title: Mechanisms underlying fibronectin-induced up-regulation of P2X4R expression in microglia: distinct roles of PI3K-Akt and MEK-ERK signalling pathways.
abstract: 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(4)R) is a sub-type of the purinergic ion-channel receptors expressed in microglia. P2X(4)R 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(4)R in microglia remains unknown. In the present study, we investigated the intracellular signal transduction pathway that promotes P2X(4)R expression in microglia in response to fibronectin, an extracellular matrix protein that has previously been shown to stimulate P2X(4)R 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(4)R 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(4)R expression. Conversely, an inhibitor of p53 caused increased expression of P2X(4)R, implying a negative regulatory role of p53. On the other hand, inhibiting MEK-ERK signalling activated by fibronectin suppressed an increase in P2X(4)R protein but interestingly did not affect the level of P2X(4)R 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(4)R protein. Together, these results suggest that the PI3K-Akt and MEK-ERK signalling cascades have distinct roles in the up-regulation of P2X(4)R expression in microglia at transcriptional and post-transcriptional levels, respectively.
authors.affiliation: Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Tsuda
authors.forename: Emika
authors.initials: E
authors.lastname: Toyomitsu
authors.forename: Miho
authors.initials: M
authors.lastname: Kometani
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Tozaki-Saitoh
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Inoue
mesh_terms: D000818:Animals; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry; D007150:Immunohistochemistry / Q000379:methods; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D017628:Microglia / Q000378:metabolism*; D019636:Neurodegenerative Diseases / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D051381:Rats; D017208:Rats, Wistar; D058848:Receptors, Purinergic P2X4 / Q000378:metabolism*; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D005353:Fibronectins; D058848:Receptors, Purinergic P2X4; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases