Survey of ochratoxin A in cereals from Puglia and Basilicata.
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Edible Grain↗
Biomedical subjects
V Quaranta
Publications and source records attributed to V Quaranta.
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.
Animals↗
Defining signal thresholds in DNA microarrays: exemplary application for invasive cancer.
BACKGROUND: Genome-wide or application-targeted microarrays containing a subset of genes of interest have become widely used as a research tool with the prospect of diagnostic application. Intrinsic variability of microarray measurements poses a major problem in defining signal thresholds for absent/present or differentially expressed genes. Most strategies have used fold-change threshold values, but variability at low signal intensities may invalidate this approach and it does not provide information about false-positives and false negatives. RESULTS: We introduce a method to filter false-positives and false-negatives from DNA microarray experiments. This is achieved by evaluating a set of positive and negative controls by receiver operating characteristic (ROC) analysis. As an advantage of this approach, users may define thresholds on the basis of sensitivity and specificity considerations. The area under the ROC curve allows quality control of microarray hybridizations. This method has been applied to custom made microarrays developed for the analysis of invasive melanoma derived tumor cells. It demonstrated that ROC analysis yields a threshold with reduced missclassified genes in microarray experiments. CONCLUSIONS: Provided that a set of appropriate positive and negative controls is included on the microarray, ROC analysis obviates the inherent problem of arbitrarily selecting threshold levels in microarray experiments. The proposed method is applicable to both custom made and commercially available DNA microarrays and will help to improve the reliability of predictions from DNA microarray experiments.
Journal Article↗
Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development.
Branching morphogenesis of the ureteric bud (UB) [induced by the metanephric mesenchyme (MM)] is necessary for normal kidney development. The role of integrins in this complex developmental process is not well understood. However, the recent advent of in vitro model systems to study branching of UB cells and isolated UB tissue makes possible a more detailed analysis of the integrins involved. We detected integrin subunits alpha3, alpha6, beta1, and beta4 in both the UB and cells derived from the early UB. Blocking the function of each of these integrin subunits individually markedly inhibited branching morphogenesis in cell culture models. However, inhibiting individual integrin function with blocking antibodies in whole kidney and isolated UB culture only partially inhibited UB branching morphogenesis, suggesting that, in these more complex in vitro systems, multiple integrins are involved in the branching program. In whole organ and isolated bud culture, marked retardation of UB branching was observed only when both alpha3 and alpha6 integrin subunits were inhibited. The alpha6 integrin subunit can be expressed as both alpha6beta1 and alpha6beta4, and both of these beta subunits are important for UB branching morphogenesis in both cell and organ culture. Furthermore, laminin-5, a common ligand for integrins alpha3beta1 and alpha6beta4, was detected in the developing UB and shown to be required for normal UB branching morphogenesis in whole embryonic kidney organ culture as well as isolated UB culture. Together, these data from UB cell culture, organ culture, and isolated UB culture models indicate that both integrin alpha3 and alpha6 subunits play a direct role in UB branching morphogenesis, as opposed to being modulators of the inductive effects of mesenchyme on UB development. Furthermore the data are consistent with a role for laminin-5, acting through its alpha3beta1 and/or alpha6beta4 integrin receptors, in UB branching during nephrogenesis. These data may help to partially explain the renal phenotype seen in integrin alpha3 and alpha3/alpha6 subunit-deficient animals.
Animals↗
Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.
Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.
Cell Adhesion Molecules↗
The LG3 module of laminin-5 harbors a binding site for integrin alpha3beta1 that promotes cell adhesion, spreading, and migration.
Laminins are a family of extracellular matrix glycoproteins involved in cell adhesion and migration. A major obstacle to understanding their structure-function relationships is the lack of small laminin domains capable of replicating integrin-binding, cell-adhesive, and migratory functions of the intact molecule. Here, we show that the recombinant LG3 (rLG3) module (26 kDa) of laminin-5 (Ln-5) alpha(3) chain replicated key Ln-5 activities. rLG3 but not rLG1 or rLG2 supported cell adhesion and migration of at least two distinct cell lines, in an integrin alpha(3)beta(1)-dependent manner. Cell adhesion to rLG3 was regulated by divalent cations and accompanied by cell spreading and tyrosine phosphorylation of FAK focal adhesion kinase. The integrin binding activity of rLG3 was confirmed by rLG3 affinity chromatography of detergent cell lysates, which resulted in specific purification of integrin alpha(3)beta(1). To our knowledge, this is the first report directly demonstrating that a recombinant laminin LG module is an active domain capable of supporting integrin-dependent cell adhesion and migration.
Amino Acid Sequence↗
Human hepatocellular carcinoma (HCC) cells require both alpha3beta1 integrin and matrix metalloproteinases activity for migration and invasion.
Hepatocellular carcinoma (HCC) is the most frequent malignant tumor of the liver; prognosis depends on the tendency to metastasize. Cancer cell invasion is regulated by proteolytic remodeling of extracellular matrix components and by integrin expression. We have shown that matrix metalloproteinase-2 (MMP-2) and membrane-type-1 matrix metalloproteinase (MT1-MMP) cleave Laminin-5 (Ln-5), stimulating cell migration. Here we report that all HCC cells express MT1-MMP, migrate on Ln-1 and Collagen IV, whereas only HCC cells that express alpha3beta1 integrin secrete detectable levels of gelatinases, migrate on Ln-5, and invade through a reconstituted basement membrane (BM). Migration on Ln-5 is blocked by BB-94, an MMP inhibitor, and by MIG1, a monoclonal antibody that hinders migration on MMP-2-cleaved Ln-5. Invasion through a reconstituted BM is also inhibited by BB-94. HCC alpha3beta1-negative cells migrate on Ln-1 and Collagen IV, but not on Ln-5, and do not invade through a reconstituted BM, although they express MT1-MMP. Anti-alpha3beta1 blocking antibodies inhibit gelatinase activation, cell motility, and cell invasion through MATRIGEL: In vivo, alpha3beta1 integrin and Ln-5 are expressed in HCC tissue but not in normal liver. In conclusion, our data suggest that both alpha3beta1 integrin and gelatinase activity are required for HCC migration and invasion.
Basement Membrane↗
Characterization of morphological and cytoskeletal changes in MCF10A breast epithelial cells plated on laminin-5: comparison with breast cancer cell line MCF7.
The extracellular matrix regulates functional and morphological differentiation of mammary epithelial cells both in vivo and in culture. The MCF10A human breast epithelial cell line is ideal for studying these processes because it retains many characteristics of normal breast epithelium. We describe a distinct set of morphological changes occurring in MCF10A cells plated on laminin-5, a component of the breast gland basement membrane extracellular matrix. MCF10A cells adhere and spread on laminin-5 about five times more rapidly than on fibronectin or uncoated surfaces. Within 10 minutes from plating on laminin-5, they send out microfilament-rich filopodia and by 30 minutes acquire a cobblestone appearance with microfilaments distributed around the cell periphery. At 90 minutes, with or without serum, > 75% of the MCF10A cells plated on laminin-5 remain in this stationary cobblestone phenotype, while the remainder takes on a motile appearance. Even after 18 hours, when the culture is likely entering an exponential growth phase, the majority of cells maintain a stationary cobblestone appearance, though motile cells have proportionally increased. In contrast, the fully transformed, malignant human breast epithelial cells, MCF7, never acquire a stationary cobblestone appearance, do not organize peripheral microfilaments, and throughout the early time points up to 120 min appear to be constantly motile on laminin-5. We propose that changes in morphology and microfilament organization in response to laminin-5 may represent a benchmark for distinguishing normal vs. malignant behavior of epithelial cells derived from the mammary gland. This may lead to better model systems for studying the interactions between breast epithelium and the basement membrane extracellular matrix, which appear to be deregulated in processes like carcinogenesis and metastasis.
Breast↗
Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells.
Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.
Adult↗
Cell migration through extracellular matrix: membrane-type metalloproteinases make the way.
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Animals↗
Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5.
Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the gamma2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2- cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2- cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.
Animals↗
Antibody-induced activation of beta1 integrin receptors stimulates cAMP-dependent migration of breast cells on laminin-5.
The beta1 integrin-stimulating antibody TS2/16 induces cAMP-dependent migration of MCF-10A breast cells on the extracellular matrix protein laminin-5. TS2/16 stimulates a rise in intracellular cAMP within 20 min after plating. Pertussis toxin, which inhibits both antibody-induced migration and cAMP accumulation, targets the Galphai3 subunit of heterotrimeric G proteins in these cells, suggesting that Galphai3 may link integrin activation and migration via a cAMP signaling pathway.
Adenosine Diphosphate Ribose↗
Laminin 5 in the human thymus: control of T cell proliferation via alpha6beta4 integrins.
Laminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, we observed that while alpha3beta1 are known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation.
Antibodies, Monoclonal↗
Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale Delphinapterus leucas.
Given the importance of the cell surface recognition protein, CD4, in immune function, the cloning and characterization of CD4 at the molecular level from an odontocete cetacean, the white whale (Delphinapterus leucas), was carried out. Whale CD4 cDNA contains 2662 base pairs and translates into a protein containing 455 amino acids. Whale CD4 shares 64% and 51% identity with the human and mouse CD4 protein, respectively, and is organized in a similar manner. Unlike human and mouse, however, the cytoplasmic domain, which is highly conserved, contains amino acid substitutions unique to whale. Moreover, only one of the seven potential N-linked glycosylation sites present in whale is shared with human and mouse. Evolutionarily, the whale CD4 sequence is most similar to pig and structurally similar to dog and cat, in that all lack the cysteine pair in the V2 domain. These differences suggest that CD4 may have a different secondary structure in these species, which may affect binding of class II and subsequent T-cell activation, as well as binding of viral pathogens. Interestingly, as a group, species with these CD4 characteristics all have high constitutive expression of class II molecules on T lymphocytes, suggesting potential uniqueness in the interaction of CD4, class II molecules, and the immune response. Molecular characterization of CD4 in an aquatic mammal provides information on the CD4 molecule itself and may provide insight into adaptive evolutionary changes of the immune system.
Adaptation, Biological↗
Expression of matrix metalloprotease-2-cleaved laminin-5 in breast remodeling stimulated by sex steroids.
The extracellular matrix plays an important role in breast remodeling. We have shown that matrix metalloprotease-2 (MMP2) cleaves laminin-5 (Ln-5), a basement membrane component, generating a fragment called gamma2x. Human breast epithelial cells, while constitutively immobile on intact Ln-5, acquire a motile phenotype on MMP2-cleaved Ln-5. We hypothesize that this mechanism may underlie cell mobilization across the basement membrane during branching morphogenesis in breast development regulated by sex steroids. We report that the expression of MMP2 and cleavage of Ln-5 correlate well with tissue remodeling and epithelial rearrangement of the breast both in vivo and in vitro. Thus, the Ln-5 gamma2x fragment was detected by immunoblotting in sexually mature, pregnant, and postweaning, but not in prepubertal or lactating mammary glands. Furthermore, cleaved Ln-5, as well as MMP2, became detectable in remodeling glands from sexually immature rats treated with sex steroids. In rat mammary gland explants, epithelial reorganization and luminal cell morphological changes were induced by the addition of exogenous MMP2, in parallel to the appearance of cleaved Ln-5. Similar effects were observed in epithelial monolayers plated on human Ln-5 and exposed to MMP2. These results suggest that cleavage of Ln-5 by MMP2 might be regulated by sex steroids and that it may contribute to breast remodeling under physiological and possibly pathological conditions.
Animals↗
Evidence that laminin-5 is a component of the tooth surface internal basal lamina, supporting epithelial cell adhesion.
