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The Reversion-inducing Cysteine-rich Protein with Kazal Motifs (RECK) Interacts with Membrane Type 1 Matrix Metalloproteinase and CD13/Aminopeptidase N and Modulates Their Endocytic Pathways

2007; Elsevier BV; Volume: 282; Issue: 16 Linguagem: Inglês

10.1074/jbc.m610948200

ISSN

1083-351X

Autores

Takao Miki, Yujiro Takegami, Katsuya Okawa, Teruyuki Muraguchi, Makoto Noda, Chiaki Takahashi,

Tópico(s)

Signaling Pathways in Disease

Resumo

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is anchored to the cell surface via glycosylphosphatidylinositol. This molecule antagonizes the function of membrane type 1 matrix metalloproteinase (MT1-MMP) to promote proMMP-2 maturation. Here, we attempt to clarify the mechanism underlying RECK functions. First, we found that RECK forms a complex with MT1-MMP and inhibits its proteolytic activity. Notably, RECK increases the amount of MT1-MMP that associates with detergent-resistant membranes during sucrose gradient ultracentrifugation. Furthermore, perturbation of membrane cholesterol significantly affected the function of RECK in suppressing MT1-MMP function. These findings indicate that RECK possibly regulates MT1-MMP function by modulating its behavior on the cell surface as well as by enzymatic action; this prompted us to find another molecule whose behavior in detergent-resistant membranes is influenced by RECK. Subsequently, we found that RECK interacts with CD13/aminopeptidase N. Further, we found that RECK inhibits the proteolytic activity of CD13 in a cholesterol perturbation-sensitive manner. Finally, we examined whether RECK influences the behavior of MT1-MMP and CD13 during their internalization from the cell surface. In the absence of RECK, MT1-MMP and CD13 were internalized along with the markers of clathrin- or caveolae-dependent endocytosis. However, interestingly, in the presence of RECK these molecules were internalized preferentially with an endocytic marker that is neither clathrinnor caveolae-dependent, indicating that RECK modulates endocytic pathways of MT1-MMP and CD13. This modulation was correlated with the accelerated internalization and decay of MT1-MMP and CD13. This study unveils the novel function and target molecules of RECK. The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is anchored to the cell surface via glycosylphosphatidylinositol. This molecule antagonizes the function of membrane type 1 matrix metalloproteinase (MT1-MMP) to promote proMMP-2 maturation. Here, we attempt to clarify the mechanism underlying RECK functions. First, we found that RECK forms a complex with MT1-MMP and inhibits its proteolytic activity. Notably, RECK increases the amount of MT1-MMP that associates with detergent-resistant membranes during sucrose gradient ultracentrifugation. Furthermore, perturbation of membrane cholesterol significantly affected the function of RECK in suppressing MT1-MMP function. These findings indicate that RECK possibly regulates MT1-MMP function by modulating its behavior on the cell surface as well as by enzymatic action; this prompted us to find another molecule whose behavior in detergent-resistant membranes is influenced by RECK. Subsequently, we found that RECK interacts with CD13/aminopeptidase N. Further, we found that RECK inhibits the proteolytic activity of CD13 in a cholesterol perturbation-sensitive manner. Finally, we examined whether RECK influences the behavior of MT1-MMP and CD13 during their internalization from the cell surface. In the absence of RECK, MT1-MMP and CD13 were internalized along with the markers of clathrin- or caveolae-dependent endocytosis. However, interestingly, in the presence of RECK these molecules were internalized preferentially with an endocytic marker that is neither clathrinnor caveolae-dependent, indicating that RECK modulates endocytic pathways of MT1-MMP and CD13. This modulation was correlated with the accelerated internalization and decay of MT1-MMP and CD13. This study unveils the novel function and target molecules of RECK. The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) has been identified as a negative transcriptional target for various oncogene products, including activated Ras (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar, 2Sasahara R.M. Takahashi C. Noda M. Biochem. Biophys. Res. Commun. 