Masao Hayashi

Snail Regulates Cell-Matrix Adhesion by Regulation of the Expression of Integrins and Basement Membrane Proteins

Snail, a transcriptional repressor of E-cadherin expression, plays a role in the process of epithelial-mesenchymal transition. However, the molecular basis of the role of snail in epithelial-mesenchymal transition has not been fully clarified. Here we show that the expression of snail in epithelial Madin-Darby canine kidney (MDCK) and A431 cells enhances both cell detachment and attachment. Snail did not confer resistance to anoikis induced by loss of contact but instead enhanced cell attachment to extracellular matrices such as fibronectin. This attachment was inhibited by Arg-Gly-Asp (RGD) peptides. Up-regulation of the promoter activity of integrin αV was observed in snail-expressing MDCK (MDCK/snail) cells. Snail also enhanced MDCK cell migration toward osteopontin that is a ligand for integrin αVβ3. We confirmed the reduction of basement membrane proteins such as laminin (LN) α3, β3, and γ2 (laminin-5/LN-5) and of receptors for LN-5 such as integrins α3, α6, or β4 in MDCK/snail or in snail-expressing A431 (A431/snail) cells. Nevertheless, suppression of LN-α3 chain by transient transfection of small interference RNAs resulted in no enhancement of cell detachment. We also found an induction of matrix metalloproteinase-3 in MDCK/snail and A431/snail cells. However, the inhibition of matrix metalloproteinase-3 showed no significant effect on the detachment of MDCK/snail cells. These results suggest that snail enhances cell detachment by multiple mechanism and leads to cell migration and reattachment at a second site, at least in part, by changing the expression of integrins in the cells.

Vitronectin exists in two structurally and functionally distinct forms in human plasma

Vitronectin (serum spreading factor, S-protein or epibolin) is a plasma glycoprotein implicated in cell adhesion, as well as in the regulation of complement-mediated cytolysis and antithrombin III function. Vitronectin was found to exist in fresh human plasma as a heterogeneous mixture consisting of 2% heparin-binding form and the remainder as a non-binding species. Heparin-binding vitronectin consisted of 6.5 S aggregates with a Stokes radius of 5.6 nm, which was enriched in the 65 kDa polypeptide, with a high content of molecules and a putative unfolded conformation. In contrast, non-heparin-binding vitronectin was a 4.2 S monomer with a Stokes radius of 3.9 nm, which appeared to be in a folded conformation with an immunologically cryptic site. Both vitronectins displayed similar activities in mediating the spreading of BHK fibroblastic cells on substrates. During blood coagulation, 5% more of the non-heparin-binding vitronectin was converted into the heparin-binding form, producing a greater than 3.5-fold increase in this species. Our results indicate that vitronectin normally exists in circulating blood in at least two structurally and functionally distinct forms which may serve different functions.

In vivo guidance of regenerating nerve by laminin-coated filaments

Laminin is reportedly an extremely potent neurite-promoting agent in vitro. We investigated the effect of laminin on regeneration of axons in vivo, using a cord of polyester filaments coated with laminin as a guide, replacing a 10-mm segment of the rat sciatic nerve. After 4 weeks the cross section of the nerve guide at its midportion was examined under an electron microscope. Many regenerated axons were seen in the laminin-coated group, whereas no axon was observed in the controls. We conclude that laminin guides the regeneration of axons in vivo.

Vitronectin diversity in evolution but uniformity in ligand binding and size of the core polypeptide

We isolated vitronectins from the plasma or sera of 14 animal species including mouse and rat by heparin affinity chromatography. They cross-reacted with anti-vitronectin antibody and their amino terminal sequences showed strong homology. They also promoted spreading of BHK cells and were bound to heparin and collagen in the same way. Therefore, these properties appear to be essential for vitronectin function. However, the apparent molecular weights of these vitronectins varied considerable from 59 to 78 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, the number of bands also varied from 1 to 3. To search for the uniformity of vitronectin polypeptide, vitronectins were deglycosylated and examined by Ferguson plot analysis. The size of the polypeptide portion of vitronectins was estimated to range from 40 to 57 kDa which was 19-26 kDa smaller than original values. Supposing a possible cleavage site at 5-13 kDa far from the carboxyl terminus, all vitronectin polypeptides were speculated to be synthesized de novo in the size range of 50-57 kDa. Proteins reacting with anti-vitronectin antibody were also detected on the immunoblot of 13 more species including Drosophila and Physarum. Almost all of these vitronectin-like proteins showed marked species-specific variations in their apparent molecular weights from 51 to 96 kDa in SDS-PAGE.

Mouse integrin αv promoter is regulated by transcriptional factors Ets and Sp1 in melanoma cells

A 17-bp region between the -31 and -15 bp region of the mouse integrin alphav gene is known to be one of the cis-acting elements for promoter activity. Experimental binding of nuclear proteins to the -31/-15 region reveals that the -27/-16 region mediates the binding. The -27/-16 region, GGCTCCTCCTCC, has a TCCTCC motif, one of the Sp1 binding motifs. An anti-Sp1 IgG and an Sp1-binding oligonucleotide interfered with the binding of nuclear proteins to the -27/-16 oligonucleotide, demonstrating that Sp1 binds to the -27/-16 region. In addition to the -27/-16 region, two other regions, -108/-89 and -64/-44, were found to bind to nuclear proteins within the -108/+1 alphav promoter region. An oligonucleotide containing the Ets-binding consensus sequence of CAGGAAGT interfered with their binding, indicating that both regions have a functional Ets-binding site; which is ACGGAAGT from -106 to -99 bp and ACTTCCTC from -61 to -54 bp, as deduced from the sequence. Mutations in or deletions from any one of three cis-acting elements, the two Ets-binding sites or one Sp1-binding site, remarkably decreased the promoter activity detected in the -108/+1 region. Cotransfection of both Sp1 and Ets-1 cDNAs with the -108/+1 region into B16F10 cells increased the promoter activity 2.9-fold. These results demonstrate that Sp1 and Ets cooperate to activate the -108/+1-alphav promoter region.

