ABSTRACT
Essentials Phosphoinositide 3-kinase and MAPK pathways crosstalk via PDK1. PDK1 is required for adenosine diphosphate-induced platelet activation and thromboxane generation. PDK1 regulates RAF proto-oncogene Ser/Thr kinase (Raf1) activation in the MAPK pathway. Genetic ablation of PDK1 protects against platelet-dependent thrombosis in vivo. SUMMARY: Background Platelets are dynamic effector cells with functions that span hemostatic, thrombotic and inflammatory continua. Phosphoinositide-dependent protein kinase 1 (PDK1) regulates protease-activated receptor 4-induced platelet activation and thrombus formation through glycogen synthase kinase3ß. However, whether PDK1 also signals through the ADP receptor and its functional importance in vivo remain unknown. Objective To establish the mechanism of PDK1 in ADP-induced platelet activation and thrombosis. Methods We assessed the role of PDK1 on 2MeSADP-induced platelet activation by measuring aggregation, thromboxane generation and phosphorylation events in the presence of BX-795, which inhibits PDK1, or by using platelet-specific PDK1 knockout mice and performing western blot analysis. PDK1 function in thrombus formation was assessed with an in vivo pulmonary embolism model. Results PDK1 inhibition with BX-795 reduced 2-methylthio-ADP (2MeSADP)-induced aggregation of human and murine platelets by abolishing thromboxane generation. Similar results were observed in pdk1-/- mice. PDK1 was also necessary for the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2, and cytosolic phospholipase A2, indicating that PDK1 regulates an upstream kinase in the mitogen-activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf-1, a serine/threonine kinase that is necessary for the phosphorylation of MEK1/2, as pharmacological inhibition and genetic ablation of PDK1 were sufficient to prevent Raf1 phosphorylation. Furthermore, in vivo inhibition or genetic ablation of PDK1 protected mice from collagen/epinephrine-induced pulmonary embolism. Conclusion PDK1 governs thromboxane generation and thrombosis in platelets that are stimulated with 2MeSADP by regulating activation of the MAPK pathway.
Subject(s)
3-Phosphoinositide-Dependent Protein Kinases/metabolism , Blood Platelets/enzymology , Mitogen-Activated Protein Kinases/blood , Platelet Aggregation/drug effects , Proto-Oncogene Proteins c-raf/blood , Pulmonary Embolism/enzymology , Thrombosis/enzymology , Thromboxanes/blood , 3-Phosphoinositide-Dependent Protein Kinases/antagonists & inhibitors , 3-Phosphoinositide-Dependent Protein Kinases/blood , 3-Phosphoinositide-Dependent Protein Kinases/deficiency , 3-Phosphoinositide-Dependent Protein Kinases/genetics , Animals , Blood Platelets/drug effects , Disease Models, Animal , Humans , Mice, Knockout , Phosphorylation , Platelet Aggregation Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Mas , Pulmonary Embolism/blood , Pulmonary Embolism/genetics , Pulmonary Embolism/prevention & control , Pyrimidines/pharmacology , Signal Transduction , Thiophenes/pharmacology , Thrombosis/blood , Thrombosis/genetics , Thrombosis/prevention & controlABSTRACT
microRNA (miR)-138 has been recognized as a potential tumor suppressor via regulating 3-phosphoinositide-dependent protein kinase-1 (PDK1) expression in non-small cell lung cancer (NSCLC) cells. The aim of this study was to investigate miR-138 and PDK1 mRNA expression in serum of NSCLC and their associations with patients' prognosis. miR-138 and PDK1 mRNA expressions in 100 NSCLCs and 100 healthy control sera were detected by quantitative real-time PCR. miR-138 expression level was significantly lower in NSCLC serum samples than in healthy control serum samples (P < 0.001), while PDK1 mRNA expression level was significantly increased in NSCLC serum samples compared to healthy control serum samples (P < 0.001). In addition, miR-138 downregulation and PDK1 upregulation were both significantly associated with advanced tumor-node-metastasis (TNM) stage (both P = 0.002) and positive lymph node metastasis (both P = 0.01) of NSCLC patients. Moreover, the overall survival of NSCLC patients with low miR-138 expression or high PDK1 mRNA expression was obviously shorter than those with high miR-138 expression or low PDK1 mRNA expression (both P < 0.001). Notably, NSCLC patients with combined miR-138 downregulation and PDK1 upregulation (miR-138-low/PDK1-high) had shortest overall survival (P < 0.001). Furthermore, multivariate analysis showed that miR-138 expression (P = 0.01), PDK1 expression (P = 0.01), and combined expression of miR-138 and PDK1 (miR-138/PDK1, P = 0.001) were all independent prognostic factors for overall survival in NSCLC patients. Deregulation of miR-138/PDK1 cascade may be implicated in carcinogenesis and cancer progression of human NSCLC. More importantly, miR-138 and PDK1 may synergistically predict patients' prognosis and their combination may represent a promising prognostic biomarker of human NSCLC.