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Keywords:

  • nuclear gelatinolysis;
  • oxidative DNA damage;
  • poly-ADP-ribose polymerase-1;
  • stroke;
  • X-ray cross-complementary factor 1

Abstract

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

J. Neurochem. (2010) 112, 134–149.

Abstract

Increased matrix metalloproteinase (MMP) activity is implicated in proteolysis of extracellular matrix in ischemic stroke. We recently observed intranuclear MMP activity in ischemic brain neurons at early reperfusion, suggesting a possible role in nuclear matrix proteolysis. Nuclear proteins, poly-ADP-ribose polymerase-1 (PARP-1) and X-ray cross-complementary factor 1 (XRCC1), as well as DNA repair enzymes, are important in DNA fragmentation and cell apoptosis. We hypothesized that intranuclear MMP activity facilitates oxidative injury in neurons during early ischemic insult by cleaving PARP-1 and XRCC1, interfering with DNA repair. We induced a 90-min middle cerebral artery occlusion in rats. Increase activity of MMP-2 and -9, detected in the ischemic neuronal nuclei at 3 h, was associated with DNA fragmentation at 24 and 48 h reperfusion. The intranuclear MMPs cleaved PARP-1. Treatment of the rats with a broad-spectrum MMP inhibitor, BB1101, significantly attenuated ischemia-induced PARP-1 cleavage, increasing its activity. Degradation of XRCC1 caused by ischemic insult in rat brain was also significantly attenuated by BB1101. We found elevation of oxidized DNA, apurinic/apyrimidinic sites, and 8-hydroxy-2′-deoxyguanosine, in ischemic brain cells at 3 h reperfusion. BB1101 markedly attenuated the early increase of oxidized DNA. Using tissue from stroke patients, we found increased intranuclear MMP expression. Our data suggest that intranuclear MMP activity cleaves PARP-1 and XRCC1, interfering with oxidative DNA repair. This novel role for MMPs could contribute to neuronal apoptosis in ischemic injuries.

Abbreviations used:
8-OHdG

8-hydroxy-2′-deoxyguanosine

aMMP

active MMP

AP site

apurinic/apyrimidinic (abasic) sites

BBB

blood–brain barrier

BER

base excision repair

ISZ

in situ zymography

MCAO

middle cerebral artery occlusion

MMP

matrix metalloproteinase

MT1-MMP

membrane type 1-metalloproteinase

PARP-1

poly-ADP-ribose polymerase-1

PFC

piriform cortex

pMMP

proMMP

TUNEL

terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

XRCC1

X-ray cross-complementary factor 1

Matrix metalloproteinases (MMPs) play fundamental roles in regulation of the extracellular matrix and have been linked to blood–brain barrier (BBB) opening and neurodegeneration associated with ischemia and neuroinflammation (Rosenberg et al. 1996; Heo et al. 1999; Planas et al. 2001; Lee et al. 2004; Rosenberg and Yang 2007; Candelario-Jalil et al. 2009). During an earlier study of the role of MMP-2 in the early reversible opening of the BBB, we unexpectedly observed gelatinase activity in the nuclei of ischemic neurons at 3 h after reperfusion (Yang et al. 2007). As nuclear matrix proteolysis is implicated in numerous activities such as apoptosis, cell cycle, and DNA fragmentation, the early increased intranuclear gelatinase activity in ischemic neurons suggested a possible role in nuclear matrix proteolysis, which may be involved in neuronal death or survival in focal ischemia with reperfusion.

Poly-ADP-ribose polymerase 1 (PARP-1) is a nuclear chromatin-associated multifunctional enzyme (Hassa and Hottiger 2008) that acts as a sensor protein to detect oxidative DNA stress. DNA strand breaks induce PARP-1 activity, which plays an important role in repair of oxidized DNA and cell survival (Tanaka et al. 2005). DNA base excision repair (BER) machinery is the main mechanism in mammalian neuronal nuclei to repair various types of oxidative DNA damage. PARP-1 is required for efficient BER function by recruiting X-ray cross-complementary factor 1 (XRCC1) to oxidized DNA bases (Martin 2008). XRCC1 plays a central role in the DNA BER pathway by interacting with major DNA repair enzymes in the BER pathway (Vidal et al. 2001).

Accumulation of oxidative DNA damage is associated with ischemia and, if not repaired promptly, triggers cell death. Appearance of oxidative DNA, apurinic/apyrimidinic (AP) sites and 8-hydroxy-2′-deoxyguanosine (8-OHdG), and BER reduction occurs as early as 30 min after the onset of reperfusion in 2 h middle cerebral artery occlusion (MCAO) model (Lan et al. 2003; Luo et al. 2007). A recent study demonstrated that gelatinases are activated 15 min after reperfusion start in a MCAO model (Amantea et al. 2008). Expression of low level of PARP-1 partially prevented neuronal apoptosis and PARP-1 inhibition enhanced neuronal vulnerability to apoptosis (Nagayama et al. 2000; Diaz-Hernandez et al. 2007). MMP-2 is also detected in the nucleus of human cardiac myocytes and purified MMP-2 is capable of cleaving PARP-1 in vitro (Kwan et al. 2004). Activation of MMP-2 is an early and key event in oxidative stress injury to the heart after ischemic reperfusion injury (Ali and Schulz 2009). Considering the early induction of oxidative DNA lesion and reduction of BER function, we hypothesized that the early increased activity of MMPs in the nucleus after stroke degraded PARP-1 and XRCC1, contributing to a reduction of DNA BER function and an accumulation of oxidized DNA bases in neurons, triggering their death.

To test the hypothesis, we evaluated the role of intranuclear MMP activity on the cleavage of PARP-1 and XRCC1. We show for the first time in brain that shortly after the injury MMPs in a nuclear fraction of ischemic brain tissue cleave PARP-1 and XRCC1, which facilitates accumulation of oxidized DNA at an early stage after an ischemic insult. Moreover, we show that treatment with a broad-spectrum MMP inhibitor, BB1101, significantly attenuates ischemia-induced cleavage of PARP-1 and XRCC1 degradation and protects against oxidative DNA damage.

