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Keywords:

  • astrocyte migration;
  • astrogliosis;
  • integrins;
  • P2 nucleotide receptors;
  • uridine 5′-triphosphate

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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.

Abbreviations used
Akt

protein kinase B

DMEM

Dulbecco's modified Eagle's medium

ERK

extracellular signal-regulated kinase

FBS

fetal bovine serum

G3PDH

glyceraldehyde 3-phosphate dehydrogenase

GFAP

glial fibrillary acidic protein

HRP

horseradish peroxidase

IL-1β

interleukin-1β

MEK/ERK

mitogen-activated protein kinase/extracellular signal-regulated kinase

MEK

mitogen-activated protein kinase

OPN

osteopontin

PBS

phosphate-buffered saline

PI3-K

phosphatidylinositol-3-kinase

P2Y2R

P2Y2 nucleotide receptor

P2Y2RsiRNA

small interference RNA of P2Y2R

RGD

arginine-glycine-aspartic acid

SDS

sodium dodecyl sulfate

TGF-β

Transforming growth factor-β

TNF-α

tumor necrosis factor-α

UDP

uridine 5′-diphosphate

UTP

uridine 5′-triphosphate

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 αVβ35 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 αVβ35. 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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Antibodies and reagents

Goat anti-rat αV, anti-rat α4, anti-rat β3 and anti-rat β5, rabbit anti-rat ERK, anti-rat GFAP (H50), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated monkey anti-goat IgG antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LY294002, U0126, rabbit anti-rat phospho-p44/42 ERK1/2, rabbit anti-rat Akt and rabbit anti-rat phospho-Akt (Ser473) antibodies were obtained from Cell Signaling Biotechnology (Beverly, MA, USA). Rabbit anti-rat αV antibody was obtained from Chemicon (Temecula, CA, USA). Rabbit anti-rat actin antibody was obtained from Cytoskeleton (Denver, CO, USA). Nucleotides and other chemicals were from Sigma (St Louis, MO, USA).

Primary rat astrocyte cell culture

Astrocytes were isolated from the cerebral cortices of post-natal 2–3-day-old Sprague–Dawley rat pups and cultured as previously described (McCarthy and de Vellis 1980) with minor modifications. Briefly, dissected cerebral cortices were placed in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, USA). The meninges were removed, and the cortices were cut into small pieces and incubated with 0.25% (w/v) trypsin-EDTA at 37°C for 7 min. The suspension was then filtered through 85 µm nylon mesh and centrifuged at 1000 rev/min (approximately 250 g) for 5 min. The cell pellet was re-suspended in culture medium comprising DMEM plus 10% (v/v) fetal bovine serum (FBS), 100 IU/mL penicillin, 100 µg/mL streptomycin and 7.5 µg/mL fungizone, and transferred to T75 culture flasks (Techno Plastic Products, Trasadingen, Switzerland). Cells were maintained in an atmosphere of 5% CO2 and 95% air at 37°C, and the medium was changed twice a week. When cells reached approximately 80–90% confluence, flasks were shaken at 225 rev/min for 6 h at room temperature (15–25°C) to remove microglial cells. Then, cells were washed with phosphate-buffered saline (PBS), removed from flasks by treatment with 0.05% (w/v) trypsin-EDTA at 37°C for 4 min, and seeded at 1 × 106 cells in 60 mm culture dishes for RT-PCR experiments. Approximately 1 × 105 cells/well were cultured in 12-well plates for immunoblot analysis. Astrocytes were identified by GFAP staining (more than 95% of the total cells were GFAP positive).

Astrocyte treatments

Primary rat astrocytes (1 × 105 cells/well) were maintained in 12-well plates with culture medium for 2 days, followed by serum-free medium for 1 day. Astrocytes were subsequently incubated with 10 µg/mL anti-αV or anti-α4 antibody for 2 h, and stimulated with UTP at the concentrations and times indicated in the figure legends. For immunoblot analysis, cells were washed with ice-cold PBS and lysed with 2× Laemmli sample buffer [120 mm Tris-HCl, pH 6.8, 2% (w/v) sodium dodecyl sulfate (SDS), 10% (w/v) sucrose, 1 mm EDTA, 50 mm dithiothreitol, and 0.003% (w/v) bromophenol blue]. The cell lysate was used for immunoblot analysis.

