Non-activating ITGB3 mutation in β3 cytoplasmic region causes macrothrombocytopenia with impaired αIIbβ3/RhoA pathway
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ABSTRACT:
PROVIDER: PRJNA1103825 | ENA |
REPOSITORIES: ENA
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Project description:Almost all mutations of ITGA2B or ITGB3 identified in congenital macrothrombocytopenia induce constitutive activation of αIIbβ3. However, whether concomitant αIIbβ3 activation is essential for macrothrombocytopenia development remains unknown. Recently, we identified the β3(R734C) mutation that does not induce αIIbβ3 activation in macrothrombocytopenia. Here, we generated β3(R734C) knock-in (KI) mice and investigated the abnormalities in the platelet/megakaryocyte biology of KI mice and a family with β3(R734C)-related macrothrombocytopenia. Macrothrombocytopenia was observed in heterozygous β3(R734C) subjects with reduced expression of αIIbβ3. Compared to those in the wild-type (WT) mice, platelet counts decreased to 76 and 40% in heterozygous (Hetero) and homozygous (Homo) KI mice, respectively. Platelet αIIbβ3 expression was decreased in KI mice, and αIIbβ3 activation was not detected on non-stimulated KI platelets. Platelet aggregation, agonist-induced JON/A binding and P-selectin expression were impaired in KI mice. Platelet spreading on fibrinogen was also impaired in KI mice with ADP or thrombin stimulation. Additionally, filopodia and lamellipodia formation was impaired in fibrinogen-adhered megakaryocytes of KI mice. Moreover, RhoA activation was significantly impaired in fibrinogen-adhered megakaryocytes of KI mice. Proplatelet formation in KI mice was impaired with abnormal morphology. Platelet lifespan was shortened in Homo mice. Our data newly revealed that constitutive αIIbβ3 activation is not essential for the development of macrothrombocytopenia. We also showed that β3(R734C) mutation impairs the inside-out and outside-in signaling of αIIbβ3, and abnormal actin rearrangement and impaired RhoA activation may play major roles in leading to macrothrombocytopenia.
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Project description:We sought to transcriptomically characterize the effect of integrin β3 expression on breast cancer cell responses to the microtubule-inhibiting chemotherapeutic agent docetaxel. Experiments were performed in PyMT-BO1 murine breast cancer cells with CRISPR mediated deletion of the Itgb3 gene. Cells were subsequently retrovirally rescued using either an empty vector construct (pMx), a functional human integrin β3 construct (hβ3), or a signaling-deficient integrin β3 mutant.
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Project description:Layer V pyramidal neurons, particularly pyramidal tract (PT) neurons, are essential for cortical output. Genetic studies have linked ITGB3, the gene encoding the cell-adhesion molecule β3 integrin, to autism spectrum disorder (ASD), yet its role in brain function remains poorly understood. Here, we investigate β3 integrin function in the prefrontal cortex, revealing its selective regulation of PT neuron excitability. β3 integrin modulates action potential (AP) discharge through its interaction with Ca2+-activated SK2 channels. Genetic ablation of Itgb3 reduces SK2 channel surface expression, impairing the gain, adaptation and precision of AP discharge in PT neurons. Phosphoproteomic analysis reveals altered protein kinase A (PKA)-dependent phosphorylation of Tau in Itgb3 KO mice, while PKA inhibition restores SK2 channel currents, linking phosphorylation changes to excitability defects. These findings identify a novel β3 integrin-dependent signaling pathway as a selective key modulator of PT neuron function, and provide insight into the cellular mechanisms linking dysfunction in cortical output to ASD.
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Project description:We sought to transcriptomically characterize the effect of integrin β3 expression on breast cancer cell responses to the microtubule-inhibiting chemotherapeutic agent docetaxel. Experiments were performed in 4T1 murine breast cancer cells with or without CRISPR mediated deletion of the Itgb3 gene.
