PI3K/Akt-mediated PDGFRalpha signaling in craniofacial development

描述（由申请人提供）：颅面发育是一个复杂的形态发生过程，其中断导致人类出生缺陷非常普遍。血小板源性生长因子（PDGF）信号传导在颅面发育中起着关键作用，这一点可以通过该途径突变的小鼠模型的表型来证明，其范围从腭裂到额鼻肿块缩短和完全的面部裂开。虽然PDGF信号已经从细胞和分子水平的生物化学角度进行了广泛的分析，但这种生长因子信号通路及其下游效应物在颅面发育中的作用才刚刚开始探索。Soriano实验室以前的工作已经确定PI 3 K是小鼠颅面发育过程中PDGFR信号传导的主要下游效应子，然而，进一步的下游途径目前尚不清楚。由于PI 3 K/Akt信号传导影响在增殖、细胞生长或代谢中具有如此不同功能的信号传导途径，因此建立在现有知识的基础上并在定义体内颅面发育中定义由PDGFR调节的细胞内信号传导途径是至关重要的。这些研究将提供一个更完整的了解PDGF信号传导的机制，在中线的发展和它的中断在人类疾病。该提案的目的是确定和表征颅面发育中PI 3 K介导的PDGFR信号传导下游的细胞内途径。我们将联合收割机最先进的蛋白质组学方法与体外细胞活性测定和体内遗传相互作用研究相结合，以表征Akt磷酸化靶点在中线发育过程中的作用。首先，通过蛋白质印迹分析在各种PDGFR信号传导背景下在原代小鼠胚胎腭间充质（MEPM）细胞中检查Akt下游的蛋白质磷酸化，并通过质谱鉴定差异磷酸化的蛋白质。其次，通过转染表达质粒或siRNA双链体，体外分析Akt磷酸化靶点错误表达对MEPM细胞增殖、生长和迁移的影响。最后，Akt磷酸化靶点在颅面发育中的作用将通过PDGFR和靶基因突变小鼠之间的遗传上位性实验在体内进行表征。本文提出的创新研究将通过表征中线发育中PI 3 K介导的PDGFR信号传导下游的细胞内途径，为PDGF信号传导在体内的作用提供关键的见解。我们的研究策略将利用可用于这种生长因子信号通路的大量试剂来探索颅面生物学的新方面，并最终提供旨在预防颅面出生缺陷的新治疗方向。

DESCRIPTION (provided by applicant): Craniofacial development is a complex morphogenic process, disruptions in which result in highly prevalent birth defects in humans. Platelet-derived growth factor (PDGF) signaling plays a critical role in craniofacial development, as evidenced by the phenotypes of mouse models with mutations in the pathway, which range from a cleft palate to shortening of the frontonasal masses and complete facial clefting. While PDGF signaling has been extensively analyzed from a biochemical perspective at the cellular and molecular level, the role of this growth factor signaling pathway and its downstream effectors in craniofacial development have just begun to be explored. Previous work by the Soriano laboratory has identified PI3K as the main downstream effector of PDGFR¿ signaling during craniofacial development in the mouse, however, further downstream pathways are currently unknown. Because PI3K/Akt signaling impacts signaling pathways that have such disparate functions in proliferation, cell growth or metabolism, it is critical to build on the present knowledge and defie the intracellular signaling pathways regulated by PDGFR¿ in defining craniofacial development in vivo. These studies will provide a more complete understanding of the mechanisms of PDGF signaling in midline development and its disruption in human disease. The aim of this proposal is to identify and characterize the intracellular pathways downstream of PI3K-mediated PDGFR¿ signaling in craniofacial development. We will combine state-of-the art proteomic approaches with in vitro cell activity assays and in vivo genetic interaction studies to characterize the role of Akt phosphorylation targets during midline development. First, protein phosphorylation downstream of Akt will be examined in primary mouse embryonic palatal mesenchyme (MEPM) cells under various PDGFR¿ signaling contexts by Western blot analysis and differentially phosphorylated proteins will be identified by mass spectrometry. Secondly, the effect of Akt phosphorylation target misexpression on MEPM cell proliferation, growth and migration will be analyzed in vitro via transfection of expression plasmids or siRNA duplexes. Finally, the role of Akt phosphorylation targets in craniofacial development will be characterized in vivo through genetic epistasis experiments between PDGFR¿ and target gene mutant mice. The innovative studies proposed here will provide critical insight into the role of PDGF signaling in vivo, by characterizing the intracellular pathways downstream of PI3K-mediated PDGFR¿ signaling in midline development. Our research strategy will take advantage of the vast array of reagents available for this growth factor signaling pathway to explore novel aspects of craniofacial biology and ultimately, provide new therapeutic directions aimed at the prevention of craniofacial birth defects.