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PubMedDate not supplied

Decreased expression of alpha3 and beta1 integrin subunits is responsible for differentiation-associated changes in cells behavior in terminally differentiated human oral keratinocytes.

Primary normal human oral keratinocytes (NHOKs) terminally differentiate in serial subculture. To investigate whether this subculture-induced differentiation of NHOKs affects integrin expression and cell-matrix interaction, we studied the expression levels of integrin subunits and cellular response to the extracellular matrix (ECM) proteins in NHOKs at different population doublings. The phosphorylation statuses of focal adhesion kinase (FAK), extracellular signal regulated kinase (ERK), p38, and c-Jun amino-terminal kinase (JNK) were also determined in NHOK cells cultured on ECM proteins, to evaluate the functions of integrins with respect to cellular responses to ECM proteins. The expression levels of alpha3 and beta1 integrin subunits progressively decreased in NHOKs undergoing terminal differentiation. The ability of NHOKs to spread upon laminin and type I collagen significantly decreased in terminally differentiated oral keratinocytes. Keratinocyte migration was significantly increased on type I collagen for terminally differentiated NHOKs. Similar results were seen following preincubation of rapidly proliferating NHOKs with function-blocking antibodies to alpha3 or beta1 integrin subunit. In contrast, fibronectin had no effect on cellular responses in NHOKs, which were almost negligible in the expression levels of alpha5 integrin subunits. The extent of FAK phosphorylation in terminally differentiated NHOKs was notably lower than that of rapidly proliferating cells, but was enhanced in terminally differentiated cells that were cultured on type I collagen. Our results indicate that decreased expression of alpha3 and beta1 integrin subunits is responsible for differentiation-associated changes in cells behavior in terminally differentiated oral keratinocytes. Our data also show that the abrogation of the alpha5beta1 integrin function caused by omitting alpha5 subunit is linked to the loss of a cell-fibronectin interaction in human oral keratinocytes.

Antibodies, Monoclonal↗
PubMed2006

Platelet-derived growth factor BB-mediated normalization of dermal interstitial fluid pressure after mast cell degranulation depends on beta3 but not beta1 integrins.

Interstitial fluid pressure (PIF) is one of the determinants of transcapillary fluid flux and thereby interstitial fluid volume. Cell-mediated control of PIF regulates fluid content in the loose interstitial connective tissues that surround the capillary bed. To maintain a normal PIF in dermis, beta1 integrins mediate the tensile strength applied by connective tissue cells on the extracellular matrix. Platelet-derived growth factor (PDGF)-BB normalizes anaphylaxis-induced reduction of PIF. Anti-beta3 integrin IgG and a cyclic RGD peptide that inhibits the alphaVbeta3 integrin blocked the ability of PDGF-BB to normalize the lowered PIF resulting from mast cell degranulation. PDGF-BB was unable to normalize PIF lowered as a result of mast cell degranulation in beta3-negative mice. Monoclonal anti-beta3 integrin IgG had no effect on PIF in normal mouse dermis. In contrast, administration of anti-beta1 integrin IgM lowered PIF in normal dermis but had no effect on PDGF-BB-induced normalization of PIF after anaphylaxis. Furthermore, collagen gel contraction mediated by wild-type mouse embryonal fibroblasts were only marginally affected by function-blocking anti-beta1 integrin antibodies, especially in the presence of PDGF-BB. In contrast, contraction mediated by alphaV-negative mouse embryonic fibroblasts was completely blocked by anti-beta1 integrin antibodies, even after stimulation with PDGF-BB. These results show a previously unrecognized in vivo function for the alphaVbeta3 integrin, as a participant in the control of PIF during inflammatory reactions. Furthermore, our data demonstrate that PDGF-BB induces connective tissue cells to generate tensile forces via alphaVbeta3 during such reactions.

Animals↗
PubMed2004

Conditional beta1-integrin gene deletion in neural crest cells causes severe developmental alterations of the peripheral nervous system.

Integrins are transmembrane receptors that are known to interact with the extracellular matrix and to be required for migration, proliferation, differentiation and apoptosis. We have generated mice with a neural crest cell-specific deletion of the beta1-integrin gene to analyse the role of beta1-integrins in neural crest cell migration and differentiation. This targeted mutation caused death within a month of birth. The loss of beta1-integrins from the embryo delayed the migration of Schwann cells along axons and induced multiple defects in spinal nerve arborisation and morphology. There was an almost complete absence of Schwann cells and sensory axon segregation and defective maturation in neuromuscular synaptogenesis. Thus, beta1-integrins are important for the control of embryonic and postnatal peripheral nervous system development.

