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PubMed2003

Retraction of the Drosophila germ band requires cell-matrix interaction.

Integrins and laminins are important mediators of cell-matrix interactions in both vertebrates and invertebrates. Here, we show that germ-band retraction in the Drosophila embryo, during which the tail end of the embryo retracts to its final posterior position, allows the investigation of cell spreading and lamellipodia formation in real time in vivo. We demonstrate that alpha1, 2 laminin and alphaPS3betaPS integrin are required for the spreading of a small group of cells of the amnioserosa epithelium over the tail end of the germ band. We further implicate a role for this spreading in the process of germ-band retraction.

Animals↗
PubMed1997

The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix.

We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti-MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the alpha2 integrin subunit but not by antibodies against the alpha1 or alpha3 subunits. We propose that interaction of the alpha2beta1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.

Animals↗
PubMed2001

Requirement for tyrosine kinase-ERK1/2 signaling in alpha 1 beta 1 integrin-mediated collagen matrix remodeling by rat mesangial cells.

Abnormal mesangial extracellular matrix remodeling by mesangial cells (MCs) is the hallmark of progressive glomerulonephritis (GN). We recently showed, using a type I collagen gel contraction assay, that alpha 1 beta 1 integrin-dependent MC adhesion and migration are necessary cell behaviors for collagen matrix remodeling. To further determine the mechanism of alpha 1 beta 1 integrin-mediated collagen remodeling, we studied the signaling pathways of MCs that participate in the regulation of collagen gel contraction. Immunoprecipitation and phosphotyrosine detection revealed that gel contraction is associated with the enhanced activity and phosphorylation of ERK1/2 by MCs. The tyrosine kinase inhibitors herbimycin and genistein inhibited collagen gel contraction dose dependently. Furthermore, targeting ERK1/2 activity with a MEK inhibitor, PD98059, and antisense ERK1/2 hindered gel contraction in a dose-dependent manner. Similar inhibitory effects on gel contraction and ERK1/2 phosphorylation were observed when MC-mediated gel contraction was performed in the presence of function-blocking anti-alpha1 or anti-beta1 integrin antibodies. However, cell adhesion and migration assays indicated that PD98059 and antisense ERK1/2 blocked alpha 1 beta 1 integrin-dependent MC migration, but did not interfere with collagen adhesion, although there was a marked decrease in ERK1/2 phosphorylation and ERK1/2 protein expression in cell adhesion on type I collagen. None of the above could affect membrane expression of alpha 1 beta 1 integrin. These results suggested that ERK1/2 activation is critical for the alpha 1 beta 1 integrin-dependent MC migration necessary for collagen matrix reorganization. We therefore conclude that ERK1/2 may serve as a possible target for pharmacological inhibition of pathological collagen matrix formation in GN.

Animals↗
PubMed1998

Migration of breast epithelial cells on Laminin-5: differential role of integrins in normal and transformed cell types.

We examined the role of Laminin-5 (Ln-5) an extracellular matrix component of breast gland basement membrane, in supporting migration of normal (HUMEC), immortalized (MCF-10A), and malignant breast epithelial cells that exhibit different degrees of metastatic potential (MDA-MB-435>MDA-MB-231>MCF-7). HUMEC, MCF-10A, and MCF-7 cells all adhered to purified Ln-5 through the alpha3beta1 integrin receptor in adhesion assays. However, HUMEC and MCF-10A cells remained statically adherent, while MCF-7 cells migrated on Ln-5 in Transwell and colloidal gold displacement assays. Anti-alpha3 integrin antibodies blocked migration of MCF-7 cells on Ln-5. MDA-MB-231 and MDA-MB-435 cells bound and migrated on Ln-5 through a beta1 integrin receptor that is insensitive to antibodies that block the function of alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphaV integrin subunits. Migration of all cell types tested was blocked by CM6, a monoclonal antibody directed to a cell adhesion site on the alpha3 chain of Ln-5. Thus, Ln-5 may play an important role in regulating adhesion and migration in normal and transformed breast epithelium. Our results indicate that the type of integrin utilized by breast cells to interact with Ln-5, as well as its functional state, may determine whether cells will be statically adherent or migratory on Ln-5.

