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The P2X7 receptor is an ion-gated channel, which is activated by high extracellular concentrations of adenosine triphosphate (ATP). Activation of P2X7 receptors has been shown to induce neuroinflammatory changes associated with several neurological conditions. The matrix metalloproteinases (MMPs) are a family of endopeptidases that have several functions including degradation of the extracellular matrix, cell migration and modulation of bioactive molecules. The actions of MMPs are prevented by a family of protease inhibitors called tissue inhibitors of metalloproteinases (TIMPs). In this study, we show that ATP-treated glial cultures from neonatal C57BL/6 mice release and increase MMP-9 activity, which is coupled with a decrease in release of TIMP-1 and an increase in activated cathepsin B within the extracellular space. This process occurs independently of NLRP3-inflammasome formation. Treatment with a P2X7 receptor antagonist prevents ATP-induced MMP-9 activity, inhibition of active cathepsin B release and allows for TIMP-1 to be released from the cell. We have shown that cathepsin B degrades TIMP-1, and inhibition of cathepsin B allows for release of TIMP-1 and inhibits MMP-9 activity. We also present data that indicate that ATP or cell damage induces glial cell migration, which is inhibited by P2X7 antagonism, depletion of MMP-9 or inhibition of cathepsin B.
Microglia are regarded as the immunocompetent cells of the central nervous system and thus play an important role in response to any brain injury. Under resting conditions, microglia are found in a motile ramified state where they constantly extend processes to sample the surrounding parenchyma (Nimmerjahn et al. 2005). Upon stimulation, for example in response to injury, microglia adopt an activated phenotype. They retract their processes and migrate towards the site of injury, where they release pro- and anti-inflammatory cytokines with the objective of limiting damage by phagocytosing cellular debris and subsequently, to exert a restorative effect by initiating tissue repair.
The purine adenosine 5′-triphosphate (ATP) has previously been shown to act as a potent chemoattractant for microglia when present in the extracellular milieu (Choi et al. 2010). Under normal physiological conditions, extracellular concentrations of ATP are maintained between 400 and 700 nM. However, under pathological conditions, for example ischaemia, damaged cells release ATP and increase extracellular levels to concentrations in the high millimolar range (Melani et al. 2005). At these concentrations, ATP acts as a danger-associated molecular pattern (DAMP) and can act on members of the purinergic receptor family. The P2X7 receptor is a member of this family of receptors (Bianchi 2007). It has previously been shown to be present on immune cells including macrophages (Qu et al. 2007) and microglia (Murphy et al. 2012).
For cell migration to occur, proteases must cleave cell–cell interactions or cell–matrix interactions. Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteolytic enzymes that have previously been shown to be involved in cell migration (Palmisano and Itoh 2010). MMPs have several functions including cell migration, morphogenesis, cell proliferation, apoptosis and modulation of the activity of enzymes or other biologically active molecules such as growth factors. As proteases, matrix metalloproteinases have the potential to be destructive to surrounding tissue and therefore their expression and activity is tightly regulated. They are expressed as inactive zymogens and must be processed to expose their active site. They are also regulated by a family of endogenous inhibitors called tissue inhibitors of matrix metalloproteinases (TIMPs). The family consists of four members, TIMP-1, -2, -3 and -4. TIMPs are 20–30kDa in size and interact directly with MMPs through a small number of amino acids. All MMPs can be inhibited by TIMPs once activated, but the MMPs can also form complexes with TIMPs in their latent form.
Another family of proteases, the cathepsins, which are cysteine peptidases, have also been implicated in the process of cell migration. The cathepsins are a family of 15 proteins, which are located predominantly within lysosomes. However, in response to neurological insults (Fogarty et al. 2010) (Kilinc et al. 2010), the lysosomal membrane can become destabilized and result in the release of cathepsins into the surrounding extracellular space. Their function in the extracellular space is still debated, but a role in cell migration has been suggested (Veeravalli et al. 2010).
