RECK Forms Cowbell-shaped Dimers and Inhibits Matrix Metalloproteinase-catalyzed Cleavage of Fibronectin

Abstract

The membrane-anchored protease regulator RECK plays important roles in mammalian development and tumor suppression. The biochemical bases of these bioactivities, however, remain poorly understood. Here we report on the properties of a recombinant RECK protein expressed in mouse fibroblasts and purified to near homogeneity. Multiple lines of evidence indicate that RECK forms dimers. Single particle reconstruction using transmission electron microscopy revealed a unique cowbell-like shaped RECK dimer. RECK is cleaved by MMP-2 and MMP-7 and competitively inhibits MMP-7-catalyzed cleavage of fibronectin. Forced RECK expression in HT1080 cells, whose endogenous RECK expression is minimal, leads to an increase in the amount of fibronectin associated with the cell. Our data demonstrate the ability of RECK to protect fibronectin from MMP-mediated degradation. The membrane-anchored protease regulator RECK plays important roles in mammalian development and tumor suppression. The biochemical bases of these bioactivities, however, remain poorly understood. Here we report on the properties of a recombinant RECK protein expressed in mouse fibroblasts and purified to near homogeneity. Multiple lines of evidence indicate that RECK forms dimers. Single particle reconstruction using transmission electron microscopy revealed a unique cowbell-like shaped RECK dimer. RECK is cleaved by MMP-2 and MMP-7 and competitively inhibits MMP-7-catalyzed cleavage of fibronectin. Forced RECK expression in HT1080 cells, whose endogenous RECK expression is minimal, leads to an increase in the amount of fibronectin associated with the cell. Our data demonstrate the ability of RECK to protect fibronectin from MMP-mediated degradation. RECK (reversion-inducing cysteine-rich protein with Kazal motifs) was initially identified as a transformation suppressor gene (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 13221-13226Google Scholar). It encodes a glycoprotein of 971 amino acid residues (∼125 kDa) anchored to the membrane through C-terminal glycosylphosphatidylinositol (GPI) 2The abbreviations used are: GPI, glycosylphosphatidylinositol; MMP, matrix metalloproteinase; FN, fibronectin; TEM, transmission electron microscopy; PI-PLC, phosphatidylinositol-specific phospholipase C; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; RT, reverse transcription; MEF, mouse embryo fibroblast; pFN, plasma fibronectin. modification. RECK-deficient mice die around embryonic day 10.5 (E10.5) with reduced extracellular matrix integrity, premature blood vessels, and precocious neuronal differentiation, and some of these phenotypes have been attributed to the deregulation of metalloproteases such as MMP-2 and ADAM10 (2Oh J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. Alexander D.B. Ide C. Horan T.P. Arakawa T. Yoshida H. Nishikawa S. Itoh Y. Seiki M. Itohara S. Takahashi C. Noda M. Cell.. 2001; 107: 789-800Google Scholar, 3Muraguchi T. Takegami Y. Ohtsuka T. Kitajima S. Chandana E.P. Omura A. Miki T. Takahashi R. Matsumoto N. Ludwig A. Noda M. Takahashi C. Nat. Neurosci.. 2007; 10: 838-845Google Scholar). RECK may also be involved in other developmental events such as myogenesis, chondrogenesis, and neuromuscular junction formation (4Echizenya M. Kondo S. Takahashi R. Oh J. Kawashima S. Kitayama H. Takahashi C. Noda M. Oncogene.. 2005; 24: 5850-5857Google Scholar, 5Kawashima S. Imamura Y. Chandana E.P. Noda T. Takahashi R. Adachi E. Takahashi C. Noda M. J. Neurochem.. 2008; 104: 376-385Google Scholar, 6Kondo S. Shukunami C. Morioka Y. Matsumoto N. Takahashi R. Oh J. Atsumi T. Umezawa A. Kudo A. Kitayama H. Hiraki Y. Noda M. J. Cell Sci.. 2007; 120: 849-857Google Scholar). Although RECK is expressed ubiquitously in adult human tissues, it is down-regulated in many kinds of solid tumors (7Noda M. Takahashi C. Cancer Sci.. 2007; 98: 1659-1665Google Scholar). Restored expression of RECK in tumor cells suppresses tumor angiogenesis, invasion, and metastasis in animal models (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 13221-13226Google Scholar, 2Oh J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. Alexander D.B. Ide C. Horan T.P. Arakawa T. Yoshida H. Nishikawa S. Itoh Y. Seiki M. Itohara S. Takahashi C. Noda M. Cell.. 2001; 107: 789-800Google Scholar), and the