Laminin Binding Research Articles

Galectin-1 Modulates Human Melanoma Cell Adhesion to Laminin

Galectins constitute a gene family of β-galactoside-specific lectins that show high homology in their carbohydrate-binding site. They have been postulated to be involved in many biological events, but their specific functions are not yet well defined. Galectin-1 is a laminin binding protein that recognizes poly N-acetyllactosamine chains on this major basement membrane glycoprotein. In this study, we analyzed the possibility that galectin-1 could modulate interactions between human melanoma cells and laminin. We demonstrated that A375 and A2058 cell lines express galectin-1 both intracellularly and on the cell surface. In an in vitro assay, recombinant galectin-1 increased melanoma cell attachment to laminin in a dose-dependent manner. This effect was abolished by lactose. Anti-galectin-1 inhibited adhesion of melanoma cells to laminin in a dose-dependent fashion. However, neither galectin-1 nor anti-galectin-1 antibody affected melanoma cell spreading on laminin in vitro. These data indicate that galectin-1 might participate in melanoma cell adhesion to laminin and therefore could be a modulator of invasion and metastasis.

Protein Synthesis Is Required for Laminin-Induced Expression of the 67-kDa Laminin Receptor and Its 37-kDa Precursor

The high affinity 67-kDa laminin receptor (67LR) is highly expressed in metastatically active human cancers. A 37- kDa polypeptide has been identified as its precursor (37LRP). Antibodies raised against 37LRP-derived synthetic peptides were used in immunogold electron microscopy and immunoblot studies to assess the effect of laminin on expression of the 67LR and the 37LRP. Laminin (15 μg/ml) treatment of suspended A2058 human melanoma cells doubled the expression of both 37LRP and the 67LR. Fibronectin had no effect. There was no effect of laminin on the expression of actin or galectin-3. Cycloheximide treatment of cells prior to laminin abrogated its inducible effect. The results suggest that binding of laminin by cell surface laminin receptors induces synthesis of the 37LRP and mature 67LR, with a consequent delivery to the cell surface of more laminin binding proteins for potentiated attachment of the melanoma cell to the basement membrane during invasion and metastasis.

Expression of a novel integrin beta 1 chain epitope and anti-beta 1 antibody-mediated enhancement of fibronectin binding are dependent on the stage of T cell differentiation.

Beta 1 integrins are a family of alpha beta heterodimers that serve as cell surface receptors for extracellular matrix proteins. We demonstrate that the anti-mouse integrin beta 1 chain mAb KMI6 selectively recognizes a beta 1 epitope that is constitutively expressed by certain immature thymocytes and is induced only slightly on mature thymocytes and peripheral T cells by activation with Con A. Because virtually all cells examined expressed beta 1 integrins on their surface, expression of the KMI6 epitope is T cell differentiation stage specific. Most CD3-4-8- thymocytes were KMI6+, with the lowest level of staining observed on the earliest CD44+IL-2R- cells within this subset. Expression was down-regulated during the CD3-4-8- to CD3-4-8+ transition, and lost by the CD4+8+ stage. Mature single positive thymocytes and resting peripheral T cells were also KMI6-. In contrast with the loss of the epitope before TCR expression by other thymocytes, most CD3+4-8- and certain CD8+ gamma delta TCR+ thymocytes were KMI6+ Addition of KMI6 to cell adhesion assays enhanced CD4-8- thymocyte, but not activated mature thymocyte or peripheral T cell, binding to fibronectin (via alpha 4 beta 1 and alpha 5 beta 1), whereas laminin binding (via alpha 6 beta 1) was unaffected. These properties distinguish the KMI6 epitope from other epitopes involved in beta 1 integrin activation in mice and other species. The unique selectivity of KMI6 recognition of beta 1 integrins, and its selective enhancement of ligand binding suggest that beta 1 integrin structure and factors that regulate beta 1 integrin binding are correlated with the stage of T cell differentiation.

Expression of binding of plasminogen, thrombospondin, vitronectin, and fibrinogen, and adhesive properties by Escherichia coli strains isolated from patients with colonic diseases.