P2Y2 receptor siRNA transfection

Vector-based, P2Y2R-specific siRNAs were designed using Genscript's siRNA design target and construct builder (http://www.genscript.com). The consecutive sequences for P2Y2R antisense, loop and P2Y2R sense cDNA, 5′-AGGCCGCATACAGTGCATCAGTTGATATCCGCTGATGCACTGTATGCGGCCT-3′, were cloned into the pRNA-U6.1/Neo plasmid vector. Empty vector was used as a negative control. Transfection of primary rat astrocytes was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, astrocytes at approximately 70–80% confluence were cultured for 1 day in culture medium without antibiotics. Lipofectamine 2000 was mixed in DMEM for 5 min at room temperature (15–25°C), incubated for 20 min with plasmid DNA diluted in DMEM, and incubated with astrocytes for 24 h at 37°C in a cell culture incubator. The efficiency of siRNA transfection of astrocytes was determined by the reduction in P2Y2R mRNA levels detected by RT-PCR.

RNA extraction and RT-PCR

Total RNA was isolated from primary astrocytes using the Qiagen Rneasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. cDNA was synthesized from total RNA using the First Strand cDNA Synthesis Kit and an oligo-dT primer (Roche Diagnostics Corporation, Indianapolis, IN, USA). Specific primers were used to amplify P2Y2R, P2Y4R and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNA by RT-PCR, as previously described (Gendron et al. 2003a). PCR was performed as follows: 94°C for 2 min, 35 cycles at 94°C for 1 min, 60°C for 1 min and 72°C for 1 min, with a final elongation step at 72°C for 7 min. PCR products were analyzed by electrophoresis on 1.5% agarose gels with 10 µg/mL ethidium bromide, and bands were visualized under UV light. The ratio of P2Y2R/G3PDH product was determined using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Chemokinesis assay

This assay was adapted from a protocol described by Albrecht-Buehler (1977). Coverslips (22 × 22 mm) and gold particles were prepared as described. Primary rat astrocytes (1 × 105 cells/mL) in serum-free medium were seeded onto coverslips. Then, cells were incubated with or without UTP or ATP at the concentration indicated in the figure legends for 18 h at 37°C. When indicated, inhibitors or other agents were added to the astrocytes 2 h before addition of UTP or ATP. Then, cells were washed with PBS and fixed with 4% (v/v) formaldehyde for 15 min. Fixed cells were observed under an inverted microscope, images were captured by a digital camera (Qimaging, Burnaby, BC, Canada) and analyzed with Northern Eclipse 6.0 software (Empix Imaging Inc. Mississauga, ON, Canada). As cells migrated on the coverslips, they displaced and endocytosed gold particles, leaving white phagokinetic tracks that were recorded.

Chemotaxis assay

Primary rat astrocytes (transfected or untransfected) were suspended with trypsin, washed, and re-suspended in serum-free medium at 5 × 105 cells/mL. Transwell chambers (Becton Dickinson Labware, Franklin Lakes, NJ, USA) with 8 µm pore size polycarbonate membrane inserts were placed into wells of 24-well culture plates. The lower Transwell chamber was filled with 600 µL serum-free medium containing UTP or ATP, as indicated, and a 100 µL cell suspension was added to the upper chamber. In some cases, antibodies were added to the lower chamber, and inhibitors to both chambers, 2 h prior to nucleotide addition. After 18 h at 37°C in a 5% CO2 incubator, the cells remaining in the upper chamber were removed by scraping the membrane with a cotton swab. Cells attached to the bottom of the membrane were fixed with ice-cold methanol for 15 min, washed with PBS and stained with hematoxylin for 15 min. Stained cells observed under a microscope were counted in 10 fields and represented chemotactic cells that had migrated from the upper to the lower chamber in response to nucleotides in the lower chamber.

