Integrin α6Aβ1 Induces CD81-dependent Cell Motility without Engaging the Extracellular Matrix Migration Substrate

It is well established that integrins and extracellular matrix (ECM) play key roles in cell migration, but the underlying mechanisms are poorly defined. We describe a novel mechanism whereby the integrin α6β1, a laminin receptor, can affect cell motility and induce migration onto ECM substrates with which it is not engaged. By using DNA-mediated gene transfer, we expressed the human integrin subunit α6A in murine embryonic stem (ES) cells. ES cells expressing α6A (ES6A) at the surface dimerized with endogenous β1, extended numerous filopodia and lamellipodia, and were intensely migratory in haptotactic assays on laminin (LN)-1. Transfected α6A was responsible for these effects, because cells transfected with control vector or α6B, a cytoplasmic domain α6 isoform, displayed compact morphology and no migration, like wild-type ES cells. The ES6A migratory phenotype persisted on fibronectin (Fn) and Ln-5. Adhesion inhibition assays indicated that α6β1 did not contribute detectably to adhesion to these substrates in ES cells. However, anti-α6 antibodies completely blocked migration of ES6A cells on Fn or Ln-5. Control experiments with monensin and anti-ECM antibodies indicated that this inhibition could not be explained by deposition of an α6β1 ligand (e.g., Ln-1) by ES cells. Cross-linking with secondary antibody overcame the inhibitory effect of anti-α6 antibodies, restoring migration or filopodia extension on Fn and Ln-5. Thus, to induce migration in ES cells, α6Aβ1 did not have to engage with an ECM ligand but likely participated in molecular interactions sensitive to anti-α6β1 antibody and mimicked by cross-linking. Antibodies to the tetraspanin CD81 inhibited α6Aβ1-induced migration but had no effect on ES cell adhesion. It is known that CD81 is physically associated with α6β1, therefore our results suggest a mechanism by which interactions between α6Aβ1 and CD81 may up-regulate cell motility, affecting migration mediated by other integrins.

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae)1 : III. Organization of Fucoglucuronogalactans within the Adhesive Stalks of Achnanthes longipes

Achnanthes longipes is a marine, biofouling diatom that adheres to surfaces via adhesive polymers extruded during motility or organized into structures called stalks that contain three distinct regions: the pad, shaft, and collar. Four monoclonal antibodies (AL.C1–AL.C4) and antibodies from two uncloned hybridomas (AL.E1 and AL.E2) were raised against the extracellular adhesives of A. longipes. Antibodies were screened against a hot-water-insoluble/hot-bicarbonate-soluble-fraction. The hot-water-insoluble/hot-bicarbonate-soluble fraction was fractionated to yield polymers in three size ranges: F1, ≥ 20,000,000 Mr; F2, ≅100,000 Mr; and F3, <10,000 Mr relative to dextran standards. The ≅100,000-Mr fraction consisted of highly sulfated (approximately 11%) fucoglucuronogalactans (FGGs) and low-sulfate (approximately 2%) FGGs, whereas F1 was composed of O-linked FGG (F2)-polypeptide (F3) complexes. AL.C1, AL.C2, AL.C4, AL.E1, and AL.E2 recognized carbohydrate complementary regions on FGGs, with antigenicity dependent on fucosyl-containing side chains. AL.C3 was unique in that it had a lower affinity for FGGs and did not label any portion of the shaft. Enzyme-linked immunosorbent assay and immunocytochemistry indicated that low-sulfate FGGs are expelled from pores surrounding the raphe terminus, creating the cylindrical outer layers of the shaft, and that highly sulfated FGGs are extruded from the raphe, forming the central core. Antibody-labeling patterns and other evidence indicated that the shaft central-core region is related to material exuded from the raphe during cell motility.

Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly.

The human immunodeficiency virus type 1 (HIV-1) matrix protein forms a structural shell associated with the inner viral membrane and performs other essential functions throughout the viral life cycle. The crystal structure of the HIV-1 matrix protein, determined at 2.3 angstrom resolution, reveals that individual matrix molecules are composed of five major helices capped by a three-stranded mixed beta-sheet. Unexpectedly, the protein assembles into a trimer in three different crystal lattices, burying 1880 angstrom2 of accessible surface area at the trimer interfaces. Trimerization appears to create a large, bipartite membrane binding surface in which exposed basic residues could cooperate with the N-terminal myristoyl groups to anchor the protein on the acidic inner membrane of the virus.

Estrogens increase the expression of fibulin-1, an extracellular matrix protein secreted by human ovarian cancer cells.

Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens. In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins. This study was aimed at identifying a protein secreted by ovarian cancer cells whose level was increased by estradiol [Galtier-Dereure, F., Capony, F., Maudelonde, T. & Rochefort, H. (1992) J. Clin. Endocrinol. Metab. 75, 1497-1502]. By using microprotein sequencing, the 110-kDa protein was identified as fibulin-1, a protein of the extracellular matrix that binds to fibronectin, laminin, and nidogen. The amount of immunoprecipitated fibulin-1 secreted into the medium and present in the cell extract was increased up to 10-fold by estradiol in three estrogen-responsive ovarian cancer cell lines. By immunohistochemistry fibulin-1 was located in the stroma of several ovarian cancers and cysts. The findings highlight a potential role for fibulin-1 in the spread of ovarian cancer in the peritoneal cavity and/or in distal metastases.

The nuclear matrix protein NMP-1 is the transcription factor YY1.

NMP-1 was initially identified as a nuclear matrix-associated DNA-binding factor that exhibits sequence-specific recognition for the site IV regulatory element of a histone H4 gene. This distal promoter domain is a nuclear matrix interaction site. In the present study, we show that NMP-1 is the multifunctional transcription factor YY1. Gel-shift and Western blot analyses demonstrate that NMP-1 is immunoreactive with YY1 antibody. Furthermore, purified YY1 protein specifically recognizes site IV and reconstitutes the NMP-1 complex. Western blot and gel-shift analyses indicate that YY1 is present within the nuclear matrix. In situ immunofluorescence studies show that a significant fraction of YY1 is localized in the nuclear matrix, principally but not exclusively associated with residual nucleoli. Our results confirm that NMP-1/YY1 is a ubiquitous protein that is present in both human cells and in rat osteosarcoma ROS 17/2.8 cells. The finding that NMP-1 is identical to YY1 suggests that this transcriptional regulator may mediate gene-matrix interactions. Our results are consistent with the concept that the nuclear matrix may functionally compartmentalize the eukaryotic nucleus to support regulation of gene expression.

Neonatal calyceal dilation and renal fibrosis resulting from loss of Adamts-1 in mouse kidney is due to a developmental dysgenesis

Background. A disintegrin and metalloproteinase with thrombospondin motifs 1, Adamts-1, is important for the development and function of the kidney. Mice lacking this protein present with renal lesions comprising enlarged calyces, and reduced cortex and medulla layers. Our current findings are consistent with the defect occurring due to a developmental dysgenesis. Methods. We generated Adamts-1 null mice, and further investigated their kidney phenotype in a time course study ranging from E18.5 to 12 months of age. Immunohistochemistry was used to assess the localization of type IV collagen, TGF-beta and F4/80-positive macrophages in the kidneys of Adcants-1 null mice compared to wild-type control animals. The expression of Adamts-1 mRNA was determined in metanephric kidney explants by in situ hybridization. Results. Adamts-1 null mice have a gross kidney defect. At day 18.5 of gestation, the Adcants-1 null kidney has a normal appearance but at birth when the kidney begins to function, the defect becomes evident. During development of the kidney Adamts-1 expression was specifically detected in the developing loops of Henle, as well as in the proximal and distal convoluted tubules. Expression was not detected in the ureter, ureteric bud or its derivatives as had been previously suggested. At 6 months and I year of age, the Adamts-1 null mice displayed interstitial fibrosis in the cortical and medullary regions of the kidney. At I year of age, the Adamts-1 null mice displayed mild interstitial matrix expansion associated with increased collagen type IV expression, without apparent tubular dilatation, compared to wild-type animals. Immunohistochemical analysis demonstrated TGF-beta protein localized to infiltrating macrophages and glomeruli of Adamts-1 null mice. Conclusions. Adamts-1 is required for the normal development of the kidney. The defect observed in its absence results from a dysgenic malformation affecting the medulla that becomes apparent at birth, once the kidneys start to function.

RGD-independent cell adhesion via a tissue transglutaminase-fibronectin matrix promotes fibronectin fibril deposition and requires syndecan-4/2 and α5β1 integrin co-signaling

Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and ß1 integrin co-signaling pathway. By using a5 null cells, ß1 integrin functional blocking antibody, and a a5ß1 integrin targeting peptide A5-1, we demonstrate that the a5 and ß1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCa is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.

Increased TG2 expression can result in induction of transforming growth factor β1, causing increased synthesis and deposition of matrix proteins, which can be regulated by nitric oxide

In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 3T3 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-kappaB activation, resulting in the increased expression of transforming growth factor (TGF) beta(1). In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFbeta(1) in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-kappaB and TGFbeta1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor S-nitroso-N-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NF-kappaB and TGFbeta(1) production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment.

