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PubMed1998

The involvement of the fibronectin type II-like modules of human gelatinase A in cell surface localization and activation.

Recombinant collagen-binding domain (rCBD) comprising the three fibronectin type II-like modules of human gelatinase A was found to compete the zymogen form of this matrix metalloproteinase from the cell surface of normal human fibroblasts in culture. Upon concanavalin A treatment of cells, the induced cellular activation of gelatinase A was markedly elevated in the presence of the rCBD. Therefore, the mechanistic aspects of gelatinase A binding to cells by this domain were further studied using cell attachment assays. Fibroblasts attached to rCBD-coated microplate wells in a manner that was inhibited by soluble rCBD, blocking antibodies to the beta1-integrin subunit but not the alpha2-integrin subunit, and bacterial collagenase treatment. Addition of soluble collagen rescued the attachment of collagenase-treated cells to the rCBD. As a probe on ligand blots of octyl-beta-D-thioglucopyranoside-solubilized cell membrane extracts, the rCBD bound 140- and 160-kDa protein bands. Their identities were likely procollagen chains being both bacterial collagenase-sensitive and also converted upon pepsin digestion to 112- and 126-kDa bands that co-migrated with collagen alpha1(I) and alpha2(I) chains. A rCBD mutant protein (Lys263 --> Ala) with reduced collagen affinity showed less cell attachment, whereas a heparin-binding deficient mutant (Lys357 --> Ala), heparinase treatment, or heparin addition did not alter attachment. Thus, a cell-binding mechanism for gelatinase A is revealed that does not involve the hemopexin COOH domain. Instead, an attachment complex comprising gelatinase A-native type I collagen-beta1-integrin forms as a result of interactions involving the collagen-binding domain of the enzyme. Moreover, this distinct pool of cell collagen-bound proenzyme appears recalcitrant to cellular activation.

Antibodies↗
PubMed1998

Laminin alpha1 chain G domain peptide, RKRLQVQLSIRT, inhibits epithelial branching morphogenesis of cultured embryonic mouse submandibular gland.

Active sequences from the laminin alpha1 and alpha2 chain carboxyl-terminal globular domains (G domain) have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al. [1995] J. Biol. Chem. 270:20583-20590; Nomizu et al. [1996] FEBS Lett. 396:37-42). We have tested the activity of these peptides in submandibular gland explants of embryonic day 13 mice to determine the functional sites involved in organ development. The laminin alpha1 chain peptide, RKRLQVQLSIRT (residues 2719-2730 and designated AG-73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin alpha1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG-73, a homologue of AG-73 from the laminin alpha2 chain, did not inhibit the branching. The alpha2 chain peptide had no effect, which may be due to the low levels of this laminin chain in day 13 mice. Laminin alpha2 chain-specific monoclonal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelium. The expression of E-cadherin and alpha6 integrin, as detected by immunofluorescence, were unchanged in both AG-73 and control scramble peptide-treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG-73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG-73 sequence of the laminin alpha1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG-73-treated explants may indicate an important role for this region in basement membrane assembly.

Animals↗
PubMed1997

Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell spreading.

Cell adhesion to amino acids 2179-2198 (SN-peptide) of the laminin-1 alpha1-chain is required for lung alveolar formation in vitro (M. L. Matter and G. W. Laurie. J. Cell Biol. 124: 1083-1090, 1994). The nature of the SN-peptide receptor(s) was probed with neutralizing anti-integrin monoclonal antibodies (MAb), cells lacking integrin subunits, soluble heparin, and SN-peptide columns. Cell adhesion and spreading studies confirmed the specificity of SN-peptide and revealed adhesion to be unaffected by inclusion of anti-beta1-, anti-alpha(2-6)- or anti-alpha(V)beta5-integrin MAb. Cells lacking beta1- or alpha6-integrin subunits were fully adherent. Adhesion was heparin, but not chondroitin sulfate or heparinase, sensitive, much as is alpha-dystroglycan-laminin-1 binding. Heparin eluted approximately 155- and 180-kDa cell-surface proteins from SN-peptide columns. An additional approximately 91-kDa protein was eluted by EDTA. All were unrecognized by anti-beta1-integrin MAb. SN-peptide therefore interacts with three cell-surface proteins for which the identity remains to be determined.