Escherichia coli strains isolated from patients with colonic disorders (n = 27) and strains isolated from the rectal mucosa of healthy subjects (n = 24) were compared with respect to expression of cell surface hydrophobicity, carriage of intestinal virulence factors, adhesion to tissue culture cells, and expression of binding of extracellular matrix proteins and plasma proteins. Strains isolated from patients with colonic disease did not express a more hydrophobic cell surface than strains from healthy subjects. Few strains from both groups carried genes encoding for recognised virulence factors of E coli. Only one strain, carrying the eae gene induced actin polymerisation in tissue culture cells. Strains from patients with colonic diseases adhered to HT29 cells, which are of intestinal origin, to a higher extent than E coli from healthy subjects. Significantly more strains from patients with colonic disorders than E coli from healthy subjects expressed binding of fibronectin, collagens, laminin, vitronectin, plasminogen, throbospondin, and fibrinogen. Expression of binding of these proteins may influence the pathogenesis of colonic disease by mediating binding to ulcerated tissue, preventing complement induced lysis of bacteria and by exerting proteolytic activity. There was no correlation between serotype, expression of cell surface hydrophobicity, and binding of extracellular matrix and plasma proteins.

FIBRONECTIN AND FIBRONECTIN-RECEPTORS OF THE INTEGRIN TYPE IN NORMAL COLON MUCOSA, ADENOMA AND CARCINOMA

Integrin alpha 3, alpha 4, alpha 5, and alpha v subunits heterodimerize with beta 1 and beta 3 chains to form functional fibronectin (FN) receptors. Two of them, alpha 4 beta 1 and alpha 5 beta 1, recognize FN as the only matrix molecule. Additional binding of laminin, collagen, and entactin is achieved by alpha 3 beta 1 while alpha v beta 1 and alpha v beta 3 also bind vitronectin. We investigated immunohistochemically the tissue distribution of FN and the expression of FN receptor alpha- and beta-chains in 20 normal colon tissues, 10 adenomas, 90 carcinomas, and 10 liver metastases derived thereof. In normal and adenomatous colon tissues FN was detected in association with reticular and fibrillar structures of the gut wall. The tumor stroma of carcinomas and liver metastases contained abnormally high amounts of FN which were detectable as chaotic deposits. Normal and adenomatous mucosa were alpha 3(+), alpha 4(-), alpha 5(-), alpha v(+/-), beta 1(+), beta 3(-) indicating that only promiscuous FN receptors were expressed. 21 of 90 carcinomas had a focal or complete loss of alpha 3 which was more often found in Dukes C/D tumors (p<0.005). The liver metastases more often had lower levels of alpha 3 expression compared to the primary tumor. The alpha v-expression was inconsistent and often weak. These losses of FN receptor constituents in carcinomas were not paralleled by any noticeable differences in stromal content or distribution pattern of FN, Enhanced expression/induction of FN receptors was found in only 2 of 90 carcinomas which focally neo-expressed the alpha 5-chain. This aberrant alpha 5 expression, however, was flow cytometrically found in 3/4 colon carcinoma cell lines which otherwise had receptor profiles corresponding to the in situ situation. FN-adherence of cell lines was variable. Anti-alpha 3 had no blocking effect on FN adherence of SW480, SW620, and COLO 205 but a minor effect on FN binding of HT-29. Anti-alpha 5 blocked FN adhesion whenever alpha 5 was expressed. Anti-av had no blocking effect while anti-beta 1 reduced FN adherence of all cell lines. The net biological effect(s) of aberrant expression of integrin type FN receptors in colon carcinomas is unpredictable as yet.

Inhibitory Effect of Oversulfated Fucoidan on Invasion through Reconstituted Basement Membrane by Murine Lewis Lung Carcinoma