Materials and methods

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

A detailed description of all methods utilized in this study is found in the Appendix S1.

Middle cerebral artery occlusion with reperfusion

The study was approved by the University of New Mexico Animal Care Committee and conformed to the National Institutes of Health Guidelines for use of animals in research. Male spontaneous hypertensive rats (300–320 g of body weight) were subjected to 90 min of MCAO (Rosenberg et al. 2001) and reperfusion for 3, 24, and 48 h.

In situ zymography for rat and human brains

Gelatinolytic activity in frozen, non-fixed sections (14 μm) from ischemic rat brains was examined, using EnzCheck Collagenase Kit (Molecular Probes, Eugene, OR, USA) as previously described (Yang et al. 2007). After in situ gelatinolysis, some sections were processed for immunohistochemistry to detect the expression of proteins co-localized with active gelatinases in cells.

Frozen non-fixed human brain sections (12 μm) from five deceased stroke patients were examined using EnzCheck Collagenase Kit to detect gelatinolysis. This study was approved by the Ethics Committee of the Hospital Vall d’Hebron and informed consent was acquired from all relatives prior to the autopsy.

Preparation of nuclear extracts

Nuclear extracts from rat brains having undergone 90 min MCAO with 3 h reperfusion were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents and Nuclear Enrichment Kit for Tissue (Pierce, Rockford, IL, USA) following manufacturer’s protocol.

Gelatin zymography

The expression of MMP-2 and -9 in nuclear extracts from ischemic rat brains was measured using gelatin zymography as previously described (Yang et al. 2007).

Immunohistochemistry

Ten micrometer sections from rat brain tissues fixed with 2%p-formaldehyde were used for immunohistochemical analysis.

Immunoblotting

Nuclear proteins extracted from tissues of ischemic and non-ischemic cortex of rats were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis. Coomassie blue staining on the same polyvinylidene difluoride membranes was used for loading and transfer control. The results are reported as normalized band intensity for quantifying relative protein expression.

In vitro degradation assay of PARP-1 and XRCC1

In vitro degradation of PARP-1 and XRCC1 by recombinant rat MMP-2, murine MMP-9 (R&D Systems, Minneapolis, MN, USA), gelatinase extracts or total nuclear extracts was examined in an assay buffer.

Poly-ADP-ribose polymerase-1 activity assay

Poly-ADP-ribose polymerase-1 activity assay in nuclear extracts of ischemic brain tissues was measured with HT Universal Colorimetric PARP Assay Kit (Trevigen, Gaithersburg, MD, USA) following manufacturer’s protocol.

Assessment of ischemia-induced DNA damage

8-Hydroxy-2′-deoxyguanosine was evaluated using an oxidative DNA ELISA kit (Cell Biolabs, San Diego, CA, USA) following the kit instructions. AP sites in genomic DNA were detected using the OxiSelect Oxidative DNA damage quantification kit (AP sites) (Cell Biolabs).

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was carried out to identify the extent of DNA fragmentation by using the NeuroTACS II kit (Trevigen). For dual immunofluorescence, the slides were then exposed to the primary antibody and streptavidin fluorescein and prepared as described above.

Statistical analysis

Statistical comparisons among groups were done using anova with post hoc analysis for multiple t-test (prism 4.0; GraphPad Software Incorporated, San Diego, CA, USA). All data are presented as mean ± SEM. Statistical significance was set at < 0.05.

Results

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

Gelatinase activity in neuronal nuclei in both ischemic rat and human brains

The subcellular localization of active gelatinases was determined using in situ zymography (ISZ) with a reaction buffer containing FITC-labeled DQ-gelatin (Molecular Probes). Gelatin-FITC is cleaved by gelatinases, yielding peptides whose green fluorescence is representative of net proteolytic activity (Fig. 1). With ISZ, we showed previously that there was a marked increase in gelatinase activity in the ischemic hemisphere after 3 h of reperfusion, which was seen in ischemic vessels, cells, and cell nuclei (Yang et al. 2007). Intense gelatinolytic activity was seen in brain cells within 4′-6-diamidino-2-phenylindole (DAPI)-positive nuclei of the ischemic cortex at 3 h after reperfusion (Fig. 1a). Confocal microscopy confirmed the strong gelatinolytic activity in the nuclei (Fig. 1b). Minimal fluorescence was detected in both sham-operated rats and contralateral (non-ischemic) hemisphere (Fig. 1a). The nuclei with gelatinase activity co-localized with the neuronal marker, NEUronal Nuclei (NeuN), suggesting that the increased proteolytic activity from MMPs was in ischemic neurons (Fig. 1c).

image

Figure 1.  Gelatinase activity in the nuclei of ischemic brain cells in rat and human infarct preparations. (a) In situ zymography (ISZ) was performed in MCAO and sham-operated rat brains with 3 h post-ischemic reperfusion. Gelatinase activity is visualized with green fluorescence. DAPI (blue) shows nuclear localization. Scale bars, 50 μm. (b) Z-stack confocal images show gelatinase activity is co-localized with DAPI (blue) in nucleus. Scale bars, 5 μm. (c) Z-stack confocal images show that gelatinase activity in cell nuclei is co-localized with NeuN (red), a neuronal marker. Scale bars, 5 μm. (d) Gelatinase activity in the nuclei of brain cells in human infarct preparations. 1,10-phenanthroline (1-10 Phe) eliminated proteolytic activity displayed in infarct preparations. Scale bars, 50 μm.

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We also detected the proteolytic activity of gelatinases in human brains with an ischemic stroke within the previous range from 12 to 96 h using ISZ. Proteolytic activity was found in the cell nucleus within the infarction in brain parenchyma samples from deceased stroke patients (Fig. 1d). In contrast, nuclear proteolytic activity was rarely detected in the contralateral hemisphere. To ensure the signal was because of MMP activity, samples from the infarct core were treated with the MMP inhibitor 1,10-phenanthroline that is known to reversibly chelate zinc ions, essential for MMP activity. Phenanthroline-treated preparations had very little signal (Fig. 1d).

