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Biomedical subjects

Z Werb

Publications and source records attributed to Z Werb.

At least 91 records · Page 5Linked to original sources
PubMed1996

Focalized proteolysis: spatial and temporal regulation of extracellular matrix degradation at the cell surface.

Cells respond to changes in their microenvironment by altering their cell surface and extracellular matrix proteins. Rapid and irreversible changes in these proteins are possible through their degradation or activation by proteolysis. By focalizing the proteolytic events at or near the cell surface, these processes can be effective even in the presence of high concentrations of inhibitors. Evidence is emerging that secreted and transmembrane matrix metalloproteinases, metalloproteinases of the adamalysin and astacin (tolloid) families, and serine proteinases are crucial in development, differentiation, cell motility and invasion, and cell-extracellular decisions.

Animals↗
PubMed1996

Matrix metalloproteinases and the development of cancer.

Proteolytic remodeling of the extracellular matrix is an important aspect of the creation and progression of cancer. Matrix metalloproteinases are important at several points during multi-stage neoplastic progression in tumor cells and responding blood vessels, inflammatory cells and stroma.

Enzyme Activation↗
PubMed1996

Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene.

We have used transgenic mice overexpressing the human tissue inhibitor of metalloproteinases (TIMP)-1 gene under the control of the ubiquitous beta-actin promoter/enhancer to evaluate matrix metalloproteinase (MMP) function in vivo in mammary gland growth and development. By crossing the TIMP-1 transgenic animals with mice expressing an autoactivating stromelysin-1 transgene targeted to mammary epithelial cells, we obtained a range of mice with genetically engineered proteolytic levels. The alveolar epithelial cells of mice expressing autoactivating stromelysin-1 underwent unscheduled apoptosis during late pregnancy. When stromelysin-1 transgenic mice were crossed with mice overexpressing TIMP-1, apoptosis was extinguished. Entactin (nidogen) was a specific target for stromelysin-1 in the extracellular matrix. The enhanced cleavage of basement membrane entactin to above-normal levels was directly related to the apoptosis of overlying mammary epithelial cells and paralleled the extracellular MMP activity. These results provide direct evidence for cleavage of an extracellular matrix molecule by an MMP in vivo.

Animals↗
PubMed1996

The role of stromelysin-1 in stromal-epithelial interactions and cancer.

Stromelysin-1 was one of the first proteinases found to be associated with cancer. In this review we describe the role of stromelysin-1 in normal mammary gland involution. When stromelysin-1 is overexpressed in transgenic mice the mammary gland undergoes precocious involution and is predisposed to forming a reactive stroma resembling that of a wound site or a tumor. Stromelysin-1 may act as an oncogene because transgenic mice expressing an active form of the enzyme develop mammary tumors. These observations suggest that stromelysin-1 and other matrix metalloproteinases may be useful targets for therapeutic intervention in cancer.

Animals↗
PubMed1996

Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways.

Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when beta-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when beta-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1 beta converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin-1 and uPA were weakly induced, if at all, in hydrocortisone-treated mice. Furthermore, mRNA for membrane-type matrix metalloproteinase decreased after hydrocortisone treatment and paralleled the almost complete inhibition of activation of latent gelatinase A. Concomitantly, the gland filled with an overabundance of milk. Our data support the hypothesis that there are at least two distinct phases of involution: an initial phase, characterized by induction of the apoptosis-associated genes SGP-2 and ICE and apoptosis of fully differentiated mammary epithelial cells without visible degradation of the extracellular matrix, and a second phase, characterized by extracellular matrix remodeling and altered mesenchymal-epithelial interactions, followed by apoptosis of cells that are losing differentiated functions.

Animals↗
PubMed1996

Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation.

