Growth defects induced by perturbation of beta1-integrin function in the mammary gland epithelium result from a lack of MAPK activation via the Shc and Akt pathways

Adhesion to extracellular matrix (ECM) induces intracellular signals that modulate cell proliferation, survival and differentiation. To study signalling events triggered by cell–ECM interactions in vivo we used transgenic mice exhibiting reduced mammary epithelial cell proliferation and increased apoptosis rates during the growth phase in pregnancy and lactation due to expression of a β1-integrin dominant-negative mutant in the mammary gland epithelium. Here we show that ERK and JNK MAPKs were markedly less activated in lactating transgenic glands thereby accounting for the growth defects. The FAK pathway was not affected suggesting a mechanism of activation additional to the ECM signal. On the contrary, the significant decrease of Shc phosphorylation, Grb2 recruitment and the reduced phosphorylation level of Akt Thr308 and Akt substrates FKHR and Bad detected in transgenic glands show that activation of the Shc and the Akt pathways require intact cell–ECM interactions. These results provide an insight into the mechanisms of growth control by integrin-mediated adhesion that operate in vivo.     

Mammary gland ECM remodeling, stiffness, and mechanosignaling in normal development and tumor progression

Cells of the mammary gland are in intimate contact with other cells and with the extracellular matrix (ECM), both of which provide not only a biochemical context, but a mechanical context as well. Cell-mediated contraction allows cells to sense the stiffness of their microenvironment, and respond with appropriate mechanosignaling events that regulate gene expression and differentiation. ECM composition and organization are tightly regulated throughout development of the mammary gland, resulting in corresponding regulation of the mechanical environment and proper tissue architecture. Mechanical regulation is also at play during breast carcinoma progression, as changes in ECM deposition, composition, and organization accompany breast carcinoma. These changes result in stiffer matrices that activate mechanosignaling pathways and thereby induce cell proliferation, facilitate local tumor cell invasion, and promote progression. Thus, understanding the role of forces in the mammary gland is crucial to understanding both normal developmental and pathological processes.     

Identification of a Novel Gene (ECM2) Encoding a Putative Extracellular Matrix Protein Expressed Predominantly in Adipose and Female-Specific Tissues and Its Chromosomal Localization to 9q22.3

We isolated by the differential display technique a novel gene that was expressed abundantly in adipose and female-specific tissues. The cDNA contained an open reading frame of 2097 nucleotides encoding a 699-amino-acid peptide. The predicted protein showed homology to several known extracellular matrix (ECM) proteins such as proteoglycan, keratocan, and decorin. Moreover, the amino acid sequence contained several possible functional domains that would participate in protein–protein interactions, including an RGD sequence, a von Willebrand factor domain (VWFC), and a leucine-rich repeat. These findings suggest that this novel protein functions in cell–cell and/or cell–ECM recognition processes. Northern blot analysis revealed expression predominantly in adipose tissue as well as female-specific organs such as mammary gland, ovary, and uterus among 20 human adult tissues examined. We assigned the gene to chromosome 9q22.3 by means of fluorescencein situhybridization.     

The contribution of adhesion signaling to lactogenesis

The mammary gland undergoes hormonally controlled cycles of pubertal maturation, pregnancy, lactation, and involution, and these processes rely on complex signaling mechanisms, many of which are controlled by cell–cell and cell–matrix adhesion. The adhesion of epithelial cells to the extracellular matrix initiates signaling mechanisms that have an impact on cell proliferation, survival, and differentiation throughout lactation. The control of integrin expression on the mammary epithelial cells, the composition of the extracellular matrix and the presence of secreted matricellular proteins all contribute to essential adhesion signaling during lactogenesis. In vitro and in vivo studies, including the results from genetically engineered mice, have shed light on the regulation of these processes at the cell and tissue level and have led to increased understanding of the essential signaling components that are regulated in temporal and cell specific manner during lactogenesis. Recent studies suggest that a secreted matricellular protein, CTGF/CCN2, may play a role in lactogenic differentiation through binding to β1 integrin complexes, enhancing the production of extracellular matrix components and contributions to cell adhesion signaling.     

Hyaluronan concentration within a 3D collagen matrix modulates matrix viscoelasticity, but not fibroblast response

The use of 3D extracellular matrix (ECM) microenvironments to deliver growth-inductive signals for tissue repair and regeneration requires an understanding of the mechanisms of cell–ECM signaling. Recently, hyaluronic acid (HA) has been incorporated in collagen matrices in an attempt to recreate tissue specific microenvironments. However, it is not clear how HA alters biophysical properties (e.g. fibril microstructure and mechanical behavior) of collagen matrices or what impact these properties have on cell behavior. The present study determined the effects of varying high molecular weight HA concentration on 1) the assembly kinetics, fibril microstructure, and viscoelastic properties of 3D type I collagen matrices and 2) the response of human dermal fibroblasts, in terms of morphology, F-actin organization, contraction, and proliferation within the matrices. Results showed increasing HA concentration up to 1 mg/ml (HA:collagen ratio of 1:2) did not significantly alter fibril microstructure, but did significantly alter viscoelastic properties, specifically decreasing shear storage modulus and increasing compressive resistance. Interestingly, varied HA concentration did not significantly affect any of the measured fibroblast behaviors. These results show that HA-induced effects on collagen matrix viscoelastic properties result primarily from modulation of the interstitial fluid with no significant change to the fibril microstructure. Furthermore, the resulting biophysical changes to the matrix are not sufficient to modulate the cell–ECM mechanical force balance or proliferation of resident fibroblasts. These results provide new insight into the mechanisms by which cells sense and respond to microenvironmental cues and the use of HA in collagen-based biomaterials for tissue engineering.     