Amino Acid Sequence↗
PubMed2003

Processing of alpha4 integrin by the proprotein convertases: histidine at position P6 regulates cleavage.

The proprotein convertases (PCs) participate in the limited proteolysis of integrin alpha4 subunit at the H(592)VISKR(597) downward arrow ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and downward arrow indicates the cleavage site), since this cleavage is inhibited by the serpin alpha1-PDX (alpha1-antitrypsin Portland). Co-expression of alpha4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-alpha4 convertases. In agreement, processing of endogenous pro-alpha4 in human lymphoblastoid CEM-T4 cells was enhanced greatly in stable transfectants overexpressing either enzyme. In many leucocyte cell lines, the expression of furin closely correlated with the endogenous processing efficacy, suggesting that furin is a candidate pro-alpha4 convertase. Mutational analysis showed that replacement of P1 Arg(597) with alanine (R597A) abrogated cleavage, whereas the P6 mutant H592R is even better processed by the endogenous convertases of Chinese-hamster ovary CHO-K1 cells. In vitro kinetic studies using synthetic peptides confirmed the importance of a positively charged residue at P6 and showed that wild-type alpha4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-alpha4 and its H592R and H592K mutants in the presence or absence of the weak base, NH(4)Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-alpha4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg downward arrow, our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4.

Amino Acids↗
PubMed2002

Differential gene expression in human abdominal aorta: aneurysmal versus occlusive disease.

OBJECTIVE: Inflammation and atherosclerosis are present in both abdominal aortic aneurysm (AAA) and arterial occlusive disease (AOD). Changes in gene expression that underlie the development of AAA versus AOD are poorly defined. This study evaluated differences in gene expression in AAA, AOD, and control aortic tissue with human gene array technology. METHODS: RNA was isolated from human aortic specimens (seven AAA, five AOD, and five control), and complementary DNA (cDNA) probes were generated. The cDNA probes were hybridized to a human cell interaction array of 265 genes and quantitated with phosphorimaging. The data were corrected for background and were standardized to housekeeping genes. Statistical differences in individual gene expression were determined with the Kruskal-Wallis test. RESULTS: Of 265 genes studied, 11 showed statistically different expression in diseased aorta as compared with control. The following three genes were downregulated in AAA: collagen VI alpha1 (P <.037), glycoprotein IIIA (P <.006), and alpha2-macroglobulin (P <.020). The following two genes were upregulated in AOD: laminin alpha4 (P <.034) and insulin-like growth factor 2 receptor (P <.049). The following three genes were upregulated in both AAA and AOD: matrix metalloproteinase-9 (MMP-9; P <.005), intercellular adhesion molecule-1 (P <.012), and tumor necrosis factor--beta receptor (P <.022). The following three genes were downregulated in both AAA and AOD: integrin alpha5 (P <.012), ephrin A5 (P <.037), and rho/rac guanine nucleotide exchange factor (P <.028). Of 16 MMPs evaluated, only MMP-9 was significantly (P <.005) upregulated in both AAA and AOD. Evaluation results of four tissue inhibitors of metalloproteinases showed no significant difference in expression for all tissue types, although tissue inhibitor of metalloproteinase-1 trended toward upregulation in AAA (P =.053). Eight of the fifteen most highly expressed genes in all the groups were extracellular matrix or secreted proteins. Of these, only collagen VI alpha1 (P <.037) showed a significant change, although biglycan trended toward downregulation in AAA (P =.076). CONCLUSION: This study used cDNA array technology in the comparison of human control and pathologic aortic tissue. Six genes had similar differential expression in both AAA and AOD as compared with control. Even more interesting were differences between AAA and AOD in the expression of five genes. These data suggest a similarity in genetic expression for both AAA and AOD, with altered expression of several genes playing a role in disease differentiation.

