Localization Of Membrane-type 1 Matrix Metalloproteinase Research Articles

Coordination of two kinesin superfamily motor proteins, KIF3A and KIF13A, is essential for pericellular matrix degradation by membrane-type 1 matrix metalloproteinase (MT1-MMP) in cancer cells

MT1-MMP plays a crucial role in promoting the cellular invasion of cancer cells by degrading the extracellular matrix to create a path for migration. During this process, its localization at the leading edge of migrating cells is critical, and it is achieved by targeted transport of MT1-MMP-containing vesicles along microtubules by kinesin superfamily motor proteins (KIFs). Here we identified three KIFs involved in MT1-MMP vesicle transport: KIF3A, KIF13A, and KIF9. Knockdown of KIF3A and KIF13A effectively inhibited MT1-MMP-dependent collagen degradation and invasion, while knockdown of KIF9 increased collagen degradation and invasion. Our data suggest that KIF3A/KIF13A dependent MT1-MMP vesicles transport takes over upon KIF9 knockdown. Live-cell imaging analyses have indicated that KIF3A and KIF13A coordinate to transport the same MT1-MMP-containing vesicles from the trans-Golgi to the endosomes, and KIF13A alone transports the vesicle from the endosome to the plasma membrane. Taken together, we have identified a unique interplay between three KIFs to regulate leading edge localization of MT1-MMP and MT1-MMP-dependent cancer cell invasion.

Abstract 1953: Expression and activity of MT1-MMP in endothelial cells is regulated by Semaphorin 4D

Abstract The semaphorins and plexins, originally shown to control axon growth cone guidance, are now known to influence many aspects of cell adhesion and migration. Our group and others have demonstrated that carcinomas over express Semaphorin 4D (S4D), particularly under conditions of hypoxia, for the purpose of supporting tumor-induced angiogenesis. However, S4D is a transmembrane protein that must be proteolytically cleaved to attract endothelial cells into a malignancy, a process that in head and neck squamous cell carcinoma (HNSCC) is controlled by membrane type 1-matrix metalloproteinase (MT1-MMP). We wanted to test the possibility that S4D acts through its receptor, Plexin-B1 (PB1), to enhance activity of endothelial cell MT1-MMP, facilitating extracellular matrix degradation and further promoting angiogenesis. We observed upregulated transcription, expression and cell surface localization of MT1-MMP in endothelial cells treated with S4D, occurring in a PB1, RhoA and NF-κB-dependent manner. We also demonstrated that S4D-induced endothelial cell collagen degradation, migration and capillary tube formation are suppressed when PB1 is silenced or its downstream effectors RhoA and NF-κB are inhibited. Endothelial MT1-MMP contributes to the ability of these cells to digest their way through the basement membrane and extracellular matrix, a requirement for the creation of the vascular networks that supply a developing tumor with oxygen and nutrients and allow it access to the systemic circulation for metastasis. These results demonstrate that S4D promotes angiogenesis at multiple levels, and suggest that it would be a valid target for anti-angiogenic intervention in the treatment of solid malignancies. Citation Format: Amr Bugshan, Zaid Khoury, Asma Buhamrah, John R. Basile. Expression and activity of MT1-MMP in endothelial cells is regulated by Semaphorin 4D [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1953.

MDia1 regulates breast cancer invasion by controlling membrane type 1-matrix metalloproteinase localization.

Mammalian diaphanous-related formin 1 (mDia1) expression has been linked with progression of malignant cancers in various tissues. However, the precise molecular mechanism underlying mDia1-mediated invasion in cancer cells has not been fully elucidated. In this study, we found that mDia1 is upregulated in invasive breast cancer cells. Knockdown of mDia1 in invasive breast cancer profoundly reduced invasive activity by controlling cellular localization of membrane type 1-matrix metalloproteinase (MT1-MMP) through interaction with microtubule tracks. Gene silencing and ectopic expression of the active form of mDia1 showed that mDia1 plays a key role in the intracellular trafficking of MT1-MMP to the plasma membrane through microtubules. We also demonstrated that highly invasive breast cancer cells possessed invasive activity in a 3D culture system, which was significantly reduced upon silencing mDia1 or MT1-MMP. Furthermore, mDia1-deficient cells cultured in 3D matrix showed impaired expression of the cancer stem cell marker genes, CD44 and CD133. Collectively, our findings suggest that regulation of cellular trafficking and microtubule-mediated localization of MT1-MMP by mDia1 is likely important in breast cancer invasion through the expression of cancer stem cell genes.