Immunoblot analysis

The detection of integrin subunits, GFAP, phospho-ERK1/2, ERK1/2, phospho-Akt, Akt and actin was achieved by immunoblot analysis. For these assays, primary rat astrocytes were grown to about 70–80% confluence in 6-well plates, cultured in serum-free medium for 2 days, and stimulated with UTP in the absence or presence of inhibitors (added 2 h before the UTP), as indicated in the figure legends. Then, cells were washed with ice-cold PBS and lysed in 100 µL 2× Laemmli sample buffer. Cell lysates were sonicated with 2 s blasts of 4 pulses using a Sonic Dismembrator (Fisher, Pittsburgh, PA, USA), heated for 5 min at 96°C, subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane. Western blot analysis was performed by incubation of membranes with a 1 : 1000 dilution of the indicated primary antibody at 4°C overnight, followed by a 1 : 2000 dilution of horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG antibody for 1 h at room temperature (15–25°C). Chemiluminescence of antibody-labeled proteins was detected on X-ray films and quantitated with Quantity One software (Bio-Rad Laboratories). Then, the antibodies were stripped from the membranes by a 30 min incubation at 60°C in stripping buffer [62.5 mm Tris-HCl, pH 6.8, 100 mm 2-mercaptoethanol and 2% (w/v) SDS], and the membranes were re-probed with anti-actin, anti-ERK or anti-Akt antibody to determine the relative amounts of protein loaded in each lane.

Immunoprecipitation

Primary rat astrocytes grown to approximately 80% confluence in 100 mm tissue culture dishes were cultured in serum-free medium for 48 h and stimulated with 100 µm UTP for 0, 8, 18 or 24 h. Then, cells were lysed for 15 min at 4°C in RIPA buffer [50 mm Tris-HCl, pH 7.4, 1% (v/v) NP-40, 0.25% (v/v) Na-deoxycholate, 150 mm NaCl, 1 mm EDTA] supplemented with proteinase inhibitor cocktail (Roche). Insoluble membrane and unlysed cells were removed from the lysate by centrifugation at 13 000 g for 20 min at 4°C. Then, 5 µg/mL anti-αV antibody was added to 800 µL lysate and incubated at 4°C with gentle rocking overnight, followed by incubation with 100 µL Protein A agarose beads (Upstate Biotechnology, Waltham, MA, USA) at 4°C, with gentle rocking, overnight. The mixture was washed three times with 800 µL PBS and immunoblot analysis was performed with anti-αV, anti-β3 or anti-β5 antibodies, as described above.

Statistical analysis

Data indicate the means ± SEM of results from at least three experiments. Results were analyzed by one-way anova with a Bonferroni post-test for comparing more than two groups, and unpaired Student's t-test for comparing two groups, where p < 0.05 was considered to be significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

P2Y2Rs mediate migration of primary rat cerebral cortical astrocytes

GFAP, an intermediate filament protein expressed in astrocytes of the central nervous system, is a specific marker of astrocytes (Eng et al. 2000). Increased expression of GFAP in astrocytes has been used as an indicator of reactive astrogliosis (Rutka et al. 1997). Stimulation of primary rat astrocytes with UTP (Fig. 1a) or ATP (Neary et al. 1994b) caused a time-dependent increase in GFAP expression (Fig. 1a), suggesting that extracellular nucleotides induce reactive astrogliosis. Incubation of primary astrocytes with ATP or UTP also caused an increase in the area of chemokinetic tracks (Fig. 1b), an indication of migration of reactive astrocytes. In addition, ATP or UTP added to the lower chamber of cell culture dishes containing Transwells with semi-permeable polycarbonate membrane inserts caused a dose-dependent increase in the transmembrane chemotactic migration of astrocytes that were plated in the upper chamber (Fig. 1c). Uridine 5′-diphosphate (UDP), which is not a P2Y2R agonist (King et al. 2001), had no significant effect on astrocyte migration in either assay (data not shown). Together, these data demonstrate that ATP and UTP, equipotent agonists of the P2Y2R subtype (Parr et al. 1994; Weisman et al. 1999; King et al. 2001), activate the migration of primary astrocytes.