The data here propose a mechanism by which ATP or cell injury induces cell migration. We demonstrate that ATP, acting through the P2X7 receptor, induces release of cathepsin B into the extracellular space where it degrades TIMP-1, permitting the MMP-9-dependent migration of glial cells.
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- Experimental procedure
In this study, we have demonstrated that ATP, acting via the P2X7 receptor induces increased activity of the matrix metalloproteinase, MMP-9, in glial cells. The activation of MMP-9 occurs as a result of degradation of its endogenous inhibitor, TIMP-1, by cathepsin B and this series of events provides a mechanism for the ATP-induced migration of cells. The data indicate that the specific P2X7 receptor antagonist inhibits migration of cells by blocking the ATP-induced activity of MMP-9.
Extracellular ATP is a ‘find me’ signal that dying cells release to recruit phagocytic cells to the area of damage (Elliott et al. 2009), and here we demonstrate that its ability to influence migration of glia is inhibited by a specific antagonist at the P2X7 receptor. Importantly, the data show that the ATP-induced migration is dependent on MMP-9 activity. The link between MMP-9 activity and cell migration has been widely studied in endothelial cells (Regina et al. 2003), leucocytes (Khandoga et al. 2006) and monocytes (Zhou et al. 2012). To date, relatively few studies have investigated the role of MMP-9 in glial migration, although it was suggested that the migration of microglial cells in response to urokinase-type plasminogen activator (uPA) is mediated by MMP-9 (Shin et al. 2010). Similarly, stress-inducible protein 1 (STI1) activates MMP-9 and induces cell migration in microglial cultures (da Fonseca et al. 2012). Several processes are necessary for cell migration including extension and projection of the cell membrane, as well as cell-to-extracellular matrix adhesion, which relies on ligation of adhesion receptors by components of the extracellular matrix. Although the mechanism by which MMP-9 modulates cell migration is not well understood, it has been shown to modulate the interaction between the receptor CD44, which is present in astrocytes and microglia, and hyaluronan in the ECM leading to increased motility of glioblastoma cells (Chetty et al. 2012).
Our data indicate that MMP-9 activation occurs downstream of P2X7 receptor activation and is evident in mixed glia as well as isolated microglia and isolated astrocytes; this broadly supports previous data, which indicate that extracellular ATP induces MMP-9 release from peripheral blood mononuclear cells (Gu and Wiley 2006) and increases its expression in mesangial (Huwiler et al. 2003) and epithelial (Wesley et al. 2007) cells. ATP-induced release of MMP-9 from microglia and BV-2 cells has also been reported (Choi et al. 2010). The authors reported that ATP-induced migration of BV2 cells was MMP-9 dependent, and argued that the effect was mediated by activation of P2Y1 and P2Y12 receptors as it was blocked by pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonate (PPADS) and clopidogrel. However, it has been suggested that PPADS is not a specific P2Y1 receptor antagonist and that it can also act as an antagonist of P2X receptors (Oliveira et al. 2011) (Prasai et al. 2011). These other purinergic receptors, however, may play a role in the secretion of MMP-9 from the cell into the extracellular space. Although our data indicate that inhibition of the P2X7 receptor inhibits MMP-9 activation, there is no evidence of any change in the secretion of MMP-9 from the cell when this receptor is antagonized. Activation of P2Y2 by ATP has been shown to induce release of the chemokine MCP-1 from immune cells (Stokes and Surprenant 2007) and this, in turn, can induce the secretion of MMP-9 (Cross and Woodroofe 1999). Similarly, another inducer of MMP-9 secretion, RANTES (Cross and Woodroofe 1999), is released from the cell in response to ATP activation of P2Y1 (Pastore et al. 2007). These reports, taken together with the evidence presented here, suggest that purinergic receptors other than P2X7, play a role in MMP-9 secretion.