KIF1B promotes glioma migration and invasion via cell surface localization of MT1-MMP.

Malignant glioma is notorious for its aggressiveness and poor prognosis, and the invasiveness of glioma cells is the major obstacle. Accumulating evidence indicates that kinesin superfamily proteins (KIFs) may play key roles in tumor invasiveness, but the mechanisms remained unresolved. Our previous study demonstrated that membrane type 1-matrix metalloproteinase (MT1-MMP) was involved in Kinesin family member 1B (KIF1B)-modulated invasion of gastric cancer cells. Therefore, the role of KIF1B in glioma cell invasion and its relationship with MT1-MMP were explored in the present study. We found that aberrantly increased expression of KIF1B was associated with worse WHO pathological classification and Karnofsky performance status (KPS), which also showed a trend towards worse prognosis. In the transwell assay, knockdown of KIF1B using siRNA repressed U87MG and A172 glioma cell migration and invasion. Silencing KIF1B inhibited expression of membranal MT1-MMP; however, the amount of MT1-MMP in the whole cell lysate was not affected. In conclusion, targeting KIF1B may be an option for anti-invasive therapies targeting glioma.

The tetraspanin network modulates MT1-MMP cell surface trafficking

The membrane-type 1 matrix metalloproteinase (MT1-MMP) drives fundamental physiological and pathophysiological processes. Among other substrates, MT1-MMP cleaves components of the extracellular matrix and activates other matrix-cleaving proteases such as MMP-2. Trafficking is a highly effective means to modulate MT1-MMP cell surface expression, and hence regulate its function. Here, we describe the complex interaction of MT1-MMP with tetraspanins, their effects on MT1-MMP intracellular trafficking and proteolytic function. Tetraspanins are credited as membrane organizers that form a network within the membrane to regulate the trafficking of associated proteins. In short, we found MT1-MMP to interact with the tetraspanin-associated EWI-2 protein by a yeast two-hybrid screen. Immunoprecipitation analysis confirmed this interaction and further revealed that MT1-MMP also stably interacts with distinct tetraspanins (CD9, CD37, CD53, CD63, CD81, and CD82) and the tetraspanin-like MAL protein. By using different MT1-MMP truncation constructs and mutants, we observed that all tetraspanins and MAL associated with the hemopexin domain of MT1-MMP. Moreover, this interaction was independent of O-glycosylation of MT1-MMP and exclusively occurred in the endoplasmic reticulum. Here, the respective subcellular compartment was identified by fitting the MT1-MMP interaction pattern to a model for post-translational processing of MT1-MMP. In addition, tetraspanins differentially affected the cell surface localization of MT1-MMP, its capacity to activate pro-MMP-2, and the collagen invasion capacity. Interestingly, the degree of tetraspanin-MT1-MMP association did not correlate with its impact on MT1-MMP function. Tetraspanins thus distinctly affect MT1-MMP subcellular localization and function, and may constitute an effective mechanism to control MT1-MMP-dependent proteolysis at the cell surface.

Leptin-mediated regulation of MT1-MMP localization is KIF1B dependent and enhances gastric cancer cell invasion

