ADAM15 mediates upregulation of Claudin-1 expression in breast cancer cells.

A Disintegrin and Metalloproteinase-15 (ADAM15) is a transmembrane protein involved in protein ectodomain shedding, cell adhesion and signalling. We previously cloned and characterised alternatively spliced variants of ADAM15 that differ in their intracellular domains and demonstrated correlation of the expression of specific variants with breast cancer prognosis. In this study we have created isogenic cell panels (MDA-MB-231 and MCF-7) expressing five ADAM15 variants including wild-type and catalytically inactive forms. The expression of ADAM15 isoforms in MDA-MB-231 cells led to cell clustering to varying degree, without changes in EMT markers vimentin, slug and E-cadherin. Analysis of tight junction molecules revealed ADAM15 isoform specific, catalytic function dependent upregulation of Claudin-1. The expression of ADAM15A, and to a lesser degree of C and E isoforms led to an increase in Claudin-1 expression in MDA-MB-231 cells, while ADAM15B had no effect. In MCF-7 cells, ADAM15E was the principal variant inducing Claudin-1 expression. Sh-RNA mediated down-regulation of ADAM15 in ADAM15 over-expressing cells reduced Claudin-1 levels. Additionally, downregulation of endogenous ADAM15 expression in T47D cells by shRNA reduced endogenous Claudin-1 expression confirming a role for ADAM15 in regulating Claudin-1 expression. The PI3K/Akt/mTOR pathway was involved in regulating Claudin-1 expression downstream of ADAM15. Immunofluorescence analysis of MDA-MB-231 ADAM15A expressing cells showed Claudin-1 at cell-cell junctions, in the cytoplasm and nuclei. ADAM15 co-localised with Claudin-1 and ZO1 at cell-cell junctions. Immunoprecipitation analysis demonstrated complex formation between ADAM15 and ZO1/ZO2. These findings highlight the importance of ADAM15 Intra Cellular Domain-mediated interactions in regulating substrate selection and breast cancer cell phenotype.

Metalloproteinase-dependent and -independent processes contribute to inhibition of breast cancer cell migration, angiogenesis and liver metastasis by a disintegrin and metalloproteinase with thrombospondin motifs-15.

The ADAMTS proteinases are a family of secreted, matrix-associated enzymes that have diverse roles in the regulation of tissue organization and vascular homeostasis. Several of the 19 human family members have been identified as having either tumor promoting or suppressing roles. We previously demonstrated that decreased ADAMTS15 expression correlated with a worse clinical outcome in mammary carcinoma (e.g., Porter et al., Int J Cancer 2006;118:1241-7). We have explored the effects of A Disintegrin and Metalloproteinase with Thrombospondin motifs-15 (ADAMTS-15) on the behavior of MDA-MB-231 and MCF-7 breast cancer cells by stable expression of either a wild-type (wt) or metalloproteinase-inactive (E362A) protein. No effects on mammary cancer cell proliferation or apoptosis were observed for either form of ADAMTS-15. However, both forms reduced cell migration on fibronectin or laminin matrices, though motility on a Type I collagen matrix was unimpaired. Knockdown of syndecan-4 attenuated the inhibitory effects of ADAMTS-15 on cell migration. In contrast to its effects on cell migration, wt ADAMTS-15 but not the E362A inactive mutant inhibited endothelial tubulogenesis in 3D collagen gels and angiogenesis in the aortic ring assay. In experimental metastasis assays in nude mice, MDA-MB-231 cells expressing either form of ADAMTS-15 showed reduced spread to the liver, though lung colonization was enhanced for cells expressing wt ADAMTS-15. These studies indicate that extracellular ADAMTS-15 has multiple actions on tumor pathophysiology. Via modulation of cell-ECM interactions, which likely involve syndecan-4, it attenuates mammary cancer cell migration independent of its metalloproteinase activity; however, its antiangiogenic action requires catalytic functionality, and its effects on metastasis in vivo are tissue niche-dependent.

Membrane type 1 matrix metalloproteinase (MT1-MMP) ubiquitination at Lys581 increases cellular invasion through type I collagen.

