Takahisa Takino

Membrane-type matrix metalloproteinase (MT-MMP), which we have identified recently, is unique in its transmembrane (TM) domain at the C terminus and mediates activation of pro-gelatinase A on the cell surface (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65; [Medline] Takino, T., Sato, H., Yamamoto, E., and Seiki, M.(1995) Gene (Amst.) 115, 293-298). In addition to MT-MMP, a novel MMP-related cDNA of 2.1 kilobases was isolated from a human placenta cDNA library. The cDNA contains an open reading frame for a new MMP. The deduced protein composed of 604 amino acids was closely related to MT-MMP in the amino acid sequence (66% homology at the catalytic domains) and has a potential TM domain at the C terminus. Monoclonal antibodies raised against the synthetic peptide recognized a 64-kDa protein as the major product in the transfected cells. TIMP-1 fused with the potential TM domain was localized on the cell surface while native TIMP-1 is in the culture medium. Thus, we called the second membrane-type MMP, MT-MMP-2 and renamed MT-MMP, MT-MMP-1. MT-MMP-1 and −2 are thought to form a distinct membrane-type subclass in the MMP family since all the others are secreted as soluble forms. Like MT-MMP-1, expression of MT-MMP-2 induced processing of pro-gelatinase A (68-kDa in gelatin zymography) into the activated form of 62-kDa fragments through a 64-kDa intermediate form. Expression of MT-MMP-2 mRNA was at the highest levels in the brain where MT-MMP-1 was at the lowest level compared to other tissues. MT-MMP-1 and −2 are thought to be utilized for extracellular matrix turnover on the surface of cells under different genetic controls. Membrane-type matrix metalloproteinase (MT-MMP), which we have identified recently, is unique in its transmembrane (TM) domain at the C terminus and mediates activation of pro-gelatinase A on the cell surface (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65; [Medline] Takino, T., Sato, H., Yamamoto, E., and Seiki, M.(1995) Gene (Amst.) 115, 293-298). In addition to MT-MMP, a novel MMP-related cDNA of 2.1 kilobases was isolated from a human placenta cDNA library. The cDNA contains an open reading frame for a new MMP. The deduced protein composed of 604 amino acids was closely related to MT-MMP in the amino acid sequence (66% homology at the catalytic domains) and has a potential TM domain at the C terminus. Monoclonal antibodies raised against the synthetic peptide recognized a 64-kDa protein as the major product in the transfected cells. TIMP-1 fused with the potential TM domain was localized on the cell surface while native TIMP-1 is in the culture medium. Thus, we called the second membrane-type MMP, MT-MMP-2 and renamed MT-MMP, MT-MMP-1. MT-MMP-1 and −2 are thought to form a distinct membrane-type subclass in the MMP family since all the others are secreted as soluble forms. Like MT-MMP-1, expression of MT-MMP-2 induced processing of pro-gelatinase A (68-kDa in gelatin zymography) into the activated form of 62-kDa fragments through a 64-kDa intermediate form. Expression of MT-MMP-2 mRNA was at the highest levels in the brain where MT-MMP-1 was at the lowest level compared to other tissues. MT-MMP-1 and −2 are thought to be utilized for extracellular matrix turnover on the surface of cells under different genetic controls. INTRODUCTIONMatrix metalloproteinases (MMPs) 1The abbreviations used are: MMPsmatrix metalloproteinasesmAbmonoclonal antibodyMT-MMPmembrane-type MMPPCRpolymerase chain reactionTIMPtissue inhibitors of metalloproteinasesTMtransmembraneDMEMDulbecco's modified Eagle's mediumkbkilobase(s). are a family of enzymes that share a common domain structure composed of propeptide, catalytic, hinge, and hemopexin-like domains (exception is MMP-7•matrilysin, which lacks a hemopexin-like domain)(3Woessner J.F.J. FASEB J. 1991; 5: 2145-2154Google Scholar, 4Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Google Scholar). These enzymes are responsible for the turnover of extracellular matrix by degrading native macromolecules, including a variety of collagens and glycoproteins such as fibronectin and laminin, and play crucial roles in tissue remodeling during morphogenesis, wound healing, angiogenesis, and also in many pathological conditions such as tumor invasion and rheumatoid arthritis(5Matrisian L.M. Trends Genet. 1990; 6: 121-125Google Scholar, 6Liotta L.A. Steeg P.S. Stetler-Stevenson W.G. Cell. 1991; 64: 327-336Google Scholar, 7Matrisian L.M. BioEssays. 1992; 14: 455-463Google Scholar, 8Sato H. Kida Y. Mai M. Endo Y. Sasaki T. Tanaka J. Seiki M. Oncogene. 1992; 7: 77-83Google Scholar, 9Tsuchiya Y. Sato H. Endo Y. Okada Y. Mai M. Sasaki T. Seiki M. Cancer Res. 1993; 53: 1397-1402Google Scholar, 10Tsuchiya Y. Endo Y. Sato H. Okada Y. Mai M. Sasaki T. Seiki M. Int. J. Cancer. 1994; 56: 46-51Google Scholar). 11 MMPs encoded by different genes are known as the MMP family members, and they have different substrate specificity against extracellular matrix macromolecules. 10 of them are produced by cells as a soluble zymogen form, but the last one, which we recently discovered, has a transmembrane domain at the C terminus and is expressed as a membrane protein(1Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Google Scholar, 2Takino T. Sato H. Yamamoto E. Seiki M. Gene (Amst.). 1995; 115: 293-298Google Scholar, 11Cao J. Sato H. Takino T. Seiki M. J. Biol. Chem. 1995; 270: 801-805Google Scholar). Thus, we called this ectoenzyme membrane-type MMP (MT-MMP).All of the MMPs are expressed as inactive zymogens and need proteolytic activation for them to function. Although serine proteases such as plasmin, neutrophil elastase, and trypsin can activate several MMP zymogens in a test tube(12He C.S. Wilhelm S.M. Pentland A.P. Marmer B.L. Grant G.A. Eisen A.Z. Goldberg G.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2632-2636Google Scholar, 13Nagase H. Enghild J.J. Suzuki K. Salvesen G. Biochemistry. 1990; 29: 5783-5789Google Scholar), little is known about the activators in tissue. MMP-2•gelatinase A cannot be activated by these serine proteinases (14Okada Y. Morodomi T. Enghild J.J. Suzuki K. Yasui A. Nakanishi I. Salvesen G. Nagase H. Eur. J. Biochem. 