Yun-Ge Zhao

Endometase/Matrilysin-2 in Human Breast Ductal Carcinoma in Situ and Its Inhibition by Tissue Inhibitors of Metalloproteinases-2 and -4 A Putative Role in the Initiation of Breast Cancer Invasion

Local disruption of the integrity of both the myoepithelial cell layer and the basement membrane is an indispensable prerequisite for the initiation of invasion and the conversion of human breast ductal carcinoma in situ (DCIS) to infiltrating ductal carcinoma (IDC). We previously reported that human endometase/matrilysin-2/matrix metalloproteinase (MMP) 26-mediated pro-gelatinase B (MMP-9) activation promoted invasion of human prostate carcinoma cells by dissolving basement membrane proteins (Y. G. Zhao et al., J. Biol. Chem., 278: 15056–15064, 2003). Here we report that tissue inhibitor of metalloproteinases (TIMP)-2 and TIMP-4 are potent inhibitors of MMP-26, with apparent Ki values of 1.6 and 0.62 nm, respectively. TIMP-2 and TIMP-4 also inhibited the activation of pro-MMP-9 by MMP-26 in vitro. The expression levels of MMP-26, MMP-9, TIMP-2, and TIMP-4 proteins in DCIS were significantly higher than those in IDC, atypical intraductal hyperplasia, and normal breast epithelia adjacent to DCIS and IDC by immunohistochemistry and integrated morphometry analysis. Double immunofluorescence labeling and confocal laser scanning microscopy revealed that MMP-26 was colocalized with MMP-9, TIMP-2, and TIMP-4 in DCIS cells. Higher levels of MMP-26 mRNA were also detected in DCIS cells by in situ hybridization.

Spatio-Temporal Expression of Matrix Metalloproteinase-26 in Human Placental Trophoblasts and Fetal Red Cells During Normal Placentation

Abstract The processes of implantation and placentation involve the degradation and remodeling of extracellular matrix, cellular proliferation, apoptosis, and differentiation. Evidence indicates that members of the matrix metalloproteinase (MMP) family play crucial roles in these processes. In the present study, we identified the expression and localization of MMP26/endometase/ matrilysin-2 in human placentae at different stages of gestation using methods of reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry. MMP26 was widely localized to villous cytotrophoblast cells, syncytiotrophoblast cells, and to column trophoblasts during early pregnancy. The mRNA and protein level of MMP26 in chorionic villi was highest at Weeks 6–7, and decreased thereafter, reaching its lowest level at the second trimester. The mRNA level was significantly up-regulated in term placenta, while the immunoreactivity remained undetectable. Notably, intense expression of MMP26 was found in fetal nucleated red cells inside the villous capillaries during gestational Weeks 6–9. Strong expression of MMP26 mRNA was also demonstrated in fetal red cells isolated from the whole blood of fetuses at midpregnancy. The expression patterns of MMP26 in human placenta suggests complicated roles for MMP26 during the processes of placentation and hematopoiesis, perhaps working in concert with other members of the MMP family, such as MMP9.

Protein engineering and properties of human metalloproteinase and thrombospondin 1.

This work generated many truncated proteins and Glu(385) to Ala (E(385)/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E(385)/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH(2)-terminal sequence of L(33)GRPSEEDEE. A species at 115 kDa and some other protein bands began with F(236)VSSHRYV(243), indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R(235)-F(236) peptide bond. This cleavage was not an autocatalytic process since the E(385)/A mutants were also processed. Furthermore, a 52 kDa band with an NH(2)-terminal sequence of L(800)KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.

Effect of matrix metalloproteinase-26 (MMP-26) during embryo implantation in the mouse

Matrix metalloproteinase-26 (MMP-26, endometase and matrilysin-2), a novel member of the MMPs family, is detected not only in the placenta and uterus, but is widely expressed in malignant tumors from different sources as well as in diverse tumor cell lines. However, the function of MMP-26 in the reproductive system has never been reported. Expression of MMP-26 in mouse embryos and the function of the MMP-26 antibody during mouse embryo implantation was examined for the first time by injecting the uterine horn, immunohistochemistry,in situ hybridization, co-culture of mouse blastocysts and uterine monolayer epithelial cells, Western blot, RT-PCR, Northern blot and zymography. Our results show that there is strong expression of MMP-26 mRNA and protein in the mouse embryo. Furthermore, the MMP-26 antibody dramatically inhibited mouse embryo implantation and significantly inhibited adhesion and outgrowth of mouse blastocysts onin vitro uterine monolayer epithelial cells. At the same time, the MMP-26 antibody inhibited the expression of integrin αV mRNA and protein in a dose-dependent manner. These data suggest that MMP-26 may play a role in some of the tissue-remodeling events associated with the invasion of the endometrium by trophoblast cells and facilitate successfully embryo implantation.