Effect of Lumican on the Migration of Human Mesenchymal Stem Cells and Endothelial Progenitor Cells: Involvement of Matrix Metalloproteinase-14

Background

Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful in vivo administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC).

Methodology/Principal Findings

Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression.

Conclusion/Significance

Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration via mechanisms that involve a decrease of MMP-14 expression and activity.     

Number of binding sites of DNA and polynucleotides with tris(2,2'-dipyridyl)ruthenium(II) cations

The binding of tris(2,2′-bipyridyl)ruthenium(II) cations [Ru(bpy)] with single- and double-stranded (ss and ds) DNA, and the polynucleotides poly(A), poly(C), poly(G), poly(I), poly(I) · poly(C), and poly(U), was studied in aqueous solution. Steady-state electrical conductivity measurements with the polynucleotides, ssDNA, and dsDNA reveal that approximately three nucleotides offer one binding site. This may be compared with the ratio [nucleotide]/[Mg²⁺] of 2.4 : 1 for dsDNA. After laser excitation (353 nm), the luminescence of Ru(bpy) bound to nucleic acids shows two decay components. The contribution of the fast component, which is interpreted as resulting from quenching processes of the absorbed ruthenium complex, exhibits a maximum with increasing [nucleotide]/[Ru(bpy)] at a ratio of about three to one. Bound Ru(bpy) can be released from the strand by addition of NaClO₄ [half-concentration: 2.5 and ≤ 10 mM for poly(U) and dsDNA, respectively].     

Brain Histamine in Rats with Hepatic Encephalopathy

Chronic liver failure induced by portocaval anastomosis (PCA) in Wistar rats resulted in a dramatic increase in histamine concentration in hypothalamus and a smaller, but clearly pronounced, elevation in the rest of brain. Between 10 and 120 days following surgery, shunted rats exhibited a histamine level 2.4- to 13-fold higher in hypothalamus and 1.5- to 2.5-fold higher in the rest of brain as compared to their control, sham-operated pairs. There were no significant changes in histamine concentration in the other examined tissues. The increase in brain histamine could not be attributed to the inhibition of its degradation, because activity of histamine N-methyltransferase remained unchanged for at least 40 days. Although the activity of histidine decarboxylase also remained unchanged when measured at a saturating concentration of L-histidine, the increase in histamine content in brain seems to be due to its enhanced synthesis brought about by increased availability of L-histidine in the tissue, as indicated by two to four times higher concentrations of this amino acid in PCA rats.     

Recombinant Human and Rat Ciliary Neurotrophic Factors

The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20-40% and 60-70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF.     

Application of chemically desialylated and degalactosylated human glycophorin for induction and characterization of anti-Tn monoclonal antibodies

Human erythrocyte glycophorin was desialylated by mild acid hydrolysis and degalactosylated by Smith degradation. Two monoclonal antibodies (Tn5 and Tn56) obtained by immunization of mice with this artificial Tn antigen were characterized and compared in some experiments with two antibodies (BRIC111 and LM225) obtained in other laboratories by immunization with Tn erythrocytes. The specific binding of the antibodies to glycophorins desialylated and degalactosylated on the nitrocellulose blot and to asialo-agalactoglycophorin-coated ELISA plates, and reactions with authentic Tn antigen served for identification of their anti-Tn specificity. The antibodies were further characterized in inhibition assay with various glycoproteins. The antibody Tn5 (similar to BRIC111) was shown to be specific for human erythrocyte Tn antigen, whereas Tn56 reacted strongly with different glycoproteins carrying O-linked GalNAc- residues, and was strongly bound to the murine adenocarcinoma cell line Ta3-Ha. The antibodies Tn5, Tn56 and BRIC111 were similarly inhibited by ovine submaxillary mucin (OSM) and asialoOSM, but the antibody LM225 showed a distinct preference in reaction with OSM (sialosyl-Tn antigen). The results show that Tn antigen, obtained by chemical modifications of human glycophorin, enables the preparation and characterization of anti-Tn monoclonal antibodies, without using rare Tn erythrocytes.Abbreviations HuGph human erythrocyte glycophorin - HoGph horse erythrocyte glycophorin - OSM ovine submaxillary mucin - mAb monoclonal antibody - ELISA enzyme-linked immunosorbent assay - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - PBS phosphate buffered saline (0.01m Na₂HPO₄/0.15m NaCl, pH 7.2) - BSA bovine serum albumin - TBS 0.05m Tris-HCl/0.15m NaCl, pH 7.2 - TGr transformation grade     

Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent

Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process.     

