MT1-MMP Is Required for Myeloid Cell Fusion via Regulation of Rac1 Signaling

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Highlights? Myeloid cells fuse during osteoclast and giant cell formation ? MT1-MMP is required for this fusion in vitro and in vivo ? MT1-MMP regulates the GTPase Rac1 through p130Cas binding ? MT1-MMP proteolytic activity is not required     

Functional extracellular matrix hydrogel modified with MSC‐derived small extracellular vesicles for chronic wound healing

ObjectivesDiabetic wound healing remains a global challenge in the clinic and in research. However, the current medical dressings are difficult to meet the demands. The primary goal of this study was to fabricate a functional hydrogel wound dressing that can provide an appropriate microenvironment and supplementation with growth factors to promote skin regeneration and functional restoration in diabetic wounds.Materials and MethodsSmall extracellular vesicles (sEVs) were bound to the porcine small intestinal submucosa‐based hydrogel material through peptides (SC‐Ps‐sEVs) to increase the content and achieve a sustained release. NIH3T3 cell was used to evaluate the biocompatibility and the promoting proliferation, migration and adhesion abilities of the SC‐Ps‐sEVs. EA.hy926 cell was used to evaluate the stimulating angiogenesis of SC‐Ps‐sEVs. The diabetic wound model was used to investigate the function/role of SC‐Ps‐sEVs hydrogel in promoting wound healing.ResultsA functional hydrogel wound dressing with good mechanical properties, excellent biocompatibility and superior stimulating angiogenesis capacity was designed and facilely fabricated, which could effectively enable full‐thickness skin wounds healing in diabetic rat model.ConclusionsThis work led to the development of SIS, which shows an unprecedented combination of mechanical, biological and wound healing properties. This functional hydrogel wound dressing may find broad utility in the field of regenerative medicine and may be similarly useful in the treatment of wounds in epithelial tissues, such as the intestine, lung and liver.

Schematic illustration showing synthesis of the SC‐Ps scaffold dressing and nanoscale sEVs loaded SC‐Ps scaffold dressing and the potential application of the dressings in diabetic wound healing and skin reconstruction.     

Centrosomal BRCA2 is a target protein of membrane type-1 matrix metalloproteinase (MT1-MMP)

BRCA2 localizes to centrosomes between G1 and prophase and is removed from the centrosomes during mitosis, but the underlying mechanism is not clear. Here we show that BRCA2 is cleaved into two fragments by membrane type-1 matrix metalloproteinase (MT1-MMP), and that knockdown of MT1-MMP prevents the removal of BRCA2 from centrosomes during metaphase. Mass spectrometry mapping revealed that the MT1-MMP cleavage site of human BRCA2 is between Asn-2135 and Leu-2136 (²¹³²LSNN/LNVEGG²¹⁴¹), and the point mutation L2136D abrogated MT1-MMP cleavage. Our data demonstrate that MT1-MMP proteolysis of BRCA2 regulates the abundance of BRCA2 on centrosomes.     

When MT1-MMP meets ADAMs

MT1-MMP is a membrane-tethered enzyme capable of remodeling extracellular matrix. MT1-MMP-deficient mice exhibit systematic defects during development, especially in craniofacial development characterized by retarded calvarial bone formation. Recently, we identified MT1-MMP as a critical positive modulator of FGF signaling during intramembranous ossification. MT1-MMP cleaves ADAM9 to protect FGFR2 from ectodomain shedding. Depletion of ADAM9 in MT1-MMP-deficient mice significantly rescued the calvarial defects via restoring FGF signaling. Interestingly, this regulatory mechanism seems to be highly tissue-specific, as defective FGF2-induced corneal angiogenesis in Mmp14^?/? mice could not be rescued by removal of ADAM9. In addition, MT1-MMP also cleaves another ADAM family member, ADAM15. Our current findings not only present a novel regulatory mechanism for FGF signaling but also reveal a functional crosstalk between MMP and ADAM families. Better understanding of the interplay between ADAMs and MT1-MMP and its consequences for signaling pathways will provide new insights into therapeutic approaches for the management of developmental disorders and various diseases, such as cancer.     

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.     

