Activation of protease-activated receptor-1 triggers astrogliosis after brain injury

Abstract

We have studied the involvement of the thrombin receptor [protease-activated receptor-1 (PAR-1)] in astrogliosis, because extravasation of PAR-1 activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of CNS disorders. PAR1-/- animals show a reduced astrocytic response to cortical stab wound, suggesting that PAR-1 activation plays a key role in astrogliosis associated with glial scar formation after brain injury. This interpretation is supported by the finding that the selective activation of PAR-1 in vivo induces astrogliosis. The mechanisms by which PAR-1 stimulates glial proliferation appear to be related to the ability of PAR-1 receptor signaling to induce sustained extracellular receptor kinase (ERK) activation. In contrast to the transient activation of ERK by cytokines and growth factors, PAR-1 stimulation induces a sustained ERK activation through its coupling to multiple G-protein-linked signaling pathways, including Rho kinase. This sustained ERK activation appears to regulate astrocytic cyclin D1 levels and astrocyte proliferation in vitro and in vivo. We propose that this PAR-1-mediated mechanism underlying astrocyte proliferation will operate whenever there is sufficient injury-induced blood-brain barrier breakdown to allow extravasation of PAR-1 activators.

Figure 1.

Cortical stab wound involves breakdown of the blood-brain barrier. C57BL/6 mice received an intrajugular injection of 100 μl of 2% Evan's Blue 20 min before receiving a cortical stab wound. A, B, Two hours later, animals were perfused, and the brain was removed (A) and sectioned at 350 μm on a vibratome (B). C, Evan's Blue extravasation was clearly evident in the area surrounding the lesion.

Figure 2.

Effect of PAR-1 activation on astrogliosis. A, GFAP immunohistochemistry was performed on brain sections from wild-type and PAR1^-/- mice 7 d after nitrocellulose filter insertion. High levels of GFAP reactivity can be seen in astrocytes at the surface of the filter only when the implantation of filter was performed in cerebral cortex of wild-type (WT) mice (n = 6 mice for each condition). B, GFAP immunohistochemistry was performed on the brain sections from wild-type and PAR1^-/- mice 7 d after cortical stab wound without nitrocellulose. High levels of GFAP reactivity can be seen in astrocytes extending radially away from the lesion in cerebral cortex of wild-type but not PAR1^-/- mice (n = 3 WT; n = 2 PAR1^-/- mice). C, GFAP immunohistochemistry was performed on 20 μm mouse brain sections 5 d after an intracortical injection (I) of TFLLR (10 nmol; left and middle panels) or vehicle (PBS-0.1% BSA; right panels) in a volume of 0.5 μl over 5 min using a Hamilton syringe (n = 2-4 animals per condition). TFLLR injection caused increased GFAP reactivity in the ipsilateral cortex of wild-type animals compared with the ipsilateral cortex of PAR1^-/- animals, the contralateral cortex of wild-type animals (data not shown), and vehicle injections. The bottom row shows a representative region at 40×.

Figure 3.

