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. 2014 May 1;28(9):1005-17.
doi: 10.1101/gad.238709.114.

PI3K-mediated PDGFRα Signaling Regulates Survival and Proliferation in Skeletal Development Through p53-dependent Intracellular Pathways

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Free PMC article

PI3K-mediated PDGFRα Signaling Regulates Survival and Proliferation in Skeletal Development Through p53-dependent Intracellular Pathways

Katherine A Fantauzzo et al. Genes Dev. .
Free PMC article

Abstract

Previous studies have identified phosphatidylinositol 3-kinase (PI3K) as the main downstream effector of PDGFRα signaling during murine skeletal development. Autophosphorylation mutant knock-in embryos in which PDGFRα is unable to bind PI3K (Pdgfra(PI3K/PI3K)) exhibit skeletal defects affecting the palatal shelves, shoulder girdle, vertebrae, and sternum. To identify proteins phosphorylated by Akt downstream from PI3K-mediated PDGFRα signaling, we immunoprecipitated Akt phosphorylation substrates from PDGF-AA-treated primary mouse embryonic palatal mesenchyme (MEPM) lysates and analyzed the peptides by nanoliquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS). Our analysis generated a list of 56 proteins, including 10 that regulate cell survival and proliferation. We demonstrate that MEPM cell survival is impaired in the presence of a PI3K inhibitor and that Pdgfra(PI3K/PI3K)-derived MEPMs do not proliferate in response to PDGF-AA treatment. Several of the identified Akt phosphorylation targets, including Ybox1, mediate cell survival through regulation of p53. We show that Ybox1 binds both the Trp53 promoter and the p53 protein and that expression of Trp53 is significantly decreased upon PDGF-AA treatment in MEPMs. Finally, we demonstrate that introduction of a Trp53-null allele attenuates the vertebral defects found in Pdgfra(PI3K/PI3K) neonates. Our findings identify p53 as a novel effector downstream from PI3K-engaged PDGFRα signaling that regulates survival and proliferation during skeletal development in vivo.

Keywords: PDGFRα; PI3K; p53; proliferation; skeleton; survival.