Membrane type 1 matrix metalloproteinase (MT1-MMP/MMP14) is a zinc-dependent type I transmembrane metalloproteinase playing pivotal roles in the regulation of pericellular proteolysis and cellular migration. Elevated expression levels of MT1-MMP have been demonstrated to correlate with a poor prognosis in cancer. MT1-MMP has a short intracellular domain (ICD) that has been shown to play important roles in cellular migration and invasion, although these ICD-mediated mechanisms remain poorly understood. In this study, we report that MT1-MMP is mono-ubiquitinated at its unique lysine residue (Lys(581)) within the ICD. Our data suggest that this post-translational modification is involved in MT1-MMP trafficking as well as in modulating cellular invasion through type I collagen matrices. By using an MT1-MMP Y573A mutant or the Src family inhibitor PP2, we observed that the previously described Src-dependent MT1-MMP phosphorylation is a prerequisite for ubiquitination. Taken together, these findings show for the first time an additional post-translational modification of MT1-MMP that regulates its trafficking and cellular invasion, which further emphasizes the key role of the MT1-MMP ICD.

MT1-MMP regulates VEGF-A expression through a complex with VEGFR-2 and Src.

Membrane-type-1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent type-I transmembrane metalloproteinase involved in pericellular proteolysis, migration and invasion, with elevated levels correlating with a poor prognosis in cancer. MT1-MMP-mediated transcriptional regulation of genes in cancer cells can contribute to tumour growth, although this is poorly understood at a mechanistic level. In this study, we investigated the mechanism by which MT1-MMP regulates the expression of VEGF-A in breast cancer cells. We discovered that MT1-MMP regulates VEGFR-2 cell surface localisation and forms a complex with VEGFR-2 and Src that is dependent on the MT1-MMP hemopexin domain and independent of its catalytic activity. Although the localisation of VEGFR-2 was independent of the catalytic and intracellular domain of MT1-MMP, intracellular signalling dependent on VEGFR-2 activity leading to VEGF-A transcription still required the MT1-MMP catalytic and intracellular domain, including residues Y573, C574 and DKV582. However, there was redundancy in the function of the catalytic activity of MT1-MMP, as this could be substituted with MMP-2 or MMP-7 in cells expressing inactive MT1-MMP. The signalling cascade dependent on the MT1-MMP-VEGFR-2-Src complex activated Akt and mTOR, ultimately leading to increased VEGF-A transcription.

Peptide aptamers as new tools to modulate clathrin-mediated internalisation--inhibition of MT1-MMP internalisation.

BACKGROUND: Peptide aptamers are combinatorial protein reagents that bind to targets with a high specificity and a strong affinity thus providing a molecular tool kit for modulating the function of their targets in vivo. RESULTS: Here we report the isolation of a peptide aptamer named swiggle that interacts with the very short (21 amino acid long) intracellular domain of membrane type 1-metalloproteinase (MT1-MMP), a key cell surface protease involved in numerous and crucial physiological and pathological cellular events. Expression of swiggle in mammalian cells was found to increase the cell surface expression of MT1-MMP by impairing its internalisation. Swiggle interacts with the LLY573 internalisation motif of MT1-MMP intracellular domain, thus disrupting the interaction with the mu2 subunit of the AP-2 internalisation complex required for endocytosis of the protease. Interestingly, swiggle-mediated inhibition of MT1-MMP clathrin-mediated internalisation was also found to promote MT1-MMP-mediated cell migration. CONCLUSIONS: Taken together, our results provide further evidence that peptide aptamers can be used to dissect molecular events mediated by individual protein domains, in contrast to the pleiotropic effects of RNA interference techniques.

Golgi reassembly stacking protein 55 interacts with membrane-type (MT) 1-matrix metalloprotease (MMP) and furin and plays a role in the activation of the MT1-MMP zymogen.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a proteinase involved in the remodelling of extracellular matrix and the cleavage of a number of substrates. MT1-MMP is synthesized as a zymogen that requires intracellular post-translational cleavage to gain biological activity. Furin, a member of the pro-protein convertase family, has been implicated in the proteolytic removal of the MT1-MMP prodomain sequence. In the present study, we demonstrate a role for the peripheral Golgi matrix protein GRASP55 in the furin-dependent activation of MT1-MMP. MT1-MMP and furin were found to co-localize with Golgi reassembly stacking protein 55 (GRASP55). Further analysis revealed that GRASP55 associated with the cytoplasmic domain of both proteases and that the LLY(573) motif in the MT1-MMP intracellular domain was crucial for the interaction with GRASP55. Overexpression of GRASP55 was found to enhance the formation of a complex between MT1-MMP and furin. Finally, we report that disruption of the interaction between GRASP55 and furin led to a reduction in pro-MT1-MMP activation. Taken together, these data suggest that GRASP55 may function as an adaptor protein coupling MT1-MMP with furin, thus leading to the activation of the zymogen.

