A new role of the membrane-type matrix metalloproteinase 16 (MMP16/MT3-MMP) in neural crest cell migration.

Neural crest cells (NCCs) are a transient population of neuroectodermal-originated cells that populate the dorsal neural tube (dNT), before migrating and giving rise to multiple cell lineages in the developing embryo. Prior to their migration, NCCs undergo epithelial-to-mesenchymal-transition (EMT) through which they lose cell contacts and detach from the dNT to invade their surrounding environment. Multiple signals and transcription factors have been identified to regulate these events. Yet, less is known regarding effectors that act downstream to execute the actual NCC separation and migration. Matrix metalloproteinases (MMPs) are a family of proteases that degrade the extracellular matrix as well as other pericellular proteins during processes of tissue remodeling, angiogenesis and metastasis. Previously, we and others have demonstrated the role of the gelatinases MMP2 and MMP9 during the onset of NCC migration. Several evidences link the cleavage and activation of these secreted gelatinases to the activity of membrane-type MMPs (MT-MMP), such as MMP14 and MMP16, which are tethered to plasma membrane and affect various cellular behaviors. The aim of this study was to investigate whether MMP16 acts in NCCs. Here we demonstrate the expression of MMP16 mRNA and protein in cranial NCCs in avian embryos. Knockdown of MMP16 inhibited NCC migration. This inhibition was rescued by the addition of recombinant MMP16, which was also sufficient to increase proper NCC migration. Furthermore, excess MMP16 caused enhanced NCC EMT, concomitant with degradation of dNT-related proteins, laminin and N-cadherin. Altogether, these results uncover MMP16 as a new effector participating in EMT and in the migration of NCCs.

MMP16 Mediates a Proteolytic Switch to Promote Cell-Cell Adhesion, Collagen Alignment, and Lymphatic Invasion in Melanoma.

Lymphatic invasion and accumulation of continuous collagen bundles around tumor cells are associated with poor melanoma prognosis, but the underlying mechanisms and molecular determinants have remained unclear. We show here that a copy-number gain or overexpression of the membrane-type matrix metalloproteinase MMP16 (MT3-MMP) is associated with poor clinical outcome, collagen bundle assembly around tumor cell nests, and lymphatic invasion. In cultured WM852 melanoma cells derived from human melanoma metastasis, silencing of MMP16 resulted in cell-surface accumulation of the MMP16 substrate MMP14 (MT1-MMP) as well as L1CAM cell adhesion molecule, identified here as a novel MMP16 substrate. When limiting the activities of these trans-membrane protein substrates toward pericellular collagen degradation, cell junction disassembly, and blood endothelial transmigration, MMP16 supported nodular-type growth of adhesive collagen-surrounded melanoma cell nests, coincidentally steering cell collectives into lymphatic vessels. These results uncover a novel mechanism in melanoma pathogenesis, whereby restricted collagen infiltration and limited mesenchymal invasion are unexpectedly associated with the properties of the most aggressive tumors, revealing MMP16 as a putative indicator of adverse melanoma prognosis.

Membrane-type matrix metalloproteinase-3 regulates neuronal responsiveness to myelin through Nogo-66 receptor 1 cleavage.

Nogo-66 receptor 1 (NgR1) is a glycosylphosphatidylinositol-anchored receptor for myelin-associated inhibitors that restricts plasticity and axonal regrowth in the CNS. NgR1 is cleaved from the cell surface of SH-SY5Y neuroblastoma cells in a metalloproteinase-dependent manner; however, the mechanism and physiological consequence of NgR1 shedding have not been explored. We now demonstrate that NgR1 is shed from multiple populations of primary neurons. Through a loss-of-function approach, we found that membrane-type matrix metalloproteinase-3 (MT3-MMP) regulates endogenous NgR1 shedding in primary neurons. Neuronal knockdown of MT3-MMP resulted in the accumulation of NgR1 at the cell surface and reduced the accumulation of the NgR1 cleavage fragment in medium conditioned by cortical neurons. Recombinant MT1-, MT2-, MT3-, and MT5-MMPs promoted NgR1 shedding from the surface of primary neurons, and this treatment rendered neurons resistant to myelin-associated inhibitors. Introduction of a cleavage-resistant form of NgR1 reconstitutes the neuronal response to these inhibitors, demonstrating that specific metalloproteinases attenuate neuronal responses to myelin in an NgR1-dependent manner.

Secreted versus membrane-anchored collagenases: relative roles in fibroblast-dependent collagenolysis and invasion.

Fibroblasts degrade type I collagen, the major extracellular protein found in mammals, during events ranging from bulk tissue resorption to invasion through the three-dimensional extracellular matrix. Current evidence suggests that type I collagenolysis is mediated by secreted as well as membrane-anchored members of the matrix metalloproteinase (MMP) gene family. However, the roles played by these multiple and possibly redundant, degradative systems during fibroblast-mediated matrix remodeling is undefined. Herein, we use fibroblasts isolated from Mmp13(-/-), Mmp8(-/-), Mmp2(-/-), Mmp9(-/-), Mmp14(-/-) and Mmp16(-/-) mice to define the functional roles for secreted and membrane-anchored collagenases during collagen-resorptive versus collagen-invasive events. In the presence of a functional plasminogen activator-plasminogen axis, secreted collagenases arm cells with a redundant collagenolytic potential that allows fibroblasts harboring single deficiencies for either MMP-13, MMP-8, MMP-2, or MMP-9 to continue to degrade collagen comparably to wild-type fibroblasts. Likewise, Mmp14(-/-) or Mmp16(-/-) fibroblasts retain near-normal collagenolytic activity in the presence of plasminogen via the mobilization of secreted collagenases, but only Mmp14 (MT1-MMP) plays a required role in the collagenolytic processes that support fibroblast invasive activity. Furthermore, by artificially tethering a secreted collagenase to the surface of Mmp14(-/-) fibroblasts, we demonstrate that localized pericellular collagenolytic activity differentiates the collagen-invasive phenotype from bulk collagen degradation. Hence, whereas secreted collagenases arm fibroblasts with potent matrix-resorptive activity, only MT1-MMP confers the focal collagenolytic activity necessary for supporting the tissue-invasive phenotype.

