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Abstract

The expression profile of the protease-activated receptor-2 (PAR-2) and effects of PAR-2 gene knockout (PAR-2 KO) on the infarct size were investigated after 60 minutes of transient middle cerebral artery occlusion (tMCAO) in mice in relation to phosphorylated extracellular signal-regulated kinase (p-ERK) and astrocyte activation. PAR-2 was normally distributed mainly in neurons of the central nervous system (CNS), and strongly upregulated at 8–24 hours after tMCAO. Deficiency of PAR-2 gene significantly increased the infarct volume and the number of TUNEL-positive cells at 24 hours of reperfusion. The strong neuronal expression of p-ERK was induced at 5 minutes as a peak after reperfusion in wild-type mice, but the signal change was significantly reduced in PAR-2 KO mice. Astroglial activation was also greatly inhibited at 24 hours after tMCAO in PAR-2 KO mice. These results show that the deficiency of PAR-2 gene increases the acute ischemic cerebral injury associating with suppression of neuronal ERK activation and reactive astroglial activation.

Keywords

ERK GFAP ischemia knockout mice NeuN PAR-2

Introduction

Protease-activated receptor-2 (PAR-2), a member of seven transmembrane G protein-coupled receptors, is widely distributed in the central nervous system (CNS) (Striggow et al, 2001), and is known to play important roles in response to injury, inflammation, neurodegeneration, ischemia, neuronal signaling and pain pathway (Smith-Swintosky et al, 1997; Steinhoff et al, 2000; Striggow et al, 2001; Macfarlane et al, 2001; Vergnolle et al, 2001a, 2001b; Fiorucci and Distrutti, 2002; Oliver and Hill, 2002).

PAR-2 is first activated within proteolytic cleavage of the extracellular N-terminal domain by proteases such as trypin and tryptase (Nystedt et al, 1994; Macfarlane et al, 2001), couples to G_αq/11, and then results in activation of intracellular signals, such as mobilization of Ca²⁺, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (Macfarlane et al, 2001; Cottrell et al, 2003). The activated MAPK/ERK plays critical roles in regulating cell growth, differentiation, inflammation and promoting survival under normal and pathological conditions (Bonni et al, 1999; Lee et al, 2000; Han and Holtzman, 2000; Wang et al, 2000; Gu et al, 2001; von Gise et al, 2001; Morita et al, 2003). A number of recent reports suggest that activation of PAR-2 induces activating of MAPK/ERK signaling pathway (DeFea et al, 2000b; Macfarlane et al, 2001; Koo et al, 2002; Cottrell et al, 2003).

Astrocytes are the most numerous cell type in the CNS. They provide structural, trophic and metabolic support to neurons and thus modulate synaptic activity under normal conditions. On brain injury, astrocytes are transformed into the activated form, and critically influence neuronal survival by affecting glutamate reuptake, free radical scavenging, water transport, and the production of cytokines and nitric oxide (Stoll et al, 1998; Chen and Swanson, 2003). Neurons cannot survive in the brain without the close interaction with astrocytes, both under normal and pathological conditions. PAR-2 is also involved in activation of astroglia in vitro (Ubl et al, 1998; Wang et al, 2002), and glial fibrillary acidic protein (GFAP) expression is strikingly enhanced in the trypsinogen IV transgenic mice (Minn et al, 1998). However, the exact relationship between PAR-2 and astroglial activation is unclear.

In recent years, there has been increasing interest in functions of PAR-2 as a mediator of neurogenic inflammation and nociception. However, many studies performed to date have shown only the effect of PAR-2 by administrating PAR-2 agonists or performing in vitro study, while the intracerebral targets and pathological roles of PAR-2 in vivo are largely unknown. In this study, therefore, we examined an expression profile of the PAR-2 after transient focal brain ischemia, and the effect of PAR-2 gene knockout on the infarct volume in relation to ERK and astrocyte activations.

Materials and methods

Generation of Protease-Activated Receptor-2-Deficient Mouse

The PAR-2-deficient mice with C57BL/6 background, which replaced almost the entire exon 2 coding sequence with a cassette TAG3/IRES/LacZpA/MC1neopA (Figure 1A), were generated as described previously (Ferrell et al, 2003). The PAR-2-deficient strain was maintained by backcrossing a heterozygous (PAR-2^+/−) male with C57BL/6 females at each generation at Kowa Tokyo New Drug Research Laboratories. The genotype of the mice was confirmed by Southern blot analysis (Figure 1B). Homozygous (PAR-2^−/−) and wild-type (WT; PAR-2^+/+) female mice generated from male and female PAR-2^+/− mice at backcross generation eight were used at 8 weeks of age for the experiments. Saliva-secreting response to the PAR-2-activating peptide SLIGRL-NH₂ was measured and compared with control mice to confirm the PAR-2^−/− phenotype (Figure 1C, PAR-2 knockout (PAR-2 KO)). The PAR-2^−/− mice were of the same body size and vitality when compared with PAR- 2^+/− and PAR-2^+/+ littermates, and, as previously reported, they exhibited no evidence of macroscopic abnormalities (Lindner et al, 2000).

Figure 1

Generation of PAR-2-deficient mice. (A) Gene-targeting strategy. The wild-type PAR-2 allele (top), structure of the PAR-2 targeting vector (middle), and targeted allele (bottom) resulting from replacement recombination at the dashed crosses. The null allele was created by substitution of a reporter/selection cassette (dark box) for most of the exon 2 sequence (gray box). Non-exon-containing chromosome is showed by a thin black line. The targeted allele was identified by Kpnl digestion and hybridization with the 5′ and 3′ flanking probe fragments. (B) Southern blot analysis of Kpnl-digested genomic DNA prepared from pups from PAR-2^+/− matings using 5′ flanking probe. Targeting introduced a new kpnl site. The 12- and 7.1-kb bands correspond to wild-type and targeted alleles, respectively. (C) Loss of secretive response to the PAR-2-activating peptide SLIGRL-NH₂ in the PAR-2^−/− mice. The secretion of saliva was measured during 3 minutes after intravenous injection of PAR-2-activating peptide SLIGRL-NH₂ (100 μmol/kg), or saline, and expressed as the mean±s.e.m. ***P<0.001.

