Hypersensitivity to DNA damage leads to increased apoptosis during early mouse development.

Gastrulation in mice is associated with the start of extreme proliferation and differentiation. The potential cost to the embryo of a very rapid proliferation rate is a high production of damaged cells. We demonstrate a novel surveillance mechanism for the elimination of cells damaged by ionizing radiation during mouse gastrulation. During this restricted developmental window, the embryo becomes hypersensitive to DNA damage induced by low dose irradiation (<0.5 Gy) and undergoes apoptosis without cell cycle arrest. Intriguingly, embryonic cells, including germ cell progenitors, but not extraembryonic cells, become hypersensitive to genotoxic stress and undergo Atm- and p53-dependent apoptosis. Thus, hypersensitivity to apoptosis in the early mouse embryo is a cell fate-dependent mechanism to ensure genomic integrity during a period of extreme proliferation and differentiation.

Inflammatory mast cells up-regulate angiogenesis during squamous epithelial carcinogenesis.

Expression of HPV16 early region genes in basal keratinocytes of transgenic mice elicits a multistage pathway to squamous carcinoma. We report that infiltration by mast cells and activation of the matrix metalloproteinase MMP-9/gelatinase B coincides with the angiogenic switch in premalignant lesions. Mast cells infiltrate hyperplasias, dysplasias, and invasive fronts of carcinomas, but not the core of solid tumors, where they degranulate in close apposition to capillaries and epithelial basement membranes, releasing mast-cell-specific serine proteases MCP-4 (chymase) and MCP-6 (tryptase). MCP-6 is shown to be a mitogen for dermal fibroblasts that proliferate in the reactive stroma, whereas MCP-4 can activate progelatinase B and induce hyperplastic skin to become angiogenic in an in vitro bioassay. Notably, premalignant angiogenesis is abated in a mast-cell-deficient (KITW/KITWWv) HPV16 transgenic mouse. The data indicate that neoplastic progression in this model involves exploitation of an inflammatory response to tissue abnormality. Thus, regulation of angiogenesis during squamous carcinogenesis is biphasic: In hyperplasias, dysplasias, and invading cancer fronts, inflammatory mast cells are conscripted to reorganize stromal architecture and hyperactivate angiogenesis; within the cancer core, upregulation of angiogenesis factors in tumor cells apparently renders them self-sufficient at sustaining neovascularization.

Expression of autoactivated stromelysin-1 in mammary glands of transgenic mice leads to a reactive stroma during early development.

Extracellular matrix and extracellular matrix-degrading matrix metalloproteinases play a key role in interactions between the epithelium and the mesenchyme during mammary gland development and disease. In patients with breast cancer, the mammary mesenchyme undergoes a stromal reaction, the etiology of which is unknown. We previously showed that targeting of an autoactivating mutant of the matrix metalloproteinase stromelysin-1 to mammary epithelia of transgenic mice resulted in reduced mammary function during pregnancy and development of preneoplastic and neoplastic lesions. Here we examine the cascade of alterations before breast tumor formation in the mammary gland stroma once the expression of the stromelysin-1 transgene commences. Beginning in postpubertal virgin animals, low levels of transgene expression in mammary epithelia led to increased expression of endogenous stromelysin-1 in stromal fibroblasts and up-regulation of other matrix metalloproteinases, without basement membrane disruption. These changes were accompanied by the progressive development of a compensatory reactive stroma, characterized by increased collagen content and vascularization in glands from virgin mice. This remodeling of the gland affected epithelial-mesenchymal communication as indicated by inappropriate expression of tenascin-C starting by day 6 of pregnancy. This, together with increased transgene expression, led to basement membrane disruption starting by day 15 of pregnancy. We propose that the highly reactive stroma provides a prelude to breast epithelial tumors observed in these animals.

Metalloproteinases regulate parietal endoderm differentiating and migrating in cultured mouse embryos.