We investigated the effects of native, Oversulfated, and desulfated fucoidans and heparin on the invasion of 3LL cells through Matrigel. Of the four polysaccharides tested, Oversulfated fucoidan was the most potent inhibitor of tumor cell invasion and inhibited most potently and specifically the tumor cell adhesion to laminin. Sodium dodecyl sulfate‐polyacrylamide gel electrophoretic analysis of the binding of elastase‐cleaved laminin to fucoidan‐ and heparin‐Sepharoses showed that both polysaccharides bound to the 62 and 56 kDa fragments. Pretreatment of 3LL cells with native or Oversulfated fucoidan reduced their adhesive potency to laminin. The two fucoidans inhibited further the laminin binding of 3 LL cells which had been pretreated with a laminin‐based pentapeptide, YIGSR. These results suggest that fucoidan specifically binds to not only the heparin binding domain(s) of laminin but also site(s) other than the cell surface laminin receptor. 3 LL cells secreted a 50 kDa form of urokinase‐type plasminogen activator (u‐PA). The extracellular level of u‐PA activity was increased 1.7 times by addition of laminin but not type IV collagen. Oversulfated fucoidan most potently reduced the increased u‐PA levels. Therefore, the reduction in in vitro invasiveness of 3 LL cells in response to either fucoidan or its Oversulfated derivative may result from an inhibition of physical interaction between the tumor cells and the Matrigel (laminin), followed by a suppression of the laminin‐induced increase in extracellular u‐PA.

Glycolipid receptors for attachment of Mycoplasma hyopneumoniae to porcine respiratory ciliated cells.

Glycolipid receptors for Mycoplasma hyopneumoniae attachment were analyzed by using a thin-layer chromatography (TLC) overlay assay. M. hyopneumoniae bound specifically to sulfatide, globoside, and monosialoganglioside GM3. No binding to sphingomyelin, cerebroside, lactosyl ceramide, ceramide trihexoside, monosialogangliosides GM1 and GM2, disialogangliosides (GD1a, GD1b, and GD3), trisialoganglioside (GT1b), cholesterol, cholesterol sulfate, palmitic acid, tripalmitin, or cholesteryl palmitate was detected. Total lipids extracted from cilia of the swine respiratory epithelium, the natural targets of M. hyopneumoniae infection, were also separated on TLC plates and overlaid with mycoplasmas. M. hyopneumoniae bound specifically to three ciliary glycolipids identified as La, Lb, and Lc. Binding to Lc was stronger than to La and Lb. All three lipids were believed to be sulfated glycolipids, as determined by laminin binding and staining with azure A. Lc was identified as a putative sulfatide because it has a mobility similar to that of authentic sulfatide and comigrated with sulfatide on TLC plates. Laminin bound to La, Lb, and Lc and produced dose-dependent inhibition of adherence of the mycoplasma to the three ciliary receptors. Binding of the mycoplasma to sulfatide, La, Lb, and Lc was partially inhibited by dextran sulfate, heparin, fucoidan, mucin, and chondroitin sulfate B. These substances blocked the adherence of M. hyopneumoniae to cilia and ciliated cells as shown in a previous study (Q. Zhang, T. F. Young, and R. F. Ross, Infect. Immun. 62:1616-1622, 1994). These results indicate that La, Lb, and Lc are the major native receptors for M. hyopneumoniae adherence to ciliated cells.

Dystroglycan is a binding protein of laminin and merosin in peripheral nerve

α-Dystroglycan, a 156 kDa dystrophin-associated glycoprotein, binds laminin in skeletal muscle. Here we demonstrate that α-dystroglycan is a binding protein of laminin (A/B1/B2) and merosin (M/B1/B2) in peripheral nerve. Immunocytochemical analysis demonstrates the localization of α-dystroglycan and merosin surrounding myelin sheath of peripheral nerve fibers. Biochemical analysis demonstrates that the 120 kDa peripheral nerve α-dystroglycan binds merosin as well as laminin. The binding of laminin and merosin is Ca 2+ dependent and is inhibited by NACl and heparin. Recently, merosin was shown to be deficient in the peripheral nerve of dy mice which have defects in myelination. The interaction between α-dystroglycan and merosin may play a role in the regulation of Schwann cell myelination and/or maintenance of myelin sheath.

Interaction of lipopolysaccharides of Helicobacter pylori with basement membrane protein laminin.