Focal Cerebral Ischemia

The PAR-2 KO and WT male mice (20–22 g) were subjected to transient focal cerebral ischemia by intraluminal middle cerebral artery (MCA) blockade with a nylon suture, as described previously (Connolly, Jr et al, 1996). Anesthesia was induced with 2% halothane in a mixture of 70% nitrous oxide and 30% oxygen delivered by face mask. The rectal temperature was controlled at 37°C, and the regional CBF (rCBF) of the right front parietal cortex was continually monitored during surgical procedure. Under an operating microscope, the right common carotid artery (CCA) and external carotid artery (ECA) were exposed through a midline neck incision and its branches were electrocoagulated. An 11-mm 6–0 monofilament nylon suture, blunted at the end, was inserted via the proximal ECA into the internal carotid artery (ICA), occluding MCA. After 60 minutes of MCA occlusion (MCAO), blood flow was restored by withdrawal of the nylon suture. The CBF was confirmed down to 10±5% of basal level after MCA occlusion, and returned to 90% of normal CBF level after reperfusion. The experimental protocol and procedures were approved by the Animal Committee of the Okayama University Medical School.

Calculation of Infarction Volume

The animals (n=6) were killed and the brains were removed 24 hours after MCAO reperfusion. Fourteen serial 10 μm-thick slice coronal sections from each brain were cut at 600 μm intervals beginning at 1 mm from the front using a cryostat. Each slice was stained with cresyl violet, and the infarction area was measured with the use of computer-assisted image analysis software SigmaScan pro 5 (SPSS Inc.). Total infarct volume was calculated by adding the area of infarction of each slice and multiplying by the distance between each slice.

Single Immunohistochemistry

Mice brains were removed at 5 minutes, 8 hours and 24 hours after MCAO reperfusion in both PAR-2^+/+ and PAR-2^−/− groups (each n=4), and quickly frozen in liquid nitrogen. Coronal sections at the caudate level were cut on a cryostat at −20°C to 10 μm thickness, and collected on glass slides. With these sections, immunohistochemistry for PAR-2, phosphorylated ERK (p-ERK), and histochemistry for TUNEL were performed. The fresh-frozen sections were fixed by 4% paraformaldehyde (PFA) and rinsed three times in PBS (pH 7.4). After blocking with 3% to 10% bovine serum albumin (BSA) for 1 hour, the slides were incubated with primary antibodies overnight at 4°C. The primary antibodies used and the dilutions for each were as follows: goat polyclonal anti-PAR-2 antibody at 1:100 (Santa Cruz Biotechnology), rabbit polyclonal anti-p-ERK antibody at 1:100 (Cell Signaling, CA, USA) and goat polyclonal anti-GFAP antibody at 1:200 (Santa Cruz Biotechnology). The sections were then washed and incubated for 1 hour with relevant biotinylated anti-goat or anti-rabbit IgG at 1:400 (Vector Laboratories, CA, USA), followed by incubation with avidin–biotin–peroxidase complex (Vectastatin ABC kit, Vector Laboratories) for 30 minutes. Diaminobenzidine tetrahydrochloride (DAB) was used as a color substrate. A set of sections was stained in a similar way without the primary antibody.

Double Fluorescent Immunohistochemistry of Protease-Activated Receptor-2 with NeuN or GFAP

We performed double staining for PAR-2 with NeuN or GFAP to investigate the expression of PAR-2 on the neuron and astrocyte. For the immunofluorescent detection, the coronal brain sections fixed by 4% PFA as above were first immunostained with the PAR-2 antibody, followed by fluorescein isothiocyanate (FITC)-conjugated rabbit anti-goat IgG antibody (Vector) at a dilution of 1:500; then, the sections were treated with an MOM kit (BMK-2202, Vector Laboratories, Bulingme, CA, USA) to prevent nonspecific binding, and finally reacted with mouse monoclonal anti-NeuN antibody (Chemicon, Temecula, CA, USA) at a dilution of 1:200 or mouse monoclonal anti-GFAP antibody (61011, PROGEN Biotechnik GmbH) at a dilution of 1:10, followed by Rhodamine-conjugated goat anti-mouse IgG antibody (Chemicon) at a dilution of 1:500. Subsequently, the slides were covered with VECTASHIELD mounting medium with 4′,6′-diamidino-2-phenylindole (DAPI) (H-1200; Vector Laboratories). Signals were examined using an Olympus microscope (BX51; Olympus, Japan) equipped with FITC and rhodamine filter set.

Double Fluorescent Immunohistochemistry of p-ERK with NeuN or GFAP

We performed double staining for p-ERK with NeuN or GFAP to investigate the expression of p-ERK on the neuron and astrocyte. For double staining of p-ERK and NeuN, the sections were immunostained with p-ERK antibody, followed by FITC-conjugated donkey anti-rabbit IgG (Chemicon) at a dilution of 1:500. Subsequently, the sections treated with the MOM kit, and reacted with anti-NeuN antibody and secondary antibody, as described above. For double staining of p-ERK and GFAP, the sections were incubated together with p-ERK antibody and goat polyclonal anti-GFAP antibody, followed by FITC-conjugated donkey anti-rabbit IgG (Chemicon) and Alexa fluor 546-conjugated donkey anti-goat IgG (Molecular Probes) antibody at a dilution of 1:500. After mounting, fluorescence was observed, as mentioned above.

