Intracerebroventricular Administration of an Endothelin ETB–Receptor Agonist Increases Expression of Matrix Metalloproteinase-2 and -9 in Rat Brain

J Pharmacol Sci 114, 433 – 443 (2010)

Journal of Pharmacological Sciences ©2010 The Japanese Pharmacological Society

Full Paper

Intracerebroventricular Administration of an Endothelin ETB–Receptor Agonist Increases Expression of Matrix Metalloproteinase-2 and -9 in Rat Brain Yutaka Koyama1,* and Kazuhiro Tanaka1 1

Laboratory of Pharmacology, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-Kita, Tonda-bayashi, Osaka 584-8540, Japan

Received July 23, 2010; Accepted October 13, 2010

Abstract. Matrix metalloproteinases (MMPs), a family of zinc-endopeptidases, have a critical role in the pathophysiological responses in damaged brains. MMPs are up-regulated in brain pathologies. To clarify the extracellular signals involved in brain MMP production, the effects of endothelins (ETs), a family of vasoconstricting peptides, were examined. Intracerebroventricular administration of 500 pmol/day Ala1,3,11,15-ET-1, an ETB-receptor agonist, increased the mRNAs of MMP2 and MMP9 in rat hippocampus and cerebrum. Ala1,3,11,15-ET-1 did not affect mRNA levels of MMP 1, 12, and 14. Administration of Ala1,3,11,15-ET-1 for 7 days also increased the protein content and proteolytic activities of MMP2 and MMP9 in the cerebrum. Immunohistochemical observations showed that astrocytes in the hippocampus and the cerebrum of ET-infused rats had MMP2 and MMP9 reactivities. In rat cultured astrocytes, both Ala1,3,11,15-ET-1 (100 nM) and ET-1 (100 nM) increased MMP2 and MMP9 mRNAs. ET-1 stimulated the protein releases and the proteolytic activities of MMP2 and MMP9 from cultured astrocytes. BQ788, an ETB antagonist, inhibited the effects of ET-1 on astrocytic MMP2 and MMP9. The ET-induced expression of MMP9, but not MMP2, was inhibited by pyrrolidine dithiocarbamate, proteasome inhibitor I, and MG132. These results suggest that ET stimulates astrocytic MMP2 and MMP9 production through ETB receptors. Keywords: endothelin-1, matrix metalloproteinase (MMP), astrocyte, brain injury, gene expression Introduction

sive activations of MMPs often occur in pathological conditions and reveal detrimental actions on damaged tissues (3, 4). In the CNS, expressions of MMP 2, 3, 9, 12, and 14 have been detected (5, 6). The expression and proteolytic activities of brain MMPs are up-regulated in brain ischemia, neurodegenerative disease, and head trauma (7 – 13). Previous studies showed that synthetic MMP inhibitors reduced the disruption of the blood– brain barrier (BBB), brain edema formation, and neuronal degradation after brain ischemia (14 – 16). MMP9-null mice were resistant to neuronal damages after brain ischemia and head trauma (17 – 19). These findings suggest pathological roles of MMPs in aggravations of nerve injuries after brain insults. Some signal molecules have been suggested to regulate the expression of MMPs (20 – 23). However, the regulation of MMP expression in damaged brains has not been fully understood.

Matrix metalloproteinases (MMPs) are a family of zinc-endopeptidases consisting of at least 20 members. Some members of MMPs are responsible for the degradation of extracellular matrix (ECM) molecules such as collagen, laminin, and fibronectin (1). The degradation of ECM molecules by MMPs is implicated in diverse biological processes, including embryonic development, inflammation, and tissue repair. Because of the importance in multiple cellular functions, proteolytic activities of MMPs are tightly controlled by transcriptional and post-translational mechanisms (1, 2). However, exces*Corresponding author. [email protected] Published online in J-STAGE on November 26, 2010 (in advance) doi: 10.1254/jphs.10195FP