Extracellular matrix (ECM) adhesion and proteolysis play important roles in embryonic development. In previous work (Behrendtsen et al. [1992] Development 114:447-456) we showed that gelatinase B activity is rate-limiting for trophoblast-mediated invasion and degradation of ECM in culture. In the present study, we show that metalloproteinases (MMPs) have distinct roles in migration along ECM as opposed to invasion through ECM. We investigated the role of ECM proteolysis in the differentiation and migration of parietal endoderm (PE), the first embryonic migratory cell type, adhering to ECM surfaces. Gelatinase B was the major MMP of PE; mRNA and protein were detected in PE of 7.5- and 8.5-day embryos. Using cultures of inner cell masses (ICMs) isolated from mouse blastocysts, we found that inhibitors of metalloproteinases, specifically, tissue inhibitor of metalloproteinases (TIMP)-1 and a peptide hydroxamic acid stimulated outgrowth and differentiation of PE from ICMs cultured on fibronectin, but inhibitors of plasminogen activators did not. TIMP-1 increased the number of PE cells and mean distance migrated and increased expression of the PE differentiation marker vimentin; the increase in cell number was not at the expense of other cell types. The stimulatory effect of TIMP-1 was most marked on low concentrations of substrate fibronectin, decreasing as concentrations of fibronectin increased. TIMP-1 also stimulated the outgrowth of PE in blastocyst cultures and in ICM/trophectoderm co-cultures; in ICM/trophectoderm co-cultures TIMP-1 stimulated PE differentiation on higher concentrations of fibronectin than was permissive for ICMs cultured alone. These data indicate that metalloproteinase inhibitors preserved the migration-inducing status of the ECM. We conclude that metalloproteinases have distinct roles in invasive activity through ECM barriers and migratory activity along ECM surfaces.

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.

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.

Role of zinc-binding- and hemopexin domain-encoded sequences in the substrate specificity of collagenase and stromelysin-2 as revealed by chimeric proteins.

The relationship of enzyme structure to substrate specificity for the matrix metalloproteinases interstitial collagenase and stromelysin-2 has been investigated by analysis of the cleavage specificity of recombinant human collagenase-stromelysin-2 hybrid proteins and C terminally truncated collagenase and stromelysin-2. Two series of chimeric proteins were devised by progressive substitution of exon-encoded domains. The recombinant proteins were expressed in COS-7 cells as protein A-fusion proteins and purified on an IgG affinity matrix. Treatment with 4-amino-phenylmercuric acetate released active metalloproteinase of the sizes predicted for the chimeric proteins. Active forms of both the chimeric protein series and the short form enzymes expressed both casein- and gelatin-degrading activities. Like stromelysin, the catalytic activity of stromelysin-2 was contained in the N-terminal domain (encoded by exons 1-5) and was apparently independent of the C-terminal domain (encoded by exons 6-10). Only full-length collagenase displayed a triple helicase (collagenolytic) activity; no combination of N- or C-terminal collagenase domains fused with stromelysin-2 domains had such activity. This suggests that the triple helicase activity is a composite of elements derived from both halves of the collagenase molecule. C terminally truncated collagenase (exons 1-5) and a hybrid of collagenase exons 1-5 and stromelysin-2 exons 6-10 cleaved denatured type I collagen (gelatin) to generate diagnostic peptides in gelatin fingerprint assays. When exon 5 (the exon encoding the zinc-binding domain) was derived from stromelysin-2, the enzyme specificity in the fingerprint assay changed to that of native stromelysin-2. In contrast, when exon 5 was derived from collagenase, the specificity reflected that of the parent enzyme. Our data also suggest that mismatching of exons 2 and 5 destabilizes the enzyme, presumably by altering the geometry of the propeptide-zinc-binding site interaction. We conclude that the loss of triple helicase collagenolytic activity is not accompanied by a shift to the broad specificity characteristic of stromelysin. Rather, the zinc-binding domain confers a distinct cleavage specificity on each metalloproteinase.

Insulin-like growth factor II acts through an endogenous growth pathway regulated by imprinting in early mouse embryos.

