Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix

Abstract

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...

Signaling by integrin receptors

“…Inhibition of a v b 3 function during angiogenesis with anti-a v b 3 antibodies both inhibited proliferation and induced apoptosis, whereas treatment with antibodies that block b 1 integrin function did not (Brooks et al, 1994). Similarly, prevention of interactions of mammary epithelium with the matrix by anti-b 1 integrin antibodies both inhibited proliferation and induced apoptosis (Boudreau et al, 1995). These results suggest that the same integrin might connect to di erent pathways in di erent cell types and that a given integrin could provide a survival signal in one cell type and an anoikis signal in another.…”

“…Other integrins such as a v b 1 or a v b 3 and various b 1 integrins fail to rescue cells from apoptosis under these conditions. It is possible that under other conditions cells depend on other integrins for survival (Brooks et al, 1994;Boudreau et al, 1995). Di erent cell types may di er in their integrin dependence under the same conditions.…”

Overexpression of Bcl-2 inhibits alveolar cell apoptosis during involution and accelerates c-myc-induced tumorigenesis of the mammary gland in transgenic mice

“…This is consistent with the discrimination of two independent phases of involution, one characterized by apoptosis of alveolar cells, and a later where the activation of extracellular matrix (ECM)-degrading matrix metalloproteinases leads to alveolar collapse and remodelling of the mammary gland (Lund et al, 1996). Therefore, it is unlikely that the disruption of the ECM triggers the apoptosis of alveolar cells as has been concluded from in vitro experiments where ECM provided a survival signal to mammary epithelial cells (Boudreau et al, 1995(Boudreau et al, , 1996Pullan et al, 1996). However, in the WAP-bcl-2 transgenic mice the increased apoptosis observed at day four of involution could well be explained by this, as transgene expression dropped at this timepoint and alveolar structures were disrupted, most likely due to degradation of the ECM.…”

The matrix metalloproteinase stromelysin-1 acts as a natural mammary tumor promoter

“…Either way, Str1 could act as an apoptotic stimulus. Indeed, Str1 does induce apoptosis in di erentiated mammary alveolar epithelial cells in culture and in vivo, however it also promotes the proliferation and branching of ductal epithelium Boudreau et al, 1995;Sympson et al, 1994;Thomasset et al, 1998;Witty et al, 1995). These seemingly contradictory e ects can be reconciled by noting that ductal epithelial cells normally divide during branching morphogenesis and persist throughout involution, whereas alveolar epithelial cells do not.…”