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Endothelins (ETs), a vasoconstrictor peptide family, are present in the brain. Production of brain ETs is increased by brain insults; these increases in brain ETs affect several pathophysiological responses of nerve tissues (24, 25). Receptors for ETs are classified into ETA and ETB receptors. Activation of brain ETA receptors, which are present in brain vascular smooth muscles, causes vasospasm and aggravates ischemic brain injuries (26, 27). ETB receptors are predominantly expressed in astrocytes (28). In damaged brains, phenotypic conversion of resting astrocytes to “reactive” ones commonly occurs. Reactive astrocytes produce several signal molecules, including growth factors, chemokines, and cytokines, and release them at injured sites (29, 30). By this function, reactive astrocytes affect pathophysiological responses of injured nerve tissues. We previously showed that administration of an ETB-selective agonist in the rat brain promoted conversion to reactive astrocytes (31, 32). In this study, to clarify the pathophysiological significance of ETB receptors in the brain, Ala1,3,11,15-ET-1, an ETB-selective agonist, was continuously administered into the rat cerebroventricle, and the effects on the expressions of brain MMPs were examined. Materials and Methods Intracerebroventricular administration of an ETB-receptor agonist All experimental protocols conformed to the Guiding Principles for the Care and Use of Animals approved by The Japanese Pharmacological Society. Male-Wistar rats, weighing 250 – 300 g, were anesthetized with sodium pentobarbital (50 mg/kg) and placed in a stereotaxic frame. Intracerebroventricular administration of an ETBreceptor agonist was performed as described before (32). Briefly, Ala1,3,11,15-ET-1 (Peninsula Labs, San Carlos, CA, USA), an ETB agonist, was dissolved in sterile artificial CSF (150 mM NaCl, 1.2 mM MgSO4, 1 mM K2HPO4, and 10 mM D-glucose) and put into a miniosmotic pump (Alzet 2002 and 28 gage stainless cannula brain infusion kit; Durect Co., Cupertino, CA, USA). After the rat’s skull was exposed, a burr hole was made at 0.5-mm posterior and 2.0-mm right lateral to the bregma. A mini-osmotic pump was implanted in the subcutaneous tissue and a stainless-steel cannula was inserted through the burr hole to infuse the Ala1,3,11,15ET-1 solution into the cerebral ventricle (4.0 mm under the surface of the skull). Ala1,3,11,15-ET-1 was continuously administered at a dosage of 500 pmol/day. Control animals were infused with artificial CSF in the same manner. For determination of the levels of MMP mRNA and protein, rats were decapitated under deep ether anesthesia. After the brains were removed, the hippocampi

were dissected from the right hemispheres. The caudate putamen was dissected from coronal blocks of the right hemisphere at approximately similar levels between 2.0 and −2.0 mm from the bregma. Cerebral tissue was dissected from the left hemisphere between 1.0 and −5.0 mm from the bregma. Dissected brain tissues were stored at −80°C. For the immunohistochemistry, rats were intracardially perfused with 3% paraformaldehyde under pentobarbital anesthesia. Brains were removed, immersed in 30% sucrose for 2 – 3 days and stored at −80°C. Measurement of MMP mRNA levels by quantitative RTPCR Total RNA was extracted from cultured astrocytes and brain tissue according to an acid-phenol method followed by repeated isopropanol precipitation as described previously (32). First strand cDNA was synthesized from total RNA (1 μg) using MMLV reverse transcriptase (200 U; Invitrogen, Carlsbad, CA, USA), random hexanucleotides (0.2 μg, Invitrogen), and RNase inhibitor (20 U; Takara, Ohtsu) in 10 μl of a buffer supplied by the enzyme manufacturer. MMP mRNA levels in each sample were determined by real-time PCR using the SYBR Green fluorescent probe. The reverse transcription product was included in the SYBR Green Master Mix (Toyobo, Osaka) with rat MMP primers, and the mixture was applied to a real-time PCR thermal cycler (Opticom 2; MJ Research, Waltham, MA, USA). The following primer pairs were used: MMP1: 5′-GCCATTACTAGTC TCCGAGGA-3′ and 5′-GGAATTCGTTGGCATGACT CTCAC-3′, MMP2: 5′-CTATTCTGTCAGCACTTTGG3′ and 5′-CAGACTTTGGTTCTCCAACTT-3′, MMP9: 5′-AAATGTGGGTGTACACAGGC-3′ and 5′-TTCAC CCGGTTGTGGAAACT-3′, MMP12: 5′-AACAACTT GGTACCAGAGCC-3′ and 5′-TGGTGACACGATCC AGTAAG-3′, MMP14 (MT-MMP): 5′-TGGGTAGCG ATGAAGTCTTC-3′ and 5′-AGTAAAGCAGTCGCTT GGGT-3′, glyceraldehyde-3-phosphate dehydrogenase (G3PDH): 5′-CTCATGACCACAGTCCATGC-3′ and 5′-TACATTGGGGGTAGGAACAC-3′. As a standard for copy numbers of PCR products, serial concentrations of each PCR fragment were amplified in the same manner. The amount of MMP cDNA was calculated as the copy numbers in each reverse transcription product equivalent to 1 μg total RNA and normalized to the values for G3PDH. In some experiments, the PCR products were separated in a 1.5% agarose gel and stained with 1 μg/ml ethidium bromide to be visualized under UV light. Immunoblotting of MMP2 and MMP9 To determine the amount of MMP protein expression in rat brains, brain tissues were homogenized in ice-cold