1999; 264: 668-675Crossref PubMed Scopus (95) Google Scholar). Overexpression of this molecule partially suppressed phenotypes induced by ras transformation in murine fibroblasts (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar) and efficiently blocked matrix invasion and remote metastasis of malignant tumor cells (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar). Subsequently, it was demonstrated that RECK functionally antagonizes multiple matrix metalloproteinases (MMPs) 3The abbreviations used are: MMP, matrix metalloproteinase; MT1-MMP, membrane type 1 MMP; GPI, glycosylphosphatidylinositol; APN, aminopeptidase N; TIMP, tissue inhibitor of metalloproteinase; GEEC, GPI-anchored protein enriched early endosomal compartment; MBCD, methyl-β cyclodextrin; MEF, mouse embryonic fibroblast; HUVEC, human umbilical vein endothelial cell; Tf, transferrin; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; DRM, detergent-resistant membrane; FACS, fluorescence-activated cell sorter; F-RECK, FLAG-tagged RECK; siRNA, short interference RNA.3The abbreviations used are: MMP, matrix metalloproteinase; MT1-MMP, membrane type 1 MMP; GPI, glycosylphosphatidylinositol; APN, aminopeptidase N; TIMP, tissue inhibitor of metalloproteinase; GEEC, GPI-anchored protein enriched early endosomal compartment; MBCD, methyl-β cyclodextrin; MEF, mouse embryonic fibroblast; HUVEC, human umbilical vein endothelial cell; Tf, transferrin; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; DRM, detergent-resistant membrane; FACS, fluorescence-activated cell sorter; F-RECK, FLAG-tagged RECK; siRNA, short interference RNA. including MMP-9, MMP-2, and MT1-MMP (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar, 3Oh 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: 789-800Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). The phenotypes exhibited by RECK-deficient mice, such as increased activation of proMMP-2 and aberrant metabolism of type I collagen, strongly supported this belief (3Oh 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: 789-800Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar), although we recently noticed that the vascular phenotype that appeared in these mice (3Oh 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: 789-800Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar) can be explained by the functional interaction with yet unidentified target molecules. 4T. Muraguchi and C. Takahashi, unpublished observation.4T. Muraguchi and C. Takahashi, unpublished observation. Clinically, it has been revealed that in many types of cancer the expression level of RECK is highly correlated with invasiveness of the tumor and prognosis of the tumor host (see references in Ref. 4Rabien A. Burkhardt M. Jung M. Fritzsche F. Ringsdorf M. Schicktanz H. Loening S.A. Kristiansen G. Jung K. Eur. Urol. 2006; 51: 1259-1266Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). RECK promoter was recently found to be frequently methylated in metastatic non-small cell lung carcinomas (5Chang H.C. Cho C.Y. Hung W.C. Cancer Sci. 2007; 98: 169-173Crossref PubMed Scopus (58) Google Scholar). In addition, RECK has been implicated in chronic inflammatory diseases (6van Lent P.L. Span P.N. Sloetjes A.W. Radstake T.R. van Lieshout A.W. Heuvel J.J. Sweep C.G. van den Berg W.B. Ann. Rheum. Dis. 2005; 64: 368-374Crossref PubMed Scopus (31) Google Scholar, 7Paulissen G. Rocks N. Quesada-Calvo F. Gosset P. Foidart J.M. Noel A. Louis R. Cataldo D.D. Mol. Med. 2006; 12: 171-179Crossref PubMed Scopus (41) Google Scholar). The structural basis of RECK function is currently under extensive investigation. RECK contains domains that are similar to those shared by Kazal-type serine protease inhibitors; however, the electric charge of the amino acid residues constituting the prospective P1 site in these domains of RECK is completely different from that of others, and thus far there is no evidence of serine protease inhibitor function of 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: 13221-13226Crossref PubMed Scopus (418) Google Scholar). RECK has been shown to suppress the extracellular release of proMMP-9 (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar) and competitively inhibit the proteolytic activity of active MMP-9 (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar) and MMP-2 (3Oh 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: 789-800Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Subsequently, the current investigation implicates that RECK contains domains functionally similar to the substrates of MMPs 5Y. Takegami and C. Takahashi, unpublished observation.5Y. Takegami and C. Takahashi, unpublished observation. as in the case of α2-macroglobulin, which is a major plasma inhibitor of metalloproteases (8Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (969) Google Scholar). As compared with soluble MMP inhibitors represented by tissue inhibitor of metalloproteinases (TIMPs) (8Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (969) Google Scholar), the most distinguishing feature of RECK is its ability to covalently anchor to the membrane surface via a post-translational modification (glycosylphosphatidylinositol (GPI) anchor) that is conserved among species (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar). In contrast to RECK-deficient mice, deletion of any TIMP genes had little impact on embryonic development (3Oh 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: 789-800Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 8Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (969) Google Scholar). In this work, we hypothesize that the membrane anchoring of RECK assigns unknown active roles to this molecule in addition to rendering it highly accessible to membrane-bound MMPs. In consonance, a soluble mutant of RECK that lacks the hydrophobic domain at the carboxyl-terminal failed to attenuate the proteolytic activity of MT1-MMP when introduced into HT1080 cells, 6C. Takahashi, unpublished observation.6C. Takahashi, unpublished observation. suggesting that the membrane anchoring is required for RECK to exert its function to antagonize MT1-MMP. MT1-MMP directly degrades various components of the extracellular matrix (9Itoh Y. Seiki M. J. Cell Physiol. 2006; 206: 1-8Crossref PubMed Scopus (414) Google Scholar). CD44, αv-integrin, and syndecan-1 are also substrates of MT1-MMP; the shedding of these molecules significantly affects cell motility through various mechanisms (9Itoh Y. Seiki M. J. Cell Physiol. 2006; 206: 1-8Crossref PubMed Scopus (414) Google Scholar). Furthermore, MT1-MMP directly processes proMMP-2 and proMMP-13 and brings about their activation (9Itoh Y. Seiki M. J. Cell Physiol. 2006; 206: 1-8Crossref PubMed Scopus (414) Google Scholar, 10Osenkowski P. Toth M. Fridman R. J. Cell Physiol. 2004; 200: 2-10Crossref PubMed Scopus (166) Google Scholar). TIMP-2 has been identified as its soluble inhibitor (8Baker A.H. Edwards D.R. Murphy G. J. Cell Sci. 2002; 115: 3719-3727Crossref PubMed Scopus (969) Google Scholar). Further, TIMP-2 facilitates the activation of proMMP-2 by MT1-MMP via ternary complex formation (11Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1435) Google Scholar). Because RECK overexpression significantly suppressed MT1-MMP-dependent activation of proMMP-2 in cultured cells (3Oh 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: 789-800Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar), RECK has been proposed to be an inhibitor of MT1-MMP; however, no direct evidence and mechanism have been provided. Because of the membrane-tethering ability of MT1-MMP, it undergoes a unique mode of post-translational regulation that is initiated by internalization. Recent studies indicate that MT1-MMP internalization is controlled by a clathrin- or caveolae-dependent endocytic pathway or by a combination of these two pathways (12Annabi B. Lachambre M. Bousquet-Gagnon N. Page M. Gingras D. Beliveau R. Biochem. J. 2001; 353: 547-553Crossref PubMed Scopus (135) Google Scholar, 13Remacle A. Murphy G. Roghi C. J. Cell Sci. 2003; 116: 3905-3916Crossref PubMed Scopus (212) Google Scholar, 14Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar, 15Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar, 16Galvez B.G. Matias-Roman S. Yanez-Mo M. Vicente-Manzanares M. Sanchez-Madrid F. Arroyo A.G. Mol. Biol. Cell. 2004; 15: 678-687Crossref PubMed Scopus (149) Google Scholar, 17Wu X. Gan B. Yoo Y. Guan J.L. Dev. Cell. 2005; 9: 185-196Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). These mechanisms contribute to the selective internalization of MT1-MMP from a specific compartment of the cell membrane and its sorting to endosomes for subsequent degradation or recycling (9Itoh Y. Seiki M. J. Cell Physiol. 