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形成过程，其中断导致人类高度普遍的出生缺陷。血小板衍生生长因子（PDGF）信号在颅面发育中起着至关重要的作用，正如该途径突变的小鼠模型的表型所证明的那样，其范围从腭裂到额鼻块缩短和完全面裂。虽然PDGF信号已经从生物化学的角度在细胞和分子水平上进行了广泛的分析，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实验室之前的工作已经确定PI3K是小鼠颅面发育过程中PDGFR信号传导的主要下游效应物，然而，进一步的下游途径目前尚不清楚。由于PI3K/Akt信号通路影响着在增殖、细胞生长或代谢中具有不同功能的信号通路，因此在现有知识的基础上，确定由PDGFR调控的细胞内信号通路在体内定义颅面发育是至关重要的。这些研究将为PDGF信号在中线发育中的机制及其在人类疾病中的破坏提供更完整的理解。本提案的目的是鉴定和表征颅面发育中pi3k介导的PDGFR信号下游的细胞内通路。我们将结合最先进的蛋白质组学方法、体外细胞活性测定和体内遗传相互作用研究来表征Akt磷酸化靶点在中线发育过程中的作用。首先，在不同PDGFR信号背景下，通过Western blot分析小鼠胚胎腭间质（MEPM）细胞中Akt下游的蛋白磷酸化，并通过质谱分析鉴定差异磷酸化蛋白。其次，通过转染表达质粒或siRNA双链，分析Akt磷酸化靶蛋白错表达对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pi3k介导的PDGFR信号在中线发育中的下游细胞内通路，为PDGF信号在体内的作用提供关键的见解。我们的研究策略将利用大量可用于这一生长因子信号通路的试剂，探索颅面生物学的新方面，并最终为预防颅面出生缺陷提供新的治疗方向。

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. PUBLIC HEALTH RELEVANCE: Platelet-derived growth factor (PDGF) signaling has been shown to regulate numerous processes throughout the body, both during development and adult life, and aberrant PDGF signaling has been implicated in defects in craniofacial development, tumors, vascular disorders and fibrotic diseases. Defects in craniofacial development, including cleft lip and palate (CL/P), comprise the most prevalent birth defects in humans, with an estimated incidence of 1/500 to 1/2,000 live births. The PDGF signaling studies proposed here will impart valuable insight into the intracellular pathways and molecular mechanisms contributing to craniofacial development, and ultimately, provide new therapeutic directions aimed at the prevention of craniofacial birth defects.