Adult↗
PubMed2001

Expression of integrins in human cultured mesothelial cells: the roles in cell-to-extracellular matrix adhesion and inhibition by RGD-containing peptide.

Integrins play key roles in cell-to-cell and cell-to-extracellular matrix (ECM) adhesion. We investigated integrin expression on pleural mesothelial cells (PMCs) and the inhibitory effect of arginine-glycine-asparate (RGD)-containing peptide on the adhesion of PMCs to fibronectin and collagen. Using flow cytometry and immunostaining, PMCs freshly isolated from pleural effusions and one mesothelial cell line were screened for different integrins. Intact pleural tissue was evaluated by immunohistochemistry. The adhesion of Met-5A cells to fibronectin and collagen types I, III and IV was assayed with prior treatment of various concentrations of glycine-arginine-glycine aspartate-serine (GRGDS). On primary PMCs, alpha2, alpha3, alpha5, beta1, beta3 and alphavbeta3 were highly expressed (>70%); alpha1 expression was intermediate (30-70%); and alpha4 and alpha6 expressions were low (< 30%). On Met-SA cells, alpha3, alpha5, alpha6 and beta1 were highly expressed (>70%); alpha1 was intermediate (30-70%); and alpha2, alpha4, beta3 and alphavbeta3 were low (<30%). The patterns of immunostaining on pleural tissues were similar to the results of flow cytometry for primary PMCs except for beta3. There was no statistically different expression in various disease states (transudate vs. exudate, benign vs. malignant). The inhibitory effect of GRGDS peptide on Met-5A cell adhesion to all four matrix proteins was dose-dependent.

Adolescent↗
PubMed2000

Laminin alpha4 and integrin alpha6 are upregulated in regenerating dy/dy skeletal muscle: comparative expression of laminin and integrin isoforms in muscles regenerating after crush injury.

The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin alpha2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and "preactivated" myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin alpha1 was not detectable in skeletal muscle. Laminin alpha2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin alpha2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin alpha chains in early myogenic differentiation, such as laminin alpha4 and alpha5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin beta1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin alpha3 was not expressed in uninjured or regenerating muscle, while integrin alpha6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin alpha6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin alpha6 occurred on some newly formed myotubes. Integrin alpha7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin alpha7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin alpha7 negative. No marked difference was observed in integrin alpha7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin alpha4 and integrin alpha6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin alpha4 and integrin alpha6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin alpha2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.

Animals↗
PubMed2004

The binding sites for competitive antagonistic, allosteric antagonistic, and agonistic antibodies to the I domain of integrin LFA-1.

We explore the binding sites for mAbs to the alpha I domain of the integrin alphaLbeta2 that can competitively inhibit, allosterically inhibit, or activate binding to the ligand ICAM-1. Ten mAbs, some of them clinically important, were mapped to species-specific residues. The results are interpreted with independent structures of the alphaL I domain determined in seven different crystal lattices and in solution, and which are present in three conformational states that differ in affinity for ligand. Six mAbs bind to adjacent regions of the beta1-alpha1 and alpha3-alpha4 loops, which show only small (mean, 0.8 angstroms; maximum, 1.8 angstroms) displacements among the eight I domain structures. Proximity to the ligand binding site and to noncontacting portions of the ICAM-1 molecule explains competitive inhibition by these mAbs. Three mAbs bind to a segment of seven residues in the beta5-alpha6 loop and alpha6 helix, in similar proximity to the ligand binding site, but on the side opposite from the beta1-alpha1/alpha3-alpha4 epitopes, and far from noncontacting portions of ICAM-1. These residues show large displacements among the eight structures in response to lattice contacts (mean, 3.6 angstroms; maximum, 9.4 angstroms), and movement of a buried Phe in the beta5-alpha6 loop is partially correlated with affinity change at the ligand binding site. Together with a lack of proximity to noncontacting portions of ICAM-1, these observations explain variation among this group of mAbs, which can either act as competitive or allosteric antagonists. One agonistic mAb binds distant from the ligand binding site of the I domain, to residues that show little movement (mean, 0.5 angstroms; maximum, 1.0 angstroms). Agonism by this mAb is thus likely to result from altering the orientation of the I domain with respect to other domains within an intact integrin alphaLbeta2 heterodimer.