1990; 194: 721-730Google Scholar, 15Nagase H. Suzuki K. Morodomi T. Enghild J.J. Salvesen G. Matrix. 1992; 1: 237-244Google Scholar) but is activated on the surfaces of fibroblasts (16Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Google Scholar) and tumor cell lines treated with 12-O-tetradecanoylphorbol-13-acetate or concanavalin A (17Brown P.D. Levy A.T. Margulies I.M. Liotta L.A. Stetler-Stevenson W.G. Cancer Res. 1990; 50: 6184-6191Google Scholar, 18Azzam H.S. Arand G. Lippman M.E. Thompson E.W. J. Natl. Cancer Inst. 1993; 85: 1758-1764Google Scholar, 19Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Google Scholar). Thus, a unique activator on the cell surface was expected to be responsible for gelatinase A activation, and it turned out to be MT-MMP. Expression of MT-MMP in the transfected cells induced specific activation of gelatinase A in a cell-mediated manner(1Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Google Scholar). Appearance of the activated form of gelatinase A in the tissue is also a characteristic feature of invasive carcinomas(20Brown P.D. Bloxidge R.E. Anderson E. Howell A. Clin. Exp. Metastasis. 1993; 11: 183-189Google Scholar, 21Brown P.D. Bloxidge R.E. Stuart N.S.A. Gatter K.C. Carmichael J. J. Natl. Cancer Inst. 1993; 85: 574-578Google Scholar). Expression of MT-MMP was detected there, and the product was immunolocalized in and on the carcinoma cells(1Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Google Scholar). Since gelatinase A is an important enzyme for basement membrane invasion by degrading type IV collagen, MT-MMP on the tumor cell surface is thought to play a critical role in the invasive phenotype of tumor cells.MT-MMP cDNA was isolated from a human placenta cDNA library using a MMP-related gene fragment as a probe(2Takino T. Sato H. Yamamoto E. Seiki M. Gene (Amst.). 1995; 115: 293-298Google Scholar). The probe with a possible new MMP gene sequence was obtained from the amplified cDNA fragments by polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to the conserved sequences within the MMP family. The structural characteristic of MT-MMP is the transmembrane domain at the C terminus and two more additional insertions. One is the insertion of 8 amino acids in the catalytic domain, and the other is the insertion of 11 amino acids between the propeptide and the catalytic domain. MMP-11•stromelysin-3 (22Basset P. Bellocq J.P. Wolf C. Stoll I. Hutin P. Limacher J.M. Podhajcer O.L. Chenard M.P. Rio M.C. Chambon P. Nature. 1990; 348: 699-704Google Scholar) has a similar insertion of 10 amino acids between propeptide and catalytic domain, and RXKR sequences, potential recognition sites for subtilisin-like processing enzymes(23Hosaka M. Nagahama M. Kim W.S. Watanabe T. Hatsuzawa K. Ikemizu J. Murakami K. Nakayama K. J. Biol. Chem. 1991; 266: 12127-12130Google Scholar), are conserved between them. No insertion corresponding to the 8 amino acids of MT-MMP was found in the other members of the MMP family.Both MT-MMP and stromelysin-3 were identified by cDNA cloning instead of the conventional biochemical purification of the enzymes. Thus, new MMP members may be obtained further by survey of the MMP-related genes. To identify yet unknown MMP members, we extended our previous study to survey the MMP-related cDNAs amplified from various human tissues by reverse transcription-PCR. In this study, we identified a fragment of another new MMP-related gene from a human melanoma tissue. This fragment was used as a probe to screen a human placenta cDNA library, and a cDNA fragment that encodes a new MMP having a TM domain at the C terminus was obtained. Thus, MT-MMPs form a distinct subgroup in the MMP family.DISCUSSIONIn addition to MT-MMP-1 (MT-MMP in the previous paper), which we previously reported(1Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Google Scholar, 2Takino T. Sato H. Yamamoto E. Seiki M. Gene (Amst.). 1995; 115: 293-298Google Scholar), we identified a new MMP gene that was expressed in a human oral malignant melanoma and a human placenta. The isolated 2.1-kilobase pair cDNA contained a sufficient coding frame for MT-MMP-2. However, the transcript in tissues or cell lines was at 12 kb in size. Since the cDNA lacks the typical polyadenylation signal (AATAAA), the 3′-non-coding region of the gene is thought to be missing in the cloned fragment. MT-MMP-1 and −2 are approximately the same in their molecular weights, but they are encoded by 4.5- and 12-kb transcripts, respectively. The most plausible explanation for the difference of the mRNA sizes is the different length of non-coding regions at their 3′-ends.MT-MMP-2 is the most closely related to MT-MMP-1 in the amino acid sequence (66% homology at the catalytic domain) and in the characteristic insertions compared to other MMPs. These insertions may be important for their function and regulation. For example, the first insertions (IS-1) between the propeptide and catalytic domain contain the conserved RXKR sequences. A similar insertion also exists in stromelysin-3 but not in others (Fig. 2B). These sequences may be the recognition site for processing, since immediately downstream of the RXKR sequences is the reported N terminus of the processed forms of stromelysin 3 and MT-MMP-1(34Murphy G. Segain J.P. O'Shea M. Cockett M. Ioannou C. Lefebvre O. Chambon P. Basset P. J Biol Chem. 1993; 268: 15435-15441Google Scholar, 35Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Google Scholar). Since RXKR is the consensus sequence for subtilisin-like enzymes, autocatalytic activation mechanism, which is common for other MMPs, may not be applicable for these three MMPs. Indeed, 4-aminophenylmercuric acetate or SDS that induces autocatalytic activation of pro-MMPs cannot activate stromelysin-3 and MT-MMPs.There exist 8 amino acid insertions (IS-2) in the catalytic domains of MT-MMP-1 and −2 at the same position. Three of the eight amino acids at both ends were conserved. Although the significance of