As ISZ cannot distinguish between MMP-2 and -9, several experiments were performed to identify the source of gelatinase activity. With co-localization of immunolabeling for MMP-2 and -9 with ISZ, we found higher levels of MMP-2 and -9 immunoreaction in the brain cells with increased gelatinase activity in ischemic brain (Fig. 2a). Confocal analysis showed that immunoreaction of both MMP-2 and -9 was co-localized with the proteolytic activity in nuclei (Fig. 2b). To validate the finding, brain sections were pre-incubated with MMP-2 and -9 antibodies, broad-spectrum MMP inhibitor 1,10-phenanthroline and selective MMP-2/9 inhibitor II, prior to an ISZ analysis. Pre-incubation with MMP-2 and -9 antibodies and MMP inhibitors reduced the fluorescence intensity in ischemic brain tissue, suggesting that both MMP-2 and -9 were involved in the gelatinase activity (Fig. 2c). For quantitation of the gelatinase activity, fluorescence intensity in fields of ischemic cortex, piriform cortex, and striatum in each section derived from bregma area were measured using imagej software (http://www.rsbweb.nih.gov/ij). The data strongly suggest that MMP-2 and -9 contribute to the intranuclear gelatinase activity in ischemic neurons.

image

Figure 2.  MMP-2 and -9 contribute to intranuclear gelatinase activity in rat brain at 3 h post-ischemic reperfusion. (a) Double staining of in situ zymography (ISZ) (green) and MMP-2 and -9 immunohistochemistry (red) was performed in MCAO and sham-operated rat brains with 3 h post-ischemic reperfusion. DAPI (blue) shows nuclear localization. Scale bars, 100 μm. Inset shows example of negative control for immunostaining with normal IgGs and secondary antibody conjugated with Cy-3 and no specific immunolabeling was exhibited. (b) Representative confocal images show immunostaining for MMP-2 and -9 (red) co-localized with ISZ in nuclei in ischemic cells. Scale bars, 10 μm. (c) Pre-incubation with specific antibodies for MMP-2 and -9 (MMP-2 AB and MMP-9 AB), 1,10-phenanthroline (Phen), and MMP-2/9 inhibitor II (Inhibitor II) block the gelatinase activity by ISZ in nuclei of ischemic cells; *p < 0.05 and **p < 0.01 versus ISZ vehicle, n = 4 in each group. Fluorescent intensity of gelatinase activity was measured with imagej program. Scale bar, 50 μm.

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We next performed gelatin zymography, a common method for examining and quantifying MMP-2/-9, with ischemic rat brains. Supernatants of human fibrosarcoma HT-1080 cells were used as gelatinase standards for MMP-2 and -9 (Fig. S1). Zymography showed increased expression of MMP-2 and -9 in nuclear extracts from ischemic rat brains compared with sham-operated controls and non-ischemic brains (p < 0.001 and 0.01, respectively) (Fig. 3a and b). A statistically significant increase in active bands of MMP-2 (aMMP; p < 0.05) was seen in the ischemic hemisphere. We used an inhibitor of MMP activity, BB1101, to determine the role of intranuclear MMP activity on the nuclear matrix proteolysis. Treatment with BB1101 significantly reduced proMMP-2 and MMP-9 (pMMP-2 and -9) in the ischemic brains compared with ischemic vehicle-treated control (p < 0.05) (Fig. 3a and b).

image

Figure 3.  ProMMP-9, MMP-2, and active MMP-2 (pMMP-9, pMMP-2, and aMMP-2) in nuclear preparations of rat brains at 3 h post-ischemic reperfusion with and without BB1101 treatment. (a) Gelatin zymography analysis was performed in nuclear preparations of rat brains. Human HT-1080 was used as a standard for MMP-2 and -9; pMMP-9, MMP-2 and aMMP-2 were detected in MCAO brains. (b) Relative intensity of pMMP-9 (95 kDa), pMMP-2 (68 kDa) and aMMP-2 (62 kDa). 3 h R, ischemic hemispheres; 3 h L, non-ischemic hemispheres; BB1101 R, ischemic hemisphere with BB1101 treatment; BB1101 L, non-ischemic hemisphere with BB1101 treatment; Sham, sham-operated brains. Sham group n = 4, 3 h group n = 8, BB1101 group n = 5; pMMP-9 level: ***p < 0.001 and *p < 0.05 versus 3 h L, BB1101 L, and sham, respectively; #p < 0.05 versus 3 h R; pMMP-2 level: **p < 0.01 versus 3 h L, BB1101 L, and sham; *p < 0.05 versus sham; #p < 0.05 versus 3 h R; aMMP-2 level: *p < 0.05 versus 3 h L, BB1101 L, and sham.

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Zymography revealed aMMP-2 bands in the nucleus. To confirm the nuclear presence of proteolytic activity of MMP-2, we analyzed the expression of membrane type 1-MMP (MT1-MMP), a major activator of MMP-2, in ischemic rat brains. With immunohistochemistry of MT1-MMP and ISZ, co-localization of MT1-MMP and gelatinolytic activity was seen in the ischemic cells (Fig. 4a). Z-stack and 3D confocal images showed that the expression of MT1-MMP was detected in both cellular membrane and nucleus (Fig. 4a and b), with greater MT1-MMP staining seen in the nuclei. The nuclear presence of MT1-MMP was confirmed by western blot analysis in nuclear extracts (Fig. 4c). We also detected the expression and location of furin, an activator of MT1-MMP, using immunohistochemistry and confocal microscopy (Fig. 4d). The co-localization of MT1-MMP and furin was seen in ischemic nuclei.

image

Figure 4.  Expression and location of MT1-MMP and furin in nuclei at 3 h post-ischemic reperfusion. DAPI shows nucleus localization. (a) Colocalization of MT1-MMP immunohistochemistry with in situ zymography (ISZ) in ischemic (top panel) and non-ischemic (bottom panel) brain sections. Scale bars, 5 μm. (b) Z-stack confocal analysis for the co-localization of MT1-MMP and gelatinase activity in nucleus (i). (ii) Image from top panel corresponding to z-axis consecutive images (16 sections). In all sections MT1-MMP co-localizes with ISZ in the nucleus. A three-dimensional image shows the reconstruction from the stack of 16 optical sections. Scale bars, 5 μm. (c) Western blot analysis for MT1-MMP in nuclear extract preparations; *p < 0.05 versus non-ischemic hemispheres, n = 4 in each group. (d) Immunostaining for furin is co-localized with MT1-MMP (i). Scale bar, 20 μm. Z-stack confocal images show furin is co-localized with MT1-MMP in nucleus (ii). (iii) Image from top panel corresponding to z-axis consecutive images (10 sections). A three-dimensional image with a 90° rotation shows the reconstruction from the stack of 10 optical sections. Scale bars, 5 μm.

