PI3K/Akt-mediated PDGFRalpha signaling in craniofacial development

描述(申请人提供)：颅面发育是一个复杂的形态发生过程，其中的干扰导致非常普遍的出生缺陷在人类。血小板衍生生长因子(PDGF)信号在颅面发育中起着关键作用，该通路突变的小鼠模型的表型就证明了这一点，突变的范围从腭裂到额鼻部肿块缩短和完全性面部裂隙。虽然人们已经从细胞和分子水平从生化角度对PDGF信号进行了广泛的分析，但该生长因子信号通路及其下游效应因子在颅面部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信号的主要下游效应器，然而，更多的下游途径目前尚不清楚。由于PI3K/Akt信号影响在增殖、细胞生长或代谢中具有不同功能的信号通路，因此在定义活体颅面发育时，建立在现有知识的基础上并挑战PDGFR调控的细胞内信号通路是至关重要的。这些研究将更全面地了解PDGF信号在中线发育中的机制及其在人类疾病中的破坏作用。这项建议的目的是确定和表征PI3K介导的PDGFR信号下游在颅面发育中的细胞内通路。我们将结合最先进的蛋白质组学方法与体外细胞活性分析和体内遗传相互作用研究，以表征Akt磷酸化靶标在中线发育中的作用。首先，通过蛋白质印迹分析检测不同PDGFR信号环境下原代小鼠胚胎腭间充质(MEPM)细胞Akt下游蛋白的磷酸化情况，并通过质谱仪鉴定差异磷酸化的蛋白质。其次，通过转染表达载体或siRNA双链，在体外分析Akt磷酸化靶向错误表达对MEPM细胞增殖、生长和迁移的影响。最后，通过PDGFR和靶基因突变小鼠之间的遗传上位性实验，将在体内表征Akt磷酸化靶标在头面部发育中的作用。这里提出的创新性研究将通过表征PI3K介导的PDGFR信号下游的细胞内通路在中线发育中对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这一生长因子信号通路可用的大量试剂来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。 公共卫生相关性：血小板衍生生长因子(PDGF)信号已被证明在发育期间和成年生活中调节体内的许多过程，PDGF信号异常与颅面发育缺陷、肿瘤、血管疾病和纤维性疾病有关。包括唇腭裂(CL/P)在内的颅面部发育缺陷是人类最常见的出生缺陷，估计活产儿的发病率为1/500至1/2000。PDGF信号转导机制的研究将对颅面部发育的细胞内途径和分子机制提供有价值的见解，并最终为预防颅面部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.