Western Blotting Analysis

Whole-cell protein extraction was performed. Samples were obtained from both the cortex and caudate on the sham controls and ischemic sides after 5 mins, 8 hours, 24 hours (n=4 each) of reperfusion. The brain tissues were homogenized in 10 vol of cold protein extraction buffer (complete tablet; 1697498, Roche Molecular Biochemicals, Mannheim, Germany). The homogenate was centrifuged at 10,000g for 20 mins at 4°C, and the supernatant was used for the analysis. After adding the 2 vol of Laemmli sample buffer (Bio-Rad Laboratories) to the supernatant, equal amounts of the samples were loaded per lane (4 μg protein). The primary antibodies were a 1:1000 dilution of rabbit polyclonal antibody (Cell Signaling) against p-ERK1/2 (Thr202/Tyr204), and a 1:1000 dilution of monoclonal PAR-2 antibody (Santa Cruz). Western blots were performed with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Amersham, Buckinghamshire, UK) or anti-mouse IgG (PI-2000, Sigma, Buringame, USA) using a chemiluminescence Western blotting detection system kit (ECLTM-RPN 2106, Amersham Pharmacia Biotech, Uppsala, Sweden). The results were scanned with a LAS-1000 image analyzer (LAS-1000-mini, FUJIFILM, Tokyo), and quantified using Scion Image software (NIH).

Apoptosis Assay

For detection of DNA fragmentation, terminal deoxynucleotidyl dUTP nick-end labeling (TUNEL) was performed using an Apoptag In Situ Apoptosis Detection kit (TACS in situ apoptosis detection kit #4810–30k, Trevigen, Gaithers-burg, MD, USA) at 24 hours after the reperfusion. Briefly, the sections fixed by 4% paraformaldehyde were incubated with dioxigenin-labeled dUTP in the presence of terminal deoxynucleotidyl transferase (TdT) at 37°C for 1 hour, then incubated with HRP-conjugated anti-digoxigenin antibody for 1 hour at room temperature and stained with DAB. The positive cell stainings were counted in the three 1 × 1 mm² regions of the inner boundary zone of subsequent infarction.

Data analyses and Statistics

The data are expressed as mean±s.d., except the extra notation. Statistical analysis was performed using the Student's t-test as P<0.05 statistically significant.

Results

Expression of Protease-Activated Receptor-2 in the Normal WT Mice

In all sham control brain areas examined, PAR-2 immunoreactivity was detected mainly in the cells with the morphological appearance of neurons, where the reaction product was mainly localized in neuronal perikarya (Figure 2). The staining of PAR-2 was slightly stronger in the cerebral cortex (Figure 2A) than that in the caudate putamen (Figure 2B). No immunoreactivity was found in the whole brain sections without the primary antibody (data not shown).

Figure 2

Immunoreactivity for PAR-2 on the cerebral cortex (A) and the caudate putamen (B) in the sham control brains. A typical neuronal staining with PAR-2 at each area is shown in the inset. Note that the stainings of PAR-2 appeared mainly in the neuronal perikarya, and the stainings were slightly stronger in the cortex than those in the caudate putamen. Scale bar: 100 μm.

Protease-Activated Receptor-2 Upregulation in the WT Mice after Transient MCAO

At 5 mins after transient MCAO (tMCAO), no significant difference was observed for the expression of PAR-2 compared with the sham control (data not shown). However, the expression of PAR-2 became increased significantly in the ischemic territory at 8 hours after reperfusion (Figure 3), and stronger staining was observed mostly in the larger cells of the peri-ischemic area of the cerebral cortex (Figure 3B), then the caudate (Figure 2E). The strong immunoreactivity of PAR-2 remained until 24 hours after reperfusion in the penumbra of the ischemic cortex (Figure 3C), where the number of PAR-2-positive cells also remained. In contrast, the number of PAR-2-positive cells was significantly reduced at 24 hours after reperfusion in the ischemic caudate putamen, where the dense stained cells were still detectable (Figure 3F, arrow).

Figure 3

Immunoreactivity for PAR-2 on the WT mice cortex (A, B, C) and the caudate putamen (D, E, F) in the peri-ischemic area in the sham control (A, D), and after 8 hours (B, E) and 24 hours (C, F) of reperfusion. Note that the expression of PAR-2 with weaker staining in the sham control (A, D) was enhanced at 8 hours (B, E), and remained until 24 hours in the cortex (C), but the number of positive cells was reduced in the caudate putamen (F). The stronger staining was seen mostly in the larger cells (arrowed). Scale bar: 30 μm.

With Western blot analysis, PAR-2 immunoreactivity was evident as a band with a molecular mass of 55 kDa (Figure 4), and no bands were detected without primary antibody. After tMACO, the level of PAR-2 increased in the cerebral cortex (Figure 4A) at 8 and 24 hours, while that in the caudate putamen showed a transient increase at 8 hours (Figure 4B).

Figure 4

Western bolt analysis of PAR-2 in the cerebral cortex (A) and the caudate putamen (B) at 5 minutes, 8 and 24 hours of reperfusion or sham control. The expression of PAR-2 was increased at 8 to 24 hours in the cortex (A), but transiently increased at 8 hours in the caudate putamen (B). *P<0.05.