this insertion is not clear, it may modulate substrate specificity of the enzymes from its position in the catalytic domain like the gelatin-binding domain of two gelatinases(36Murphy G. Nguyen Q. Cockett M.I. Atkinson S.J. Allan J.A. Knight C.G. Willenbrock F. Docherty A.J. J. Biol. Chem. 1994; 269: 6632-6636Google Scholar).Additional sequences (IS-3) containing the TM domains are found downstream of the hemopexin-like domains of MT-MMPs. Both MT-MMPs are expressed as membrane proteins embedded into the plasma membrane through the TM domains at the C terminus. Thus, these two MMPs form a unique membrane-type subclass in the MMP family, while the others are expressed as a soluble form. We previously aligned the most C-terminal portion of the cysteine residue of MT-MMP-1 to that of the hemopexin-like domain of other MMPs and thought that the TM domain is an insertion in the hemopexin-like domain(1Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Google Scholar, 2Takino T. Sato H. Yamamoto E. Seiki M. Gene (Amst.). 1995; 115: 293-298Google Scholar). However, it is more appropriate that the IS-3 locate downstream of the hemopexin-like domains of MT-MMP-1 and −2 rather than splitting the domain as shown in the alignment in Fig. 2. With this alignment, it becomes possible for MT-MMPs to form a cysteine bridge in the hemopexin-like domain, the conserved structure among the MMPs, outside of the cells.Expression of MT-MMP-2 in cells, like that of MT-MMP-1, induced activation of the pro-gelatinase A to the fully activated form (62 kDa) through the intermediate form (64 kDa). Thus, both MT-MMPs have similar biochemical activities at least in part, though it remains to be elucidated whether these MT-MMPs have different substrate specificity or not. If both MT-MMPs are similar in function, why are two separate genes required for the organism? The organism may have to utilize these MT-MMPs in different tissue environment or situations. Consistent with this idea, the tissue distribution of the MT-MMP-1 and MT-MMP-2 mRNAs was different in the human tissues. In contrast to MT-MMP-1, which is expressed widely in various tissues, MT-MMP-2 is expressed in only restricted tissues such as brain, heart, and placenta. In particular, brain tissue expresses MT-MMP-2 at the highest level but expresses MT-MMP-1 at the lowest level. The high level expression of MT-MMP-2 in brain may suggest a specific role of the product in the central nervous system. In cell lines, squamous cell carcinoma OSC-19 and human embryonal lung fibroblasts express MT-MMP-1 mRNA at higher levels but MT-MMP-2 mRNA at lower levels. The reverse was also the case; the MT-MMP-1 mRNA level was low in T24 cells where MT-MMP-2 mRNA was expressed predominantly.Since MT-MMP-2 was originally detected in oral malignant melanoma, whether MT-MMP-2 is also involved in activation of gelatinase A in tumor tissues like MT-MMP-1 is of interest. Our preliminary findings with lung carcinomas where MT-MMP-1 was overexpressed indicated that MT-MMP-2 was not expressed frequently there. 2T. Takino, H. Sato, A. Shinagawa, and M. Seiki, unpublished results. In addition to the activation of pro-gelatinase A, some of the proteins, such as β-amyloid precursor protein, tumor necrosis factor-α, and V-2 vasopressin receptor, are reported to be processed on the cell membrane by MMP-like activities(37Miyazaki K. Hasegawa M. Funahashi K. Umeda M. Nature. 1993; 362: 839-841Google Scholar, 38Mohler K.M. Sleath P.R. Fitzner J.N. Cerretti D.P. Alderson M. Kerwar S.S. Torrance D.S. Otten-Evans C. Greenstreet T. K. M. M. Nature. 1994; 370: Scholar, A.J. P. M. M. J. M. K. P. K. S. G. L.M. K. Nature. 1994; 370: Scholar, E. F. J. Biol. Chem. 1995; 270: Scholar). MT-MMPs may be responsible for the processing of many important proteins on the cell surface as a These to be this study, we reported MT-MMP-2 as a new of the MMP family. Since both MT-MMP-1 and −2 have TM domains at the C we a new subclass for MT-MMPs in the MMP family. of MT-MMPs to the cell surface extracellular matrix turnover and INTRODUCTIONMatrix metalloproteinases (MMPs) 1The abbreviations used are: MMPsmatrix metalloproteinasesmAbmonoclonal antibodyMT-MMPmembrane-type MMPPCRpolymerase chain reactionTIMPtissue inhibitors of metalloproteinasesTMtransmembraneDMEMDulbecco's modified Eagle's mediumkbkilobase(s). are a family of enzymes that share a common domain structure composed of propeptide, catalytic, hinge, and hemopexin-like domains (exception is MMP-7•matrilysin, which lacks a hemopexin-like domain)(3Woessner J.F.J. FASEB J. 1991; 5: 2145-2154Google Scholar, 4Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Google Scholar). These enzymes are responsible for the turnover of extracellular matrix by degrading native macromolecules, including a variety of collagens and glycoproteins such as fibronectin and laminin, and play crucial roles in tissue remodeling during morphogenesis, wound healing, angiogenesis, and also in many pathological conditions such as tumor invasion and rheumatoid arthritis(5Matrisian L.M. Trends Genet. 1990; 6: 121-125Google Scholar, 6Liotta L.A. Steeg P.S. Stetler-Stevenson W.G. Cell. 1991; 64: 327-336Google Scholar, 7Matrisian L.M. BioEssays. 1992; 14: 455-463Google Scholar, 8Sato H. Kida Y. Mai M. Endo Y. Sasaki T. Tanaka J. Seiki M. Oncogene. 1992; 7: 77-83Google Scholar, 9Tsuchiya Y. Sato H. Endo Y. Okada Y. Mai M. Sasaki T. Seiki M. Cancer Res. 1993; 53: 1397-1402Google Scholar, 10Tsuchiya Y. Endo Y. Sato H. Okada Y. Mai M. Sasaki T. Seiki M. Int. J. Cancer. 1994; 56: 46-51Google Scholar). 11 MMPs encoded by different genes are known as the MMP family members, and they have different substrate specificity against extracellular matrix macromolecules. 10 of them are produced by cells as a soluble zymogen form, but the last one, which we recently discovered, has a transmembrane domain at the C terminus and is expressed as a membrane protein(1Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Google Scholar, 2Takino T. Sato H. Yamamoto E. Seiki M. Gene (Amst.). 