Proactive for invasion: Reuse of matrix metalloproteinase for structural memory

Migratory cells translocate membrane type-1 matrix metalloproteinase (MT1-MMP) to podosomes or invadosomes to break extracellular matrix barriers. In this issue, El Azzouzi et al. (2016. J. Cell. Biol. http://dx.doi.org/10.1083/jcb.201510043) describe an unexpected function for the MT1-MMP cytoplasmic domain in imprinting spatial memory for podosome reformation via assembly in membrane islets.Invasion of most normal and cancer cells across basement membranes and collagen-rich interstitial tissues involves degradation of the ECM by membrane type-1 matrix metalloproteinase (MT1-MMP/MMP14; Willis et al., 2013). To fulfill this activity, MT1-MMP is transported to podosomes, the specialized ECM-degrading membrane protrusions found in highly migratory cells such as activated macrophages, osteoclasts, endothelial cells, and smooth muscle cells (Murphy and Courtneidge, 2011). In cancer cells, MT1-MMP is transported to ECM-degrading invasive structures called invadopodia (Poincloux et al., 2009). Both these membrane protrusions, collectively called invadosomes, are composed of an actin-rich core surrounded by scaffold and adhesion proteins, and numerous mechanisms of invadosome assembly, maturation, and dynamics have been identified (Poincloux et al., 2009; Murphy and Courtneidge, 2011). MT1-MMP activity is regulated at multiple levels to achieve targeted ECM degradation, cell surface protein processing, and protease activation (Sato et al., 1994; Osenkowski et al., 2004; Sugiyama et al., 2013; Willis et al., 2013; Itoh, 2015). Potential regulatory functions of MT1-MMP toward the cytoskeleton have, however, remained unclear. In this issue, El Azzouzi et al. describe an unexpected and novel function for MT1-MMP that goes beyond its traditional proteolytic activity: they show that MT1-MMP accumulates in membrane islets that provide macrophages with spatial information, or memory, in sites of podosome dissolution so as to enable efficient podosome reassembly.El Azzouzi et al. (2016) first used total internal reflection fluorescence microscopy and a pH-sensitive version of MT1-MMP devised to fluoresce only when the MT1-MMP ectodomain is exposed to the extracellular environment’s pH. With this approach, they show that, on the ventral surface of cultured human macrophages, MT1-MMP is localized at two different membrane compartments: underneath the podosome core, as previously suggested based on matrix degradation and colocalization with podosome markers, and in distinct islets devoid of other podosome components, CD44, or integrin-mediated adhesion to the ECM (Fig. 1; Osiak et al., 2005). MT1-MMP islets were dependent on intact cortical actin, but became more apparent and persisted after podosome disruption by pharmacological perturbation of key components of podosome assembly and maturation, such as integrin adhesion, Src kinase activity, and the Arp2/3 complex essential for actin nucleation and branched actin cytoskeleton. Podosomes often reemerge at sites of previous podosome localization, and El Azzouzi et al. (2016) hypothesized that MT1-MMP islets might mark sites of podosome formation. They treated cells with an Arp2/3 inhibitor to disrupt podosomes and induce the appearance of MT1-MMP membrane clusters, and used time-lapse imaging to track what happens upon washout and podosome reformation. Interestingly, they show that these novel MT1-MMP structures serve as remarkably immobile cell membrane anchors capable of rerecruiting the podosomal actin cores/scaffolds to the same islets.Open in a separate windowFigure 1.MT1-MMP islets as memory sites for podosome reformation. Migratory cells translocate MT1-MMP (red) to podosomes or invadosomes to degrade the ECM (green fibers). These membrane structures are composed of an actin-rich core (brown) surrounded by adhesion and scaffold proteins (beige) such as integrins (blue). El Azzouzi et al. (2016) show a function for MT1-MMP accumulation in membrane ”islets” (1), where they imprint spatial memory for podosome reemergence after podosome disassembly (2). Unlike dynamic mature podosomes (3), MT1-MMP assembles in stable islets via anchorage to cortical actin. Future work in the fields of inflammation, cancer, and angiogenesis will need to address the nature of the cytoskeletal dynamics mediating islet formation, the involvement of microtubules in islet formation, the exact islet protein composition, and the relevance of these memory sites to 2D or 3D environments and to other cell types beyond macrophages, including endothelial cells and invasive cancer cells.Further, by expressing mutant MT1-MMP proteins in cells silenced for the endogenous proteinase and using a podosome reformation assay (based on pharmacological dissolution of podosomes via Src inhibition, followed by podosome reformation after washout), El Azzouzi et al. (2016) pinpointed the region of MT1-MMP critical for islet formation, the LLY-sequence in its cytoplasmic domain. Moreover, when attached to the membrane by the MT1-MMP transmembrane domain, the 20–amino acid cytoplasmic tail appeared necessary and sufficient to form the islets. Considering the LLY sequence–dependent actin-binding ability of MT1-MMP (Yu et al., 2012) coupled with the observed necessity of cortical actin for islet appearance and podosome reformation, the direct interaction with unbranched cortical actin was suggested by the authors as a likely decisive mechanism for the remarkable MT1-MMP islet stabilization in podosome-free areas, although a possible indirect interaction was not ruled out. Actin binding through the MT1-MMP cytosolic tail was likewise suggested as a potential means for podosome rerecruitment by MT1-MMP memory islets.Although cortical actin is instrumental for the emergence of the spatially and temporally stable MT1-MMP islets upon podosome dissolution in macrophages and direct actin–MT1-MMP interaction has been proven in vitro and suggested as a means for retaining MT1-MMP in invadopodia, a Src-regulated interaction between MT1-MMP’s cytoplasmic domain and the actin-binding scaffold protein palladin has also been shown to regulate MT1-MMP targeting into invadopodia (Yu et al., 2012; von Nandelstadh et al., 2014). Moreover, the LLY sequence in MT1-MMP’s cytoplasmic tail harbors a Src substrate sequence and mediates an interaction between MT1-MMP and AP-2 that is important for MT1-MMP internalization and dynamics in cell migration and invasion (Uekita et al., 2001; Nyalendo et