Gelatinase B, a matrix metalloproteinase (MMP) of high specific activity, is highly expressed and activated by mouse blastocysts in culture, and inhibition of this enzyme activity inhibits lysis of extracellular matrix (Behrendtsen, O., Alexander, C. M. and Werb, Z. (1992) Development 114, 447-456). Because gelatinase B expression is linked to invasive potential, we studied the expression of gelatinase B mRNA and protein in vivo, in implanting trophoblast giant cells, and found that it was expressed and activated during colonization of the maternal decidua. mRNAs for several other MMPs (stromelysin-1, stromelysin-3 and gelatinase A) and MMP inhibitors (TIMP-1 and TIMP-2) were expressed in the undifferentiated stroma toward the outside of the decidua, and TIMP-3 mRNA was expressed in primary and some mature decidual cells during their differentiation. Both mRNA and TIMP-3 protein were present at high concentrations transiently, and declined from 6.5 days post coitum onward, as the cells underwent apoptosis during the main period of gelatinase B expression and ectoplacental growth and expansion. To assess the function of MMPs during implantation and decidual development, we either injected a peptide hydroxamate MMP inhibitor into normal mice or studied transgenic mice overexpressing TIMP-1. In both cases, decidual length and overall size were reduced, and the embryo was displaced mesometrially. Embryo orientation was less strictly regulated in inhibitor-treated deciduae than in control deciduae. Morphogenesis and development of oil-induced deciduomas were also slowed in the presence of the inhibitor. We conclude that administration of MMP inhibitors retards decidual remodeling and growth, and we suggest that the MMPs expressed in precursor stromal cells promote their differentiation and expansion.

Animals↗
PubMed1996

Extracellular matrix remodeling and the regulation of epithelial-stromal interactions during differentiation and involution.

An intact basement membrane is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or remodeling of the basement membrane occurs during normal development as well as in the disease state. To examine the importance of basement membrane during development in vivo, we altered the matrix metalloproteinase and tissue inhibitor of metalloproteinases balance in mammary gland. Inhibition of matrix metalloproteinase synthesis by glucocorticoids or implants or transgenic overexpression of tissue inhibitor of metalloproteinases -1 delays matrix degradation and the involution process after weaning. The mammary glands from transgenic mice that inappropriately express auto-activating isoforms of stromelysin-1 are both functionally and morphologically altered throughout development. Transgenic mammary glands have supernumerary branches, and show precocious development of alveoli that express beta-casein expression and undergo unscheduled apoptosis during pregnancy. This is accompanied by progressive development of an altered stroma, which becomes fibrotic after postweaning involution, and by development of neoplasias. These data suggest that metalloproteinases and disruption of the basement membrane may play key roles in branching morphogenesis of mammary gland, cell cycle, apoptosis, and stromal fibrosis as well as in induction and progression of breast cancer.

Animals↗
PubMed1996

Extracellular matrix remodeling as a regulator of stromal-epithelial interactions during mammary gland development, involution and carcinogenesis.

An intact basement membrane is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or remodeling of the basement membrane occurs during normal development as well as in the disease state. Stromelysin-1 (SL-1), a member of the matrix metalloproteinase (MMP) family, was one of the first proteinases found to be associated with cancer. In this review we describe the role of MMPs in normal mammary gland involution. To examine the importance of basement membrane during development in vivo, we altered the MMP and tissue inhibitor of metalloproteinases (TIMP) balance in mammary gland. Inhibition of MMP synthesis by glucocorticoids or implants or transgenic overexpression of TIMP-1 delays matrix degradation and the involution process after weaning. The mammary glands from transgenic mice that inappropriately express autoactivating isoforms of SL-1 are both functionally and morphologically altered throughout development. Transgenic mammary glands have supernumerary branches, and show precocious development of alveoli that express beta-casein expression and undergo unscheduled apoptosis during pregnancy. This is accompanied by progressive development of an altered stroma, which resembles that of a wound site or a tumor, and becomes fibrotic after postweaning involution, and by development of neoplasias. These data suggest that MMPs and disruption of the basement membrane may play key roles in branching morphogenesis of mammary gland, apoptosis, and stromal fibrosis as well as in induction and progression of breast cancer. These observations suggest that SL-1 and other MMPs may be useful targets for therapeutic intervention in cancer.

Animals↗
PubMed1995

Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor.

Since the discovery that epidermal growth factor (EGF) can accelerate opening of the eyelids, the EGF receptor (EGF-R) has been extensively studied and is now considered to be a prototype tyrosine kinase receptor. Binding of EGF or of transforming growth factor-alpha (TGF-alpha) or other related factors activates the receptor and induces cell proliferation and differentiation. Although it is not found on haematopoietic cells, the EGF-R is widely expressed in mammals and has been implicated in various stages of embryonic development. Here we investigate the developmental and physiological roles of this receptor and its ligands by inactivating the gene encoding EGF-R. We find that EGF-R-/- mice survive for up to 8 days after birth and suffer from impaired epithelial development in several organs, including skin, lung and gastrointestinal tract.

