Silencing of the MT1-MMP/ G6PT axis suppresses calcium mobilization by sphingosine-1-phosphate in glioblastoma cells

The contributions of membrane type-1 matrix metalloproteinase (MT1-MMP) and of the glucose-6-phosphate transporter (G6PT) in sphingosine-1-phosphate (S1P)-mediated Ca(2+) mobilization were assessed in glioblastoma cells. We show that gene silencing of MT1-MMP or G6PT decreased the extent of S1P-induced Ca(2+) mobilization, chemotaxis, and extracellular signal-related kinase phosphorylation. Chlorogenic acid and (-)-epigallocatechin-3-gallate, two diet-derived inhibitors of G6PT and of MT1-MMP, respectively, reduced S1P-mediated Ca(2+) mobilization. An intact MT1-MMP/G6PT signaling axis is thus required for efficient Ca(2+) mobilization in response to bioactive lipids such as S1P. Targeted inhibition of either MT1-MMP or G6PT may lead to reduced infiltrative and invasive properties of brain tumor cells.     

Ack1 mediates Cdc42-dependent cell migration and signaling to p130Cas

We previously showed that activation of the small GTPase Cdc42 promotes breast cell migration on a collagen matrix. Here we further define the signaling pathways that drive this response and show that Cdc42-mediated migration relies on the adaptor molecule p130(Cas). Activated Cdc42 enhanced p130(Cas) phosphorylation and its binding to Crk. Cdc42-driven migration and p130(Cas) phosphorylation were dependent on the Cdc42 effector Ack1 (activated Cdc42-associated kinase). Ack1 formed a signaling complex that also included Cdc42, p130(Cas), and Crk, formation of which was regulated by collagen stimulation. The interaction between Ack1 and p130(Cas) occurred through their respective SH3 domains, while the substrate domain of p130(Cas) was the major site of Ack1-dependent phosphorylation. Signaling through this complex is functionally relevant, because treatment with either p130(Cas) or Ack1 siRNA blocked Cdc42-induced migration. These results suggest that Cdc42 exerts its effects on cell migration in part through its effector Ack1, which regulates p130(Cas) signaling.     

Invasive matrix degradation at focal adhesions occurs via protease recruitment by a FAK-p130Cas complex

Tumor cell migration and the concomitant degradation of extracellular matrix (ECM) are two essential steps in the metastatic process. It is well established that focal adhesions (FAs) play an important role in regulating migration; however, whether these structures contribute to matrix degradation is not clear. In this study, we report that multiple cancer cell lines display degradation of ECM at FA sites that requires the targeted action of MT1-MMP. Importantly, we have found that this MT1-MMP targeting is dependent on an association with a FAK-p130Cas complex situated at FAs and is regulated by Src-mediated phosphorylation of Tyr 573 at the cytoplasmic tail of MT1. Disrupting the FAK-p130Cas-MT1 complex significantly impairs FA-mediated degradation and tumor cell invasion yet does not appear to affect invadopodia formation or function. These findings demonstrate a novel function for FAs and also provide molecular insights into MT1-MMP targeting and function.     

MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21^WAF1 and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin α_vβ₃ were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.     

Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5

Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the gamma2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2- cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2- cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.     

Membrane-type 1 matrix metalloproteinase modulates focal adhesion stability and cell migration

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in extracellular matrix-induced cell migration and the activation of extracellular signal-regulated kinase (ERK). We showed here that transfection of the MT1-MMP gene into HeLa cells promoted fibronectin-induced cell migration, which was accompanied by fibronectin degradation and reduction of stable focal adhesions, which function as anchors for actin-stress fibers. MT1-MMP expression attenuated integrin clustering that was induced by adhesion of cells to fibronectin. The attenuation of integrin clustering was abrogated by MT1-MMP inhibition with a synthetic MMP inhibitor, BB94. When cultured on fibronectin, HT1080 cells, which endogenously express MT1-MMP, showed so-called motile morphology with well-organized focal adhesion formation, well-oriented actin-stress fiber formation, and the lysis of fibronectin through trails of cell migration. Inhibition of endogenous MT1-MMP by BB94 treatment or expression of the MT1-MMP carboxyl-terminal domain, which negatively regulates MT1-MMP activity, resulted in the suppression of fibronectin lysis and cell migration. BB94 treatment promoted stable focal adhesion formation concomitant with enhanced phosphorylation of tyrosine 397 of focal adhesion kinase (FAK) and reduced ERK activation. These results suggest that lysis of the extracellular matrix by MT1-MMP promotes focal adhesion turnover and subsequent ERK activation, which in turn stimulates cell migration.     