Animals↗
PubMed2004

PDGF-BB enhances alpha1beta1 integrin-mediated activation of the ERK/AP-1 pathway involved in collagen matrix remodeling by rat mesangial cells.

Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the pathogenesis of progressive glomerulonephritis (GN). Previous studies have reported that PDGF-BB stimulates mesangial cells (MCs)-induced collagen matrix remodeling through enhancement of alpha1beta1 integrin-dependent migratory activity. To determine the cell signaling pathway responsible for abnormal MC-related mesangial matrix remodeling in progressive GN, we studied the involvement of the extracellular signal-regulated kinase (ERK)/activator protein-1 (AP-1) pathway in PDGF-BB-enhanced collagen gel contraction. Western blotting and gel shift assay revealed that MC-induced gel contraction resulted in ERK activation in parallel with that of AP-1 binding, peaking at 4 h and lasting at least for 24 h. Application of the MEK inhibitor, U0126, and the c-jun/AP-1 inhibitor, curcumin, inhibited gel contraction and AP-1 activity, respectively, dose dependently. PDGF-BB enhanced not only gel contraction but ERK phosphorylation and AP-1 activity by MCs. Marked inhibitory effects on PDGF-BB-induced gel contraction and ERK/AP-1 activity were observed in the presence of either function blocking anti-alpha1- or anti-beta1-integrin antibody or U0126. Consistently, AP-1-inactive MCs expressing a dominant-negative mutant of c-jun showed a significant decrease of PDGF-BB-induced gel contraction as compared with mock-transfected MCs. Finally, migration assay showed that ERK/AP-1 activity is required for PDGF-BB-stimulated alpha1beta1 integrin-dependent MC migration to collagen I. These results indicated that PDGF-BB enhances alpha1beta1 integrin-mediated collagen matrix reorganization through the activation of the ERK/AP-1 pathway that is crucial for MC migration. We conclude that the ERK/AP-1 pathway plays an important role in PDGF-BB-induced alpha1beta1 integrin-dependent collagen matrix remodeling; therefore, the inhibition of its pathway may provide a novel approach to regulate abnormal collagen matrix remodeling in progressive GN.

Animals↗
PubMed1998

Effect of ligand conformation on melanoma cell alpha3beta1 integrin-mediated signal transduction events: implications for a collagen structural modulation mechanism of tumor cell invasion.

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the alpha3beta1 integrin to bind to models of the alpha1(IV)531-543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by alpha3beta1 integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125(FAK), consistent with prior studies correlating beta1 integrin subunit binding to collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3 integrin subunit monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of alpha1(IV)531-543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125(FAK). Induction of p125(FAK) Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125(FAK) phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, alpha1(IV)531-543, can induce alpha3beta1 integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several alpha3beta1 integrin-mediated signal transduction events. The effects of ligand conformation suggest that a "collagen structural modulation" mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.

Cell Adhesion↗
PubMed2003

Altered expression of integrins in RSV-transformed chick epiphyseal chondrocytes.

Chondrocytes have been shown to express both in vivo and in vitro a number of integrins of the beta1-, beta3- and beta5-subfamilies (Biorheology 37 (2000) 109). Normal and v-Src-transformed chick epiphyseal chondrocytes (CEC) display different adhesion properties. While normal CEC with time in culture tends to increase their adhesion to the substrate by organizing focal adhesions and actin stress fibers, v-Src-transformed chondrocytes display a refractile morphology and disorganization of actin cytoskeleton. We wondered whether the reduced adhesion and spreading of v-Src-transformed chondrocytes could be ascribed to changes in integrin expression and/or function. Integrin expression by normal CEC is studied and compared to v-Src-transformed chick chondrocytes, using monoclonal and polyclonal antibodies to integrins alpha- and beta-chains. We show the presence of alpha1-, alpha3-, alphav-, alpha6-, beta1- and beta3-chains on CEC, with very low levels of alpha2- and alpha5-chains. Alphav chain associates with multiple beta subunits in normal and transformed chondrocytes. With the exception of alpha1- and alpha2-chains, the levels of the integrin chains analyzed are higher in transformed chondrocytes as compared with normal chondrocytes. In spite of the increased levels of integrin expression, transformed chondrocytes exhibit loss of focal adhesion and actin stress fibers and low adhesion activity on several extracellular matrix constituents. These observations raise the possibility that, in addition to its effects on global pattern of integrin expression, v-Src can influence integrin function in chondrocytes.