Use of a solvent-free dry matrix coating for quantitative matrix-assisted laser desorption ionization imaging of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate in rat brain and quantitative analysis of the drug from laser microdissected tissue regions

A dry matrix application for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was used to profile the distribution of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate, monohydrochloride (BDNC, SSR180711) in rat brain tissue sections. Matrix application involved applying layers of finely ground dry alpha-cyano-4-hydroxycinnamic acid (CHCA) to the surface of tissue sections thaw mounted onto MALDI targets. It was not possible to detect the drug when applying matrix in a standard aqueous-organic solvent solution. The drug was detected at higher concentrations in specific regions of the brain, particularly the white matter of the cerebellum. Pseudomultiple reaction monitoring imaging was used to validate that the observed distribution was the target compound. The semiquantitative data obtained from signal intensities in the imaging was confirmed by laser microdissection of specific regions of the brain directed by the imaging, followed by hydrophilic interaction chromatography in combination with a quantitative high-resolution mass spectrometry method. This study illustrates that a dry matrix coating is a valuable and complementary matrix application method for analysis of small polar drugs and metabolites that can be used for semiquantitative analysis.

Identification Of Corynebacterium Spp. Isolated From Bovine Intramammary Infections By Matrix-assisted Laser Desorption Ionization-time Of Flight Mass Spectrometry.

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n=180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n=156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp.

Matrix Support And Institutional Support: Analyzing Their Construction.

This is an analysis of the theoretical and practical construction of the methodology of Matrix Support by means of studies on Paideia Support (Institutional and Matrix Support), which is an inter-professional work of joint care in recent literature and official documents of the Unified Health System (SUS). An attempt was made to describe methodological concepts and strategies. A comparative analysis of Institutional Support and Matrix Support was also conducted using the epistemological framework of Field and Core Knowledge and Practices.

Theobromine Increases Nad⁺/sirt-1 Activity And Protects The Kidney Under Diabetic Conditions.

Reduction in sirtuin 1 (Sirt-1) is associated with extracellular matrix (ECM) accumulation in the diabetic kidney. Theobromine may reduce kidney ECM accumulation in diabetic rats. In the current study, we aimed to unravel, under diabetic conditions, the mechanism of kidney ECM accumulation induced by a reduction in Sirt-1 and the effect of theobromine in these events. In vitro, we used immortalized human mesangial cells (iHMCs) exposed to high glucose (HG; 30 mM), with or without small interfering RNA for NOX4 and Sirt-1. In vivo, spontaneously hypertensive rats (SHR) were rendered diabetic by means of streptozotocin and studied after 12 wk. The effects of treatment with theobromine were investigated under both conditions. HG leads to a decrease in Sirt-1 activity and NAD(+) levels in iHMCs. Sirt-1 activity could be reestablished by treatment with NAD(+), silencing NOX4, and poly (ADP-ribose) polymerase-1 (PARP-1) blockade, or with theobromine. HG also leads to a low AMP/ATP ratio, acetylation of SMAD3, and increased collagen IV, which is prevented by theobromine. Sirt-1 or AMPK blockade abolished these effects of theobromine. In diabetic SHR, theobromine prevented increases in albuminuria and kidney collagen IV, reduced AMPK, elevated NADPH oxidase activity and PARP-1, and reduced NAD(+) levels and Sirt-1 activity. These results suggest that in diabetes mellitus, Sirt-1 activity is reduced by PARP-1 activation and NAD(+) depletion due to low AMPK, which increases NOX4 expression, leading to ECM accumulation mediated by transforming growth factor (TGF)-β1 signaling. It is suggested that Sirt-1 activation by theobromine may have therapeutic potential for diabetic nephropathy.

Key Participants Of The Tumor Microenvironment Of The Prostate: An Approach Of The Structural Dynamic Of Cellular Elements And Extracellular Matrix Components During Epithelial-stromal Transition.

Cancer is a multistep process that begins with the transformation of normal epithelial cells and continues with tumor growth, stromal invasion and metastasis. The remodeling of the peritumoral environment is decisive for the onset of tumor invasiveness. This event is dependent on epithelial-stromal interactions, degradation of extracellular matrix components and reorganization of fibrillar components. Our research group has studied in a new proposed rodent model the participation of cellular and molecular components in the prostate microenvironment that contributes to cancer progression. Our group adopted the gerbil Meriones unguiculatus as an alternative experimental model for prostate cancer study. This model has presented significant responses to hormonal treatments and to development of spontaneous and induced neoplasias. The data obtained indicate reorganization of type I collagen fibers and reticular fibers, synthesis of new components such as tenascin and proteoglycans, degradation of basement membrane components and elastic fibers and increased expression of metalloproteinases. Fibroblasts that border the region, apparently participate in the stromal reaction. The roles of each of these events, as well as some signaling molecules, participants of neoplastic progression and factors that promote genetic reprogramming during epithelial-stromal transition are also discussed.