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Astrocytes become activated in response to brain injury, as characterized by increased expression of glial fibrillary acidic protein (GFAP) and increased rates of cell migration and proliferation. Damage to brain cells causes the release of cytoplasmic nucleotides, such as ATP and uridine 5′-triphosphate (UTP), ligands for P2 nucleotide receptors. Results in this study with primary rat astrocytes indicate that activation of a G protein-coupled P2Y2 receptor for ATP and UTP increases GFAP expression and both chemotactic and chemokinetic cell migration. UTP-induced astrocyte migration was inhibited by silencing of P2Y2 nucleotide receptor (P2Y2R) expression with siRNA of P2Y2R (P2Y2R siRNA). UTP also increased the expression in astrocytes of αVβ3/5 integrins that are known to interact directly with the P2Y2R to modulate its function. Anti-αV integrin antibodies prevented UTP-stimulated astrocyte migration, suggesting that P2Y2R/αV interactions mediate the activation of astrocytes by UTP. P2Y2R-mediated astrocyte migration required the activation of the phosphatidylinositol-3-kinase (PI3-K)/protein kinase B (Akt) and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways, responses that also were inhibited by anti-αV integrin antibody. These results suggest that P2Y2Rs and their associated signaling pathways may be important factors regulating astrogliosis in brain disorders.
Astrocytes, a type of glial cell in the central nervous system, regulate water and electrolyte transport, local pH and ionic equilibrium, and neurotransmitter uptake (Svendsen 2002). Astrocytes can become reactive under a variety of pathological conditions, a process termed astrogliosis characterized by increased expression of glial fibrillary acidic protein (GFAP) and enhanced cell migration and proliferation (Norton et al. 1992). In cerebral ischemia, reactive astrocytes migrate to the edge of an injured area and form a barrier between damaged and healthy tissue (Ellison et al. 1998). Although there are indications that reactive astrocytes can protect undamaged tissue and limit secondary injury, excessive or chronic accumulation of astrocytes can produce deleterious effects and prevent neuronal regeneration within the damaged area (Rutka et al. 1997; Gahtan and Overmier 1999). Reactive astrogliosis is associated with increased production of cytokines and other pro-inflammatory agents that can damage neurons (McGraw et al. 2001). Release from astrocytes of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) has been shown to precede neuronal degeneration (Sheng et al. 1996; Loos et al. 2003). Therefore, a greater understanding of the mechanisms involved in reactive astrogliosis should provide new insights into ways to prevent irreversible brain damage in neurological disorders.
Astrocyte migration during reactive astrogliosis requires cell cytoskeletal rearrangements involving the extracellular matrix (McGraw et al. 2001). It has been shown that osteopontin (OPN), an extracellular matrix protein, is up-regulated during the formation of glial scars after focal ischemia (Ellison et al. 1999). A receptor for OPN, the integrin αVβ3, is also up-regulated in reactive astrocytes that localize to the peri-infarct area 5 days after ischemia and to an OPN-rich, glial barrier 15 days post-ischemia, suggesting that αVβ3 and its extracellular ligands are involved in reactive astrogliosis (Ellison et al. 1998). The αVβ3 and αVβ5 integrins play essential roles in cell migration by interacting with extracellular ligands containing an arginine-glycine-aspartic acid (RGD) motif including OPN, vitronectin, fibronectin and thrombospondin (Carriero et al. 1999; Cirulli et al. 2000; Kappert et al. 2001; Manes et al. 2003).
When tissues are damaged, cytoplasmic nucleotides such as ATP and uridine 5′-triphosphate (UTP) are released from injured cells. These nucleotides can activate cell surface P2 nucleotide receptors and trigger cell proliferation, migration or apoptosis (Wilden et al. 1998; Coutinho-Silva et al. 1999; Chaulet et al. 2001). Previous studies indicate that stretch- or stab-induced injury causes the activation of a G protein-coupled P2Y nucleotide receptor in astrocytes and, in turn, increases extracellular signal-regulated kinase (ERK) activation, GFAP expression and astrocyte proliferation (Neary et al. 1994a,b, 1999, 2003; Franke et al. 1999). The present study investigated the role of the P2Y2 nucleotide receptor (P2Y2R) subtype in the activation of primary rat astrocytes. Among the human nucleotide receptor subtypes, G protein-coupled P2Y2Rs are unique in that they are activated by either ATP or UTP, and contain an RGD motif in the first extracellular loop that enables P2Y2Rs to interact directly with αVβ3/β5 integrins (Erb et al. 2001). In this study, we tested the hypothesis that activation of P2Y2Rs leads to increased astrocyte migration, a feature of astrogliosis. The results indicate that P2Y2R activation induces migration of primary astrocytes associated with an increase in the expression of GFAP and αVβ3/β5. Furthermore, activation of intracellular signaling pathways involving phosphatidylinositol-3-kinase (PI3-K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) were required for P2Y2R-mediated astrocyte migration, and these responses were inhibited by anti-αV antibodies. The results demonstrate a pathway whereby activation of P2Y2Rs by nucleotides released from damaged or stressed cells can trigger astrogliosis associated with brain injuries, suggesting potential targets for the prevention of neurodegeneration.