Actin Cytoskeleton↗
PubMed1996

Adenomatous transformation of the human anterior pituitary is associated with alterations in integrin expression.

Integrins are heterodimeric transmembrane molecules that mediate cell-cell and cell-substratum adhesion. Because alterations in the adhesive properties of tumor cells influence tumor growth and progression, the distribution of different alpha and beta integrin subunits was studied in both the parenchyma and the connective tissue in 6 normal and 25 adenomatous human anterior pituitaries. All normal parenchymal cells expressed the alpha3beta1 and alpha6beta4 integrins. By contrast, in adenomatous parenchymal cells the expression of alpha3beta1 was down-regulated and that of alpha6beta4 abrogated. Neoexpression of alphavbeta3 Occurred in the parenchyma of a subset of adenomas. All normal connective tissue cells expressed the alpha1 and beta1 subunits, a third subunit (alpha5) being present in the normal endothelium. By comparison, all adenomatous stromal cells expressed many more integrin subunits (alpha1, alpha3, alpha5, alphav, beta1 and beta3), adenomatous endothelial cells bearing additional subunits (alpha6, beta4 and beta5). Vitronectin, absent from the normal connective tissue, was constantly observed in the adenomatous stroma. To conclude, compared with cells of the normal gland, adenomatous anterior pituitary cells display a decreased expression of integrins whereas the adenomatous stroma expresses a rich repertoire of integrins. These changes are not related to the secretory type, grade or invasiveness of the adenoma. The resulting alterations in the adhesive properties of adenomatous cells could facilitate their dissemination. Enrichment of the integrin repertoire expressed by the adenomatous vasculature is indicative of its dual nature, systemic and tumoral.

Adenoma↗
PubMed1997

Bone morphogenetic protein-2 is a regulator of cell adhesion.

Bone morphogenetic proteins (BMPs) are a group of peptide growth factors closely related to transforming growth factors-beta. The BMPs are suggested to play an essential role in bone development and they are strong candidate molecules to be used clinically to improve fracture healing. BMPs are also involved in the differentiation of several other tissues during embryogenesis. Here, we show that human recombinant BMP-2 regulates cell-matrix interactions by modifying the expression of integrin-type receptors. The synthesis of alpha3 integrin was down-regulated by BMP-2 in two osteogenic sarcoma-derived cell lines, Saos-2 and HOS, and also in human fetal chondrocytes. BMP-2 had no effect on the expression of alpha1, alpha2, alpha5, alpha6, and alphaV integrins. BMP-2 reduced the expression of alpha3 integrin subunit at mRNA level. Laminin-5 was shown to be the ligand for alpha3beta1 integrin on Saos cells and BMP-2 decreased the ability of Saos cells to attach to it. These results suggest that BMP-2 has a specific effect on the alpha3beta1 integrin-mediated cell adhesion to laminin-5. Given the fact that BMP-2 is expressed in osteosarcomas, in addition to in bone, this mechanism is putatively important especially in bone development and tumors. We also studied the effect of BMP-2 on a human keratinocyte cell line, HaCaT. In HaCaT cells, the expression of alpha2 integrin was strongly down-regulated by BMP-2, whereas its effect on the expression of alpha3 integrin was smaller. We suggest that the effects of BMP-2 may be partially mediated by specifically altered cell adhesion.

Antigens, CD↗
PubMed2004

Integrins in the mouse myotome: developmental changes and differences between the epaxial and hypaxial lineage.