Leptin overexpression is closely correlated with gastric cancer (GC) invasion, but its exact effect and the underlying mechanism in tumorigenesis remain poorly understood. Membrane type 1-matrix metalloproteinase (MT1-MMP), a surface-anchored 'master switch' proteinase, is overexpressed and plays crucial roles in tumor invasion. Here, we characterized the influence of leptin on the generation and surface localization of MT1-MMP in GC and elucidated its molecular mechanisms. Our results revealed that leptin promoted GC cell invasion in vitro by upregulating MT1-MMP expression. Furthermore, cell surface biotinylation assay and flow cytometry demonstrated that the surface expression of MT1-MMP was also enhanced by leptin, and knockdown of kinesin family member 1B (KIF1B, a microtubule plus end-directed monomeric motor protein) by small interference RNA inhibited this process. Notably, coimmunoprecipitation analysis indicated that leptin enhanced the interaction of MT1-MMP with KIF1B in a time-dependent manner, which consequently contributed to GC cell invasion. Moreover, leptin increased MT1-MMP or KIF1B expression by the protein kinase B (AKT) pathway and extracellular signal-regulated kinase 1/2 partially participated in this process. However, only AKT was implicated in the leptin-mediated membrane localization of MT1-MMP. Immunohistochemistry analysis revealed that leptin, MT1-MMP and KIF1B are overexpressed in GC tissues, and they positively correlated with clinical stage and lymph node metastasis. These observations indicate that this regulatory network exists in vivo. Taken together, our findings suggest that leptin is an effective intracellular stimulator of MT1-MMP and that leptin-enhanced cell surface localization of MT1-MMP is dependent on KIF1B, which consequently plays a critical role in GC invasion.

Fluid Shear Stress and Sphingosine 1-Phosphate Activate Calpain to Promote Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Membrane Translocation and Endothelial Invasion into Three-dimensional Collagen Matrices

The vascular endothelium continually senses and responds to biochemical and mechanical stimuli to appropriately initiate angiogenesis. We have shown previously that fluid wall shear stress (WSS) and sphingosine 1-phosphate (S1P) cooperatively initiate the invasion of human umbilical vein endothelial cells into collagen matrices (Kang, H., Bayless, K. J., and Kaunas, R. (2008) Am. J. Physiol. Heart Circ. Physiol. 295, H2087-2097). Here, we investigated the role of calpains in the regulation of endothelial cell invasion in response to WSS and S1P. Calpain inhibition significantly decreased S1P- and WSS-induced invasion. Short hairpin RNA-mediated gene silencing demonstrated that calpain 1 and 2 were required for WSS and S1P-induced invasion. Also, S1P synergized with WSS to induce invasion and to activate calpains and promote calpain membrane localization. Calpain inhibition results in a cell morphology consistent with reduced matrix proteolysis. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been shown by others to regulate endothelial cell invasion, prompting us to test whether calpain acted upstream of MT1-MMP. S1P and WSS synergistically activated MT1-MMP and induced cell membrane localization of MT1-MMP in a calpain-dependent manner. Calpain activation, MT1-MMP activation and MT1-MMP membrane localization were all maximal with 5.3 dynes/cm(2) WSS and S1P treatment, which correlated with maximal invasion responses. Our data show for the first time that 5.3 dynes/cm(2) WSS in the presence of S1P combine to activate calpains, which direct MT1-MMP membrane localization to initiate endothelial sprouting into three-dimensional collagen matrices.

NHE1 mediates migration and invasion of HeLa cells via regulating the expression and localization of MT1‐MMP

Na(+)/H(+) exchanger 1 (NHE1), acting as an important regulator of intracellular pH (pH(i)) and extracellular pH (pH(e)), has been known to play a key role in the metastasis of many solid tumours. However, the exact mechanism underlying these processes, especially in cervical cancer, is still poorly understood. In the current study, we first showed that the inhibition of NHE1 activity by the specific inhibitor cariporide could suppress migration and invasion of HeLa cells in vitro. Moreover, cariporide also reversed the enhanced migration and invasion in HeLa cells by overexpressed membrane-type 1 matrix metalloproteinase (MT1-MMP). Subsequently, our results showed that NHE1 regulated the expression of MT1-MMP at both messenger RNA and protein levels as well as its localization. Meanwhile, we observed slight modification in the morphology of HeLa cell after treating with cariporide. The present work indicates that NHE1 mediates HeLa cell metastasis via regulating the expression and localization of MT1-MMP and provides a theoretical basis for the development of novel therapeutic strategies targeting cervical cancer.