image

Figure 1. Extracellular UTP or ATP stimulates GFAP expression and astrocyte migration. (a) UTP stimulates GFAP expression. Quiescent astrocytes (maintained in serum-free medium for 48 h at 37°C) were stimulated with 50 µm UTP for the indicated times at 37°C. Then, cell lysates were prepared and subjected to 10% SDS–PAGE, and immunoblot analysis was performed as described in Materials and methods. GFAP was detected with anti-GFAP antibody and membranes were stripped and re-probed with anti-actin antibody to verify the equivalence of protein loading between lanes. Data represent the means ± SEM of results from three experiments where **p < 0.01 indicates a significant difference from untreated control. (b) UTP or ATP stimulates astrocyte chemokinesis. Primary rat astrocytes (black dots) were seeded on coverslips coated with gold particles and incubated in serum-free medium, with or without 100 µm ATP or UTP, for 18 h at 37°C. Then, cells were washed with PBS, fixed, observed under a microscope, the images captured with a digital camera and analyzed by Northern Eclipse software. The area of the white phagokinetic tracks indicates the extent of cell migration. Bar = 100 µm. (c) UTP or ATP stimulates astrocyte chemotaxis. A modified, open-well Transwell chamber with a polycarbonate membrane insert was used to separate two chambers in 24-well plates. Primary rat astrocytes (5 × 104 cells per chamber) in serum-free medium were seeded onto the membrane in the upper chamber, whereas the lower chamber contained 0, 1, 10 or 100 µm UTP or ATP. Cells were incubated in a cell culture incubator for 18 h at 37°C. Then, membranes were washed with PBS and cells that had migrated from the upper to the lower chamber were quantitated as described in Materials and methods. Data represent the means ± SEM of results from at least three experiments, where *p < 0.05 and **p < 0.01 indicate significant differences from untreated controls.

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To examine the role of the P2Y2R in extracellular nucleotide-induced astrocyte migration, we used P2Y2R siRNA to inhibit the expression of the receptor. RT-PCR showed that P2Y2R siRNA significantly reduced P2Y2R but not P2Y4R mRNA expression in astrocytes (Fig. 2a,b). Pre-treatment of astrocytes with P2Y2R siRNA also resulted in the inhibition of UTP-induced astrocyte migration (Fig. 2c). This result demonstrates that P2Y2Rs mediate UTP-induced migration of primary rat astrocytes.

image

Figure 2. P2Y2R siRNA inhibits UTP-induced astrocyte migration. (a) P2Y2R siRNA decreases P2Y2R mRNA expression. Primary rat astrocytes incubated for 1 day in DMEM without antibiotics were treated with Lipofectamine 2000, with or without 3 µg P2Y2R siRNA or empty vector, as described in Materials and methods. After 24 h, total RNA was isolated and expression of P2Y2R, P2Y4R (negative control) or G3PDH mRNA was determined by RT-PCR, as described in Materials and methods. Lane 1: untreated control; lane 2: Lipofectamine 2000 control; lane 3: P2Y2R siRNA; lane 4: empty vector control. (b) Data represent the means ± SEM of results from three experiments, where **p < 0.01 indicates a significant difference from untreated controls. (c) P2Y2R siRNA inhibits UTP-induced astrocyte migration. Cells treated with P2Y2R siRNA or empty vector in Lipofectamine 2000 for 24 h as in (a) were removed with trypsin, washed three times with serum-free medium and used for chemotactic assays, with or without 100 µm UTP, as described for Fig. 1(c). After 18 h, cell migration was determined, as described in Materials and methods. Data represent the means ± SEM of results from three experiments, where **p < 0.01 indicates a significant difference from UTP-treated controls.

