Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations that leads to stable epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells Page: 3 of 28
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The extracellular matrix (ECM) microenvironment of cells is critical for determination of
cellular behavior and differentiation (for review see Adams and Watt, 1993; Lochter and Bissell,
1995; Ashkenas et al., 1996). Matrix metalloproteinases (MMPs) modify the cellular
microenvironment thereby playing important roles during development and tumor progression.
Experimental manipulation of net MMP activity can alter signaling through ECM receptors with
dramatic effects on cell migration, invasion, apoptosis, neurite extension, epithelial branching
morphogenesis, and expression of tissue-specific genes (for review see MacDougall and
Matrisian, 1995; Coussens and Werb, 1996). Although most MMPs are secreted extracellularly,
cell surface-bound MMPs catalyze focal dissolution of ECM (Monsky et al., 1993; for review
see Basbaum and Werb, 1996; Brooks et al., 1996). A wide variety of ECM proteins and certain
cell surface molecules are substrates for MMPs (Gearing et al., 1994; Ochieng et al., 1994; Levi
et al., 1996; Preece et al., 1996). Whereas MMPs are thought to shape cellular function by
remodeling ECM, targeting of cell surface molecules may also play a role in their mode of
action, although the latter possibility has not been elucidated.
To understand MMP function in mammary epithelial cells, we have focused on stromelysin-1
(SL-1), because of its diverse molecular targets (Lochter and Bissell, 1997). In the normal
mammary gland, SL-1 is synthesized by fibroblasts but not by epithelial cells (Witty et al.,
1995). Although SL-1 is produced at all stages of postnatal mammary gland development, its
expression is highest during involution (Talhouk et al., 1992; Li et al., 1994; Witty et al.,
1995). Intermediate levels of SL-1 are associated with phases of ductal branching and alveolar
morphogenesis. The lowest level of SL-1 expression is found in the resting mammary gland.
Targeting of an autoactivating mutant of SL-1 to mammary epithelia in vivo increases branching
morphogenesis during puberty and impairs lactogenic differentiation during pregnancy
(Sympson et al., 1994). Later in life, SL-1 transgenic mice develop preneoplastic lesions
and invasive breast tumors (Sympson et al., 1995). In culture, overexpression of SL-1 in a
mammary cell strain that contains both epithelial and mesenchymal cell types produces an
involutionlike phenotype with induction of apoptosis (Boudreau et al., 1995). Furthermore, SL-1
is used by mammary tumor cells for invasion (Lochter et al., 1997) and has been cloned a
number of times as a metastasisspecific gene (for review see MacDougall and Matrisian,
1995; Coussens and Werb, 1996). But how expression of SL-1 could lead to such diverse
consequences has remained undetermined. In this study, we have transfected the functionally
normal mouse mammary epithelial cell line SCp2 (Desprez et al., 1993) with a cDNA coding for
an autoactivating rat SL-1 transgene under the control of a tetracycline (Tet)-regulated promoter.
We describe the resulting cascade of molecular and functional alterations in the absence of a
Materials and Methods
Generation of Expression Vector for Epitope-tagged SL-1 and Transfection of SCp2 Cells
HA. 11, a hemagglutinin epitope tag comprising the amino acid sequence YDVPDYA and the
nucleotide sequence TACGACGTACCAGACTACGCA was added to the 3' end of the cDNA
coding for the autoactivating M1 mutant (Val92-Gy92 mutation) (Sanchez-Lopez et al., 1988;
Sympson et al., 1994) of the rat SL-1 cDNA (SL-1.M1) by PCR. The PCR product generated
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Lochter, A.; Galosy, S.; Muschler, J.; Freedman, N.; Werb, Z. & Bissell, M.J. Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations that leads to stable epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells, article, August 11, 1997; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc829363/m1/3/: accessed January 21, 2019), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.