Double immunofluorescence for PAR-2 (green) (Figures 5A, 5D, 5G and 5J) and NeuN (red) (Figures 5B, 5E and 5H) showed that the expression of PAR-2 colocalized with some neurons in the sham control brain (Figure 5C). The PAR-2 + NeuN double-positive neurons are the cells with stronger NeuN stainings (Figure 5B). After tMCAO, strong induction of PAR-2 (Figures 5D and 5G) shows double positive cells with NeuN (Figures 5F and 5I), where double-positive cells are the cells with weaker NeuN stainings (Figures 5E and 5H). Although GFAP (Figure 5K, red) is not generally colocalized (Figure 5L) with PAR-2 (Figure 5J) in the control and postischemic brains, a few cells which appeared typical reactive astrocytes were double stained in the peri-ischemic area (Figures 5M, 5N and 5O).

Figure 5

Double immunostaining for PAR-2 (green, A, D, G, J, M) with NeuN (red, B, E, H) or GFAP (red, K, N) in peri-ischemic cortex of WT mice after tMCAO. The weaker PAR-2-positive cells are colocalized with NeuN in the sham control (A–C), and the expression of PAR-2 was increased at 8 and 24 hours (D, G, F, I) in the weaker NeuN stained large cells (E, H). Although PAR-2 is not generally colocalized with GFAP (J, K, L), a few cells, which appeared to be typical reactive astrocytes were double stained (M, N, O). Scale bar: 30 μm.

Evaluation of Infarct Volume in Protease-Activated Receptor-2 KO Mice

As for physiological parameters such as pH, PO₂, PCO₂, body temperature, and CBF, there was no significant difference between PAR-2 KO and WT groups before and after MCAO. The infarct volumes of the PAR-2 KO group (38.9±14.4 mm³) (Figures 6B and 6C) were significantly increased at 24 hours of reperfusion compared with the WT group (23.8±7.7 mm³) (Figures 6A and 6C) (n=6, *P<0.05).

Figure 6

Effects of PAR-2 deficiency on the infarct volume at 24 hours after 60 minutes of tMCAO. The infarct volume of PAR-2 KO mice (B, C) was increased compared with WT mice (A, C). *P<0.05.

Neuronal p-ERK1/2 Expression in WT and Protease-Activated Receptor-2 KO Mice

p-ERK was only minimally detectable in the neuronal cells of cerebral cortex in the sham control brain (Figures 7A and 7D). At 5 mins after tMCAO, p-ERK became strongly positive in the ischemic cortex of the MCA territory of WT mice (Figures 7B and 8A) with immunoreactivity in the neuronal nuclei. Comparatively, p-ERK moderately increased in the caudate putamen (Figure 7E). In the PAR-2 KO mice, the staining of p-ERK in the cortex and caudate putamen was significantly reduced compared with that in the WT mice at the same time points (Figures 7C, 7F and 8D). With Western blot analysis, the bands of p-ERK were observed at 44 kDa (p-ERK1) and 42 kDa (p-ERK2) (Figure 7J) in the sham control brains of WT and KO mice. With reperfusion after tMCAO, protein level of p-ERK1/2 was significantly increased at 5 mins and 8 hours in the ischemic cortex with a peak at 5 mins, while that in the caudate putamen only showed a slight increase at 5 mins in WT mice. In the PAR-2 KO mice, the expression of p-ERK in the ischemic cortex was reduced compared with that in the WT mice, and did not change in the caudate putamen compared with the sham control. Schematic illustration (Figures 7G–7I) showed spatial distribution of p-ERK positive cells in the sham, WT, and PAR-2 KO mice, respectively. The expression of p-ERK in the contralateral hemisphere after reperfusion was not different from that in the normal brain (data not shown).

Figure 7

Immunoreactivity for p-ERK on the cerebral cortex (A, B, C) and the caudate putamen (D, E, F) in the sham control (A, D, G) and after 5 minutes of tMCAO in WT (B, E, H) and PAR-2 KO (C, F, I) mice. Western blot analysis for p-ERK1/2 (J) on the cortex (top) and the caudate putamen (bottom) in WT and PAR-2 KO mice after tMCAO. Minimal signals of p-ERK in the sham control (A, D) were increased in the WT mice cortex after 5 minutes (B, H, J) and 8 hours (J-top) of tMCAO with peak at 5 minutes (J-top), but slightly increased at 5 minutes in the caudate putamen (E, H, J—bottom). p-ERK in the PAR-2 KO mice cortex was reduced more than that in the WT mice (C, I, J—top), and did not show significant change in the caudate putamen (F, I, J). Schematic illustration shows the distribution of p-ERK positive cells (G, H, I). Scale bar: 100 μm.

Figure 8

Double immunostaining for p-ERK (green, A, D, G) with NeuN (red, B, E) or GFAP (red, H) on the peri-ischemic cortex after 5 minutes of tMCAO in WT (A–C, G–I) and PAR-2 KO (D–F) mice. As compared with WT mice (C), p-ERK plus NeuN double-positive cells were significantly reduced in the PAR-2 KO mice (F). p-ERK-positive cells did not coexpress with GFAP (I). Scale bar: 30 μm.

With double immunofluorescence, the staining of p-ERK (green) was colocalized (Figures 8C and 8F) with the neuronal marker NeuN (red) (Figures 8B and 8E) at 5 mins of reperfusion, but did not coexpress with glial marker GFAP (red) (Figures 8G–8I), showing that p-ERK is expressed primarily in neurons after tMCAO.

Expression of GFAP in the Protease-Activated Receptor-2 KO Mice

In the sham control brain, GFAP immunoreactivity was principally located in the hippocampus and corpus callosum. At 24 hours after tMCAO, GFAP immunoreactivitity was strongly found in the peri-ischemic area, with thick numerous processes as typical reactive astrocytes (Figure 9A). In contrast, the expression of GFAP was greatly reduced in PAR-2 KO mice at 24 hours of reperfusion with reduced numbers of positive cells and processes (Figure 9B). The integral densitometric analysis for GFAP staining (intensity plus number) was significantly decreased in PAR-2 KO mice quantified using SigmaScan pro 5 software (Figure 9C) (*P<0.05).