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PARP-1 expression and degradation by intranuclear MMPs in vivo and in vitro

To investigate whether the increase in nuclear MMP-2 and -9 is involved in delayed cell death, we compared MMP-2 and -9 immunohistochemistry with the spatial patterns of DNA fragmentation (TUNEL expression) in brain cells. At 24 h, we detected scattered TUNEL-positive cells in the ischemic hemispheres that co-localized with MMP-2 (Fig. 5a). By 48 h, we observed extensive TUNEL-positive cells in the entire lesion area of the ischemic hemisphere but not in the non-ischemic side. Most TUNEL-positive cells expressed MMP-2 (Fig. 5a) and co-localized with NEUronal Nuclei (NeuN) (Fig. 5c). We found MMP-9 expression is co-localized with TUNEL expression in some brain cells in the lesion area of the ischemic hemisphere at 24 and 48 h. However, compared with MMP-2, the association between the MMP-9 expression and DNA fragmentation is complicated at 48 h. We detected some TUNEL-positive cells without presence of MMP-9 expression. In contrast, some cells that expressed MMP-9 were TUNEL negative. In addition, we also detected the expression of MMP-9 in vessels in the lesion area of the ischemic hemisphere at 48 h (Fig. 5b).

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Figure 5.  Cell apoptosis and expression of MMP-2 and PARP-1. (a and b) Representative double staining of MMP-2 or MMP-9 with TUNEL in rat brain at 24 and 48 h post-ischemic reperfusion. (a) Arrows indicate few TUNEL-positive cells with the expression of MMP-2 at 24 h. Scale bar, 20 μm. At 48 h reperfusion, extensive colocalization of TUNEL-positive cells with MMP-2 expression was seen in ischemic brain section. Arrows and inset: TUNEL-positive cells show very weak DAPI staining that labels nuclei. Scale bars, 50 μm. (b) Arrows indicate TUNEL-positive cell with the expression of MMP-9 at 24 h. Scale bar, 20 μm. Expression of MMP-9 and TUNEL in ischemic and non-ischemic brain cells at 48 h reperfusion (middle and bottom panels). Scale bar, 50 μm. Arrows: TUNEL-positive cells with MMP-9 expression. Arrowheads: TUNEL-negative cells with MMP-9 expression. Stars: TUNEL-positive cells without MMP-9 expression. V: vessel. (c) Representative confocal images of double staining for TUNEL with NeuN immunoreaction reveal that most of TUNEL-positive cells are neurons in ischemic hemisphere at 48 h reperfusion. Scale bars, 50 μm. (d) Representative confocal analysis of expression of PARP-1 and ISZ in rat brain at 3 h post-ischemic reperfusion. The z-stack image shows the co-localization of PARP-1 and gelatinase activity in nucleus. A three-dimensional image with a 90° rotation shows the same cells with ISZ and PARP-1 in nuclei. Scale bars, 20 μm. (e) Representative co-localization of MMP-2 and PARP-1 in cell nuclei of rat brain at 3 h post-ischemic reperfusion. DAPI shows nucleus localization. Scale bars, 20 μm.

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We next examined the relationship of PARP-1 expression in cells to gelatinase activity using an antibody that recognizes both full-length (116 kDa) and cleaved PARP-1. Compared with the non-ischemic tissue, higher PARP-1 immunostaining was seen in the ISZ-positive cells in the ischemic tissue (Fig. 5d). This co-localization was confirmed by double staining of MMP-2 and PARP-1 (Fig. 5e).

To determine whether the increase in PARP-1 staining reflected either increased accumulation of PARP-1 or differential cleavage in the lesions, or possibly both, western blot analyses and densitometry were carried out. Nuclear extracts from ischemic rat brains with or without BB1101 treatment were used to determine the involvement of MMPs in changes of PARP-1 expression and degradation. After 3 h of reperfusion, the full-length form of PARP-1 (116 kDa) was detected in nuclear extracts from both ischemic and non-ischemic hemispheres (Fig. 6a). However, in ischemic nuclear extracts, cleavage products of PARP-1 yielded an 89 kDa band and a 43 kDa band that were significantly increased compared with the non-ischemic side (Fig. 6a). More importantly, even though not all rats showed the increased 89 kDa band, all animals had an additional 43 kDa band. To determine whether the cleavage of PARP-1 was a consequence of the early nuclear gelatinase proteolysis, we examined PARP-1 cleavage in ischemic nuclear extracts prepared from rats treated with BB1101. Treatment with BB1101 significantly reduced the degradation of PARP-1 as shown by a significant reduction in the bands migrating at 89 and 43 kDa (Fig. 6a). It is well known that PARP-1 cleavage by caspase 3 yields 89- and 24-kDa fragments during apoptosis. However, very low immunostaining of cleaved caspase 3 was detected in the cells that showed active gelatinase at 3 h reperfusion after ischemia. Increased cleaved caspase 3 was detected at 72 h reperfusion when excessive apoptosis appears in the ischemic area (Fig. 6b).