2006; 206: 1-8Crossref PubMed Scopus (414) Google Scholar, 10Osenkowski P. Toth M. Fridman R. J. Cell Physiol. 2004; 200: 2-10Crossref PubMed Scopus (166) Google Scholar). This machinery appeared to be implicated in the control of proMMP-2 maturation and cell motility (14Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar, 15Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar). β1 integrin is co-localized with MT1-MMP on the surface of human endothelial cells. This interaction appeared to modulate the mechanism of MT1-MMP internalization when the cells were exposed to β1 integr in-dependent extracellular matrices (18Galvez B.G. Matias-Roman S. Yanez-Mo M. Sanchez-Madrid F. Arroyo A.G. J. Cell Biol. 2002; 159: 509-521Crossref PubMed Scopus (181) Google Scholar). These findings indicate that cell surface molecules can participate in regulating MT1-MMP internalization and thereby influence cell behavior. CD13/aminopeptidase N (APN) is another membrane protease whose gene transcription is up-regulated by the ras oncogene product (19Bhagwat S.V. Petrovic N. Okamoto Y. Shapiro L.H. Blood. 2003; 101: 1818-1826Crossref PubMed Scopus (95) Google Scholar). This molecule functions as a metal-dependent ectopeptidase that is involved in processing angiotensins, met-enkephalin, neurokinin A, somatostatin, monocyte chemotactic protein (MCP)-1, and macrophage inhibitory protein (MIP)-1, and it also acts as a receptor for viruses, including coronavirus 229E that, in turn, mediates infection by human cytomegalovirus (20Riemann D. Kehlen A. Langner J. Immunol. Today. 1999; 20: 83-88Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 21Bauvois B. Oncogene. 2004; 23: 317-329Crossref PubMed Scopus (144) Google Scholar). Hence, this molecule is expected to play pivotal roles in tumor progression, angiogenesis, the cardiovascular system, immune systems, and in viral infection. The treatment of animals or cells using synthetic inhibitors of CD13 (e.g. bestatin, amastatin, and actinonin) or the anti-CD13 antibody resulted in abnormalities in various biological systems, i.e. cell proliferation and survival, blood pressure, cytokine levels, angiogenesis, and vasculogenesis (20Riemann D. Kehlen A. Langner J. Immunol. Today. 1999; 20: 83-88Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 21Bauvois B. Oncogene. 2004; 23: 317-329Crossref PubMed Scopus (144) Google Scholar, 22Bhagwat S.V. Lahdenranta J. Giordano R. Arap W. Pasqualini R. Shapiro L.H. Blood. 2001; 97: 652-659Crossref PubMed Scopus (265) Google Scholar). Although the mechanism is unclear, the anti-CD13 antibody suppresses type IV collagen degradation by tumor cells (HT1080 cells) and thereby inhibits their invasion (23Saiki I. Fujii H. Yoneda J. Abe F. Nakajima M. Tsuruo T. Azuma I. Int. J. Cancer. 1993; 54: 137-143Crossref PubMed Scopus (330) Google Scholar). Unlike MT1-MMP, no endogenous inhibitor of CD13 has been discovered thus far. Here, we report that RECK forms complex with MT1-MMP and CD13, competitively inhibits their proteolytic activities, and influences their behavior on the cell surface. In addition, this study demonstrates that RECK is internalized most likely via a recently identified novel endocytic pathway that involves the GPI-anchored protein enriched early endosomal compartments (GEECs) (24Sabharanjak S. Sharma P. Parton R.G. Mayor S. Dev. Cell. 2002; 2: 411-423Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). Previous reports indicated that the internalization of MT1-MMP is clathrin- and/or caveolae-dependent (13Remacle A. Murphy G. Roghi C. J. Cell Sci. 2003; 116: 3905-3916Crossref PubMed Scopus (212) Google Scholar, 16Galvez B.G. Matias-Roman S. Yanez-Mo M. Vicente-Manzanares M. Sanchez-Madrid F. Arroyo A.G. Mol. Biol. Cell. 2004; 15: 678-687Crossref PubMed Scopus (149) Google Scholar) and that of CD13 is caveolae-dependent (25Nomura R. Kiyota A. Suzaki E. Kataoka K. Ohe Y. Miyamoto K. Senda T. Fujimoto T. J. Virol. 