Figure 9

Immunoreactivity for GFAP on the peri-ischemic area in WT (A) and PAR-2 KO (B) mice after 24 hours of tMCAO. Each value is represented as a mean±s.d. of densimetoric scores. The expression of GFAP was increased in the WT mice with thick numerous processes as typical reactive astrocyte (A, C), but the signals were greatly reduced in the PAR-2 KO mice (B, C). Scale bar: 100 μm. *P<0.05.

TUNEL Staining after Transient Middle Cerebral Artery Occlusion in Protease-Activated Receptor-2 KO Mice

TUNEL staining at 24 hours of reperfusion showed that positive cells were mainly distributed in the border of the occluded MCA area in both WT and PAR-2 KO mice. TUNEL labeling was essentially found in the nuclei of cells (Figures 10A and 10B). As compared with WT mice (261.2±41.3, n=6) (Figures 10A and 10C), the number of TUNEL-positive cells in PAR-2 KO mice was significantly increased (356.7±71.8, n=6; *P<0.05) (Figures 10B and 10C). TUNEL staining was negative in the sham control (data not shown).

Figure 10

Staining of TUNEL on the border of the occluded MCA area in WT (A) and PAR-2 KO (B) mice after 24 hours of tMCAO. The number of TUNEL-positive cells was significantly increased in PAR-2 KO mice (C). Scale bar: 100 μm. *P<0.05.

Discussion

The current study shows the important roles of PAR-2 in the transient focal cerebral ischemia with the PAR-2 KO mice. We observed that, first, PAR-2 was widely distributed in the sham control brains and significantly upregulated after 8 and 24 hours of tMCAO in the WT mice. Second, the infarct volume in the PAR-2 KO mice was increased after 24 hours of ischemia compared with WT mice. Third, the neuronal expression of p-ERK was induced after 5 minutes of tMCAO in both WT and PAR-2 KO mice, but the signals were significantly reduced in the PAR-2 KO mice. Fourth, the astrocyte activation was greatly reduced in the PAR-2 KO mice. Fifth, the number of TUNEL positive cells was increased in the PAR-2 KO mice.

The PAR-2 was widely distributed in the CNS and had important effects on distinct cerebral processes under physiological conditions such as learning and memory (Striggow et al, 2001). The expression of PAR-2 was enhanced by ischemic stimulation (Smith-Swintosky et al, 1997; Ubl et al, 1998; Striggow et al, 2001). In the current study, the basal neuronal expression of PAR-2 was widely distributed in the WT sham controls (Figure 2), reflecting the physiological activity of PAR-2 in the normal brain. Upregulation of PAR-2 at 8 and 24 hours after tMCAO (Figures 3 and 4) indicates the role of PAR-2 under ischemic conditions. Of interest is the fact that the strong expression of PAR-2 was found mainly in the large neurons of the peri-ischemic area (Figures 5D and 5G), and those cells were double positive with the weak NeuN positive cell (Figures 5E, 5F, 5H and 5I). These large neurons are known to have a high metabolic rate and the weak expression of NeuN was considered to be induced due to axonal injury (McPhail et al, 2004), suggesting that PAR-2 might be closely involved in the repair or cell-protective mechanism in these cells.

Activation of PAR-2 mobilizes intracellular Ca²⁺, and induces the assembly of a MAPK/ERK signaling module by a mechanism that depends on β-arrestins (Dery et al, 1998; DeFea et al, 2000a, 2000b; Macfarlane et al, 2001). Previous studies indicate that PAR-2 is involved in the activation of ERK subfamily of MAPK relatively selectively by forming a multiprotein signaling complex that contains PAR-2, β-arrestin, Raf-1 and p-ERK1/2 (Belham et al, 1996; Defea et al, 2000a, 2000b; Cottrell et al, 2003). Extracellular signal-regulated kinase is abundant in the CNS, and is activated by phosphorylation, which requires elevation of intracellular Ca²⁺ during various physiological and pathological events such as brain ischemia and epilepsy (Fukunaga and Miyamoto, 1998). P-ERK plays a critical role in mediating intracellular signal transduction in response to a variety of stimulation, and morphological differentiation and inflammatory response, and in promoting survival in neurons (Fukunaga and Miyamoto, 1998; Erhardt et al, 1999; Irving et al, 2000; Han and Holtzman, 2000; Fahlman et al, 2002; Hasegawa et al, 2003; Obata and Noguchi, 2004; Tian et al, 2004). Our previous reports also showed that the total ERK and p-ERK were induced after tMCAO and promoted neuronal survival in the ischemic brain (Kitagawa et al, 1999; Li et al, 2002). In the present study, the strong expression of p-ERK was observed predominantly in the peri-ischemic neurons from the very early period of reperfusion (Figure 7), suggesting that the neural cells in the peri-ischemic area were more supported by p-ERK than those in the ischemic core. In contrast, the activation of p-ERK was significantly reduced in the PAR-2 KO mice compared with the WT mice (Figures 7 and 8). These results indicate that PAR-2 was involved in activation of the p-ERK pathway after tMCAO, so that the activation of p-ERK by PAR-2 was inhibited, and the neuronal supporting induced by p-ERK was reduced in the PAR-2 KO mice. Although both our results and accumulating reports show p-ERK benefits to cell survival, some papers show that p-ERK does not have neuronal protective effects (Alessandrini et al, 1999). The functional role of p-ERK may differ depending on types of cells or kinds of insults. In the present study, the strong expression of p-ERK showed earlier than PAR-2 protein induction. This must be the reason why these signaling events occurred rapidly without inducing additional PAR-2 protein expression, which usually needs time after ischemic stimulation.

