Mechanisms of Cytosolic Targeting of Matrix Metalloproteinase-2 MOHAMMAD A.M. ALI, 1 AVA K. CHOW, 2 ARULMOZHI D. KANDASAMY, 1 XIAOHU FAN, 2 LORI J. WEST, 2 BRYAN D. CRAWFORD, 3 THOMAS SIMMEN, 4 * AND RICHARD SCHULZ 1,2 ** 1 Department of Pharmacology, Cardiovascular Research Centre, Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta, Canada 2 Department of Pediatrics, Cardiovascular Research Centre, Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta, Canada 3 Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada 4 Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada Matrix metalloproteinase-2 (MMP-2) is best understood for its biological actions outside the cell. However, MMP-2 also localizes to intracellular compartments and the cytosol where it has several substrates, including troponin I (TnI). Despite a growing list of cytosolic substrates, we currently do not know the mechanism(s) that give rise to the equilibrium between intracellular and secreted MMP-2 moieties. Therefore, we explored how cells achieve the unique distribution of this protease. Our data show that endogenous MMP-2 targets inefficiently to the endoplasmic reticulum (ER) and shows significant amounts in the cytosol. Transfection of canonical MMP-2 essentially reproduces this targeting pattern, suggesting it is the quality of the MMP-2 signal sequence that predominantly determines MMP-2 targeting. However, we also found that human cardiomyocytes express an MMP-2 splice variant which entirely lacks the signal sequence. Like the fraction of ER-excluded, full-length MMP-2, this variant MMP-2 is restricted to the cytosol and specifically enhances TnI cleavage upon hypoxia-reoxygenation injury in cardiomyocytes. Together, our findings describe for the first time a set of mechanisms that cells utilize to equilibrate MMP-2 both in the extracellular milieu and intracellular, cytosolic locations. Our results also suggest approaches to specifically investigate the overlooked intracellular biology of MMP-2. J. Cell. Physiol. 227: 3397–3404, 2012. ß 2011 Wiley Periodicals, Inc. Matrix metalloproteinase-2 (MMP-2) is widely considered to be a secreted, zinc-dependent protease that primarily targets extracellular matrix proteins. However, an increasing number of studies shows that MMP-2 can also cleave cytosolic substrates and thereby modulate diverse cell functions including cardiomyocyte contractility, proliferation, apoptosis, and transcription (Schulz, 2007; Butler and Overall, 2009; Cauwe and Opdenakker, 2010). For example, upon oxidative stress injury in the heart, MMP-2 proteolyzes cytosolic targets including troponin I (TnI) (Wang et al., 2002; Bergman et al., 2007), a-actinin (Sung et al., 2007) and titin (Ali et al., 2010). Cleavage of these and other substrates by MMP-2 results in acute myocardial contractile dysfunction, underscoring the importance of a portion of MMP-2 that is not entering the secretory pathway. The cytosolic enzymatic activity of MMP-2 is consistent with multiple demonstrations that MMP-2 itself is localized to the cardiac sarcomere (Wang et al., 2002; Bergman et al., 2007). Moreover, MMP-2 activity has been detected in mitochondria (Wang et al., 2002) and nuclei (Kwan et al., 2004). Evidence for all of these localizations has been provided by immunogold electron microscopy, confocal microscopy, immunoprecip- itation experiments and zymographic analysis (see (Schulz, 2007) for review). Surprisingly however, to date no biological mechanism has been described that could account for this unusual intracellular and extracellular distribution. Yet, MMP-2 is a known substrate for cytosol-associated post-translational modifications including phosphorylation (Sariahmetoglu et al., 2007) and glutathiolation (Viappiani et al., 2009), which alter its enzymatic activity. Together with the ever-increasing number of its known intracellular substrates, these posttranslational modifications suggest that a significant portion of MMP-2 fails to enter the secretory pathway and instead remains in the cytosol. MMP-2 possesses a signal sequence which allows its entry into the endoplasmic reticulum (ER) lumen for secretion (Massova et al., 1998). Here, we examined whether the presence and quality of the MMP-2 signal sequence could account for these intracellular moieties of MMP-2 that are Abbreviations: ER, endoplasmic reticulum; HA, hemagglutinin; MMP, matrix metalloproteinase; SS, signal sequence; TnI, troponin I. Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: CIHR; Contract grant number: MOP-66953. Contract grant sponsor: NSERC; Contract grant number: 386757. Contract grant sponsor: CCSRI; Contract grant number: 700306. *Correspondence to: Thomas Simmen, Department of Cell Biology, 5-65 MSB, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. E-mail: thomas.simmen@ualberta.ca **Correspondence to: Richard Schulz, Cardiovascular Research Centre, 4-62 HMRC, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. E-mail: richard.schulz@ualberta.ca Manuscript Received: 21 July 2011 Manuscript Accepted: 19 December 2011 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 29 December 2011. DOI: 10.1002/jcp.24040 ORIGINAL RESEARCH ARTICLE 3397 Journal of Journal of Cellular Physiology Cellular Physiology ß 2011 WILEY PERIODICALS, INC.