MT1-MMP downregulation via the PI3K/Akt signaling pathway is required for the mechanical stretching-inhibited invasion of bone-marrow-derived mesenchymal stem cells

Mobilization from the bone marrow and the migration of bone-marrow-derived mesenchymal stem cells (BMSCs) through the peripheral circulation to injured tissue sites are regulated by multiple mechanical and chemical factors. We previously demonstrated that mechanical stretching promotes the migration but inhibits the invasion of BMSCs. However, the involved mechanisms, especially the mechanism of stretching-inhibited BMSC invasion, have not been thoroughly elucidated to date. In this study, we found that mechanical stretching with a 10% amplitude at a 1-Hz frequency for 8 hr significantly reduces BMSC invasion and downregulates the expression of membrane type-1 matrix metalloproteinases (MT1-MMP) at both the messenger RNA and protein levels. The overexpression of MT1-MMP restores mechanical stretching-reduced BMSC invasion. Moreover, phosphatidylinositol 3-kinase (PI3K)-dependent Akt phosphorylation in BMSCs was found to be inactivated by mechanical stretching. Pharmacological inhibitors of PI3K/Akt signaling (LY294002 or A443654) reduced the expression of MT1-MMP and impaired BMSC invasion. In addition, the upregulation of Akt phosphorylation by a pharmacological activator (SC79) increased MT1-MMP expression and suppressed mechanical stretching-reduced BMSC invasion. Taken together, our results suggest that mechanical stretching inhibits BMSC invasion by downregulating MT1-MMP expression by suppressing the PI3K/Akt signaling pathway.     

Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.     

Phosphorylation of membrane type 1-matrix metalloproteinase (MT1-MMP) and its vesicle-associated membrane protein 7 (VAMP7)-dependent trafficking facilitate cell invasion and migration

In multicellular organisms, uncontrolled movement of cells can contribute to pathological conditions, such as multiple sclerosis and cancer. In highly aggressive tumors, the expression of matrix metalloproteinases (MMPs) is linked to the capacity of tumor cells to invade surrounding tissue and current research indicates that the membrane-anchored membrane type 1-matrix metalloproteinase (MT1-MMP) has a central role in this process. Endocytosis and trafficking of MT1-MMP are essential for its proper function, and here we examine the phosphorylation, internalization, and recycling of this enzyme, and the associated biochemical signaling in HeLa and HT-1080 fibrosarcoma cells. Activation of protein kinase C with phorbol 12-myristate 13-acetate resulted in phosphorylation of endogenous MT1-MMP at Thr(567) in vivo. Mutation of Thr(567) to alanine (to mimic non-phosphorylated MT1-MMP) reduced internalization of MT1-MMP, whereas mutation of Thr(567) to glutamic acid (to mimic phosphorylation) resulted in decreased levels of MT1-MMP on the cell surface. The endosomal trafficking and recycling of MT1-MMP was found to be dependent upon Rab7 and VAMP7, and blocking the function of these proteins reduced cell migration and invasion. Intracellular trafficking of MT1-MMP was observed to be coupled to the trafficking of integrin α5 and phosphorylation of ERK that coincided with this was dependent on phosphorylation of MT1-MMP. Together, these results reveal important roles for MT1-MMP phosphorylation and trafficking in both cell signaling and cell invasion.     