1995; 115: 293-298Google Scholar, 11Cao J. Sato H. Takino T. Seiki M. J. Biol. Chem. 1995; 270: 801-805Google Scholar). Thus, we called this ectoenzyme membrane-type MMP (MT-MMP).All of the MMPs are expressed as inactive zymogens and need proteolytic activation for them to function. Although serine proteases such as plasmin, neutrophil elastase, and trypsin can activate several MMP zymogens in a test tube(12He C.S. Wilhelm S.M. Pentland A.P. Marmer B.L. Grant G.A. Eisen A.Z. Goldberg G.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2632-2636Google Scholar, 13Nagase H. Enghild J.J. Suzuki K. Salvesen G. Biochemistry. 1990; 29: 5783-5789Google Scholar), little is known about the activators in tissue. MMP-2•gelatinase A cannot be activated by these serine proteinases (14Okada Y. Morodomi T. Enghild J.J. Suzuki K. Yasui A. Nakanishi I. Salvesen G. Nagase H. Eur. J. Biochem. 1990; 194: 721-730Google Scholar, 15Nagase H. Suzuki K. Morodomi T. Enghild J.J. Salvesen G. Matrix. 1992; 1: 237-244Google Scholar) but is activated on the surfaces of fibroblasts (16Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Google Scholar) and tumor cell lines treated with 12-O-tetradecanoylphorbol-13-acetate or concanavalin A (17Brown P.D. Levy A.T. Margulies I.M. Liotta L.A. Stetler-Stevenson W.G. Cancer Res. 1990; 50: 6184-6191Google Scholar, 18Azzam H.S. Arand G. Lippman M.E. Thompson E.W. J. Natl. Cancer Inst. 1993; 85: 1758-1764Google Scholar, 19Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Google Scholar). Thus, a unique activator on the cell surface was expected to be responsible for gelatinase A activation, and it turned out to be MT-MMP. Expression of MT-MMP in the transfected cells induced specific activation of gelatinase A in a cell-mediated manner(1Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Google Scholar). Appearance of the activated form of gelatinase A in the tissue is also a characteristic feature of invasive carcinomas(20Brown P.D. Bloxidge R.E. Anderson E. Howell A. Clin. Exp. Metastasis. 1993; 11: 183-189Google Scholar, 21Brown P.D. Bloxidge R.E. Stuart N.S.A. Gatter K.C. Carmichael J. J. Natl. Cancer Inst. 1993; 85: 574-578Google Scholar). Expression of MT-MMP was detected there, and the product was immunolocalized in and on the carcinoma cells(1Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Google Scholar). Since gelatinase A is an important enzyme for basement membrane invasion by degrading type IV collagen, MT-MMP on the tumor cell surface is thought to play a critical role in the invasive phenotype of tumor cells.MT-MMP cDNA was isolated from a human placenta cDNA library using a MMP-related gene fragment as a probe(2Takino T. Sato H. Yamamoto E. Seiki M. Gene (Amst.). 1995; 115: 293-298Google Scholar). The probe with a possible new MMP gene sequence was obtained from the amplified cDNA fragments by polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to the conserved sequences within the MMP family. The structural characteristic of MT-MMP is the transmembrane domain at the C terminus and two more additional insertions. One is the insertion of 8 amino acids in the catalytic domain, and the other is the insertion of 11 amino acids between the propeptide and the catalytic domain. MMP-11•stromelysin-3 (22Basset P. Bellocq J.P. Wolf C. Stoll I. Hutin P. Limacher J.M. Podhajcer O.L. Chenard M.P. Rio M.C. Chambon P. Nature. 1990; 348: 699-704Google Scholar) has a similar insertion of 10 amino acids between propeptide and catalytic domain, and RXKR sequences, potential recognition sites for subtilisin-like processing enzymes(23Hosaka M. Nagahama M. Kim W.S. Watanabe T. Hatsuzawa K. Ikemizu J. Murakami K. Nakayama K. J. Biol. Chem. 1991; 266: 12127-12130Google Scholar), are conserved between them. No insertion corresponding to the 8 amino acids of MT-MMP was found in the other members of the MMP family.Both MT-MMP and stromelysin-3 were identified by cDNA cloning instead of the conventional biochemical purification of the enzymes. Thus, new MMP members may be obtained further by survey of the MMP-related genes. To identify yet unknown MMP members, we extended our previous study to survey the MMP-related cDNAs amplified from various human tissues by reverse transcription-PCR. In this study, we identified a fragment of another new MMP-related gene from a human melanoma tissue. This fragment was used as a probe to screen a human placenta cDNA library, and a cDNA fragment that encodes a new MMP having a TM domain at the C terminus was obtained. Thus, MT-MMPs form a distinct subgroup in the MMP family.

Cell migration is modulated by regulatory molecules such as growth factors, oncogenes, and the tumor suppressor PTEN. We previously described inhibition of cell migration by PTEN and restoration of motility by focal adhesion kinase (FAK) and p130 Crk-associated substrate (p130(Cas)). We now report a novel pathway regulating random cell motility involving Shc and mitogen-activated protein (MAP) kinase, which is downmodulated by PTEN and additive to a FAK pathway regulating directional migration. Overexpression of Shc or constitutively activated MEK1 in PTEN- reconstituted U87-MG cells stimulated integrin- mediated MAP kinase activation and cell migration. Conversely, overexpression of dominant negative Shc inhibited cell migration; Akt appeared uninvolved. PTEN directly dephosphorylated Shc. The migration induced by FAK or p130(Cas) was directionally persistent and involved extensive organization of actin microfilaments and focal adhesions. In contrast, Shc or MEK1 induced a random type of motility associated with less actin cytoskeletal and focal adhesion organization. These results identify two distinct, additive pathways regulating cell migration that are downregulated by tumor suppressor PTEN: one involves Shc, a MAP kinase pathway, and random migration, whereas the other involves FAK, p130(Cas), more extensive actin cytoskeletal organization, focal contacts, and directionally persistent cell motility. Integration of these pathways provides an intracellular mechanism for regulating the speed and the directionality of cell migration.

