A role of chondroitin sulfate glycosaminoglycan binding site in alpha4beta1 integrin-mediated melanoma cell adhesion

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.     

Brain-specific receptor-type protein-tyrosine phosphatase RPTP beta is a chondroitin sulfate proteoglycan in vivo

By using polyclonal antiserum, which recognizes multiple proteoglycan core proteins, we isolated a cDNA species for an unknown chondroitin sulfate proteoglycan in bovine brain. Unexpectedly, DNA sequencing revealed that the cDNA encodes an open reading frame highly homologous to the human receptor-type protein-tyrosine phosphatase, RPTP beta. To prove that RPTP beta is a proteoglycan, we raised three polyclonal antibodies against extracellular and cytoplasmic domains of human RPTP beta. These antibodies have been shown to react with a smear band ranging from 350 to 500 kDa in human brain extracts. Digestion with chondroitinase ABC eliminated this smear and gave rise to a 310/300-kDa doublet band that was not detected without digestion, indicating that almost all of the RPTP beta molecules in the brain contain chondroitin sulfate chains. In the cerebellum, immunofluorescence staining of chondroitinase-treated sections revealed pericellular localization of RPTP beta in the external and internal granular layers. These data establish that RPTP beta is expressed constitutively as a chondroitin sulfate proteoglycan in the brain, and suggest that chondroitin sulfates may be an essential component for the physiological function of RPTP beta in vivo.     

A chondroitin sulfate proteoglycan on human neutrophils specifically binds platelet factor 4 and is involved in cell activation

Platelet factor 4 (PF-4), a member of the alpha-chemokine subfamily of cytokines, activates human neutrophils independently of intracellular free calcium mobilization or binding to IL-8R. In the present study, we have identified and partially characterized a receptor for PF-4 on human neutrophils, which displays weak cross-reactivity with the IFN-gamma-inducible protein 10, but not with other alpha-chemokines such as IL-8, neutrophil-activating peptide 2, or melanoma growth-stimulatory activity (GRO alpha). Binding studies revealed that human neutrophils express a high number of receptors (Bmax approximately 7.6 x 10(6) sites/cell) of moderate affinity (Kd approximately 650 nM). The kinetics of PF-4-binding correlates with the proportion of PF-4 tetramers in solution and with the activation of neutrophils for exocytosis. Reduction of PF-4 binding and PF-4-induced exocytosis in the presence of various glycosaminoglycans or following treatment of cells with chondroitinase ABC (but not other glycosaminoglycan-degrading enzymes) altogether demonstrates that the PF-4 receptor is a proteoglycan of the chondroitin sulfate class. Cross-linking experiments with radiolabeled PF-4 revealed a receptor-ligand complex of approximately 250 kDa. Taken together, our data show that a distinct chondroitin sulfate proteoglycan represents specific receptors for tetrameric PF-4 on human neutrophils.     

Retroviral gene transfer is inhibited by chondroitin sulfate proteoglycans/glycosaminoglycans in malignant pleural effusions

Gene therapy may be an important adjuvant for treating cancer in the pleural space. The initial results of retroviral gene transfer to cancer cells in malignant pleural effusions revealed that transduction was markedly inhibited, and studies to characterize the inhibitory factor(s) were performed. The inhibition was contained within the soluble, rather than cellular, components of the effusions and was demonstrated with amphotropic, gibbon ape leukemia virus, and vesicular stomatitis virus-glycoprotein pseudotyped retroviral vectors. After excluding complement proteins, a series of studies identified chondroitin sulfates (CSs) as the inhibitory substances. First, treatment of the effusions with mammalian hyaluronidase or chondroitinases, but not Streptomyces hyaluronidase, abolished the inhibitory activity. Second, addition of exogenous CS glycosaminoglycans mimicked the inhibition observed with pleural effusions. Third, immunoassays and biochemical analyses of malignant pleural effusion specimens revealed CS in relevant concentrations within pleural fluid. Fourth, proteoglycans/glycosaminoglycans isolated from the effusions inhibited retroviral gene transfer. Analyses of the mechanism of inhibition indicate that the chondroitin sulfates interact with vector in solution rather than at the target cell surface. These results suggest that drainage of the malignant pleural effusion, and perhaps enzymatic pretreatment of the pleural cavity, will be necessary for efficient retroviral vector mediated gene delivery to pleural metastases.     

