title: LEOPARD-type SHP2 mutant Gln510Glu attenuates cardiomyocyte differentiation and promotes cardiac hypertrophy via dysregulation of Akt/GSK-3β/β-catenin signaling.
abstract: LEOPARD syndrome (LS) is an autosomal dominant inherited multisystemic disorder. Most cases involve mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase Src homology 2-containing protein phosphatase 2 (SHP2). LS frequently causes severe hypertrophic cardiomyopathy (HCM), even from the fetal period. However, the molecular pathogenesis has not been clearly elucidated. Here, we analyzed the roles of the LS-type SHP2 mutant Gln510Glu (Q510E), which showed the most severe type of HCM in LS, in cardiomyocyte differentiation, and in morphological changes. We generated mutant P19CL6 cell lines, the most convenient cardiomyocyte differentiation model, which continuously expressed SHP2-Q510E, SHP2-D61N (Noonan-type mutant), wild-type SHP2, and green fluorescent protein (native SHP2 expression only). SHP2-Q510E mutant P19CL6 cells showed significant attenuation of myofibrillogenesis, with increased proliferative activity. Mature cardiomyocytes from the SHP2-Q510E mutant were significantly larger than those of controls and the other mutants. However, expression of cardiac-specific transcriptional factors (Gata4, Tbx5, and Nkx2.5) did not differ significantly between the LS-type SHP2-Q510E mutants and the other mutants and controls. Our results indicate that SHP2-Q510E mutants can differentiate into cardiac progenitors but are inhibited from undergoing terminal differentiation into mature cardiomyocytes. In contrast, Akt and glycogen synthase kinase (GSK)-3β phosphorylation were upregulated, and nuclear β-catenin at the late stage of differentiation was highly accumulated in SHP2-Q510E mutant P19CL6 cells. Supplementation with the phosphoinositide 3-kinase/Akt inhibitor LY-294002 during the late stage of differentiation was found to partially restore myofibrillogenesis while suppressing the increase in size of individual mature cardiomyocytes derived from the SHP2-Q510E mutants. Our findings suggest that dysregulation of the Akt/GSK-3β/β-catenin pathway can contribute to the pathogenesis of HCM in LS patients, not only through hypertrophic changes in individual cardiac cells but also via the expansion of cardiac progenitors.
authors.affiliation: Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Ishida
authors.forename: Shigetoyo
authors.initials: S
authors.lastname: Kogaki
authors.forename: Jun
authors.initials: J
authors.lastname: Narita
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Ichimori
authors.forename: Nobutoshi
authors.initials: N
authors.lastname: Nawa
authors.forename: Yoko
authors.initials: Y
authors.lastname: Okada
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Takahashi
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ozono
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D006332:Cardiomegaly / Q000235:genetics / Q000503:physiopathology* / Q000517:prevention & control; D002454:Cell Differentiation / Q000235:genetics*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D004791:Enzyme Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D044542:LEOPARD Syndrome / Q000235:genetics*; D051379:Mice; D009154:Mutation / Q000502:physiology; D032383:Myocytes, Cardiac / Q000502:physiology*; D009210:Myofibrils / Q000187:drug effects; D051058:Oncogene Protein v-akt / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D051176:beta Catenin / Q000502:physiology*
chemical_list: D004791:Enzyme Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051176:beta Catenin; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D051058:Oncogene Protein v-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; C516725:Ptpn11 protein, mouse