Endothelin-Induced MMP Production

20 mM Tris/HCl pH 7.4 containing 1% SDS, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, and 20 μg/ ml aprotinin. The tissue lysate was centrifuged at 15,000 × g for 15 min and protein contents of the supernatant were measured with a BCA protein assay kit (Pierce, Rockford, IL, USA). The lysate (20 μg protein) was subjected to SDS-PAGE and blotted to polyvinylidene difluoride membranes. The membranes were probed with rabbit antibodies against MMP2 and MMP9 (Santa Cruz Biotech, Santa Cruz, CA, USA) and subsequently incubated with a peroxidase-conjugated secondary antibody. Immunoreactive bands were detected with an ECL kit (GE Healthcare, Buckinghamshire, UK). The exposed X-ray films were scanned and densities of protein bands were measured by Image J. To detect MMP proteins released from cultured astrocytes, proteins in culture medium were concentrated by acetone precipitation. After treatment of cultured astrocytes with ETs in serum-free MEM, the culture medium was collected in a centrifuge tube. The medium was mixed with four volumes of −20°C acetone and incubated at −20°C for 60 min. The mixture was centrifuged at 15,000 × g for 15 min and the resulting pellet was solubilized in 1 × SDSPAGE buffer (33). The acetone precipitation was subjected to SDS-PAGE as well. Measurement of MMP2 and MMP9 activities To assay the MMP activity, rat cerebrums were homogenized in 50mM Tris/HCl pH7.6, 150 mM NaCl, 50mM CaCl2, 0.05% Brij-35, and 1% Triton-X 100. MMP9 activities in the astrocytic culture medium and rat cerebrum were determined using a MMP9 activity assay kit (SensoLyte 520; AnaSpec, San Jose, CA, USA). Prior to the activity determination, MMP9 proteins in the culture medium and the brain extract were concentrated by immunoprecipitation. Either the culture medium or the brain extract was incubated with an anti-MMP9 antibody (Santa Cruz Biotech) and protein A/G-agarose at 4°C for 6 h. After centrifugation, the agarose pellets were suspended in an MMP9 assay buffer supplemented by the assay kit, and MMP9 activity was determined. Activities of MMP2 in the culture medium and brain extract were determined using an MMP2 activity assay kit (GE Healthcare) according to the supplier’s protocol. Histological observations Serial frozen coronal sections (15-μm-thick) of ETinfused and control rat brain were made and mounted onto gelatin-coated slides. For MMP staining, rabbit antibodies against MMP2 (Santa Cruz Biotech) and MMP9 (Santa Cruz Biotech) were used. Neurons and astrocytes were labeled with anti-NeuN (Chemicon, Temecula, CA, USA) and anti-glial fibrillary acidic