Membrane type-1 matrix metalloproteinase activity is regulated by the endocytic collagen receptor Endo180.

The molecular interactions leading to organised, controlled extracellular matrix degradation are of central importance during growth, development and tissue repair, and when deregulated contribute to disease processes including cancer cell invasion. There are two major pathways for collagen degradation: one dependent on secreted and membrane-bound collagenases, the other on receptor-mediated collagen internalisation and intracellular processing. Despite the established importance of both pathways, the functional interaction between them is largely unknown. We demonstrate here, that the collagen internalisation receptor Endo180 (also known as CD280, uPARAP, MRC2) is a novel regulator of membrane-bound matrix metalloproteinase (MT1-MMP) activity, MT1-MMP-dependent MMP-2 activation and urokinase plasminogen activator (uPA) activity. We show close correlation between Endo180 expression, collagen accumulation and regulation of MT1-MMP cell-surface localisation and activity. We directly demonstrate, using collagen inhibition studies and non-collagen-binding mutants of Endo180, that the molecular mechanism underlying this regulation is the ability of Endo180 to bind and/or internalise collagens, rather than by acting as an interaction partner for pro-uPA and its receptor uPAR. These studies strongly support a functional interaction between two distinct collagen degradation pathways, define a novel mechanism regulating MT1-MMP activity and might have important implications for organised collagen clearance in the pericellular environment.

Membrane-type 1 matrix metalloproteinase stimulates cell migration through epidermal growth factor receptor transactivation.

Proteolysis of extracellular matrix proteins by membrane-type 1 matrix metalloproteinase (MT1-MMP) plays a pivotal role in tumor and endothelial cell migration. In addition to its proteolytic activity, several studies indicate that the proinvasive properties of MT1-MMP also involve its short cytoplasmic domain, but the specific mechanisms mediating this function have yet to be fully elucidated. Having previously shown that the serum factor sphingosine 1-phosphate stimulates MT1-MMP promigratory function through a process that involves its cytoplasmic domain, we now extend these findings to show that this cooperative interaction is permissive to cellular migration through MT1-MMP-dependent transactivation of the epidermal growth factor receptor (EGFR). In the presence of sphingosine 1-phosphate, MT1-MMP stimulates EGFR transactivation through a process that is dependent upon the cytoplasmic domain of the enzyme but not its catalytic activity. The MT1-MMP-induced EGFR transactivation also involves G(i) protein signaling and Src activities and leads to enhanced cellular migration through downstream extracellular signal-regulated kinase activation. The present study, thus, elucidates a novel role of MT1-MMP in signaling events mediating EGFR transactivation and provides the first evidence of a crucial role of this receptor activity in MT1-MMP promigratory function. Taken together, our results suggest that the inhibition of EGFR may represent a novel target to inhibit MT1-MMP-dependent processes associated with tumor cell invasion and angiogenesis.

Src-dependent phosphorylation of membrane type I matrix metalloproteinase on cytoplasmic tyrosine 573: role in endothelial and tumor cell migration.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr(573)) and that this phosphorylation requires the kinase Src. Using phosphospecific antibodies recognizing MT1-MMP phosphorylated on Tyr(573), we observed that tyrosine phosphorylation of the enzyme is rapidly induced upon stimulation of tumor and endothelial cells with the platelet-derived chemoattractant sphingosine-1-phosphate, suggesting a role in migration triggered by this lysophospholipid. Accordingly, overexpression of a nonphosphorylable MT1-MMP mutant (Y573F) blocked sphingosine-1-phosphate-induced migration of Human umbilical vein endothelial cells and HT-1080 (human fibrosarcoma) cells and failed to stimulate migration of cells lacking the enzyme (bovine aortic endothelial cells). Altogether, these findings strongly suggest that the Src-dependent tyrosine phosphorylation of MT1-MMP plays a key role in cell migration and further emphasize the importance of the cytoplasmic domain of the enzyme in this process.

