PI3K/Akt-mediated PDGFRalpha signaling in craniofacial development

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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.