The transmembrane heparan sulfate proteoglycan syndecan-1 was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with syndecan-1 in HEK293T cells promoted syndecan-1 shedding, and concentration of cell-associated syndecan-1 was reduced. Treatment of cells with MMP inhibitor BB-94 or tissue inhibitor of MMP (TIMP)-2 but not TIMP-1 interfered with the syndecan-1 shedding promoted by MT1-MMP expression. In contrast, syndecan-1 shedding induced by 12-O-tetradecanoylphorbol-13-acetate treatment was inhibited by BB-94 but not by either TIMP-1 or TIMP-2. Shedding of syndecan-1 was also induced by MT3-MMP but not by other MT-MMPs. Recombinant syndecan-1 core protein was shown to be cleaved by recombinant MT1-MMP or MT3-MMP preferentially at the Gly245-Leu246 peptide bond. HT1080 fibrosarcoma cells stably transfected with the syndecan-1 cDNA (HT1080/SDC), which express endogenous MT1-MMP, spontaneously shed syndecan-1. Migration of HT1080/SDC cells on collagen-coated dishes was significantly slower than that of control HT1080 cells. Treatment of HT1080/SDC cells with BB-94 or TIMP-2 induced accumulation of syndecan-1 on the cell surface, concomitant with further retardation of cell migration. Substitution of Gly245 of syndecan-1 with Leu significantly reduced shedding from HT1080/SDC cells and cell migration. These results suggest that the shedding of syndecan-1 promoted by MT1-MMP through the preferential cleavage of Gly245-Leu246 peptide bond stimulates cell migration. The transmembrane heparan sulfate proteoglycan syndecan-1 was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with syndecan-1 in HEK293T cells promoted syndecan-1 shedding, and concentration of cell-associated syndecan-1 was reduced. Treatment of cells with MMP inhibitor BB-94 or tissue inhibitor of MMP (TIMP)-2 but not TIMP-1 interfered with the syndecan-1 shedding promoted by MT1-MMP expression. In contrast, syndecan-1 shedding induced by 12-O-tetradecanoylphorbol-13-acetate treatment was inhibited by BB-94 but not by either TIMP-1 or TIMP-2. Shedding of syndecan-1 was also induced by MT3-MMP but not by other MT-MMPs. Recombinant syndecan-1 core protein was shown to be cleaved by recombinant MT1-MMP or MT3-MMP preferentially at the Gly245-Leu246 peptide bond. HT1080 fibrosarcoma cells stably transfected with the syndecan-1 cDNA (HT1080/SDC), which express endogenous MT1-MMP, spontaneously shed syndecan-1. Migration of HT1080/SDC cells on collagen-coated dishes was significantly slower than that of control HT1080 cells. Treatment of HT1080/SDC cells with BB-94 or TIMP-2 induced accumulation of syndecan-1 on the cell surface, concomitant with further retardation of cell migration. Substitution of Gly245 of syndecan-1 with Leu significantly reduced shedding from HT1080/SDC cells and cell migration. These results suggest that the shedding of syndecan-1 promoted by MT1-MMP through the preferential cleavage of Gly245-Leu246 peptide bond stimulates cell migration. Syndecans are transmembrane heparan sulfate proteoglycans expressed on all adherent cells (1Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2361) Google Scholar, 2Rapraeger A.C. Ott V.L. Curr. Opin. Cell. Biol. 1998; 10: 620-628Crossref PubMed Scopus (102) Google Scholar) and have been proposed to play an important role in tissue morphogenesis by virtue of their ability to bind, via their covalently attached glycosaminoglycan chains, to a variety of extracellular adhesive molecules including fibronectin, thrombospondin, various collagens, and heparin-binding growth-associated molecules and growth factors such as basic fibroblast growth factor (3Perrimon N. Bernfield M. Nature. 2000; 404: 725-728Crossref PubMed Scopus (667) Google Scholar, 4Park P.W. Reizes O. Bernfield M. J. Biol. Chem. 2000; 275: 29923-29926Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 5Rapraeger A.C. J. Cell. Biol. 2000; 149: 995-998Crossref PubMed Scopus (177) Google Scholar, 6Perrimon N. Bernfield M. Semin. Cell Dev. Biol. 2001; 12: 65-67Crossref PubMed Scopus (102) Google Scholar, 7Rapraeger A.C. Semin. Cell Dev. Biol. 2001; 12: 107-116Crossref PubMed Scopus (109) Google Scholar, 8Woods A. J. Clin. Invest. 2001; 107: 935-941Crossref PubMed Scopus (115) Google Scholar). Since the expression of syndecans appears to be controlled during both development and the progression of tumor cells to the metastatic phenotype, it has been proposed that syndecans are important regulators of the migratory and invasive behaviors of both normal and transformed cells (9Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-393Crossref PubMed Scopus (988) Google Scholar, 10Inki P. Jalkanen M. Ann. Med. 1996; 28: 63-67Crossref PubMed Scopus (105) Google Scholar). The syndecan family is composed of four closely related proteins (syndecan-1, -2, -3, and -4) encoded by four different genes. Syndecan-1 is abundant in normal epithelial cells and tissues, localizing to both basal and suprabasal cell layers (1Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2361) Google Scholar). Disruption of syndecan-1 expression in cultured cells leads to an epithelial mesenchymal transformation, with associated changes in cell polarity and cell-cell adhesion and altered epithelium-specific gene expression (7Rapraeger A.C. Semin. Cell Dev. Biol. 2001; 12: 107-116Crossref PubMed Scopus (109) Google Scholar, 11Dobra K. Andang M. Syrokou A. Karamanos N.K. Hjerpe A. Exp. Cell Res. 2000; 258: 12-22Crossref PubMed Scopus (65) Google Scholar). The intact ectodomain of each syndecan is constitutively shed from cultured cells (12Kim C.W. Goldberger O.A. Gallo R.L. Bernfield M. Mol. Biol. Cell. 1994; 5: 797-805Crossref PubMed Scopus (358) Google Scholar, 13Spring J. Paine-Saunders S.E. Hynes R.O. Bernfield M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3334-3338Crossref PubMed Scopus (122) Google Scholar) as part of normal cell surface heparan sulfate proteoglycan turnover (14Yanagishita M. Hascall V.C. J. Biol. Chem. 1992; 267: 9451-9454Abstract Full Text PDF PubMed Google Scholar). Ectodomain shedding appears to contribute to diverse pathophysiological events such as host defense, wound healing, arthritis, and Alzheimer's disease, but how shedding is regulated remains largely unknown (15Kiessling L.L. Gordon E.J. Chem. Biol. 1998; 5: R49-R62Abstract Full Text PDF PubMed Scopus (32) Google Scholar, 16Merlos-Suarez A. Arribas J. Biochem. Soc. Trans. 