We present evidence that insulin-like growth factor II (IGF-II) mediates growth in early mouse embryos and forms a pathway in which imprinted genes influence development during preimplantation stages. mRNA and protein for IGF-II were expressed in preimplantation mouse embryos, but the related factors IGF-I and insulin were not. IGF-I and insulin receptors and the IGF-II/mannose-6-phosphate receptor were expressed. Exogenous IGF-II or IGF-I increased the cell number in cultured blastocysts, but a mutant form of IGF-II that strongly binds only the IGF-II receptor did not. Reduction of IGF-II expression by antisense IGF-II oligonucleotides decreased the rate of progression to the blastocyst stage and decreased the cell number in blastocysts. Preimplantation parthenogenetic mouse embryos expressed mRNA for the IGF-II receptor but not for either IGF-II ligand or the IGF-I receptor, indicating that the latter genes are not expressed when inherited maternally. These data imply that some growth factors and receptors, regulated by genomic imprinting, may control cell proliferation from the earliest stages of embryonic development.

Metalloproteinases mediate extracellular matrix degradation by cells from mouse blastocyst outgrowths.

The maintenance and developmental remodeling of extracellular matrix is crucial to such processes as uterine implantation and the cell migratory events of morphogenesis. When mouse blastocysts are placed in culture they adhere to extracellular matrix, and trophoblast giant cells migrate out onto the matrix and degrade it. The secretion of functional proteinases by developing mouse embryos increases dramatically at the time of implantation. By zymography we identified the major secreted gelatin-degrading proteinase, also known as type IV collagenase, as one migrating at 92 x 10(3) Mr. Several casein-degrading proteinases were also secreted. The tissue inhibitor of metalloproteinases (TIMP) inhibited all of the embryo-derived proteinases detected by gelatin gel zymography, indicating that they are metalloproteinases, whereas TIMP did not inhibit all of the caseinases. Urokinase was also secreted. Addition of TIMP at 5-500 nM effectively inhibited the degradation of matrix by the trophoblast outgrowths. Blocking antibodies directed against 92 x 10(3) Mr gelatinase abolished matrix degradation by the trophoblast cells. These observations suggest that several metalloproteinases are regulated in early development and that 92 x 10(3) Mr gelatinase, in particular, has a rate-limiting function in degradation of the maternal extracellular matrix by trophoblast cells.

Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression.

We have investigated the effects of ligation of the fibronectin receptor (FnR) on gene expression in rabbit synovial fibroblasts. Monoclonal antibodies to the FnR that block initial adhesion of fibroblasts to fibronectin induced the expression of genes encoding the secreted extracellular matrix-degrading metalloproteinases collagenase and stromelysin. That induction was a direct consequence of interaction with the FnR was shown by the accumulation of mRNA for stromelysin and collagenase. Monoclonal antibodies to several other membrane glycoprotein receptors had no effect on metalloproteinase gene expression. Less than 2 h of treatment of the fibroblasts with anti-FnR in solution was sufficient to trigger the change in gene expression, and induction was blocked by dexamethasone. Unlike other inducers of metalloproteinase expression, including phorbol diesters and growth factors, addition of the anti-FnR in solution to cells adherent to serum-derived adhesion proteins or collagen produced no detectable change in cell shape or actin microfilament organization. Inductive effects were potentiated by cross-linking of the ligand. Fab fragments of anti-FnR were ineffective unless cross-linked or immobilized on the substrate. Adhesion of fibroblasts to native fibronectin did not induce metallo-proteinases. However, adhesion to covalently immobilized peptides containing the arg-gly-asp sequence that were derived from fibronectin, varying in size from hexapeptides up to 120 kD, induced collagenase and stromelysin gene expression. This suggests that degradation products of fibronectin are the natural inductive ligands for the FnR. These data demonstrate that signals leading to changes in gene expression are transduced by the FnR, a member of the integrin family of extracellular matrix receptors. The signaling of changes in gene expression by the FnR is distinct from signaling involving cell shape and actin cytoarchitecture. At least two distinct signals are generated: the binding of fibronectin-derived fragments and adhesion-blocking antibodies to the FnR triggers events different from those triggered by binding of the native fibronectin ligand. Because the genes regulated by this integrin are for enzymes that degrade the extracellular matrix, these results suggest that information transduced by the binding of various ligands to integrins may orchestrate the expression of genes regulating cell behavior in the extracellular environment.