Laminin-5 (Ln-5) is an extracellular matrix (ECM) glycoprotein found in epithelial basal laminae. We studied its expression on the surface of rat molars, in relationship to the location of the internal basal lamina (IBL) of the junctional epithelium (JE). In order to avoid disruption of the JE-tooth interface as much as possible, the surface of molars was prepared by mechanical removal of tissue debris and detergent/osmotic lysis of epithelial cell layers, and directly stained by immunohistochemistry, without sectioning. Antibodies to Ln-5 specifically stained a narrow band in the region of the cemento-enamel junction (CEJ), consistent with the expected location of the IBL. Western blotting of ECM material detergent--solubilized from the prepared tooth surfaces confirmed the molecular nature of Ln-5 identified by immunohistochemistry. By the use of a high-definition 3-D microscope, it appeared that Ln-5 coated the most apical part of the enamel and the most coronal portion of the cementum, on either side of the CEJ. In adhesion assays performed directly on tooth surfaces, epithelial cells adhered preferentially to the Ln-5 coated area of the tooth compared to the root surface, which is coated by other ECM components. Adhesion to the Ln-5 coated surface was specifically inhibited by a function-blocking monoclonal antibody to Ln-5. These results suggest that Ln-5 is a component of the IBL, and that it may be important in promoting adhesion of JE cells onto the tooth surface.
Animals↗
Mode of adsorption and orientation of an extracellular matrix protein affect its cell-adhesion-promoting activity.
Cell adhesion to extracellular matrix contributes to the organization of tissues and modulates cell behavior. In conventional cell adhesion assays, plastic wells are coated with matrix proteins and assayed for their adhesion-promoting activity. We show here that factors such as sample composition, coating buffers, and manufacturers' plastic treatment markedly affect cell adhesion to the extracellular matrix protein laminin-5 (Ln-5). These factors were shown to affect adsorption efficiency as determined by measuring total adsorbed protein with a polyclonal anti-Ln-5 antiserum. They also influence the availability of the epitope for an adhesion-blocking anti-Ln-5 monoclonal antibody, suggesting that coating conditions affect the orientation of Ln-5. Generally, cell adhesion correlates more strongly with the availability of the epitope for the adhesion-blocking antibody than with total adsorbed Ln-5. Our data further indicate that cell adhesion to other matrix proteins may be influenced by similar factors. Adding Ln-5 samples to plastic wells that had been precoated with non-adhesion-blocking anti-Ln-5 antibodies made cell adhesion independent of factors such as sample composition, coating buffers, and source of plastic. Thus, the control of adsorption efficiency and orientation of extracellular matrix proteins is essential for creation of reliable and reproducible conditions in cell adhesion assays.
Adsorption↗
Characterization of a tight molecular complex between integrin alpha 6 beta 4 and laminin-5 extracellular matrix.
In many adult epithelia, e.g., epidermis or intestine, adhesion of epithelial cells to basement membrane requires the integrin alpha6 beta4 and laminin-5 (Ln-5). In the absence of one or the other, extensive blistering and exfoliation occur. While alpha6 beta4 was reported to be a receptor for Ln-5, this interaction is poorly understood. We characterize complexes between alpha6 beta4 and Ln-5 in cell-free preparations of extracellular matrix (ECM) from the epithelial cell line, 804G. By microsequencing, Ln-5 and alpha6 beta4 were the major proteins in this ECM and were likely engaged in receptor/ligand complexes because, by immunofluorescence, alpha6 beta4 was colocalized with Ln-5 both in cell monolayers and in cell-free ECM preparations, but they disappeared after preincubation of the monolayers with alpha6 beta4 or Ln-5 function-blocking antibodies. The alpha6 beta4/Ln-5 complexes were resistant to dissociation by extreme pH, urea, chaotropes, eDTA, non-ionic detergents, and b-mercaptoethanol. They were only dissociated by strong anionic detergents, e.g., 1% SDS, suggesting receptor/ligand interactions based on high affinity or avidity. We propose that these alpha6 beta4/Ln-5 complexes may provide links between plasma membrane and basement membrane that resist mechanical stress and support epithelial integrity.
Amino Acid Sequence↗