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protein (GFAP) (Sigma, St. Louis, MO, USA) mouse antibodies, respectively. Labeled cells were visualized by secondary antibodies conjugated to FITC or rhodamine. Microglia were labeled with biotin-conjugated B4-isolectin from Griffonia simplicifolia (Sigma) and visualized with Texas Red-conjugated streptavidin. Preparation of primary cultured astrocytes from rat brain Astrocytes were prepared from the cerebra of 1 – 2-day-old Wistar rats as described previously (32). Cells were seeded at 1 × 104 cells/cm2 in 75 cm2 culture flasks and grown in minimum essential medium (MEM) supplemented with 10% fetal calf serum. To remove small process-bearing cells (mainly oligodendrocyte progenitors and microglia from the protoplasmic cell layer), culture flasks were shaken at 250 rpm overnight at 10 – 14 days after seeding. The monolayer cells were trypsinized and plated into 6-well culture plates and further cultured for 7 – 10 days. At this stage, approximately 95% of the cells showed immunoreactivity for GFAP. Results Intracerebroventricular administration of an ETB-agonist increased production of MMP2 and MMP9 in rat brain Although several types of MMPs were detected in the rat brains (5, 6), a comparison of each MMP expression level has not been examined. Therefore, we first determined the copy numbers of MMP mRNAs in rat brain. Among MMP 1, 2, 9, 12, and 14, the copy numbers of MMP2 and MMP9 were relatively high (>105 copies/μg total RNA) in the rat cerebrum, while those of MMP 1, 12, and 14 were low (MMP1: 0.40 ± 0.06, MMP2: 19.54 ± 6.12, MMP9: 24.45 ± 4.98, MMP12: 0.11 ± 0.03, MMP14: 2.33 ± 0.36, G3PDH: 6982.5 ± 508.9 in × 104/ μg total RNA, means ± S.E.M. of 6 – 8 different preparations). The effects of an ETB agonist on brain MMP productions were examined. Administration of 500 pmol/ day Ala1,3,11,15-ET for 3 or 7 days increased MMP2 and MMP9 mRNA levels in the cerebrum (Fig. 1: A and B). In the hippocampus, significant increases in MMP2 and MMP9 mRNAs were obtained by the administration of Ala1,3,11,15-ET-1 for 7 days (Fig. 1B). MMP2 and MMP9 mRNAs in the caudate putamen did not increase with Ala1,3,11,15-ET-1 administration. Ala1,3,11,15-ET-1 did not largely affect the mRNA levels of MMP1, MMP12, and MMP14 in any of the brain regions examined. Immunoblot analysis showed that the administration of Ala1,3,11,15ET-1 for 7 days increased the protein contents of MMP2 and MMP9 in the cerebrum, while the protein contents of β-actin was not changed (Fig. 2A). Along with the in-

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l l -1 -1 -1 ol tro ET ntro ET ntr ET n o o o a a a C C C Al Al Al

(A) MMP2 MMP9 MMP14 G3PDH

MMP2

MMP1/G3PDH mRNA (% of control)

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MMP14/G3PDH mRNA (% of control)

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caudate-putamen

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hippocampus

MMP9

CE

300

hippocampus MMP2/G3PDH mRNA (% of control)

MMP1

CP

HP

(B)

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cerebrum 3 days

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cerebrum 3 days

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500 pmol/day Ala-ET-1

7 days

Fig. 1. Effects of intracerebroventricular administration of Ala1,3,11,15-ET-1 on expression of MMP mRNAs in the rat brain. A) Detection of MMP 2, 9, and 14 mRNAs by RT-PCR. Either Ala1,3,11,15-ET-1 (500 pmol/day) or artificial CSF was infused into rat brain for 7 days. Each primer pair for the detection of MMPs showed a single PCR product corresponding to MMP2, MMP9, or MMP14 cDNA fragments. HP: hippocampus, CP: caudate putamen, CE: cerebrum. B) Determination of MMP2, MMP9, and MMP14 mRNA levels by quantitative RT-PCR. Either Ala1,3,11,15ET-1 (500 pmol/day) or artificial CSF (control) was infused into rat brain for 3 or 7 days. The copy numbers of MMP1, MMP2, MMP9, MMP12, and MMP14 mRNAs in the brain regions were determined, and the values were normalized to the copy numbers of G3PDH. Results are means ± S.E.M. of 7 – 9 rats and are presented as a percentage of the level in control rats. *P < 0.05 vs. control rats by Student’s t-test.

Endothelin-Induced MMP Production

creases in the MMP proteins, the proteolytic activities of MMP2 and MMP9 in the cerebrum were increased by the administration of Ala1,3,11,15-ET-1 for 7 days (Fig. 2B).