level of residual RECK expression in tumor tissues often correlates with better prognosis (7Noda M. Takahashi C. Cancer Sci.. 2007; 98: 1659-1665Google Scholar). Despite the accumulating evidence indicating the importance of RECK in mammalian development and tumor suppression, the molecular basis of its actions remains poorly understood. Several characteristics of RECK, such as high cysteine content, requirement of carbohydrate modification for its bioactivity (8Simizu S. Takagi S. Tamura Y. Osada H. Cancer Res.. 2005; 65: 7455-7461Google Scholar), and susceptibility to proteolysis, make it difficult to produce a large quantity of this protein in a laboratory. In addition, many RECK mutants and fragments have been found biologically inactive, most likely because of misfolding. It is therefore imperative to establish a system to produce full-length RECK protein satisfactory for biochemical studies. Matrix metalloproteinases (MMPs) (∼23 in humans) typically consist of a propeptide, catalytic domain, linker peptide, and hemopexin domain (9Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol.. 2001; 17: 463-516Google Scholar, 10Overall C.M. Mol. Biotechnol.. 2002; 22: 51-86Google Scholar, 11Nagase H. Visse R. Murphy G. Cardiovasc. Res.. 2006; 69: 562-573Google Scholar). Noncatalytic domains of MMPs contribute to their interaction with other molecules, including their specific substrates. MMP-7 (also known as matrilysin and pump-1) is one of the smallest members of the family, consisting only of a propeptide and the catalytic domain. Some MMP family members are up-regulated in cancer cells, and their expression is often associated with poor prognosis (12Fingleton B. Front. Biosci.. 2006; 11: 479-491Google Scholar). Enhanced production of MMP-7, for instance, is found in endometrial carcinoma, and its expression is correlated with invasion and metastasis (13Shiomi T. Okada Y. Cancer Metastasis Rev.. 2003; 22: 145-152Google Scholar). MMP-2-deficient mice show reduced progression of inoculated tumors (14Itoh T. Tanioka M. Yoshida H. Yoshioka T. Nishimoto H. Itohara S. Cancer Res.. 1998; 58: 1048-1051Google Scholar). Previous studies using a partially purified, soluble form of RECK expressed in CHO cells demonstrated competitive inhibition of gelatinases (MMP-2 and MMP-9) and MT1-MMP in vitro (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 13221-13226Google Scholar, 2Oh J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. Alexander D.B. Ide C. Horan T.P. Arakawa T. Yoshida H. Nishikawa S. Itoh Y. Seiki M. Itohara S. Takahashi C. Noda M. Cell.. 2001; 107: 789-800Google Scholar, 15Miki T. Takegami Y. Okawa K. Muraguchi T. Noda M. Takahashi C. J. Biol. Chem.. 2007; 282: 12341-12352Google Scholar). The spectrum and modes of inhibition by RECK on other MMP family members remain to be elucidated. Fibronectin (FN) is one of the major extracellular matrix components that can be cleaved by several MMPs (9Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol.. 2001; 17: 463-516Google Scholar). FN is mainly composed of three repeating units (types I–III), expressed in multiple cell types, and plays important roles in cell adhesion, migration, proliferation, and differentiation (16Pankov R. Yamada K.M. J. Cell Sci.. 2002; 115: 3861-3863Google Scholar, 17Mao Y. Schwarzbauer J.E. Matrix Biol.. 2005; 24: 389-399Google Scholar). FN was initially identified as a glycoprotein lost in malignant transformation, and later studies revealed the involvement of both transcriptional and post-transcriptional regulation in this event (18Olden K. Yamada K.M. Cell.. 1977; 11: 957-969Google Scholar). Whether RECK down-regulation in malignant transformation plays any part in this FN loss is an interesting question yet to be addressed. In this study, we established a system to produce a highly purified, soluble form of human RECK protein. Using this protein, we could uncover its dimeric nature and unique three-dimensional structure. We also identified MMP-7 as its novel potential target and FN as an MMP substrate protected by RECK. Plasmid Construction—The retroviral vector, pL(RECK-His)SB, was constructed by inserting, by three parts ligation, the MluI-EagI fragment of human RECK cDNA, and the double-stranded oligonucleotides encoding the C-terminal 7 amino acids (954AHHHHHH) between the MluI and ApaI site of pLXSB, a derivative of pLXSN (19Miller A.D. Rosman G.J. BioTechniques.. 