al., 2007). Intriguingly, El Azzouzi et al. (2016) did not find evidence of involvement of dynamin-dependent membrane trafficking events in the ability of MT1-MMP islets to function as memory sites. However, their results after treatment with the microtubule inhibitor nocodazole indicated that although the islets themselves remained intact, podosome reappearance was mislocalized, suggesting that microtubules contribute by as yet undefined mechanisms to the ability of MT1-MMP islets to provide spatial memory and to facilitate podosome reassembly. Therefore, further identification of drivers and specific regulatory mechanisms of the actin–MT1-MMP interaction dynamics in podosomes, of the stable actin–MT1-MMP interaction and structures in podosome-free areas, and of microtubule-dependent podosome reassembly will be of interest.A striking observation of this study is that MT1-MMP islets do not display degradative activity in matrix degradation assays. In addition, inhibition of the proteolytic activity of MT1-MMP through pharmacological agents or via an inactivating mutation did not impact islet appearance or podosome reemergence at sites of MT1-MMP clustering. Overall, on the extracellular side of the plasma membrane, the apparent lack of contact and degradation of the ECM as well as the relatively minor impact of the N-terminal MT1-MMP ectodomain on islet formation and podosome reemergence are peculiar features of the MT1-MMP islets. However, El Azzouzi et al. (2016) show evidence for somewhat impaired islet formation in cells expressing an MT1-MMP mutant lacking the entire ectodomain, and they demonstrate that endogenous MT1-MMP must be silenced for the LLY MT1-MMP mutant to disrupt islet localization. Based on these results, the authors suggest the possible influence of MT1-MMP oligomerization and of MT1-MMP–ECM binding on islet recruitment and stabilization. Nevertheless, these observations altogether indicate that the adhesive and degradative activities of MT1-MMP memory islets toward the ECM are minor and, intriguingly, do not influence the structure or function of these islets as currently characterized in 2D cultures.Furthermore, the aforementioned results raise questions about the possible contribution of the different molecular forms of MT1-MMP (e.g., cleaved or uncleaved and inhibitor bound or not) to the stabilization and podosome reassembly function of MT1-MMP islets. In cells and conditions in which MT1-MMP activity is high, MT1-MMP turnover is typically fast via autocatalytic cleavage or shedding of the N-terminal catalytic domain (Lehti et al., 1998; Yana and Weiss, 2000; Itoh et al., 2001; Osenkowski et al., 2004). After interaction with inhibitors such as tissue inhibitors of metalloproteinases, active endocytosed MT1-MMP may dissociate from the bound inhibitor to be recycled to the plasma membrane (Jiang et al., 2001; Remacle et al., 2003). However, in the absence of interaction with a protease inhibitor or collagen/matrix substrate, MT1-MMP oligomerization facilitates MT1-MMP turnover via autocatalytic inactivating cleavage (Itoh et al., 2001; Lehti et al., 2002; Osenkowski et al., 2004). In the current study, El Azzouzi et al. (2016) used MT1-MMP proteins with a pH sensor inserted N-terminally to the transmembrane domain, so that the probe is located extracellularly on the surface-exposed protease. The fluorescence signal from these constructs is not expected to be affected by proteolytic processing or shedding of the catalytic domain, so it is unclear whether the MT1-MMP proteins clustered in islets are cleaved or not. However, FRAP results showed that the turnover of MT1-MMP molecules within the islets is relatively slow. It thus remains to be clarified if and how the proteolytically active or possibly processed or protease inhibitor–bound inactive forms of MT1-MMP are stabilized in these MT1-MMP islets.As posodomes are highly dynamic protrusions, their rapid turnover implicates a constant disassembly at the rear and formation at the front of migrating macrophages. Assembly and dissassembly are known to depend on Arp2/3-mediated actin nucleation and fission of preexisting podosomes, respectively (Linder et al., 2000). Both of these mechanisms may contribute to podosome reassembly at MT1-MMP memory sites. Considering that these islets are laterally immobile and overall stable in at least unpolarized cells, it is unclear how migrating cells coordinate their actin and microtubule cytoskeletons for podosome reassembly at the front using islets formed upon podosome dissolution at the rear of the cell (Fig. 1). Moreover, the structural and functional features of MT1-MMP islets in the scenario of 3D cell–ECM microenvironments is intriguing and will need to be investigated at high resolution, as cytoskeletal dynamics, cell polarity, and matrix stiffness greatly differ in 3D tissues and matrices from the simple 2D setting of cultured cells, and all are known to influence cell behavior. Although the transient nature of these MT1-MMP islets in bridging podosome disassembly and reassembly exemplifies and reflects the efficiency of podosome reusage, probing the protein composition of these islets as well as the dynamics of podosome reassembly will likely be challenging. Future studies comparing MT1-MMP state, dynamics, reuse, and turnover in different types of invadosomes, islets, and other subcellular compartments will be instrumental to better understand how cells integrate the different types of MT1-MMP membrane structures and cell–ECM communication with other cellular signals and with drivers of cytoskeletal dynamics.The identification of the molecular mechanisms of structural and functional podosome memory are not only relevant to the fields of macrophage biology and inflammation but also more broadly to those of tissue invasion and matrix remodeling. For instance, endothelial cells, smooth muscle cells, and cancer cells are also known to target MT1-MMP to podosomes or related invadosomes. Examining MT1-MMP memory in such specialized subcellular compartments will be interesting beyond the podosome field, as the podosome counterparts in cancer cells may display and use MT1-MMP or other metalloproteinases in a similar manner. By shedding light on the mechanisms of dynamic protrusion formation and function, this paper not only opens new avenues of investigation into the cellular structures marking protrusion sites as “memory devices” but also brings about a new concept to the fields of cell invasion, angiogenesis, and cancer.     