FAK-mediated src phosphorylation of endophilin A2 inhibits endocytosis of MT1-MMP and promotes ECM degradation

Focal adhesion kinase (FAK) is an important mediator of integrin signaling in the regulation of cell proliferation, survival, migration, and invasion. To understand how FAK contributes to cell invasion, we explored the regulation of matrix metalloproteinases (MMPs) by FAK. We found that v-Src-transformed cells activate a FAK-dependent mechanism that attenuates endocytosis of MT1-MMP. This in turn increases cell-surface expression of MT1-MMP and cellular degradation of extracellular matrix. Further, we identified an interaction between FAK's second Pro-rich motif and endophilin A2's SH3 domain. This interaction served as an autophosphorylation-dependent scaffold to allow Src phosphorylation of endophilin A2 at Tyr315. Tyr315 phosphorylation inhibited endophilin/dynamin interactions, and blockade of Tyr315 phosphorylation promoted endocytosis of MT1-MMP. Together, these results suggest a regulatory mechanism of cell invasion whereby FAK promotes cell-surface presentation of MT1-MMP by inhibiting endophilin A2-dependent endocytosis.     

BST-2 binding with cellular MT1-MMP blocks cell growth and migration via decreasing MMP2 activity

MT1-MMP (membrane type 1-matrix metalloproteinase) plays important roles in cell growth and tumor invasion via mediating cleavage of MMP2/gelatinase A and a variety of substrates including type I collagen. BST-2 (bone marrow stromal cell antigen 2) is a membrane tetherin whose expression dramatically reduces the release of a broad range of enveloped viruses including HIV from infected cells. In this study, we provided evidence that both transient and IFN-α induced BST-2 could decrease the activity of MMP2 via binding to cellular MT1-MMP on its C-terminus and inhibiting its proteolytic activity; and finally block cell growth and migration. Zymography gel and Western blot experiments demonstrated that BST-2 decreased MMP2 activity, but no effect on the expression of MMP2 and MT1-MMP genes. Confocal and immunoprecipitation data showed that BST-2 co-localized and interacted with MT1-MMP. This interaction inhibited the proteolytic enzyme activity of MT1-MMP, and blocked the activation of proMMP2. Experimental results of C-terminus deletion mutant of MT1-MMP showed that activity of MMP2 was no change and also no interaction existed between the mutant and BST-2 after co-transfection with the mutant and BST-2. It meant that C-terminus of MT1-MMP played a key role in the interaction with BST-2. In addition, cell growth in 3D type I collagen gel lattice and cell migration were all inhibited by BST-2. Taken together, BST-2, as a membrane protein and a tetherin of enveloped viruses, was a novel inhibitor of MT1-MMP and could be considerable as an inhibitor of cancer cell growth and migration on clinic.     

CD44 directs membrane-type 1 matrix metalloproteinase to lamellipodia by associating with its hemopexin-like domain

Membrane-type 1 matrix metalloproteinase (MT1- MMP) localizes at the front of migrating cells and degrades the extracellular matrix barrier during cancer invasion. However, it is poorly understood how the polarized distribution of MT1-MMP at the migration front is regulated. Here, we demonstrate that MT1-MMP forms a complex with CD44H via the hemopexin-like (PEX) domain. A mutant MT1-MMP lacking the PEX domain failed to bind CD44H and did not localize at the lamellipodia. The cytoplasmic tail of CD44H, which comprises interfaces that associate with the actin cytoskeleton, was important for its localization at lamellipodia. Overexpression of a CD44H mutant lacking the cytoplasmic tail also prevented MT1-MMP from localizing at the lamellipodia. Modulation of F-actin with cytochalasin D revealed that both CD44H and MT1-MMP co-localize closely with the actin cytoskeleton, dependent on the cytoplasmic tail of CD44H. Thus, CD44H appears to act as a linker that connects MT1-MMP to the actin cytoskeleton and to play a role in directing MT1-MMP to the migration front. The PEX domain of MT1-MMP was indispensable in promoting cell migration and CD44H shedding.     