PAR-1 activation induces astrocyte proliferation. A, Images of BrdU (FITC) and GFAP (Texas Red) immunofluorescence show that all BrdU-containing cells are immunoreactive for GFAP. B, Fluorescent image analysis of cortical astrocytes labeled for BrdU in control condition or after treatment with TFLLR (30 μm) suggests that application of TFLLR increases the number of BrdU-positive cells only in wild-type (wt), but not in PAR1^-/-, astrocytes. C, Proliferation is shown as the percentage change in the number of BrdU-positive cells compared with control (mean ± SEM; ^*p < 0.05; ^#p < 0.01; unpaired t test; n = 10). D, Cortical astrocytes were treated with TFLLR (30 μm) or forskolin (50 μm) for 12, 24, or 48 h. Five micrograms of protein were separated by SDS-PAGE, transferred to nitrocellulose filters, and probed with anti-GFAP antibody. The application of TFLLR did not significantly modify the protein level of GFAP (n = 3). In this experiment, forskolin treatment served as a positive control (^*p < 0.05; n = 3). The immunoblot shown is representative of three independent experiments; the results of densitometry are tabulated. E, A monolayer of astrocytes was wounded by scratching and treated with serum, TFLLR (30 μm), or buffer, all in the presence of AraC (see Materials and Methods). Photomicrographs show the migration of astrocytes to the wound area at 0 h (e1, e3) and at 24 h (e2, e4) after TFLLR (e1, e2) or 10% FBS (e3, e4). F, There was no significant difference (p > 0.05; unpaired t test) in wound closure (percentage of area recovered after 24 h of treatment) between vehicle and TFLLR in the absence of serum. Serum treatment served as a positive control and strongly stimulated repopulation of the injured area (p < 0.001; unpaired t test). Data shown are percentage of area recovered (n = 10-12). N.S., Not significant. G, TFLLR or vehicle was injected into the right striatum (anterior, 0.9 mm; lateral, 2.0 mm; and ventral, 2.5 mm from bregma) in a volume of 0.5 μl over 5 min using a Hamilton syringe (33 gauge needle). The animal received one intraperitoneal injection of 50 μg/g BrdU immediately after surgery and the nevery 12 h for 5 d, for a total of 11 injections. Animals were perfusion fixed at 5 d. GFAP and BrdU immunohistochemistry was performed on brain sections (20 μm) 5 d after injection. With this procedure, the cytoplasm of astrocyte cells was stained red (g1, g2) and the nuclei of proliferating cells were stained green (g2, g3). H, Cell counts were performed in five fields of five independent slices per animal (separated by a distance of 50 μm around the injection site). The number sign indicates a significant difference from PBS/BSA injection. All experimentation and analysis were performed blind. A total of three TFLLR-treated mice plus three PBS-0.1% BSA-treated control animals were analyzed. Error bars represent SEM.

Figure 4.

ERK activation is involved in PAR-1-induced astrocyte proliferation. A₁, Serum-starved primary cortical astrocytes were treated with TFLLR (30 μm) for indicated times. Aliquots of whole-cell lysates were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with the anti-p-ERK antibody. The immunoblots are representative of three independent experiments. Con, Control. A₂, Total RNA was extracted from cultured cortical astrocytes treated with vehicle or TFLLR (30 μm) and subjected to RT-PCR for PAR-1 and β-actin transcripts. This figure is representative of three experiments. B₁, Primary cortical astrocytes were treated for 15 min with TFLLR (30 μm) in the presence of U0126 (10 μm; 30 min before and during). Aliquots of whole-cell lysates were separated by SDS-PAGE, transferred to membranes, and incubated with the p-ERK. A representative immunoblot is shown demonstrating the specific blockage of ERK phosphorylation by U0126 treatment (n = 3). B₂, Treatment (30 min before and during 24 h) of cortical astrocytes with U0126 (10 μm) reduced the TFLLR-induced proliferation, as determined by counting of BrdU-positive cells. The proliferation is given as the percentage change compared with control (n = 6; ^*p < 0.001; unpaired t test). C₁, Primary cortical astrocytes were treated for 15 min with TFLLR (30 μm) in the presence of PTX (1 μg/ml; overnight before). Aliquots of whole-cell lysates were separated by SDS-PAGE, transferred to membranes, and incubated with the p-ERK. A representative immunoblot shows a reduction of ERK phosphorylation by PTX treatment (n = 3). C₂, Pretreatment (24 h) of primary cortical astrocytes with PTX (1 μg/ml) reduced TFLLR-induced proliferation, as determined by counting of BrdU-positive cells (n = 10; ^*p < 0.05; unpaired t test). D₁, Cortical astrocytes were pretreated with vehicle control (C) or Y27632 (Y; 10 μm) for 12 h before TFLLR (T; 30 μm). Aliquots of whole-cell lysates at indicated time of TFLLR incubation were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with the p-ERK. A representative immunoblot is shown demonstrating that pretreatment with Y27632 had no effect on the early phase of TFLLR-induced ERK activation (after 1 h of treatment), but completely blocks the sustained ERK activation (12 h) (n = 3). D₂, The pretreatment (24 h) of cortical astrocytes with Y27632 (10 μm) reduced the TFLLR-induced proliferation, as determined by counting of BrdU-positive cells (n = 6; ^*p < 0.05; unpaired t test). Error bars represent SEM.

Figure 5.

