Regulation of ATPase Activity of Transglutaminase 2 by MT1-MMP: Implications for Mineralization of MC3T3-E1 Osteoblast Cultures YUKIKO NAKANO, 1 JENNIFER FORSPRECHER, 1 AND MARI T. KAARTINEN 1,2 * 1 Division of Biomedical Sciences, Faculty of Dentistry, McGill University, Montreal, Quebec, Canada 2 Division of Experimental Medicine, Department of Medicine, Faculty of Medicine, McGill University, Montreal, Quebec, Canada A pro-mineralization function for transglutaminase 2 (TG2) has been suggested in numerous studies related to bone, cartilage, and vascular calcification. TG2 is an enzyme which can perform protein crosslinking functions, or act as a GTPase/ATPase depending upon different stimuli. We have previously demonstrated that TG2 can act as an ATPase in a Ca 2þ -rich environment and that it can regulate phosphate levels in osteoblast cultures. In this study, we investigate the role MT1-MMP in regulating the ATPase activity of TG2. We report that proteolytic cleavage of TG2 by MT1-MMP in vitro results in nearly a 3-fold increase in the ATPase activity of TG2 with a concomitant reduction in its protein-crosslinking activity. We show that MC3T3-E1 osteoblasts secreted full-length TG2 and major smaller fragments of 66 and 56 kDa, the latter having ATP-binding abilities. MT1-MMP inhibition by a neutralizing antibody suppressed mineralization of osteoblast cultures to 35% of control, and significantly reduced phosphate levels in conditioned medium (CM). Furthermore, MT1-MMP inhibition abolished two of TG2 fragments in the cultures, one of which, the 56-kDa fragment, has ATPase activity. Neutralization of MT1-MMP at early phases of mineralization significantly reduced mineral deposition, but had no effect in later phases implying MT1-MMP and TG2 might contribute to the initiation of mineralization. The cleavage of TG2 by MT1-MMP likely occurs on the cell surface/pericellular matrix where MT1-MMP and TG2 were co-localized. Based on these data, we propose that MT1-MMP modulates the extracellular function TG2 as part of a regulatory mechanism activates the pro-mineralization function of TG2. J. Cell. Physiol. 223: 260–269, 2010. ß 2010 Wiley-Liss, Inc. Normal physiologic mineralization and the formation of hydroxyapatite [Ca 10 (PO 4 ) 6 (OH) 2 ] (HA) is restricted to bone, cartilage, and tooth indicating that mineralization is tightly regulated by the cellular and extracellular components of these tissues (Bonucci, 1967; Matsuzawa and Anderson, 1971; McKee et al., 2005; Addison et al., 2007). Extracellular matrix (ECM) components in mineralized tissues, such as collagen type I and fibronectin (FN), form a fibrous and structural scaffold for mineral deposition. Inorganic phosphate (Pi) and pyrophosphate (PPi) (a mineralization inhibitor) regulating molecules such as tissue-nonspecific alkaline phosphatase (TNAP, TNSALP, AKP2) (Moursi et al., 1996; Murshed et al., 2005), ectonucleotide pyrophosphatase phosphodiesterase1 (ENPP1) (Solan et al., 1996; Goding et al., 1998; Johnson et al., 1999), and ANK (Hakim et al., 1984; Ho et al., 2000) control hydroxyapatite formation by regulating local Pi and PPi levels. TNSALP is highly expressed by osteoblasts (Watson et al., 1989; Bonucci et al., 1992) in bone, and is important for hard tissue mineralization (Bonucci et al., 1992; Whyte, 1994; Anderson et al., 1997; Narisawa et al., 1997; Beertsen et al., 1999; Fedde et al., 1999; Hessle et al., 2002; Tesch et al., 2003; Anderson et al., 2004). In addition to hydrolyzing PPi, TNSALP can also hydrolyze adenosine 5 0 -triphosphate (ATP), pyridoxal-5 0 -phosphate (PLP), and phosphoethanolamine (PEA) which likewise might provide Pi for mineralization (Smith, 1982; Waymire et al., 1995; Whyte et al., 1995). Although it is clear that TNSALP is the main mineralization-driving enzyme as demonstrated by the severe hypomineralization states of both Tnsalp-deficient mice and hypophosphatasia patients lacking TNSALP, it nevertheless appears not to be an absolute requirement for bone mineralization (Whyte et al., 1995; Beertsen et al., 1999). This indicates that other phosphatases likely contribute to mineralization by elevating local Pi levels. We have previously demonstrated that ATP can act as a significant Pi source for mineralization in MC3T3-E1 osteoblast cultures indicating that ATP-hydrolyzing enzymes could induce mineral deposition together with TNSALP in normal bone. In our previous report we demonstrated that the enzyme transglutaminase 2 (TG2, tissue transglutaminase, tTG) could act as such a phosphatases, and could be involved in ATP hydrolysis in the osteoblast cultures contributing to the elevation in Pi levels required for mineral deposition (Nakano et al., 2007a). Contract grant sponsor: Canadian Institutes of Health Research (CIHR); Contract grant numbers: MOP-89827, MOP-85024. Contract grant sponsor: CIHR Institute for Musculoskeletal Health and Arthritis (IMHA); Contract grant number: IMH-62713. Contract grant sponsor: Fonds de la Recherche en Sante ´ du Que ´bec (FRSQ). Contract grant sponsor: FRSQ Re ´seau de recherche en sante ´ buccodentaire et osseuse (RSBO). Contract grant sponsor: CIHR Strategic Training Program in Applied Oral Health Research. Contract grant sponsor: McGill University Health Centre (MUHC) Research Institute. *Correspondence to: Mari T. Kaartinen, McGill University, Strathcona Bldg, Room M72, 3640 University Street, Montreal, Quebec H3A 2B2, Canada. E-mail: mari.kaartinen@mcgill.ca Received 13 August 2009; Accepted 19 November 2009 Published online in Wiley InterScience (www.interscience.wiley.com.), 4 January 2010. DOI: 10.1002/jcp.22034 ORIGINAL ARTICLE 260 Journal of Journal of Cellular Physiology Cellular Physiology ß 2010 WILEY-LISS, INC.