(A)

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Immunohistochemical observations of MMP expression in rat brain after Ala1,3,11,15-ET-1 administration To identify cellular origins of ET-induced MMP production in the rat brain, MMP-positive cells were labeled with markers for neurons (NeuN), astrocytes (GFAP), and microglial cells (B4-isolectin). In the hippocampus and the cerebrum of saline-infused rats, NeuN-positive neurons had no immunoreactivity for MMP2 (data not shown). Administration of 500 pmol/day Ala1,3,11,15-ET-1

Ala-ET-1

MMP2 MMP9 Actin

MMP2 **

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0.5

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MMP9/ -actin protein (Arbitrary Units)

MMP2/ -actin protein (Arbitrary Units)

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0

MMP2

0.3 0.2 0.1

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500 pmol/day Ala-ET-1

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MMP2 activity (Arbitrary Units)

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(B)

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500 pmol/day Ala-ET-1

Fig. 2. Effects of intracerebroventricular administration of Ala1,3,11,15-ET-1 on production and activities of MMPs in the rat cerebrum. A) Determination of MMP2 and MMP9 proteins in the rat cerebrum. Either Ala1,3,11,15-ET-1 (500 pmol/day) or artificial CSF was infused into rat brain for 7 days. Brain lysates were prepared as described in the Materials and Methods. Typical immunoblots of MMPs and β-actin from two different rats in each group are presented. After detection of MMP2 and MMP9 proteins by immunoblotting, the protein bands in X-ray films were subjected to densitometry analyses. The densities of MMPs were normalized to that of β-actin in the same blots. Results are means ± S.E.M. of 5 – 6 rats and are presented as ratios of MMP/β-actin proteins. **P < 0.01 vs. control rats by Student’s t-test. B) Effects of Ala1,3,11,15-ET-1 on MMP2 and MMP9 activities in the rat cerebrum. Either Ala1,3,11,15-ET-1 (500 pmol/day) or artificial CSF was infused into rat brain for 7 days. The activities of MMP2 and MMP9 in the brain lysates were then measured. Results are means ± S.E.M. of 6 different preparations. ***P < 0.001 vs. control rats by Student’s t-test.

Fig. 3. Immunohistochemical observations of MMP2 in Ala1,3,11,15ET-1–infused rat brain. Ala1,3,11,15-ET-1 (500 pmol/day) was infused into rat brains for 7 days. Brain sections from control or Ala1,3,11,15ET-1–infused rats were labeled with an antibody against MMP2. Neurons, astrocytes, and microglia were visualized with their cell markers, NeuN, GFAP, and B4-isolectin, respectively. NeuN-positive neurons in the hippocampus (A) and cerebrum (C) were labeled with an MMP2 antibody (B, D). GFAP-positive astrocytes in the hippocampus (E) and cerebrum (G) were labeled with an MMP2 antibody (F, H). Scale bars are 50 μm for A – D and 25 μm for E – H.

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for 7 days did not induce MMP2 reactivity in the hippocampal and the cerebral NeuN-positive cells (Fig. 3: A – D). On the other hand, GFAP-positive cells in the hippocampus and the cerebrum of ET-infused rats showed immunoreactivity for MMP2 (Fig. 3: E – H). A small number of B4-isolectin labeled activated microglia appeared in the brain of ET-infused rats (32). B4isolectin–labeled cells in the hippocampus and the cerebrum had no MMP2 reactivity (data not shown). Although diffuse and faint immunoreactivity for MMP9 was observed along hippocampal neuronal cell layers of the control rats (Fig. 1: A and B). NeuN-positive cells in the cerebrum had no MMP9 reactivity (Fig. 4: C and D). Administration of Ala1,3,11,15-ET-1 did not affect the MMP9 reactivities in the hippocampal (Fig. 4: E and F) and the cerebral (data not shown) neurons. GFAP-positive cells in the hippocampus and cerebrum of ET-infused rats demonstrated MMP9 reactivity (Fig. 4: G – J). B4isolectin–labeled cells in the cerebrum did not show MMP9 reactivity with Ala1,3,11,15-ET-1 administration (Fig. 4: K and L).

Effects of ET-1 on MMP expression in rat cultured astrocytes Immunohistochemical observations of Ala1,3,11,15-ET1–infused rats showed that MMP2 and MMP9 reactivities were present in astrocytes. To clarify whether ETs stimulate the MMP productions directly through astrocytic ET receptors, the effects of ET-1 in rat cultured astrocytes were examined. Treatment with ET-1 or Ala1,3,11,15-ET-1 increased mRNA levels of astrocytic MMP2 and MMP9 (Fig. 5A). ET-1 did not affect mRNA levels of MMP 1, 12, and 14 (Fig. 5A). The ET-induced increases in the MMP mRNAs were reduced by BQ788, an ETB antagonist, while FR139317, an ETA antagonist, did not show obvious inhibition (Fig. 5B). Immunoblot analysis showed that treatment of cultured astrocytes with 100 nM ET-1 increased MMP2 and MMP9 protein contents in the culture medium, but did not affect MMP1 proteins (Fig. 6A). Along with the increases in the MMP proteins, the proteolytic activities of MMP2 and MMP9 in the culture medium were increased by 100 nM ET-1 (Fig. 6B).