1989; 7 (984–986, 989–990): 980-982Google Scholar) carrying the bsd marker. The virus was propagated using EcoPack 293 (Clontech). Establishing RECK-His Producer—MEFs were prepared as described (2Oh J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. Alexander D.B. Ide C. Horan T.P. Arakawa T. Yoshida H. Nishikawa S. Itoh Y. Seiki M. Itohara S. Takahashi C. Noda M. Cell.. 2001; 107: 789-800Google Scholar). R–M2–MT– MEFs were infected with L(RECK-His)SB virus and selected with blasticidin S (8 μg/ml). A transfectant clone (TOM2–6) secreting RECK-His at a high level was expanded to 20–40 150-mm dishes using selection medium. When the culture reached confluence, the medium was replaced with α-minimum essential medium containing 5% fetal calf serum and harvested every 48 h for several weeks. Purification of RECK-His—The outline is presented in the supplemental material. Transmission Electron Microscopy (TEM)—Purified proteins were adsorbed to thin carbon films and negatively stained with 2% uranyl acetate. Micrographs were taken using a JEOL 100CX transmission electron microscope (JEOL) at ×52,000 magnification with 100-kV acceleration voltage, and digitized using Scitex Leafscan 45 (Leaf System Inc) with a pixel size of 1.92 Å (specimen level). Automated Particle Selection and Image Analysis—Three-dimensional reconstruction (20van Heel M. Gowen B. Matadeen R. Orlova E.V. Finn R. Pape T. Cohen D. Stark H. Schmidt R. Schatz M. Patwardhan A. Q. Rev. Biophys.. 2000; 33: 307-369Google Scholar, 21Frank J. Three-dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State. Oxford University Press, New York2006Crossref Scopus (395) Google Scholar) was performed using our SPINNS techniques (22Ogura T. Iwasaki K. C. J. Biol.. 2003; Scholar, T. C. J. Biol.. Scholar, T. C. J. Biol.. Scholar, T. C. J. Biol.. 2006; Scholar) and the (20van Heel M. Gowen B. Matadeen R. Orlova E.V. Finn R. Pape T. Cohen D. Stark H. Schmidt R. Schatz M. Patwardhan A. Q. Rev. Biophys.. 2000; 33: 307-369Google Scholar). The outline is presented in the supplemental material. of proteins in with only were by for RECK, for membrane and using a mouse RECK FN is full-length human FN FN C-terminal and were used for was performed using System and were in with substrate in the and at cell of purified RECK-His was in and with New in of in A. M. 10: proteins were by from the and in with of the were performed by using an of RECK and FN with HT1080 have been described (2Oh J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. Alexander D.B. Ide C. Horan T.P. Arakawa T. Yoshida H. Nishikawa S. Itoh Y. Seiki M. Itohara S. Takahashi C. Noda M. Cell.. 2001; 107: 789-800Google Scholar). the cells were for and in protease the cells were in containing extracellular the cells were with for at by and with cell the cells were with and at for h in of for h in of containing of and was using was performed using with the system used are and for FN and the for were as 45 in were in in and on the day stained with mouse FN and plasma was using The were with medium with and with a microscope a microscope and Purification of RECK that a recombinant human RECK protein in the C-terminal was replaced by could be expressed as a protein in cells and that this protein could be cleaved by MMP-2 and MT1-MMP protein, however, was as as RECK in mammalian cells In an to produce and RECK-His in large we established a from an animal carrying in three and When the RECK-His was expressed in this R–M2–MT– using a retroviral vector, near full-length RECK-His could be in the culture We a from this and to RECK-His to with a of from of culture of the RECK-His is in cysteine and Kazal with cysteine residues (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 13221-13226Google Scholar). When we the RECK protein in the of HT1080 cells RECK by a major of was found and a of was found the of A was the purified recombinant RECK-His was and and and In to the several and kDa) were in the of the the C-terminal of RECK was used and of these were in the cells with a spectrum MMP of the major are to of the RECK-His fragments by MMPs in vitro that the of the RECK-His was with that the of was between the purified RECK-His and the RECK in cell of the molecular of RECK-His by A and and by and by the molecular of RECK-His is and the of carbohydrate is typically in a RECK (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 13221-13226Google Scholar, S. Takagi