Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycine-arginine-glycine-aspartic acid- serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed intense staining of the cell membrane with occasional small unstained areas, probably corresponding to the sealing zones. Immunoelectron microscopy confirmed the results obtained by light microscopy showing specific labeling only at the ruffled borders and basolateral membranes (0.82 and 2.43 gold particles/microns of membrane, respectively), but not at the sealing zone areas (0.06 gold particles/microns of membrane). Both alpha v and beta 3 subunits of the vitronectin receptor were similarly localized. These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface.     

MT-LOOP-dependent Localization of Membrane Type I Matrix Metalloproteinase (MT1-MMP) to the Cell Adhesion Complexes Promotes Cancer Cell Invasion

Localization of membrane type I matrix metalloproteinase (MT1-MMP) to the leading edge is thought to be a crucial step during cancer cell invasion. However, its mechanisms and functional impact on cellular invasion have not been clearly defined. In this report, we have identified the MT-LOOP, a loop region in the catalytic domain of MT1-MMP (¹⁶³PYAYIREG¹⁷⁰), as an essential region for MT1-MMP to promote cellular invasion. Deletion of the MT-LOOP effectively inhibited functions of MT1-MMP on the cell surface, including proMMP-2 activation, degradation of gelatin and collagen films, and cellular invasion into a collagen matrix. This is not due to loss of the catalytic function of MT1-MMP but due to inefficient localization of the enzyme to β1-integrin-rich cell adhesion complexes at the plasma membrane. We also found that an antibody that specifically recognizes the MT-LOOP region of MT1-MMP (LOOP_Ab) inhibited MT1-MMP functions, fully mimicking the phenotype of the MT-LOOP deletion mutant. We therefore propose that the MT-LOOP region is an interface for molecular interactions that mediate enzyme localization to cell adhesion complexes and regulate MT1-MMP functions. Our findings have revealed a novel mechanism regulating MT1-MMP during cellular invasion and have identified the MT-LOOP as a potential exosite target region to develop selective MT1-MMP inhibitors.     

Membrane type 1-matrix metalloproteinase (MMP)-associated MMP-2 activation increases in the rat ovary in response to an ovulatory dose of human chorionic gonadotropin

Gonadotropins stimulate ovarian proteolytic enzyme activity that is believed to be important for the remodeling of the follicular extracellular matrix. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been identified in vitro as an activator of pro-MMP-2 by forming a complex with tissue inhibitors of metalloproteinase-2 (TIMP-2). In the present study, the expression pattern of MT1-MMP mRNA and the role of MT1-MMP were examined in the ovary using the gonadotropin-treated immature rat model. Ovaries were collected at selected times after eCG or hCG. RNase protection assays revealed a transient increase in MT1-MMP mRNA beginning 4 h after hCG. High expression of MT1-MMP mRNA was localized to the theca-interstitial layer of developing and preovulatory follicles, while low expression was observed in the granulosa cell layer of developing follicles by in situ hybridization. The localization pattern of MT1-MMP mRNA was compared with TIMP-2 mRNA. Both MMP-2 and TIMP-2 mRNA were expressed in the theca layer of preovulatory follicles, showing a similarity to MT1-MMP mRNA expression. To further determine whether MT1-MMP activates pro-MMP-2 in the ovary, crude plasma membrane fractions from preovulatory ovaries were analyzed by gelatin zymography. In plasma membrane fractions, pro-MMP-2 increased around the time of ovulation. Upon incubation, pro-MMP-2 was activated with the highest levels of activation at 12 h post-hCG. The addition of MT1-MMP antibody or excess TIMP-2 to membrane fractions inhibited pro-MMP-2 activation. The increase in MT1-MMP mRNA may be an important part of the mechanism necessary for the efficient generation of active MMP-2 during the ovulatory process.     