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.Malignant tumor cells grow invasively and form distant metastases after moving through multiple tissue barriers. Invasion requires cell locomotion together with degradation of the extracellular matrix (ECM)² by matrix metalloproteinases (MMPs) (1). MT1-MMP (MMP-14) is an integral membrane protease that degrades a variety of protein components within the extracellular milieu (2). The substrates of MT1-MMP include a variety of components of the ECM, membrane proteins including cell adhesion molecules, and growth factors and cytokines (3). To degrade the ECM barrier in advance of an invading cell, MT1-MMP localizes to the leading edge of invasion (4) and cellular protrusions called invadopodia (5–7). Therefore, it is of particular interest how reorganization of actin structures is regulated at sites where MT1-MMP localizes.During mass spectrometric analysis of proteins co-purified with MT1-MMP, we identified a protein of unknown function (8). Although this protein did not affect MT1-MMP activity, we observed that enhanced expression or down-regulation of this protein affected activation of RhoA. Thus, we became interested in the possibility that this protein mediates focal reorganization of actin structures close to sites where MT1-MMP localizes.RhoA plays a pivotal role in signal transduction pathways that regulate reorganization of actin structures and does so by assuming active GTP-bound and inactive GDP-bound states, with the transition between the two forms finely regulated by many cellular proteins (9, 10). In addition to the classical modulators, recent studies have revealed that p27^kip1 also regulates activation of RhoA and Rac1 (11, 12). p27^kip1 has been characterized as a cyclin-dependent kinase inhibitor localized to the nucleus, but phosphorylation of p27^kip1 by protein kinase B/Akt or kinase-interacting stathmin (KIS) mediates its translocation from the nucleus to the cytoplasm. Cytoplasmic p27^kip1 binds RhoA and prevents activation of RhoA by GEFs (12, 13). However, it is not known how inhibition of RhoA by p27^kip1 is released to allow activation. The protein we identified binds p27^kip1, thereby preventing its binding to RhoA (schematically illustrated in supplemental Fig. S1). We named this protein p27RF-Rho (p27^kip1 releasing factor from RhoA) based on this activity.     