Ku80 as a novel receptor for thymosin beta4 that mediates its intracellular activity different from G-actin sequestering

Our data demonstrate that increased intracellular expression of thymosin beta4(Tbeta4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced Tbeta4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tbeta4((AcSDKPT/4A)), Tbeta4((KLKKTET/7A)), and Tbeta4((K16A)) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tbeta4. Overexpression of Tbeta4, Tbeta4((AcSDKPT/4A)), and Tbeta4((K16A)) affected intracellular formation of actin filaments. As expected, Tbeta4((K16A)) uptake by nuclei was impaired. On the other hand, overexpression of Tbeta4((KLKKTET/7A)) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular Tbeta4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both Tbeta4 and Tbeta4((AcSDKPT/4A)) induced the PAI-1 gene to the same extent, whereas mutants Tbeta4((KLKKTET/7A)) and Tbeta4((K16A)) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with Tbeta4. Ku80 and Tbeta4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tbeta4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for Tbeta4 and mediates its intracellular activity.     

Interaction and functional association of protein disulfide isomerase with alphaVbeta3 integrin on endothelial cells

Adhesive properties of endothelial cells are influenced by the thioldisulfide balance. However, the molecular mechanism of this effect is unclear, although recent observations indicate that integrin receptors may be direct targets for redox modulation. The purpose of this study was to examine whether protein disulfide isomerase (PDI) is directly involved in this process. As manganese ions are known to affect the thioldisulfide balance and activate integrins to maximal affinity, we searched for PDI interactions with integrins, particularly with alpha(V)beta(3), in Mn(2+)-treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn(2+) resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and alpha(V)beta(3), and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI-alpha(V)beta(3) complex, which dissociates upon reduction. In addition, PDI in a complex with alpha(V)beta(3) induces conversion of the integrin to the ligand-competent high-affinity state, as evidenced by increased binding of vitronectin. The membrane-impermeable sulfhydryl blockers 3-N-maleimidylpropionyl biocytin 3-N-maleimidylpropionyl biocytin and p-chloromercuriphenyl sulfonate, as well as the PDI inhibitors bacitracin, MA3 018, and MA3 019, abolished the binding of vitronectin and LM609 to endothelial cells that is activated by Mn(2+). Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI-alpha(V)beta(3) stoichiometric complex was further demonstrated by surface plasmon resonance analysis, which showed that the initial reversible binding of PDI becomes irreversible in the presence of Mn(2+), probably mediated by disulfide bonds. Thus, we show that Mn(2+) simultaneously modulates the thiol isomerase activity of PDI that is bound to alpha(V)beta(3) and induces its transition to the ligand-competent state, suggesting an alternative mechanism of integrin regulation.     

Ceramide enrichment of the plasma membrane induces CD81 internalization and inhibits hepatitis C virus entry

Virus entry is a major step in which host-cell lipids can play an essential role. In this report, we investigated the importance of sphingolipids in hepatitis C virus (HCV) entry. For this purpose, sphingomyelin present in the plasma membrane of target cells was hydrolysed into ceramide by sphingomyelinase treatment. Interestingly, ceramide enrichment of the plasma membrane strongly inhibited HCV entry. To understand how ceramide affected HCV entry, we analysed the effect of ceramide enrichment of the plasma membrane on three cell-surface molecules identified as entry factors for HCV: CD81 tetraspanin, scavenger receptor BI and Claudin-1. These proteins, which we found to be mainly associated with detergent-soluble membranes in Huh-7 cells, were not relocated in detergent-resistant microdomains after sphingomyelin hydrolysis into ceramide. Importantly, ceramide enrichment of the plasma membrane led to a 50% decrease in cell-surface CD81, which was due to its ATP-independent internalization. Our results strongly suggest that the ceramide-induced internalization of CD81 is responsible for the inhibitory effect of ceramide on HCV entry. Together, these data indicate that some specific lipids of the plasma membrane are essential for HCV entry and highlight plasma membrane lipids as potential targets to block HCV entry.