1999; 27: 243-246Crossref PubMed Scopus (17) Google Scholar, 17Fitzgerald M.L. Wang Z. Park P.W. Murphy G. Bernfield M. J. Cell Biol. 2000; 148: 811-824Crossref PubMed Scopus (352) Google Scholar). Matrix metalloproteinases (MMPs) 1The abbreviations used are: MMP, matrix metalloproteinase; GST, glutathione S-transferase; MT-MMP, membrane-type matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; BB-94, [4-(N-hydroxyamino)-2R-iso-butyl-3-S-(thienylthiomethyl)-succinyl]-l-phenylalanine-N-methylamide. are a family of Zn2+-dependent enzymes that are known to cleave extracellular matrix proteins in normal and pathological conditions (18Birkedal H.H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal H.B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2677) Google Scholar, 19Woessner J.F.J. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3125) Google Scholar, 20Seiki M. APMIS. 1999; 107: 137-143Crossref PubMed Scopus (276) Google Scholar). To date, more than 20 mammalian MMPs have been identified by cDNA cloning, and they can be subgrouped into soluble type and membrane type MMPs (MT-MMPs) (20Seiki M. APMIS. 1999; 107: 137-143Crossref PubMed Scopus (276) Google Scholar, 21Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3937) Google Scholar). MMPs are overexpressed in various human malignancies and have been thought to contribute to tumor invasion and metastasis by degrading extracellular matrix components (18Birkedal H.H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal H.B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2677) Google Scholar, 22Stetler S.W. Aznavoorian S. Liotta L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1536) Google Scholar). Thus, the level of MMP expression correlates with the invasiveness or malignancy of tumors (23Nomura H. Sato H. Seiki M. Mai M. Okada Y. Cancer Res. 1995; 55: 3263-3266PubMed Google Scholar, 24Ueno H. Nakamura H. Inoue M. Imai K. Noguchi M. Sato H. Seiki M. Okada Y. Cancer Res. 1997; 57: 2055-2060PubMed Google Scholar). Particularly, MT1-MMP, MMP-2, MMP-7, and MMP-9 have been reported to be most closely associated with tumor invasion and metastasis. Whereas degradation of extracellular matrix is an important aspect of MMP biology, growing evidence has demonstrated specific processing/activation or degradation of cell surface receptors and ligands. Fas ligand (25Powell W.C. Fingleton B. Wilson C.L. Boothby M. Matrisian L.M. Curr. Biol. 1999; 9: 1441-1447Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar), tumor necrosis factor-α (26Gearing A.J. Beckett P. Christodoulou M. Churchill M. Clements J. Davidson A.H. Drummond A.H. Galloway W.A. Gilbert R. Gordon J.L. Nature. 1994; 370: 555-557Crossref PubMed Scopus (1118) Google Scholar), the ectodomain of the fibroblast growth factor receptor-1 (27Levi E. Fridman R. Miao H.Q. Ma Y.S. Yayon A. Vlodavsky I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7069-7074Crossref PubMed Scopus (300) Google Scholar), the heparin-binding epidermal growth factor (28Suzuki M. Raab G. Moses M.A. Fernandez C.A. Klagsbrun M. J. Biol. Chem. 1997; 272: 31730-31737Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), and interleukin-8 (29Van den Steen P.E. Proost P. Wuyts A. Van Damme J. Opdenakker G. Blood. 2000; 96: 2673-2681Crossref PubMed Google Scholar) were reported to be released or activated by MMPs. MMPs also cleave and inactivate interleukin-1β (30Ito A. Mukaiyama A. Itoh Y. Nagase H. Thogersen I.B. Enghild J.J. Sasaguri Y. Mori Y. J. Biol. Chem. 1996; 271: 14657-14660Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), insulin-like growth factor-binding proteins (31Fowlkes J.L. Enghild J.J. Suzuki K. Nagase H. J. Biol. Chem. 1994; 269: 25742-25746Abstract Full Text PDF PubMed Google Scholar), fibrinogen and factor XII (32Hiller O. Lichte A. Oberpichler A. Kocourek A. Tschesche H. J. Biol. Chem. 2000; 275: 33008-33013Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), the CC chemokine MCP-3 (33McQuibban G.A. Gong J.H. Tam E.M. McCulloch C.A. Clark-Lewis I. Overall C.M. Science. 2000; 289: 1202-1206Crossref PubMed Scopus (654) Google Scholar), and the CXC chemokines stromal cell-derived factor-1 α and β (34McQuibban G.A. Butler G.S. Gong J.H. Bendall L. Power C. Clark-Lewis I. Overall C.M. J. Biol. Chem. 2001; 276: 43503-43508Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar, 35McQuibban G.A. Gong J.H. Wong J.P. Wallace J.L. Clark-Lewis I. Overall C.M. Blood. 2002; 100: 1160-1167Crossref PubMed Google Scholar). Recently, matrilysin (MMP-7) was shown to mediate shedding of syndecan-1/a CXC chemokine (KC) complex from the mucosal surface, which directs and confines to in Park P.W. Wilson C.L. W.C. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). have an expression cloning method to the of which not by MT1-MMP but also as of MT1-MMP H. Y. H. Itoh Y. Seiki M. Sato H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. H. J. Sato H. Cancer Res. 2001; Google Scholar, H. H. M. Okada Y. Seiki M. Sato H. PubMed Scopus Google Scholar). In have identified syndecan-1 as a of MT1-MMP and demonstrated that shedding of syndecan-1 by MT1-MMP stimulates cell migration. was from were by human placenta cDNA library in the expression was from Recombinant MT1-MMP and MT3-MMP with at the were as Sato H. Okada A. E. Imai K. Okada Y. Seiki M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Nakamura H. E. Y. Y. Sato H. Seiki M. Okada Y. J. Biochem. 