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Reactive astrogliosis is critical for tissue remodeling and repair in the central nervous system (McGraw et al. 2001), and it occurs in various brain pathologies such as trauma, stroke and Alzheimer's disease (Ridet et al. 1997). Chronic astrogliosis is also thought to have deleterious effects such as the inhibition of neuronal regeneration in brain injury (Rutka et al. 1997). Previous studies have indicated that nucleotides can increase GFAP expression in astrocytes, indicative of reactive astrogliosis (Neary et al. 1994b; Franke et al. 1999). This study was undertaken to determine the P2 receptor subtype and signaling pathways involved in nucleotide-induced reactive astrogliosis.
Extracellular nucleotides are released from aggregating platelets, degranulating macrophages, excitatory neurons and injured or stressed cells in response to ischemia, hypoxia or mechanical stretch (Bergfeld and Forrester 1992; Beigi et al. 1999; Ciccarelli et al. 1999; Pedersen et al. 1999; Ahmed et al. 2000; Bodin and Burnstock 2001; Ostrom et al. 2001; Joseph et al. 2003). Release of nucleotides has been proposed to occur by exocytosis of ATP/UTP-containing vesicles, facilitated diffusion by putative ABC transporters, cytoplasmic leakage, and by electrodiffusional movements through ATP/nucleotide channels (Zimmermann and Braun 1996). The released nucleotides can activate cell surface P2 nucleotide receptors that mediate physiological functions such as neurotransmission and cell proliferation, migration and apoptosis (Bodin and Burnstock 2001; Ciccarelli et al. 2001). It has been found that extracellular ATP and UTP can induce smooth muscle and endothelial cell migration (Chaulet et al. 2001; Goepfert et al. 2001; Pillois et al. 2002; Seye et al. 2002), and ATP and ADP have been shown to induce chemotaxis in a microglial cell line (Honda et al. 2001). However, it has not been established whether extracellular nucleotides can stimulate the migration of primary astrocytes. Furthermore, the subtype of P2 receptor that promotes reactive astrogliosis is not known and the mechanisms involved have not been fully elucidated. The results of the present study indicate that extracellular UTP activates a G protein-coupled P2Y2R subtype in primary rat astrocytes, and induces the phenotype of reactive astrogliosis that is characterized by increased expression of GFAP and an enhanced rate of cell migration. In addition, we have identified novel and complex signaling pathways that regulate nucleotide-induced astrocyte migration through the interaction of the P2Y2R with αvβ3/β5 integrins and the stimulation of the downstream signaling molecules, PI3-K and MEK.
The P2Y2R subtype of G protein-coupled P2Y nucleotide receptor has been proposed to play an essential role in immune responses and injury (Koshiba et al. 1997; Seye et al. 1997, 2002; Turner et al. 1998). P2Y2R up-regulation occurs in response to vascular injury; this leads to neointimal hyperplasia and inflammation in arteries through processes involving smooth muscle cell migration/proliferation and the endothelial-dependent adherence of monocytes, respectively, responses associated with atherogenesis and atherosclerosis (Seye et al. 2002, 2003, 2004). In the present study, we investigated the ability of P2Y2Rs to mediate reactive astrogliosis. Results indicate that the equi-potent and equi-efficacious P2Y2R agonists, ATP and UTP (Parr et al. 1994; Weisman et al. 1999), stimulate astrocyte chemokinesis and chemotaxis (Fig. 1). Among the P2 nucleotide receptors, only the P2Y2, P2Y4 and P2Y6 receptor subtypes can be activated by uridine nucleotides (Nicholas et al. 1996; Abbracchio and Burnstock 1998), and primary astrocytes express mRNA for these three P2YR subtypes (Fumagalli et al. 2003; data not shown). Among these receptors, only the P2Y2R can be fully activated by ATP as well as UTP, whereas P2Y6 receptors are more sensitive to UDP than UTP (Nicholas et al. 1996). We have determined that UDP does not induce astrocyte migration (data not shown), eliminating a role for the P2Y6R in responses to UTP that could have occurred upon UTP degradation to UDP by ecto-NTPDases (Gendron et al. 2002). P2Y4Rs are preferentially activated by UTP, and are relatively insensitive to ATP (Communi et al. 1995, 1996; Nguyen et al. 1995). Therefore, the ability of similar concentrations of UTP and ATP to induce astrocyte migration is most characteristic of the pharmacological profile of P2Y2Rs. Nonetheless, the finding that P2Y2R siRNA, which suppresses P2Y2R but not P2Y4R mRNA expression, can prevent UTP-induced astrocyte migration (Fig. 2) unambiguously demonstrates the role of P2Y2Rs in this process. Unfortunately, the unavailability of specific anti-P2Y2R antibodies precludes an attempt to evaluate whether P2Y2R siRNA inhibits P2Y2R protein expression in primary astrocytes.