Polymorphism of the human vitronectin gene causes vitronectin blood type

Human blood plasma/sera are classified into three distinct vitronectin types based on the relative amount of the 75 kDa polypeptide to its cleavage product of 65 kDa. We asked whether the vitronectin blood types correlated with the polymorphism of the vitronectin gene. A portion of the vitronectin gene was amplified by using polymerase chain reaction and digested with a restriction enzyme PmaC I which may distinguish the base sequence causing the polymorphic change at the amino acid position 381. Amplified DNAs of the blood type I (75 kDa-rich), II (75/65 kDa-even), and III (65 kDa-rich) were shown to be resistant, moderately sensitive and completely sensitive to PmaC I, respectively. These results suggest that Thr at position 381 is essential for the cleavage of the vitronectin 75 kDa polypeptide and that three possible combinations of two codominant alleles of vitronectin determine three vitronectin blood types.

Activation of the collage-binding of endogenous serum vitronectin by heating, urea and glycosaminoglycans

The present study describes that the collagen-binding activity of vitronectin in human serum increases by treatment with heparin, heating and urea. Vitronectin purified from human serum was bound to native collagen, whereas endogenous vitronectin in the serum was not. We have examined the conditions to change the collagen-binding activity of endogenous vitronectin. Endogenous vitronectin in human serum became considerably bound to collagen when the serum was boiled in 4-8 M urea for 5 min and mixed with heparin (0.5-5 micrograms/ml). Each treatment of heating, urea or heparin alone, and any combination of the two factors, inefficiently activated the binding. Dextran sulfate could substitute for heparin, but dermatan sulfate, keratan sulfate, chondroitin sulfate A and C, heparan sulfate and hyaluronan could not. Possible explanations for the activation of endogenous vitronectin are discussed.

Physarum vitronectin-like protein: An ArgGlyAsp-dependent cell-spreading protein with a distinct NH2-terminal sequence☆

A 70-kDa protein cross-reacted with anti-bovine vitronectin was isolated from slime mold Physarum polycephalum. The NH2-terminal amino acid sequence of the protein, referred to as Physarum vitronectin-like protein, did not share any homology with those of animal vitronectins. It had cell-spreading activity, which was specifically inhibited by an Arg-Gly-Asp (RGD)-containing peptide.

Cell-type dependency of two Foxa/HNF3 sites in the regulation of vitronectin promoter activity.

The mouse vitronectin promoter has two consensus sequences of the Foxa/hepatocyte nuclear factor (HNF) 3-binding site (from -34 to -25, site A, and +15 to +26 base pairs (bp), site B). Site-directed mutagenesis of site B inhibited binding of nuclear proteins from mouse neuroblastoma Neuro2a and reduced the promoter activity to 4.6% in a 101-bp fragment (from -48 to +53 bp) in Neuro2a cells. The nuclear proteins of site B were identified as the Foxa1/HNF3alpha and Foxa2/HNF3beta proteins by supershift assay. Next, we examined site A. Mutation of site A in Neuro2a cells did not affect the promoter activity, and binding of nuclear proteins was not detected. Overexpression of Foxa1 or Foxa2 protein activated the mutated site B promoter, but failed to activate the sites A and B double-mutated promoter in Neuro2a cells, indicating that site A is a potential transcription regulatory site. Recombinant Foxa1 and Foxa2 proteins and nuclear extract from mouse liver bound not only to site B, but also to site A. In human hepatoma HepG2 cells, mutation of sites A and B decreased the promoter activity to 82% and 38%, respectively, in the wild promoter, and double mutation of sites A and B decreased the wild promoter activity to 5%, indicating that sites A and B contribute to the promoter activity in HepG2 cells. These results demonstrate that the two Foxa-binding sites regulate the vitronectin promoter activity in cell type-dependent manner.

Characterization of a zinc finger protein ZAN75: nuclear localization signal, transcriptional activator activity, and expression during neuronal differentiation of P19 cells.

The ZAN75 cDNA was first identified in NIH 3T3 cells and codes for a DNA-binding protein with two zinc finger motifs. In this study, we characterized the nuclear localization signal of ZAN75, tested if ZAN75 regulates transcription, and examined its expression during embryonic development and neuronal differentiation of P19 mouse embryonal carcinoma cells. By examining the cellular localization of deletion mutants of ZAN75 fused to green fluorescence protein, ZAN75 was revealed to have a bipartite nuclear localization signal sequence upstream of the zinc finger domains. The N-terminal region of ZAN75, when fused to the GAL4 DNA-binding domain, strongly activated transcription. The expression of ZAN75 mRNA was found to be developmentally regulated, showing the highest expression in E11.5 embryos. In situ hybridization experiments using E11.5 embryos showed a high expression of the transcripts in neuronal tissues such as brain and neural tube. The expression of ZAN75 was transiently increased at both the mRNA and the protein levels when P19 cells were treated with retinoic acid to induce neuronal differentiation. Taken together, these results indicate that ZAN75 is a transcriptional activator with a bipartite nuclear localization signal and may play a role in neuronal differentiation.