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Figure 6.  PARP-1 cleavage by MMPs in vivo and in vitro. (a) Western blot analyses for PARP-1 cleavage in nuclear preparations of rat brain at 3 h post-ischemic reperfusion. 3 h R, ischemic hemispheres; 3 h L, non-ischemic hemispheres; BB1101 R, ischemic hemisphere with BB1101 treatment; BB1101 L, non-ischemic hemisphere with BB1101 treatment; n = 5 in each group. Full-length PARP-1 was visualized as a 116 kDa band. Cleavage of PARP-1 yielded two bands at 89 and 43 kDa – 89 kDa band: *p < 0.05, 3 h R versus 3 h L and BB1101 L and #p < 0.01 BB1101 R versus 3 h R – 43 kDa band: ***p < 0.001 3 h R versus 3 h L and BB1101 L and ###p < 0.001 BB1101 R versus 3 h R. (b) Representative confocal analysis of expression of cleaved caspase 3 and ISZ in rat brain at 3 h post-ischemic reperfusion. Bottom panel shows the positive immunostanining for cleaved caspase 3 in ISZ-positive cells in ischemic brain section at 72 h reperfusion. DAPI shows nucleus localization. Scale bars, 5 μm. (c, d, e, and f) In vitro degradation of PARP-1 by recombinant rat MMP-2 (c), MMP-9 (d), gelatinase extracts prepared from ischemic nuclear extracts (e and f) ischemic nuclear extracts. (c) n = 4, **p < 0.01, PARP-1 (116 kDa) positive control versus PARP-1 incubated with MMP-2; *p < 0.05, PARP-1 incubated with MMP-2 versus BB1101 inhibition. (d) n = 4, **p < 0.01, PARP-1 (116 kDa) positive control versus PARP-1 incubated with MMP-9. (e) n = 5, **p < 0.01 PARP-1 (116 kDa) positive control versus PARP-1 incubated with gelatinase extracts; *p < 0.05, PARP-1 incubated with gelatinase extracts versus BB1101 inhibition. (f) n = 6, **p < 0.01, PARP-1 (116 kDa) positive control versus PARP-1 incubated with nuclear extracts (NE); *p < 0.05, PARP-1 (116 kDa) incubated with NE versus inhibition of MMP-2/9 inhibitor II (Inhibitor II); *p < 0.05, PARP-1 (43 kDa) positive control versus PARP-1 incubated with NE; #p < 0.05, PARP-1 (43 kDa) incubated with NE versus inhibition of MMP-2/9 inhibitor II. (g) PARP activity assay in nuclear extract preparations at 3 h post-ischemic reperfusion. Treatment with BB1101 significantly increased the PARP activity in ischemic nuclear extracts: *p < 0.05 versus 3 h R, # #p < 0.01 versus 3 h L, BB1101 L, and sham. Sham group n = 3, 3 h group n = 6, and BB1101 group n = 5.

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In in vitro studies, incubation of purified bovine PARP-1 with recombinant rat MMP-2 and -9 or with gelatinase extracts prepared from nuclear fractions of rat brain using gelatin beads also showed the loss of full-length PARP-1 (116 kDa) by western blot (Fig. 6c, d, and e). After 1 h of incubation with MMP-2 and -9, there was appearance of a cleaved product of PARP-1 around 43 kDa (Fig. 6c and d). Co-incubation with BB1101 inhibited the PARP-1 cleavage caused by both MMPs and gelatinase extracts. MMP inhibitors GM6001 and 1,10-phenanthroline were also tested and these compounds significantly inhibited PARP-1 cleavage caused by both rat MMP-2 and gelatinase extracts (data not shown). Western blot did not detect any cleaved band of PARP-1 after incubation with gelatinase extracts (Fig. 6e). This could be because of the action of other MMPs or proteases that may be present in the bead extracts. These proteases could degrade PARP-1 into fragments that are not recognized by the PARP-1 antibody.

To further demonstrate the cleavage of PARP-1 by nuclear MMPs, the pure bovine PARP-1 was incubated with total nuclear extracts from ischemic brains in assay buffer (Fig. 6f). In addition to pure PARP-1, we added total nuclear extracts alone as another control since the total nuclear extracts contain PARP-1, XRCC1, and other proteases. Immunoblotting showed a significant loss of full-length PARP-1 (116 kDa) and a significant increase in the 43-kDa cleavage product of PARP-1 after incubation with the ischemic nuclear extracts. Co-incubation with selective MMP-2/9 inhibitor II attenuated PARP-1 cleavage, suggesting the involvement of nuclear MMP-2 and -9 in this process. We observed the cleaved product of PARP-1 at 89 kDa (Fig. 6f), but treatment with MMP-2/9 inhibitor II failed to inhibit its appearance.

Poly-ADP-ribose polymerase-1 is activated by DNA strand breaks caused by oxidative stress. We measured PARP activity in nuclear extracts obtained from ischemic rat brains to determine whether the cleavage of PARP-1 by early active gelatinases may affect PARP-1 activity. There was a significant reduction in PARP-1 activity in ischemic brains compared with those treated with BB1101 (Fig. 6g).

Reduction of XRCC1 in ischemic brain by nuclear gelatinase activity

The nuclear protein XRCC1 plays a central role in the DNA BER pathway. We therefore investigated the expression of XRCC1 in ischemic rat brain with 3 h reperfusion using immunohistochemistry and western blot.

Immunohistochemistry showed the constitutive expression in the nucleus of XRCC1 in the entire region of the normal rat brain (data not shown) and it was mainly seen in the nucleus (Fig. 7a). At 3 h after reperfusion, reduction of XRCC1 was observed in the lesion areas, including the ischemic piriform cortex (PFC) when compared with the non-ischemic PFC. Treatment with BB1101 reversed the reduction of XRCC1 in the ischemic PFC. Double staining with XRCC1 and ISZ showed the spatial relationship between XRCC1 loss and nuclear gelatinase activity (Fig. 7a, inset). Using an antibody against XRCC1 that recognizes the full-length XRCC1, western blot detected two bands around 95.74 kDa in the nuclear extracts from rat brain and in the positive control XRCC1, which represent the full-length XRCC1, as well as several bands around 43 kDa (Fig. 7b). All bands of XRCC1 detected were significantly decreased in the nuclear extracts of ischemic brains compared with the non-ischemic brain. The decrease of XRCC1 in the ischemic side was significantly reversed by BB1101 treatment (Fig. 7b).

