Takahisa Takino

Cleavage of Syndecan-1 by Membrane Type Matrix Metalloproteinase-1 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.

Roles of membrane-type matrix metalloproteinase-1 in tumor invasion and metastasis.

Degradation of extracellular matrix (ECM) is one of the first steps in tumor invasion and metastasis. Matrix metalloproteinases (MMP) have been strongly implicated in this step. Membrane‐type MMP‐1 (MT1‐MMP) was first identified as an activator of proMMP‐2 expressed on the surface of tumor cells and later, not only ECM macromolecules but also various biologically important molecules, were shown to serve as substrates for MT1‐MMP. Accumulated lines of evidence have demonstrated that MT1‐MMP expression level is closely associated with invasiveness and malignancy of tumors, suggesting that MT1‐MMP is one of the most critical factors for tumor invasion and metastasis. Despite enthusiasm for MMP inhibitors, phase III trials have not yet demonstrated significance in overall survival and side‐effects remain an issue. An understanding of the functions of MT1‐MMP could supply clues for developing novel therapeutic strategies targeting MT1‐MMP. (Cancer Sci 2005; 96: 212u2003–217)

Membrane Type 1 Matrix Metalloproteinase Regulates Collagen-Dependent Mitogen-Activated Protein/Extracellular Signal-Related Kinase Activation and Cell Migration

Mitogen-activated protein kinase-extracellular signal-related kinase (ERK) kinase 1 (MEK1)/ERK signaling has been implicated in the regulation of tumor cell invasion and metastasis. Migration of HT1080 cells on type I collagen was suppressed by the matrix metalloproteinase (MMP) inhibitors BB94 and tissue inhibitor of metalloproteinase (TIMP)-2 but not by TIMP-1. TIMP-2-specific inhibition suggests that membrane type 1 MMP (MT1-MMP) is likely involved in this process. Activation of ERK was induced in HT1080 cells adhered on dishes coated with type I collagen, and this was inhibited by BB94. MMP-2 processing in HT1080 cells, which also was stimulated by cultivation on type I collagen, was inhibited by MEK inhibitor PD98059. Expression of a constitutively active form of MEK1 promoted MMP-2 processing concomitant with the increase of MT1-MMP levels, suggesting that MT1-MMP is regulated by MEK/ERK signaling. In addition, expression of the hemopexin-like domain of MT1-MMP in HT1080 cells interfered with MMP-2 processing, ERK activation, and cell migration, implying that the enzymatic activity of MT1-MMP is involved in collagen-induced ERK activation, which results in enhanced cell migration. Thus, adhesion of HT1080 cells to type I collagen induces MT1-MMP-dependent ERK activation, which in turn causes an increase in MT1-MMP levels and subsequent cell migration.

Cleavage of lumican by membrane-type matrix metalloproteinase-1 abrogates this proteoglycan-mediated suppression of tumor cell colony formation in soft agar.

The small leucine-rich proteoglycan lumican 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). Coexpression of MT1-MMP with lumican in HEK293T cells reduced the concentration of lumican secreted into culture medium, and this reduction was abolished by addition of the MMP inhibitor BB94. Lumican protein from bovine cornea and recombinant lumican core protein fused to glutathione S-transferase was shown to be cleaved at multiple sites by recombinant MT1-MMP. Transient expression of lumican in HEK293 cells induced expression of tumor suppressor gene product p21/Waf-1, which was abrogated by the coexpression of MT1-MMP concomitant with a reduction in lumican concentration in culture medium. Stable expression of lumican in HeLa cells induced expression of p21 and reduction of colony formation in soft agar, which were both abolished by the expression of MT1-MMP. HT1080 fibrosarcoma cells stably transfected with the lumican cDNA (HT1080/Lum), which express endogenous MT1-MMP, secreted moderate levels of lumican; however, treatment of HT1080/Lum cells with BB94 resulted in accumulation of lumican in culture medium. The expression levels of p21 in HT1080/Lum were proportional to the concentration of secreted lumican and showed reverse corelation with colony formation in soft agar. These results suggest that MT1-MMP abrogates lumican-mediated suppression of tumor cell colony formation in soft agar by degrading this proteoglycan, which down-regulates it through the induction of p21.