Allosteric Site↗
PubMed2000

The collagen-binding A-domains of integrins alpha(1)beta(1) and alpha(2)beta(1) recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens.

We have previously assigned an integrin alpha(2)beta(1)-recognition site in collagen I to the sequence, GFOGERGVEGPOGPA (O = Hyp), corresponding to residues 502-516 of the alpha(1)(I) chain and located in the fragment alpha(1)(I)CB3 (Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Messent, A. J., Smethurst, P. A., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (1998) J. Biol. Chem. 273, 33287-33294). In this study, we show that recognition is entirely contained within the six-residue sequence GFOGER. This sequence, when in triple-helical conformation, readily supports alpha(2)beta(1)-dependent cell adhesion and exhibits divalent cation-dependent binding of isolated alpha(2)beta(1) and recombinant alpha(2) A-domain, being at least as active as the parent collagen. Replacement of E by D causes loss of recognition. The same sequence binds integrin alpha(1) A-domain and supports integrin alpha(1)beta(1)-mediated cell adhesion. Triple-helical GFOGER completely inhibits alpha(2) A-domain binding to collagens I and IV and alpha(2)beta(1)-dependent adhesion of platelets and HT 1080 cells to these collagens. It also fully inhibits alpha(1) A-domain binding to collagen I and strongly inhibits alpha(1)beta(1)-mediated adhesion of Rugli cells to this collagen but has little effect on either alpha1 A-domain binding or adhesion of Rugli cells to collagen IV. We conclude that the sequence GFOGER represents a high-affinity binding site in collagens I and IV for alpha(2)beta(1) and in collagen I for alpha(1)beta(1). Other high-affinity sites in collagen IV mediate its recognition of alpha(1)beta(1).

Amino Acid Sequence↗
PubMed2002

Lymphocytes expressing alpha1beta1 integrin (very late antigen-1) in peripheral blood of patients with arthritis are a subset of CD45RO(+) T-cells primed for rapid adhesion to collagen IV.

We report that very late antigen-1 (VLA-1(+)) CD3(+)CD45RO(+) T-cells are selectively segregated from VLA-1(-) peripheral blood (PB) mononuclear cells (MC), in which CD3(+) T-cells are evenly CD45RO(+) and CD45RO(-), when PBMC are stained with a monoclonal antibody (mAb) to VLA-1 and passaged on immunomagnetic columns. In contrast, both VLA-1(+) and VLA-1(-) MC isolated from synovial fluid (SF) are mainly CD45RO(+)CD3(+) T-cells. VLA-1(+) MC formed 13 +/- 5.3% of MC eluting from columns loaded with PBMC of patients with seropositive rheumatoid arthritis (n = 6) and 2.3 +/- 1.6% of patients (n = 4) with other arthritides (P < 0.022). Importantly, only the VLA-1(+) MC from PB and SF adhered to collagen IV upon triggering with phorbol 12-myristate 13-acetate. Moreover, adhesion and migration on collagen IV were preferentially maintained in lines cultured from VLA-1(+) T-cells, and both were inhibited by mAb to the VLA-1 alpha1 I domain. These results suggest that VLA-1(+) CD45RO(+) T-cells in patients with arthritis could play a role in both systemic and local inflammation by rapidly adhering to collagen IV.

Antibodies, Monoclonal↗
PubMed1999

Expression of laminins and their integrin receptors in different conditions of synovial membrane and synovial membrane-like interface tissue.