A prominent response of glia to many types of injuries or insults in the CNS is the activation of astrocytes, which is typically characterized by an upregulation of GFAP expression (Norton et al, 1992). Reactive astrogliosis is a key component of the cellular response to CNS injury (Ridet et al, 1997). During acute brain injury, astrocytes support neuronal survival by glutamate reuptake, release of metabolic intermediates, scavenging of oxygen-free radical, buffering of extracellular K⁺ and H⁺, and secreting neurotrophic factor and cytokines (Goss et al, 1998; Hailer et al, 2001; Chen and Swanson, 2003; Dhandapani et al, 2003). Early astroglial dysfunction plays a critical role in determining the outcome of acute hypoxic–ischemic injury by compromising neuronal–glial interactions (Liu et al, 1999). Wang et al (2002) reported a PAR-2-induced proliferation of astrocytes in rat cultured cells. Minn et al (1998) reported that the expression of GFAP was strikingly enhanced in the trypsinogen IV transgenic mice. In the present study, the expression of GFAP was greatly enhanced at 24 hours after tMCAO in the WT mice (Figure 9A), and this was significantly inhibited in the PAR-2 KO mice (Figure 9B). Consequently, the number of TUNEL-positive cells and the infarction volume increased in the PAR-2 KO mice (Figures 6 and 10), suggesting that the increase of brain injury was also partly attributable to astroglial dysfunction induced by deficient PAR-2.

One mechanism for astrocyte activation by PAR-2 was considered via intracellular signalings such as Ca²⁺, PKC and ERK in astrocytes (Yu et al, 1997; Cazaubon et al, 1997; Abe and Saito, 2000; Wang et al, 2002; Fiorucci et al, 2003). However, in the present study, the PAR-2 was expressed mainly in the neurons (Figures 5C, 5F and 5I), whereas only a few activated large astrocytes became positive with PAR-2 at 24 hours after tMCAO (Figures 5M and 5O). A recent report also showed that PAR-2 was expressed in limited astrocytes after the brain injury by the trimethyltin (Pompili et al, 2004). These results suggest a possible involvement of other indirect mechanisms in astroglial activation. Microglia is known to mediate astrocyte activation by the secretion of inflammation cytokines after ischemic injury. In fact, we also investigated the activation of microglia and macrophage in the WT and KO mice by immunostaining, but we did not find significant difference in the two groups (data not shown), similar to previous reports (Minn et al, 1998; Pompili et al, 2004). However, the accumulating evidence indicates that neuronal regulation of astrocyte function by neurotransmitters, cytokines and calcium oscillations was an important mechanism of astroglial activation (Hu and Levin, 1994; Matsutani and Yamamoto, 1997; Acarin et al, 2000; Zonta and Carmignoto, 2002; Zonta et al, 2003). In our study, the neuronal response for ischemic injury was found in the very early period of reperfusion as p-ERK induction (Figures 7 and 8). The early neuronal activation probably plays an important role in remodeling astrocyte processes. The exact mechanism on the relationship between PAR-2 and astrocyte activation remains to be determined further.

Some studies report that PAR-2 is associated with neurodegeneration or mediation of neurotoxic factors such as tumor necrosis factor alpha (TNF-α) (Smith-Swintosky et al, 1997; Kim et al, 2002). The neuroprotective or neurodegenerative effects may be concomitant in the pathological processes, leading to the final outcome. The current study showed that the deficiency of PAR-2 leads to the increase of the infarction volume after acute transient cerebral focal ischemia, and that neuronal p-ERK activation and astroglial activation were involved in the mechanism. Further investigations of cellular signal transduction mechanisms and their long-term effect are necessary for understanding the role of PAR-2 further.

Footnotes

Acknowledgements

This work was partly supported by Grant-in-Aid for Scientific Research (B) 15390273 and (Hoga) 15659338 and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Culture and Sports of Japan, by grants (Itoyama Y, Kimura I and Kuzuhara S) from the Ministry of Health and Welfare of Japan, and by grants from Kanae Foundation for Life and Socio-Medical Science.

References

Abe

Saito

(2000) The p44/42 mitogen-activated protein kinase cascade is involved in the induction and maintenance of astrocyte stellation mediated by protein kinase C. Neurosci Res 36:251–7

Acarin

Gonzalez

Castellano

(2000) Neuronal, astroglial and microglial cytokine expression after an excitotoxic lesion in the immature rat brain. Eur J Neurosci 12:3505–20

Alessandrini

Namura

Moskowitz

Bonventre

(1999) MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia. Proc Natl Acad Sci USA 96:12866–9

Belham

Tate

Scott

Pemberton

Miller

Wadsworth

Gould

Plevin

(1996) Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases. Biochem J 320(Part 3):939–46

Bonni

Brunet

West

Datta

Takasu

Greenberg

(1999) Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286:1358–62

Cazaubon

Chaverot

Romero

Girault

Adamson

Strosberg

Couraud

(1997) Growth factor activity of endothelin-1 in primary astrocytes mediated by adhesion-dependent and -independent pathways. J Neurosci 17:6203–12

Chen

Swanson

(2003) Astrocytes and brain injury. J Cereb Blood Flow Metab 23:137–49

Connolly

Jr Winfree

Stern

Solomon

Pinsky

(1996) Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia. Neurosurgery 38:523–31

Cottrell

Amadesi

Schmidlin

Bunnett

(2003) Protease-activated receptor 2: Activation, signalling and function. Biochem Soc Trans 31:1191–7

10.

Defea

Schmidlin

Dery

Grady

Bunnett

(2000a) Mechanisms of initiation and termination of signalling by neuropeptide receptors: A comparison with the proteinase-activated receptors. Biochem Soc Trans 28:419–26

11.