1999; PubMed Scopus Google Scholar). Recombinant TIMP-2 and were from and were from and was a from Cell HEK293T and fibrosarcoma HT1080 cells were from and cultured in with cloning to the of which with MMP-2, or MT1-MMP, was as H. Y. H. Itoh Y. Seiki M. Sato H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). and an expression syndecan-1 was with either MT1-MMP or control into cells cultured in dishes to the was with and cells were a further Syndecan-1 was from the or cell by as K. N. Kato H. K. H. C. J. A. Cancer Res. 1995; 55: Google Scholar, C. M. A. J. 1999; PubMed Scopus Google Scholar). syndecan-1 was on and membrane The membrane was with in and with in Syndecan-1 was with as a and with as a in The was by an expression syndecan-1 was with either MT1-MMP or control into cells cultured in as The and cell were by the method M.L. Wang Z. Park P.W. Murphy G. Bernfield M. J. Cell Biol. 2000; 148: 811-824Crossref PubMed Scopus (352) Google Scholar, Fitzgerald M.L. Bernfield M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), and syndecan-1 was as HT1080 cells stably syndecan-1 were by cells transfected with syndecan-1 cDNA in in syndecan-1 and MT1-MMP were with or control into cells cultured in dishes with with type syndecan-1 was and with as H. Y. H. Itoh Y. Seiki M. Sato H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). was with was of Recombinant Syndecan-1 cDNA was by a with an at and a with an at cDNA a syndecan-1 protein in which Gly245 was with Leu was by of syndecan-1 cDNA which The was with and and into the and of H. H. M. Okada Y. Seiki M. Sato H. PubMed Scopus Google Scholar). protein was by a cDNA at the of the as H. H. M. Okada Y. Seiki M. Sato H. PubMed Scopus Google Scholar). The was transformed with and the protein expression was induced by were and in Syndecan-1 protein with was from the by a Syndecan-1 proteins were to the of the of Syndecan-1 syndecan-1 protein was with recombinant MT1-MMP in of at and were on and to membrane The of each was the Migration HT1080 cells or cells were dishes with were with a in to cells and cultured in further cells were and at each H. H. M. Okada Y. Seiki M. Sato H. PubMed Scopus Google Scholar). of cDNA from the human placenta cDNA library was with MMP-2, and MT1-MMP cDNA into and cell were by with MMP-2, and MT1-MMP cDNA into cells a of a of and a of of a of cDNA of to the cDNA of from cDNA were by a of which induced to the not The of all cDNA was and the of the cDNA was that cDNA the transmembrane heparan sulfate proteoglycan syndecan-1 Shedding of Syndecan-1 by of by MT1-MMP by the expression of syndecan-1 was it a MT1-MMP and syndecan-1. Thus, syndecan-1 as a of Syndecan-1 was with MT1-MMP in and the shedding of syndecan-1 into or with cells was by and Syndecan-1 was spontaneously shed into from cells transfected with the syndecan-1 as a from to more than of the syndecan-1 was in cell which slower than that in the Co-expression of MT1-MMP syndecan-1 shedding and reduced level in the cell Treatment of cells with the MMP inhibitor BB-94 the shedding of syndecan-1 by MT1-MMP expression. Treatment of cells with also shedding of syndecan-1 as reported M.L. Wang Z. Park P.W. Murphy G. Bernfield M. J. Cell Biol. 2000; 148: 811-824Crossref PubMed Scopus (352) Google Scholar, A.J. Biochem. J. 1997; PubMed Scopus Google Scholar). To syndecan-1 shedding induced by and MT1-MMP, of MMP BB-94, and TIMP-2 were and Shedding of syndecan-1 induced by either treatment or MT1-MMP expression was inhibited by the of BB-94, but TIMP-1 In contrast, the of recombinant TIMP-2 syndecan-1 shedding induced by MT1-MMP expression but not that by These results that a endogenous of syndecan-1 shedding in cells that is different from Shedding of syndecan-1 induced by and MT1-MMP was also by of cells The surface of cells transfected with the syndecan-1 cDNA was syndecan-1 with Syndecan-1 was also on the surface of cells with the MT1-MMP Treatment with BB-94 a syndecan-1 on cells with or with Co-expression of TIMP-2 or syndecan-1 of cells with the MT1-MMP but of TIMP-1 of syndecan-1 by treatment was by the expression of but not by the expression of TIMP-1 or TIMP-2. These results further that the syndecan-1 shedding is to different from MT1-MMP were with type their to shedding of syndecan-1 MT1-MMP in not syndecan-1 of the or shedding, but of the with that of not MT1-MMP also to syndecan-1 These results suggest that MT1-MMP with syndecan-1 through and that cell surface of MT1-MMP is the shedding of syndecan-1. other of the family were their to shedding of syndecan-1 the MT1-MMP and MT3-MMP promoted syndecan-1 shedding with a of Syndecan-1 by syndecan-1 ectodomain protein with at the was with recombinant MT1-MMP and was on The product of and a of were of was by the the that syndecan-1 ectodomain protein with at the is preferentially cleaved at the to the To the cleavage recombinant syndecan-1 ectodomain protein to was and with recombinant MT1-MMP The product was a the of which demonstrated the cleavage of Gly245-Leu246 peptide bond of syndecan-1. protein in which Gly245 was with Leu was with recombinant MT1-MMP, which not a but a of The of the by of or the cleavage of peptide bond of syndecan-1 by of protein with MT3-MMP a including other not Syndecan-1 Shedding Cell syndecan-1 cDNA was stably transfected into HT1080 fibrosarcoma cells (HT1080/SDC), which express of endogenous MT1-MMP H. Okada Y. J. A. E. Seiki M. Nature. 1994; 370: PubMed Scopus Google Scholar), and syndecan-1 shed into or associated with cells was by and HT1080/SDC cells shed a level of which was by as a slower that that from and the syndecan-1 concentration in cell was treatment of HT1080/SDC cells induced further syndecan-1 shedding and reduced the level of cell-associated syndecan-1. In contrast, treatment of cells with BB-94 syndecan-1 shedding, in syndecan-1 accumulation in the cells. shedding of syndecan-1 from HT1080/SDC cells was also by TIMP-2 but not by TIMP-1 as shown by shedding from HT1080/SDC cells was inhibited by HT1080/SDC cells were also by syndecan-1 Syndecan-1 was on HT1080/SDC cell surface, and treatment with BB-94 induced accumulation of syndecan-1 on the cell of was in HT1080/SDC cells but not in cells not The of TIMP-2 but not TIMP-1 significantly cell surface of syndecan-1. of HT1080/SDC cells on collagen-coated dishes was in the of HT1080 cells was inhibited by the of BB-94 or TIMP-2 and of Migration of HT1080/SDC cells was of cells. Treatment of HT1080/SDC cells with BB-94 or TIMP-2 further to and of the HT1080/SDC Syndecan-1 Shedding through the of Gly245-Leu246 syndecan-1 and with an of Gly245 with Leu were shedding by MT1-MMP expression Shedding of induced by MT1-MMP expression or treatment was significantly than that of syndecan-1. shedding from HT1080 cells stably was than that from HT1080/SDC and the level of cell-associated syndecan-1 in cells was than that of HT1080/SDC cells The concentration of syndecan-1 in cells by BB-94 treatment was at a with the level of cell-associated of cells was significantly slower than that of HT1080/SDC cells BB-94 of HT1080/SDC but of was not significantly by have identified and as molecules that with MMPs by the expression cloning H. Y. H. Itoh Y. Seiki M. Sato