Fig. 4. Immunohistochemical observations of MMP9 in control and Ala1,3,11,15ET-1–infused rat brain. Ala1,3,11,15-ET-1 (500 pmol/day) was infused into rat brain for 7 days. Brain sections from the control or Ala1,3,11,15-ET-1–infused rats were labeled with an antibody against MMP9. Neurons, astrocytes, and microglia were visualized with their cell markers, NeuN, GFAP, and B4-isolectin, respectively. A – D) Control rat: NeuN-positive neurons in the hippocampus (A) and cerebrum (C) were labeled with an MMP9 antibody (B, D). E – L) Ala1,3,11,15-ET-1–infused rats: NeuN-positive neurons in the hippocampus (E) showed MMP9 reactivity (F). GFAP-positive astrocytes in the hippocampus (G) and cerebrum (I) were labeled with the MMP9 antibody (H, J). B4-Isolectin–labeled microglia in the cerebrum (arrowheads in K) were not stained by the MMP9 antibody (L). Scale bars are 50 μm for A – F and 25 μm for G – L.

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(A) MMP1

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Effects of signal-transduction Inhibitors on ET-induced MMP expression ET-induced expression of astrocytic MMP2 and MMP9 was decreased by chelation of intra- and extracellular Ca2+ (30 μM BAPTA/AM and 0.5 mM EGTA), a protein kinase C (PKC) inhibitor (100 nM staurosporine), and an inhibitor of MEK/ERK signal (50 μM PD98059) (Table 1). Pyrrolidine dithiocarbamate (PDTC), which inhibits transcriptional activity of NFκB, and proteasome inhibitors (1 μM proteasome inhibitor I and 5 μM MG132) reduced the effect of ET-1 on MMP9 expression. The ET-induced astrocytic MMP2 mRNA expression was not affected by PDTC and these proteasome

control

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Fig. 5. Effects of ETs on MMP mRNA expression in cultured cells. A) Timecourse of changes in astrocytic MMP mRNA levels induced by ETs. Serumstarved astrocytes were treated with 100 nM ET-1 and 100 nM Ala1,3,11,15-ET-1 for the indicated length of time. The expressions of MMP 1, 2, 9, 12, and 14 mRNAs were normalized to that of G3PDH. Each primer pair for the detection of MMPs showed a single PCR product (above the graphs). Results are means ± S.E.M. of 8 – 12 experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the 0-time by one-way ANOVA followed by the Dunnett test. B) Effects of ET-receptor antagonists. Serumstarved astrocytes were treated with 10 nM ET-1 for 1 and 6 h to determine MMP2 and MMP9 mRNA levels, respectively. BQ788 (1 μM) or FR139317 (1 μM) was included in the medium 30 min before the treatments with ET-1. The expression of MMP2 and MMP9 mRNAs was normalized to that of G3PDH. Results are means ± S.E.M. of 4 – 6 experiments. ***P < 0.001 vs. control with no antagonist, #P < 0.05, ##P < 0.01 vs. ET-1 alone. Data were statistically analyzed by one-way ANOVA followed by the Tukey test.

inhibitors. Discussion In this study, a continuous intracerebroventricular administration of Ala1,3,11,15-ET-1 increased the mRNA levels of MMP2 and MMP9 in rat brain (Fig. 1). Accompanying the increases in mRNAs, Ala1,3,11,15-ET-1 also increased the protein contents and proteolytic activities of MMP2 and MMP9 (Fig. 2). These findings indicate that activation of brain ETB receptors stimulated the production of MMP2 and MMP9. In normal adult brain, some populations of neurons in the hippocampus, cere-

