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.
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- Materials and methods
- 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.
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.
- Top of page
- Materials and methods
- 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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