Apatite-mediated actin dynamics in resorbing osteoclasts

The actin cytoskeleton is essential for osteoclasts main function, bone resorption. Two different organizations of actin have been described in osteoclasts, the podosomes belt corresponding to numerous F-actin columns arranged at the cell periphery, and the sealing zone defined as a unique large band of actin. To compare the role of these two different actin organizations, we imaged osteoclasts on various substrata: glass, dentin, and apatite. Using primary osteoclasts expressing GFP-actin, we found that podosome belts and sealing zones, both very dynamic actin structures, were present in mature osteoclasts; podosome belts were observed only in spread osteoclasts adhering onto glass, whereas sealing zone were seen in apico-basal polarized osteoclasts adherent on mineralized matrix. Dynamic observations of several resorption cycles of osteoclasts seeded on apatite revealed that 1) podosomes do not fuse together to form the sealing zone; 2) osteoclasts alternate successive stationary polarized resorption phases with a sealing zone and migration, nonresorption phases without any specific actin structure; and 3) apatite itself promotes sealing zone formation though c-src and Rho signaling. Finally, our work suggests that apatite-mediated sealing zone formation is dependent on both c-src and Rho whereas apico-basal polarization requires only Rho.     

Distinctive Subdomains in the Resorbing Surface of Osteoclasts

We employed a novel technique to inspect the substrate-apposed surface of activated osteoclasts, the cells that resorb bone, in the scanning electron microscope. The surface revealed unexpected complexity. At the periphery of the cells were circles and crescents of individual or confluent nodules. These corresponded to the podosomes and actin rings that form a ‘sealing zone’, encircling the resorptive hemivacuole into which protons and enzymes are secreted. Inside these rings and crescents the osteoclast surface was covered with strips and patches of membrane folds, which were flattened against the substrate surface and surrounded by fold-free membrane in which many orifices could be seen. Corresponding regions of folded and fold-free membrane were found by transmission electron microscopy in osteoclasts incubated on bone. We correlated these patterns with the distribution of several proteins crucial to resorption. The strips and patches of membrane folds corresponded in distribution to vacuolar H+-ATPase, and frequently co-localized with F-actin. Cathepsin K localized to F-actin-free foci towards the center of cells with circular actin rings, and at the retreating pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined band immediately inside the actin ring, peripheral to vacuolar H+-ATPase. The sealing zone of osteoclasts is permeable to molecules with molecular mass up to 10,000. Therefore, ClC-7 might be distributed at the periphery of the resorptive hemivacuole in order to prevent protons from escaping laterally from the hemivacuole into the sealing zone, where they would dissolve the bone mineral. Since the activation of resorption is attributable to recognition of the αVβ3 ligands bound to bone mineral, such leakage would, by dissolving bone mineral, release the ligands and so terminate resorption. Therefore, ClC-7 might serve not only to provide the counter-ions that enable proton pumping, but also to facilitate resorption by acting as a ‘functional sealing zone’.     

Caveolae are a novel pathway for membrane-type 1 matrix metalloproteinase traffic in human endothelial cells

The extracellular matrix (ECM) distinctly modulates membrane type 1-matrix metalloproteinase (MT1-MMP) in human endothelial cells (ECs). Herein, ECM-dependent RhoA activation is shown to regulate MT1-MMP localization and activity as well as clathrin-independent internalization in confluent ECs. In this regard, caveolae are revealed as the major MT1-MMP endocytic pathway in human ECs. Thus, MT1-MMP is present at caveolae with caveolin-1 and both proteins together with alpha v beta 3 integrin colocalize at endothelial motility-associated extensions. Remarkably, caveolae traffic is required for proper MT1-MMP localization, activity, and function in migratory ECs as demonstrated by both treatment with caveolae-disrupting agents or selective targeting caveolin-1 expression by interference RNA. Thus, caveolae-mediated traffic constitutes a novel mechanism for MT1-MMP regulation in ECs during angiogenesis.     

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.     

Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris

A soluble, C-terminal truncated form of human membrane type 1 matrix metalloproteinase (MT1-MMP) containing the hemopexin-like domain was expressed in Pichia pastoris strain KM71. High levels of secreted protein were detected. Although the c-DNA for the proenzyme (Ala(21)-Glu(523) called DeltaTM-MT1-MMP) was cloned, almost only active MT1-MMP (Tyr(112)-Glu(523)) with identical N-terminus as described for the wild-type enzyme was isolated. This active enzyme was highly purified and characterized with respect to its biochemical properties. The recombinant protein showed high stability against autolysis and proteolysis by yeast proteases, although the calculated in vivo half-life is rather low. The biochemical properties of this new MT1-MMP species were compared with the well-characterized catalytic domain (Ile(114)-Ile(318)) of MT1-MMP. The novel form of MT1-MMP exhibited a higher stability against autolysis than the isolated catalytic domain (Ile(114)-Ile(318)).     