Animals↗
PubMed1998

Perturbation of beta1-integrin function alters the development of murine mammary gland.

The expression of a transgene coding for a chimeric molecule, containing the cytoplasmic and transmembrane domains of the beta1-integrin chain and the extracellular domain of the T-cell differentiation antigen CD4, was targeted to the mouse mammary gland by the mouse mammary tumor virus (MMTV) promoter. The chimera does not interact with the extracellular ligands; however, its expression in cultured cells was shown to interfere with focal adhesion kinase (FAK) phosphorylation following ligation of endogenous beta1-integrin. Therefore, expression of the transgenic protein on the cell surface should uncouple adhesion from intracellular events associated with the beta1-cytoplasmic domain and thus perturb beta1-integrin functions. Although most of the transgenic females were able to lactate, their mammary glands had a phenotype clearly distinct from that of wild-type mice. At mid-pregnancy and the beginning of lactation, transgenic glands were underdeveloped and the epithelial cell proliferation rates were decreased, while the apoptosis levels were higher than in wild-type glands. In lactation, the amounts of the whey acidic protein (WAP) and beta-casein gene transcripts were diminished, and the basement membrane component, laminin and the beta4-integrin chain accumulated at the lateral surface of luminal epithelial cells, revealing defects in polarization. Our observations prove that in vivo, beta1-integrins are involved in control of proliferation, apoptosis, differentiation and maintenance of baso-apical polarity of mammary epithelial cells, and therefore are essential for normal mammary gland development and function.

Animals↗
PubMed2003

Beta1-integrin and IL-1alpha expression as bystander effect of medium from irradiated cells: the pilot study.

Bystander effects have been proposed as a third action pathway of ionising radiation besides direct and indirect effects. The purpose of the study was to investigate whether expression of interleukin-1alpha (IL-1alpha) and beta1-integrin is elevated in bystander cells as a marker for bystander effects in comparison with classical markers such as the clonogenic assay, apoptosis and the presence of micronuclei. The hybrid cell line E.A. hy.926 obtained by fusion of HUVEC cells with the epithelial cell line A 459 was irradiated with 0-5 Gy. Bystander effects were established via medium transfer at 45 min and 4 h after irradiation from irradiated to nonirradiated cell populations. In order to exclude effects of the irradiated medium itself, irradiated medium only was also used for transfer to nonirradiated cells. Then, cells were fixed at 1, 2, 6, and 24 h after irradiation or medium transport and IL-1alpha and beta1-integrin were detected and evaluated. A higher number of beta1-integrin-positive cells was observed in both irradiated and bystander cell populations than in the control group at 1 and 24 h after irradiation with 1 Gy or medium transfer. Significantly higher numbers of IL-1alpha-positive cells were found at 1, 2, and 6 h after irradiation with 1 Gy or medium transfer as well as at 2 and 6 h after irradiation with 5 Gy or medium transfer. Clonogenic survival decreased dependently on the dose in irradiated cells but did not show any significant difference between the bystander cell populations and sham-irradiated cells. The irradiated medium itself did not have any effect. It is concluded that beta1-integrin and IL-1alpha expression may serve as more sensitive markers of post-irradiation responses in bystander cell populations than the classical radiobiological markers. Moreover, overexpression of beta1-integrin and IL-1alpha may induce increased susceptibility to inflammation of bystander cells.

Apoptosis↗
PubMed1996

Regulation of beta1-integrin function in cultured human vascular smooth muscle cells.

Avidity modulation and function of beta1-integrin receptors in cultured human vascular smooth muscle cells (SMCs) were investigated using monoclonal antibody (mAb) 8A2, which binds to the beta1 subunit of integrin heterodimers and induces a high avidity state. The adhesion of SMCs to extracellular matrix proteins, but not to poly-L-lysine, was enhanced by pretreatment with mAb 8A2. A qualitative alteration of beta1 integrin was assessed with mAb 15/7, which binds to an activation-dependent epitope on the beta1 subunit. Binding of mAb 15/7 was enhanced by mAb 8A2 in a dose-dependent manner. Arg-Gly-Asp peptide and soluble fibronectin also enhanced expression of the 15/7 epitope, suggesting that the 15/7 epitope is closely related to the ligand-occupied state of beta1 integrin. Platelet-derived growth factor (PDGF)-AA and -BB increased SMC adhesion to type I collagen but did not augment mAb 15/7 binding, suggesting that PDGFs increase binding avidity by a postreceptor mechanism. In addition, mAb 8A2 inhibited PDGF-BB-induced SMC migration through Matrigel-coated filters. These results suggest that avidity modulation of beta1 integrin may play an important role in the function of SMCs.