Internal Cleavages of the Autoinhibitory Prodomain Are Required for Membrane Type 1 Matrix Metalloproteinase Activation,although Furin Cleavage Alone Generates Inactive Proteinase

The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD↓L⁵⁰ site initiates the MT1-MMP activation, whereas the ¹⁰⁸RRKR¹¹¹↓Y¹¹² cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.     

Identification and Characterization of Lutheran Blood Group Glycoprotein as a New Substrate of Membrane-type 1 Matrix Metalloproteinase 1 (MT1-MMP): A SYSTEMIC WHOLE CELL ANALYSIS OF MT1-MMP-ASSOCIATING PROTEINS IN A431 CELLS*

Membrane-type 1 matrix metalloproteinase 1 (MT1-MMP) is a potent modulator of the pericellular microenvironment and regulates cellular functions in physiological and pathological settings in mammals. MT1-MMP mediates its biological effects through cleavage of specific substrate proteins. However, our knowledge of MT1-MMP substrates remains limited. To identify new substrates of MT1-MMP, we purified proteins associating with MT1-MMP in human epidermoid carcinoma A431 cells and analyzed them by mass spectrometry. We identified 163 proteins, including membrane proteins, cytoplasmic proteins, and functionally unknown proteins. Sixty-four membrane proteins were identified, and they included known MT1-MMP substrates. Of these, eighteen membrane proteins were selected, and we confirmed their association with MT1-MMP using an immunoprecipitation assay. Co-expression of each protein together with MT1-MMP revealed that nine proteins were cleaved by MT1-MMP. Lutheran blood group glycoprotein (Lu) is one of the proteins cleaved by MT1-MMP, and we confirmed the cleavage of the endogenous Lu protein by endogenous MT1-MMP in A431 cells. Mutation of the cleavage site of Lu abrogated processing by MT1-MMP. Lu protein expressed in A431 cells bound to laminin-511, and knockdown of MT1-MMP in these cells increased both their binding to laminin-511 and the amount of Lu protein on the cell surface. Thus, the identified membrane proteins associated with MT1-MMP are an enriched source of physiological MT1-MMP substrates.Cells in tissues are surrounded by an extracellular cellular matrix that interacts with cells to regulate their activity (1, 2). Matrix metalloproteinases (MMPs)³ are endopeptidases responsible for extracellular matrix degradation and thereby regulate turnover of the extracellular matrix. However, recent studies have demonstrated that substrates of MMPs are expanded to a variety of pericellular proteins.MT1-MMP/MMP14 is an integral membrane proteinase that cleaves multiple proteins in the pericellular milieu and thereby regulates various cell functions. Substrates of MT1-MMP identified to date include extracellular matrix proteins (type I collagen, fibronectin, vitronectin, laminin-1 and -5, and others), cell adhesion molecules (CD44, syndecan-1, and αv integrin), cytokines (SDF-1 and transforming growth factor-β and others), and latent forms of pro-MMPs (pro-MMP-2 and pro-MMP13) (3–5). Processing of these proteins by MT1-MMP alters their activities and thereby regulates a variety of cellular functions, such as motility, invasion, growth, differentiation, and apoptosis. Consistent with these functions, forced expression of MT1-MMP in tumor cells enhances behavior consistent with increased malignancy, such as rapid tumor growth, invasion, and metastasis (6). However, MT1-MMP is normally expressed in various types of cell and mice deficient in MT1-MMP expression (MT1^−/−) display pleiotropic defects (7–10). However, we as yet have only limited knowledge of the physiological substrates of MT1-MMP that could explain such pleiotropic effects.Proteases interact with their substrates at least transiently, but in some cases such interaction is more stable. For instance, type I collagen binds MT1-MMP via a hemopexin-like domain and is cleaved (11, 12). Cleavage of collagen by MT1-MMP regulates cell growth and invasion in a collagen-rich environment (13). CD44, a hyaluronic acid receptor, also binds to the hemopexin of MT1-MMP and is cleaved (14). Expression of CD44 and MT1-MMP in tumor cells promotes cell migration, accompanied by the shedding of CD44 by MT1-MMP (14, 15). pro-MMP-2, which is cleaved by MT1-MMP for activation, forms a tri-molecular complex with MT1-MMP and TIMP-2 (3, 16). Therefore, screening of proteins that associate with MT1-MMP may provide a systematic method to identify potential substrates of MT1-MMP in cells. In addition, these proteins may also be regulatory proteins of MT1-MMP.To identify proteins associating with MT1-MMP in different types of tumor cells, we first studied conditions for cell lysis using malignant melanoma A375 cells and following purification method of the proteins as reported recently (17). Proteins purified in this manner were analyzed by high-throughput proteomic analysis (18–21). Interestingly, approximately one-half of the membrane proteins identified in our previous study could be cleaved by MT1-MMP at least in vitro. Here, we applied this approach to human carcinoma cells (A431) that originate from epidermoid cells and further validated the systemic whole cell analysis method. To evaluate whether the MT1-MMP-associated membrane proteins so identified include physiological targets of MT1-MMP activity, we select one of them, Lutheran blood group glycoprotein (Lu), and evaluate its processing in A431 cells.     