Direct regulation of membrane type 1 matrix metalloproteinase following myocardial infarction causes changes in survival, cardiac function, and remodeling.

The membrane type 1 matrix metalloproteinase (MT1-MMP) is increased in left ventricular (LV) failure. However, the direct effects of altered MT1-MMP levels on survival, LV function, and geometry following myocardial infarction (MI) and the proteolytic substrates involved in this process remain unclear. MI was induced in mice with cardiac-restricted overexpression of MT1-MMP (MT1-MMPexp; full length human), reduced MT1-MMP expression (heterozygous; MT1-MMP(+/-)), and wild type. Post-MI survival was reduced with MT1-MMPexp and increased with MT1-MMP(+/-) compared with WT. LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT post-MI and was higher in the MT1-MMP(+/-) mice. In vivo localization of MT1-MMP using antibody-conjugated microbubbles revealed higher MT1-MMP levels post-MI, which were the highest in the MT1-MMPexp group and the lowest in the MT1-MMP(+/-) group. LV collagen content within the MI region was higher in the MT1-MMPexp vs. WT post-MI and reduced in the MT1-MMP(+/-) group. Furthermore, it was demonstrated that MT1-MMP proteolytically processed the profibrotic molecule, latency-associated transforming growth factor-1-binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by over fourfold in the post-MI MT1-MMPexp group and reduced in the MT1-MMP(+/-) group, which was directionally paralleled by phospho-Smad-3 levels, a critical signaling component of the profibrotic transforming growth factor pathway. We conclude that modulating myocardial MT1-MMP levels affected LV function and matrix structure, and a contributory mechanism for these effects is through processing of profibrotic signaling molecules. These findings underscore the diversity of biological effects of certain MMP types on the LV remodeling process.

A role for MT1-MMP as a cell death sensor/effector through the regulation of endoplasmic reticulum stress in U87 glioblastoma cells

Recent findings in cell death signalling show that membrane type 1 matrix metalloproteinase (MT1-MMP), an MMP known for its involvement in cancer cell invasion and metastasis, can act as a "bioswitch" in the invasion versus cell death decision in brain tumour cells. Given that the endoplasmic reticulum (ER) is a subcellular compartment involved in metabolic control and cell death signalling and that cytoskeleton disruption, as encountered during cancer cell invasion, can lead to ER stress, we questioned whether MT1-MMP contributes to ER stress. We found that MT1-MMP gene silencing or pharmacological inhibition of vesicular trafficking with Brefeldin-A abrogated MT1-MMP cell surface-mediated proMMP-2 activation by the lectin Concanavalin-A (ConA) in U87 glioblastoma cells. ConA, also known to trigger the expression of pro-inflammatory cyclooxygenase (COX)-2 through MT1-MMP signalling from the plasma membrane, failed to do so when MT1-MMP was prevented from reaching the cell surface by Brefeldin-A. Gene silencing of MT1-MMP antagonized the expression of ConA-induced COX-2 and of the ER stress marker glucose-related protein 78 (GRP78), further suggesting that plasma membrane localization of MT1-MMP contributes to signalling ER stress. MT1-MMP maturation, which partially occurs during its trafficking from the ER to the plasma membrane, showed correlation of the 60kDa MT1-MMP with GRP78 expression. Finally, Brefeldin-A treatment of glioblastoma cells led to Akt dephosphorylation; this effect was reversed when MT1-MMP was silenced. Collectively, our results provide a molecular rationale for a new role for MT1-MMP in the regulation of cancer cell death processes through ER stress signalling.