Block of ERK by delayed U0126 inhibits PAR-1-induced proliferation. Serum-starved murine primary cortical astrocytes were treated with TFLLR (30 μm) for 24 h, and U0126 (10 μm) was added at indicated times after TFLLR application (top). Delayed blockade of ERK activation with U0126 blocks TFLLR-induced proliferation, as measured with BrdU incorporation (middle; n = 8; ^*,#p < 0.001; ANOVA). A representative immunoblot is shown below demonstrating the blockade of TFLLR-induced ERK phosphorylation by delayed U0126 treatment (bottom). Con, Control. Error bars represent SEM.

Figure 6.

PAR-1 activation increases cyclin D1 expression. A, B, RT-PCR is shown for cyclin D1, p27^kip1, and β-actin transcripts. Total mRNA from control (C) or TFLLR (T; 30 μm)-treated astrocyte cultures in serum-free medium was harvested 1, 3, 6, 12, and 24 h after treatment and subjected to RT-PCR (see Materials and Methods). PAGE for one of three independent experiments shows cyclin D1 upregulation at 12 and 24 h, with no change in p27^kip1 mRNA levels. B, Left, Total p27^kip1 mRNA levels were not altered by TFLLR treatment. Right, Total mRNA obtained from TFLLR-treated astrocyte cultures with or without U0126 (30 min before and during TFLLR treatment). TFLLR-induced cyclin D1 expression is blocked by treatment with U0126 (top). The results of densitometry analysis of cyclin D1 mRNA expression (▵) are summarized below (E). C, D, Representative immunoblots of cyclin D1 protein expression from three independent experiments. An immunoblot of cortical astrocytes at 1, 3, 6, or 12 h after control or TFLLR treatment shows cyclin D1 protein upregulation from 3 to 12 h (C; n = 2). An immunoblot of cortical astrocytes pretreated with U0126 or Y27632 and treated with TFLLR for 24 h shows blockade of cyclin D1 protein upregulation (D). E, The results of densitometry analysis of cyclin D1 mRNA expression (▵) show a correlation to the time course of astrocytic proliferation (▪) (n = 8) after TFLLR treatment, as measured by cell counting of BrdU-positive cells. The asterisks indicate a significant difference from control in the same time for cyclin D1 expression; the number sign indicates a significant difference from control in the same time for BrdU incorporation. F, Cortical astrocytes from wild-type mice were cultured for 12 h in the presence of TFLLR (30 μm), and conditioned media were collected and replaced by fresh medium. The asterisk indicates a significant difference from control condition (without TFLLR). Proliferation assays were performed 12 h later by evaluating BrdU incorporation. Cortical astrocytes from PAR1^-/- mice were subsequently incubated with conditioned media from wild-type (WT) astrocytes treated with TFLLR for 24 h, and a proliferation assay by BrdU incorporation was performed (n = 8). Error bars represent SEM.

Figure 7.

Effect of microglial cells on thrombin-induced astrocyte proliferation in vitro. A, Immunostaining for GFAP (Marina Blue 460) and BrdU incorporation (Alexa Fluor 488) in control and α-thrombin-treated cocultures of wild-type astrocytes and wild-type microglia illustrate increased BrdU-positive astrocytes in response to α-thrombin (1 U/ml) (red arrows). Distinguishing GFAP-positive, BrdU-positive astrocytes (red arrows) from GFAP-negative, BrdU-positive cells (white arrows) allowed for identification and counting of proliferating astrocytes in cocultures. B, Compared with untreated wild-type (WT) as trocytes (marked with a dotted line at 100%), all α-thrombin (1 U/ml)-treated wild-type astrocytes exhibited significantly increased BrdU incorporation, regardless of microglial cell absence or the presence of wild-type or PAR1^-/- [knock-out (KO)] microglial cells (n = 6; ^*p < 0.0001; unpaired t test). In contrast, all PAR1^-/- astrocyte cultures were similar to untreated wild-type astrocytes. Cocultures of wild-type astrocytes with PAR1^-/- microglia modestly increased BrdU-positive astrocytes compared with cocultures of wild-type astrocytes and wild-type microglia (n = 6; ^#p < 0.005; unpaired t test). Error bars represent SEM.