The αVβ3/β5 integrins are receptors for RGD-containing extracellular matrix proteins (Nakamura et al. 2003) that have important roles in angiogenesis and inflammation. The αVβ3 integrin is up-regulated in reactive astrocytes; it plays a key role in tissue remodeling and limits the extent of brain injury (Ellison et al. 1998, 1999). The αVβ5 integrin and its extracellular ligands have also been linked to cell migration in astrocytes, breast carcinoma cells, endocrine progenitor cells and smooth muscle cells (Faber-Elman et al. 1995; Carriero et al. 1999; Cirulli et al. 2000; Kappert et al. 2001). Inhibition of αVβ3 and αVβ5 integrin activities by peptidic or non-peptidic antagonists was found to decrease UTP-induced smooth muscle cell migration associated with OPN expression (Chaulet et al. 2001), which is mediated by P2Y2Rs (Pillois et al. 2002). Recently, the P2Y2R was shown to interact directly with αVβ3/β5 integrins via an RGD motif in its first extracellular loop (Erb et al. 2001). Furthermore, our studies indicate that interactions between the P2Y2R and αV integrins are critical for the P2Y2R to activate G12 and G0, and stimulate G12- and G0-mediated signaling events that lead to cell migration in human astrocytoma 1321N1 cells expressing a recombinant P2Y2R (Erb et al. 2001; unpublished data). Results from the present study indicate that UTP induced expression of αVβ3 and αVβ5 integrin complexes (Fig. 3), and that anti-αV antibody significantly inhibited P2Y2R-mediated migration of primary astrocytes (Fig. 4). Thus, these results indicate for the first time that UTP induces the up-regulation of αVβ3 and αVβ5 in astrocytes, and demonstrate a role for these integrins in P2Y2R-mediated astrocyte migration.
PI3-K/Akt and ERK are critical signaling molecules that regulate cell migration (Sotsios and Ward 2000; Stahle et al. 2003) and integrin-mediated cell migration (Hood and Cheresh 2002). It has been reported that PI3-K is involved in the migration of reactive astrocytes (Tezel et al. 2001), and a PI3-K inhibitor significantly prevented P2Y2R-mediated astrocyte migration (Fig. 5). The MEK/ERK pathway is involved in nucleotide-induced reactive astrogliosis, and mediates the elongation of cellular processes and the up-regulation of cyclooxygenase-2 (Brambilla et al. 2001, 2002, 2003). We also found that a MEK inhibitor prevented UTP-stimulated astrocyte chemokinesis and chemotaxis (Fig. 5). UTP-induced phosphorylation of Akt was completely inhibited by anti-αV antibody (Fig. 6), whereas the antibody partially inhibited UTP-induced ERK phosphorylation. These results are consistent with a role for P2Y2Rs and αVβ3/β5 integrin interactions in both Akt and ERK activation, although P2Y2R activation of ERK can also occur through G protein-dependent activation of phospholipase C and Src-dependent transactivation of growth factor receptors (Soltoff 1998; Erb et al. 2001; Liu et al. 2004). Nonetheless, it appears that astrocyte migration in response to UTP-induced activation of PI3-K/Akt and MEK/ERK is dependent upon P2Y2R-mediated interactions with αVβ3/β5 integrins.
Previous studies have shown that extracellular nucleotides cause rapid release from astrocytes of the wound-related factor Transforming growth factor-β (TGF-β) (Gendron et al. 2003b). Our recent studies also indicate that P2Y2R activation by UTP rapidly induces the transactivation of vascular endothelial growth factor receptors that mediate sustained increases in the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells (Seye et al. 2003, 2004). Similarly, P2Y2Rs and extracellular nucleotides cause increases in GFAP and αVβ3/β5 integrin expression in primary astrocytes. These results suggest that P2Y2Rs may represent an early mediator of reactive astrogliosis, and may prove to be novel targets for therapies that minimize the deleterious effects of chronic astrogliosis associated with brain injury or disease.
The effects of reactive astrogliosis on neurological functions have been described in both positive and negative terms. In the initial stages, reactive astrogliosis can be beneficial in limiting brain damage in Alzheimer's disease by promoting the clearance of β-amyloid (Monsonego and Weiner 2003). In the chronic stages, reactive astrogliosis leads to the formation of glial scars that prevent neuronal cell regeneration, or have neurotoxic effects that promote the formation of astrocyte-derived amyloid plaques (Nagele et al. 2004). Although the long-term effects of reactive astrogliosis require further elucidation, our data provide insights into the mechanisms underlying the initiation of reactive astrogliosis due to P2Y2R activation. We also have found that activation of P2Y2Rs in astrocytic cells promotes cell survival mechanisms, and conditioned medium from these UTP-treated cells stimulates outgrowth of neurites in PC-12 cells (Chorna et al. 2004). Thus, the dual role of P2Y2Rs in promoting neuroprotection and neurodegeneration warrants further investigation.