S. Tamura Y. Osada H. Cancer Res.. 2005; 65: 7455-7461Google Scholar), the molecular is to the for a RECK also a molecular we a of cells to A major at the to the was We also prepared the of the cells in the of phosphatidylinositol-specific phospholipase this a large of RECK the on the cell the medium and a major at the dimeric in a major of RECK molecules, including on the cell are in the dimeric Three-dimensional of the high and of the RECK-His we its three-dimensional by and shaped of of RECK-His at were The of these were and to a three-dimensional dimeric using our SPINNS and the The reconstruction of the selected In are with their the and the of the reconstruction and Despite the large in the of the a level of was in their and with the and the from the three-dimensional structure. three-dimensional reconstruction from the particle A of the of the that RECK-His was on the The (20van Heel M. Gowen B. Matadeen R. Orlova E.V. Finn R. Pape T. Cohen D. Stark H. Schmidt R. Schatz M. Patwardhan A. Q. Rev. Biophys.. 2000; 33: 307-369Google Scholar, 21Frank J. Three-dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State. Oxford University Press, New York2006Crossref Scopus (395) Google Scholar) was Å at the of to the G. Heel M. Scholar) The that the RECK-His is a with an that at the an the at the and are and The molecular is Å a of Å from the can be and and The three-dimensional is using and a to by a protein of is of the dimeric RECK kDa) from the amino acid We also RECK-His with of this to the was indicating that the we were found the dimeric of of RECK-His by with the data to the protein expressed in cells and supplemental RECK-His purified in this could be cleaved by MMP-2 the was the substrate major fragments and was these RECK-His could also be cleaved by MMP-7 the was in a major fragment of that MMP-2 and MMP-7 RECK-His at to the of RECK-His on the of we highly purified RECK-His can We to plasma fibronectin a substrate known to be cleaved by both MMP-2 and MMP-7 (9Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol.. 2001; 17: 463-516Google Scholar, K. Y. H. M. Cancer Res.. Scholar). MMP-2 at several as by was partially by RECK-His its was the of both MMP-2 and RECK-His could also the cleavage of by MMP-7 The indicate that RECK can at the a substrate of this is we the susceptibility of and RECK-His to MMP-7 and MMP-7-catalyzed cleavage of was a of substrate the cleavage of RECK-His was RECK-His could only be cleaved at MMP-7 indicating that RECK-His is a poor substrate for In studies such as the one in we that it was to the of MMP-mediated cleavage by and We therefore at in the of RECK-His and The lines the data in the and of near the competitive and the for cleavage by MMP-7, and for inhibition of this by of of by MMP-7 in the of an by the of a molecular The the remains for h in to RECK been of fragments was also was with MMP-2 in the of RECK-His The FN fragments could be by a C-terminal specific by an specific for the of of by MMP-7 also to a and of these that the to the of the FN and that a to a site near the C-terminal of FN The size between the is because of the of the in is with the known of the only one the the FN fragments kDa) that in the of RECK-His are of the domain, mainly of and of RECK on FN with HT1080 the of our in we the of FN associated with HT1080 cells with the FN both in the cell and culture were by in cells as with cells and the FN fragments are in this of endogenous FN was the involvement of post-transcriptional The MMP also the level of FN as by of cells and by the involvement of protein degradation. The major FN was up-regulated in RECK it that the increase in the level of FN an increase in the ability of the cells to we the of FN by In the we the cells with and the of FN associated with the cells the level of FN in the was also in cells of cells a plasma membrane that the FN in culture can be found both and the plasma membrane RECK to the of FN both and the Using a expression we could produce a recombinant RECK protein of Despite the the system its as their and to their for a of to a and cells from can be we used RECK of Although the events it to the cell may be to by RECK, on several was between RECK and in by of MMP and the ability to form dimers. our that RECK-His is for the properties of RECK protein. proteins are for because and often Although of is one to this this often the Single particle reconstruction