The ability of hemagglutinating and poorly hemagglutinating strains of the gastroduodenal pathogen Helicobacter pylori to bind 125I-radiolabelled laminin was quantitated in a liquid phase assay. Although all strains bound laminin, some hemagglutinating strains were good binders of laminin (maximum of 31% binding), whereas poorly hemagglutinating strains bound intermediate to small amounts of laminin (minimum of 6% binding). Since a hydrophobic component of the bacterium has been reported to be involved in binding of laminin (T. J. Trust, P. Doig, L. Emödy, Z. Kienle, T. Wadström, and P. O'Toole, Infect. Immun. 59:4398-4404, 1991), we investigated the role of lipopolysaccharide (LPS) in the interaction of both types of strains with laminin. Although the extent of inhibition varied among strains, laminin binding to hemagglutinating and poorly hemagglutinating strains was inhibited with homologous and heterologous smooth-form LPS. The ability of heterologous rough-form LPS to produce inhibition comparable to that shown by smooth-form LPS indicated that the O side chain of H. pylori LPS was not involved in the interaction. Further inhibition experiments with dephosphorylated LPS, isolated core oligosaccharide, and free lipid A suggested that a phosphorylated structure in the core oligosaccharide mediates the interaction of a hemagglutinating strain of H. pylori with laminin, whereas a conserved nonphosphorylated structure in the core oligosaccharide mediates the interaction of a poorly hemagglutinating strain. Furthermore, we showed that the interaction of H. pylori LPS with 125I-radiolabelled laminin in a solid phase assay was saturable, specific, and inhibitable with unlabelled laminin. It was postulated that the initial recognition and binding of laminin by H. pylori may occur through LPS and that subsequently a more specific interaction with a lectin-like adhesin on the bacterial surface occurs.

Growth factors and decidualization in vitro.

Growth factors are believed to act as local regulators of endometrial cyclic activity, but there is limited information on their regulation of decidual differentiation and function. Cell cultures of human endometrial stroma treated with progesterone (P) undergo morphologic, proliferative and secretory changes characteristic of decidualizing endometrium. In the presence of P, different growth factors can stimulate cell proliferation, but decidual differentiation is induced specifically by EGF, as shown by the production of prolactin (PRL), fibronectin, laminin, and insulin-like growth factor binding protein 1 (IGFBP-1). The present study investigates the effects of the insulin-like growth factors (IGF-I, IGF-II) on decidualization in vitro, as indicated by a P-dependent growth response and by the secretion of PRL and IGFBP-1. IGFs were required together with EGF and P to stimulate stromal cell proliferation. In contrast, PRL (38 +/- 4 ng/day/10(6) cells) and IGFBP-1 (26 +/- 3 micrograms/day/10(6) cells) were secreted by in vitro decidualized cells in the absence of exogenous IGFs. However, IGFs regulated both IGFBP-1 and PRL secretion in a dose-dependent biphasic manner. Stimulation of IGFBP-1 (200-250%) and PRL (243-324%) peaked at 1 ng/ml for IGF-I, and 10 ng/ml for IGF-II, followed by inhibition at higher peptide concentrations (ED50s 3 and 30 ng/ml, respectively). Maximal physiological doses (100 ng/ml) of IGF-I and IGF-II virtually abolished IGFBP-1 secretion (1% and 2% of basal levels, respectively), but did not cause total suppression of PRL secretion (8% and 22% of basal levels). IGF-induced mitogenesis was inversely correlated with endogenous IGFBP-1 levels in in vitro decidualized stromal cultures. Our studies show that growth factor interactions regulate decidual function, and that specific cellular functions associated with the decidual response are differentially regulated by growth factor interactions. Our findings support a role for the IGF system in autocrine/paracrine interactions during decidualization and early pregnancy. It is speculated that IGF-II may constitute one of the embryonic signaling mechanisms during early postimplantation stages.

Dystroglycan-α, a dystrophin-associated glycoprotein, is a functional agrin receptor

Aggregation of acetylcholine receptors (AChRs) on skeletal muscle fibers is thought to be mediated by the basal lamina protein agrin. Structural similarities shared by agrin and laminin suggested that the laminin receptor dystroglycan-α, part of a dystrophin-receptor complex, might also bind agrin. We show here that dystroglycan-α and dystrophin-related protein (DRP/utrophin) are concentrated within AChR aggregates in cultures of C2 myotubes and that agrin binds specifically to dystroglycan-α in in vitro assays. This binding is calcium dependent and is inhibited by monoclonal antibody (MAb) IIH6 against dystroglycan-α, heparin, and laminin, but not by fibronectin. In S27 cells, which do not aggregate AChRs spontaneously, agrin and laminin binding to dystroglycan-α are dramatically decreased. Moreover, MAb IIH6 significantly inhibits agrin-induced AChR aggregation on C2 cells. We conclude that dystroglycan-α is an agrin-binding protein and part of a dystrophin-receptor complex involved in AChR aggregation.

