描述的配体和/或药物抑制剂来分析c-Met和基质金属蛋白酶参与的相对作用。意义我们先前的工作确定了β-聚糖影响依赖于转化生长因子的转录。然而，Betaglycan对生长因子的具体调控以及对肺发育的基质金属蛋白酶依赖的调控仍不清楚。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(S)，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 TGFß receptors and enhances TGFß-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 TGFß-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.