2004; 78: 8701-8708Crossref PubMed Scopus (135) Google Scholar). This study demonstrates that endocytic pathways for these molecules can be changed to one that is preferred by RECK. Cell Culture and Transfection—HT1080 cells were transfected with the mammalian expression vector pCXN2neo, pCXN2neo containing human RECK (hRECK) cDNA (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar), pLXSB, or pLXSB containing FLAG- or Myc-tagged hRECK cDNA (see below); they were selected for 10 days in the presence of 1.0 mg/ml G418 or 8 μg/ml blastcidin. The selected cells were immediately pooled or used for the following experiments within several passages. RECK-/- mouse embryonic fibroblasts (MEFs) were described previously (3Oh 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: 789-800Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). MT1-MMP-/- MEFs were prepared from MT1-MMP-/- mice (a gift from M. Seiki) at embryonic day 13.0, infected with retrovirus generated by transfecting EcoPak293 cells (Clontech) with pLXSB-MT1-MMP, and selected with 8 μg/ml blastcidin (26Oh J. Takahashi R. Adachi E. Kondo S. Kuratomi S. Noma A. Alexander D.B. Motoda H. Okada A. Seiki M. Itoh T. Itohara S. Takahashi C. Noda M. Oncogene. 2004; 23: 5041-5048Crossref PubMed Scopus (109) Google Scholar). pSG5-FLAG-MT1-MMP was presented by M. Seiki (15Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar). Human umbilical vein endothelial cells (HUVECs) were obtained from CAM-BREX (CC2517), maintained according to the provider's protocol, transfected with pSG5-FLAG-MT1-MMP using FuGENE 6 (11814443001; Roche Applied Sciences), and sorted by using FACS Aria (BD Biosciences). Construction of Plasmids—To generate Myc- or FLAG-tagged hRECK, a mutation (sense primer, 5′-CCCGCACTGTCCGGAGCCGGGCCCCCT-3′) was induced in pLXSB-hRECK (27Echizenya M. Kondo S. Takahashi R. Oh J. Kawashima S. Kitayama H. Takahashi C. Noda M. Oncogene. 2005; 24: 5850-5857Crossref PubMed Scopus (44) Google Scholar) to generate two novel restriction enzyme sites (ApaI and BspEI) that facilitate the insertion of a DNA cassette encoding a FLAG (Pro-Thr-Met-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) or a Myc (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu-Leu-Ala) epitope between Gly27 and Asp29 in hRECK. The amino-terminal signal peptide of hRECK ends at Gly26 (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar). Immunoprecipitation and Immunoblotting—The cells were lysed in a solution containing 25 mm Hepes, pH 7.5, 0.15 m NaCl, 1% Nonidet P-40, 0.25% deoxycholate, 10% glycerol, 10 mm MgCl2, and 1 mm EDTA in the presence of protease inhibitor mixture (03969-21; Nacalai tesque). FLAG-tagged RECK was immunoprecipitated with rabbit anti-FLAG polyclonal antibody (F7425; Sigma), MT1-MMP with mouse anti-MT1-MMP antibody (1D8; a gift from M. Seiki) (28Suenaga N. Mori H. Itoh Y. Seiki M. Oncogene. 2005; 24: 859-868Crossref PubMed Scopus (83) Google Scholar), and CD13 with rabbit anti-CD13 antibody (a gift from S. Roffler) (29Chang Y.W. Chen S.C. Cheng E.C. Ko Y.P. Lin Y.C. Kao Y.R. Tsay Y.G. Yang P.C. Wu C.W. Roffler S.R. Int. J. Cancer. 2005; 116: 243-252Crossref PubMed Scopus (60) Google Scholar). The immunoprecipitates were collected on protein G-agarose beads (45210; Pierce), washed five times with lysis buffer, and eluted. The cell lysates or immunoprecipitates were separated by SDS-PAGE and used for immunoblotting as described previously (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar). Production and Purification of Recombinant RECK—293F (Invitrogen) cells were transfected with pCXN2-hRECK1-941-His6, and the culture supernatants were recovered as the protein source. Purification of the product was performed by using a Hi-trap chelating column (GE Healthcare) and FPLC (Bio-Rad). The purity of this material was ∼97%. Kinetics of MT1-MMP Inhibition—To study the kinetics of MT1-MMP inhibition, 58 ng of recombinant soluble catalytic domain of MT1-MMP (S.E.