Regulation of vimentin by SIP1 in human epithelial breast tumor cells.

The expression of Smad interacting protein-1 (SIP1; ZEB2) and the de novo expression of vimentin are frequently involved in epithelial-to-mesenchymal transitions (EMTs) under both normal and pathological conditions. In the present study, we investigated the potential role of SIP1 in the regulation of vimentin during the EMT associated with breast tumor cell migration and invasion. Examining several breast tumor cell lines displaying various degrees of invasiveness, we found SIP1 and vimentin expression only in invasive cell lines. Also, using a model of cell migration with human mammary MCF10A cells, we showed that SIP1 is induced specifically in vimentin-positive migratory cells. Furthermore, transfection of SIP1 cDNA in MCF10A cells increased their vimentin expression both at the mRNA and protein levels and enhanced their migratory abilities in Boyden Chamber assays. Inversely, inhibition of SIP1 expression by RNAi strategies in BT-549 cells and MCF10A cells decreased vimentin expression. We also showed that SIP1 transfection did not activate the TOP-FLASH reporter system, suggesting that the beta-catenin/TCF pathway is not implicated in the regulation of vimentin by SIP1. Our results therefore implicate SIP1 in the regulation of vimentin observed in the EMT associated with breast tumor cell migration, a pathway that may contribute to the metastatic progression of breast cancer.

Vimentin contributes to human mammary epithelial cell migration.

Vimentin expression in human mammary epithelial MCF10A cells was examined as a function of their migratory status using an in vitro wound-healing model. Analysis of the trajectories of the cells and their migratory speeds by time lapse-video microscopy revealed that vimentin mRNA and protein expression were exclusively induced in cells at the wound's edge which were actively migrating towards the center of the lesion. Actin labeling showed the reorganization of actin filaments in cells at the wound's edge which confirmed the migratory phenotype of this cell subpopulation. Moreover, the vimentin protein disappeared when the cells became stationary after wound closure. Using cells transfected with the vimentin promoter controlling the green fluorescent protein gene, we also demonstrated the specific activation of the vimentin promoter in the migratory cells at the wound's edge. Transfection of the antisense vimentin cDNA into MCF10A cells clearly reduced both their ability to express vimentin and their migratory speed. Taken together, these observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.

Expression of gelatinases A and B and their tissue inhibitors by cells of early and term human placenta and gestational endometrium.

BACKGROUND: Human placentation is mediated by fetal trophoblastic cells that invade the maternal uterine endometrium. Trophoblast invasion requires a precisely regulated secretion of specific proteolytic enzymes able to degrade the endometrial basement membrane and extracellular matrix. EXPERIMENTAL DESIGN: Several studies have documented the key roles of matrix metalloproteinases and their tissue inhibitors in the invasion of various matrices by cultured trophoblasts. In vitro studies suggest that placentation could result from a balance between the secretion of these enzymes by trophoblast cells and their inhibition by the natural tissue inhibitors (TIMPs) produced by maternal decidual cells. The precise localization and levels of expression of these proteins that account for and control invasion during human placentation in vivo however, have not been described. We have evaluated, in vivo, by immunohistochemistry, Northern blot analysis and in situ hybridization, the expression of two metalloproteinases (gelatinases A and B) and their two tissue inhibitors (TIMPs 1 and 2) in placental villi and placental beds of first and third trimesters of normal pregnancy. RESULTS: Human first trimester intermediate trophoblast produced both gelatinases A and B; these two gelatinases were respectively less and no more detected at term in these cells. We found that both TIMP1 and 2 were also expressed in maternal decidual cells with a dramatic increase of TIMP1 at the term of pregnancy. In floating villi, gelatinase A and TIMP1 were localized in the stromal compartment, whereas gelatinase B and TIMP2 were codistributed in trophoblast cells. CONCLUSIONS: The gelatinases A and B and their tissue inhibitors are thus expressed by specific cells in early and late placental beds and villi. This pattern of expression varies during pregnancy. Therefore, our morphologic study supports biologic findings suggesting that these proteins may participate in placentation.

Down-Regulation of MT1-MMP expression by the alpha3 chain of type IV collagen inhibits bronchial tumor cell line invasion.

The basement membrane (BM) is the first barrier encountered by tumor cells when they become invasive. Moreover, some invasive tumor clusters are surrounded by a remnant or neosynthetized BM material. We have previously reported the presence of a particular alpha chain of type IV collagen, the alpha3(IV) chain, in bronchopulmonary carcinomas. This chain was not detected in the normal bronchial epithelium, but was found around some invasive tumor cluster BM. In the present study, we examined the effects of the alpha3(IV) chain on the invasive properties of bronchial tumor cell lines, with special emphasis on their expression of matrix metalloproteinase-2 (MMP-2) and its activator, membrane type 1-matrix metalloproteinase (MT1-MMP), which is largely involved in tumor progression. Two epithelial bronchial cell lines (16HBE14o- and BZR), showing different invasive abilities, were evaluated. Using the Boyden chamber invasion assay, we demonstrated that the alpha3(IV) chain inhibits the invasive properties of BZR cells and modifies their morphology by inducing an epithelial cell shape. In the presence of the recombinant NC1 domain of the alpha3(IV) chain, the expression of MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) was not modified in either cell line. The NC1 alpha3(IV) domain did not modulate the MT1-MMP expression of noninvasive 16HBE14o- cells, whereas a 50% decrease of MT1-MMP mRNA was observed in invasive BZR cells. Accordingly, Western blot analyses showed a disappearance of the 45-kd MT1-MMP form when BZR cells were treated with the recombinant NC1 alpha3(IV) domain. These findings suggest that the alpha3 chain of type IV collagen may play a role in tumor invasion, at least by decreasing the expression and synthesis of MT1-MMP.

Contribution of MT1-MMP and of human laminin-5 gamma2 chain degradation to mammary epithelial cell migration.

Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a membrane-anchored matrix metalloproteinase (MMP) that is frequently associated with processes involving tissue remodelling and cell migration. We have examined MT1-MMP expression and subcellular distribution as a function of MCF10A mammary epithelial cell migration using an in vitro outgrowth migration assay. Stronger expression of MT1-MMP was observed at the mRNA and at the protein level in cells at the periphery of the outgrowth. As shown by videomicroscopy, these cells were involved in an orientated cell migration, in contrast to stationary cells distant from the periphery. Furthermore, MT1-MMP was mainly distributed in lamellipodia of migratory cells, as well as at their basal surface in contact with the substrate. Laminin-5 (Ln-5), a recently described substrate for MT1-MMP, was deposited preferentially in the matrix by migratory cells. Fragments of the gamma2 subunit of Ln-5 were also identified in migratory cultures of MCF10A cells, attesting to its proteolytic degradation. These fragments corresponded in size to those we observed after incubation of purified human Ln-5 with the recombinant catalytic domain of human MT1-MMP. We also show that anti-Ln5 blocking antibodies, MMP inhibitors (BB94 and TIMP-2) and MT1-MMP antisense oligonucleotides significantly decreased MCF10A cell migration. Taken together, these observations demonstrate that MT1-MMP is spatially and temporally regulated during MCF10A cell migration, and suggest that MT1-MMP-mediated pericellular proteolysis of Ln-5 gamma2 chain could contribute to this process.