NHE1 mediates MDA-MB-231 cells invasion through the regulation of MT1-MMP

Na⁺/H⁺ exchanger 1 (NHE1), an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been shown to play a key role in breast cancer metastasis. However, the exact mechanism by which NHE1 mediates breast cancer metastasis is not yet well known. We showed here that inhibition of NHE1 activity, with specific inhibitor Cariporide, could suppress MDA-MB-231 cells invasion as well as the activity and expression of MT1-MMP. Overexpression of MT1-MMP resulted in a distinguished increase in MDA-MB-231 cells invasiveness, but treatment with Cariporide reversed the MT1-MMP-mediated enhanced invasiveness. To explore the role of MAPK signaling pathways in NHE1-mediated breast cancer metastasis, we compared the difference of constitutively phosphorylated ERK1/2, p38 MAPK and JNK in non-invasive MCF-7 cells and invasive MDA-MB-231cells. Interestingly, we found that the phosphorylation levels of ERK1/2 and p38 MAPK in MDA-MB-231 cells were higher than in MCF-7 cells, but both MCF-7 cells and MDA-MB-231 cells expressed similar constitutively phosphorylated JNK. Treating MDA-MB-231 cells with Cariporide led to decreased phosphorylation level of both p38 MAPK and ERK1/2 in a time-dependent manner, but JNK activity was not influenced. Supplementation with MAPK inhibitor (MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125) or Cariporide all exhibited significant depression of MDA-MB-231 cells invasion and MT1-MMP expression. Furthermore, we co-treated MDA-MB-231 cells with MAPK inhibitor and Cariporide. The result showed that Cariporide synergistically suppressed invasion and MT1-MMP expression with MEK inhibitor and p38 MAPK inhibitor, but not be synergistic with the JNK inhibitor. These findings suggest that NHE1 mediates MDA-MB-231 cells invasion partly through regulating MT1-MMP in ERK1/2 and p38 MAPK signaling pathways dependent manner.     

CrkI and p130(Cas) complex regulates the migration and invasion of prostate cancer cells

Prostate cancer metastasis is often associated with poor prognosis. The molecular coupling of the adaptor protein Crk to the docking protein p130(Cas) serves as a switch that regulates cell migration in several invasive cancer cells and Ack appears to act upstream of CrkII to modulate the cell motility. However, the precise role of Ack, Crk and p130(Cas) complex in prostate cancer migration remains unknown. In this study we examined the expression of Crk and p130(Cas) in prostate cancer cell lines, and found that CrkI and p130(Cas) protein level was higher in highly invasive PC-3M and PC-3 cell lines than in moderately invasive DU-145 cells. Upon shRNA mediated knockdown of CrkI and p130(Cas) in PC-3M cells, cell migration and invasion were significantly inhibited as analyzed by wound healing assay and transwell invasion assay. Furthermore, co-immunoprecipitation assay showed that p130(Cas) interacted with CrkI in PC-3M cells and the stability of p130(Cas) and CrkI depended on each other. AckI interacted with both CrkI and p130(Cas) and the interaction of AckI with CrkI seemed to be independent of p130(Cas) . Taken together, our results demonstrate the high expression of CrkI and p130(Cas) in invasive prostate cancer cells and the important role of CrkI/p130(Cas) complex in the migration and invasion of prostate cancer cells. These data suggest that CrkI/p130(Cas) could be exploited as potential molecular therapeutic target for prostate cancer metastasis.     