Involvement of syntaxin 4 in the transport of membrane-type 1 matrix metalloproteinase to the plasma membrane in human gastric epithelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) localized on the plasma membrane plays a central role in various normal biological responses including tissue remodeling, wound heeling, and angiogenesis and in cancer cell invasion and metastasis, by functioning as a collagenase and activating other matrix metalloproteinases. In order to elucidate the molecular mechanism of the MT1-MMP targeted localization on the plasma membrane, we examined the participation of syntaxin proteins in MT1-MMP intracellular transport to the plasma membrane in human gastric epithelial AGS cells. Western blotting showed that syntaxin 3 and 4 proteins, which are known to function in intracellular transport towards the plasma membrane, were expressed in AGS cells. Immunocytochemistry revealed that transient transfection of AGS cells with dominant-negative mutant syntaxin 4 decreased plasma membrane MT1-MMP expression. In contrast, transient transfection with either dominant-negative mutant syntaxin 3 or 7 did not affect MT1-MMP localization on the plasma membrane. Cell surface biotinylation assay and Matrigel chamber assay demonstrated that stable transfection with dominant-negative mutant syntaxin 4 decreased the amount of MT1-MMP on the plasma membranes and inhibited the cell invasiveness. We suggest that syntaxin 4 is involved in the intracellular transport of MT1-MMP toward the plasma membrane.     

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.     

Inhibition of β1 integrin induces its association with MT1-MMP and decreases MT1-MMP internalization and cellular invasiveness

Integrin signaling plays a fundamental role in the establishment of focal adhesions and the subsequent formation of invadopodia in malignant cancer cells. Invadopodia facilitate localized adhesion and degradation of the extracellular matrix (ECM), which promote tumour cell invasion and metastasis. Degradation of ECM components is often driven by membrane type-1 matrix metalloproteinase (MT1-MMP), and we have recently shown that regulation of enzyme internalization is dependent on signaling downstream of β1 integrin. Phosphorylation of the cytoplasmic tail of MT1-MMP is required for its internalization and delivery to Rab5-marked early endosomes, where it is then able to be recycled to new sites of invadopodia formation and promote invasion. Here we found that inhibition of β1 integrin, using the antibody AIIB2, inhibited the internalization and recycling of MT1-MMP that is necessary to support long-term cellular invasion. MT1-MMP and β1 integrin were sequestered at the cell surface when β1-integrin was inhibited, and their association under these conditions was detected using immunoprecipitation and mass spectrometry analyses. Sequestration of β1 integrin and MT1-MMP at the cell surface resulted in the formation of large invadopodia and local ECM degradation; however, the impaired internalization and recycling of MT1-MMP and β1 integrin ultimately led to a loss of invasive behaviour.     

Progesterone differentially regulates the membrane-type matrix metalloproteinase-1 (MT1 -MMP) compartment of proMMP-2 activation in MG-63 cells.

Osteoblast-derived matrix metalloproteinases (MMPs) are considered to play a crucial role in bone formation and initiation of bone resorption by degrading the bone matrix. MMP-2 is constitutively secreted in a latent zymogen by osteoblasts, and requires the process of activation mediated by membrane-type matrix metalloproteinase-1 (MT1-MMP)/tissue inhibitor of metalloproteinase (TIMP-2) complex in the cell surface. Bone is one target tissue for progestins. In the present study, we observed the effects of progesterone on proMMP-2 activation and MT1-MMP expression, and also TIMP-2 levels in osteoblastic MG-63 cells. Gelatin zymograms and ELISA showed that progesterone have no effects on proMMP-2 activation. Using Western immunoblot analysis, we unexpectedly found that treatment with increasing doses of progesterone in MG-63 cells caused a dose-dependent increase in expression of MT1-MMP protein, and after 48h treatment, progesterone at 10(-8)M increased MT1-MMP protein level. Confocal immunohistochemistry analysis also confirmed that progesterone induced MT1-MMP expression in MG-63 cells. The results of Northern blot analysis showed that progesterone at 10(-8)M increased MT1-MMP protein levels after 48 h treatment. We also found that TIMP-2 levels were undetectable in MG-63 cells. In conclusion, progesterone increases MT1-MMP protein and mRNA levels in MG-63 cells, but has no effects on proMMP-2 activation, which is partly attributable to the undetectable levels of tissue inhibitor of metalloproteinase-2 (TIMP-2). Our studies suggest that TIMP-2 is involved in proMMP-2 activation, and regulation of MT1-MMP by progesterone may contribute to its actions on bone formation.     