Tetraspanin CD63 promotes targeting and lysosomal proteolysis of membrane-type 1 matrix metalloproteinase.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is known to be internalized from cell surface, however, the fate of internalized MT1-MMP is still unknown. Here we demonstrate that at least a part of internalized MT1-MMP is targeted for lysosomal proteolysis. Treatment with an inhibitor of lysosomal proteinases chloroquine suppressed degradation of internalized MT1-MMP and induced accumulation of MT1-MMP in CD63-positive lysosomes. Ectopic expression of CD63 accelerated degradation of MT1-MMP, which was blocked by chloroquine. MT1-MMP, and CD63 were shown to form a complex through hemopexin-like domain of MT1-MMP and N-terminal region of CD63, and thus accelerated degradation of MT1-MMP was not observed with mutants lacking these domains. CD63 mutant lacking lysosomal targeting motif was unable to promote MT1-MMP degradation. These results suggest that CD63 regulates MT1-MMP by targeting to lysosomes.

Tyrosine phosphorylation of the CrkII adaptor protein modulates cell migration

CrkII belongs to a family of adaptor proteins that become tyrosine phosphorylated after various stimuli. We examined the role of CrkII tyrosine phosphorylation in fibronectin-induced cell migration. Overexpression of CrkII inhibited dephosphorylation of focal adhesion components such as p130 Crk-associated substrate (p130cas) and paxillin by protein tyrosine phosphatase 1B (PTP1B). Tyrosine-phosphorylated CrkII was dephosphorylated by PTP1B both in vitro and in vivo, showing for the first time that PTP1B directly dephosphorylates CrkII. A CrkII mutant in which tyrosine residue 221 was substituted by phenylalanine (CrkII-Y221F) could not be tyrosine phosphorylated, and it showed significantly increased binding to p130cas and paxillin. Enhanced binding of CrkII to p130cas has been reported to promote cell migration. Nonphosphorylated CrkII-Y221F promoted HT1080 cell migration on fibronectin, whereas wild-type CrkII did not at moderate expression levels. Moreover, co-expression of CrkII and PTP1B promoted HT1080 cell migration on fibronectin and retained tyrosine phosphorylation and binding of p130cas to CrkII, whereas paxillin tyrosine phosphorylation was reduced. These findings support the concepts that CrkII binding activity is regulated by tyrosine kinases and phosphatases, and that tyrosine phosphorylation of CrkII can downmodulate cell migration mediated by the focal adhesion kinase/p130cas pathway.

A scaffold protein in the c-Jun N-terminal kinase signaling pathway is associated with focal adhesion kinase and tyrosine-phosphorylated.

Focal adhesion kinase (FAK) becomes activated and tyrosine-phosphorylated in response to cell adhesion to extracellular matrix proteins in a variety of cell types, and associates with a number of signaling molecules, structural proteins, and β integrin cytoplasmic domains. Here we demonstrated that c-Jun N-terminal kinase (JNK)/stress activated protein kinase-associated protein 1 (JSAP1), a scaffold factor in the mitogen-activated protein kinase (MAPK) cascades, forms a complex with the N-terminus of FAK. The complex formation was further stimulated by c-Src, in which JSAP1 was tyrosine-phosphorylated and other FAK/Src signaling molecules were recruited. Fibronectin (FN) stimulation of cells expressing JSAP1 induced its tyrosine phosphorylation concomitant with association with FAK. Expression of JSAP1 in Hela cells facilitated formation of well-organized focal contacts and actin stress fibers, and promoted cell spreading onto FN. Taken together, these results suggest that JSAP1 is involved an integrin-mediated signaling pathway through FAK/Src by recruiting other signaling molecules, resulting in promotion of cell spreading onto FN.

Increased matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase activity and expression in heterotopically transplanted murine tracheas.

