Dog as the experimental model: Laboratory uses of dogs in the United States.

Dogs are the experimental model in many types of biomedical research. Each year, hundreds of publications report the use of dogs in invasive biomedical procedures, often without sufficient explanation of the purpose and justification for selecting dog as the experimental model. The European Union requires detailed reporting of animal use that includes research purpose, but animal use reporting in the United States, overseen by the USDA, does not require this information. The ability to replace dogs with alternative models begins by understanding how they are used. Therefore, this study was undertaken to investigate the types of invasive biomedical procedures that dogs are subjected to in US laboratories. Well-defined sets of research publications and grants were accessed to obtain information on the types of biomedical research using dogs. USDA databases provided additional information. An ontology to categorize biomedical research uses of dogs identified the most common as translational studies for cardiovascular, cancer, nervous/mental, and mus-culoskeletal disorders. Information typically reported for experimental animals was sometimes missing or incomplete in publications, including the number, source, fate, species justification, and pain management of dogs, suggesting that many journals have not adopted the ARRIVE guidelines on animal use reporting. It was not possible to identify the research purpose for all dogs used by US institutions because (a) not all dog use is published and (b) animal research purpose is not required reporting in the US. These findings should be informative to future initiatives to replace, reduce, and refine the use of dogs in research.

The inactive 44-kDa processed form of membrane type 1 matrix metalloproteinase (MT1-MMP) enhances proteolytic activity via regulation of endocytosis of active MT1-MMP.

Membrane type 1 (MT1) matrix metalloproteinase (MMP-14) is a membrane-tethered MMP considered to be a major mediator of pericellular proteolysis. MT1-MMP is regulated by a complex array of mechanisms, including processing and endocytosis that determine the pool of active proteases on the plasma membrane. Autocatalytic processing of active MT1-MMP generates an inactive membrane-tethered 44-kDa product (44-MT1) lacking the catalytic domain. This form preserves all other enzyme domains and is retained at the cell surface. Paradoxically, accumulation of the 44-kDa form has been associated with increased enzymatic activity. Here we report that expression of a recombinant 44-MT1 (Gly(285)-Val(582)) in HT1080 fibrosarcoma cells results in enhanced pro-MMP-2 activation, proliferation within a three-dimensional collagen I matrix, and tumor growth and lung metastasis in mice. Stimulation of pro-MMP-2 activation and growth in collagen I was also observed in other cell systems. Expression of 44-MT1 in HT1080 cells is associated with a delay in the rate of active MT1-MMP endocytosis resulting in higher levels of active enzyme at the cell surface. Consistently, deletion of the cytosolic domain obliterates the stimulatory effects of 44-MT1 on MT1-MMP activity. In contrast, deletion of the hinge turns the 44-MT1 form into a negative regulator of enzyme function in vitro and in vivo, suggesting a key role for the hinge region in the functional relationship between active and processed MT1-MMP. Together, these results suggest a novel role for the 44-kDa form of MT1-MMP generated during autocatalytic processing in maintaining the pool of active enzyme at the cell surface.

Prostate cancer-associated membrane type 1-matrix metalloproteinase: a pivotal role in bone response and intraosseous tumor growth.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a major mediator of collagen I degradation. In human samples, we show that prostate cancer cells in skeletal metastases consistently express abundant MT1-MMP protein. Because prostate cancer bone metastasis requires remodeling of the collagen-rich bone matrix, we investigated the role of cancer cell-derived MT1-MMP in an experimental model of tumor-bone interaction. MT1-MMP-deficient LNCaP human prostate cancer cells were stably transfected with human wild-type MT1-MMP (MT1wt). Furthermore, endogenous MT1-MMP was down-regulated by small interfering RNA in DU145 human prostate cancer cells. Intratibial tumor injection in severe combined immunodeficient mice was used to simulate intraosseous growth of metastatic tumors. LNCaP-MT1wt cells produced larger osseous tumors than Neo control cells and induced osteolysis, whereas DU145 MT1-MMP-silenced transfectants induced osteogenic changes. In vitro assays showed that MT1wt overexpression enhanced collagen I degradation, whereas MT1-MMP-silencing did the opposite, suggesting that tumor-derived MT1-MMP may contribute directly to bone remodeling. LNCaP-MT1wt-derived conditioned medium stimulated in vitro multinucleated osteoclast formation. This effect was inhibited by osteoprotegerin, a decoy receptor for receptor activator of nuclear factor kappaB ligand, and by 4-[4-(methanesulfonamido) phenoxy] phenylsulfonyl methylthiirane, an MT1-MMP inhibitor. Our findings are consistent with the hypothesis that prostate cancer-associated MT1-MMP plays a direct and/or indirect role in bone matrix degradation, thus favoring intraosseous tumor expansion.