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.     

Interruption of endothelin signaling modifies membrane type 1 matrix metalloproteinase activity during ischemia and reperfusion

The matrix metalloproteinases (MMPs), in particular, membrane type 1 MMP (MT1-MMP), are increased in the context of myocardial ischemia and reperfusion (I/R) and likely contribute to myocardial dysfunction. One potential upstream induction mechanism for MT1-MMP is endothelin (ET) release and subsequent protein kinase C (PKC) activation. Modulation of ET and PKC signaling with respect to MT1-MMP activity with I/R has yet to be explored. Accordingly, this study examined in vivo MT1-MMP activation during I/R following modification of ET signaling and PKC activation. With the use of a novel fluorogenic microdialysis system, myocardial interstitial MT1-MMP activity was measured in pigs (30 kg; n = 9) during I/R (90 min I/120 min R). Local ET(A) receptor antagonism (BQ-123, 1 microM) and PKC inhibition (chelerythrine, 1 microM) were performed in parallel microdialysis probes. MT1-MMP activity was increased during I/R by 122 +/- 10% (P < 0.05) and was unchanged from baseline with ET antagonism and/or PKC inhibition. Selective PKC isoform induction occurred such that PKC-betaII increased by 198 +/- 31% (P < 0.05). MT1-MMP phosphothreonine, a putative PKC phosphorylation site, was increased by 121 +/- 8% (P < 0.05) in the I/R region. These studies demonstrate for the first time that increased interstitial MT1-MMP activity during I/R is a result of the ET/PKC pathway and may be due to enhanced phosphorylation of MT1-MMP. These findings identify multiple potential targets for modulating a local proteolytic pathway operative during I/R.     

