Showing papers by "Zena Werb published in 2011"

Extracellular Matrix Degradation and Remodeling in Development and Disease

Abstract: The extracellular matrix (ECM) serves diverse functions and is a major component of the cellular microenvironment. The ECM is a highly dynamic structure, constantly undergoing a remodeling process where ECM components are deposited, degraded, or otherwise modified. ECM dynamics are indispensible during restructuring of tissue architecture. ECM remodeling is an important mechanism whereby cell differentiation can be regulated, including processes such as the establishment and maintenance of stem cell niches, branching morphogenesis, angiogenesis, bone remodeling, and wound repair. In contrast, abnormal ECM dynamics lead to deregulated cell proliferation and invasion, failure of cell death, and loss of cell differentiation, resulting in congenital defects and pathological processes including tissue fibrosis and cancer. Understanding the mechanisms of ECM remodeling and its regulation, therefore, is essential for developing new therapeutic interventions for diseases and novel strategies for tissue engineering and regenerative medicine.

Zeppo1 is a novel metastasis promoter that represses E-cadherin expression and regulates p120-catenin isoform expression and localization

Abstract: Amplification of 8p11-12 in human breast cancers is associated with increased proliferation and tumor grade and reduced metastasis-free patient survival. We identified Zeppo1 (zinc finger elbow-related proline domain protein 1 )( FLJ14299/ZNF703) within this amplicon as a regulator of cell adhesion, migration, and proliferation in mammary epithelial cells. Overexpression of Zeppo1 reduces cell–cell adhesion and stimulates migration and proliferation. Knockdown of Zeppo1 induces adhesion and lumen formation. Zeppo1 regulates transcription, complexing with Groucho and repressing E-cadherin expression and Wnt and TGFb reporter expression. Zeppo1 promotes expression of metastasis-associated p120-catenin isoform 1 and alters p120-catenin localization upon cell contact with the extracellular matrix. Significantly, Zeppo1 overexpression in a mouse breast cancer model increases lung metastases, while reducing Zeppo1 expression reduces both tumor size and the number of lung metastases. These results indicate that Zeppo1 is a key regulator of breast cancer progression.

A whole-organism screen identifies new regulators of fat storage

Abstract: The regulation of energy homeostasis integrates diverse biological processes ranging from behavior to metabolism and is linked fundamentally to numerous disease states. To identify new molecules that can bypass homeostatic compensatory mechanisms of energy balance in intact animals, we screened for small-molecule modulators of Caenorhabditis elegans fat content. We report on several molecules that modulate fat storage without obvious deleterious effects on feeding, growth and reproduction. A subset of these compounds also altered fat storage in mammalian and insect cell culture. We found that one of the newly identified compounds exerts its effects in C. elegans through a pathway that requires previously undescribed functions of an AMP-activated kinase catalytic subunit and a transcription factor previously unassociated with fat regulation. Thus, our strategy identifies small molecules that are effective within the context of intact animals and reveals relationships between new pathways that operate across phyla to influence energy homeostasis.

Impaired remodeling phase of fracture repair in the absence of matrix metalloproteinase-2

Abstract: SUMMARY The matrix metalloproteinase (MMP) family of extracellular proteases performs crucial roles in development and repair of the skeleton owing to their ability to remodel the extracellular matrix (ECM) and release bioactive molecules. Most MMP-null skeletal phenotypes that have been previously described are mild, thus permitting the assessment of their functions during bone repair in the adult. In humans and mice, MMP2 deficiency causes a musculoskeletal phenotype. In this study, we assessed the role of MMP2 during mouse fracture repair and compared it with the roles of MMP9 and MMP13. Mmp2 was expressed at low levels in the normal skeleton and was broadly expressed in the fracture callus. Treatment of wild- type mice with a general MMP inhibitor, GM6001, caused delayed cartilage remodeling and bone formation during fracture repair, which resembles the defect observed in Mmp9-/- mice. Unlike Mmp9- and Mmp13-null mutations, which affect both cartilage and bone in the callus, the Mmp2- null mutation delayed bone remodeling but not cartilage remodeling. This remodeling defect occurred without changes in either osteoclast recruitment or vascular invasion of the fracture callus compared with wild type. However, we did not detect changes in expression of Mmp9, Mmp13 or Mt1-Mmp (Mmp14) in the calluses of Mmp2-null mice compared with wild type by in situ hybridization, but we observed decreased expression of Timp2 in the calluses of Mmp2-, Mmp9- and Mmp13-null mice. In keeping with the skeletal phenotype of Mmp2-null mice, MMP2 plays a role in the remodeling of new bone within the fracture callus and impacts later stages of bone repair compared with MMP9 and MMP13. Taken together, our results indicate that MMPs play unique and distinct roles in regulating skeletal tissue deposition and remodeling during fracture repair.

