Transmembrane/cytoplasmic, rather than catalytic, domains of Mmp14 signal to MAPK activation and mammary branching morphogenesis via binding to integrin ß1.

Epithelial cell invasion through the extracellular matrix (ECM) is a crucial step in branching morphogenesis. The mechanisms by which the mammary epithelium integrates cues from the ECM with intracellular signaling in order to coordinate invasion through the stroma to make the mammary tree are poorly understood. Because the cell membrane-bound matrix metalloproteinase Mmp14 is known to play a key role in cancer cell invasion, we hypothesized that it could also be centrally involved in integrating signals for mammary epithelial cells (MECs) to navigate the collagen 1 (CL-1)-rich stroma of the mammary gland. Expression studies in nulliparous mice that carry a NLS-lacZ transgene downstream of the Mmp14 promoter revealed that Mmp14 is expressed in MECs at the tips of the branches. Using both mammary organoids and 3D organotypic cultures, we show that MMP activity is necessary for invasion through dense CL-1 (3 mg/ml) gels, but dispensable for MEC branching in sparse CL-1 (1 mg/ml) gels. Surprisingly, however, Mmp14 without its catalytic activity was still necessary for branching. Silencing Mmp14 prevented cell invasion through CL-1 and disrupted branching altogether; it also reduced integrin ß1 (Itgb1) levels and attenuated MAPK signaling, disrupting Itgb1-dependent invasion/branching within CL-1 gels. FRET imaging revealed that Mmp14 associates directly with Itgb1. We identified a domain of Mmp14 that is required for modulating the levels of Itgb1, MEC signaling and the rate of invasion within CL-1. These results shed light on hitherto undescribed non-proteolytic activities of Mmp14 that are necessary for the Itgb1-dependent biochemical and mechanical signals that regulate branching in the mammary epithelium.

Molecular and biochemical mechanisms in teratogenesis involving reactive oxygen species.

Developmental pathologies may result from endogenous or xenobiotic-enhanced formation of reactive oxygen species (ROS), which oxidatively damage cellular macromolecules and/or alter signal transduction. This minireview focuses upon several model drugs (phenytoin, thalidomide, methamphetamine), environmental chemicals (benzo[a]pyrene) and gamma irradiation to examine this hypothesis in vivo and in embryo culture using mouse, rat and rabbit models. Embryonic prostaglandin H synthases (PHSs) and lipoxygenases bioactivate xenobiotics to free radical intermediates that initiate ROS formation, resulting in oxidation of proteins, lipids and DNA. Oxidative DNA damage and embryopathies are reduced in PHS knockout mice, and in mice treated with PHS inhibitors, antioxidative enzymes, antioxidants and free radical trapping agents. Thalidomide causes embryonic DNA oxidation in susceptible (rabbit) but not resistant (mouse) species. Embryopathies are increased in mutant mice deficient in the antioxidative enzyme glucose-6-phosphate dehydrogenase (G6PD), or by glutathione (GSH) depletion, or inhibition of GSH peroxidase or GSH reductase. Inducible nitric oxide synthase knockout mice are partially protected. Inhibition of Ras or NF-kB pathways reduces embryopathies, implicating ROS-mediated signal transduction. Atm and p53 knockout mice deficient in DNA damage response/repair are more susceptible to xenobiotic or radiation embryopathies, suggesting a teratological role for DNA damage, consistent with enhanced susceptibility to methamphetamine in ogg1 knockout mice with deficient repair of oxidative DNA damage. Even endogenous embryonic oxidative stress carries a risk, since untreated G6PD- or ATM-deficient mice have increased embryopathies. Thus, embryonic processes regulating the balance of ROS formation, oxidative DNA damage and repair, and ROS-mediated signal transduction may be important determinants of teratological risk.

Reduced tumorigenesis in p53 knockout mice exposed in utero to low-dose vitamin E.

BACKGROUND: The limited antioxidative capacity of the fetus renders it more susceptible to reactive oxygen species (ROS), and possibly to ROS-mediated cancer initiation or promotion in utero. METHODS: To test this hypothesis, pregnant cancer-prone p53 knockout mice were prenatally supplemented with a low dietary dose of the antioxidant vitamin E (VE) (0.1% all-rac-alpha-tocopherol-acetate), and the homozygous (-/-) and heterozygous (+/-) p53-deficient and wild-type (+/+) offspring were examined for VE levels, oxidative DNA damage, chromosomal stability, cellular viability and postnatal tumorigenesis. RESULTS: In utero exposure to VE reduced spontaneous postnatal tumorigenesis in p53 +/- offspring, and increased VE levels and reduced fetal DNA oxidation in some but not all tissues of p53-deficient fetuses. Survival of VE-exposed p53 +/- offspring at the end of the study was double that of the +/- controls (45% vs 23%). In primary culture of skin fibroblasts from VE-exposed fetuses, VE did not alter chromosomal ploidy, but reduced cell death, indicating that its protective effect did not involve chromosomal stability. CONCLUSIONS: The tissue-selective increase in fetal VE levels and reduced DNA oxidation, together with a concomitant reduction in postnatal tumorigenesis, suggest that in utero oxidative stress contributes to some postnatal cancers, and the risk can be reduced by maternal dietary supplementation with low-dose VE.