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(A)

none

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Fig. 6. Effects of ET-1 on releases of MMP proteins in cultured astrocytes. A) Effects of ET-1 on the release of MMP proteins from cultured astrocytes. Cells were incubated in serum-free MEM in the absence or presence of 100 nM ET-1 for the indicated times. The amounts of MMP proteins in the medium were detected by immunoblotting. Typical patterns of immunoblots for MMP1, MMP2, and MMP9 are indicated above the graphs. The graphs show densitometric analysis of MMP2 and MMP9 proteins, and the results are means ± S.E.M. of 4 – 6 different preparations. *P < 0.05, **P < 0.01 vs. none by one-way ANOVA followed by the Tukey test. B) Effects of ET-1 on MMP2 and MMP9 activities released from cultured astrocytes. Cells were incubated in serumfree MEM in the absence or presence of 100 nM ET-1 for the indicated times. The activities of astrocytic MMP2 and MMP9 released into the medium were then measured. Results are means ± S.E.M. of 6 different preparations. *P < 0.05, **P < 0.01 vs. none by one-way ANOVA followed by the Tukey test.

Table 1. Effects of signal transduction inhibitors on the ET-induced expressions of astrocytic MMP2 and MMP9 mRNA Ratio of MMP/G3PDH mRNA copy numbers (×10−3) MMP2 None

1.35 ± 1.37 (19)

100 nM ET-1

3.06 ± 0.53 (19)*

MMP9 7.04 ± 1.37 (26) 22.97 ± 3.55 (25)***

+ 30 μM BAPTA / 0.5 mM EGTA

0.53 ± 0.13 (6)#

5.56 ± 1.93 (8)###

+ 10 nM staurosporine

0.55 ± 0.13 (6)#

6.99 ± 4.07 (6)#

+ 50 μM PD98059

0.57 ± 0.22 (6)#

3.50 ± 1.46 (8)###

+ 100 μM PDTC

2.47 ± 0.41 (6)

10.66 ± 2.24 (14)##

+ 1 μM proteasome inhibitor I

3.13 ± 0.65 (6)

3.96 ± 0.57 (12)###

+ 5 μM MG-132

3.45 ± 0.41 (6)

3.84 ± 0.59 (12)###

Astrocytes were treated with 100 nM ET-1 in the presence of the indicated signal transduction inhibitors. For determination of mRNA levels of MMP2 and MMP9, total RNA was extracted after 2 and 6 h, respectively. The inhibitors were included in the serum-free medium 30 min before the addition of ET-1. The copy numbers of MMP2 and MMP9 mRNA was normalized to G3PDH. Results are means ± S.E.M. and the numbers of experiments are in parentheses. *P < 0.05, ***P < 0.001 vs. none; # P < 0.05, ##P < 0.01, ###P < 0.001 vs. 100 nM ET-1 by one-way ANOVA followed by the Tukey test.

Endothelin-Induced MMP Production

bellum, and cerebral cortex have been reported to produce MMP2 and MMP9 (7, 34, 35), although the MMP expression levels were low. On the other hand, previous immunohistochemical observations on damaged brains followed by ischemia and head trauma showed that neurons, astrocytes, and microglia all had an ability to produce MMP2 and MMP9 (7 – 10). In the present study, immunohistochemical observations of Ala1,3,11,15-ET-1– infused rats showed MMP2 was present in GFAP-positive reactive astrocytes, but not in neuronal cells and microglia (Fig. 3). Similar to the observation by Szklarczyk et al. (35), MMP9 immunoreactivity was observed along the neuronal cell layer of the hippocampus (Fig. 4: B and F). MMP9 was also produced by reactive astrocytes in the hippocampus and the cerebrum of ET-infused rats (Fig. 4: H and J). Activation of brain ETB receptors are involved in the phenotypic conversion to reactive astrocytes upon brain injury (36, 37). Our previous observations showed that an intracerebroventricular administration of Ala1,3,11,15-ET-1 at the same dosage used in this study increased GFAP-positive reactive astrocytes in rat brain (32). While production of MMP9 by the hippocampal neurons was not excluded, the present immunohistochemical observations suggest that reactive astrocytes are sources of the increased MMP2 and MMP9 by the intracerebroventricular administration of Ala1,3,11,15ET-1. The production and proteolytic activities of MMP2 and MMP9 are increased in nerve tissues injured by brain ischemia and head trauma, where reactive astrocytes are the main sources of the increased MMPs (7 – 10). Administration of ET-1, which is an endogenous ET and activates both ETA and ETB receptors, induced spasm of brain vessels and reduction of cerebral blood flow (26, 27). The reduction of blood flow by ET-1 aggravated brain ischemia and caused neuronal degradation (24). Studies of ET-receptor antagonists showed that the ETinduced ischemic brain damage was mediated by ETA receptors (38, 39). In contrast to ET-1, intracerebroventricular administration of Ala1,3,11,15-ET-1 did not induce neuronal degeneration and microglial activation (32), which was shown in the present study (Figs. 3 and 4). The absence of neuronal damage with the administration of Ala1,3,11,15-ET-1 indicates that the increases in astrocytic MMP2 and MMP9 productions were not consequences of nerve injuries. In the brain, ETB receptors are predominantly expressed in astrocytes. In the examinations of cultured astrocytes, ET-1 and Ala1,3,11,15-ET-1 increased MMP2 and MMP9 production (Figs. 5 and 6). The effects of ET-1 on the MMP expressions in cultured astrocytes were antagonized by BQ788, suggesting an involvement of ETB receptors. Recently, Wang et al. (40) also reported that ET-1 stimulated MMP9 production in

