Enamelysin is a tooth-specific matrix metalloproteinase that is expressed during the early through middle stages of enamel development. The enamel matrix proteins amelogenin, ameloblastin, and enamelin are also expressed during this same approximate developmental time period, suggesting that enamelysin may play a role in their hydrolysis. In support of this interpretation, recombinant enamelysin was previously demonstrated to cleave recombinant amelogenin at virtually all of the precise sites known to occur in vivo. Thus, enamelysin is likely an important amelogenin-processing enzyme. To characterize the in vivo biological role of enamelysin during tooth development, we generated an enamelysindeficient mouse by gene targeting. Although mice heterozygous for the mutation have no apparent phenotype, the enamelysin null mouse has a severe and profound tooth phenotype. Specifically, the null mouse does not process amelogenin properly, possesses an altered enamel matrix and rod pattern, has hypoplastic enamel that delaminates from the dentin, and has a deteriorating enamel organ morphology as development progresses. Our findings demonstrate that enamelysin activity is essential for proper enamel development.Dental enamel covers the crown of the tooth and is unique among mineralized tissues because of its high mineral content, large crystals, and organized prism pattern. Other mineralized tissues such as bone, dentin, and cementum are composed of ϳ20% organic material. In contrast, mature enamel has less than 1% organic matter by weight (1, 2). Moreover, enamel crystallites possess a volume that is 100 times greater than the volume of crystallites found in other mineralizing tissues. These enamel crystallites form enamel rods that, in turn, form a unique interlacing (decussating) prism pattern. As a result, dental enamel is the hardest substance in the body. Its hardness is intermediate between that of iron and carbon steel, yet it also has a high elasticity (3).Although mature enamel is a very hard protein-free tissue, it does not start this way. Enamel development (amelogenesis) consists of several stages that include the secretory, transition, and maturation stages. During the secretory stage, enamel crystallites elongate into long thin ribbons that are only a few apatitic unit cells in thickness (about 10 nm) with a width of ϳ30 nm (4, 5). The ribbons are evenly spaced, are oriented parallel to each other, and grow in length but very little in width and thickness. Ultimately, enamel crystal length determines the final thickness of the enamel layer as a whole (for review, see Ref. 6). It is during the secretory stage that the columnar-shaped ameloblast cells, located adjacent to the forming enamel, secrete specialized enamel proteins into the enamel matrix. These proteins include amelogenin (7), ameloblastin (8), and enamelin (9). Amelogenin is the predominant component and comprises ϳ90% of total enamel matrix protein (10). Interestingly, the full-length enamel proteins are found only at the mineralizing front, su...

To understand the biologic function of TIMP-2, a member of the tissue inhibitors of metalloproteinases family, an inactivating mutation was introduced in the mouse Timp-2 gene by homologous recombination. Outbred homozygous mutants developed and procreated indistinguishably from wild type littermates, suggesting that fertility, development, and growth are not critically dependent on TIMP-2. Lack of functional TIMP-2, however, dramatically altered the activation of proMMP-2 both in vivo and in vitro. Fully functional TIMP-2 is essential for efficient activation of proMMP-2 in vivo. No evidence of successful functional compensation was observed. The results illustrate the duality of TIMP-2 function, i.e. at low concentrations, TIMP-2 exerts a "catalytic" or enhancing effect on cell-mediated proMMP-2 activation, whereas at higher concentrations, TIMP-2 inhibits the activation and/or activity of MMP-2.

Peri-cellular remodeling of mesenchymal extracellular matrices is considered a prerequisite for cell proliferation, motility and development. Here we demonstrate that membrane-type 3 MMP, MT3-MMP, is expressed in mesenchymal tissues of the skeleton and in peri-skeletal soft connective tissue. Consistent with this localization, MT3-MMP-deficient mice display growth inhibition tied to a decreased viability of mesenchymal cells in skeletal tissues. We document that MT3-MMP works as a major collagenolytic enzyme, enabling cartilage and bone cells to cleave high-density fibrillar collagen and modulate their resident matrix to make it permissive for proliferation and migration. Collectively, these data uncover a novel extracellular matrix remodeling mechanism required for proper function of mesenchymal cells. The physiological significance of MT3-MMP is highlighted in mice double deficient for MT1-MMP and MT3-MMP. Double deficiency transcends the combined effects of the individual single deficiencies and leads to severe embryonic defects in palatogenesis and bone formation incompatible with life. These defects are directly tied to loss of indispensable collagenolytic activities required in collagen-rich mesenchymal tissues for extracellular matrix remodeling and cell proliferation during embryogenesis.

Enamelysin is a recently isolated member of the matrix metalloproteinase (MMP) family of extracellular matrix (ECM)-degrading enzymes. Here we describe the isolation and characterization of the mouse enamelysin cDNA. Expression of mouse enamelysin was detectable only in ameloblasts and odontoblasts of developing teeth. Characterization of mouse enamelysin demonstrated that it is highly conserved in both its sequence content and pattern of expression relative to the porcine, human, and bovine homologues previously described.

IntroductionAfter embryo implantation and decidualization, a critical step in placental development is the formation of the placental labyrinth (LA), which enables nutrient and gas exchange between the embryonic vasculature and the maternal blood supply. 1-5 LA formation is associated with substantial tissue remodeling and cell differentiation, as well as ingrowth of the embryonic vasculature through the chorion to a point of immediate proximity with the maternal blood supply. During this process, chorionic trophoblasts (CHs) differentiate into 2 perivascular cell populations that form distinct bilaminar envelopes of syncytiotrophoblasts in immediate contact with the fetal vascular endothelium. 6 Multiple transcription factors, growth factors, adhesion molecules, and gap junction molecules are known to influence the formation of the LA. 7 Interestingly, few if any proteolytic enzymes have so far been proven essential for LA formation although tissue remodeling is considered an integral part of this morphogenetic process. Several of the matrix metalloproteinases (MMPs), cathepsins, and serine proteinases are expressed in the placenta during development, however to date none have proven indispensable for development of the LA and in turn development of the embryo to term. [8][9][10][11][12] Among the 6 known membrane-type MMP (MT-MMP) molecules in the mouse, MT1-MMP and MT3-MMP possess pericellular collagenase activity and are required for both prenatal and postnatal remodeling of the major fibrillar collagen types, cell surface receptors, and signaling molecules. [13][14][15] Ablation of MT1-MMP deprives cells of the ability to migrate through and process several both permanent and provisional extracellular matrices, and in vivo leads to severe defects in postnatal remodeling of connective tissues. [16][17][18][19] Moreover, MT1-MMP is required in the stromal compartment for efficient dissemination of malignant epithelial cells in mouse mammary carcinoma. 20 MT1-MMP deficiency is partially mitigated by the activity of the molecular relative, MT3-MMP, which shares at least some overlapping substrate specificity with MT1-MMP. Accordingly, incremental loss of alleles encoding each molecule markedly exacerbates the cellular matrix remodeling deficit in a gene dosage dependent manner, and double deficiency results in severe developmental deficits and perinatal death, but importantly, not preterm loss of embryos. 15 As expected from these observations, MT1-MMP and MT3-MMP are frequently coexpressed in the same tissue compartments. The placental LA however is devoid of MT3-MMP expression but displays conspicuous expression of another MT-MMP family member, MT2-MMP. 11,21 So far, the function of MT2-MMP has been determined biochemically and in cell-based assays. Under these conditions, MT2-MMP displays the ability to process basement membrane components and fibrillar collagen matrices whereas its role in vivo has remained unexplored. 22,23 Here we demonstrate that MT2-MMP deficiency in the mouse is compatible with develop...

No abstract

No abstract

Using homology-based database screening, we have identified the mouse homologue for the recently described matrix metalloproteinase-19 (MMP-19). Sequencing of mouse MMP-19 resulted in a putative open reading frame (ORF) of 527 amino acids showing 84% identity to the human homologue. In mouse, MMP-19 appears to be most highly expressed in the liver; however, there is a detectable level of expression in all tissues analyzed. The major mouse MMP-19 transcript is almost twice as long as that of human. The COOH-terminal serine and threonine-rich domain is considerably longer in the mouse homologue. The mouse MMP-19 gene maps to very distal end of mouse chromosome 10.