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ABSTRACT: P2X4 receptors (P2X4Rs), a subtype of the purinergic P2X family, play important roles in regulating neuronal and glial functions in the nervous system. We have previously shown that the expression of P2X4Rs is upregulated in activated microglia after peripheral nerve injury and that activation of the receptors by extracellular ATP is crucial for maintaining nerve injury-induced pain hypersensitivity. However, the regulation of P2X4R expression on the cell surface of microglia is poorly understood. Here, we identify the CC chemokine receptor CCR2 as a regulator of P2X4R trafficking to the cell surface of microglia. In a quantitative cell surface biotinylation assay, we found that applying CCL2 or CCL12, endogenous ligands for CCR2, to primary cultured microglial cells, increased the levels of P2X4R protein on the cell surface without changing total cellular expression. This effect of CCL2 was prevented by an antagonist of CCR2. Time-lapse imaging of green fluorescent protein (GFP)-tagged P2X4R in living microglial cells showed that CCL2 stimulation increased the movement of P2X4R-GFP particles. The subcellular localization of P2X4R immunofluorescence was restricted to lysosomes around the perinuclear region. Notably, CCL2 changed the distribution of lysosomes with P2X4R immunofluorescence within microglial cells and induced release of the lysosomal enzyme β-hexosaminidase, indicating lysosomal exocytosis. Moreover, CCL2-stimulated microglia enhanced Akt phosphorylation by ATP applied extracellularly, a P2X4R-mediated response. These results indicate that CCL2 promotes expression of P2X4R protein on the cell surface of microglia through exocytosis of P2X4R-containing lysosomes, which may be a possible mechanism for pain hypersensitivity after nerve injury.
Purinergic Signalling 01/2012; 8(2):301-10. · 3.16 Impact Factor
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ABSTRACT: Up-regulation of P2X4 receptors in spinal cord microglia is crucial for tactile allodynia, an untreatable pathological pain reaction occurring after peripheral nerve injury. How nerve injury in the periphery leads to this microglia reaction in the dorsal horn of the spinal cord is not yet understood. It is shown here that CCL21 was rapidly expressed in injured small-sized primary sensory neurons and transported to their central terminals in the dorsal horn. Intrathecal administration of a CCL21-blocking antibody diminished tactile allodynia development in wild-type animals. Mice deficient for CCL21 did not develop any signs of tactile allodynia and failed to up-regulate microglial P2X4 receptor expression. Microglia P2X4 expression was enhanced by CCL21 application in vitro and in vivo. A single intrathecal injection of CCL21 to nerve-injured CCL21-deficient mice induced long-lasting allodynia that was undistinguishable from the wild-type response. This effect of CCL21 injection was strictly dependent on P2X4 receptor function. Since neuronal CCL21 is the earliest yet identified factor in the cascade leading to tactile allodynia, these findings may lead to a preventive therapy in neuropathic pain.
The EMBO Journal 03/2011; 30(9):1864-73. · 9.20 Impact Factor
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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 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.
Journal of Cellular and Molecular Medicine 08/2009; 13(9b):3251 - 3259. · 4.13 Impact Factor
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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.
Journal of Cellular and Molecular Medicine 03/2009; 13(9B):3251-9. · 4.13 Impact Factor
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ABSTRACT: We have previously shown that activation of the ATP-gated ion channel subtype P2X(4) receptors (P2X(4)Rs) in the spinal cord, the expression of which is upregulated in microglia after nerve injury, is necessary for producing neuropathic pain. The upregulation of P2X(4)Rs in microglia is, therefore, a key process in neuropathic pain, but the mechanism remains unknown. Here, we find a fibronectin/integrin-dependent mechanism in the upregulation of P2X(4)Rs. Microglia cultured on dishes coated with fibronectin, an extracellular matrix molecule, expressed a higher level of P2X(4)R protein when compared with those cultured on control dishes. The increase was suppressed by echistatin, a peptide that selectively blocks beta(1) and beta(3)-containing integrins, and with a function-blocking antibody of beta(1) integrin. In in vivo studies, the upregulation of P2X(4)Rs in the spinal cord after spinal nerve injury was significantly suppressed by intrathecal administration of echistatin. Tactile allodynia in response to nerve injury and intrathecal administration of ATP- and fibronectin-stimulated microglia was inhibited by echistatin. Furthermore, intrathecal administration of fibronectin in normal rats increased the level of P2X(4)R protein in the spinal cord and produced tactile allodynia. Moreover, the fibronectin-induced allodynia was not observed in mice lacking P2X(4)R. Taken together with the results of our previous study showing an increase in the spinal fibronectin level after nerve injury, the present results suggest that the fibronectin/integrin system participates in the upregulation of P2X(4)R expression after nerve injury and subsequent neuropathic pain.
Glia 05/2008; 56(5):579-85. · 4.82 Impact Factor
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ABSTRACT: Neuropathic pain, a debilitating chronic pain following nerve damage, is a reflection of the aberrant functioning of a pathologically altered nervous system. One hallmark is abnormal pain hypersensitivity to innocuous stimuli (tactile allodynia), for which effective therapy is lacking, and the underlying mechanisms of which remain to be determined. Here we show that Lyn, a member of the Src family kinases (SFKs), plays an important role in the pathogenesis of neuropathic pain. Nerve injury, but not peripheral inflammation, increased immunoreactivity for active SFKs that were autophosphorylated in the kinase domain (phospho-SFK-IR) in spinal microglia. In spinally derived microglial cells, we identified Lyn as the predominant SFK among the five members (Src, Fyn, Yes, Lck, and Lyn) known to be expressed in the CNS. Lyn expression in the spinal cord was highly restricted to microglia, and its level was increased after nerve injury. We found that mice lacking lyn (lyn(-/-)) exhibit a striking reduction in the levels of phospho-SFK-IR and tactile allodynia after nerve injury, without any change in basal mechanical sensitivity or inflammatory pain. Importantly, lyn(-/-) mice displayed impaired upregulation of the ionotropic ATP receptor subtype P2X(4) receptors (P2X(4)R) in the spinal cord after nerve injury, which is crucial for tactile allodynia. Microglial cells from lyn(-/-) mice showed a deficit in their ability to increase P2X(4)R expression in response to fibronectin, a factor implicated as a microglial P2X(4)R upregulator in allodynia. Together, our findings suggest that Lyn may be a critical kinase mediating nerve injury-induced P2X(4)R upregulation and neuropathic pain.
Glia 02/2008; 56(1):50-8. · 4.82 Impact Factor
