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Matrix metalloproteinase-2 (MMP-2) is present in the nucleus of cardiac myocytes and is capable of cleaving poly (ADP-ribose) polymerase (PARP) in vitro¹

JENNIFER A. KWAN^*, COSTAS J. SCHULZE, WENJIE WANG, HERNANDO LEON^*, MELTEM SARIAHMETOGLU, MIRANDA SUNG, JOLANTA SAWICKA, DAVID E. SIMS, GRZEGORZ SAWICKI and RICHARD SCHULZ^*,,2

^* Departments of Pediatrics and
Pharmacology, Cardiovascular Research Group, University of Alberta, Edmonton, Alberta, Canada; and
Department of Anatomy and Physiology, Atlantic Veterinary College, University of Prince Edward Island, Prince Edward Island, Canada

Correspondence: Departments of Pediatrics and Pharmacology, 462 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. E-mail: richard.schulz{at}ualberta.ca

SPECIFIC AIMS

Matrix metalloproteinase-2 (MMP-2) is a zinc-dependent endopeptidase best known to be involved in remodeling extracellular matrix, yet recent evidence suggests novel non-matrix as well as intracellular substrates for this protease. Here we characterize for the first time the presence of nuclear MMP-2 activity and hypothesize that its role may include degradation of the nuclear matrix protein poly (ADP-ribose) polymerase (PARP).

PRINCIPAL FINDINGS

1. MMP-2 in the nucleus
Subcellular localization of MMP-2 was determined using immunogold electron microscopy and two different MMP-2 antibodies. There was a significant presence of MMP-2 within the nuclei with a relatively homogeneous distribution pattern and a degree of association with condensed chromatin. Negative control using an unrelated IgG showed no intracellular staining.

Both human heart and rat liver nuclear extracts were found by Western blot analysis to be uncontaminated by plasma membrane, mitochondrial, and cytosolic fractions as no positive staining was found using antibodies against sodium/potassium ATPase, muscle carnitine palmitoyltransferase-1, or soluble guanylate cyclase, respectively. These nuclear extracts did display gelatinolytic activities corresponding to both MMP-2 and MMP-9 (Fig. 1 a) with greater overall gelatinolytic activities seen in the human heart nuclear extract. Both extracts showed MMP-2 activity predominantly in 72 kDa form with a lesser amount of activity in 64 kDa form. Western blots of the 64 kDa form of MMP-2 confirmed its presence in both extracts (Fig. 1b ). In accordance with zymographic results, there was less MMP-2 present in rat liver nuclear extract than that of the human heart. In addition, human heart nuclear extract displayed an additional unknown gelatinolytic activity at a higher molecular weight of 130 kDa (Fig. 1a ). Immunoprecipitation of human heart nuclear extract with anti PARP antibody showed 72 kDa gelatinolytic activity; there was no activity in samples immunoprecipitated with control mouse IgG₁ (Fig. 1c ).

View larger version (42K): [in this window] [in a new window]   Figure 1. Gelatinase activities and MMP-2 expression in nuclear extracts. A) Both the human heart and rat liver nuclear extracts (10 µg protein/lane) show gelatinase activities corresponding to both MMP-2 and MMP-9, as seen by gelatin zymography (n=5). std indicates standard which is culture media from HT 1080 cells which possesses both MMP-2 and MMP-9 activities. Although both extracts express activity, rat liver extract does so to a lesser extent. MMP-2 also appears most predominantly in the 72 kDa form, with a lesser amount of 64 kDa form present. B) MMP-2 (64 kDa form) is present in both extracts as seen by Western blotting. Blots are representative of n = 4 extracts each. C) Gelatin zymography analysis of human heart nuclear extracts immunoprecipitated with either anti PARP antibody or IgG1.

In the presence of synthetic MMP inhibitors doxycycline (Doxy, 100 µM) or phenanthroline (Phen, 100 µM), all gelatinolytic activities in both extracts were inhibited. Upon densitometric analysis, doxycycline, the less potent MMP inhibitor, caused partial inhibition of gelatinolytic activities of human heart nuclear extract whereas phenanthroline nearly abolished all gelatinolytic activities.

2. MMP-2 can degrade PARP
Comparison of the amino acid sequence of bovine PARP with various MMP-2 cleavage recognition sequences revealed three sites of interest. Sites centered around amino acids 308 and 385 showed a 62.5% homology with sequences recognized by MMP-2. The site at amino acid 500 showed a 75% homology to MMP-2 cleavage recognition sites.

Incubation of purified bovine PARP with human MMP-2 for either 20 min or 2 h at 37°C showed a loss of the 116 kDa PARP band (Fig. 2 a). Following 2 h of incubation, there was also an appearance of a 66 kDa degradation product by silver staining as well as a <48 kDa band as shown by Western blotting (Fig. 2b ).

