Maspin (Mp) is a member of the serpin family with inhibitory functions against cell migration, metastasis and angiogenesis. To identify its role in embryonic development in vivo, we generated maspin knockout mice by gene targeting. In this study, we showed that homozygous loss of maspin expression was lethal at the peri-implantation stage. Maspin was specifically expressed in the visceral endoderm after implantation; deletion of maspin interfered with the formation of the endodermal cell layer, thereby disrupting the morphogenesis of the epiblast. In vitro, the ICM of the Mp–/– blastocysts failed to grow out appropriately. Data from embryoid body formation studies indicated that the Mp–/– EBs had a disorganized, endodermal cell mass and lacked a basement membrane layer. We showed that the embryonic ectoderm lineage was lost in the Mp–/– EBs,compared with that of the Mp+/+ EBs. Re-expression of maspin partially rescued the defects observed in the Mp–/– EBs, as evidenced by the appearance of ectoderm cells and a layer of endoderm cells surrounding the ectoderm. In addition, a maspin antibody specifically blocked normal EB formation,indicating that maspin controls the process through a cell surface event. Furthermore, we showed that maspin directly increased endodermal cell adhesion to laminin matrix but not to fibronectin. Mp+/–endodermal cells grew significantly slower than Mp+/+endodermal cells on laminin substrate. We conclude that deletion of maspin affects VE function by reducing cell proliferation and adhesion, thereby controlling early embryonic development.
Maspin is a unique serpin with the ability to suppress certain types of malignant tumors. It is one of the few p53-targeted genes involved in tumor invasion and metastasis. With this in mind, we attempted to study the molecular mechanism behind this tumor suppression. Maspin-expressing mammary tumors are more susceptible to apoptosis in both implanted mammary tumors in vivo, a three-dimensional spheroid culture system, as well as in monolayer cell culture under lowered growth factors. Subcellular fractionation shows that a fraction of maspin (in both TM40D-Mp and mutant maspin⌬N cells) translocates to the mitochondria. This translocation of maspin to the mitochondria is linked to the opening of the permeability transition pore, which in turn causes the loss of transmembrane potential, thus initiating apoptotic degradation. This translocation is absent in the other mutant, maspin⌬RSL. It fails to cause any loss of membrane potential and also shows decreased caspase 3 levels, proving that translocation to the mitochondria is a key event for this increase in apoptosis by maspin. Suppression of maspin overexpression by RNA interference desensitizes cells to apoptosis. Our data indicate that maspin inhibits tumor progression through the mitochondrial apoptosis pathway. These findings will be useful for maspin-based therapeutic interventions against breast cancer.
Background: Maspin is a member of serpin family with tumor suppressing activity. Recent studies of maspin in animal models strongly support maspin's role as an inhibitor against the growth of primary tumor sand the process of metastasis. However, the molecular mechanism underlying this inhibition has not been fully elucidated. In this report, we analyze the effect of maspin on tumor cell apoptosis under several stress conditions.
Maspin has been shown to possess tumor-suppressing activity against breast tumor growth and metastasis. To test the therapeutic value of the maspin gene (SERPINB5) in breast cancer, we established a syngeneic breast tumor metastasis model. This model involved the implantation of mammary tumor cells orthotopically to mammary gland and allowed tumors to grow within the gland and become metastatic to other organs. The mammary tumor cells were initially isolated from MMTV-polyoma virus middle T transgenic mice and were selected in vitro for high invasiveness. Here, we demonstrate that the mammary tumor cells were highly invasive and metastatic. Overall, 100% of tumor-transplanted mice developed lung metastasis. Using nonviral liposome as a carrier, we delivered SERPINB5 to mice bearing mammary tumors. Our data showed that both primary tumor growth and metastasis were significantly inhibited in this syngeneic metastasis model. Such inhibition is mediated by SERPINB5 transgene through increased apoptosis in SERPINB5-treated tumors. Thus, SERPINB5 can be used in gene therapy against breast tumor growth and metastasis.
Maspin is a unique member of the serpin family, which functions as a class II tumor suppressor gene. Despite its known activity against tumor invasion and motility, little is known about maspin's functions in normal mammary gland development. In this paper, we show that maspin does not act as a tPA inhibitor in the mammary gland. However, targeted expression of maspin by the whey acidic protein gene promoter inhibits the development of lobular-alveolar structures during pregnancy and disrupts mammary gland differentiation. Apoptosis was increased in alveolar cells from transgenic mammary glands at midpregnancy. However, the rate of proliferation was increased in early lactating glands to compensate for the retarded development during pregnancy. These findings demonstrate that maspin plays an important role in mammary development and that its effect is stage dependent.
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