using TEM, in on a protein are is an J. Three-dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State. Oxford University Press, New York2006Crossref Scopus (395) Google Scholar). this of of The molecular size of RECK as a however, is near the for particle this was by our including T. C. J. Biol.. Scholar, T. C. J. Biol.. Scholar). Three-dimensional reconstruction of with a of is also a in the this was by the of the T. C. J. Biol.. 2006; Scholar). The RECK-His a from its molecular most because of an large The of the C-terminal specific are are to on one of the is with the nature of the the with one that the C-terminal domain is on the the that the RECK to the membrane at its and its with for and with MMPs and other in and of are to this our three-dimensional a for studies on the actions of RECK at the molecular and Our data that MMP-2 and MMP-7 RECK to major fragments and kDa) as by C-terminal specific fragments are often in of cells and the of at of RECK that are to as one near the and one near the The of these on RECK to be in studies. Our in vitro also indicate that RECK-His inhibits MMP-7 cleavage of pFN, through a competitive several important MMP-7 is known to be involved in many (9Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol.. 2001; 17: 463-516Google Scholar, B. J. 2000; Scholar, A. Werb Z. Nat. Rev. Mol. Cell Biol.. 2007; RECK to be as a potential regulator of these MMP-7 is composed of the MMP catalytic RECK may therefore with this domain, and the spectrum of inhibition MMP family members may be we the expression of both RECK and FN is known to be down-regulated by this may the of the expression of these mice and mice phenotypes with in the and H. some of the phenotypes of RECK-deficient mice may be to FN fragments is with MMP-2 MMP-7 in the of RECK-His these fragments the the major domain Although the fragments were in the HT1080 culture system such fragments may a in other cells tissues in A acid from the FN domain been to show and by M. E. Proc. Natl. Acad. Sci. U. S. A.. 2001; 98: Scholar, A. Z. E. Scholar). Several MMPs can FN fragments with to differentiation and M. N. H. T. Y. K. T. Scholar). It therefore be interesting to the FN fragments have any It is also important to these fragments the C-terminal cysteine residues of FN and are essential for formation and T. Y. J. R. N. K. K. H. K. J. Biol. Chem.. 2003; Scholar). The MMP-7 cleavage site in FN the fragments is in the between FN and FN and is known to be to some other including and 2000; Scholar). Previous studies using several MMP-7 including R. H. J. Biol. Chem.. 2001; Scholar), B. M. Biol.. Scholar, T. B. Biophys.. 2002; Scholar), N. A. Y. E. J. Biol. Chem.. 2002; Scholar), Y. H. A. J. Biol. Chem.. 2003; Scholar, T. Scholar), and S. K. S. G. T. Y. K. M. T. A. Cancer Res.. Scholar), indicate that cleavage between a amino acid and at and a amino acid and at The MMP-7 target site we identified in FN is in its in its The data from of MMP-7 using M. J. Biol. Chem.. Scholar) indicate a for at for at and of and in the of the target is the in our these residues contribute to the interaction with MMP-7 and RECK this cleavage at this site are interesting to be in studies. RECK expression is known to be down-regulated by several including (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 13221-13226Google Scholar, R.M. Takahashi C. Noda M. Scholar). HT1080 cells and show RECK expression (1Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. Ikawa Y. Ratzkin B.J. Arakawa T. Noda M. Proc. Natl. Acad. Sci. U. S. A.. 1998; 95: 13221-13226Google Scholar, R.M. Takahashi C. Noda M. Scholar). FN are in cells through several including reduced FN reduced FN and FN (18Olden K. Yamada K.M. Cell.. 1977; 11: 957-969Google Scholar). Our data indicate that expression of RECK in HT1080 cells some in the amount of FN both and the cells a of RECK is associated with some the cell Morioka and M. and at a part of it to be involved in T. Takegami Y. Okawa K. Muraguchi T. Noda M. Takahashi C. J. Biol. Chem.. 2007; 282: 12341-12352Google Scholar). The nature and of RECK the cell and their to the FN found the cells are also important to be in studies. We are to Maki and for Itohara for Seiki for MT1-MMP catalytic domain and and for their on recombinant protein expression and three-dimensional to the for important on and to Alexander for of the We also and for Oh for the in and and for and with