Collagen fibril growth during chicken tendon development: matrix metalloproteinase-2 and its activation.

The role of matrix metalloproteinases (MMPs) in collagen fibrillogenesis during development has been studied in the well-characterized chicken metatarsal tendon. Collagen fibrils are initially assembled as intermediates, and the mature fibrils assemble by linear and lateral growth from these intermediates. We hypothesize that this involves the turnover of fibril-associated molecules mediated by the expression and activation of matrix metalloproteinase-2 (MMP-2). We demonstrate changes in the ratio of full-length to truncated MMP-2 during tendon development, consistent with enzyme activation. The level of full-length proMMP-2 remains relatively unchanged, although the truncated form of MMP-2 is highest prior to and during fibril growth. Membrane-type matrix metalloproteinase-3 (MT3-MMP, MMP-16) is fibroblast-associated and involved in the regulation of MMP-2 and in direct matrix turnover. The ratio of full-length proMT3-MMP/truncated (active) MT3-MMP has a pattern similar to that of full-length proMMP-2/truncated (active) MMP-2 during tendon development. Regulation of proMMP-2 activation involves complex formation with active MT3-MMP and TIMP-2. The constantly low TIMP-2 expression seen in tendon development is consistent with this role. Isolation of collagen fibrils from pre-fibril growth tendons (14 day) in the presence of activated MMP-2 is associated with premature fibril growth observed as increased fibril diameters compared with controls. These data implicate MMP-2/MT3-MMP in the initiation and progression of fibril growth, matrix assembly, and tendon development. This may involve the turnover of fibril-associated molecules involved in regulating linear and lateral growth, such as small leucine-rich proteoglycans and fibril-associated collagens. Activation of proMMP-2 dependent on MT3-MMP would allow the focal control of turnover.

Matrix metalloproteinase gene polymorphisms and bronchopulmonary dysplasia: identification of MMP16 as a new player in lung development.

BACKGROUND: Alveolarization requires coordinated extracellular matrix remodeling, a process in which matrix metalloproteinases (MMPs) play an important role. We postulated that polymorphisms in MMP genes might affect MMP function in preterm lungs and thus influence the risk of bronchopulmonary dysplasia (BPD). METHODS AND FINDINGS: Two hundred and eighty-four consecutive neonates with a gestational age of <28 weeks were included in this prospective study. Forty-five neonates developed BPD. Nine single-nucleotide polymorphisms (SNPs) were sought in the MMP2, MMP14 and MMP16 genes. After adjustment for birth weight and ethnic origin, the TT genotype of MMP16 C/T (rs2664352) and the GG genotype of MMP16 A/G (rs2664349) were found to protect from BPD. These genotypes were also associated with a smaller active fraction of MMP2 and with a 3-fold-lower MMP16 protein level in tracheal aspirates collected within 3 days after birth. Further evaluation of MMP16 expression during the course of normal human and rat lung development showed relatively low expression during the canalicular and saccular stages and a clear increase in both mRNA and protein levels during the alveolar stage. In two newborn rat models of arrested alveolarization the lung MMP16 mRNA level was less than 50% of normal. CONCLUSIONS: MMP16 may be involved in the development of lung alveoli. MMP16 polymorphisms appear to influence not only the pulmonary expression and function of MMP16 but also the risk of BPD in premature infants.

Cell surface chondroitin sulfate glycosaminoglycan in melanoma: role in the activation of pro-MMP-2 (pro-gelatinase A).

We previously reported that CS (chondroitin sulfate) GAG (glycosaminoglycan), expressed on MCSP (melanoma-specific CS proteoglycan), is important for regulating MT3-MMP [membrane-type 3 MMP (matrix metalloproteinase)]-mediated human melanoma invasion and gelatinolytic activity in vitro. In the present study, we sought to determine if CS can directly enhance MT3-MMP-mediated activation of pro-MMP-2. Co-immunoprecipitation studies suggest that MCSP forms a complex with MT3-MMP and MMP-2 on melanoma cell surface. When melanoma cells were treated with betaDX (p-nitro-beta-D-xylopyranoside) to inhibit coupling of CS on the core protein, both active form and proform of MMP-2 were no longer co-immunoprecipitated with either MCSP or MT3-MMP, suggesting a model in which CS directly binds to MMP-2 and presents the gelatinase to MT3-MMP to be activated. By using recombinant proteins, we determined that MT3-MMP directly activates pro-MMP-2 and that this activation requires the interaction of the C-terminal domain of pro-MMP-2 with MT3-MMP. Activation of pro-MMP-2 by suboptimal concentrations of MT3-MMP is also significantly enhanced in the presence of excess C4S (chondroitin 4-sulfate), whereas C6S (chondroitin 6-sulfate) or low-molecular-mass hyaluronan was ineffective. Affinity chromatography studies using CS isolated from aggrecan indicate that the catalytic domain of MT3-MMP and the C-terminal domain of MMP-2 directly bind to the GAG. Thus the direct binding of pro-MMP-2 with CS through the C-domain would present the catalytic domain of pro-MMP-2 to MT3-MMP, which facilitates the generation of the active form of MMP-2. These results suggest that C4S, which is expressed on tumour cell surface, can function to bind to pro-MMP-2 and facilitate its activation by MT3-MMP-expressing tumour cells to enhance invasion and metastasis.