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Figure 7.  XRCC1 degradation by MMPs in vivo and in vitro. (a) XRCC1 immunostaining in ISZ-positive cells of non-ischemic, ischemic brains and ischemic brains with BB1101 treatment at 3 h post-ischemic reperfusion. The bottom panel from the inset shows less XRCC1 in the cells that expressed high gelatinase activity (arrows). DAPI shows nucleus localization. Scale bar, 50 μm. (b) Western blot analysis for degradation of XRCC1 in nuclear extract preparation. XRCC1 immunoblotting detected a band 95.74 kDa in the positive control XRCC1 (Abnova) and two bands around 96 kDa in the nuclear extracts, which represent the full-length XRCC1, as well as several bands around 43 kDa. All bands of XRCC1 detected were quantified, n = 5 in each group, *p < 0.05 versus 3 h L and BB1101 L; **p < 0.01 versus 3 h R. (c, d, e, and f) In vitro degradation of XRCC1 by recombinant rat MMP-2 (c), MMP-9 (d), gelatinase extracts prepared from ischemic nuclear extracts (e), and (f) ischemic nuclear extracts. All bands of XRCC1 detected were quantified unless indicated. (c) n = 4 in each group, **p < 0.01 versus positive control; *p < 0.05 versus XRCC1 incubated with MMP-2. (d) n = 5 in each group, *p < 0.05 versus XRCC1 positive control. (e) n = 5 in each group, ***p < 0.001, versus XRCC1 positive control; ##p < 0.02 versus XRCC1 positive control; ###p < 0.001 versus XRCC1 incubated with gelatinase extracts. (f) n = 4 in each group, ***p < 0.001 versus XRCC1 positive control; ##p < 0.01 versus XRCC1 positive control; #p < 0.05 versus XRCC1 incubated with nuclear extracts (NE). Inhibitor II, MMP-2/9 inhibitor II.

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These results from both immunohistochemistry and western blot strongly suggest that the early nuclear gelatinase proteolysis is involved in the early reduction of XRCC1 induced by transient focal cerebral ischemia. To provide additional evidence in support of this conclusion, we next performed a study of XRCC1 degradation in vitro by recombinant rat MMP-2, murine MMP-9 or with gelatinase extracts prepared from nuclear fractions of rat brain using gelatin beads. Western blots showed that incubation of recombinant XRCC1 with synthetic rat MMP-2 or with gelatinase extracts significantly reduced the level of XRCC1 protein (Fig. 7c and e). Co-incubation with BB1101 inhibited the XRCC1 cleavage caused by both MMP-2 and gelatinase extracts.

Matrix metalloproteinase-9 produced a significant reduction in full-length XRCC1 (96 kDa) as well as in the bands migrating in the 43-55 kDa range (Fig. 7d). Compared with MMP-2 or gelatinase extracts, MMP-9-mediated cleavage of XRCC1 was unique in that the lowest size XRCC1 band was increased compared with the positive control and BB1101-treated samples. MMP inhibitors GM6001 and 1,10-phenanthroline were also tested and these compounds significantly inhibited XRCC1 cleavage caused by both human MMP-2/9 and gelatinase extracts (data not shown).

Similarly to PARP-1, incubation of XRCC1 with total nuclear extracts produced a significant degradation of all XRCC1 bands. This was significantly inhibited by the selective MMP-2/-9 inhibitor II (Fig. 7f). This strongly suggests that gelatinases present in the nuclear extracts of the ischemic brain are involved in the degradation of XRCC1.

Nuclear gelatinase activity enhances the induction of oxidative DNA damage

Based on the observation that the early nuclear gelatinase activity was involved in the proteolysis of PARP-1 and XRCC1, which mediate repair of oxidized DNA, we next measured the formation of AP sites, one of the prevalent lesions of oxidative DNA damage, and 8-OHdG, a ubiquitous marker of oxidative stress, in ischemic rat brains at 3 h after reperfusion.

An antibody against 8-OHdG was used to detect whether there was an induction of oxidative DNA damage at 3 h after reperfusion. Double staining with 8-OHdG and ISZ showed that a strong signal of 8-OHdG was seen in nuclei of the cells that expressed active gelatinases (Fig. 8a).

image

Figure 8.  Analysis of oxidative DNA lesion in rat brain at 3 h post-ischemic reperfusion. (a) Representative confocal analysis of 8-OHdG (red) induction in ISZ-positive cells of ischemic brain section. Scale bar, 10 μm. (b) Induction of AP sites in nuclear DNA of ischemic brain cells. 3 h R, ischemic hemispheres; 3 h L, non-ischemic hemispheres; BB1101 R, ischemic hemisphere with BB1101 treatment; ***p < 0.001 and *p < 0.05 versus sham R and L, 3 h L, and BB1101 L, respectively; ##p < 0.01 versus 3 h R. Sham group n = 4, 3 h and BB1101 groups n = 5. (c) Induction of 8-OHdG in nuclear DNA of ischemic brain cells. The level of 8-OHdG is presented as ratio of ischemic/non-ischemic 8-OHdG levels. **p < 0.01 versus sham; *p < 0.05 3 h post-ischemic reperfusion. Sham n = 4, 3 h group n = 7, BB1101 group n = 5. (d) Representative immunohistochemistry for 8-OHdG induction (brown) in the treated and non-treated piriform cortex in rat brain at 48 h post-ischemic reperfusion. Scale bar, 50 μm.

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Figure 8b and c illustrate the profiles of AP sites and 8-OHdG induction, respectively, in the ischemic rat brains at 3 h after reperfusion. Both AP sites and 8-OHdG were significantly increased in the ischemic brains compared with the sham-operated animal and non-ischemic side. In contrast, in BB1101-treated animals the number of AP sites was significantly decreased compared with ischemic animals receiving the vehicle. We did not find a significant difference of 8-OHdG between the ischemic animals and BB1101-treated animals using absolute levels of 8-OHdG in each group. However, the 8-OHdG ratio of ischemic/non-ischemic showed BB1101 treatment significantly decreased the 8-OHdG levels when compared with ischemic control. 3,3′-diaminobenzidine (DAB) immunohistochemical staining showed fewer 8-OHdG-positive cells in areas of piriform cortex in BB1101-treated animals (Fig. 8d). These results suggested that the early nuclear gelatinase activity enhanced the nuclear accumulation of oxidative DNA damage in the ischemic rat brain with reperfusion injury.

