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

Tsuda M, Toyomitsu E, Kometani M, Tozaki-Saitoh H, Inoue K - J. Cell. Mol. Med. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.

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.

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Schematic representation of the hypothetical mechanisms involved in fibronectin-induced P2X4R up-regulation in microglia. Extracellular fibronectin binds to α5β1 integrins on microglial cells, which leads to activation of the PI3K–Akt and MEK–ERK signalling cascades through SFKs, presumably Lyn tyrosine kinase. Signalling through the PI3K–Akt pathway induces degradation of p53 via MDM2 in a proteasome-dependent manner. The reduction of the repressive effect of p53 may enhance P2X4R gene expression. Activated MEK–ERK signalling in microglia exposed to fibronectin enhances eIF4E phosphorylation status via activated MNK1, which may play a role in regulating P2X4R expression at translational levels. SFK, Src-family kinase; PI3K, phosphatidylinositol 3-kinase; MDM2, mouse double minute 2; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; MNK1, MAPK-interacting protein kinase-1; eIF4E, eukaryotic translation initiation factor 4E.
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fig07: Schematic representation of the hypothetical mechanisms involved in fibronectin-induced P2X4R up-regulation in microglia. Extracellular fibronectin binds to α5β1 integrins on microglial cells, which leads to activation of the PI3K–Akt and MEK–ERK signalling cascades through SFKs, presumably Lyn tyrosine kinase. Signalling through the PI3K–Akt pathway induces degradation of p53 via MDM2 in a proteasome-dependent manner. The reduction of the repressive effect of p53 may enhance P2X4R gene expression. Activated MEK–ERK signalling in microglia exposed to fibronectin enhances eIF4E phosphorylation status via activated MNK1, which may play a role in regulating P2X4R expression at translational levels. SFK, Src-family kinase; PI3K, phosphatidylinositol 3-kinase; MDM2, mouse double minute 2; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; MNK1, MAPK-interacting protein kinase-1; eIF4E, eukaryotic translation initiation factor 4E.

Mentions: In summary, we propose the following molecular machinery for the up-regulation of P2X4R expression in microglia in response to fibronectin (Fig. 7). Extracellular fibronectin, by binding to α5β1 integrins on microglial cells, activates the PI3K–Akt and MEK–ERK signalling cascades through SFKs, presumably Lyn tyrosine kinase. The signalling through the PI3K–Akt pathway induces degradation of p53 via MDM2 in a proteasome-dependent manner. The consequence of an attenuated repressive effect of p53 may be associated with enhanced P2X4R gene expression. On the other hand, activated MEK–ERK signalling in microglia exposed to fibronectin enhances eIF4E phosphorylation status via activated MNK1, which may play a role in regulating P2X4R expression at translational levels. The physiological and pathological relevance of these pathways regulating P2X4R expression in microglia in vivo remains to be elucidated, but the role of several proteins in these pathways in CNS diseases has been reported. In neuropathic pain, to which P2X4R critically contributes [10, 13, 20–22], mice lacking Lyn tyrosine kinase exhibit impaired pain behaviour and attenuated P2X4R up-regulation in microglia after nerve injury [31]. Inhibitors of PI3K–Akt signalling and of the proteasome have been reported to attenuate neuropathic pain [68, 69]. Furthermore, ERK activation occurs in microglia after nerve injury, and inhibiting the activation suppresses nerve injury–induced pain [70]. Interestingly, ERK activation in microglia is observed from 2 to 10 days after nerve injury [70], which is similar to the time course of fibronectin up-regulation in the spinal cord, which contributes to initiation of P2X4R expression in microglia [29, 30]. Therefore, the signalling pathways underlying fibronectin-induced up-regulation of P2X4R expression in microglia that we have demonstrated here may provide clues for understanding the molecular machinery for the up-regulation of P2X4R expression in microglia in vivo and for understanding pathological CNS disorders that are associated with microglial P2X4R.