Animals↗
PubMed1995

Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix.

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 beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta 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.

Animals↗
PubMed1995

Mammary gland tumor formation in transgenic mice overexpressing stromelysin-1.

An intact basement membrane (BM) is essential for the proper function, differentiation and morphology of many epithelial cells. The disruption or loss of this BM occurs during normal development as well as in the disease state. To examine the importance of BM during mammary gland development in vivo, we generated transgenic mice that inappropriately express autoactivating isoforms of the matrix metalloproteinase stromelysin-1. The mammary glands from these mice are both functionally and morphologically altered throughout development. We have now documented a dramatic incidence of breast tumors in several independent lines of these mice. These data suggest that overexpression of stromelysin-1 and disruption of the BM may be a key step in the multi-step process of breast cancer.

Animals↗
PubMed1995

Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin.

Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors.

Amino Acid Sequence↗
PubMed1995

Components of the nuclear signaling cascade that regulate collagenase gene expression in response to integrin-derived signals.

We have shown previously that the expression of collagenase is upregulated in rabbit synovial fibroblasts cultured on a substrate of antibody to the alpha 5 chain of the alpha 5 beta 1 integrin fibronectin receptor or on the 120-kD cell-binding chymotryptic fragment of plasma fibronectin, but remains at basal levels in cells plated on intact plasma fibronectin. We now have identified some of the components of a signaling pathway that couples the fibronectin receptor to the induction of collagenase transcription. We studied the control of collagenase gene expression in cells adhering to the 120-kD fragment of fibronectin, to antifibronectin receptor antibody, or to plasma fibronectin by transiently introducing promoter-reporter constructs into rabbit synovial fibroblasts before plating cells on these matrices. The constructs contained segments of the human collagenase promoter regulating transcription of chloramphenicol acyl transferase. Expression of constructs containing the -1200/-42-bp segment or the -139/-42-bp segment of the collagenase promoter inserted upstream from the reporter gene was induced to similar extents in cells plated on the 120-kD fragment of fibronectin or on anti-fibronectin receptor antibody, relative to that in fibroblasts plated on fibronectin. The expression of the construct containing the -66/-42-bp segment of the promoter was not regulated and was similar to that of the parent pBLCAT2 plasmid, suggesting that the -139/-67 region of the collagenase promoter, which contains PEA3- and AP1-binding sites, regulates the transcription of collagenase caused by integrin-derived signals. Expression of a reporter construct containing only the PEA3 and AP1 sites in the collagenase promoter (-90/-67) also increased in cells plated on the 120-kD fragment of fibronectin or on anti-fibronectin receptor antibody, relative to that in cells plated on fibronectin. Mutations in either the AP1 or PEA3 site of this minimal promoter abrogated its activity in cells plated on these inductive ligands. Expression of c-fos mRNA increased within 1 h of plating cells on the 120-kD fibronectin fragment or on anti-fibronectin receptor antibody, relative to that in cells plated on fibronectin. c-Fos protein accumulated in the nuclei of fibroblasts within 10 min of plating on the 120-kD fibronectin fragment. The increase in c-Fos was required for the increase in collagenase in cells plated on the 120-kD fibronectin fragment: incubation of cells with antisense, but not sense, c-fos oligonucleotides diminished both basal and induced expression of the -139/-42 collagenase promoter-reporter construct and decreased expression of the endogenous collagenase gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Cooperative interactions between extracellular matrix, integrins and parathyroid hormone-related peptide regulate parietal endoderm differentiation in mouse embryos.