Discussion

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

We demonstrated an increase in intranuclear gelatinase activation in neurons in rat brain at 3 h of post-ischemic reperfusion. This increase of nuclear gelatinolytic activity is also observed in human brain after stroke. We showed that both MMP-2 and -9 contribute to the early nuclear gelatinase activity, which produced cleavage of PARP-1 in ischemic rat brain leading to a decrease of PARP-1 activity. Moreover, we demonstrated that the nuclear gelatinase proteolysis is involved in the early reduction of XRCC1 induced by the ischemic insult. Finally, we report that the early oxidative DNA damage caused by transient focal cerebral ischemia was attenuated in animals treated with the MMP inhibitor, BB1101. Our results provide the first experimental evidence that early intranuclear aMMPs promote neuronal accumulation of oxidative DNA after an ischemic injury by cleaving PARP-1 and BER enzymes, such as XRCC1, reducing their ability to mediate DNA repair.

Although best known for their role in the proteolysis of extracellular protein targets, recent studies have revealed that MMPs are also localized to various intracellular sites including nucleus (Si-Tayeb et al. 2006; Schulz 2007; Ali and Schulz 2009). Nuclear gelatinolytic activity in transient focal ischemic models has been previously observed (Gasche et al. 2001; Yang et al. 2007; Amantea et al. 2008). Up-regulation of aMMP-13 in the nuclei of both rat and human ischemic brains was recently reported and the intranuclear MMP-13 activity is detectable as early as 30 min after the occlusion onset (Cuadrado et al. 2009). Our present ISZ results confirmed the early activation of gelatinases in neuronal nuclei in rat, as well as in human brain cells after an ischemic insult.

In experimental focal cerebral ischemia, MMP-2 increases initially followed by a later increase in MMP-9 expression (Rosenberg et al. 1996; Heo et al. 1999; Chang et al. 2003; Yang et al. 2007). In the present study, gelatin zymography revealed increases in pMMP-9 and aMMP-2 in the nucleus; pMMP-2 is activated by a molecular cascade that involves a trimolecular complex made up of MMP-2, tissue inhibitor of metalloproteinase-2 (TIMP-2), and MT1-MMP (Ratnikov et al. 2002). We observed that MT1-MMP was induced not only along the cytosolic membrane but also in the nuclei of ischemic brain cells, which was co-localized with active gelatinases, as well as with furin, an activator of MT1-MMP (McMahon et al. 2005). Immediate up-regulation of the activation system for pMMP-2 has been demonstrated in a focal cerebral ischemic model (Chang et al. 2003). This atypical location of MT1-MMP that co-localized with MMP-2 in the nucleus is also observed in hepatocellular carcinoma (Ip et al. 2007). The finding that the enzymes involved in the activation of MMP-2 are present in the nucleus along with MMP-2 supports the presence of nuclear MMP-2 activity.

Matrix metalloproteinases cleave most components of the extracellular matrix including fibronectin, laminin, proteoglycans, type IV collagen, and tight junction proteins (Sternlicht and Werb 2001; Rosenberg 2002; Yang et al. 2007). The intranuclear location of MMP activity after stroke suggests a novel role in nuclear matrix proteolysis. Proteolysis of nuclear matrix is implicated in numerous processes such as apoptosis, cell cycle, and DNA fragmentation. Oxidative stress generated during stroke is a critical event leading to BBB disruption and apoptosis. The BER pathway is a critical mechanism for repair of oxidative DNA lesions in the brain after ischemia (Fujimura et al. 1999; Dutra et al. 2006; Li et al. 2007). The active PARP-1 triggers the recruitment of XRCC1 (Schreiber et al. 2002). During BER and single-strand break repair, a functional XRCC1 is critical for the accurate repair of damaged bases, abasic (AP) sites (Campalans et al. 2005; Nazarkina et al. 2007). XRCC1 is involved in all the steps of the repair of oxidized bases by interacting with the DNA repair enzymes (Marsin et al. 2003; Wiederhold et al. 2004). Oxidative DNA damage as shown by AP sites and 8-OHdG, and BER reduction occurred as early as 15–30 min after the onset of reperfusion in a 2 h MCAO model (Lan et al. 2003; Luo et al. 2007). Strong 8-OHdG was observed 3–6 h after reperfusion (Ohtaki et al. 2007). Considering the compatible timing of when the oxidative DNA lesion and BER reduction occurred and when the intranuclear aMMPs were first seen after ischemia and reperfusion, we reasoned that early intranuclear MMPs may cleave nuclear enzymes like PARP-1 and XRCC1 and interfere with oxidized DNA repair, which could promote apoptosis after an ischemic injury.

We found MMP-2 associated with TUNEL-positive neurons, suggesting a possible involvement of MMP-2 in neuronal apoptosis. In vivo and in vitro studies showed that nuclear MMP activity cleaves PARP-1, reducing PARP-1 activity in ischemic cell nuclei of rat brain. We used the MMP inhibitors to confirm the role of MMPs on the nuclear matrix cleavage. Both the PARP-1 cleavage (in vivo and in vitro) and the reduction of PARP-1 activity could be reversed by MMP inhibitor treatment. The PARP-1 cleavage by nuclear MMP-2 produced a 43 kDa band, which was also seen in a previous in vitro study of PARP-1 degradation by purified human MMP-2 (Kwan et al. 2004). We detected very low level of active caspase 3 in cells with gelatinase activity, suggesting that PARP-1 cleavage was mainly because of MMPs. In addition to PARP-1 cleavage, we found a significant reduction of XRCC1 in the nuclear extracts from ischemic rat brain at 3 h reperfusion, which was reversed by MMP inhibitors in vivo and in vitro.

