Molecular Mechanisms Driven by MT4-MMP in Cancer Progression.

MT4-MMP (or MMP-17) belongs to the membrane-type matrix metalloproteinases (MT-MMPs), a distinct subset of the MMP family that is anchored to the cell surface, in this case by a glycosylphosphatidylinositol (GPI) motif. Its expression in a variety of cancers is well documented. However, the molecular mechanisms by which MT4-MMP contributes to tumor development need further investigation. In this review, we aim to summarize the contribution of MT4-MMP in tumorigenesis, focusing on the molecular mechanisms triggered by the enzyme in tumor cell migration, invasiveness, and proliferation, in the tumor vasculature and microenvironment, as well as during metastasis. In particular, we highlight the putative substrates processed and signaling cascades activated by MT4-MMP that may underlie these malignancy processes and compare this with what is known about its role during embryonic development. Finally, MT4-MMP is a relevant biomarker of malignancy that can be used for monitoring cancer progression in patients as well as a potential target for future therapeutic drug development.

Emerging roles of MT-MMPs in embryonic development.

Membrane-type matrix metalloproteinases (MT-MMPs) are cell membrane-tethered proteinases that belong to the family of the MMPs. Apart from their roles in degradation of the extracellular milieu, MT-MMPs are able to activate through proteolytic processing at the cell surface distinct molecules such as receptors, growth factors, cytokines, adhesion molecules, and other pericellular proteins. Although most of the information regarding these enzymes comes from cancer studies, our current knowledge about their contribution in distinct developmental processes occurring in the embryo is limited. In this review, we want to summarize the involvement of MT-MMPs in distinct processes during embryonic morphogenesis, including cell migration and proliferation, epithelial-mesenchymal transition, cell polarity and branching, axon growth and navigation, synapse formation, and angiogenesis. We also considered information about MT-MMP functions from studies assessed in pathological conditions and compared these data with those relevant for embryonic development.

Dynamic Expression of Membrane Type 1-Matrix Metalloproteinase (Mt1-mmp/Mmp14) in the Mouse Embryo.

MT1-MMP/MMP14 belongs to a subgroup of the matrix metalloproteinases family that presents a transmembrane domain, with a cytosolic tail and the catalytic site exposed to the extracellular space. Deficient mice for this enzyme result in early postnatal death and display severe defects in skeletal, muscle and lung development. By using a transgenic line expressing the LacZ reporter under the control of the endogenous Mt1-mmp promoter, we reported a dynamic spatiotemporal expression pattern for Mt1-mmp from early embryonic to perinatal stages during cardiovascular development and brain formation. Thus, Mt1-mmp shows expression in the endocardium of the heart and the truncus arteriosus by E8.5, and is also strongly detected during vascular system development as well as in endothelial cells. In the brain, LacZ reporter expression was detected in the olfactory bulb, the rostral cerebral cortex and the caudal mesencephalic tectum. LacZ-positive cells were observed in neural progenitors of the spinal cord, neural crest cells and the intersomitic region. In the limb, Mt1-mmp expression was restricted to blood vessels, cartilage primordium and muscles. Detection of the enzyme was confirmed by Western blot and immunohistochemical analysis. We suggest novel functions for this metalloproteinase in angiogenesis, endocardial formation and vascularization during organogenesis. Moreover, Mt1-mmp expression revealed that the enzyme may contribute to heart, muscle and brain throughout development.

Tracing Gene Expression Through Detection of ß-galactosidase Activity in Whole Mouse Embryos.

The Escherichia coli LacZ gene, encoding ß-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, ß-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of ß-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for ß-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Developmental expression of membrane type 4-matrix metalloproteinase (Mt4-mmp/Mmp17) in the mouse embryo.

Matrix metalloproteinases (MMPs) constitute a large group of endoproteases that play important functions during embryonic development, tumor metastasis and angiogenesis by degrading components of the extracellular matrix. Within this family, we focused our study on Mt4-mmp (also called Mmp17) that belongs to a distinct subset that is anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety and with the catalytic site exposed to the extracellular space. Information about its function and substrates is very limited to date, and little has been reported on its role in the developing embryo. Here, we report a detailed expression analysis of Mt4-mmp during mouse embryonic development by using a LacZ reporter transgenic mouse line. We showed that Mt4-mmp is detected from early stages of development to postnatal stages following a dynamic and restricted pattern of expression. Mt4-mmp was first detected at E8.5 limited to the intersomitic vascularization, the endocardial endothelium and the dorsal aorta. Mt4-mmpLacZ/+ cells were also observed in the neural crest cells, somites, floor plate and notochord at early stages. From E10.5, expression localized in the limb buds and persists during limb development. A strong expression in the brain begins at E12.5 and continues to postnatal stages. Specifically, staining was observed in the olfactory bulb, cerebral cortex, hippocampus, striatum, septum, dorsal thalamus and the spinal cord. In addition, LacZ-positive cells were also detected during eye development, initially at the hyaloid artery and later on located in the lens and the neural retina. Mt4-mmp expression was confirmed by quantitative RT-PCR and western blot analysis in some embryonic tissues. Our data point to distinct functions for this metalloproteinase during embryonic development, particularly during brain formation, angiogenesis and limb development.