A Novel Protein Associated with Membrane-type 1 Matrix Metalloproteinase Binds p27kip1 and Regulates RhoA Activation, Actin Remodeling, and Matrigel Invasion

Pericellular proteolysis by membrane-type 1 matrix metalloproteinase (MT1-MMP) plays a pivotal role in tumor cell invasion. Localization of MT1-MMP at the invasion front of cells, e.g. on lamellipodia and invadopodia, has to be regulated in coordination with reorganization of the actin cytoskeleton. However, little is known about how such invasion-related actin structures are regulated at the sites where MT1-MMP localizes. During analysis of MT1-MMP-associated proteins, we identified a heretofore uncharacterized protein. This protein, which we call p27RF-Rho, enhances activation of RhoA by releasing it from inhibition by p27(kip1) and thereby regulates actin structures. p27(kip1) is a well known cell cycle regulator in the nucleus. In contrast, cytoplasmic p27(kip1) has been demonstrated to bind GDP-RhoA and inhibit GDP-GTP exchange mediated by guanine nucleotide exchange factors. p27RF-Rho binds p27(kip1) and prevents p27(kip1) from binding to RhoA, thereby freeing the latter for activation. Knockdown of p27RF-Rho expression renders cells resistant to RhoA activation stimuli, whereas overexpression of p27RF-Rho sensitizes cells to such stimulation. p27RF-Rho exhibits a punctate distribution in invasive human tumor cell lines. Stimulation of the cells with lysophosphatidic acid induces activation of RhoA and induces the formation of punctate actin structures within foci of p27RF-Rho localization. Some of the punctate actin structures co-localize with MT1-MMP and cortactin. Down-regulation of p27RF-Rho prevents both redistribution of actin into the punctate structures and tumor cell invasion. Thus, p27RF-Rho is a new potential target for cancer therapy development.

Increased Aggressiveness of Human Prostate PC‐3 Tumor Cells Expressing Cell Surface Localized Membrane Type‐1 Matrix Metalloproteinase (MT1‐MMP)

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a multidomain transmembrane endopeptidase with a major role in physiological and pathological processes through proteolysis of extracellular matrix and other pericellular proteins. We examined cell surface function of MT1-MMP in PC-3 human prostate tumor cells selected for metastasis in nude mice (PC-3-LN4), or transfected with the full-length wild-type (WT) MT1-MMP or with the mutant form lacking the cytoplasmic tail (Delta C-MT1-MMP). Enhanced cell surface MT1-MMP was determined by fluorescence-activated cell sorting analysis and evidenced mechanistically by increased activation of proMMP-2 and invasion into type-I collagen gels. PC-3 cells overexpressing MT1-MMP grew faster than mock-transfected control cells subcutaneously in nude mice. MT1-MMP localized in caveolae, as judged by immunofluorescence microscopy and sucrose-gradient, detergent-resistant cell fractionation. Delta C-MT1-MMP was strongly associated with caveolae, whereas the WT form was present in both caveolae and noncaveolae fractions. The role of plasma membrane MT1-MMP was supported by localization of MT1-MMP by immunofluorescence microscopy at the cell surface of tumor cells in primary prostate cancers. Increased plasma membrane localization of MT1-MMP, either in caveolae or in other lipid raft structures, is a mechanism to localize this proteinase in contact with extracellular matrix and other pericellular proteins, the cleavage of which can facilitate prostate cancer cell invasion and metastasis.

Membrane Type-1 Matrix Metalloproteinase Potentiates Basic Fibroblast Growth Factor-Induced Corneal Neovascularization

Corneal neovascularization is one of the leading causes of blindness. The aim of this study was to evaluate the pro-angiogenic role of corneal fibroblast-derived membrane type-1 matrix metalloproteinase (MT1-MMP) on basic fibroblast growth factor (bFGF)-induced corneal neovascularization in vivo and in vitro. Immunohistochemical studies demonstrated that MT1-MMP was expressed in keratocytes and immortalized corneal fibroblast cell lines. Vascular endothelial growth factor protein levels were increased after bFGF-stimulation of wild-type fibroblast cells compared with MT1-MMP knockout fibroblast cells. Corneal vascularization was significantly increased after a combination of bFGF pellet implantation and naked MT1-MMP DNA injection in wild-type mouse corneas compared with either bFGF pellet implantation or naked MT1-MMP DNA-injected corneas. Western blotting analysis of the phosphorylation levels of the key signaling molecules (p38, JNK, and ERK) demonstrated that phosphorylation levels of both p38 and JNK were diminished after bFGF stimulation of MT1-MMP knockout cells compared with wild-type and MT1-MMP knockin cells. These results suggest that MT1-MMP potentiates bFGF-induced corneal neovascularization, likely by modulating the bFGF signal transduction pathway.