Inhibition of binding of malaria-infected erythrocytes by a tetradecasaccharide fraction from chondroitin sulfate A

Adherence of parasite-infected erythrocytes (IEs) to the microvascular endothelium of various organs, a process known as sequestration, is a feature of Plasmodium falciparum malaria. This event is mediated by specific adhesive interactions between parasite proteins, expressed on the surface of IEs, and host molecules. P. falciparum IEs can bind to purified chondroitin sulfate A (CS-A), to the proteoglycan thrombomodulin through CS-A side chains, and to CS-A present on the surface of brain and lung endothelial cells and placental syncytiotrophoblasts. In order to identify structural characteristics of CS-A important for binding, oligosaccharide fragments ranging in size from 2 to 20 monosaccharide units were isolated from CS-A and CS-C, following controlled chondroitin lyase digestion, and used as competitive inhibitors of IE binding to immobilized ligands. Inhibition of binding to CS-A was highly dependent on molecular size: a CS-A tetradecasaccharide fraction was the minimum length able to almost completely inhibit binding. The effect was dose dependent and similar to that of the parent polysaccharide, and the same degree of inhibition was not found with the CS-C oligosaccharides. There was no effect on binding of IEs to other ligands, e.g., CD36 and intercellular adhesion molecule 1. Hexadeca- and octadecasaccharide fractions of CS-A were required for maximum inhibition of binding to thrombomodulin. Analyses of oligosaccharide fractions and polysaccharides by electrospray mass spectrometry and high-performance liquid chromatography suggest that the differences between the activities of CS-A and CS-C oligosaccharides can be attributed to differences in sulfate content and sulfation pattern and that iduronic acid is not involved in IE binding.     

Pasteurella multocida toxin is a mitogen for bone cells in primary culture

The effect of recombinant Pasteurella multocida toxin (PMT) on primary cultures of embryonic chick bone-derived osteoblastic cells was investigated. It was found that PMT was a potent mitogen for primary derived chicken osteoblasts. The toxin stimulated DNA synthesis and cell proliferation in quiescent osteoblasts at the first passage and accelerated cell growth in subconfluent cultures. Cell viability was not affected by PMT, even at relatively high concentrations. Osteoblast numbers increased in a dose-dependent manner in response to PMT. Intracellular inositol phosphates were elevated in response to PMT, but no elevation in cyclic AMP (cAMP) levels was evident. Indeed, PMT inhibited cAMP elevation in osteoblasts in response to cholera toxin at a stage before other PMT-mediated events take place. In addition to increased cell turnover, PMT down-regulated the expression of several markers of osteoblast differentiation. Both alkaline phosphatase and type I collagen were reduced, but osteonectin was not affected. The in vitro deposition of mineral in cultures of primary osteoblasts and osteoblast-like osteosarcoma cells was also inhibited by the presence of PMT. This suggests that PMT interferes with differentiation at a preosteoblastic stage.     

Anandamide hydrolysis by human cells in culture and brain

Anandamide (arachidonylethanolamide; AnNH) has important neuromodulatory and immunomodulatory activities. This lipid is rapidly taken up and hydrolyzed to arachidonate and ethanolamine in many organisms. As yet, AnNH inactivation has not been studied in humans. Here, a human brain fatty-acid amide hydrolase (FAAH) has been characterized as a single protein of 67 kDa with a pI of 7.6, showing apparent Km and Vmax values for AnNH of 2.0 +/- 0.2 microM and 800 +/- 75 pmol.min-1.mg of protein-1, respectively. The optimum pH and temperature for AnNH hydrolysis were 9.0 and 37 degreesC, respectively, and the activation energy of the reaction was 43.5 +/- 4.5 kJ.mol-1. Hydro(pero)xides derived from AnNH or its linoleoyl analogues by lipoxygenase action were competitive inhibitors of human brain FAAH, with apparent Ki values in the low micromolar range. One of these compounds, linoleoylethanolamide is the first natural inhibitor (Ki = 9.0 +/- 0.9 microM) of FAAH as yet discovered. An FAAH activity sharing several biochemical properties with the human brain enzyme was demonstrated in human neuroblastoma CHP100 and lymphoma U937 cells. Both cell lines have a high affinity transporter for AnNH, which had apparent Km and Vmax values for AnNH of 0.20 +/- 0.02 microM and 30 +/- 3 pmol.min-1.mg of protein-1 (CHP100 cells) and 0.13 +/- 0.01 microM and 140 +/- 15 pmol.min-1.mg of protein-1 (U937 cells), respectively. The AnNH carrier of both cell lines was activated up to 170% of the control by nitric oxide.     

Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.     