MT1-MMP hemopexin domain exchange with MT4-MMP blocks enzyme maturation and trafficking to the plasma membrane in MCF7 cells.

The hemopexin-like domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) enables MT1-MMP to form oligomers that facilitate the activation of pro-matrix metalloproteinase-2 (pro-MMP-2) at the cell surface. To investigate the role of the MT1-MMP hemopexin domain in the trafficking of MT1-MMP to the cell surface we have examined the activity of two MT1-MT4-MMP chimaeras in which the hemopexin domain of MT1-MMP has been replaced with that of human or mouse MT4-MMP. We show that MT1-MMP bearing the hemopexin domain of MT4-MMP was incapable of activating pro-MMP-2 or degrading gelatin in cell based assays. Furthermore, cell surface biotinylation and indirect immunofluorescence show that transiently expressed MT1-MT4-MMP chimaeras failed to reach the plasma membrane and were retained in the endoplasmic reticulum. Functional activity could be restored by replacing the MT4-MMP hemopexin domain with the wild-type MT1-MMP hemopexin domain. Subsequent analysis with an antibody specifically recognising the propeptide of MT1-MMP revealed that the propeptides of the MT1-MT4-MMP chimaeras failed to undergo proper processing. It has previously been suggested that the hemopexin domain of MT4-MMP could exert a regulatory mechanism that prevents MT4-MMP from activating pro-MMP-2. In this report, we demonstrate unambiguously that MT1-MT4-MMP chimaeras do not undergo normal trafficking and are not correctly processed to their fully active forms and, as a consequence, they are unable to activate pro-MMP-2 at the cell surface.

Membrane type 1-matrix metalloproteinase induces endothelial cell morphogenic differentiation by a caspase-dependent mechanism.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) has been suggested to play an essential role in angiogenesis. Based on recent evidence suggesting that the sprouting and branching of capillaries during angiogenesis involves apoptosis, we investigated the involvement of this process in MT1-MMP-dependent morphogenic differentiation of EC into capillary-like structures. We found that MT1-MMP sensitizes EC to apoptosis, since reduction of MT1-MMP expression abolished vimentin fragmentation in apoptotic HUVECs while overexpression of the enzyme induced caspase-3 activity in BAECs subjected to pro-apoptotic treatments. MT1-MMP-mediated caspase-3 activation likely occurs through the mitochondrial pathway since it was abrogated by Bcl-2, but not by CrmA overexpression. Reduction of MT1-MMP expression in HUVECs reduced morphogenic differentiation that was correlated with diminished vimentin fragmentation, whereas its overexpression in BAECs stimulated both processes. Inactivation of the catalytic activity or removal of the cytoplasmic domain of MT1-MMP markedly reduced its ability to induce both morphogenic differentiation and caspase-3 activation. The inhibitory effects of the anti-apoptotic protein Bcl-2 and the caspase inhibitor zVAD-fmk further suggested the involvement of apoptosis during MT1-MMP-mediated morphogenic differentiation. Our results show that the ability of MT1-MMP to induce EC morphogenic differentiation involves its activation of a caspase-dependent mechanism.

Src-mediated tyrosine phosphorylation of caveolin-1 induces its association with membrane type 1 matrix metalloproteinase.

We have recently shown that stimulation of endothelial cells with vascular endothelial growth factor (VEGF) induces dissociation of caveolin-1 from the VEGFR-2 receptor, followed by Src family kinase-dependent tyrosine phosphorylation of the protein (Labrecque, L., Royal, I., Surprenant, D. S., Patterson, C., Gingras, D., and Beliveau, R. (2003) Mol. Biol. Cell 14, 334-347). In this study, we provide evidence that the VEGF-dependent tyrosine phosphorylation of caveolin-1 induces interaction of the protein with the membrane-type 1 matrix metalloproteinase (MT1-MMP). This interaction requires the phosphorylation of caveolin-1 on tyrosine 14 by members of the Src family of protein kinases, such as Src and Fyn, because it is completely abolished by expression of a catalytically inactive Src mutant or by site-directed mutagenesis of tyrosine 14 of caveolin-1. Most interestingly, the association of MT1-MMP with phosphorylated caveolin-1 induced the recruitment of Src and a concomitant inhibition of the kinase activity of the enzyme, suggesting that this complex may be involved in the negative regulation of Src activity. The association of MT1-MMP with phosphorylated caveolin-1 occurs in caveolae membranes and involves the cytoplasmic domain of MT1-MMP because it was markedly reduced by mutation of Cys574 and Val582 residues of the cytoplasmic tail of the enzyme. Most interestingly, the reduction of the interaction between MT1-MMP and caveolin-1 by using these mutants also decreases MT1-MMP-dependent cell locomotion. Overall these results indicate that MT1-MMP associates with tyrosine-phosphorylated caveolin-1 and that this complex may play an important role in MT1-MMP regulation and function.