In an effort to understand the events leading up to MMP-9 activation, we examined ATP-induced release of the endogenous inhibitor of MMP-9, TIMP-1, from mixed glial cells. The data indicate that ATP decreased its release and that this effect was attenuated by inhibition of the P2X7 receptor with GSK1370319A. Similar effects were observed in both isolated microglia and isolated astrocytes, however, the effect of the P2X7 antagonist was more profound in microglia. This difference may be accounted for by our previously reported finding that the P2X7 receptor is expressed to a much greater extent on microglia than astrocytes (Murphy et al. 2012). Mirroring the changes observed here, it has been reported that release of activated MMP-9 was associated with decreased TIMP-1 release in peripheral blood mononuclear cells (Gu and Wiley 2006), although the underlying mechanism was not identified. Here we report, for the first time, that active cathepsin B is released from primary mouse glial cells in a P2X7-dependent manner. In a similar manner, activation of the P2X7 receptor by ATP has been shown to release cathepsin B from cancer cells, mouse bone marrow-derived macrophages and human lung macrophages (Lopez-Castejon et al. 2010; Jelassi et al. 2011). Lopez-Castejon et al. report that cathepsin B release in response to P2X7 receptor activation occurs in a calcium-dependent manner in bone marrow-derived macrophages. This study indicates that this process is also calcium dependent in glia. The evidence presented here suggests that cathepsin B degrades TIMP-1 and that ATP-dependent activation of MMP-9 is blocked by inhibiting cathepsin B. Therefore, we conclude that cathepsin B may play a key role in triggering the events leading to ATP-induced cell migration. Interestingly, TIMP-1, and also TIMP-2, have been identified as substrates for cathepsin B in human chondrocytes (Kostoulas et al. 1999), and a role for both cathepsin B and MMP-9 in migration of human glioma cells has been reported (Veeravalli et al. 2010). Cathepsin B is a protease, which is normally found in lysosomes. However, in response to insults, for example ischaemic injury (Qin et al. 2008), where release of ATP from damaged cells is significant, cathepsin B leaks into the cytoplasm. Indeed, the evidence suggests that its release, as a result of increased lysosomal membrane permeability, may contribute to the pathogenesis of neurodegenerative diseases (Wang and Qin 2010).
Activation of the P2X7 receptor in glial cells has been shown to result in maturation and release of the proinflammatory cytokine IL-1β from lipopolysaccharide-primed cells (Murphy et al. 2012). In this setting, activation of the P2X7 receptor results in recruitment association of three of proteins, apoptotic speck-like protein (ASC), the nod-like protein, NLRP3 and the cysteine protease, caspase 1. This assembly of proteins is known as the NLRP3 inflammasome and it drives the proteolytic cleavage of the cysteine protease, caspase 1 which, in turn, cleaves pro-IL-1β to the mature form enabling its release from the cell. We investigated whether the P2X7-mediated decrease in TIMP-1 and subsequent activation of MMP-9 was dependent on formation of this inflammasome. The data indicate that the caspase 1 inhibitor, Z-WEHD-FMK, exerted no effect on ATP-induced TIMP-1 release or MMP-9 activation, and therefore we can conclude that these ATP-associated changes which lead to cell migration are independent of inflammasome formation. Recently, it has been reported that P2X7-dependent release of cathepsin B is independent of the NLRP3 inflammasome-processed IL-1β (Lopez-Castejon et al. 2010).
We propose a novel mechanism for the migration of glia in response to injury or cell damage in the brain that is dependent on activation of the P2X7 receptor. Receptor activation induces a sequence of events initiated by release of cathepsin B that results in cell migration. Using a model of cell damage, as well as application of ATP to glia, the evidence indicates that migration is blocked by the P2X7 receptor antagonist, GSK1370319A and also by inhibiting cathepsin B and MMP-9. Targeting this pathway has the potential to provide novel pharmacological tools, which may enable repair in certain neurological disorders.