OBJECTIVE: To demonstrate the expression of laminins (Lns) and their integrin (Int) receptors in different synovial samples and synovial membrane-like interface tissues from well fixed and aseptically loosened total hip replacement (THR), and the potential role of Ln-Int interaction in the production of collagenases and cytokines. METHODS: Immunohistochemical staining was done to detect the distribution of EHS Ln, Ln alpha2, alpha3, alpha5, beta1, beta2 chains and Int alpha1, alpha2, alpha3, alpha6, beta1, beta4 subunits in different samples. Double immunofluorescence labelling was used to find colocalisation of Int alpha6 subunit and collagenase-1/collagenase-3/TNFalpha/IL6. RESULTS: General Ln immunoreactivity was detected in all specimens. Ln alpha5, beta1 and beta2, but not alpha2 and alpha3 chains were seen in the synovial lining and the basement membrane of blood vessels with the intensity/extent of labelling in the following rank order: rheumatoid arthritis (RA) loosened prostheses, osteoarthritis, well fixed prostheses, traumatic knees. Among Int subunits, staining for beta1 was usually the strongest, followed by staining for Int alpha6, alpha1, alpha3, and alpha2 subunits, with the same rank order for overall expression of Lns. Int beta4 subunit was not detectable in most of the specimens. Double labelling focused on Int alpha6 subunit disclosed its frequent colocalisation with collagenases 1 and 3 and with tumour necrosis factor alpha and interleukin 6 in synovial lining. CONCLUSION: Synovial lining contains Ln-10, Ln-11, and Int alpha6beta1 and alpha1beta1 receptors. In aseptic loosening of THR, interface tissue has a similar Ln subtype and Int receptor composition as RA synovium, which confirms its "lining-like" phenotype. Synovial lining does not contain Ln-5 (alpha3beta3gamma2) or Int alpha6beta4, which are components of epithelial hemidesmosomes. The expression of Lns and their Int receptors is upregulated in inflammation. The close spatial relation between Ln and its Int receptors in synovial lining cells containing proteinases and cytokines suggests a potential role in joint destruction and prosthetic loosening.

Adult↗
PubMed1999

Mutations of the type A domain of complement factor B that promote high-affinity C3b-binding.

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes "gain of function" mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the betaA-alpha1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G substitution, resulted in increased affinity for C3b and iC3b, a C3b derivative. These findings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding.

Amino Acid Sequence↗
PubMed2002

IgG plasma cells display a unique spectrum of leukocyte adhesion and homing molecules.

Long-lived antibody-secreting plasma cells are formed in the secondary lymphoid organs and subsequently home to the bone marrow, although the mechanisms that control this migration remain primarily unknown. In this study, we show that IgG plasma cells constitute a significant fraction of cervical lymph node cells from older mice deficient in both E- and P-selectin (E/P(-/-)), and that these cells can be prospectively isolated by phenotype. These IgG plasma cells were polyclonal, cytoplasmic Ig(+), spontaneously secreted antibody, were in the G(0)/G(1) phase of the cell cycle, and failed to express multiple B-cell surface markers. The plasma cells exhibited up-regulated cell surface expression of multiple adhesion molecules, including alpha(4) and leukocyte function-associated antigen 1 (LFA-1) integrins, CD44, and P-selectin glycoprotein ligand 1 (PSGL-1). IgG plasma cells bound to vascular cell adhesion molecule 1 (VCAM-1) significantly better than IgM(+) B cells, indicating that the alpha(4) integrins were constitutively active. A subset of IgG plasma cells also bound hyaluronic acid, the ligand for CD44. In addition, the IgG plasma cells interacted strongly with E-selectin, but poorly with P-selectin, despite elevated levels of PSGL-1 protein. The preferential interaction of plasma cells with E-selectin, but not P-selectin, correlated with elevated alpha1,3-fucosyltransferase-VII messenger RNA levels, but selective down-regulation of core 2 beta1-6-N-glucosaminyltransferase levels, compared to B cells. These results demonstrate a unique adhesion profile for murine IgG plasma cells. Furthermore, the E/P(-/-) mice represent a novel system to isolate and purify significant numbers of primary IgG plasma cells.

Animals↗
PubMed2004

Oncothanin, a peptide from the alpha3 chain of type IV collagen, modifies endothelial cell function and inhibits angiogenesis.