Multiple Laminin Binding Proteins in Human Fetal Heart: (fetal heart/laminin binding proteins/cation dependent protein/cation independen protein/developmental changes).

The possibility of occurrence of laminin binding proteins in cardiac tissue under different stages of growth was examined by affinity chromatography of the soluble fraction of human fetal myocardial plasma membrane over Ln-Sepharose. A 67 kDa protein was isolated by elution with glycine/HCl buffer containing 1 M NaCl and visualized as a coomassie stainable band on SDS gel electrophoresis under reducing conditions. Dot blot assays of the radioiodinated protein revealed the binding of 67 kDa protein with high affinity to laminin in a cation independent manner. This protein appears to be present in relatively higher amounts in tissues from early stage fetus. The occurrence of cation dependent laminin binding proteins was also examined by affinity chromatography. Electrophoresis of the EDTA eluate under reducing conditions followed by silver staining showed two prominent bands with average molecular size 130 and 174 kDa which under non-reducing conditions appeared as two bands with average molecular weight of 115 and 135 kDa. Using radioiodinated protein in dot blot assays, its binding to Ln was found to be maximum in the presence of Mn++ ions. Immunoblotting using anti-β1 integrin antibodies showed that 115 kDa protein is a β1 integrin suggesting the possibility of this protein belonging to the integrin group of receptors. The occurrence of multiple laminin binding proteins and the relative abundance of one of these proteins viz. the 67 kDa protein during early stages than in late stage tussue suggest a possible role for these proteins in cellular interactions with laminin during myocardial tissue development.

An acidic component of the heterogeneous Tc-85 protein family from the surface of Trypanosoma cruzi is a laminin binding glycoprotein

Successful infection of mammalian host by trypomastigotes of Trypanosoma cruzi is a complex event, involving host receptors and parasite ligands. Interaction of the trypomastigote stage with laminin, a component of specialized extracellular matrices, as basement membranes, is studied in this report. Binding of 125I-laminin to trypomastigotes is specific and 2−5 × 10 3 laminin binding sites were calculated to be present on the surface of live trypomastigotes. Antilaminin antibodies were able to inhibit the invasion of cultured cells by trypomastigotes (75-62%), suggesting that laminin may be involved in the adhesion of the parasite to host cells. By affinity chromatography, an 85-kDa glycoprotein was isolated (laminin binding glycoprotein, LBG) from trypomastigote lysates, but not from epimastigote lysates. It is suggested that at least fragment E8 (but not E1′) from laminin could be involved in the reaction which is independent of the carbohydrate moieties from both ligand and receptor, as suggested by glycosidase or tunicamycin treatments. It is also shown that LBG is an acidic component of the polymorphic Tc-85 protein family, a trypomastigote-specific surface membrane glycoprotein which contains several polypeptides recognized by the monoclonal antibody H1A10, and previously related with the invasion process of the parasite.

Retinoic acid enhances adhesiveness, laminin and integrin beta 1 synthesis, and retinoic acid receptor expression in F9 teratocarcinoma cells.

The teratocarcinoma-derived F9 cells respond to retinoic acid (RA) and RA plus dibutyrylcyclic adenosine monophosphate (dcAMP) by differentiating into endoderm cells, which elaborate a laminin and type IV collagen-rich matrix. We found that the induction of differentiation is accompanied by a small but consistent increase in cell adhesiveness to a variety of substrates, including laminin. Therefore we investigated biochemical mechanisms involved in this phenomenon. Endoglycosidase treatment showed that laminin contains complex and hybrid oligosaccharide structures. RA enhanced general biosynthesis of laminin without a specific increase in galactose incorporation: this sugar was mainly in polylactosamine structures in the A chain of laminin and as terminal galactose alpha 1,3 galactose in the B chain. Laminin receptor analysis showed that RA decreased laminin binding protein-37 (LBP-37) but increased the amount of beta 1 integrin, suggesting the involvement of beta 1 integrin in the attachment process. Northern blot analysis showed increased expression of retinoid receptors within hours of RA exposure. These studies demonstrate that RA increases cell to substrate interactions by increasing the biosynthesis of laminin and beta 1 integrin. These effects are most likely subsequent to the RA-induced biosynthesis of the retinoid receptors.