Up-regulation of vascular endothelial growth factor-A by active membrane-type 1 matrix metalloproteinase through activation of Src-tyrosine kinases.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF) are two key molecules involved in pericellular proteolysis and cell proliferation during tumor growth and angiogenesis. Our previous data showed that MT1-MMP overexpression in human breast carcinoma MCF7 cells induced an up-regulation of VEGF expression. This effect was associated in vivo with accelerated tumor growth and angiogenesis. We now provide evidence that MT1-MMP overexpression specifically affected VEGF-A production and failed to influence that of other VEGF family members (VEGF, B, C, D, or PlGF) or their receptors. The up-regulation of VEGF-A by MT1-MMP was related to an increased transcriptional activation rather than to a modification of mRNA stability. It was blocked by synthetic MMP inhibitors, TIMP2, but not TIMP-1 and abolished by a partial deletion of the catalytic domain or the cytoplasmic tail of MT1-MMP. Analysis of the signal transduction mechanisms demonstrated that MT1-MMP acts through a signaling pathway involving Src tyrosine kinases. Thus, our results provide new insight into the mechanisms of action of MT1-MMP during angiogenesis and suggest that the full enzymatic activity of MT1-MMP is required for a specific up-regulation of VEGF-A through an activation of Src tyrosine kinase pathways.

Membrane type I-matrix metalloproteinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is an integral type I transmembrane multidomain zinc-dependent endopeptidase involved in extracellular matrix remodelling in physiological as well as pathological processes. MT1-MMP participates in the regulated turnover of various extracellular matrix components as well as the activation of secreted metalloproteinases and the cleavage of various cell membrane components. MT1-MMP expression has been reported to correlate with the malignancy of various tumour types and is thought to be an important mediator of cell migration and invasion. Recently, it has been proposed that internalisation of the enzyme from the cell surface is a major short-term level of MT1-MMP regulation controlling the net amount of active enzyme present at the plasma membrane. In this paper we show that, in HT1080 fibrosarcoma cells, MT1-MMP is internalised from the cell surface and colocalises with various markers of the endocytic compartment. Interestingly, we observed that in these cells, internalisation occurs by a combination of both clathrin-mediated and -independent pathways, most probably involving caveolae. In addition, internalised MT1-MMP is recycled to the cell surface, which could, in addition to downregulation of the enzymatic activity, represent a rapid response mechanism used by the cell for relocalising active MT1-MMP at the leading edge during migration.

Cell cycle regulation of pEg3, a new Xenopus protein kinase of the KIN1/PAR-1/MARK family.

We report the characterization of pEg3, a Xenopus protein kinase related to members of the KIN1/PAR-1/MARK family. The founding members of this newly emerging kinase family were shown to be involved in the establishment of cell polarity and both microtubule dynamic and cytoskeleton organization. Sequence analyses suggest that pEg3 and related protein kinases in human, mouse, and Caenorhabditis elegans might constitute a distinct group in this family. pEg3 is encoded by a maternal mRNA, polyadenylated in unfertilized eggs and specifically deadenylated in embryos. In addition to an increase in expression, we have shown that pEg3 is phosphorylated during oocyte maturation. Phosphorylation of pEg3 is cell cycle dependent during Xenopus early embryogenesis and in synchronized cultured XL2 cells. In embryos, the kinase activity of pEg3 is correlated to its phosphorylation state and is maximum during mitosis. Using Xenopus egg extracts we demonstrated that phosphorylation occurs at least in the noncatalytic domain of the kinase, suggesting that this domain might be important for pEg3 function.