-259; BIOMOL) was mixed with various concentrations of a labeled synthetic peptide Mca-Lys-Pro-Leu-Gly-Leu-dpa-Ala-Arg-NH2 (0.75-6.75 nm; ES010; R&D Systems) and recombinant soluble RECK protein (0-13.3 nm) in 200 ml of assay buffer containing 100 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10 mm CaCl2, 10 mm ZnCl2, 0.1% Brij-35, and 0.1% polyethylene glycol (PEG) 6000. The output signal (excitation wavelength, 325 nm; emission wavelength, 393 nm) was recorded for 2 min at 37 °C by using SPECTRAmax (Molecular Devices). The dissociation constant Ki was calculated as described previously (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar). Sucrose Gradient Centrifugation—Protein fractionation by sucrose gradient centrifugation was performed as described previously (30Legler D.F. Micheau O. Doucey M.A. Tschopp J. Bron C. Immunity. 2003; 18: 655-664Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). The anti-clathrin (610499; BD Biosciences) and anti-caveolin-1 (610059; BD Biosciences) antibodies were used. The distribution of the proteins of interest to detergent-resistant membrane (DRM) was estimated by quantifying the intensity of the bands obtained after immunoblotting by using NIH image (ver. 1.61). In each transfectant, MBCD (C4767; Sigma)-treated cells were set to zero. Gelatin Zymography—2 × 104 cells were plated on a 12-well-type dish and incubated for 48 h before starting preparation of culture supernatants that were prepared by incubating the cells with Dulbecco's modified Eagle's medium containing 10% fetal calf serum for 2.5 h and subsequently with 0.1% fetal calf serum for another 24 h. The culture supernatants from the final 24-h incubation were analyzed as described previously (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: 13221-13226Crossref PubMed Scopus (418) Google Scholar). The cell number after the final 24-h incubation was estimated by using the cell number-counting reagent SF (07553; Nacalai tesque) that was used for adjusting loading volume. Mass Spectrometry—Mass spectrometric identification of proteins was performed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as described previously (31Oda A. Miki H. Wada I. Yamaguchi H. Yamazaki D. Suetsugu S. Nakajima M. Nakayama A. Okawa K. Miyazaki H. Matsuno K. Ochs H.D. Machesky L.M. Fujita H. Takenawa T. Blood. 2005; 105: 3141-3148Crossref PubMed Scopus (47) Google Scholar). Aminopeptidase Assay and Kinetics—Transfected cells (1 × 104 cells) were grown in a 96-well flat-bottomed microtiter plate for 24 h and successively treated with or without bestatin (B8385; Sigma) at 37 °C. After 6 h of incubation, alanine-4-methylcoumaryl-7-amide (Peptide Institute) (32Aozuka Y. Koizumi K. Saitoh Y. Ueda Y. Sakurai H. Saiki I. Cancer Lett. 2004; 216: 35-42Crossref PubMed Scopus (70) Google Scholar) was added to the wells as the substrate at a final concentration of 0.2 mm. The cells were incubated for another 10 min, and the plate was then chilled at 0 °C for 10 min. The supernatant was collected, cleared by centrifugation, and subjected to fluorometric analysis of 7-amino-4-methylcoumarin (excitation wavelength, 360 nm; emission wavelength, 440 nm) (32Aozuka Y. Koizumi K. Saitoh Y. Ueda Y. Sakurai H. Saiki I. Cancer Lett. 2004; 216: 35-42Crossref PubMed Scopus (70) Google Scholar). The nonspecific (basal) fluorescence level was determined by analyzing the sample in the presence of a saturating amount (5 μg/ml) of anti-CD13-blocking antibody (WM15: BD Biosciences), and this value was set to zero. Embryonic cells were prepared from E10.0 RECK null embryos or wild type littermates as described previously (3Oh 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: 789-800Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). Embryonic cells (1 × 104) were incubated in a 96-well flat-bottomed microtiter plate for 24 h, and the CD13 peptidase activity was then measured. For the kinetics assay, 6 ng of recombinant soluble CD13/APN (2335-ZN; R&D Systems) was mixed with various concentrations of alanine-4-methylcou-maryl-7-amide (0.025-0.15 mm) and recombinant soluble RECK protein (0-12.5 nm) in 200 μl of assay buffer (10 mm Tris at pH 7.5, 150 mm NaCl, and 0.05% Triton X-100). The output signal (440 nm) was recorded for 15 min at 37 °C by using SPECTRAmax (Molecular Devices). The dissociation constant Ki was calculated as described previously (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: 13221-13226Crossref PubMed Scop

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