Mechanisms underlying fibronectin-induced up-regulation of P2X4R expression in microglia: distinct roles of PI3K–Akt and MEK–ERK signalling pathways

摘要

AbstractMicrogliaare resident immune cells in the central nervous system that become activated and produce pro-inflammatory and neurotrophic factors upon activation of various cell-surface receptors. The P2X4 receptor (P2X4R) is a sub-type of the purinergic ion-channel receptors expressed in microglia. P2X4R expression is up-regulated under inflammatory or neurodegenerative conditions, and this up-regulation is implicated in disease pathology. However, the molecular mechanism underlying up-regulation of P2X4R in microglia remains unknown. In the present study, we investigated the intracellular signal transduction pathway that promotes P2X4R expression in microglia in response to fibronectin, an extracellular matrix protein that has previously been shown to stimulate P2X4R expression. We found that in fibronectin-stimulated microglia, activation of phosphatidylinositol 3-kinase (PI3K)–Akt and mitogen-activated protein kinase kinase (MAPK kinase, MEK)–extracellular signal-regulated kinase (ERK) signalling cascades occurred divergently downstream of Src-family kinases (SFKs). Pharmacological interference of PI3K–Akt signalling inhibited fibronectin-induced P2X4R gene expression. Activation of PI3K–Akt signalling resulted in a decrease in the protein level of the transcription factor p53 via mouse double minute 2 (MDM2), an effect that was prevented by MG-132, an inhibitor of the proteasome. In microglia pre-treated with MG-132, fibronectin failed to up-regulate P2X4R expression. Conversely, an inhibitor of p53 caused increased expression of P2X4R, implying a negative regulatory role of p53. On the other hand, inhibiting MEK–ERK signalling activated by fibronectin suppressed an increase in P2X4R protein but interestingly did not affect the level of P2X4R mRNA. We also found that fibronectin stimulation resulted in the activation of the translational factor eIF4E via MAPK-interacting protein kinase-1 (MNK1) in an MEK–ERK signalling-dependent manner, and an MNK1 inhibitor attenuated the increase in P2X4R protein. Together, these results suggest that the PI3K–Akt and MEK–ERK signalling cascades have distinct roles in the up-regulation of P2X4R expression in microglia at transcriptional and post-transcriptional levels, respectively.