Cdc42 and RhoA have opposing roles in regulating membrane type 1-matrix metalloproteinase localization and matrix metalloproteinase-2 activation

Proteolysis of the basement membrane and interstitial matrix occurs early in the angiogenic process and requires matrix metalloproteinase (MMP) activity. Skeletal muscle microvascular endothelial cells exhibit robust actin stress fibers, low levels of membrane type 1 (MT1)-MMP expression, and minimal MMP-2 activation. Depolymerization of the actin cytoskeleton increases MT1-MMP expression and MMP-2 activation. Rho family GTPases are regulators of actin cytoskeleton dynamics, and their activity can be modulated in response to angiogenic stimuli such as vascular endothelial growth factor (VEGF). Therefore, we investigated their roles in MMP-2 and MT1-MMP production. Endothelial cells treated with H1152 [an inhibitor of Rho kinase (ROCK)] induced stress fiber depolymerization and an increase in cortical actin. Both MMP-2 and MT1-MMP mRNA increased, which translated into greater MMP-2 protein production and activation. ROCK inhibition rapidly increased cell surface localization of MT1-MMP and increased PI3K activity, which was required for MMP-2 activation. Constitutively active Cdc42 increased cortical actin polymerization, phosphatidylinositol 3-kinase activity, MT1-MMP cell surface localization, and MMP-2 activation similarly to inhibition of ROCK. Activation of Cdc42 was sufficient to decrease RhoA activity. Capillary sprout formation in a three-dimensional collagen matrix was increased in cultures treated with RhoAN19 or Cdc42QL and, conversely, decreased in cultures treated with dominant negative Cdc42N17. VEGF stimulation also induced activation of Cdc42 while inhibiting RhoA activity. Furthermore, VEGF-dependent activation of MMP-2 was reduced by inhibition of Cdc42. These results suggest that Cdc42 and RhoA have opposing roles in regulating cell surface localization of MT1-MMP and MMP-2 activation.

Indian hedgehog promotes the migration of rat activated pancreatic stellate cells by increasing membrane type‐1 matrix metalloproteinase on the plasma membrane

Indian hedgehog (Ihh) is a member of hedgehog peptides family that exerts diverse effects on multiple cellular functions. Since Ihh expression is elevated in the pancreas of chronic pancreatitis patients, Ihh has been assumed to participate in the chronic pancreatic injury, especially in pancreatic fibrosis. However, its function in pancreatic fibrosis is still unknown. We thus examined Ihh effects on rat activated pancreatic stellate cells (PSCs) that play a central role in pancreatic fibrosis. Activated PSCs express both patched-1 and smoothened that are essential components of hedgehog receptor system. Ihh did not alter the PSC expression of collagen-1 or alpha-smooth muscle actin, a parameter of PSC transformation, or did not change PSC proliferation. However, Ihh enhanced PSC migration in both chemotactic and chemokinetic manners. Furthermore, Ihh increased the amount of membrane-type 1 matrix metalloproteinase (MT1-MMP) and altered its localization on the plasma membrane, which plays a stimulatory role in cellular migration. In addition, tissue inhibitor of metalloproteinase-2 (TIMP-2) attenuated Ihh-stimulated PSC migration. Since most hedgehog intracellular signals are mediated by Gli-1 transcription factor, we investigated its contribution to Ihh-enhancement of PSC migration. Ihh induced Gli-1 nuclear accumulation in PSCs, indicating that Ihh stimulates Gli-1-dependent signaling pathway in PSCs. Unexpectedly, however, adenovirus-mediated Gli-1 overexpression blocked the Ihh enhancement of both MT1-MMP localization on the plasma membrane and PSC migration. Furthermore, reduction of Gli-1 expression with RNA interference augmented Ihh-stimulated PSC migration. These data indicate that Ihh promotes PSC migration by enhancing MT1-MMP localization on the plasma membrane but is negatively regulated by Gli-1.