Previous studies from our group and the group of the Department of Biochemistry at the University of Rheims, France [corrected] have shown that basement membrane (BM) collagen from anterior lens capsule type IV collagen (ALC-COL IV) and peptides from the noncollagenous domain (NC1) of the alpha3(IV) [corrected] chain, corresponding to residues 185-203 and 179-208, inhibit tumor cell proliferation, specifically through the interaction of the -SNS- tripeptide (residues 189-191) with the CD47/alphavbeta3 integrin receptor complex. Data presented here demonstrate that the alpha3(IV)185-203 and the alpha3(IV)179-208 peptides, from here forward [corrected] designated as oncothanin, regulate endothelial cell (EC) proliferation, adhesion, and motility which [corrected] ultimately influence angiogenesis. The data also indicate that oncothanin, when used as a chemoattractant, greatly enhanced EC chemotaxis. In contrast, pretreatment of EC with oncothanin inhibited chemotaxis toward several different chemoattractants. When oncothanin was used as a substrate, it enhanced EC adhesion that was inhibited when pretreated with same. Analysis of angiogenesis by EC differentiation (tube formation), aortic ring microvessel formation [corrected] and the chorioallantoic membrane assay, [corrected] demonstrate that oncothanin, but not the control medium or peptides, inhibits angiogenesis. In the EC differentiation assay, oncothanin completely inhibited tube formation at 25 microg/ml, whereas peptides with comparable sequences, that lacked [corrected] the -SNS- sequence, from ALC-COL IV NC1 domains alpha1 and alpha2 chains failed to inhibit tube formation. The data support the hypothesis that ALC-COL IV and oncothanin inhibit angiogenesis by modulation of EC function.

Animals↗
PubMedDate not supplied

Non-natural CBP2 binding peptides and peptomers modulate carcinoma cell adhesion and invasion.

A combinatorial approach that utilized a repertoire of bacteriophage-peptides has identified a number of non-natural CBP2 binding peptides. Moreover, co-localization of some of these peptides with CBP2 in a number of tumor cell lines demonstrated that the peptides were directed to an intracellular location spatially coincident with the normal distribution of CBP2 [Sauk et al., 2000]. From among these sequences WHYPWFQNWAMA and LDSRYSLQAAMY were the most effective CBP2 binding peptides and best fulfilled the combinatorial motif containing deep hydrophobic pockets. When the hydropathic profiles of collagen alpha1(IV) and alpha2 (IV) were compared with these dodecapeptides, the hydropathic profiles of WHYPWFQNWAMA and LDSRYSLQAAMY closely matched those of alpha1(IV) 414-452 and alpha1(IV)531-543. These peptides were shown to be functional peptidomimics and possessed the ability to alter cell adhesion and invasion of human squamous cell carcinoma cell lines. Peptomers were formed of these non-natural peptides to explore the role that a repetitive peptide may have on cell adhesion. The enhanced cell adhesion observed with the peptomers required both CBP2 antibodies and integrin antibodies for inhibition. The enhanced adhesion observed even in the face of combined antibody inhibition was consistent with such complexes possessing correspondingly slower dissociation rates. Thus, suggesting that peptomers may function in a like manner to multimeric peptide MHC complexes (tetramers) binding more than one cell receptor on a specific cell. These findings evoke both peptidomimics of native ligands and their peptomers as potential reagents by which to target tumor cells for chemotherapy, imaging, or retargeting viral vectors for gene therapy.

Bacteriophages↗
PubMed1996

Expression of the human alpha2 integrin subunit in mouse melanoma cells confers the ability to undergo collagen-directed adhesion, migration and matrix reorganization.