Figures

Figure 1.
Figure 1.
Midgestation morphological abnormalities in PdgfraPI3K/PI3K mutant embryos. (AP) Gross morphology of Pdgfra+/+ versus PdgfraPI3K/PI3K embryos at E11.5–E14.5. Note the reduced size of mutant embryos at all time points. Fusion of the medial nasal processes and maxillary region is delayed (red arrows) at E11.5–E12.5 (D,H) and E13.5 (L), respectively, in PdgfraPI3K/PI3K embryos. Subepidermal blebbing (blue arrowheads) is common in the mutant embryos (J,N), where it often spans the facial midline and is accompanied by hemorrhaging (L,P; red arrowheads). Wavy neural tubes (green arrowhead) are often observed in earlier embryos and are occasionally accompanied by hemorrhaging (shown in B).
Figure 2.
Figure 2.
Akt phosphosubstrate expression in the embryo. (AH) Whole-mount immunohistochemistry of E10.5 (AE) and E13.5 (FH) embryos with an anti-Akt phosphosubstrate antibody generated against the phosphorylated Akt consensus recognition motif. Noticeably increased expression was detected in the forebrain (A,F), in the maxillary processes (B), in the tactile follicles (F,F′), in the limbs (C,F′), and along the neural tube into the tail (D,E,GH). (I,J) Immunohistochemistry performed on paraffin sections with the anti-Akt phosphosubstrate antibody. Note the increased expression in the palatal shelves (I) and the neuroepithelium of the neural tube and the dorsal root ganglia (J) at E13.5. (J, inset) No signal was detected in a secondary antibody control section of the neural tube and surrounding tissue (outlined with dashed lines) at E13.5. (FB) Forebrain; (MxP) maxillary process; (FL) forelimb bud; (HL) hindlimb bud; (NT) neural tube; (VF) vibrissae follicles; (PS) palatal shelves; (T) tongue; (DRG) dorsal root ganglia; (MN) motor neurons. Bars, 100 μm.
Figure 3.
Figure 3.
PDGF-AA treatment results in the differential phosphorylation of several Akt targets in primary MEPM cells. (A) PDGF-AA-dependent Akt and Erk1/2 phosphorylation time course in E13.5 primary MEPM lysates. Following serum starvation, low-passage MEPM cells were stimulated with 10 ng/mL PDGF-AA ligand for 2 min to 2 h. Western blot analysis of whole-cell lysates revealed peaks of both Akt and Erk1/2 phosphorylation 15 min after ligand treatment. (B) PDGF-AA-dependent Akt target phosphorylation in E13.5 primary MEPM lysates. Immunoprecipitation of Akt targets from lysates of primary MEPM cells that were untreated or treated for 15 min with 10 ng/mL PDGF-AA ligand with an anti-Akt phosphosubstrate antibody followed by Western blotting with the same antibody revealed 14 protein bands (arrowheads) that increased in intensity upon ligand stimulation. Large bands at ∼50 kDa correspond to the heavy chain of IgG. (WB) Western blot; (WCL) whole-cell lysate; (IP) immunoprecipitation.
Figure 4.
Figure 4.
Mass spectrometry-based identification of PDGF-AA-dependent Akt phosphorylation targets in primary MEPM cells. (A) Filters applied to the nano-LC-MS/MS data set. (B) Proteins identified are listed based on the ratio of the number of quantitative spectra detected in PDGF-AA-treated (+) versus untreated (−) samples. The log2 of this ratio for each protein is represented colorimetrically in a heat map at right. Additional information listed includes protein name, detection in biological replicates, coverage, protein identification probability, and quantitative spectral counts for untreated (−) and PDGF-AA-treated (+) samples as determined using Scaffold 3 software. Replicate(s), coverage, and protein identification probability pertain to the sample (untreated or PDGF-AA-treated) in which each protein had the highest number of quantitative spectra. (C) Comparison of the proteins identified in this study (56) versus previously identified Akt phosphorylation targets in humans, mice, and/or rats (167) versus proteins for which phosphorylation at an Akt consensus site is sensitive to independent treatment with the PDGFRα inhibitor Gleevec and the PI3K inhibitor Wortmannin (Wm) in H1703 cells, a non-small-cell lung cancer cell line driven by PDGFRα (17) (Moritz et al. 2010). Of the proteins identified in this study, 46 are potentially novel Akt phosphorylation targets.
Figure 5.
Figure 5.
PI3K-engaged PDGFRα signaling mediates cell survival and proliferation in primary MEPM cells. (A) Cell growth as assessed by crystal violet staining in E13.5 primary MEPM cells grown in medium containing 10% FBS and treated once with LY294002 or H2O2 and/or daily with PDGF-AA for up to 4 d. MEPM cell survival is impaired in the presence of the PI3K inhibitor LY294002 (64.88% ± 1.538% decrease in relative absorbance; P = 0.0130) regardless of PDGF-AA ligand stimulation (68.48% ± 0.2966% decrease in relative absorbance; P = 0.0197). PDGF-AA treatment provides protection from hydrogen peroxide-induced apoptosis (54.88% ± 26.34% increase in relative absorbance; P = 0.1653). (B) Cell growth as assessed by crystal violet staining in Pdgfra+/+- and PdgfraPI3K/PI3K-derived E13.5 primary MEPM cells grown in medium containing 10% or 0.1% FBS and treated with PDGF-AA for 1 d. PdgfraPI3K/PI3K-derived MEPM cells under serum starvation conditions do not proliferate in response to PDGF-AA treatment. Data are presented as mean ± SEM.
Figure 6.
Figure 6.
p53 transcription is inhibited downstream from PDGFRα signaling. (AA″) Ybx1 transcripts are robustly expressed in the facial processes, somites, and neural tube at E10.5, as determined by whole-mount in situ hybridization. (BB″) Trp53 transcripts also localize to the facial processes, somites, and neural tube at E10.5. (C) Ybox1 bound the p53 protein (arrowhead) following 30 min of PDGF-AA treatment in coimmunoprecipitation experiments using primary MEPM lysates. (WB) Western blot; (WCL) whole-cell lysate; (IP) immunoprecipitation. (D) Ybox1 bound up to two sites in the Trp53 promoter (−8/−4 and +50/+54) within the promoter region 3 (pR3) amplicon (−51/+115) exclusively following 30 min of PDGF-AA treatment in endogenous ChIP experiments in primary MEPM cells. No binding was observed in this region of the promoter in an IgG control sample or in the absence of ligand. Furthermore, no binding was observed in a coding sequence (CDS) negative control region. (E) Bar graph depicting qRT-PCR values revealing significantly reduced expression of Trp53 transcripts 30 min (48.15% ± 11.14%; P = 0.0124) to 24 h (51.99% ± 5.61%; P = 0.0008) after PDGF-AA treatment of primary MEPM cells. Data are presented as mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.
Figure 7.
Figure 7.
A subset of axial skeleton defects in PdgfraPI3K/PI3K;Trp53+/+ neonates are rescued in PdgfraPI3K/PI3K;Trp53+/− littermates. (AC) Skeletal preparations of the ribs, vertebrae, and scapular blades with acromions generated from Pdgfra+/+;Trp53+/+ (A), PdgfraPI3K/PI3K;Trp53+/+ (B), and PdgfraPI3K/PI3K;Trp53+/− (C) littermate pups. Note the asymmetric vertebral fusions of thoracic vertebrae 4–5 and the incomplete closure of the vertebral arches of thoracic vertebra 9 and lumbar vertebra 1 in a PdgfraPI3K/PI3K;Trp53+/+ skeleton (B) that are rescued in a PdgfraPI3K/PI3K;Trp53+/− skeleton (C). (T) Thoracic vertebra; (L) lumbar vertebra. (D) Pdgfra+/PI3K;Trp53+/− intercross skeletal phenotype scoring. (EG″) Alcian blue-stained micromass cultures generated from E11.5 somites (EE″), maxillary processes (FF″), and medial nasal processes (GG″). Bars, 1 mm. (H) Bar graph depicting absorbance readings from Alcian blue-stained micromass cultures. While all three cultures showed an increase in chondrogenesis upon PDGF-AA ligand treatment and a decrease in the presence of the PI3K inhibitor LY294002, these responses were more significant in the cultures derived from the facial processes (122.8% ± 3.790% increase upon PDGF-AA treatment in medial nasal processes, P = 0.0038; 94.00% ± 2.954% decrease upon LY294002 treatment in medial nasal processes, P = 0.0002) than those derived from the somites (13.74% ± 5.457% increase upon PDGF-AA treatment, P = 0.4467; 39.88% ± 5.108% decrease upon LY294002 treatment, P = 0.0336). Data are presented as mean ± SEM. (Max) Maxillary process; (MNP) medial nasal process. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

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