View larger version (46K): [in this window] [in a new window]   Figure 2. MMP-2 degrades PARP in vitro by matrix metalloproteinase activity. Analysis of the reaction between MMP-2 and PARP. A) Left: silver stained SDS-PAGE gel depicts loss of the 116 kDa PARP band and appearance of a novel 66 kDa degradation product following incubation with 30 ng MMP-2 for 2 h at 37°C. The degradation of PARP by MMP-2 was inhibited by TIMP-2 (0.5 µM) or doxycycline (Doxy, 100 µM). Right: quantitative analysis of the silver stained gels from n = 3 experiments. B) Left: Western blots for PARP reveal loss of PARP as well as appearance of a <48 kDa product. Either TIMP-2 or doxycycline prevent appearance of the <48 kDa degradation band in addition to the loss of 116 kDa band caused by MMP-2. Right: quantitative analysis of the Western blots from n = 3 experiments, *P < 0.05 compared with PARP alone, P < 0.05 compared with PARP + MMP-2.

The ability of MMP inhibitors to prevent PARP degradation by MMP-2 was also tested in the 2 h incubation protocol. Both silver stain analysis (Fig. 2a ) and Western blotting (Fig. 2b ) showed that either TIMP-2 (0.5 µM) or doxycycline (100 µM) could inhibit the loss of the 116 kDa PARP band, as well as the appearance of both the 66 kDa and <48 kDa degradation bands.

CONCLUSIONS AND SIGNIFICANCE

In this study we have shown for the first time that MMP-2 is present within purified heart and liver nuclear extracts as well as the nuclei of cardiac myocytes. As both MMP-2 and PARP are activated by pro-oxidants species such as peroxynitrite, we hypothesized that MMP-2 may be responsible for cleavage of PARP (Fig. 3 ). Indeed PARP was cleaved to novel degradation products by MMP-2.

View larger version (25K): [in this window] [in a new window]   Figure 3. Hypothetical role of MMP-2 in PARP degradation. Oxidative stress injury in cells including cardiac myocytes enhances generation of pro-oxidant species peroxynitrite (ONOO–). ONOO– causes DNA strand breaks which prompt activation of PARP, an ATP-dependent DNA repair enzyme. Excessive activation of PARP can lead to energy depletion of the cell. In addition, ONOO– can activate MMP-2 directly. MMP-2 degradation of PARP could either be detrimental as it would hinder DNA strand break repair, or beneficial in that excess PARP would be removed, sparing the cell from ATP depletion.

Both immunogold electron microscopy of rat heart ventricles and biochemical assays on human heart and rat liver nuclear extracts showed MMP-2 within the nucleus. Gelatinase activities corresponding to both MMP-2 and MMP-9 appeared in both nuclear extracts with the predominant forms being the higher molecular weight ones. We verified that these activities were indeed MMPs, as either doxycycline, a tetracycline antibiotic that inhibits MMPs independent of its antibiotic activity, or phenanthroline, a more potent MMP inhibitor, were able to attenuate gelatinase activities.

Analysis of bovine PARP amino acid sequence reveals three sites with high homology to known MMP-2 cleavage recognition sequences. Our in vitro experiments with MMP-2 and PARP show degradation products corresponding to those theoretically predicted. These products appear in a concentration-dependent manner along with a corresponding loss of the 116 kDa PARP mother band.

We also demonstrated that cleavage of PARP to these novel degradation products is due to MMP-2 activity by testing both TIMP-2 (an endogenous MMP inhibitor) or doxycycline (a synthetic inhibitor). The appearance of both degradation products, as well as the loss of the 116 kDa PARP mother band were significantly diminished in presence of either inhibitor.

The incubation time chosen for in vitro PARP degradation assay should also be considered. MMP-2 activation and release from the heart was shown to occur within 10–15 min after peroxynitrite infusion into isolated rat hearts. That significant degradation of PARP by MMP-2 occurred within 20 min is reasonable when compared with other investigations of novel proteolytic actions of this enzyme. For example, MMP-2 is capable of cleaving calcitonin gene-related peptide after 3 h in vitro incubation. Cleavage of big endothelin-1 to a novel medium endothelin by MMP-2 was shown after 6 h and MMP-2 degraded myosin heavy chain after 24 h, whereas degradation of troponin I by MMP-2 is rapid and occurs within 20 min.

In this study we identified MMP-2 in the nucleus by both immunohistochemical and biochemical methods. We have shown that MMP-2 cleaves PARP in vitro to novel degradation products. This cleavage would inactivate PARP in a manner similar to caspase-3, playing a protective role when PARP is overactivated, or a detrimental one in preventing it from repairing DNA strand breaks. Traditionally known for their role in remodeling the extracellular matrix, recent studies have discovered a number of novel substrates and thus new biological roles for matrix metalloproteinases. Although we have not found in vivo evidence of an express role for MMP-2 in the nucleus, these results suggest further investigations on the possible role of MMPs in nuclear processes.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1202fje; doi: 10.1096/fj.02-1202fje

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This Article Full Text (PDF) All Versions of this Article: 18/6/690 02-1202fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by KWAN, J. A. Articles by SCHULZ, R. Search for Related Content PubMed PubMed Citation Articles by KWAN, J. A. Articles by SCHULZ, R.