Fibronectin/integrin system is involved in P2X₄ receptor upregulation in the spinal cord and neuropathic pain after nerve injury

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.

ATP receptors gate microglia signaling in neuropathic pain

“…Also critical for upregulating expression of P2X4 receptors is the extracellular matrix molecule fibronectin, which through the Lyn kinase signaling pathway modulates the transcriptional and post-transcriptional levels of P2X4 receptor expression in microglia (Nasu-Tada et al, 2006;Tsuda et al, 2008a;Tsuda et al, 2008b;Tsuda et al, 2009c). Similarly, activation of mu-opioid receptors by morphine can drive P2X4 receptor expression in states of opioid tolerance (Horvath and DeLeo, 2009;Horvath et al, 2010) and opioid-induced hyperalgesia (Ferrini et al, 2010).…”

“…Several signaling molecules have been implicated in the upregulation of P2X4 receptors, including CCL21, a chemokine released from injured neurons (Biber et al, 2011;de Jong et al, 2005), interferon γ, a cytokine that transforms resting spinal microglia into an activated state (Tsuda et al, 2009b), and tryptase, a protease released from mast cells that activates proteinase-activated receptor 2 in microglia (Yuan et al, 2010). Also critical for upregulating expression of P2X4 receptors is the extracellular matrix molecule fibronectin, which through the Lyn kinase signaling pathway modulates the transcriptional and post-transcriptional levels of P2X4 receptor expression in microglia (Nasu-Tada et al, 2006;Tsuda et al, 2008a;Tsuda et al, 2008b;Tsuda et al, 2009c). Similarly, activation of mu-opioid receptors by morphine can drive P2X4 receptor expression in states of opioid tolerance (Horvath and DeLeo, 2009;Horvath et al, 2010) and opioid-induced hyperalgesia (Ferrini et al, 2010).…”

Microglia–neuronal signalling in neuropathic pain hypersensitivity 2.0

“…Propagation of action potentials in primary sensory neurons into the spinal cord is likely a critical step as the microglia response per se requires action potentials in primary afferents 17 . The subsequent signalling to spinal microglia has been suggested to involve a number of ligand/receptor systems, including fibronectin/integrin 18,19 , MCP1/ CCR2 20 , interferon/IFG receptor signalling 21 . Also critical for increasing expression of P2X4Rs is the tyrosine kinase Lyn 22 .…”

P2X4 purinoceptor signaling in chronic pain

“…Likewise, microglia grown in culture in the presence of the extracellular matrix molecule fibronectin show a marked increase in P2X4R levels and a consequent enhanced Ca 2+ response to ATP stimulation [52]. Fibronectin expression was elevated in the spinal cord of nerve injured animals and blockade of fibronectin receptors suppressed both the increase in P2X4Rs and tactile allodynia [52,[76][77][78]. Fibronectin induced increase in microglial P2X4R requires activity of the tyrosine kinase Lyn and downstream activation of intracellular signaling pathways involving phosphatidylinositol 3-kinase (PI3K)-Akt and mitogen-activated protein kinase kinase (MAPK kinase, MEK)-extracellular signal-regulated kinase (ERK), which have distinct roles in the up-regulation of P2X4R expression in microglia at the transcriptional and post-transcriptional levels, respectively [77].…”