Atypical localization of membrane type 1‐matrix metalloproteinase in the nucleus is associated with aggressive features of hepatocellular carcinoma

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a versatile proteinase and recent studies indicated it could be internalized. Our earlier study found that it is overexpressed in hepatocellular carcinoma (HCC) and could promote intrahepatic metastasis. The present study was conducted to examine its subcellular localization and its clinicopathological significance in HCC after curative partial hepatectomy. Localization of MT1-MMP in 101 pairs of HCCs and their adjacent liver tissues, and 8 normal liver tissues was examined by the immunohistochemical method. MT1-MMP protein was localized at membrane and cytoplasm of hepatocytes in the normal and tumor adjacent liver tissues. In contrast, the HCCs were highly heterogeneous with variable degrees of membrane, cytoplasmic, and even nuclear staining. Interestingly, patients with presence of nuclear MT1-MMP were associated with poor overall survival (log-rank test, P=0.043) and large tumor size (>5 cm) (Fisher's exact test, P=0.031). Subcellular distribution was further demonstrated by Western blotting and immunofluorescence with Hep3B stable transfectant overexpressing MT1-MMP. Western blot analyses of subcellular fractions confirmed a differential partitioning of various post-translationally modified MT1-MMP in these fractions. Different antibodies corroborated the presence of MT1-MMP in the nuclear fraction. Concomitant nuclear presence of MMP2 with MT1-MMP further indicated its potential involvement in the nuclear functions. MT1-MMP co-localized with caveolin-1 at the perinuclear region, suggesting nuclear translocation of MT1-MMP via caveolae-mediated endocytosis. In summary, the association of nuclear MT1-MMP with aggressive tumor features including poor prognosis and large tumor expands its functional repertoire and further indicates a new functional role of MMPs within nuclei of tumor cells.

Expression of MT1-MMP during deciduous tooth resorption in odontoclasts

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a membrane-bound matrix metalloproteinase capable of mediating pericellular proteolysis of extracellular matrix components. In osteoclasts, the localization of MT1-MMP has been reported at the tips of specialized membrane protrusions (podosomes and lamellipodia) so that osteoclasts might use MT1-MMP to perform focal proteolysis and move through the extracellular matrix to the bone surface. The objectives of this study were to investigate an association of MT1-MMP in physiological root resorption of the deciduous tooth by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis, and to identify MT1-MMP-producing cell during deciduous tooth resorption by in situ hybridization and immunohistochemistry. RT-PCR and Northern blot analysis revealed the exclusively high expression of MT1-MMP mRNA in bovine root-resorbing tissue, which lies between the root of the deciduous tooth and its permanent successor. Expression of MT1-MMP mRNA was seen in odontoclasts aligning in the surface layer of the root-resorbing tissue at sites of root resorption. Furthermore, immmunohistochemistry also confirmed the localization of MT1-MMP protein to the odontoclasts. The present identification of MT1-MMP in odontoclasts during deciduous tooth resorption might be relevant to the migration activity that these cells have to gain access to the root surface.

Expression and localization of MT1-MMP and furin in the glomerular wall of short- and long-term diabetic rats

Diabetic glomerulopathy has been linked to shifts in balance between the synthetic and degradative pathways of the glomerular basement membrane (GBM), a key player in the permselectivity properties of the glomerular wall. The goal of this study was to trace the expression and localization of membrane type-1 metalloprotease (MT1-MMP) and its activating enzyme furin, key proteins involved in basement membrane turnover, in short- and long-term diabetic rat renal tissues. Quantitative immunogold was carried out for MT1-MMP and furin and their expression was evaluated in renal tissues of young and old, control and diabetic rats. To corroborate immunocytochemical findings, Western blots were performed on glomerular lysates. Electron microscopy revealed that the overall expression of MT1-MMP and furin is reduced in plasma membranes of all glomerular cell types of old normoglycemic animals, a phenomenon that is exacerbated in long-term diabetic animals. This observation supports the prevailing theory that diabetes fosters acceleration in the aging process. Interestingly, while biochemical results confirmed a decrease in MT1-MMP expression, an increase in furin was observed. Immunocytochemical studies resolved this discrepancy by tracing the increased furin expression in endoplasmic reticulum and Golgi membranes of podocytes, indicating that furin is retained in the secretory pathway in a diabetic environment. Disturbances at the molecular level of the otherwise tightly regulated MT1-MMP/furin interactions found at the cell surface must account for a lack in extracellular matrix remodeling, increased deposition of GBM material, and loss of glomerular filtration integrity.