Integrins are cellular adhesion receptors that mediate signaling and play key roles in the development of multicellular organisms. However, their role in the cellular events leading to myotome formation is completely unknown. Here, we describe the expression patterns of the alpha1, alpha4, alpha5, alpha6, and alpha7 integrin subunits in the mouse myotome and correlate them with the expression of several differentiation markers. Our results indicate that these integrin subunits may be differentially involved in the various phases of myogenic determination and differentiation. A detailed characterization of the myogenic cell types expressing the alpha4 and alpha6 subunits showed a regionalization of the myotome and dermomyotome based on cell-adhesion properties. We conclude that alpha6beta1 may be an early marker of epaxial myogenic progenitor cells. In contrast, alpha4beta1 is up-regulated in the intercalated myotome after myocyte differentiation. Furthermore, alpha4beta1 is expressed in the hypaxial dermomyotome and is maintained by early hypaxial myogenic progenitor cells colonizing the myotome.

Animals↗
PubMed2002

Matrix assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation.

Laminin-1 is essential for early embryonic basement membrane assembly and differentiation. Several steps can be distinguished, i.e., the expression of laminin and companion matrix components, their accumulation on the cell surface and assembly into basement membrane between endoderm and inner cell mass, and the ensuing differentiation of epiblast. In this study, we used differentiating embryoid bodies derived from mouse embryonic stem cells null for gamma1-laminin, beta1-integrin and alpha/beta-dystroglycan to dissect the contributions of laminin domains and interacting receptors to this process. We found that (a) laminin enables beta1-integrin-null embryoid bodies to assemble basement membrane and achieve epiblast with beta1-integrin enabling expression of the laminin alpha1 subunit; (b) basement membrane assembly and differentiation require laminin polymerization in conjunction with cell anchorage, the latter critically dependent upon a heparin-binding locus within LG module-4; (c) dystroglycan is not uniquely required for basement membrane assembly or initial differentiation; (d) dystroglycan and integrin cooperate to sustain survival of the epiblast and regulate laminin expression; and (e) laminin, acting via beta1-integrin through LG1-3 and requiring polymerization, can regulate dystroglycan expression.

Animals↗
PubMed2006

Deletion of core fucosylation on alpha3beta1 integrin down-regulates its functions.

The core fucosylation (alpha1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently alpha1,6-fucosylation has been further reported to be very crucial by the study of alpha1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on alpha3beta1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in alpha3beta1 integrin-mediated events has been studied by using Fut8(+/+) and Fut8(-/-) embryonic fibroblasts, respectively. We found that the core fucosylation of alpha3beta1 integrin, the major receptor for laminin 5, was abundant in Fut8(+/+) cells but was totally abolished in Fut8(-/-) cells, which was associated with the deficient migration mediated by alpha3beta1 integrin in Fut8(-/-) cells. Moreover integrin-mediated cell signaling was reduced in Fut8(-/-) cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of alpha3beta1 integrin.

Animals↗
PubMed2005

Differential expression of endometrial integrins and progesterone receptor during the window of implantation in normo-ovulatory women treated with clomiphene citrate.

OBJECTIVE: To assess the effect of clomiphene citrate (CC) on endometrial epithelial integrins and P receptors (PR) during the window of implantation. DESIGN: Controlled, prospective, clinical study. SETTING: Teaching hospital and university research laboratory. PATIENT(S): Thirty-one fertile, normo-ovulatory women participated in this trial. Thirteen women exhibited a CC-stimulated cycle with 50 mg on days 5-9, and 18 women with spontaneous menstrual cycles served as controls. INTERVENTION(S): Endometrial biopsies in the midluteal phase. MAIN OUTCOME MEASURE(S): Immunohistochemical determination and endometrial cellular localization of alpha1, alpha v, beta3, and alpha4 epithelial integrins and PR during the window of implantation. The staining intensity was assessed by a semiquantitative index (HSCORE) and compared by nonparametric Mann-Whitney test. RESULT(S): Higher plasma levels of P and E2 and delayed histologic dating of the endometrium (38%) were features of CC-treated women. In addition, a low epithelial beta3 integrin expression and persistent PR were observed in glandular epithelial cells of "out-of-phase" endometrial biopsies from CC-treated women. In contrast, in "in-phase" biopsies, neither epithelial PR nor beta3 integrin were different from spontaneous control cycles. There was no difference in the expression of alpha1, alpha v, and alpha4 between the groups studied. CONCLUSION(S): The administration of clomiphene produces aberrant endometrial beta3 integrin expression in conjunction with a failure in the down-regulation of PR during the window of implantation in a significant number of normo-ovulatory women, notwithstanding the higher plasma P levels. Therefore, CC might affect the expression of endometrial receptivity markers.