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UTP-induced astrocyte migration is dependent on αV integrins

The αVβ3 and αVβ5 integrins play essential roles in mediating cell migration in various cell types (Carriero et al. 1999; Chaulet et al. 2001). Increased expression of αVβ3 integrin was detected in reactive astrocytes in the peri-infarct region after focal cerebral ischemia (Ellison et al. 1998, 1999). Incubation of primary rat astrocytes with 100 µm UTP induced a time-dependent increase in the expression of αV, β3 and β5 proteins (Fig. 3a). To confirm that the expressed αV, β3 and β5 integrin subunits were capable of forming αVβ3 and αVβ5 complexes, we determined that an 8–24 h treatment of primary rat astrocytes with 100 µm UTP significantly increased αV subunit association with β3 and β5 subunits, since incubation of cell lysates with anti-αV antibody could be used to immunoprecipitate β3 and β5 (Fig. 3b). These results indicate that UTP induces up-regulation of αVβ3 and αVβ5 integrin complexes associated with reactive astrogliosis. To demonstrate the involvement of αV integrins in UTP-induced astrocyte migration, we showed that addition of anti-αV antibodies to the lower chamber of Transwells nearly abolished the UTP-induced transmembrane migration of astrocytes from the upper chamber (Fig. 4).

image

Figure 3. UTP increases the expression of αVβ3 and αVβ5 integrin complexes. (a) UTP increases αV, β3 and β5 expression in a time-dependent manner. Primary rat astrocytes in serum-free medium were stimulated with 100 µm UTP for the indicated times at 37°C. Cell lysates were prepared and subjected to SDS–PAGE and immunoblot analysis, as described in Materials and methods. Detection of αV, β3 or β5 in immunoblots was performed with anti-αV, anti-β3 or anti-β5 antibodies, respectively. Membranes were stripped and re-probed with anti-actin antibody to verify the equivalence of protein loading between lanes. Bands were quantitated and expressed as the relative intensities of integrin/actin in UTP-treated cells, as compared with 0 h controls. Data represent the means ± SEM of results from at least three experiments, where *p < 0.05 and **p < 0.01 indicate significant differences. (b) UTP stimulates the expression of αVβ3 and αVβ5 integrin complexes in primary astrocytes. Cell lysates were prepared from astrocytes incubated in serum-free medium with 100 µm UTP for the indicated times at 37°C. Lysates were immunoprecipitated (IP) with anti-αV antibody and subjected to immunoblot (IB) analysis for αV, β3 or β5, as described in Materials and methods. Anti-actin antibody was used to detect actin expression to verify the equivalence of protein loading between lanes. Bands were quantitated and expressed as the relative intensities of integrin/actin in UTP-treated cells, as compared with 0 h controls. Data represent the means ± SEM of results from at least three experiments, where *p < 0.05 and **p < 0.01 indicate significant differences.

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image

Figure 4. Anti-αV antibody inhibits UTP-stimulated astrocyte chemotaxis. Anti-αV antibody, or an irrelevant IgG, was added to the lower chamber of Transwells with membrane inserts, and primary rat astrocytes were added to the upper chamber and incubated for 2 h at 37°C. When indicated, 100 µm UTP was added to the lower chamber, cells were incubated for 18 h at 37°C and the number of astrocytes that migrated into the lower chamber was determined, as described in Materials and methods. Data represent means ± SEM of results from at least three experiments, where **p < 0.01 indicates significant differences from UTP-treated controls.

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MEK/ERK and PI3-K/Akt activation are required for UTP-induced astrocyte migration

It has been reported that MEK/ERK and PI3-K/Akt are two important signaling pathways that regulate cell migration (Sotsios and Ward 2000; Chaulet et al. 2001; Stahle et al. 2003), and P2Y2Rs are known to mediate the activation of ERK and PI3-K in a variety of cell types (Erb et al. 2001; Santiago-Perez et al. 2001; Muscella et al. 2003; Liu et al. 2004; Shen et al. 2004). Results in Fig. 5 show that U0126, an inhibitor of MEK, and LY294002, an inhibitor of PI3-K, prevented UTP-induced chemokinesis (Fig. 5a) and chemotaxis (Fig. 5b) of primary rat astrocytes, whereas SB203580, an inhibitor of p38, did not (data not shown). In primary astrocytes, U0126 and LY294002, respectively, also inhibited UTP-induced phosphorylation of ERK1/2 (substrates of MEK) and Akt (a substrate of PI3-K), but not p38 (data not shown). These results suggest that P2Y2R-mediated MEK/ERK and PI3-K/Akt activation regulate UTP-induced astrocyte migration through overlapping pathways.