The core protein of the chondroitin sulfate proteoglycan phosphacan is a high-affinity ligand of fibroblast growth factor-2 and potentiates its mitogenic activity

Using a radioligand binding assay we have demonstrated that phosphacan, a chondroitin sulfate proteoglycan of nervous tissue that also represents the extracellular domain of a receptor-type protein tyrosine phosphatase, shows saturable, reversible, high-affinity binding (Kd approximately 6 nM) to fibroblast growth factor-2 (FGF-2). Binding was reduced by only approximately 35% following chondroitinase treatment of the proteoglycan, indicating that the interaction is mediated primarily through the core protein rather than the glycosaminoglycan chains. Immunocytochemical studies also showed an overlapping localization of FGF-2 and phosphacan in the developing central nervous system. At concentrations of 10 microg protein/ml, both native phosphacan and the core protein obtained by chondroitinase treatment potentiated the mitogenic effect of FGF-2 (5 ng/ml) on NIH/3T3 cells by 75-90%, which is nearly the same potentiation as that produced by heparin at an equivalent concentration. Although studies on the role of proteoglycans in mediating the binding and mitogenic effects of FGF-2 have previously focused on cell surface heparan sulfate, our results indicate that the core protein of a chondroitin sulfate proteoglycan may also regulate the access of FGF-2 to cell surface signaling receptors in nervous tissue.     

Pleiotypic response of rat heart cells in culture to serum stimulation

The pleiotypic effects of medium replacement were studied in rat heart cell cultures. After each medium change alpha-aminoisobutyric acid and glucose transport are increased, RNA and protein syntheses are activated. DNA synthesis did not begin before 12 hours and was followed by a wave of mitoses. This sequence of events suggests that the stimulated cells were in early G 1 phase. DNA synthesis, following the shift to a fresh medium, is linearly related to the amount of serum used as is protein synthesis. However when serum concentrations higher than 20 percent were used no increased protein synthesis could be observed suggesting the existence of another limiting factor, which was probably the isoleucine content of the medium. The serum stimulating factor is heat stable, dialysable and was found in both human and fetal calf sera.     

A novel cis-acting element is essential for cytokine-mediated transcriptional induction of the serum amyloid A gene in nonhepatic cells

Serum amyloid A (SAA) is a plasma protein which has been associated with several diseases, including amyloidosis, arthritis, and atherosclerosis, and its abnormal expression, particularly in nonhepatic cells, is implicated in the pathogenesis of these diseases. Transfection and DNA-binding studies were performed to investigate the mechanism controlling cytokine-induced, nonhepatic expression of the SAA gene. We have identified a novel promoter, located between positions -280 and 224, that confers interleukin-6 (IL-6) inducibility to an SAA-chloramphenicol acetyltransferase reporter gene in both nonhepatic and hepatic cells. DNase I protection assays revealed, within this region, three homologous highly pyrimidine rich octanucleotide sequence motifs, termed SAA-activating sequences (SAS). Specific mutations within these three SAS motifs severely reduced IL-6-mediated induction of the reporter gene in transfected nonhepatic cells but not in liver cells. A nuclear factor activated by IL-6 in both hepatic and nonhepatic cells efficiently interacts with the SAS. The induction kinetics and cycloheximide sensitivity of this SAS-binding factor (SAF) suggested that de novo synthesis of this factor itself or an activator protein is essential. Loss of DNA-binding ability as a result of in vitro dephosphorylation, induction of SAA-chloramphenicol acetyltransferase reporter gene activity in the presence of genistein, a protein kinase inhibitor, further indicate that a phosphorylation step is necessary for the activation of SAF. Our results suggest that SAF is a key regulator of cytokine-mediated SAA gene expression in some nonhepatic cells.     

A chondroitin sulfate/keratan sulfate proteoglycan, PG-1000, forms complexes which are concentrated in the reticular laminae of electric organ basement membranes

Previously, we identified PG-1000 as part of a disulfide-linked complex of two large proteoglycans (PG-1000 and the beta component) and three smaller proteins purified from the extracellular matrix of elasmobranch electric organ (Iwata and Carlson, 1991, J. Biol. Chem. 266: 323-333). PG-1000 is a chondroitin sulfate/keratan sulfate proteoglycan with a molecular mass of about 1.2 x 16(6) daltons. When visualized in the electron microscope, PG-1000 has the typical "bottle-brush" appearance expected for a proteoglycan with an average total length of about 345 nm and about 20 chains of approximately 110 nm (Carlson and Wight, 1987, J. Cell Biol. 105: 3075-3086). Using immunocytochemical methods, we now demonstrate that PG-1000 is a component of the interstitial extracellular matrix of the electric organ. PG-1000 immunoreactivity is found throughout the interstitial matrix, but it is highly concentrated in that region of the matrix immediately adjacent to the basal lamina, the reticular lamina. The reticular and basal laminae together form the basement membrane. PG-1000 immunoreactivity is especially apparent on basal laminae that surround nerve fibers and nerve terminals. When the disulfide-linked PG-1000 complexes are purified and examined in the electron microscope following rotary shadowing, they appear as bottle-brush structures which are often attached at a central region and radiate like spokes of a wheel. These aggregates contain two to six proteoglycan monomers. We hypothesize that the PG-1000 complexes are disulfide-stabilized parts of an extended network of linked proteoglycans in the reticular lamina.     