Adult↗
PubMed2002

Integrin activation involves a conformational change in the alpha 1 helix of the beta subunit A-domain.

The ligand-binding region of integrin beta subunits contains a von Willebrand factor type A-domain: an alpha/beta "Rossmann" fold containing a metal ion-dependent adhesion site (MIDAS) on its top face. Although there is evidence to suggest that the betaA-domain undergoes changes in tertiary structure during receptor activation, the identity of the secondary structure elements that change position is unknown. The mAb 12G10 recognizes a unique cation-regulated epitope on the beta(1) A-domain, induction of which parallels the activation state of the integrin (i.e. competency for ligand recognition). The ability of Mn(2+) and Mg(2+) to stimulate 12G10 binding is abrogated by mutation of the MIDAS motif, demonstrating that the MIDAS is a Mn(2+)/Mg(2+) binding site and that occupancy of this site induces conformational changes in the A-domain. The cation-regulated region of the 12G10 epitope maps to Arg(154)/Arg(155) in the alpha1 helix. Our results demonstrate that the alpha1 helix undergoes conformational alterations during integrin activation and suggest that Mn(2+) acts as a potent activator of beta(1) integrins because it can promote a shift in the position of this helix. The mechanism of beta subunit A-domain activation appears to be distinct from that of the A-domains found in some integrin alpha subunits.

Animals↗
PubMed2005

Molecules mediating cell-ECM and cell-cell communication in human heart valves.

The specific phenotype of different tissues depends on the interactions of cells with neighboring cells and the surrounding extracellular matrix, which is mediated by cell adhesion receptors including integrins, immunoglobulin family members, syndecans, and selectins. The aim of this study was to investigate the adhesion profile of native human valve interstitial cells (ICs) in situ and in vitro by analyzing these adhesion receptors. Flow cytometry and immunocytochemistry was used to quantify the expression of the specific receptors on ICs cultured from all human cardiac valves, and immunohistochemistry were used to profile their distribution pattern in valve tissue sections. The valve leaflets and cultured ICs from all valves expressed alpha1, alpha2, alpha3, alpha4, and alpha5 integrins to varying degrees and percentages with very little expression of alpha6 and alphaV. Valve leaflet ICs from all valves, expressed predominantly beta1 integrin but no beta3 or beta4 integrin. Syndecan-1 and Syndecan-4 were not detected. Intercellular adhesion molecule-1 was weakly detected, whereas vascular adhesion molecule-1 was barely detectable and E-selectin was not detected. This study has delineated the identity of some of the integrins synthesized and expressed by human valve ICs and the specificity of adhesion molecules with which the valve ICs interact with the extracellular matrix and mediate intercellular interactions. This pattern of expression of cell surface adhesion molecules may be considered as a basis for a fingerprint on which to base future cell alternatives and would provide useful information for valve tissue engineering.

Cell Adhesion↗
PubMed1998

Identification in collagen type I of an integrin alpha2 beta1-binding site containing an essential GER sequence.