Homology with vesicle fusion mediator syntaxin-1a predicts determinants of epimorphin/syntaxin-2 function in mammary epithelial morphogenesis.

We have shown that branching morphogenesis of mammary ductal structures requires the action of the morphogen epimorphin/syntaxin-2. Epimorphin, originally identified as an extracellular molecule, is identical to syntaxin-2, an intracellular molecule that is a member of the extensively investigated syntaxin family of proteins that mediate vesicle trafficking. We show here that, although epimorphin/syntaxin-2 is highly homologous to syntaxin-1a, only epimorphin/syntaxin-2 can stimulate mammary branching morphogenesis. We construct a homology model of epimorphin/syntaxin-2 based on the published structure of syntaxin-1a, and we use this model to identify the structural motif responsible for the morphogenic activity. We identify four residues located within the cleft between helices B and C that differ between syntaxin-1a and epimorphin/syntaxin-2; through site-directed mutagenesis of these four amino acids, we confer the properties of epimorphin for cell adhesion, gene activation, and branching morphogenesis onto the inactive syntaxin-1a template. These results provide a dramatic demonstration of the use of structural information about one molecule to define a functional motif of a second molecule that is related at the sequence level but highly divergent functionally.

Oxidative stress in developmental origins of disease: teratogenesis, neurodevelopmental deficits, and cancer.

In the developing embryo and fetus, endogenous or xenobiotic-enhanced formation of reactive oxygen species (ROS) like hydroxyl radicals may adversely alter development by oxidatively damaging cellular lipids, proteins and DNA, and/or by altering signal transduction. The postnatal consequences may include an array of birth defects (teratogenesis), postnatal functional deficits, and diseases. In animal models, the adverse developmental consequences of in utero exposure to agents like thalidomide, methamphetamine, phenytoin, benzo[a]pyrene, and ionizing radiation can be modulated by altering pathways that control the embryonic ROS balance, including enzymes that bioactivate endogenous substrates and xenobiotics to free radical intermediates, antioxidative enzymes that detoxify ROS, and enzymes that repair oxidative DNA damage. ROS-mediated signaling via Ras, nuclear factor kappa B and related transducers also may contribute to altered development. Embryopathies can be reduced by free radical spin trapping agents and antioxidants, and enhanced by glutathione depletion. Further modulatory approaches to evaluate such mechanisms in vivo and/or in embryo culture have included the use of knockout mice, transgenic knock-ins and mutant deficient mice with altered enzyme activities, as well as antisense oligonucleotides, protein therapy with antioxidative enzymes, dietary depletion of essential cofactors and chemical enzyme inhibitors. In a few cases, measures anticipated to be protective have conversely enhanced the risk of adverse developmental outcomes, indicating the complexity of development and need for caution in testing therapeutic strategies in humans. A better understanding of the developmental effects of ROS may provide insights for risk assessment and the reduction of adverse postnatal consequences.

Enhanced tumorigenesis in p53 knockout mice exposed in utero to high-dose vitamin E.

The limited antioxidative capacity of the embryo and fetus may increase their risk for cancer initiation and/or promotion by reactive oxygen species (ROS)-mediated oxidative DNA damage and/or signaling. To determine if cancer can originate in utero, a high dietary dose of the antioxidant vitamin E (VE) (10% dl-alpha-tocopherol-acetate) was given to cancer-prone p53 knockout mice throughout pregnancy. Although reducing fetal death (P < 0.05), in utero exposure to VE enhanced postnatal tumorigenesis in both +/- (P < 0.04) and -/- (P < 0.0008) p53-deficient offspring. VE did not alter maternal weights, offspring p53 genotypic distribution or tumor spectrum. Constitutive embryonic DNA oxidation in untreated -/- p53 embryos [gestational day (GD) 13] was higher than in +/- and +/+ p53 littermates (P < 0.05). VE reduced DNA oxidation in -/- p53 embryos (P < 0.05) without affecting +/- and +/+ p53 littermates. VE had contrasting, tissue-dependent effects on fetal (GD 19) DNA oxidation, with reductions in -/- and +/- p53-deficient fetal brains (P < 0.01), increases in skin (P < 0.05) and no effect in liver and thymus. The 250-fold increase in dietary VE levels produced only 1.6-6.3-fold, tissue-dependent increases in tissue concentrations. The greatest increase, in fetal skin, correlated with increased DNA oxidation in that tissue in -/- and +/- p53-deficient fetuses and enhanced tumorigenesis in these genotypes. These results show that some cancers may originate in utero and the risk can be enhanced by embryonic and fetal exposure to high dietary levels of VE. The elevated DNA oxidation in some tissues of untreated -/- p53 offspring suggests that ROS may contribute to their higher baseline tumor incidence. The limited and tissue-dependent disposition of VE indicates substantial conceptal regulation. The similarly selective and contrasting effects of VE on DNA oxidation may contribute to its controversial protective efficacy and suggest that its effects on tumorigenesis are cell-specific, possibly in high doses involving a pro-oxidative mechanism.