Inverse Expression of Two Laminin Binding Proteins, 67LR and Galectin-3, Correlates with the Invasive Phenotype of Trophoblastic Tissue

Tumor invasion of host tissues and trophoblastic penetration of the endometrium share common biological features. Both processes involve the invasion of basement membranes, an event that is initiated by adhesion of cancer or trophoblast cells to basement membrane components and particularly to laminin. Adhesion to this latter glycoprotein is mediated through a variety of cell surface receptors. We have previously shown that the 67 kD Laminin Receptor (67LR) and a 31 kD Human Laminin Binding Protein, recently renamed galectin-3, are inversely modulated as the invasive phenotype of cancer cells progresses, with up regulation of the former, and down regulation of the latter, respectively. In this study, we examined the expression of these two proteins in 27 human trophoblastic specimens at different gestational ages using Northern and Western blot techniques. Expression of the 67LR increased from 7 weeks to a maximum at 12 weeks, when invasion is maximal, and then decreased. Expression of galectin-3 was inversely modulated by the gestational age, with a minimum expression at 12 weeks. Our data demonstrate that invasive trophoblast displays the same pattern of laminin binding proteins expression than invasive cancer cells, and further demonstrates that invasion of the extracellular matrix by trophoblast and cancer cells share common molecular mechanisms.

Mercury-Induced Renal Autoimmunity in BN → LEW.1N Chimeric Rats

Repeated exposure to relatively low doses of mercuric chloride causes a variety of autoimmune responses in rats of the Brown Norway (BN) strain. These animals experience a membranous glomerulonephritis, characterized by the production of autoantibodies to renal antigens (e.g., laminin) and proteinuria. In contrast, Lewis (LEW) rats are "resistant" to the autoimmune effects of mercury. Despite extensive investigations, the mechanisms of immunoregulation in this animal model are still unknown. RT6+ T lymphocytes may have a regulatory role in both BN and LEW rats. This hypothesis is suggested by our finding of a mercury-associated decrease of RT6+ T cells in lymph nodes of BN rats exposed to mercury and the lack of such effect in similarly treated LEW rats. In the present report we show that congenic LEW.1N or BN.1L had no renal autoimmune disease after treatment with HgCl2. FCM analysis of mercury-treated LEW.1N revealed that RT6.1+ T lymphocytes were significantly decreased in both spleen and lymph nodes of these animals. Experimental depletion of RT6+ T cells (by monoclonal antibody treatment or γ irradiation) in LEW.1N and BN.1L rats did not favor the induction of renal autoimmunity after exposure to mercury. On the other hand, BN → LEW.1N chimeras (obtained by adoptive transfer of BN lymphocytes into γ-irradiated LEW.1N rats) experienced autoimmune responses to kidney antigens when treated with HgCl2. They had autoantibodies to laminin and linear binding of immunoglobulins in their kidneys as well as a decreased percentage of RT6.2+ T lymphocytes in cervical lymph nodes. Therefore, the different components of this experimental model can now be dissected using various types of BN → LEW.1N chimeras, obtained by the adoptive transfer of purified T cell subsets.

Structural determination and characterization of a 40 kDa protein isolated from rat 40 S ribosomal subunit

We have purified a 40 kDa protein from the rat 40 S ribosomal subunit and determined its primary structure by amino acid and cDNA sequencing. The amino acid sequence of the 40 kDa protein shared 29–37% homology with prokaryotic ribosomal protein S2 of eubacteria and chloroplasts, indicating that the protein is a eukaryotic counterpart to prokaryotic S2. Moreover, the amino acid sequence shared 99% identity with those deduced from cDNAs for 68 kDa laminin binding proteins of human, murine and bovine origins. The cDNAs are capable of encoding polypeptides with predicted molecular mass of 33,000 which lacked typical signal sequences, N-linked glycosylation sites and putative transmembrane domains. These results indicate that the eDNAs for 68 kDa laminin binding proteins actually code for the 40 kDa ribosomal protein.