When is a correlation matrix appropriate for factor analysis? Some decision rules.

Discusses 3 techniques for assessing the psychometric adequacy of correlation matrices; (a) computation of M. S. Bartlett's test of sphericity, (b) inspection of the off-diagonal elements of the anti-image covariance matrix, and (c) computation of the Kaiser-Meyer-Olkin 1970 measure of sampling adequacy. The advantages and disadvantages of each are compared with respect to assessment of correlation matrices prior to factor analysis. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The role of chondroitin sulfate chains of urinary trypsin inhibitor in inhibition of LPS-induced increase of cytosolic free Ca2+ in HL60 cells and HUVEC cells

Preincubation of HL60 cells and HUVEC cells with urinary trypsin inhibitor (UTI) inhibited increase of cytosolic free Ca2+ induced by LPS. In contrast, an increase of cytosolic free Ca2+ induced by LPS was not inhibited by deglycosylated UTI, UTI treated with monoclonal antibody of chondroitin sulfate. 45Ca2+ binding showed that UTI binds 45Ca2+ dose-dependently. Scatchard plot analysis showed that UTI has two binding sites for Ca2+, a high affinity binding site (Kd=15 microM) and a low affinity site (Kd=150 microM), and that UTI has more than 70 Ca2+ binding sites per molecule. The Ca2+ binding capacity of deglycosylated UTI and UTI treated with monoclonal antibody of chondroitin sulfate was markedly depressed. Furthermore, UTI forms multi-polymers in the presence of Ca2+ as demonstrated by gel filtration and agarose gel electrophoresis. These results suggest that UTI is a physiological Ca2+ chelator on the cells and that the action is due to chondroitin sulfate chains of UTI.     

Mannosylerythritol lipid is a potent inducer of apoptosis and differentiation of mouse melanoma cells in culture

Malignant melanomas are tumors that are well known to respond poorly to treatment with chemotherapeutic reagents. We report here that mannosylerythritol lipid (MEL), an extracellular glycolipid from yeast, markedly inhibited the growth of mouse melanoma B16 cells in a dose-dependent manner. Exposure of B16 cells to MEL at 10 microM and higher concentrations caused the condensation of chromatin, DNA fragmentation, and sub-G1 arrest, all of which are hallmarks of cells that are undergoing apoptosis. Analysis of the cell cycle also suggested that both the MEL-mediated inhibition of growth and apoptosis were closely associated with growth arrest in the G1 phase. Moreover, MEL exposure stimulated the expression of differentiation markers of melanoma cells, such as tyrosinase activity and the enhanced production of melanin, which is an indication that MEL triggered both apoptotic and cell differentiation programs. Forced expression of Bcl-2 protein in stably transformed B16 cells had a dual effect: it interfered with MEL-induced apoptosis but increased both tyrosinase activity and the production of melanin as compared with these phenomena in vector-transfected MEL-treated control B16 cells. These results provide the first evidence that growth arrest, apoptosis, and the differentiation of mouse malignant melanoma cells can be induced by a microbial extracellular glycolipid.     

Purification of a meningeal cell-derived chondroitin sulphate proteoglycan with neurotrophic activity for brain neurons and its identification as biglycan

Serum-free cultures of meningeal fibroblasts synthesize and release a chondroitin sulphate proteoglycan (CSPG) that markedly enhances survival but not adhesion of embryonic rat (embryonic day 15) neocortical neurons in vitro. The active molecule was purified from conditioned medium (meningeal cell-conditioned medium, MCM) in three steps by means of fast-performance liquid chromatography fractionation combined with a quantitative microphotometric bioassay: (i) preparative Q-Sepharose anion exchange chromatography under native conditions; (ii) rechromatography of biologically active Q-Sepharose fractions on a MonoQ column in the presence of 8 M urea; and (iii) final gel filtration of active MonoQ fractions on Superose 6 in the presence of 4 M guanidinium hydrochloride. Analytical sodium dodecyl sulphate-polyacrylamide gradient gel electrophoresis of active Superose 6 fractions revealed a single broad glycoprotein band with a molecular mass in the range of 220-340 kDa. Further characterization of the purified molecule with glycosaminoglycan:lyases revealed a core protein of 50 kDa and the nearly complete loss of neurotrophic activity after chondroitinase digestion, whereas heparitinase treatment changed neither electrophoretic mobility nor biological activity. Amino-terminal sequencing of the purified CSPG core protein revealed identity with the amino acid sequence of rat biglycan. Biglycan purified from bovine cartilage supported neuron survival with virtually the same activity as the CSPG purified from MCM (half-maximal activity approximate to 10(-8) M). In conclusion, we isolated a neurotrophic CSPG from meningeal cells with strong survival-enhancing activity for brain neurons that was identified as biglycan, a molecule not previously related to neural functions.