Cardiac Restricted Overexpression of Membrane Type-1 Matrix Metalloproteinase Causes Adverse Myocardial Remodeling following Myocardial Infarction

The membrane type-1 matrix metalloproteinase (MT1-MMP) is a unique member of the MMP family, but induction patterns and consequences of MT1-MMP overexpression (MT1-MMPexp), in a left ventricular (LV) remodeling process such as myocardial infarction (MI), have not been explored. MT1-MMP promoter activity (murine luciferase reporter) increased 20-fold at 3 days and 50-fold at 14 days post-MI. MI was then induced in mice with cardiac restricted MT1-MMPexp (n = 58) and wild type (WT, n = 60). Post-MI survival was reduced (67% versus 46%, p < 0.05), and LV ejection fraction was lower in the post-MI MT1-MMPexp mice compared with WT (41 ± 2 versus 32 ± 2%,p < 0.05). In the post-MI MT1-MMPexp mice, LV myocardial MMP activity, as assessed by radiotracer uptake, and MT1-MMP-specific proteolytic activity using a specific fluorogenic assay were both increased by 2-fold. LV collagen content was increased by nearly 2-fold in the post-MI MT1-MMPexp compared with WT. Using a validated fluorogenic construct, it was discovered that MT1-MMP proteolytically processed the pro-fibrotic molecule, latency-associated transforming growth factor-1 binding protein (LTBP-1), and MT1-MMP-specific LTBP-1 proteolytic activity was increased by 4-fold in the post-MI MT1-MMPexp group. Early and persistent MT1-MMP promoter activity occurred post-MI, and increased myocardial MT1-MMP levels resulted in poor survival, worsening of LV function, and significant fibrosis. A molecular mechanism for the adverse LV matrix remodeling with MT1-MMP induction is increased processing of pro-fibrotic signaling molecules. Thus, a proteolytically diverse portfolio exists for MT1-MMP within the myocardium and likely plays a mechanistic role in adverse LV remodeling.     

Full-thickness human foreskin transplantation onto nude rats as an in vivo model of acute human wound healing

Current wound-healing models do not fully duplicate the in vivo human environment. The feasibility of grafting human full-thickness foreskin onto nude rats, as a model of acute wound healing, was evaluated. Incisions were then created on the grafted skin, and wound healing was evaluated. Full-thickness human skin was obtained after elective circumcision and was grafted subcutaneously onto the dorsal thorax of nude rats. At 10 days after transplantation, graft beds were judged for graft viability, on the basis of gross appearance, texture, and adherence. Full-thickness wounds were then made in the foreskin. Graft wounds were left to close by secondary intention. The wounds were allowed to heal for 7 days. Wounds were excised and tested for breaking stress. Histological evaluations included proliferating cell nuclear antigen, factor VIII, hematoxylin and eosin, and trichrome staining. Twenty grafts were performed, with 100 percent viability. Upon incision, all grafts bled freely, indicating a rich vascular supply and tissue viability. Graft viability was confirmed by the presence of proliferating cells in the parabasal stratum of the epithelium. Furthermore, there was evidence of angiogenesis, as confirmed by staining for factor VIII. Breaking stress was evaluated by tensiometry, 7 days after wounding. Histological evaluations revealed viable grafts and active wound-healing events. Full-thickness human skin can be successfully transplanted onto nude rats, providing a larger, more physiological model of human wound healing. This model closely parallels the in vivo situation, providing a promising model for study of the complex biological processes of acute human wound healing, in a reproducible manner.     

Design,synthesis and biological evaluation of bifunctional inhibitors of membrane type 1 matrix metalloproteinase (MT1-MMP)

Collagen degradation and proMMP-2 activation are major functions of MT1-MMP to promote cancer cell invasion. Since both processes require MT1-MMP homodimerization on the cell surface, herein we propose that the use of bifunctional inhibitors of this enzyme could represent an innovative approach to efficiently reduce tumor growth. A small series of symmetrical dimers derived from previously described monomeric arylsulfonamide hydroxamates was synthesized and tested in vitro on isolated MMPs. A nanomolar MT1-MMP inhibitor, compound 6, was identified and then submitted to cell-based assays on HT1080 fibrosarcoma cells. Dimer 6 reduced MT1-MMP-dependent proMMP-2 activation, collagen degradation and collagen invasion in a dose-dependent manner with better results even compared to its monomeric analogue 4. This preliminary study suggests that dimeric MT1-MMP inhibitors might be further developed and exploited as an alternative tool to reduce cancer cell invasion.