We also detected an increase in MMP-9 in the ischemic nuclei. In vitro, 4-aminophenyl mercuric acetate (APMA)-activated MMP-9 acts similarly to MMP-2 in cleavage of PARP-1 and XRCC1, and it could have contributed to nuclear proteolysis. The co-localization of MMP-9 in TUNEL-positive cells in ischemic brain suggested the involvement in cell apoptosis. However, the association of MMP-9 expression and DNA fragmentation in ischemic brain cells is different from the pattern of MMP-2 involvement in the DNA fragmentation at 48 h reperfusion. We detected some TUNEL-positive cells without presence of MMP-9 expression and some cells that express MMP-9 were TUNEL negative. It seems that there is a delayed involvement of MMP-9 in cell death compared with MMP-2 after ischemia. It has been reported that MMP-9 co-localized with neuronal nitric oxide synthase in ischemic brain. During oxidative stress, MMP-9 is activated by peroxynitrite-induced S-nitrosylation. This may trigger proteolytic cascades to disrupt the extracellular matrix leading to apoptotic neuronal death (Okamoto et al. 2001; Gu et al. 2002). On the other hand, studies in myocardial ischemia–reperfusion injury showed intracellular location of MMP-2 that was rapidly activated by peroxynitrite and that MMP-2 may rapidly act on intracellular substrates on a minute timescale (Schulz 2007; Ali and Schulz 2009). These suggest a possibility that MMP-2 and -9 induce neuronal death via different pathways.

We demonstrated that the oxidative DNA damage as assessed by 8-OHdG levels and AP sites was markedly induced in the brain cells during the early stage of post-ischemic reperfusion. More importantly, we found that treatment with BB1101 efficiently reduced ischemia-induced oxidative DNA damage, suggesting that more oxidized DNA was repaired. To our knowledge, this is the first report of MMP proteolytic activity involvement in brain cell oxidative DNA damage in an ischemia model. Our results indicated that inhibition of the early intranuclear MMP activity could reduce neuronal DNA fragmentation and death at a later stage after ischemic insult.

Poly-ADP-ribose polymerase-1 is a repair enzyme and is involved in maintenance of nuclear homeostasis, cell survival and death. However, over-activation of PARP-1 caused by massive DNA damage may result in cell necrosis by ATP depletion, and DNA fragmentation and cell apoptosis induced by translocation of apoptosis-inducing factor (Fig. 9) (Yu et al. 2002; Kauppinen and Swanson 2007; Pacher and Szabo 2008). It has been shown that inhibition of PARP activity is neuroprotective in brain ischemia (Skaper 2003; Chiarugi 2005a,b; Tanaka et al. 2005). On the other hand, PARP-1 could act as a survival factor through its capacity to efficiently repair damaged DNA by binding to DNA and interacting with BER factors including XRCC1 (Hassa and Hottiger 2008). Activation of PARP-1 by mild genotoxic stimuli may facilitate DNA repair and cell survival. Inhibition of PARP-1 enhanced the vulnerability of neurons to apoptosis. Thus, in mild progressive damage that occurs in neurodegenerative diseases, PARP-1 activation plays a neuroprotective role and may contribute to cellular recovery following sublethal transient global ischemia (Nagayama et al. 2000; Diaz-Hernandez et al. 2007). Based on these reports and our present results, we propose that PARP-1 activity might be beneficial for neuronal survival at an early stage during ischemia/reperfusion injury when tissue has not yet become excessively damaged and PARP-1 has not yet become over-activated.

image

Figure 9.  Schematic drawing of hypothesis on how intranuclear MMPs facilitate the oxidative DNA damage in neurons after ischemic insult.

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We showed that ischemia–reperfusion triggers oxidative DNA damage, which leads to PARP-1 activation, initiating the DNA/BER mechanism for repair of oxidized DNA. When repair is successful, the neuron may be protected from apoptosis. However, excessive nuclear gelatinase proteolysis in neurons impairs the protective action of the DNA/BER pathway by cleaving PARP-1 and XRCC1, leading to neuronal DNA fragmentation and apoptosis (Fig. 9). Our results indicate that early increase in MMP activity in ischemia contributes to oxidative DNA damage in neurons, which can be reduced by MMP inhibitors. As MMPs are important in angiogenesis and neurogenesis, the beneficial effects of MMP inhibitors in the early stages of an injury need to be balanced with the later interference with recovery (Lee et al. 2006; Zhao et al. 2006). There are some limitations in this study. Based on our data, it is not possible to discern the specific role of PARP-1 in early ischemic neuronal death. PARP-1 could act as a beneficial factor or have detrimental effects in cell survival. Further experiments will be needed to provide direct evidence of whether PARP-1 has a beneficial or a detrimental role in early neuronal death in ischemia, and how MMPs could affect this balance.

In summary, the present study demonstrated a novel role for MMPs in nuclear DNA damage. We found increased MMP-2 and -9-mediated proteolysis in nuclei of neurons during the early stage of post-ischemic reperfusion both in human stroke and in an animal model. We propose that gelatinase proteolysis in the nucleus plays a role in oxidative DNA damage by cleaving nuclear matrix proteins, PARP-1 and XRCC1, reducing their ability to repair DNA damage caused by oxidative stress after stroke. More importantly, an inhibitor to MMPs protected neurons from the gelatinase-mediated oxidative DNA damage after stroke.

Acknowledgements

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

This work was supported by an American Heart Association beginning grant-in aid (0765473Z), a COBRE pilot grant and a RAC grant from University of New Mexico to YY, and by a National Institutes of Health grant (5RO1 NS04547) to GAR. Confocal images in this paper were generated in the University of New Mexico Cancer Center Fluorescence Microscopy Facility: http://www.hsc.unm.edu/crtc/microscopy.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

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

Appendix S1 Materials and methods.

Fig.  S1   The supernatant of HT-1080 human fibrosarcoma cell line contains MMP-9 and MMP-2 (pro and active forms). (a) Gelatin zymography reveals the proMMP-9 (92 kDa, pMMP-9) (Okada et  al. 1992), proMMP-2 (68 kDa, pMMP-2) and two active forms of MMP-2 (64 and 62 kDa, intermediate and aMMP-2) in HT-1080. The 64-kDa form is called activation intermediate of MMP-2, while the 62 kDa form is called fully active mature MMP-2 (Ratnikov et  al. 2002). (b) Gelatin zymography showing MMP-9 and MMP-2 in gelatinase extracts prepared from ischemic rat brain tissue. The MMP-9 comprises two bands at 94 kDa (glycosylated form) and 88 kDa (intermediate form) (Zhang et  al. 1998). The proMMP-2 at 68 kDa and active MMP-2 at 62 kDa were detected.

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