Previous studies have shown that cultured human and other mammalian cell require the alpha2 beta1 integrin receptor to reorganize and contract 3-dimensional extracellular matrix lattices containing collagen type I. This function is of prime importance for the later phases of wound healing, in which fibroblasts reorganize and contract extracellular matrix components newly deposited in the granulation tissue. It is also known that highly aggressive human melanoma cells have acquired this function, possibly enhancing their invasive potential. To further study alpha2 beta1-mediated functions, we expressed the human alpha2 and beta1 chains of integrins in mouse melanoma cells (BULT). The parental cell line was unable to exert known alpha2 beta1-mediated functions: The cells did not adhere, spread, or migrate on collagen type I, and they did not reorganize 3-dimensional collagen I lattices. Transfection of the human alpha2 chain was sufficient to confer not only specific adhesion to collagen type I in static adhesion assays, but also the ability to exert complex functions such as migration on collagen and efficient reorganization of collagen-I-containing matrices. Coexpression of the human beta1 chain did not further enhance these functions in BULT melanoma cells. This was underscored by the observation that alpha1-specific monoclonal antibodies were able to completely block the newly introduced functions, whereas beta1-specific antibodies had no such effect. Moreover, in transfectants expressing both the human alpha2 and beta1 chains, the human alpha2 chain did not preferentially associate with the human beta1 chain as compared with mouse beta-chains. This may indicate that the molecular structures guiding the physical association of the human alpha2 chain with beta chains are extremely highly conserved between the species.

Animals↗
PubMed2003

Effects of beta-adrenergic agonists on bone-resorbing activity in human osteoclast-like cells.

In the present study, we demonstrate for the first time that beta-adrenergic agonists stimulate bone-resorbing activity in human osteoclast-like multinucleated cells (MNCs). Osteoclast-like MNCs constitutively expressed mRNA for alpha1B-, alpha2B- and beta2-adrenergic receptor (AR) in addition to characteristic markers of mature osteoclast, such as calcitonin receptor (CT-R), tartrate-resistant acid phosphatase (TRAP), alphaV-chain of integrin (Int alphaV), carbonic anhydrase II (CA-II) and cathepsin K (Cathe K). Epinephrine (1 microM; alpha,beta-adrenergic agonist) up-regulated expression of Int alphaV, CA-II and Cathe K in the osteoclast-like MNCs. Osteoclastic resorbing activity was markedly increased by isoprenaline (1 microM; beta-adrenergic agonist), moderately by epinephrine, but poorly by phenylephrine (1 microM; alpha1-adrenergic agonist). The actin ring, which was suggested to be correlated with bone-resorbing activity, was clearly observed in osteoclast-like MNCs treated with isoprenaline and epinephrine, but faintly in those treated with phenylephrine. These findings suggest that beta-adrenergic agonists directly stimulate bone-resorbing activity in matured osteoclasts.

Adrenergic beta-Agonists↗
PubMed1998

Laminin-1 and laminin-2 G-domain synthetic peptides bind syndecan-1 and are involved in acinar formation of a human submandibular gland cell line.

The culture of human submandibular gland (HSG) cells on laminin-1 induces acinar differentiation. We identified a site on laminin involved in acinar differentiation using synthetic peptides derived from the C-terminal G-domain of the laminin alpha1 and alpha2 chains. The alpha1 chain peptide AG73 (RKRLQVQLSIRT) decreases the size of acini formed on laminin-1. Cells cultured with either AG73 or the homologous alpha2 chain peptide MG73 (KNRLTIELEVRT) form structures that appear acinar-like, but the cell nuclei are not polarized to the basal surface and no lumen formation occurs, indicating that additional sites on laminin are required for complete differentiation. The G-domain of laminin-1 contains both integrin and heparin binding sites, and anti-beta1-integrin antibodies disrupt acinar formation. Cell adhesion to the peptides and to E3, an elastase digest fragment of laminin-1 containing AG73, is specific, since other laminin peptides or EDTA do not compete the binding. Heparin and heparan sulfate decrease cell adhesion to AG73 and MG73 but anti-beta1-integrin antibodies have no effect. Treating the cell surface with heparitinase inhibits adhesion to both AG73 and MG73. We isolated cell surface ligands using both peptide affinity chromatography and laminin-1 affinity chromatography. Treating the material bound to the affinity columns with heparitinase and chondroitinase enriches for a core protein identified as syndecan-1 by Western blot analysis, thus identifying a syndecan-1 binding site in the globular domain of laminin-1 and laminin-2. In summary, multiple interactions between laminin and HSG cells contribute to acinar differentiation, involving both beta1-integrins and syndecan-1.

Amino Acid Sequence↗