Mechanisms underlying fibronectin-induced up-regulation of P2X ₄R expression in microglia: Distinct roles of PI3K-Akt and MEK-ERK signalling pathways

Microglia are 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 P2X ₄ receptor (P2X ₄R) is a sub-type of the purinergic ion-channel receptors expressed in microglia. P2X ₄R 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 P2X ₄R in microglia remains unknown. In the present study, we investigated the intracellular signal transduction pathway that promotes P2X ₄R expression in microglia in response to fibronectin, an extracellular matrix protein that has previously been shown to stimulate P2X ₄R 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 P2X ₄R 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 P2X ₄R expression. Conversely, an inhibitor of p53 caused increased expression of P2X ₄R, implying a negative regulatory role of p53. On the other hand, inhibiting MEK-ERK signalling activated by fibronectin suppressed an increase in P2X ₄R protein but interestingly did not affect the level of P2X ₄R 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 P2X ₄R protein. Together, these results suggest that the PI3K-Akt and MEK-ERK signalling cascades have distinct roles in the up-regulation of P2X ₄R expression in microglia at transcriptional and post-transcriptional levels, respectively.