DeFea

Zalevsky

Thoma

Dery

Mullins

Bunnett

(2000b) Beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol 148:1267–81

12.

Dery

Corvera

Steinhoff

Bunnett

(1998) Proteinase-activated receptors: Novel mechanisms of signaling by serine proteases. Am J Physiol 274:C1429–C52

13.

Dhandapani

Hadman

De Sevilla

Wade

Mahesh

Brann

(2003) Astrocyte protection of neurons: Role of transforming growth factor-beta signaling via a c-Jun-AP-1 protective pathway. J Biol Chem 278:43329–39

14.

Erhardt

Schremser

Cooper

(1999) B-Raf inhibits programmed cell death downstream of cytochrome c release from mitochondria by activating the MEK/Erk pathway. Mol Cell Biol 19:5308–15

15.

Fahlman

Bickler

Sullivan

Gregory

(2002) Activation of the neuroprotective ERK signaling pathway by fructose-1,6-bisphosphate during hypoxia involves intracellular Ca²⁺ and phospholipase C. Brain Res 958:43–51

16.

Ferrell

Lockhart

Kelso

Dunning

Plevin

Meek

Smith

Hunter

McLean

McGarry

Ramage

Jiang

Kanke

Kawagoe

(2003) Essential role for proteinase-activated receptor-2 in arthritis. J Clin Invest 111:35–41

17.

Fiorucci

Distrutti

(2002) Role of PAR2 in pain and inflammation. Trends Pharmacol Sci 23:153–5

18.

Fiorucci

Distrutti

Federici

Palazzetti

Baldoni

Morelli

Cirino

(2003) PAR-2 modulates pespinogen secretion from gastric isolated chief cells. Am J Physiol Gastrointest Liver Physiol 285:G611–620

19.

Fukunaga

Miyamoto

(1998) Role of MAP kinase in neurons. Mol Neurobiol 16:79–95

20.

Goss

O'Malley

Zou

Styren

Kochanek

DeKosky

(1998) Astrocytes are the major source of nerve growth factor upregulation following traumatic brain injury in the rat. Exp Neurol 149:301–9

21.

Jiang

Zhang

(2001) Extracellular signal-regulated kinase 1/2 activation in hippocampus after cerebral ischemia may not interfere with postischemic cell death. Brain Res 901:79–84

22.

Hailer

Wirjatijasa

Roser

Hischebeth

Korf

Dehghani

(2001) Astrocytic factors protect neuronal integrity and reduce microglial activation in an in vitro model of N-methyl-d-aspartate-induced excitotoxic injury in organotypic hippocampal slice cultures. Eur J Neurosci 14:315–26

23.

Han

Holtzman

(2000) BDNF protects the neonatal brain from hypoxic-ischemic injury in vivo via the ERK pathway. J Neurosci 20:5775–81

24.

Hasegawa

Hamada

Morioka

Yano

Kawano

Kai

Fukunaga

Ushio

(2003) Neuroprotective effect of postischemic administration of sodium orthovanadate in rats with transient middle cerebral artery occlusion. J Cereb Blood Flow Metab 23:1040–51

25.

Levin

(1994) Astrocyte growth is regulated by neuropeptides through Tis 8 and basic fibroblast growth factor. J Clin Invest 93:1820–7

26.

Irving

Barone

Reith

Hadingham

Parsons

(2000) Differential activation of MAPK/ERK and p38/SAPK in neurones and glia following focal cerebral ischaemia in the rat. Brain Res Mol Brain Res 77:65–75

27.

Kim

Choi

Kim

Nah

Kim

Lee

(2002) Agonists of proteinase-activated receptor 2 induce TNF-alpha secretion from astrocytoma cells. Cell Biochem Funct 20:339–45

28.

Kitagawa

Warita

Sasaki

Zhang

Sakai

Shiro

Mitsumoto

Mori

Abe

(1999) Immunoreactive Akt, PI3-K and ERK protein kinase expression in ischemic rat brain. Neurosci Lett 274:45–8

29.

Koo

Chung

Hwang

Kim

(2002) Factor Xa induces mitogenesis of coronary artery smooth muscle cell via activation of PAR-2. FEBS Lett 523:85–9

30.

Lee

Drabik

Van Wagoner

Lee

Choi

Dong

Benveniste

(2000) ICAM-1-induced expression of proinflammatory cytokines in astrocytes: Involvement of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways. J Immunol 165:4658–66

31.

Omori

Sato

Jin

Nagano

Manabe

Shoji

Abe

(2002) Coordinate expression of survival p-ERK and proapoptotic cytochrome c signals in rat brain neurons after transient MCAO. Brain Res 958:83–8

32.

Lindner

Kahn

Coughlin

Sambrano

Schauble

Bernstein

Foy

Hafezi-Moghadam

Ley

(2000) Delayed onset of inflammation in protease-activated receptor-2-deficient mice. J Immunol 165:6504–10

33.

Liu

Smith

Barone

Ellison

Lysko

Simpson

(1999) Astrocytic demise precedes delayed neuronal death in focal ischemic rat brain. Brain Res Mol Brain Res 68:29–41

34.

Macfarlane

Seatter

Kanke

Hunter

Plevin

(2001) Proteinase-activated receptors. Pharmacol Rev 53:245–82

35.

Matsutani

Yamamoto

(1997) Neuronal regulation of astrocyte morphology in vitro is mediated by GABAergic signaling. Glia 20:1–9

36.

McPhail

McBride

McGraw

Steeves

Tetzlaff

(2004) Axotomy abolishes NeuN expression in facial but not rubrospinal neurons. Exp Neurol 185:182–90

37.