Light- and electron-microscopic study of the distribution of axons containing substance P and the localization of neurokinin-1 receptor in bone

Substance P (SP) is a neuropeptide that is released from axons of sensory neurons and causes signal transduction through the activation of the neurokinin-1 receptor (NK1-R). The present study demonstrates the distribution of SP-like-immunoreactive (SP-LI) axons and the localization of NK1-Rs in rat bone tissue using the avidin-biotin-peroxidase complex method. Axons with SP-LI were commonly found near the trabecular bone in the temporal bone marrow, but they were only sparsely distributed in the mandible, femur, and tibia. Immunoreactivity for NK1-Rs was found on the plasma membrane and in the cytoplasm of the osteoclasts. In the osteoblasts and osteocytes, a small number of weak, punctate immunoreactive products of NK1-Rs were distributed close to the plasma membrane. At the electron-microscopic level, immunoreactivity for NK1-R was distributed mainly in the whole cytoplasm, except for the clear zone of the osteoclasts, and in pit-like structures along the plasma membrane. The NK1-R-immunoreactive structures in the cytoplasm were divided into two types of organelles, consisting of vesicular and vacuolar structures (probably transport vesicles and early endosomes). In the osteoblasts and osteocytes, the number of NK1-R-positive vesicular structures was fewer than in the osteoclasts. These results thus suggest that SP secreted by the sensory axons could directly modulate bone metabolism via NK1-Rs.     

MT1-MMP modulates the mechanosensitivity of osteocytes

Membrane-type matrix metalloproteinase-1 (MT1-MMP) is expressed by mechanosensitive osteocytes and affects bone mass. The extracellular domain of MT1-MMP is connected to extracellular matrix, while its intracellular domain is a strong modulator of cell signaling. In theory MT1-MMP could thus transduce mechanical stimuli into a chemical response. We hypothesized that MT1-MMP plays a role in the osteocyte response to mechanical stimuli. MT1-MMP-positive and knockdown (siRNA) MLO-Y4 osteocytes were mechanically stimulated with a pulsating fluid flow (PFF). Focal adhesions were visualized by paxillin immunostaining. Osteocyte number, number of empty lacunae, and osteocyte morphology were measured in long bones of MT1-MMP(+/+) and MT1-MMP(-/-) mice. PFF decreased MT1-MMP mRNA and protein expression in MLO-Y4 osteocytes, suggesting that mechanical loading may affect pericellular matrix remodeling by osteocytes. MT1-MMP knockdown enhanced NO production and c-jun and c-fos mRNA expression in response to PFF, concomitantly with an increased number and size of focal adhesions, indicating that MT1-MMP knockdown osteocytes have an increased sensitivity to mechanical loading. Osteocytes in MT1-MMP(-/-) bone were more elongated and followed the principle loading direction, suggesting that they might sense mechanical loading. This was supported by a lower number of empty lacunae in MT1-MMP(-/-) bone, as osteocytes lacking mechanical stimuli tend to undergo apoptosis. In conclusion, mechanical stimulation decreased MT1-MMP expression by MLO-Y4 osteocytes, and MT1-MMP knockdown increased the osteocyte response to mechanical stimulation, demonstrating a novel and unexpected role for MT1-MMP in mechanosensing.     

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.     