Collagen IV regulates Caco-2 cell spreading and p130Cas phosphorylation by FAK-dependent and FAK-independent pathways

We previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src-dependent p130(Cas) phosphorylation and stimulates focal adhesion kinase (FAK). However, the role of FAK and the related kinase, Pyk2, in Caco-2 spreading and migration is unclear. FAK- or Pyk2-specific siRNAs reduced protein levels by 90%. However, when detached cells were replated on collagen IV neither individual nor combined FAK and Pyk2 siRNAs affected the cell spreading rate. As combined FAK and Pyk2 siRNAs increased p130(Cas) protein levels, we cotransfected cells with 1 nm p130(Cas) siRNA to partially reduce p130(Cas) protein to control levels. Although p130(Cas) Tyr(P)(249) phosphorylation was reduced by 60%, cell spreading was unaffected. Combined siRNA reduction of FAK, Pyk2 and p130(Cas) increased cell spreading by 20% compared to p130(Cas) siRNA alone, suggesting that FAK and Pyk2 negatively regulate spreading in addition to stimulating spreading via p130(Cas). FAK-binding mutant SH3 domain-deleted rat p130(Cas) was not phosphorylated after adhesion and, unlike full-length p130(Cas), did not restore spreading after human-specific p130(Cas) siRNA knockdown of endogenous p130(Cas). Together, these data suggest that FAK positively regulates Caco-2 spreading on collagen IV via p130(Cas) phosphorylation, but also suggests that FAK may negatively regulate spreading through other mechanisms and the presence of additional FAK-independent pathways regulating p130(Cas).     

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.     

Membrane-type 1-matrix metalloproteinase regulates intracellular adhesion molecule-1 (ICAM-1)-mediated monocyte transmigration

We examined the mechanism regulating intercellular cell adhesion molecule-1 (ICAM-1)-dependent monocyte transendothelial migration. Monocyte migration through endothelial cells expressing ICAM-1 alone was comparable to that of tumor necrosis factor-alpha-treated cells. Transmigration was reduced in ICAM-1 lacking the cytoplasmic tail and in tyrosine to alanine substitutions at Tyr-485 and Tyr-474. Tissue inhibitors of matrix metalloproteinases (TIMPs) -2 and -3 blocked transmigration, whereas TIMP-1 was ineffective. This profile suggested a role for membrane-type matrix metalloproteinases (MT-MMPs) in transmigration. Inhibitory antibodies and small interference RNA directed against MT1-MMP blocked transmigration, whereas overexpression of MT1-MMP in endothelial cells or monocytes promoted transmigration. MT1-MMP mediated the ectodomain cleavage of ICAM-1 that was blocked by TIMP-2 and -3. Overexpression of MT1-MMP rescued function in ICAM-1Y485A, and to a lesser extent in the cytoplasmic tail-deleted ICAM-1. In a binding assay, wild-type ICAM-1 bound to purified MT1-MMP while ICAM-1 mutants bound poorly. MT1-MMP co-localized with ICAM-1 at distinct structures in endothelial cells. MT1-MMP localization with cells expressing ICAM-1 mutations was reduced and diffused. These results indicate that the cytoplasmic tail of ICAM-1 regulates leukocyte transmigration through MT1-MMP interaction.     

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.     

Human hemokinin-1 promotes migration of melanoma cells and increases MMP-2 and MT1-MMP expression by activating tumor cell NK1 receptors

Receptors and their regulatory peptides are aberrantly expressed in tumors, suggesting a potential tumor therapy target. Human hemokinin-1 (hHK-1) is a tachykinin peptide ligand of the neurokinin-1 (NK1) receptor which is overexpressed in melanoma and other tumor tissues. Here, we investigated the role of hHK-1 and the NK1 receptor in melanoma cell migration. NK1 receptor expression was associated with melanoma metastatic potential. Treatment with hHK-1 significantly enhanced A375 and B16F10 melanoma cell migration and an NK1 receptor antagonist L732138 blocked this effect. MMP-2 and MT1-MMP expression were up-regulated in hHK-1-treated melanoma cells and cell signaling data suggested that hHK-1 induced phosphorylation of ERK1/2, JNK and p38 by way of PKC or PKA. Kinase activation led to increased MMP-2 and MT1-MMP expression and melanoma cell migration induced by hHK-1. Thus, hHK-1 and the NK1 receptor are critical to melanoma cell migration and each may be a promising chemotherapeutic target.