A sentinel function for teat tissues in dairy cows: dominant innate immune response elements define early response to E. coli mastitis

Escherichia coli intramammary infection elicits localized and systemic responses, some of which have been characterized in mammary secretory tissue. Our objective was to characterize gene expression patterns that become activated in different regions of the mammary gland during the acute phase of experimentally induced E. coli mastitis. Tissues evaluated were from Fürstenburg’s rosette, teat cistern (TC), gland cistern (GC), and lobulo-alveolar (LA) regions of control and infected mammary glands, 12 and 24 h after bacterial (or control) infusions. The main networks activated by E. coli infection pertained to immune and inflammatory response, with marked induction of genes encoding proteins that function in chemotaxis and leukocyte activation and signaling. Genomic response at 12 h post-infection was greatest in tissues of the TC and GC. Only at 24 h post-infection did tissue from the LA region respond, at which time the response was the greatest of all regions. Similar genetic networks were impacted in all regions during early phases of intramammary infection, although regional differences throughout the gland were noted. Data support an important sentinel function for the teat, as these tissues responded rapidly and intensely, with production of cytokines and antimicrobial peptides.     

Gene Expression Profiling of Rat Cerebral Cortex Development Using cDNA Microarrays

A large amount of genetic information is devoted to brain development. In this study, the cortical development in rats at eight developmental time points (four embryonic [E15, E16, E18, E20] and four postnatal [P0, P7, P14, P21]) was studied using a rat brain 10K cDNA microarray. Significant differential expression was observed in 467 of the 9,805 genes represented on the microarray. Two major Gene Ontology classes—cell differentiation and cell–cell signaling—were found to be important for cortical development. Genes for ribosomal proteins, heterogeneous nuclear ribonucleoproteins, and tubulin proteins were up-regulated in the embryonic stage, coincidently with extensive proliferation of neural precursor cells as the major component of the cerebral cortex. Genes related to neurogenesis, including neurite regeneration, neuron development, and synaptic transmission, were more active in adulthood, when the cerebral cortex reached maturity. The many developmentally modulated genes identified by this approach will facilitate further studies of cortical functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mechanical and failure properties of extracellular matrix sheets as a function of structural protein composition

The goal of this study was to determine how alterations in protein composition of the extracellular matrix (ECM) affect its functional properties. To achieve this, we investigated the changes in the mechanical and failure properties of ECM sheets generated by neonatal rat aortic smooth muscle cells engineered to contain varying amounts of collagen and elastin. Samples underwent static and dynamic mechanical measurements before, during, and after 30 min of elastase digestion followed by a failure test. Microscopic imaging was used to measure thickness at two strain levels to estimate the true stress and moduli in the ECM sheets. We found that adding collagen to the ECM increased the stiffness. However, further increasing collagen content altered matrix organization with a subsequent decrease in the failure strain. We also introduced collagen-related percolation in a nonlinear elastic network model to interpret these results. Additionally, linear elastic moduli correlated with failure stress which may allow the in vivo estimation of the stress tolerance of ECM. We conclude that, in engineered replacement tissues, there is a tradeoff between improved mechanical properties and decreased extensibility, which can impact their effectiveness and how well they match the mechanical properties of native tissue.     

Genetic structure of farmer-managed varieties in clonally-propagated crops

The relative role of sexual reproduction and mutation in shaping the diversity of clonally propagated crops is largely unknown. We analyzed the genetic diversity of yam—a vegetatively-propagated crop—to gain insight into how these two factors shape its diversity in relation with farmers’ classifications. Using 15 microsatellite loci, we analyzed 485 samples of 10 different yam varieties. We identified 33 different genotypes organized in lineages supported by high bootstrap values. We computed the probability that these genotypes appeared by sexual reproduction or mutation within and between each lineage. This allowed us to interpret each lineage as a product of sexual reproduction that has evolved by mutation. Moreover, we clearly noted a similarity between the genetic structure and farmers’ classifications. Each variety could thus be interpreted as being the product of sexual reproduction having evolved by mutation. This highly structured diversity of farmer-managed varieties has consequences for the preservation of yam diversity.     

