Tsuda M, Toyomitsu E, Komatsu T, et al. Fibronectin/Integrin system is involved in P2X4 receptor upregulation in the spinal cord and neuropathic pain after nerve injury

Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Glia (Impact Factor: 6.03). 04/2008; 56(5):579-85. DOI: 10.1002/glia.20641
Source: PubMed


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.

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    • "P2X4 receptor upregulation was a consequence, rather than a cause of microglia activation; in fact, preventing the increase in P2X4 receptor did not affect the expression of protein markers of microglia activation, such as complement receptor 3 or the ionized calcium binding adaptor molecule-1 (Iba-1) [47, 48]. Interestingly, activation of microglial P2X4 receptor is sufficient to elicit pain, since the intrathecal injection of P2X4 receptor-stimulated cultured microglia induced mechanical allodynia in naïve animals [41, 47, 49]. Taken together, pharmacological, genetic, and behavioral findings indicate that activation of P2X4 receptor subtype expressed by spinal microglia is both necessary and sufficient to induce tactile allodynia after peripheral nerve injury. "
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    ABSTRACT: It is now well established that glial cells not only provide mechanical and trophic support to neurons but can directly contribute to neurotransmission, for example, by release and uptake of neurotransmitters and by secreting pro- and anti-inflammatory mediators. This has greatly changed our attitude towards acute and chronic disorders, paving the way for new therapeutic approaches targeting activated glial cells to indirectly modulate and/or restore neuronal functions. A deeper understanding of the molecular mechanisms and signaling pathways involved in neuron-to-glia and glia-to-glia communication that can be pharmacologically targeted is therefore a mandatory step toward the success of this new healing strategy. This holds true also in the field of pain transmission, where the key involvement of astrocytes and microglia in the central nervous system and satellite glial cells in peripheral ganglia has been clearly demonstrated, and literally hundreds of signaling molecules have been identified. Here, we shall focus on one emerging signaling system involved in the cross talk between neurons and glial cells, the purinergic system, consisting of extracellular nucleotides and nucleosides and their membrane receptors. Specifically, we shall summarize existing evidence of novel “druggable” glial purinergic targets, which could help in the development of innovative analgesic approaches to chronic pain states.
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    • "Using integrin blockers in vitro and in vivo, it was shown that fibronectin/integrin signaling was crucial for augmentation of P2X4R expression and PNI-induced tactile allodynia (Tsuda et al., 2008a). Furthermore, intrathecal injection of fibronectin to naïve animals produced tactile allodynia, a behavior not observed in P2X4R-deficient mice administered fibronectin (Tsuda et al., 2008a). Regarding intracellular signaling mechanisms underlying P2X4R upregulation by fibronectin, microglial Lyn tyrosine kinase, a member of Src-family kinases (SFKs) that belong to the non-receptor protein tyrosine kinase family, is an important molecule as upregulation of P2X4R gene expression in response to fibronectin is not observed in microglial cells lacking Lyn (Tsuda et al., 2008b). "
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    ABSTRACT: Neuropathic pain, a debilitating pain condition, is a common consequence of damage to the nervous system. Neuropathic pain is often resistant to currently available analgesics. A growing body of evidence indicates that spinal microglia react and undergo a series of changes that directly influence the establishment of neuropathic pain states. After nerve injury, P2X4 receptors (P2X4Rs) are upregulated in spinal microglia by several factors at the transcriptional and translational levels. Those include the CC chemokine CCL21 derived from damaged neurons, the extracellular matrix protein fibronectin in the spinal cord, and the transcription factor interferon regulatory factor 8 (IRF8) expressed in microglia. P2X4R expression in microglia is also regulated at the post-translational level by signaling from other cell-surface receptors such as CC chemokine receptor (CCR2). Importantly, inhibiting the function or expression of P2X4Rs and P2X4R-regulating molecules suppresses the aberrant excitability of dorsal horn neurons and neuropathic pain. These findings indicate that P2X4R-positive microglia are a central player in mechanisms for neuropathic pain. Thus, microglial P2X4Rs are a potential target for treating the chronic pain state.
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    • "Also CCL2, which instead plays an important role in microglia activation after injury [76, 77], has been suggested to participate in the P2X4R upregulation process; however, CCL2 does not seem involved in de novo expression of the protein, but rather it has been suggested to promote P2X4R trafficking from intracellular stores to the cell membrane [75]. Finally, a few nonneuronal endogenous molecules have been also identified as potential inductors of P2X4R in microglia, namely, the proinflammatory cytokines INF-γ [78], the mast cell-derived tryptase activated PAR2 [79], and fibronectin, a component of the extracellular matrix [80, 81]. At the nuclear level, the interferon regulatory factor 8 (IRF8) has been recently proposed as a key transcription factor involved in the upregulation of P2X4Rs in activated microglia [82]. "
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