BACKGROUNDnBronchiolitis obliterans syndrome (BOS) is the most common long-term cause of morbidity and mortality after heart-lung or lung transplantation. One pathologic feature of BOS is infiltration of fibroblasts and connective tissue products into the airway lumen, which form a fibrous, collagen-rich occlusion. Heterotopically transplanted allogeneic murine tracheal stenosis resemble BOS in the development of obliterans airway disease. Matrix metalloproteinases (MMPs) are key enzymes involved in tissue remodeling and, clinically, have several roles in pulmonary diseases. Among the MMP family, type IV collagenases, MMP-2 and MMP-9, have high gelatinolytic activity and are thought to play a role in several pulmonary diseases. Membrane type 1 MMP (MT1-MMP) activates the zymogen of MMP-2 (proMMP-2, 72 kd), and activated MMP-2 (active MMP-2, 62 kd) degrades type IV collagen and plays an important role in clinical pulmonary disease. In this study, we examine the expression of MMP-2, its activator MT1-MMP and MMP-9 in BOS using murine trachea transplantation models.nnnMETHODSnRats were divided into 5 experimental groups (n = 10 in each group). Group I was a control group with intact tracheas. Animals with tracheal grafts underwent heterotopically syngeneic (Groups II and III) or allogeneic (Groups IV and V) transplantation. The recipient rats were killed 7 days (Groups II and IV) or 28 days (Groups III and V) after transplantation. The harvested tracheal grafts were examined histologically. MMP activity was assessed using gelatin zymography analysis, and MMP-2 and MT1-MMP gene expression was examined by quantitative real-time polymerase chain reaction analysis. Distribution of gelatinolytic activity was studied using in situ zymography.nnnRESULTSnThere was little histologic change in the intact trachea (Group I) and in all isografts (Groups II and III). Fibrotic tissues in Group V significantly occluded the tracheal lumen, and there was severe lymphocyte infiltration in Group IV. According to gelatin zymography, proMMP-9 was faint at 7 days, but activated MMP-9 was not present in all groups. The MMP-2 gelatinolytic bands were predominant; the activation in Group V was significantly greater than that in Group IV, and in Group III it was significantly greater than that in Group II. Gene expression of both MMP-2 and MT1-MMP were significantly higher in Group V than in the other groups (p < 0.01), and MMP-2 was clearly activated. Gelatinolytic activity was localized in the fibrotic tissues or lymphocytes of thickening lumen after destruction of the epithelium by stenosis.nnnCONCLUSIONSnThese results demonstrate that MMP-2, together with its activator MT1-MMP, may have an important role in the development of BOS, which is associated with destruction of the tracheal epithelium, leading to fibrosis.

ANTI-METASTATIC EFFECT OF CAPECITABINE ON HUMAN COLON CANCER XENOGRAFTS IN NUDE MOUSE RECTUM

Capecitabine (N 4‐pentyloxycarbonyl‐5′‐deoxy‐5‐fluorocytidine) is a new fluoropyrimidine carbamate, which is converted to 5‐fluorouracil (5‐FU) by 3 sequential steps of enzyme reactions. We investigated the possibility of using capecitabine to prevent metastasis with a metastasis model of gastrointestinal cancer developed by the intrarectal injection of green fluorescent protein (GFP)‐expressing colon cancer HT‐29 cells (HT‐29‐GFP) into nude mice. Lung and lymph node metastasis in the HT‐29‐GFP rectal xenograft was assessed through both observation of GFP fluorescence and quantification of metastasis by amplification of a cancer‐related human DNA by TaqMan PCR. Furthermore, for each organ, we examined mRNA levels of cancer‐specific thymidine phosphorylase (dThdPase), which is an essential enzyme for capecitabine activation, by the quantitative RT‐PCR method. Capecitabine inhibited the HT‐29‐GFP xenograft growth by 60.8% and 43.8% in the subcutaneous and rectal xenograft models, respectively. Furthermore, it inhibited both lung and lymph node metastasis by 99.9%. dThdPase expression in the tumor cells of both the rectal xenograft and metastatic lung tumor cells was upregulated by 10.0‐ and 24.3‐fold that in the HT‐29‐GFP cells in vitro, respectively. These results indicated that capecitabine might effectively inhibit or suppress metastasis via upregulation of dThdPase expression. Capecitabine administration might be highly expected to reduce metastasis and improve survival of patients with gastrointestinal cancers.