ITP is a complex genetic trait autoimmune bleeding disease determined by multiple genetic and environmental influences [3, 14, 15]. In the plasma of ITP patients, platelet membrane proteins become antigenic and then stimulate the immune system to produce antibodies, eventually resulting in T cell immune unbalanced and thrombocytopenia .
Several DNA polymorphisms induced by SNPs played an important role in the pathogenesis of ITP [16, 17]. Rischewski’s group proposed that the existence of genetic susceptibility to ITP by describing positive familial history in pediatric ITP cases . In this study, we found several DNA missense mutations related with PI3K/Akt signaling pathway in BMBMCs from ITP patients, which may indicate the pathway is involved in the pathogenesis of ITP.
Our previous research about quantitative proteomics analysis has shown that apoptosis-related proteins (HSPA8, HSPA6, ITGB3, YWHAH, and PRDX6) and autophagy-related proteins (HSPA8, PARK7, YWHAH, ITGB3 and CSF1R) were significantly expressed abnormal in ITP BMBMC samples compared to normal controls. We found these differentially expressed proteins, excepting the expression of CSF1R was up-regulated, were significantly down-regulated using parallel reaction monitoring (PRM) technology verification . KEGG enrichment analysis showed that these differentially proteins were also closely related to the PI3K/Akt signaling pathway.
The PI3K pathway is an essential pathway for various cellular processes, it is also one of the most frequently activated signal transduction pathways in human cancer and autoimmune disease. The central role of Akt in the PI3Ks pathway makes it one of the most activated downstream effectors . Akt interacts with the cytoplasmic domain of GPIbα and transduces vWF–GPIbα interaction signaling leading to platelet activation. PI3K/Akt signaling may be antagonized by the tumor suppressor PTEN (Phosphatase and tensin homologue) that was identified as a frequently mutated gene in many types of tumors particularly endometrium, skin, brain, and prostate [21, 22]. Our previous research has shown that the perturbations of normal autophagy leads to abnormal function of platelet and megakaryocyte, which may be caused by deletion of autophagy-related genes such as ATG7 and abnormal signaling due to overexpression of mTOR. mTOR is a key kinase and negative regulator in the PI3K/Akt/mTOR signaling pathway and can regulate cell proliferation, growth, survival, and angiogenesis under physiological conditions and in the presence of environmental stress . PTEN is a key positive regulatory molecule of autophagy that blocks the inhibitory effect of PI3K/PKB on autophagy, thereby activating autophagy and forming autophagosome . In vitro experiments, indirubin was observed to restore the expression of programmed cell-death 1 (PD1) and PTEN on the CD4 + T cells of ITP patients, leading to the subsequent attenuation of the Akt/mTOR pathway and modulating the homeostasis of T cell . Thus, it may be hypothesized that PTEN mutations lead to activation of the PI3K/Akt/mTOR pathway and inhibition of autophagy, and play a role in ITP initiation and progression.
Insulin receptor (INSR) is the central mediator in the insulin response upstream of PI3K that induced tyrosine phosphroylation of insulin receptor substrate and followed by transduction of downstream enzymes of PI3K [27, 28]. Several studies have shown that PI3K/Akt pathway could be induced by insulin and act as an indispensable effector[29, 30]. As a downstream molecule of PI3K/Akt pathway, mTOR activity reduces not only influence autophagy balance by this signaling, but also increases HIF-1α (Hypoxia-inducible factor 1α) activity and production of reactive oxygen species (ROS), leading to the oxidative stress in cells . Caroline et al. have shown that insulin regulates HIF-1 subunit accumulation and activation through a PI3K/mTOR dependent pathway, resulting in increased vascular endothelial growth factor (VEGF) expression. VGEF is a key angiogenic factor involved in a wide variety of biological processes including embryonic development, tumor progression and metastasis, and regulated by platelet-derived growth factor, insulin, insulin-like growth factor-I, tumor necrosis factor [33, 34]. Functional analysis revealed that INSR mutation in ITP patients involved in the PI3K/Akt signaling pathway and HIF-1 signaling pathway in this study. Although the depth mechanism of INSR mutation in ITP patients is still uncover, the exon mutations of INSR and PTEN may be involved in the PI3K/Akt signaling pathway, further affecting the expression of downstream molecules and eventually participating in the pathogenesis of ITP.
In addition, the function clustering analysis shown that Coagulation factor C homology (COCH) participate in the platelet activation. The COCH gene is the first gene identified to cause vestibular dysfunction. COCH encodes cochlin, which contains a short-predicted signal peptide (SP), an N-terminal factor C homology (FCH or LCCL) domain and two von Willebrand factor A-like domains (vWFA1 and vWFA2)[35, 36]. vWFA domain is known for shear-induced self-aggregation and adherence to macrophages, platelets or leukocytes. PI3K associated with the cytoplasmic domain of GPIbα transduces the vWF-binding signaling, leading to Akt activation [20, 37]. Some DFNA9 (vestibular disorder) patients develop vascular diseases such as cerebral ischemia and acute myocardial infarction, and the vWFA domain has been implicated in increased shear-induced platelet aggregation (SIPA). However, the function of COCH gene in ITP pathogenesis remains to be elucidated fully. The MAM domain-containing 4 (MAMDC4) protein is associated with the unique endocytotic mechanism observed in the intestine of mammals, which may be related to the autophagy activities mediated by the PI3K/Akt signaling pathway. In addition, Fanconi Anemia-Associated Protein, 20 kDa (FAAP20) and mucin (MUC) 20 also participate in the PI3K/Akt pathway and platelet activation in most ITP samples. Further improved the missense mutation genes and related function pathway in ITP.
Wang et al. through enhanced autophagy-related protein and autophagy flux in PI3K/Akt/mTOR signaling pathway, inhibiting apoptosis and improving platelet viability, thereby alleviating platelet destruction and prolonging the life span of platelets from ITP patients. Furthermore, microRNAs acts through targeting insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), and the subsequent downregulation of insulin-like growth factor 2 (IGF-2) causes inhibition of the PI3K/Akt pathway, which is involved in the process of MSC(mesenchymal stem cells deficiency) in ITP.We reported previously that abnormal expression of multiple proteins in PI3K/Akt pathway in patient groups compared with control groups using protein profiles technology. In support of this finding, this study confirmed some exon mutations (PTEN, INSR, COCH, MAMDC4, FAAP20 and MUC20) in PI3K/Akt pathway at the gene level in ITP bone marrow samples, which further verified the important role of this signaling pathway in ITP pathogenesis. However, little is known about the concrete transcription process or protein expression pathogenesis of mutation genes leading to thrombocytopenia.
In conclusion, our findings improved the understanding of the PI3K/Akt signaling pathway and, more significantly, suggest targeted therapeutic strategies and research direction for immune thrombocytopenia caused by related genes mutation or other pathogenic factors. Future work is needed to solve the mystery that how does the transcription and translation mechanisms of key mutation genes or molecules in this pathway affect the occurrence and development in this disease.