Background: Platelets have the hemostatic function, and their aberrant activation is associated with occlusive thrombus formation. Plasma exosomes are rich in platelets containing ubiquitin-specific peptidase 15 (USP15). Herein, we aim to explore the effect of USP15 on thrombosis, as well as expounding whether USP15 acts as an upstream target of FK506 binding protein 5 (FKBP5) to regulate occlusive thrombus formation. Methods: Washed human platelets were treated with thrombin for measurement of USP15 and FKBP5 expressions. USP15 loss/gain-of-function variant in HEK293 cells was performed by cell transfection, and the interaction between USP15 and FKBP5 was examined using immunoprecipitation and ubiquitination assays. Mice with USP15-knockout platelets (Plt USP15-/-) were modeled, and subjected to calculation of bleeding time, artery thrombosis imaging and clot retraction assay. FKBP5 expression and the inhibitor of nuclear factor kappa B kinase subunit epsilon (IKBKE)/phosphatidylinositol 3-kinase (PI3K)/Rap1 pathway in wild-type and Plt USP15-/- mice-derived platelets were detected using Western blot. The activation of αIIbβ3 in washed platelets was analyzed using flow cytometry. Results: USP15 and FKBP5 expressions were upregulated in platelets after thrombin treatment. Following transfection of USP15 knockdown and USP15 overexpression plasmids into HEK293 cells, FKBP5 protein expression was downregulated by USP15 knockdown while being upregulated by USP15 overexpression. USP15 bound to FKBP5 and protected FKBP5 against ubiquitination. Knockdown of platelet USP15 prolonged bleeding time, inhibited arterial thrombosis and delayed clot retraction in mice. Knockdown of platelet USP15 also decreased protein expressions of FKBP5, IKBKE and Rap1, p-PI3K/PI3K ratio, and activation of αIIbβ3 in mice. Conclusion: USP15 knockdown in platelets affects thrombosis in mice by promoting the instability of FKBP5 to repress the activation of IKBKE/PI3K/Rap1 pathway-mediated αIIbβ3.
Ren Ting,Chen Xue,Wang Xiaozhen,Xu Yantian,Shao Na
Background The aggressive cancer known as pancreatic ductal adenocarcinoma (PDAC) has a remarkably poor response to treatment, especially gemcitabine (GEM). As a member of the noncanonical IκB kinase family, inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKE) is known to regulate tumor progression in multiple cancer types. However, its functional role in modulating chemosensitivity and its impact on cell death pathways in PDAC remain unclear. Materials and methods We examined IKBKE expression in patient tumor tissues and publicly available databases and assessed its prognostic value to investigate its function in PDAC chemoresistance. Loss-of-function approaches, including shRNA knockdown and pharmacological inhibition using amlexanox, were employed in vivo and in vitro. Furthermore, cytotoxicity along with cell death patterns induced by GEM were assessed through flow cytometry, electron microscopy, and CCK-8 assays. Co-IP and GST pull-down assays were applied to determine whether IKBKE interacts with gasdermin E (GSDME), the executioner protein of pyroptosis. Moreover, in vitro kinase assays, phosphorylation mass spectrometry, and site-specific gain-/loss-of-function mutant functional experiments were conducted to investigate the specific pathways via which IKBKE affects GSDME-mediated pyroptosis. The therapeutic potential of IKBKE targeting was further validated using patient-derived tumor organoids and xenograft models. Results IKBKE was significantly elevated in PDAC tissues and was closely linked to worse clinical outcomes. Functional studies of the signaling pathway showed that via the activation of the protein kinase B/glycogen synthase kinase-3β signaling pathway, IKBKE facilitates the aggressive phenotype of PDAC cells. IKBKE inhibition or silencing increased GEM sensitivity and caused caspase-3/GSDME-dependent pyroptosis in PDAC cells. Co-IP and GST pull-down analysis revealed IKBKE–GSDME interaction. Further in vitro cleavage and kinase assays, mass spectrometry analysis, and mutant functional studies showed that IKBKE inhibits pyroptosis by phosphorylating GSDME at Thr6, thereby hindering its cleavage by caspase-3. Treatment with amlexanox markedly suppressed tumor growth and synergized with GEM to enhance anti-tumor efficacy in organoid models by targeting IKBKE. Conclusion Our findings identify IKBKE as a key regulator of chemotherapy-induced pyroptosis in PDAC. Specifically, IKBKE phosphorylates GSDME at Thr6, which alters its conformation, thereby impeding caspase-3–mediated cleavage, ultimately contributing to GEM chemoresistance. IKBKE targeting reverses GEM resistance and enhances tumor immunogenic cell death via the caspase-3/GSDME pathway, supporting IKBKE inhibition as a promising therapeutic strategy for PDAC. Graphical The alternative text for this image may have been generated using AI.
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