Antibodies, Monoclonal↗
PubMed2006

Expression of the cytoplasmic domain of beta1 integrin induces apoptosis in adult rat ventricular myocytes (ARVM) via the involvement of caspase-8 and mitochondrial death pathway.

Stimulation of beta-adrenergic receptor (beta-AR) induces cardiac myocyte apoptosis. Integrins, a family of cell-surface receptors, play an important role in the regulation of cardiac myocyte apoptosis and ventricular remodeling. Cleavage of extracellular domain of beta1 integrin, also called integrin shedding, is observed during cardiac hypertrophy and progression to early heart failure. Here we show that stimulation of beta-AR induces beta1 integrin fragmentation in mouse heart. To examine the role of intracellular domain of beta1 integrin in cardiac myocyte apoptosis, a chimeric receptor consisting of the cytoplasmic tail domain of beta(1A) integrin and the extracellular/transmembrane domain of the interleukin-2 receptor (TAC-beta1) was expressed in adult rat ventricular myocytes (ARVM) using adenoviruses. TAC-beta1 increased the percentage of apoptotic ARVM as measured by TUNEL-staining assay. TAC-beta1-induced apoptosis was found to be associated with increased cytosolic cytochrome c and decreased mitochondrial membrane potential. TAC-beta1 increased caspase-8 activity. Z-IETD-FMK, a specific caspase-8 inhibitor, significantly inhibited TAC-beta1-induced apoptosis. TAC-beta1 expression also increased cleavage of Bid, a pro-apoptotic Bcl-2 family protein. These data suggest that shedding of beta1 integrin may be a mechanism of induction of apoptosis during beta-AR-stimulated cardiac remodeling.

Animals↗
PubMed1999

Beta1-integrins control spontaneous adhesion and motility of human progenitor thymocytes and regulate differentiation-dependent expression of the receptor for hyaluronan-mediated motility.

The functions of the receptor for hyaluronan-mediated motility (RHAMM) and beta1-integrin in adhesion and motility were analysed for human progenitor multinegative (CD3- 4- 8- 19-) thymocytes (MN Thy). Both alpha4beta1- and alpha5beta1-integrins are expressed by MN Thy, but only alpha4beta1 mediates fibronectin (FN)-dependent adhesion and motility. Freshly isolated MN Thy lack expression of RHAMM and their motility is RHAMM independent. Prolonged surface expression of RHAMM on MN Thy is dependent upon FN. RHAMM expression, which occurs prior to surface expression of CD3/T-cell receptor (TCR), was found to be inhibited by cross-linking of alpha4-, alpha5- and beta1-integrins, as was the prolonged FN-dependent phase of RHAMM expression. To confirm that RHAMM expression had been down-regulated rather than rendered cryptic by treatment with immobilized anti-integrin monoclonal antibody (MoAb), RHAMM mRNA levels were analysed. Transcription of RHAMM was decreased 7-12-fold by treatment with immobilized anti-alpha4 or anti-alpha5, and twofold by anti-beta1. Prior to expression of CD3/TCR and RHAMM, alpha4beta1 regulates migratory behaviour. After MN Thy differentiate to acquire CD3/TCR in vitro or in vivo, their motility becomes dependent upon both RHAMM and beta1-integrins. Integrins play a direct role in FN-dependent, RHAMM-independent motility of MN Thy, and an indirect role in RHAMM-dependent motility. This work shows that beta1-integrins are primary mediators and regulators of fundamental cell behaviours required during migratory phases of T-cell differentiation that occur prior to the expression of CD3/TCR.

Antibodies, Monoclonal↗
PubMed2002

The role of Aktand RAFTK in beta1 integrin mediated survival of precursor B-acute lymphoblastic leukemia cells.

In this study, we report that the related adhesion focal tyrosine kinase RAFTK, is an upstream kinase in beta1 integrin mediated activation of Akt. Stimulation through beta1 integrins by fibronectin reversed apoptosis induced by adriamycin. Inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt (LY 294002), tyrosine kinases (Herbimycin-A) and the cytotoxic agent adriamycin induced apoptosis of REH cells. beta1 integrin ligation induced activation of Akt, and tyrosine phosphorylation of RAFTK and FAK, but not SYK in REH cells. This suggested that RAFTK and FAK activation might be linked to Akt activation. Evidence that RAFTK is a modulator of Akt came from phorbol myristic acetate (PMA) stimulation. RAFTK and Akt were activated but FAK was not. Using fibroblasts from FAK -/- mice, which express high levels of RAFTK, fibronectin plating enhanced Akt activation. Pretreatment of REH cells with a P13 kinase/Akt inhibitor LY 294002 did not inhibit RAFTK tyrosine phosphorylation showing that RAFTK is upstream of P13k/Akt. Further evidence for a link between RAFTK tyrosine phosphorylation and Akt activation was the observation that the p85 subunit of P13 kinase associated with RAFTK following integrin ligation in REH cells. These results suggest that RAFTK plays an anti-apoptotic role through the activation of Akt.