H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. H. J. Sato H. Cancer Res. 2001; Google Scholar, H. H. M. Okada Y. Seiki M. Sato H. PubMed Scopus Google Scholar). In the identified that expression of the syndecan-1 gene also promoted of the to the in the by MT1-MMP in cells. Since endogenous TIMP-2 in cells are through the of complex and MT1-MMP, concentration of the complex by syndecan-1 H. Y. H. Itoh Y. Seiki M. Sato H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). the of syndecan-1 expression on by MT1-MMP was with that of expression not H. Y. H. Itoh Y. Seiki M. Sato H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). a not directs MT1-MMP to but also as a MT1-MMP M. Itoh Y. Mori H. Okada A. H. Seiki M. J. Cell Biol. 2001; PubMed Scopus Google Scholar, H. N. M. Itoh Y. Sato H. H. I. Seiki M. J. 2002; PubMed Scopus Google Scholar), which to syndecan-1 is also cleaved by Co-expression of MT1-MMP with syndecan-1 promoted shedding of and the concentration of cell surface syndecan-1 was reduced. The of syndecan-1 shedding was also with but not with other MT-MMPs. of MT1-MMP that cell surface is the shedding of syndecan-1. of the of MT1-MMP syndecan-1 shedding, which the of MT1-MMP with syndecan-1 through Syndecan-1 ectodomain shedding was also by Syndecan-1 shedding by either MT1-MMP or was inhibited by both BB-94 and TIMP-2 syndecan-1 shedding promoted by MT1-MMP, and were inhibited by These results that syndecan-1 shedding induced by is by other than shedding of syndecan-1 ectodomain results from cleavage at a in core protein M.L. Wang Z. Park P.W. Murphy G. Bernfield M. J. Cell Biol. 2000; 148: 811-824Crossref PubMed Scopus (352) Google Scholar). of the cleavage of syndecan-1 by MT1-MMP or MT3-MMP recombinant proteins was the Gly245-Leu246 peptide bond a of Gly245 with Leu significantly reduced shedding promoted by MT1-MMP expression or that the Gly245-Leu246 peptide bond is of the cleavage shedding promoted by MT1-MMP expression or Gly245-Leu246 of human syndecan-1 to in syndecan-1. remains to be syndecan-1 is cleaved at by to extracellular matrix proteins including and the migratory and invasive of both normal and transformed cells (9Bernfield M. Kokenyesi R. Kato M. Hinkes M.T. Spring J. Gallo R.L. Lose E.J. Annu. Rev. Cell Biol. 1992; 8: 365-393Crossref PubMed Scopus (988) Google Scholar, 10Inki P. Jalkanen M. Ann. Med. 1996; 28: 63-67Crossref PubMed Scopus (105) Google Scholar). expression of syndecan-1 in epithelial tumor cells an epithelial and growth S. M. Jalkanen M. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar). In syndecan-1 expression was shown to of HT1080 and treatment of cells with MMP cell surface syndecan-1 concomitant with of which in further retardation of cell migration. HT1080 cells are known to of including MT1-MMP, MMP-2, and HT1080/SDC cells constitutively shed syndecan-1 and be by TIMP-2 or BB-94 but not by The that endogenous MT1-MMP in HT1080 cells is in syndecan-1 (MMP-7) was also reported to mediate syndecan-1 shedding Park P.W. Wilson C.L. W.C. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google matrilysin appears not be in shedding from HT1080 TIMP-1 not the cell syndecan-1 expression in tissue has been to be associated with a in and A. M. P. Jalkanen M. H. J. 1999; PubMed Scopus Google Scholar, M. Jalkanen M. P. R. 1997; PubMed Scopus Google Scholar), cell A. P. M. Jalkanen M. H. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and S. K. B. J. J. Van E. J. 1998; PubMed Scopus (105) Google Scholar), and syndecan-1 expression in of tissue is associated with of in cell A. P. M. Jalkanen M. H. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), of basal cell I.B. B. J. 2000; PubMed Scopus Google Scholar), and of and of in A. M. Y. Gallo R.L. Bernfield M. Y. J. 1997; PubMed Scopus Google Scholar). syndecan-1 at were reported to be associated with in H. A. M. R. H. S. Cancer Res. 2002; Google Scholar). be that syndecan-1 ectodomain be shed by MT1-MMP matrilysin expressed in tumor expression of MT1-MMP and matrilysin are also associated with the malignancy of tumors including H. Nakamura H. Inoue M. Imai K. Noguchi M. Sato H. Seiki M. Okada Y. Cancer Res. 1997; 57: 2055-2060PubMed Google Scholar, M. Sato H. S. Okada Y. Y. Seiki M. J. 1995; PubMed Scopus Google Scholar, H. Y. Itoh S. K. M. Y. Y. M. Imai K. Cancer Res. 1999; Google Scholar, K. Nakamura K. R. 2000; PubMed Scopus (122) Google Scholar, H. S. Itoh M. Imai K. 2001; 91: PubMed Scopus Google Scholar). Syndecans are also known to to various growth factors and via glycosaminoglycan and A.C. J. Cell. Biol. 2000; 149: 995-998Crossref PubMed Scopus (177) Google Scholar, 6Perrimon N. Bernfield M. Semin. Cell Dev. Biol. 2001; 12: 65-67Crossref PubMed Scopus (102) Google Scholar, 8Woods A. J. Clin. Invest. 2001; 107: 935-941Crossref PubMed Scopus (115) Google Scholar). Thus, shedding of syndecan-1 with factors and the of shed with factors be of the important the of their in various pathophysiological Recently, matrilysin was shown to mediate shedding of a syndecan-1/a CXC chemokine (KC) complex from the mucosal surface, which directs and confines to of in Park P.W. Wilson C.L. W.C. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). MT1-MMP is also expressed in diverse pathophysiological conditions and contribute to the of from growth factors and by shedding syndecan-1 with In have shown that MT1-MMP and syndecan-1 which cell on Since syndecan-1 shedding is in diverse pathological events such as tumor wound healing, arthritis, and Alzheimer's disease, the of shedding contribute to the development of and E. of of the

The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4, 5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. We investigated molecular interactions of PTEN and FAK in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A bound wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation was retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppressed all five properties. PTEN-mutated cells were resistant to apoptosis in suspension, but most of the cells entered apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reversed the decreased FAK phosphorylation and PI 3-K activity, and it partially rescued PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. Our results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK.