Expression and localization of matrix metalloproteinases (MT1-MMP, MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) during synepitheliochorial placentation of goats (Capra hircus).

Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) play key roles during the placentation of highly invasive haemochorial type. Our knowledge is yet scanty, however, regarding the roles played by MMPs and TIMPs in the placentation of non-invasive synepitheliochorial type. In the present study, expression patterns of MT1-MMP, MMP-2 and TIMP-2 mRNAs as well as the encoded proteins in the endometrium and the placenta were examined on Days 35, 75, and 100 of pregnancy, representing roughly the 1st, 2nd and 3rd trimesters of caprine gestation, by means of quantitative RT-PCR analysis, in situ hybridization, immunoblotting, gelatin zymography and immunohistochemistry. In the endometrium and the intercotyledonal trophoblast, the expression levels of the 3 genes remained relatively uniform throughout the period of gestation examined. Curiously, however, in the placentomes, the relative expression levels of MT1-MMP mRNA increased linearly from Day 35 to Day 100, while those of MMP-2 and TIMP-2 were clearly down-regulated in Day 100 placentae. The expression levels of MT1-MMP and TIMP-2 proteins in placentomes were well correlated with those of the respective mRNAs. In the case of MMP-2, the total amount of MMP-2 protein (the combined values of the latent, the intermediate and the active forms) decreased slightly, while the levels of the active form increased markedly from Day 35 to Day 100. Immunohistochemical analysis of the placentome revealed that MT1-MMP and TIMP-2 proteins were co-localized in the binucleate trophoblast cells; expression of these 2 proteins was not detected in the uninuclear principal trophoblast cells. MMP-2 expression was detected both in the binucleate and in the uninuclear principal cells of the trophoblast and in the endometrial stromal cells of the uterine septum, regardless of the stages of gestation examined. The co-localization of MT1-MMP, MMP-2 and TIMP-2 in binucleate trophoblast cells, the cotyledonal trophoblast cells and the subsyncytial stromal cells is likely to reflect the functional coordination of the 3 proteins in these cells during trophoblastic invasion and the placental tissue remodeling in the placentome.

Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA in trophoblast and endometrial epithelial cell populations of the synepitheliochorial placenta of goats (Capra hircus).

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a membrane-bound matrix metalloproteinase, plays crucial roles in cellular migration through the matrix during embryogenesis, wound healing, and the invasion of host tissues by cancer cells. Mammalian trophoblast cells exhibit different degrees of invasiveness towards the endometrium in different species during gestation. The highly invasive trophoblast cells of primates and rodents which form hemochorial placentae have often been compared to metastatic cancer cells, and are known to express MT1-MMP at their invasive edge. So far, however, little is known about MT1-MMP expression in the placenta of non-invasive type including the synepitheliochorial placenta of bovidae. As an approach to assess the role played by MT1-MMP in the non-invasive synepitheliochorial placentation, we determined the open reading frame (ORF) base sequence of caprine MT1-MMP (DDBJ/EMBL/GenBank database: AB010921); this sequence is the first registered MT1-MMP ORF sequence of artyodactyls which develop placentae of the non-invasive type. The deduced amino acid sequence of caprine MT1-MMP exhibited 92, 87 and 89% identity with its human, mouse and rat counterparts, respectively. Availability of the cloned caprine MT1-MMP cDNA allowed us to carry out Northern blot analysis which revealed that in the placentome, the expression levels of MT1-MMP mRNA were very low on Day 35 of gestation (peri-implantation stage), while the levels gradually increased from Day 75 to Day 100. In the interplacentome regions of the placenta and the uterus, the signal levels were higher than those in the placentome, and increased from Day 35 onward, peaking on Day 75. In situ hybridization experiments revealed that the binucleate trophoblast cells reacted with the MT1-MMP cRNA probe throughout the period examined while the uninuclear principal trophoblast cells did so only on Day 100. Of particular interest is the expression of MT1-MMP transcripts in the luminal and glandular epithelial cells of the gestational endometrium, since epithelial cells in general have been noted to lack MMP expression, including MT-MMPs. The high levels of MT1-MMP expression in the endometrial epithelial cell populations might reflect extensive remodeling during gestation.

Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.