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Project description:In the context of breast cancer metastasis study, we have shown in an in vitro model of cell migration that IGDQ-exposing (IsoLeu-Gly-Asp-Glutamine type I Fibronectin motif) monolayers (SAMs) on gold sustain the adhesion of MDA-MB-231 cells by triggering Focal Adhesion Kinase by activating integrins. Such tunable scaffolds are used to mimic the extracellular environment, inducing and controlling cell migration towards an anisotropic surface. The observed migratory behavior induced by the IGDQ-bearing peptide gradient along the surface suggests the presence of cell subpopulations: “stationary” or a “migratory” phenotype. We focused on the integrins α5(β1) and (αv)β3, already known to be implicated in cell migration. To this aim, a whole proteomic analysis was performed in beta 3 integrin (ITGB3) or alpha 5 integrin (ITGA5) knock-down MDA-MB-231, in order to highlight the pathways implied into the integrin-dependent cell migration. Our results showed that i) ITGB3 depletion influenced ITGA5 mRNA expression, ii) ITGB3 and ITGA5 were both necessary for IGDQ-mediated directional single cell migration, iii) integrin (αv)β3 was activated by IGDQ fibronectin type I motif and iv) co-regulation of retrograde transport of ITGB3 by ITGA5 is potentially necessary for directional IGDQ-mediated cell migration.
2022-11-08 | PXD024540 | Pride
Project description:Background: Pulmonary immune homeostasis relies on stringent control of adaptive immunity. Integrin β3 is a critical mediator of cellular adhesion and platelet function. However, its specific role in modulating adaptive immunity, particularly B cell immune response, remains poorly understood.Results: We identified that β3⁻/⁻ mice showed a spontaneous pulmonary inflammation characterized by B cell activation and in situ immune complex deposition in alveoli. Multi-omics integration identified that the CD40-CD40 Ligand (CD40L) axis was strongly implicated in this pathology process. Mechanistically, the absence of β3 potentiates CD40L-CD40 engagement on B cells, leading to hyperactivation of the NF-κB pathway. Notably, reduced ITGB3 expression in patients with autoimmune disease correlated significantly with enhanced transcriptional signatures of B cell activation and inflammation.Methods: Histopathology characterized the pulmonary phenotype of constitutive β3-deficient (β3⁻/⁻) mice. Integrated transcriptomic and proteomic profiling of lung tissue was then performed to map the molecular landscape of spontaneous pulmonary inflammation. Related mechanisms were investigated by histopathology and functional assays, and established clinical relevance by analyzing transcriptomic datasets from patients with autoimmune diseases.Conclusions: Our findings redefine integrin β3 as a threshold regulator of the B cell activation. Consequently, the β3-CD40L-CD40 axis represents a promising therapeutic target for managing B cell-related autoimmune diseases.
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2018-05-23 | PXD008591 | Pride
Project description:Crohn’s disease is a chronic, debilitating inflammatory bowel disease. Here we report a critical role for phospholipase C-β3 (PLC-β3) in intestinal homeostasis. In PLC-β3- deficient mice, exposure to oral dextran sodium sulfate induced lethality and severe inflammation in the small intestine. The lethality was due to PLC-β3 deficiency in multiple non-hematopoietic cell types. PLC-β3 deficiency resulted in reduced Wnt/β-catenin signaling, which is essential for homeostasis and regeneration of the intestinal epithelium. PLC-β3 regulated the Wnt/β-catenin pathway in small intestinal epithelial cells (IECs) at transcriptional, epigenetic, and potentially protein-protein interaction levels. PLC-β3- deficient IECs were unable to respond to stimulation by R-spondin 1, an enhancer of Wnt/β-catenin signaling. Reduced expression of PLC-β3 and its signature genes was found in biopsies of ileal Crohn’s disease patients. PLC-β regulation of Wnt signaling was evolutionally conserved in Drosophila. Our data indicate that reduction of PLC-β3- mediated Wnt/β-catenin signaling contributes to the pathogenesis of ileal Crohn’s disease.
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