Mammary Gland Reprogramming: Metalloproteinases Couple Form with Function

Abstract: The adult mammary structure provides for the rapid growth, development, and immunological protection of the live-born young of mammals through its production of milk. The dynamic remodeling of the branched epithelial structure of the mammary gland in response to physiological stimuli that allow its programmed branching morphogenesis at puberty, cyclical turnover during the reproductive cycle, differentiation into a secretory organ at parturition, postlactational involution, and ultimately, regression with age is critical for these processes. Extracellular metalloproteinases are essential for the remodeling programs that operate in the tissue microenvironment at the interface of the epithelium and thestroma,couplingformwithfunction.Deregulatedproteolyticactivitydrivesthetransition of a physiological mammary microenvironment into a tumor microenvironment, facilitating malignant transformation.

Matrix Metalloproteinase-9 and Stromal Cell-Derived Factor-1 Act Synergistically to Support Migration of Blood-Borne Monocytes into the Injured Spinal Cord

Abstract: The infiltration of monocytes into the lesioned site is a key event in the inflammatory response after spinal cord injury (SCI). We hypothesized that the molecular events governing the infiltration of monocytes into the injured cord involve cooperativity between the upregulation of the chemoattractant stromal cell-derived factor-1 (SDF-1)/CXCL12 in the injured cord and matrix metalloproteinase-9 (MMP-9/gelatinase B), expressed by infiltrating monocytes. SDF-1 and its receptor CXCR4 mRNAs were upregulated in the injured cord, while macrophages immunoexpressed CXCR4. When mice, transplanted with bone marrow cells from green fluorescent protein (GFP) transgenic mice, were subjected to SCI, GFP+ monocytes infiltrated the cord and displayed gelatinolytic activity. In vitro studies confirmed that SDF-1α, acting through CXCR4, expressed on bone marrow-derived macrophages, upregulated MMP-9 and stimulated MMP-9-dependent transmigration across endothelial cell monolayers by 2.6-fold. There was a reduction in F4/80+ macrophages in spinal cord-injured MMP-9 knock-out mice (by 36%) or wild-type mice, treated with the broad-spectrum MMP inhibitor GM6001 (by 30%). Mice were adoptively transferred with myeloid cells and treated with the MMP-9/-2 inhibitor SB-3CT, the CXCR4 antagonist AMD3100, or a combination of both drugs. While either drug resulted in a 28-30% reduction of infiltrated myeloid cells, the combined treatment resulted in a 45% reduction, suggesting that SDF-1 and MMP-9 function independently to promote the trafficking of myeloid cells into the injured cord. Collectively, these observations suggest a synergistic partnership between MMP-9 and SDF-1 in facilitating transmigration of monocytes into the injured spinal cord.