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

Tsuda M, Toyomitsu E, Kometani M, Tozaki-Saitoh H, Inoue K - J. Cell. Mol. Med. (2009)

Schematic representation of the hypothetical mechanisms involved in fibronectin-induced P2X4R up-regulation in microglia. Extracellular fibronectin binds to α5β1 integrins on microglial cells, which leads to activation of the PI3K–Akt and MEK–ERK signalling cascades through SFKs, presumably Lyn tyrosine kinase. Signalling through the PI3K–Akt pathway induces degradation of p53 via MDM2 in a proteasome-dependent manner. The reduction of the repressive effect of p53 may enhance P2X4R gene expression. Activated MEK–ERK signalling in microglia exposed to fibronectin enhances eIF4E phosphorylation status via activated MNK1, which may play a role in regulating P2X4R expression at translational levels. SFK, Src-family kinase; PI3K, phosphatidylinositol 3-kinase; MDM2, mouse double minute 2; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; MNK1, MAPK-interacting protein kinase-1; eIF4E, eukaryotic translation initiation factor 4E.
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Related In: Results  -  Collection

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fig07: Schematic representation of the hypothetical mechanisms involved in fibronectin-induced P2X4R up-regulation in microglia. Extracellular fibronectin binds to α5β1 integrins on microglial cells, which leads to activation of the PI3K–Akt and MEK–ERK signalling cascades through SFKs, presumably Lyn tyrosine kinase. Signalling through the PI3K–Akt pathway induces degradation of p53 via MDM2 in a proteasome-dependent manner. The reduction of the repressive effect of p53 may enhance P2X4R gene expression. Activated MEK–ERK signalling in microglia exposed to fibronectin enhances eIF4E phosphorylation status via activated MNK1, which may play a role in regulating P2X4R expression at translational levels. SFK, Src-family kinase; PI3K, phosphatidylinositol 3-kinase; MDM2, mouse double minute 2; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; MNK1, MAPK-interacting protein kinase-1; eIF4E, eukaryotic translation initiation factor 4E.
Mentions: In summary, we propose the following molecular machinery for the up-regulation of P2X4R expression in microglia in response to fibronectin (Fig. 7). Extracellular fibronectin, by binding to α5β1 integrins on microglial cells, activates the PI3K–Akt and MEK–ERK signalling cascades through SFKs, presumably Lyn tyrosine kinase. The signalling through the PI3K–Akt pathway induces degradation of p53 via MDM2 in a proteasome-dependent manner. The consequence of an attenuated repressive effect of p53 may be associated with enhanced P2X4R gene expression. On the other hand, activated MEK–ERK signalling in microglia exposed to fibronectin enhances eIF4E phosphorylation status via activated MNK1, which may play a role in regulating P2X4R expression at translational levels. The physiological and pathological relevance of these pathways regulating P2X4R expression in microglia in vivo remains to be elucidated, but the role of several proteins in these pathways in CNS diseases has been reported. In neuropathic pain, to which P2X4R critically contributes [10, 13, 20–22], mice lacking Lyn tyrosine kinase exhibit impaired pain behaviour and attenuated P2X4R up-regulation in microglia after nerve injury [31]. Inhibitors of PI3K–Akt signalling and of the proteasome have been reported to attenuate neuropathic pain [68, 69]. Furthermore, ERK activation occurs in microglia after nerve injury, and inhibiting the activation suppresses nerve injury–induced pain [70]. Interestingly, ERK activation in microglia is observed from 2 to 10 days after nerve injury [70], which is similar to the time course of fibronectin up-regulation in the spinal cord, which contributes to initiation of P2X4R expression in microglia [29, 30]. Therefore, the signalling pathways underlying fibronectin-induced up-regulation of P2X4R expression in microglia that we have demonstrated here may provide clues for understanding the molecular machinery for the up-regulation of P2X4R expression in microglia in vivo and for understanding pathological CNS disorders that are associated with microglial P2X4R.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.

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.

Show MeSH