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Figure 5. MEK and PI3-K activities are required for UTP-stimulated astrocyte migration. (a) Inhibitors of MEK and PI3-K prevent UTP-stimulated astrocyte chemokinesis. Primary rat astrocytes were incubated with 10 µm U0126 or LY294002 in serum-free medium for 2 h at 37°C, and 100 µm UTP was added when indicated. After 18 h, cells were washed, fixed, and astrocyte chemokinesis was determined, as described in Materials and methods. Bar in lower left panel = 50 µm. (b) Inhibitors of MEK and PI3-K prevent UTP-stimulated astrocyte chemotaxis. Primary rat astrocytes were placed in the upper chamber of Transwells and 10 µm U0126 or LY294002 was added to both chambers for 2 h at 37°C. When indicated, 100 µm UTP was added to the lower chamber, cells were incubated for 18 h at 37°C and the number of astrocytes that migrated to the lower chamber was determined, as described in Materials and methods. Data represent the means ± SEM of results from at least three experiments, where **p < 0.01 indicates significant differences from UTP-treated controls.

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Results shown in Fig. 6 indicate that anti-αV, but not anti-α4, antibody inhibited ERK and Akt activation induced by UTP in primary rat astrocytes. Although anti-αV antibody inhibited UTP-induced ERK phosphorylation by only about 30% (Fig. 6a), UTP-induced Akt phosphorylation was almost completely inhibited (Fig. 6b). The partial inhibition of ERK phosphorylation by anti-αV antibody is consistent with previous findings showing that P2Y2R-mediated ERK activation can occur through several distinct pathways involving αVβ3 and αVβ5 integrins (Erb et al. 2001), phospholipase C (Boarder et al. 1995) and the Src-dependent transactivation of growth factor receptors (Soltoff 1998; Liu et al. 2004; Seye et al. 2004). Taken together, these results strongly suggest that αV integrin and P2Y2R interactions mediate UTP-induced PI3-K/Akt activation, and partially mediate ERK activation, responses required for astrocyte migration.

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Figure 6. Anti-αV antibody inhibits UTP-induced ERK1/2 and Akt phosphorylation. Primary rat astrocytes were incubated, with or without 10 µg/mL anti-αV or anti-α4 (negative control) antibody, in serum-free medium for 2 h at 37°C. When indicated, 100 µm UTP was added for 5 min and the phosphorylation of (a) ERK1/2 or (b) Akt was determined by Western blot analysis using anti-phospho-ERK1/2 or anti-phospho-Akt (Ser473) antibody, as described in Materials and methods. Membranes were stripped and re-probed with anti-ERK or anti-Akt antibody to verify the equivalence of protein loading between lanes. Bands were quantitated and expressed as the relative intensities of pERK/ERK or pAkt/Akt in UTP- or UTP plus antibody-treated cells, as compared with untreated controls. Data represent the means ± SEM of results from three experiments, where *p < 0.05 and **p < 0.01 indicate significant differences from UTP-treated controls.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

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 αvβ35 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 αVβ35 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 αVβ35 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 αVβ35 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 αVβ35 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 αVβ35 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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We appreciate the valuable advice of Dr Nataliya Chorna of the University of Puerto-Rico. We sincerely thank Jennifer Hamilton and Jean Camden for kindly providing primary astrocytes, and other excellent technical support. This study was supported by National Institutes of Health Grants 1 P01-AG18357 and 1 P20-RR15565, the University of Missouri-Columbia Food for the 21st Century Program, and the University of Missouri-Columbia Neurosciences Program (fellowship to MW).

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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