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Platelet-derived growth factor-induced phosphatidylinositol 3-kinase activation mediates actin rearrangements in fibroblasts.

ABSTRACT: Various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to correlate with the synthesis of PtdIns(3,4,5)P3; however, the significance of this rise in second messenger levels is not clear. We show here that wortmannin inhibits platelet-derived growth factor (PDGF)-mediated production of PtdIns(3,4,5)P3 in human foreskin fibroblasts with an IC50 of about 5 nM. A similar inhibition was observed in in vitro assays (IC50 approximately 1 nM) with phosphatidylinositol 3-kinase immunoprecipitated by antibodies directed against the 85 kDa subunit (p85). On the other hand, wortmannin did not affect PDGF-mediated phosphorylation of p85 as detected by immunoprecipitation with anti-phosphotyrosine antibodies, and did not dissociate the complex of p85 and the catalytic subunit (p110) of phosphatidylinositol 3-kinase. These results are consistent with a direct, specific inhibition of the enzyme by wortmannin at concentrations relevant for its previously reported effects on cellular responses. When stimulated with PDGF, human foreskin fibroblasts form circular structures of filamentous actin. Preincubation of these cells with wortmannin inhibits PDGF-mediated actin rearrangements, suggesting a need for PtdIns(3,4,5)P3 formation as a signal for this cell response.

SUBMITTER: Wymann M

PROVIDER: S-EPMC1137889 | biostudies-other | 1994 Mar

REPOSITORIES: biostudies-other

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Project description:BackgroundPlatelet-derived growth factor BB, a potent mitogen of pulmonary artery smooth muscle cells (PASMCs), has been implicated in pulmonary arterial remodeling, which is a key pathogenic feature of pulmonary arterial hypertension. Previous microRNA profiling in platelet-derived growth factor BB-treated PASMCs found a significantly downregulated microRNA, miR-1281, but it has not been associated with any cellular function, and we investigated the possibility.Methods and resultsReal-time quantitative reverse transcription-polymerase chain reaction assay proved that downregulation of miR-1281 was a conserved phenomenon in human and rat PASMCs. Overexpression and inhibition of miR-1281 in PASMCs promoted and suppressed, respectively, the cell proliferation and migration. Bioinformatic prediction and 3'-untranslated region reporter assay identified histone deacetylase 4 to be a direct target of miR-1281. Supporting this, proliferation and migration assay demonstrated the cellular function of histone deacetylase 4 is inversely correlated with that of miR-1281. Mechanistically, it is found that platelet-derived growth factor BB activates the phosphatidylinositol 3-kinase pathway, which then induces the expression of DNA methyltransferase 1, leading to enhanced methylation of a flanking CpG island and repressed miR-1281 expression. Finally, a reduced miR-1281 level was consistently identified in hypoxic PASMCs in vitro, in pulmonary arteries of rats with monocrotaline-induced pulmonary arterial hypertension, and in serum of patients with coronary heart disease-pulmonary arterial hypertension. These data suggest that there may be a diagnostic and therapeutic use for miR-1281.ConclusionsHerein, we report a novel regulatory axis, phosphatidylinositol 3-kinase-DNA methyltransferase 1-miR-1281-histone deacetylase 4, integrating multiple epigenetic regulators that participate in platelet-derived growth factor BB-stimulated PASMC proliferation and migration and pulmonary vascular remodeling.

Project description:Although the importance of platelet-derived growth factor receptor (PDGFR)-? signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-?-expressing cells induce ? smooth muscle actin (?-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-? activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-? signaling in differentiation of myofibroblasts from PDGFR-?-positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-? signaling was activated by administration of rosiglitazone. Changes in ?-SMA and PDGFR-? protein expression were assessed in PDGFR-?-green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-?-GFP changed dynamically during alveolar regeneration and that ?-SMA expression was induced in a subset of PDGFR-?-GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of ?-SMA in PDGFR-?-positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-? signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial-mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable epithelial-mesenchymal crosstalk regulates fibroblast phenotypes during alveolar septation.

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Platelet-derived growth factor-induced phosphatidylinositol 3-kinase activation mediates actin rearrangements in fibroblasts.

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