Expression of functional very late Antigen- α1, - α2, - α3 and - α6 integrins on Ewing's sarcoma and primitive peripheral neuroectodermal tumour cells and modulation by interferon-γ and tumour necrosis factor-α

Twelve different human primary and metastatic Ewing's sarcoma (ES) and primitive peripheral neuroectodennal tumour (pPNET) cell lines were examined by fluorocytometric analysis for the expression of α 1, α 2, α 3 and α 6 very late antigen (VLA) β 1-integrins. VLA- α 1, was abundantly expressed on all typical ES cell lines and pPNET cell lines, while absent from atypical (large cell) ES cells. VLA- α 2 was displayed on some ES and pPNET cell lines. In two different pPNET cell lines, derived from the same patient, VLA- α 2 expression was considerably higher on primary cells compared with metastatic cells. VLA- α 3 was exclusively expressed on pPNET cell lines. Expression of VLA- α 6 was higher on metastatic than on primary ES and pPNET cells. Adhesion assays on purified extracellular matrix (ECM) proteins, using monospecific adhesion-blocking antibodies, disclosed VLA-1 ( α 1 β 1) on typical ES cells and pPNET cells, and VLA-2 ( α 2 β 1) on atypical ES cells, as dual collagen type IV (COIV)/laminin (LM) binding sites, and VLA-6 ( α 6 β 1) as a specific LM binding site. Treatment of typical ES cells and pPNET cells for up to 48 h with recombinant human interferon-γ (rhIFNγ) and tumour necrosis factor-α (rhTNFα) upregulated α 1 and β 1 expression, concomitant with an increase in cell adhesion to COIV and LM. Alternatively, these cytobines downregulated the expression of α 2, α 6 and β 1 on atypical ES cells, concomitant with a decrease in the adhesion to COIV and LM. In conclusion, these findings suggest that the difference in repertory of CO and LM integrin receptors on ES cells and pPNET cells reflects tumour status and degree of differentiation. Furthermore, our data indicate that IFNγ- and TNFα-mediated alteration in the level of expression of distinct VLAs on ES and pPNET cells is correlated with changes in the adhesive behaviour of these tumour cells.

Nature and the multiple functions of the 67-kD elastin-/laminin binding protein.

Numerous cell types, including fibroblasts, vascular smooth muscle cells, chondroblasts, monocytes, neutrophils, and several tumor cells express the 67-kD galactolectin, homologous to the alternatively spliced variant of beta-galactosidase. The 67-kD protein resides on the cell surfaces and is capable of interacting with elastin, laminin and collagen type IV. This peripheral membrane protein binds its matrix ligands but only in the absence of galactosugars, whereas binding of galactosugar-containing moieties to its lectin site changes its molecular folding which causes discharge of the ligand and release of the receptor from the cell surface. This review will address the functional significance of the single receptor that interacts with multiple matrix proteins and can be shed from cell surfaces by galactosugars. I will emphasize the role of the 67-kD protein in divergent cellular processes, such as cell-matrix attachment, matrix assembly, cellular chemotaxis, and active migration through the vascular walls.

Morphological Change and Destabilization of β-Actin mRNA by Tumor Necrosis Factor in Human Microvascular Endothelial Cells

Tumor necrosis factor-α (TNF-α) produced morphological changes from a cobblestone-like shape into a spindle shape in human omental microvascular endothelial (HOME) cells and also a drastic rearrangement of actin filaments. Expression of β-actin gene was diminished in HOME cells treated with TNF-α for 24 h. Northern blot analysis of the β-actin gene demonstrated that the cellular level of β-actin mRNA was decreased at 6-12 h after exposure to TNF-α. However, there appeared to be no changes in cellular mRNA levels of β-tubulin, fibronectin, laminin B1, laminin B2, and laminin binding protein genes after treatment with TNF-α. Nuclear run-on assays showed increased transcription of the low-density lipoprotein receptor gene, but not of the β-actin gene. These data suggested that the TNF-α-induced inhibition of β-actin gene expression was not due to altered transcription activity. The degradation rates of β-actin, plasminogen activator inhibitor-1, and epidermal growth factor receptor mRNAs were examined in the presence of actinomycin D. β-Actin mRNA was found to be specifically destabilized in TNF-αtreated HOME cells, while other mRNA species were not. Coadministration of cycloheximide blocked the TNF-α-induced degradation of β-actin mRNA. The TNF-α-induced destabilization of β-actin mRNA and rearrangement of actin filaments are discussed in relation to the morphological changes in human microvascular endothelial cells.