Involvement of calpain in osteoclastic bone resorption

There is increasing evidence that calpain contributes to the reorganization of the cytoskeleton in the integrin-mediated signaling pathway. Osteoclastic bone resorption requires cell-matrix contact, an event mediated by integrin alphavbeta3, and subsequent cytoskeletal reorganization to form characteristic membrane domains such as the sealing zone and ruffled border. In this study, therefore, we investigated whether calpain is involved in osteoclastic bone resorption. Membrane-permeable calpain inhibitors suppress the resorption activity of human osteoclasts, but an impermeable inhibitor does not. Upon the attachment of osteoclasts to bone, micro-calpain is translocated from the cytosolic to the cytoskeletal fraction and is autolytically activated. Both the activation of micro-calpain and the formation of actin-rings, the cytoskeletal structures essential for bone resorption, are inhibited by membrane-permeable calpain inhibitors. The activated micro-calpain in osteoclasts selectively cleaves talin, which links the matrix-recognizing integrin to the actin cytoskeleton. These findings suggest that calpain is a regulator of the bone resorption activity of osteoclasts through reorganization of the cytoskeleton related to actin-ring formation.     

Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.     

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.     

Podosome and sealing zone: specificity of the osteoclast model

The bone resorption function of osteoclasts is dependent on the integrity of the actin cytoskeleton. Depending on the substratum upon which the osteoclasts are spread, there are two different structures of actin known as podosomes and the sealing zone. To understand the specific properties and relationship of podosomes and the sealing zone, we used live-cell imaging of cultured osteoclasts. When cultured on extracellular matrix components, podosomes in these cells are organized in higher-ordered structures. These are clustered podosomes that will arrange later into dynamic short-lived rings which finally expand to the cell periphery to form a stable long-lived podosome belt in fully differentiated cells. In osteoclasts, this specific podosome patterning is under the control of microtubules (MTs). Indeed, nocodazole treatment does not affect podosome formation but only the transition between clusters/rings and belts. During this transition, MTs accumulate a specific post-translational modification of tubulin by acetylation. This process is repressed by an inhibitory pathway involving the GTPase Rho, its effector mDIA2 and the recently discovered tubulin deacetylase HDAC6. The specific function of this acetylation is still unknown but is also observed in active osteoclasts forming a sealing zone which is also MT dependent. Thus, it appears that the podosome belt is reminiscent of the sealing zone. Indeed, podosome belts and sealing zones are characterized by their overall stability. Despite their similar behavior, a sealing zone is not formed by fusion of podosomes. The formation of a podosome belt or a sealing zone is controlled by the external environment. Indeed, only the bone mineral fraction, known as apatite crystal, is able to induce sealing zone formation in mature osteoclasts. Contact of osteoclasts with apatite stimulates the non-receptor tyrosine kinase c-Src and the GTPase Rho in order to form the sealing zone. As we will discuss in this review, it appears that podosomes and the sealing zone are strikingly linked.     

Membrane type 1-matrix metalloproteinase is activated during migration of human endothelial cells and modulates endothelial motility and matrix remodeling.

Matrix metalloproteinases are thought to play an important role in endothelial cell migration and matrix remodeling. We have used an in vitro wound healing migration model and newly generated anti-membrane type 1-matrix metalloproteinase (MT1-MMP) monoclonal antibodies (mAbs) to characterize the role of MT1-MMP during this process. First, the expression and shedding of MT1-MMP are up-regulated upon induction of migration in endothelial cells, as demonstrated by flow cytometry and Western blot analysis. Furthermore, MT1-MMP is concentrated at discrete areas in migrating endothelial cells, in contrast to the diffuse pattern observed in confluent cells. Interestingly, migration of endothelial cells results in the stimulation of MT1-MMP activity, as shown by its ability to process pro-MMP-2 and to degrade fibrinogen assessed by zymography. Moreover, MT1-MMP-mediated gelatin degradation is enriched at migration sites. mAbs generated against the MT1-MMP catalytic domain are shown to inhibit MT1-MMP enzymatic activity and to impair both phorbol 12-myristate 13-acetate-induced endothelial migration and invasion of collagen and fibrin gels. Furthermore, a reduction in the formation of capillary tubes in Matrigel is also observed when endothelial cells are pretreated with the blocking anti-MT1-MMP mAbs. Altogether, these data demonstrate that MT1-MMP plays an important role during endothelial cell migration, and its activity can modulate endothelial migration, invasion, and formation of capillary tubes during the angiogenic response.