The outgrowth of parietal endoderm (PE) cells from precursor endodermal cells is one of the first differentiation events that occur in mouse embryos. We have analyzed the molecular determinants of this process by placing isolated inner cell masses (ICMs) on defined extracellular matrix substrata in microdrop cultures. Differentiation and outgrowth of PE required a fibronectin substratum. Laminin supported the adhesion and outgrowth of visceral endoderm (VE) and actively suppressed the differentiation of PE in mixtures of fibronectin and laminin. Collagen type IV, gelatin, vitronectin or entactin supported little or no endodermal outgrowth. Trophectoderm (TE) cells have been implied to be important in PE induction in vivo. We found that recombination of ICMs in culture with TE cells, or with medium conditioned by TE cells, greatly increased the differentiation of PE. TE cells stimulated PE outgrowth on substrata other than fibronectin. One cytokine secreted by trophoblast and endodermal cells, parathyroid hormone-related peptide (PTHrP), was critical for outgrowth on any substratum. A function-perturbing antibody to PTHrP reduced the number of PE cells, whereas the addition of PTHrP increased that number. Furthermore, addition of PTHrP changed the substratum requirements for outgrowth, making laminin, vitronectin and low concentrations of fibronectin permissive for PE outgrowth. Immunostaining with anti-integrin antibodies showed that fully differentiated PE cells outgrowing on fibronectin expressed alpha 5, alpha 6 and alpha v beta 3 integrins. However, analysis of outgrowths in the presence of function-perturbing antibodies to alpha 5, alpha 6 and alpha v beta 3 integrins showed that these integrins directed PE outgrowth only on fibronectin, laminin and vitronectin substrata, respectively. We have shown that there is a cooperative interplay of extracellular matrix, integrins and PTHrP that modulates PE outgrowth.

Animals↗
PubMed1995

Stem cell defects in parthenogenetic peri-implantation embryos.

Mouse embryos containing only maternal chromosomes (parthenotes) develop abnormally in vivo, usually failing at the peri-implantation stage. We have analyzed the development of parthenote embryos by using an inner cell mass (ICM) outgrowth assay that mimics peri-implantation development. ICMs from normal embryos maintained undifferentiated stem cells positive for stage-specific embryonic antigen-1 and Rex-1 while differentiating into a variety of cell types, including visceral endoderm-like cells and parietal endoderm cells. In contrast, ICMs from parthenotes failed to maintain undifferentiated stem cells and differentiated almost exclusively into parietal endoderm. This suggests that parthenote ICMs have a defect that leads to differentiation, rather than maintenance, of the stem cells, and a defect that leads to a parietal endoderm fate for the stem cells. To test the hypothesis that the ICM population is not maintained owing to a lack of proliferation of the stem cells, we investigated whether mitogenic agents were able to maintain the ICM population in parthenotes. When parthenote blastocysts were supplied with the insulin-like growth factor-1 receptor (Igf-1r) and insulin-like growth factor-2 (Igf-2), two genes not detectable in parthenote blastocysts by in situ hybridization, the ICM population was maintained. Similarly, culture of parthenote blastocysts in medium conditioned by embryonic fibroblasts and supplemented with the maternal factor leukemia inhibitory factor maintained the ICM population. However, once this growth factor-rich medium was removed, the parthenote ICM cells still differentiated predominantly into parietal endoderm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Hxt encodes a basic helix-loop-helix transcription factor that regulates trophoblast cell development.

Trophoblast cells are the first lineage to form in the mammalian conceptus and mediate the process of implantation. We report the cloning of a basic helix-loop-helix (bHLH) transcription factor gene, Hxt, that is expressed in early trophoblast and in differentiated giant cells. A separate gene, Hed, encodes a related protein that is expressed in maternal deciduum surrounding the implantation site. Overexpression of Hxt in mouse blastomeres directed their development into trophoblast cells in blastocysts. In addition, overexpression of Hxt induced the differentiation of rat trophoblast (Rcho-1) stem cells as assayed by changes in cell adhesion and by activation of the placental lactogen-I gene promoter, a trophoblast giant cell-specific gene. In contrast, the negative HLH regulator, Id-1, inhibited Rcho-1 differentiation and placental lactogen-I transcription. These data demonstrate a role for HLH factors in regulating trophoblast development and indicate a positive role for Hxt in promoting the formation of trophoblast giant cells.

Amino Acid Sequence↗
PubMed1994

Implantation and the placenta: key pieces of the development puzzle.

The mammalian embryo cannot develop without the placenta. Its specialized cells (trophoblast, endoderm, and extraembryonic mesoderm) form early in development. They attach the embryo to the uterus (implantation) and form vascular connections necessary for nutrient transport. In addition, the placenta redirects maternal endocrine, immune, and metabolic functions to the embryo's advantage. These complex activities are sensitive to disruption, as shown by the high incidence of early embryonic mortality and pregnancy diseases in humans, as well as the numerous peri-implantation lethal mutations in mice. Integration of molecular and developmental approaches has recently produced insights into the molecules that control these processes.

Animals↗