SPARC upregulates MT1-MMP expression, MMP-2 activation, and the secretion and cleavage of galectin-3 in U87MG glioma cells.

Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas and promotes glioma invasion. We have shown by cDNA array analysis that SPARC upregulates membrane type 1-matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) transcripts. To confirm these findings at the protein level and determine whether SPARC expression correlates with increased MMP activity, we used Western blot to assess the levels of MT1-MMP, and gelatin zymography to assess MMP-2 levels and activity. We also examined the expression, secretion, and cleavage of galectin-3, a target of MT1-MMP and MMP-2. Our data confirm that SPARC upregulates MT1-MMP levels and MMP-2 activity. There was also an increase in secreted galectin-3, as well as an increase in the proteolytically processed form of galectin-3. Previous studies have demonstrated that MT1-MMP, MMP-2, and galectin-3 are increased in gliomas. Our results suggest that their upregulation and activation may be a consequence of increased SPARC expression. These data provide a provisional mechanism whereby SPARC contributes to brain tumor invasion.

Cleavage at the stem region releases an active ectodomain of the membrane type 1 matrix metalloproteinase.

MT1-MMP (membrane type 1 matrix metalloproteinase) is a membrane-anchored MMP that can be shed to the extracellular milieu. In the present study we report the primary structure and activity of the major soluble form of MT1-MMP. MS analysis of the purified 50-kDa soluble MT1-MMP form shows that the enzyme extends from Tyr112 to Val524, indicating that formation of this species requires a proteolytic cleavage within the stem region. In agreement, deletion of the entire stem region of MT1-MMP inhibited shedding of the 50-kDa species. A recombinant 50-kDa species (Tyr112-Val524) expressed in cells exhibited enzymatic activity against pro-MMP-2 and galectin-3, and thus this species is a competent protease. The recombinant 50-kDa soluble form also decreased the level of surface-associated TIMP-2 (tissue inhibitor of metalloproteinase 2) when administered to cells expressing wild-type membrane-anchored MT1-MMP, suggesting that ectodomain shedding of MT1-MMP can alter the MMP/TIMP balance on the cell surface. A approximately 53-kDa species of MT1-MMP was also isolated from a non-detergent extract of human breast carcinoma tissue and was found to lack the cytosolic tail, as determined with specific MT1-MMP domain antibodies. Together, these data show that MT1-MMP ectodomain shedding is a physiological process that may broaden MT1-MMP activity to the pericellular space.

Complex pattern of membrane type 1 matrix metalloproteinase shedding. Regulation by autocatalytic cells surface inactivation of active enzyme.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a type I transmembrane MMP shown to play a critical role in normal development and in malignant processes. Emerging evidence indicates that MT1-MMP is regulated by a process of ectodomain shedding. Active MT1-MMP undergoes autocatalytic processing on the cell surface, leading to the formation of an inactive 44-kDa fragment and release of the entire catalytic domain. Analysis of the released MT1-MMP forms in various cell types revealed a complex pattern of shedding involving two major fragments of 50 and 18 kDa and two minor species of 56 and 31-35 kDa. Protease inhibitor studies and a catalytically inactive MT1-MMP mutant revealed both autocatalytic (18 kDa) and non-autocatalytic (56, 50, and 31-35 kDa) shedding mechanisms. Purification and sequencing of the 18-kDa fragment indicated that it extends from Tyr(112) to Ala(255). Structural and sequencing data indicate that shedding of the 18-kDa fragment is initiated at the Gly(284)-Gly(285) site, followed by cleavage between the conserved Ala(255) and Ile(256) residues near the conserved methionine turn, a structural feature of the catalytic domain of all MMPs. Consistently, a recombinant 18-kDa fragment had no catalytic activity and did not bind TIMP-2. Thus, autocatalytic shedding evolved as a specific mechanism to terminate MT1-MMP activity on the cell surface by disrupting enzyme integrity at a vital structural site. In contrast, functional data suggest that the non-autocatalytic shedding generates soluble active MT1-MMP species capable of binding TIMP-2. These studies suggest that ectodomain shedding regulates the pericellular and extracellular activities of MT1-MMP through a delicate balance of active and inactive enzyme-soluble fragments.