In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry

The neuromuscular junction (NMJ), where a motor neuron intercepts and activates a muscle fiber, is a highly versatile and complex subcellular region. Genomic and proteomic approaches using the large (>1 kg) electric organ of Torpedo californica have helped advancing our understanding of this minute (30–50 μm) electric synapse. However, the majority of these studies have focused on mRNA and proteins, therefore neglecting small signaling molecules involved in muscle-nerve ‘dialogue’. We developed a novel technique, mid-infrared laser ablation electrospray ionization (LAESI) mass spectrometry (MS), with the potential of detecting a diversity of small signaling molecules in vitro. LAESI uses the native water in the tissue as the matrix to couple the laser pulse energy into the target for the ablation process and enables its direct analysis essentially without sample preparation. Here, we report the detection of metabolites from the untreated frozen tissue of the Torpedo electric organ with LAESI MS at atmospheric pressure. A total of 24 metabolites were identified by accurate mass measurements, natural isotope patterns, and tandem mass spectrometry. Most of the identified metabolites were related to the cholinergic function of the electric synapse (acetylcholine and choline), fatty acid metabolism and acetyl transfer (carnitine and acetylcarnitine), the mitigation of osmotic stress (betaine and trimethylamine N-oxide), and energy production (creatine and creatinine). The biosynthetic precursors of these metabolites and their expected degradation products were also detected indicating that LAESI MS is well suited for tissue metabolomics with the ultimate goal of imaging and in vivo studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tissue remodelling in liver diseases

Tissue remodelling is a dynamic process that occurs during fetal or adult life and involves a modification of the original organization and function of a tissue. Tissue remodelling is observed in physiological and pathological conditions such as during wound healing or in the mammary gland during the course of pregnancy. In this review we will discuss the remodelling occurring in the liver as a consequence of chronic inflammation, as observed in chronic hepatitis, or as a consequence of hepatocellular carcinoma (HCC) progression in more detail. We will consider how altered deposition and turn-over of extracellular matrix (ECM) proteins could lead to development of liver fibrosis, and how the exacerbation of fibrosis could underlie the development of cirrhosis. The involvement of inflammatory and anti-inflammatory cytokines commonly used as therapeutic agents, such as Interferon-a, is then evaluated with a particular focus on modulation of ECM proteolysis. Finally, we analyze the role of alterations of the surrounding microenvironment including ECM, growth factors, cytokines and membrane receptors for ECM ligands in the development of HCC and in its invasive behaviour.     

The extracellular matrix as an adhesion checkpoint for mammary epithelial function

The development of the mammary gland is spatially regulated by the interaction of the mammary epithelium with the extracellular matrix (ECM). Cells receive cues from the ECM through a family of adhesion receptors called integrins, consisting of alpha- and beta-chain dimers. Integrins assist cells in sensing their appropriate developmental context in response to both hormones and growth factors. Here we argue that cell adhesion to the ECM plays a key role in specific developmental checkpoints, particularly in alveolar survival, morphogenesis and function. Specific ablation of alphabeta1-integrins in the luminal epithelium of the mammary gland shows that this sub-type of receptors is required for proliferation, accurate morphological organisation, as well as milk secretion. Downstream, small Rho GTPases mediate cellular polarisation and differentiation. Current challenges in studying the integration of signals in checkpoints of mammary gland development are discussed.     

Hepsin regulates TGFβ signaling via fibronectin proteolysis

Transforming growth factor‐beta (TGFβ) is a multifunctional cytokine with a well‐established role in mammary gland development and both oncogenic and tumor‐suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFβ activity by acting as a storage compartment of latent‐TGFβ, but how TGFβ is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFβ signaling through the release of latent‐TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent‐TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell‐free and cell‐based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent‐TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.     

A Highlights from MBoC Selection: Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs

Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.     

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial–stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin^cpdm), and mice with a stromal (S100a4‐Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin^cpdm mammary epithelial cells transplanted in vivo into wild‐type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin^cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin^cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro. Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.     

Defining Life or Bringing Biology to Life

In the present, post-genomic times, systemic or holistic approaches to living phenomena are compulsory to overcome the limits of traditional strategies, such as the methodological reductionism of molecular biology. In this paper, we propose that theoretical and philosophical efforts to define life also contribute to those integrative approaches, providing a global theoretical framework that may help to deal with or interpret the huge amount of data being collected by current high-throughput technologies, in this so-called ‘omics’ revolution. We claim that two fundamental notions can capture the core of the living, (basic) autonomy and open-ended evolution, and that only the complementary combination of these two theoretical constructs offers an adequate solution to the problem of defining the nature of life in specific enough—but also encompassing enough—terms. This tentative solution should also illuminate, in its most elementary version, the leading steps towards living beings on Earth.     

Boundaries that demarcate structural and functional domains of chromatin

Understanding of how the eukaryotic genome is packaged into chromatin and what the functional consequences of this organization are has begun to emerge recently. The concept of ‘chromatin domains’ — the topologically independent structural unit — is the basis of higher order chromatin organization. The idea that this structural unit may also coincide with the functional unit, offers a useful framework in dissecting the structure-function relationship. Boundaries that define these domains have been identified and several assays have been developed to test themin vivo. We have used genetic means to identify and analyse such boundary elements in the bithorax complex ofDrosophila melanogaster. In this review we discuss chromatin domain boundaries identified in several systems using different means. Although there is no significant sequence conservation among various chromatin domain boundaries, these elements show functional conservation across the species. Finally, we discuss mechanistic aspects of how chromatin domain boundaries may function in organizing and regulating eukaryotic genome.