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rat cultured astrocytes. From these in vitro examinations, administration of Ala1,3,11,15-ET-1 is thought to stimulate the MMP production by a direct activation of astrocytic ETB receptors. Activation of astrocytic ETB receptors caused increases in cytosolic Ca2+ and activation of PKC, which lead to activation of ERK and p38 MAPKs. (36, 41). In the examination using signal transduction inhibitors (Table 1), staurosporine, Ca2+-free medium, and PD98059 inhibited the increases in MMP2 and MMP9 mRNAs by ET-1, indicating involvements of Ca2+, PKC, and ERK in the MMP expressions. The ET-induced MMP9 expression was also inhibited by PDTC and proteasome inhibitors (proteasome inhibitor I and MG132). As well as PDTC, inhibition of proteasome reduces NFκB activity by preventing the degradation of IκB. Thus, the inhibitions by PDTC and proteasome inhibitors suggest that the ETinduced astrocytic MMP9 expression is regulated by NFκB. A recent gene knock-down experiment using NFκB siRNA showed that NFκB regulated the ETinduced astrocytic MMP9 expression in the down-stream of ERK activation (40). In contrast to MMP9, the ETinduced astrocytic MMP2 expression was not affected by PDTC and the proteasome inhibitors (Table 1). NFκBindependent mechanisms may regulate the astrocytic MMP2 expression by ET-1 in the down-stream of ERK. Brain ETs are increased in brain pathologies, including brain ischemia and head trauma (24, 25). The increases in brain ETs play important roles in the regulation of several astrocytic responses (36, 37, 41). In injured nerve tissues, astrocytes become the reactive phenotype and produce MMP2 and MMP9. As for the signal molecules stimulating the astrocytic MMP productions, involvements of interleukin-1β, thrombin, plasminogen activators, and bradykinin have been suggested (20 – 23). This study indicates that ETs are one of factors stimulating astrocytic MMP2 and MMP9 productions. In addition to regulating gene transcription, the proteolytic activities of MMPs are tightly controlled by cleavages of pro-MMPs and interactions with their endogenous inhibitors called tissue-inhibitor of matrix metalloproteinases (TIMPs) (1, 2). In the brain, MMP9 is translated as an inactive proMMP9 and the N-terminus of pro-MMP9 is cleaved by MMP2 and MMP3 to reveal the proteolytic activity. Activity of MMP9 is negatively modulated by association with TIMP-1 and TIMP-3. In addition to MMP2 and MMP9, activation of ETB receptors stimulated production of MMP3, TIMP-1, and TIMP-3 in cultured astrocytes and rat brain (33, 42). Considering these functions on the brain MMP and TIMP productions, astrocytic ETB receptors are suggested to play a critical role in the regulation of brain MMP activities at the injured sites. Synthetic inhibitors of MMPs were reported to reduce

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break-down of BBB, neuroinflammation, brain edema, and neuronal degradation after brain ischemia and head trauma (14 – 16). Deletions of MMP9 genes showed lesser levels of brain damage in mouse models of global ischemia and traumatic injuries (17 – 19). These results indicate that the increases in MMP activities after brain insults have detrimental actions on injured nerve tissues. Therefore, the regulation of brain MMP activities by ETs may raise the pharmacological significance of the ETB receptors as a target for neuroprotective drugs against MMP-mediated nerve injuries. Acknowledgements

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This work was supported by a Grant-in-Aid for Scientific Research (C) from JPSP (21590108) and a Grant-in-Aid for Young Scientists (B) from MEXT (21790169).

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