The collagen type I-derived fragment alpha1(I)CB3 is known to recognize the platelet collagen receptor integrin alpha2beta1 as effectively as the parent collagen, although it lacks platelet-aggregatory activity. We have synthesized the fragment as seven overlapping peptides that spontaneously assemble into triple helices. On the basis of their capacity to bind purified alpha2 beta1 and the recombinant alpha2 A-domain, and their ability to support alpha2 beta1-mediated cell adhesion, we identified two peptides, CB3(I)-5 and -6, which contain an alpha2 beta1 recognition site. Synthesis of the peptide CB3(I)-5/6, containing the overlap sequence between peptides 5 and 6, allowed us to locate the binding site within the 15-residue sequence, GFP*GERGVEGPP*GPA (where P* represents hydroxyproline), corresponding to residues 502-516 of the collagen type I alpha1 chain. The Glu and Arg residues in the GER triplet were found to be essential for recognition since substitution of either residue with Ala caused a loss of alpha2 A-domain binding. By contrast, substitution of the Glu in GVE did not reduce binding, but rather enhanced it slightly. We were unable to detect significant recognition of alpha2 beta1 by the peptide CB3(I)-2 containing the putative alpha2 beta1 recognition sequence DGEA. Peptides CB3(I)-1 to -6, together with peptide CB3(I)-5/6, exhibited good platelet-aggregatory activity, in some cases better than collagen. However, peptide CB3(I)-7 was inactive, suggesting the presence of an inhibitory element that might account for the lack of aggregatory activity of the parent alpha1(I)CB3 fragment.

Amino Acid Sequence↗
PubMed1996

Integrin-mediated interactions between primary/T-sv40 immortalized human glomerular epithelial cells and type IV collagen.

The use of human glomerular epithelial cells (HGEC) in research has been severely restricted by several obstacles, which have been circumvented by the generation of T-SV40 immortalized human visceral glomerular epithelial cells (Delarue et al, 1991). In this work, we compared the primary and immortalized HGEC for expression of integrin and some nonintegrin surface receptors. We also studied the adhesion of both types of HGEC to glomerular basement membrane (GBM), type IV collagen (tIV), and its major noncollagenous NC1 domain. The integrins mediating adhesion of HGEC to tIV were also examined. Expression of integrin and some nonintegrin cell surface receptors was analyzed by flow cytometry. Adhesion to GBM, tIV, and its major noncollagenous NC1 domain was studied by direct solid phase cell adhesion assays. Identification of integrins mediating adhesion of HGEC to tIV was achieved by inhibition of cell adhesion using monoclonal antibodies to integrin subunits. The primary and immortalized HGEC share phenotypic characteristics, and alpha3beta1 appeared to be the major integrin present on both HGEC types. The kinetics of binding to GBM, tIV, and its noncollagenous NCI domain were similar in both the primary and immortalized HGEC, although the latter displayed a somewhat weaker binding. Both the primary and immortalized HGEC displayed significantly better adhesion to NC1-alpha3 compared with NC1-alpha1, alpha3beta1 appears to be the major integrin mediating the adhesion of HGEC to tIV. Our studies suggest that alpha3beta1 is the major integrin present on HGEC. This has been confirmed by flow cytometric analysis. In addition, we demonstrated a functional role for this integrin in mediating attachment of HGEC to tIV. Our data also demonstrate a preference in binding of HGEC to alpha3 chains of NC1 compared with alpha1 chains of NC1. These findings were seen in both the primary and immortalized HGEC. The T-SV40 immortalized HGEC can therefore serve as a very useful tool to study glomerular visceral cell biology.

Animals↗
PubMed2005

Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis.

Lebestatin, a new member of the lysine-threonine-serine (KTS)-disintegrin family, was purified to homogeneity from Tunisian snake (Macrovipera lebetina) venom. It is a single-chain polypeptide composed of 41 amino acids. The amino-acid sequence of lebestatin shows that it displays a pattern of cysteines similar to other short disintegrins, but contains the sequence KTS rather than RGD in its integrin-binding loop. Lebestatin presents a high homology with obtustatin and viperistatin. Lebestatin interacts specifically with the alpha1beta1 integrin. It was thus able to inhibit both adhesion and migration of PC12 and alpha1beta1 integrin-expressing CHO cells (CHO-alpha1) to type I and IV collagens. This disintegrin also affected adhesion and migration of endothelial cells and exhibited an anti-angiogenic effect in vivo when using the 8-day-old embryo chick chorioallantoic membrane model.

Allantois↗