Minn

Schubert

Neiss

Muller-Hill

(1998) Enhanced GFAP expression in astrocytes of transgenic mice expressing the human brain-specific trypsinogen IV. Glia 22:338–47

38.

Morita

Higuchi

Moto

Kozuka

Susuki

Itofusa

Yamashita

Kudo

(2003) Dual regulation of calcium oscillation in astrocytes by growth factors and pro-inflammatory cytokines via the mitogen-activated protein kinase cascade. J Neurosci 23:10944–52

39.

Norton

Aquino

Hozumi

Chiu

Brosnan

(1992) Quantitative aspects of reactive gliosis: A review. Neurochem Res 17:877–85

40.

Nystedt

Emilsson

Wahlestedt

Sundelin

(1994) Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 91:9208–12

41.

Obata

Noguchi

(2004) MAPK activation in nociceptive neurons and pain hypersensitivity. Life Sci 74:2643–53

42.

Oliver

Hill

(2002) Feeling below PAR: Proteinase-activated receptors and the perception of neuroinflammatory pain. Pharmacogenomics J 2:10–1

43.

Pompili

Nori

Geloso

Guadagni

Corvino

Michetti

Fumagalli

(2004) Trimethyltin-induced differential expression of PAR subtypes in reactive astrocytes of the rat hippocampus. Brain Res Mol 122:93–8

44.

Ridet

Malhotra

Privat

Gage

(1997) Reactive astrocytes: Cellular and molecular cues to biological function. Trends Neurosci 20:570–7

45.

Smith-Swintosky

Cheo-Isaacs

D'Andrea

Santulli

Darrow

Andrade-Gordon

(1997) Protease-activated receptor-2 (PAR-2) is present in the rat hippocampus and is associated with neurodegeneration. J Neurochem 69:1890–6

46.

Steinhoff

Vergnolle

Young

Tognetto

Amadesi

Ennes

Trevisani

Hollenberg

Wallace

Caughey

Mitchell

Williams

Geppetti

Mayer

Bunnett

(2000) Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism. Nat Med 6:151–8

47.

Stoll

Jander

Schroeter

(1998) Inflammation and glial responses in ischemic brain lesions. Prog Neurobiol 56:149–71

48.

Striggow

Riek-Burchardt

Kiesel

Schmidt

Henrich-Noack

Breder

Krug

Reymann

Reiser

(2001) Four different types of protease-activated receptors are widely expressed in the brain and are up-regulated in hippocampus by severe ischemia. Eur J Neurosci 14:595–608

49.

Tian

Gotoh

Tsuji

Huang

Feig

(2004) Developmentally regulated role for Ras-GRFs in coupling NMDA glutamate receptors to Ras, Erk and CREB. EMBO J 23:1567–75

50.

Ubl

Vohringer

Reiser

(1998) Co-existence of two types of [Ca2+]i-inducing protease-activated receptors (PAR-1 and PAR-2) in rat astrocytes and C6 glioma cells. Neuroscience 86:597–609

51.

Vergnolle

Bunnett

Sharkey

Brussee

Compton

Grady

Cirino

Gerard

Basbaum

Andrade-Gordon

Hollenberg

Wallace

(2001a) Proteinase-activated receptor-2 and hyperalgesia: A novel pain pathway. Nat Med 7:821–6

52.

Vergnolle

Wallace

Bunnett

Hollenberg

(2001b) Protease-activated receptors in inflammation, neuronal signaling and pain. Trends Pharmacol Sci 22:146–52

53.

von Gise

Lorenz

Wellbrock

Hemmings

Berberich-Siebelt

Rapp

Troppmair

(2001) Apoptosis suppression by Raf-1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent signals. Mol Cell Biol 21:2324–36

54.

Wang

Ubl

Reiser

(2002) Four subtypes of protease-activated receptors, co-expressed in rat astrocytes, evoke different physiological signaling. Glia 37: 53–63

55.

Wang

Martindale

Holbrook

(2000) Requirement for ERK activation in cisplatin-induced apoptosis. J Biol Chem 275:39435–43

56.

Ahmad

Schwartz

Banville

Shen

(1997) Protein-tyrosine phosphatase SHP2 is positively linked to proteinase-activated receptor 2-mediated mitogenic pathway. J Biol Chem 272:7519–24

57.

Zonta

Angulo

Gobbo

Rosengarten

Hossmann

Pozzan

Carmignoto

(2003) Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nat Neurosci 6:43–50

58.

Zonta

Carmignoto

(2002) Calcium oscillations encoding neuron-to-astrocyte communication. J Physiol Paris 96:193–8

Deficiency of PAR-2 Gene Increases Acute Focal Ischemic Brain Injury

Abstract

Keywords

Introduction

Materials and methods

Generation of Protease-Activated Receptor-2-Deficient Mouse

Focal Cerebral Ischemia

Calculation of Infarction Volume

Single Immunohistochemistry

Double Fluorescent Immunohistochemistry of Protease-Activated Receptor-2 with NeuN or GFAP

Double Fluorescent Immunohistochemistry of p-ERK with NeuN or GFAP

Western Blotting Analysis

Apoptosis Assay

Data analyses and Statistics

Results

Expression of Protease-Activated Receptor-2 in the Normal WT Mice

Protease-Activated Receptor-2 Upregulation in the WT Mice after Transient MCAO

Evaluation of Infarct Volume in Protease-Activated Receptor-2 KO Mice

Neuronal p-ERK1/2 Expression in WT and Protease-Activated Receptor-2 KO Mice

Expression of GFAP in the Protease-Activated Receptor-2 KO Mice

TUNEL Staining after Transient Middle Cerebral Artery Occlusion in Protease-Activated Receptor-2 KO Mice

Discussion

Footnotes

Acknowledgements

References