Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to β 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1β converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression.Growth, differentiation, and apoptosis are alternative cellular pathways that are each crucial to normal development and the establishment of tissue-specific function. Like growth and differentiation, apoptosis requires active and coordinated regulation of specific genes. In mammalian cells, these genes include BCL-2, a homolog of the Caenorhabditis elegans ced-9 gene, which is a potent suppressor of death (1), and ICE, a homolog of the ced-3 gene, which can actively kill cells (2). The products of the BCL-2 and ICE genes also appear to function like their C. elegans counterparts(1-3).The nature of the ECM can influence the apoptotic program in mammalian cells. Establishment of mammary gland alveolar morphology and expression of milk-specific genes are absolutely dependent on deposition of a laminin-rich ECM (4). In addition, involution of the gland, which follows expression of the lactational phenotype, is characterized by degradation of this ECM by metalloproteinases (5) and is accompanied by apoptosis (6,7). Cell attachment, mediated by integrin-ECM interactions, can suppress apoptosis in short-term two-dimensional cultures for up to 30 hours (8).Correspondence to: Nancy Boudreau. NIH Public Access Author ManuscriptScience. Author manuscript; available in PMC 2010 December 20. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptTo determine whether ECM regulates apoptosis, we compared the response of CID-9 mammary epithelial cells (MECs) plated directly on tissue culture plastic, in the absence of serum, with those plated on an exogenous basement membrane ECM. Unlike plastic, fibronectin, or type I collagen, this Englebreth-Holm-Swarm (EHS) matrix directs the cells to differentiate, as manifested by the formation of three-dimensional alveolar structures and expression of milk proteins (9). After 4 to 5 days on plastic, despite strong adhesion and spreading, the cells began to display characteristics of apoptosis including nucleosomal DNA laddering (Fig. 1A), expression of the apoptosis-associated gene SGP-2 (7) (Fig. 1B), and nuclear condensation (Fig. 1C). In situ analysis revealed that fragmented DNA was present in 10 to 20% of cells (Fig. 1D). In contrast, the cells plated on ECM did not display these apoptotic features for up to 10 days (Fig. 1, A, B, and E). Similar results were observed when the ECM was pretreated with ammoniu...

Matrix metalloproteinases (MMPs) are invariably upregulated in the stromal compartment of epithelial cancers and appear to promote invasion and metastasis. Here we report that phenotypically normal mammary epithelial cells with tetracycline-regulated expression of MMP3/stromelysin-1 (Str1) form epithelial glandular structures in vivo without Str1 but form invasive mesenchymal-like tumors with Str1. Once initiated, the tumors become independent of continued Str1 expression. Str1 also promotes spontaneous premalignant changes and malignant conversion in mammary glands of transgenic mice. These changes are blocked by coexpression of a TIMP1 transgene. The premalignant and malignant lesions have stereotyped genomic changes unlike those seen in other murine mammary cancer models. These data indicate that Str1 influences tumor initiation and alters neoplastic risk.

Abstract. The extracellular matrix (ECM) is an important regulator of the differentiated phenotype of mammary epithelial cells in culture. Despite the fact that ECM-degrading enzymes have been implicated in morphogenesis and tissue remodeling, there is little evidence for a direct role for such regulation in vivo. We generated transgenic mice that express autoactivated isoforms of the matrix metalloproteinase stromelysin-1, under the control of the whey acidic protein gene promoter, to examine the effect of inappropriate expression of this enzyme. Stromelysin-1 is implicated as the primary player in the loss of basement membrane and loss of function in the mammary gland during involution. The transgene was expressed at low levels in maramary glands of virgin female mice, leading to an unexpected phenotype: The primary ducts had supernumerary branches and showed precocious development of alveoli that expressed /3-casein at levels similar to that of an early-to midpregnant gland. Lactating glands showed high levels of transgene expression, with accumulation at the basement membrane, and a decrease in laminin and collagen IV, resulting in a loss of basement membrane integrity; this was accompanied by a dramatic alteration of alveolar morphology, with decreased size and shrunken lumina containing little/3-casein. During pregnancy, expression of endogenous whey acidic protein and r-casein was reduced in transgenic glands, confirming the observed dependence of milk protein transcription on ECM in mammary epithelial cells in culture. These data provide direct evidence that stromelysin-1 activity can be morphogenic for mammary epithelial cells, inducing hyperproliferation and differentiation in virgin animals, and that its lytic activity can, indeed, disrupt membrane integrity and reduce mammary-specific function. We conclude that the balance of ECM-degrading enzymes with their inhibitors, and the associated regulation of ECM structure, is crucial for tissue-specific gene expression and morphogenesis in vivo.T HE differentiated state is plastic, requiring continuous and active control for both its acquisition and its maintenance (Bisseli, 1981;Blau, 1992). In vertebrate cells in culture, there is an increasing body of evidence that the extracenular matrix (ECM) t plays a seminal role both in in-

Stromelysin-1 is a member of the metalloproteinase family of extracellular matrix-degrading enzymes that regulates tissue remodeling. We previously established a transgenic mouse model in which rat stromelysin-1 targeted to the mammary gland augmented expression of endogenous stromelysin-1, disrupted functional differentiation, and induced mammary tumors. A cell line generated from an adenocarcinoma in one of these animals and a previously described mammary tumor cell line generated in culture readily invaded both a reconstituted basement membrane and type I collagen gels, whereas a nonmalignant, functionally normal epithelial cell line did not. The matrix metalloproteinases (MMPs) 1 are a family of extracellular matrix (ECM)-degrading enzymes that have been implicated in a variety of normal developmental and pathological processes, including tumorigenesis (1, 2). The MMP family comprises at least 15 members with different, albeit overlapping, substrate specificities. During activation of latent MMPs, their propeptides are cleaved and they are converted to a lower molecular weight form by other enzymes, including serine proteinases, and by autocatalytic cleavage.Among the MMPs, stromelysin-1 (SL1) possesses the broadest substrate specificity (1). Despite increasing knowledge about its enzymatic properties and the regulation of its expression, little is known about its function. We have generated transgenic animals that express an autoactivating mutant of rat SL1 targeted to the epithelial compartment of the mammary gland (3). Phenotypically, SL1 transgenic mice display increased branching morphogenesis and lactogenic differentiation at prepubertal stages and premature involution during late pregnancy (3, 4). Branching morphogenesis requires the invasion of epithelial cells into the adipose tissue, a process reminiscent of invasion of stromal compartments by tumor cells. Strikingly, a large number of SL1 transgenic animals also develop mammary tumors of various histotypes, including invasive adenocarcinomas (5). 2 Because tumor development is a late response of SL1 transgenic mice to overexpression of the transgene, it remains unclear whether SL1 plays a direct role in tumor growth and/or invasion or whether the observed tumors are a consequence of other molecular alterations in the microenvironment of the mammary gland before the onset of tumor growth. 3 Studies performed with synthetic inhibitors of MMP activity and tissue inhibitors of metalloproteinases (TIMPs) have shown that suppression of MMP activity also suppresses tumor growth and metastasis (6, 7). In many cases, the level of SL1 expression in tumors of the mammary gland and other tissues is positively correlated with the degree of malignancy (8, 9). However, the only direct evidence for the nature of the MMPs involved was provided by the demonstration that function-blocking antibodies against gelatinase A and antisense inhibition of matrilysin expression decreased the invasiveness of tumor cells in a reconstituted basement membrane assay (10, 11). The...