Dynamic, Long-Term In Vivo Imaging of Tumor–Stroma Interactions in Mouse Models of Breast Cancer Using Spinning-Disk Confocal Microscopy

Abstract: Tumors contain many components in addition to the cancer cells, including blood vessels, fibroblasts, and immune cells. Genetic studies and tumor biopsies have generated insights into the importance of these stromal components for cancer progression. However, it remains a challenge to reveal the dynamic interactions among the distinct tumor components within live animals. Studies involving multiphoton microscopy allow direct imaging of cellular movement in live mice, but multiphoton microscopy is expensive, complex, and usually relies on a single excitation wavelength for all fluorophores. This article describes a method for intravital imaging using a microlensed spinning-disk confocal microscope. Although tissue penetration with spinning-disk confocal microscopy is lower than with multiphoton microscopy, image acquisition with this method is very rapid, so artifacts from respiratory motion are avoided. Photobleaching and phototoxicity are low, and multicolor acquisition is cheaper and easier than with multiphoton microscopy. This article discusses various aspects of experimental setup, as well as methods for addressing technical barriers, such as generating and working with multiple tumor microenvironments within individual live mice, image collection, and long-term anesthesia.

Dual Protective Mechanisms of Matrix Metalloproteinases 2 and 9 in Immune Defense against Streptococcus pneumoniae

Abstract: A localized and effective innate immune response to pathogenic bacterial invasion is central to host survival. Identification of the critical local innate mediators of lung defense against such pathogens is essential for a complete understanding of the mechanism(s) underlying effective host defense. In an acute model of Streptococcus pneumoniae lung infection, deficiency in matrix metalloproteinase (MMP)2 and MMP9 (Mmp2/9−/−) conferred a survival disadvantage relative to wild-type mice treated under the same conditions. S. pneumoniae-infected Mmp2/9−/− mice recruited more polymorphonuclear leukocytes to the lung but had higher bacterial burdens. Mmp2/9−/− mice showed significantly higher levels of IL-17A, IP-10, and RANTES in the lung. Although MMP2-dependent cleavage partially inactivated IL-17A, MMP9 was critical for effective bacterial phagocytosis and reactive oxygen species generation in polymorphonuclear neutrophils. These data demonstrate critical nonredundant and protective roles for MMP2 and MMP9 in the early host immune response against S. pneumoniae infection.

Preparation of mice for long-term intravital imaging of the mammary gland.

Abstract: Cold Spring Harb Protoc; Andrew J. Ewald, Zena Werb and Mikala Egeblad Preparation of Mice for Long-Term Intravital Imaging of the Mammary Gland Service Email Alerting click here. Receive free email alerts when new articles cite this article Categories Subject Cold Spring Harbor Protocols. Browse articles on similar topics from (341 articles) Visualization, general (463 articles) Visualization (164 articles) Video Imaging / Time Lapse Imaging (71 articles) Transgenic Mice (293 articles) Mouse (880 articles) Laboratory Organisms, general (300 articles) Labeling for Imaging (138 articles) In Vivo Imaging, general (274 articles) In Vivo Imaging (538 articles) Imaging/Microscopy, general (224 articles) Fluorescent Proteins (405 articles) Fluorescence (88 articles) Confocal Microscopy (462 articles) Cell Imaging (1037 articles) Cell Biology, general

Measuring matrix metalloproteinase activity in macrophages and polymorphonuclear leukocytes.

Abstract: Macrophages and polymorphonuclear cells (PMNs) represent an essential part of the innate immune system. These cells mediate a wide spectrum of immunological functions including bacterial defense, immune modulation, and inflammation; they are necessary for tissue homeostasis and also contribute to pathologies such as malignancy, autoimmunity, and chronic inflammation. Both macrophages and PMNs express a set of matrix metalloproteinases (MMPs), zinc-dependent endopeptidases that are involved in a variety of biological functions such as the turnover of extracellular matrix (ECM) components, angiogenesis, and the regulation of inflammation. Given the link between unregulated MMP function and diseases such as chronic inflammation or cancer, it is not surprising that these enzymes have been implicated as key effectors in clinical studies. Thus, it is important to widen our knowledge about the role of these enzymes in macrophage and PMN biology. Here, we briefly discuss the general role of inflammatory cell-derived MMPs and describe methods to analyze their activity in macrophages and PMN.