Cell Membrane-Associated MT1-MMP-Dependent Activation of Pro-MMP-2 in A375 Melanoma Cells

Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, can degrade extracellular matrix components under physiological conditions and during cancer invasion and metastasis. Among the MMPs, the 72 kDa type IV collagenase MMP-2 (gelatinase A) is activated in a membrane-associated manner by an activation complex composed of membrane type 1 matrix metalloproteinase (MT1-MMP), tissue inhibitor of matrixmetalloproteinase-2 (TIMP-2), and pro-MMP-2 in the presence of alphavbeta3 integrin receptor. The activation of pro-MMP-2 correlates with increased occurrence of metastases. Increased MMP-2 activity has been demonstrated in many human tumors. In the present communication, we studied cell surface-associated activation of MMP-2 (72 kDa collagenase type IV) in the moderately metastatic human melanoma cell line A375. Activation of purified 72 kDa collagenase type IV, pro-MMP-2 from cervical cancer tissue homogenate and from serum-free culture medium of HT1080 cells grown in presence of concanavalin A, by A375 cells, was shown by gelatin zymography. A375 cells activated only pro-MMP-2 from purified MMP-9/MMP-2 mixture indicating that the activation is specific for MMP-2. Activation of MMP-2 and purified collagenase type IV by A375 membrane fraction and membrane extract was also demonstrated by gelatin zymography. When A375 cells were first incubated with anti-MT1-MMP polyclonal antibody, activation of collagenase type IV was significantly decreased, indicating that membrane-associated MMP-2 activation is MT1-MMP-mediated. Immunocytochemistry showed MT1-MMP localization at focal adhesion sites. The presence of the components of activation complex-MT1-MMP and integrin alphavbeta3-were confirmed by Western blot, cell adhesion assay, and integrin subunit assay. Our experimental findings furnish another example of the unique membrane-associated MMP-2 activation mechanism in A375 melanoma cells and clearly indicate the role of MT1-MMP in MMP-2 activation. The information could help in developing new therapies designed to interfere with MMP activation and management of cancer and metastases.

Membrane Type 1-Matrix Metalloproteinase Is Regulated by Chemokines Monocyte-Chemoattractant Protein-1/CCL2 and Interleukin-8/CXCL8 in Endothelial Cells during Angiogenesis

We have investigated the putative role and regulation of membrane type 1-matrix metalloproteinase (MT1-MMP) in angiogenesis induced by inflammatory factors of the chemokine family. The absence of MT1-MMP from null mice or derived mouse lung endothelial cells or the blockade of its activity with inhibitory antibodies resulted in the specific decrease of in vivo and in vitro angiogenesis induced by CCL2 but not CXCL12. Similarly, CCL2- and CXCL8-induced tube formation by human endothelial cells (ECs) was highly dependent on MT1-MMP activity. CCL2 and CXCL8 significantly increased MT1-MMP surface expression, clustering, activity, and function in human ECs. Investigation of the signaling pathways involved in chemokine-induced MT1-MMP activity in ECs revealed that CCL2 and CXCL8 induced cortical actin polymerization and sustained activation of phosphatidylinositol 3-kinase (PI3K) and the small GTPase Rac. Inhibition of PI3K or actin polymerization impaired CCL2-induced MT1-MMP activity. Finally, dimerization of MT1-MMP was found to be enhanced by CCL2 in ECs in a PI3K- and actin polymerization-dependent manner. In summary, we identify MT1-MMP as a molecular target preferentially involved in angiogenesis mediated by CCL2 and CXCL8, but not CXCL12, and suggest that MT1-MMP dimerization might be an important mechanism of its regulation during angiogenesis.