Benzoquinones↗
PubMed2005

Identification of the beta1-integrin binding site on alpha-actinin by cryoelectron microscopy.

Cell-matrix adhesions in migrating cells are usually mediated by integrins, alpha-beta heterodimeric transmembrane proteins that link extracellular matrix molecules such as fibronectin to the cytoskeleton. We have synthesized the cytoplasmic domain of the beta1-integrin (residues H738-K778) with a histidine tag at its N-terminus. The binding of this peptide to a lipid monolayer containing a chelated-nickel group (dimyristoylphosphatidyl choline-suberimide-nitriloacetic acid:nickel salt) mimics the native environment at the cytoplasmic leaflet of the plasma membrane. A Nanogold particle was covalently linked to cysteines introduced at the C-terminus and after residue T757 on the integrin peptide, and co-crystallized with chicken smooth muscle alpha-actinin. The 2-D arrays of the beta1-integrin-alpha-actinin complex were examined by cryoelectron microscopy, with and without the gold label. Averaged projections were calculated for each specimen along with a difference map to determine the relative position of the gold-labeled beta1-integrin peptide. The difference maps indicate that the beta1-integrin cytoplasmic domain binds alpha-actinin between the first and second, 3-helix motifs in the central rod domain.

Actinin↗
PubMed1996

Widespread expression of beta1 integrins in the developing chick retina: evidence for a role in migration of retinal ganglion cells.

During extension of axons, critical neuronal interactions with extracellular matrix (ECM) and other cells are thought to be mediated in part by heterodimeric beta1 integrin receptors. In this report, we examine the expression and function of beta1 integrins in the developing chick retina. Expression of the beta1 subunit, assayed by in situ hybridization and antibody staining of dissociated cells, was widespread in undifferentiated neuroepithelial cells, before the initiation of axons. Expression persisted in most retinal cell layers throughout embryonic development, during and after axon extension. The repertoire of beta1-associated alpha subunits was examined using reverse transcription-polymerase chain reaction. In addition to the alpha6 and alpha8 subunits previously reported, chick homologues of the alpha2 and alpha4 subunits were detected. Developmental Northern blots revealed varying patterns of integrin subunit expression and showed that expression of beta1 and the mRNAs of its associated alpha subunits are not always coregulated during retinal development. The timing and distribution of expression suggested that beta1 integrins may be involved in other developmental events in addition to axon extension. To address functions carried out by beta1 integrins in the early retina, explanted eye cups were incubated in the presence of function blocking anti-beta1 antibody and migration of newly born retinal ganglion cells (RGCs) was assessed. RGC migration from the ventricular zone to the vitreal border was significantly inhibited, suggesting that beta1 integrins play a role in neuroblast migration in the retina.

Amino Acid Sequence↗
PubMed2004

Skeletal phenotype of growing transgenic mice that express a function-perturbing form of beta1 integrin in osteoblasts.

Skeletal modeling entails the deposition of large amounts of extracellular matrix (ECM) to form structures tailored to withstand increasing mechanical loads during rapid growth. Specific ECM molecules bind to integrin receptors on the cell surface, thereby triggering a cascade of signaling events that affect critical cell functions. To evaluate the role of integrins during skeletal growth, transgenic mice were engineered to express a function-perturbing fragment of beta1 integrin consisting of the transmembrane domain and cytoplasmic tail under the control of the osteocalcin promoter (TG mice). Thus, transgene expression was targeted to mature cells of the osteoblast lineage, and herein we show that cultured cells resembling osteocytes from 90-day-old TG mice display impaired adhesion to collagen I, a ligand for beta1 integrin. To determine the influence of beta1 integrin on bones that are responsible for providing structural support during periods of rapid growth, we examined the phenotype of the appendicular skeleton in TG mice compared to wild type (WT) mice. According to radiographs, bones from mice of both genotypes between 14 and 90 days of age appeared similar in gross structure and density, although proximal tibiae from 35-90 days old TG mice were less curved than those of WT mice (72-92% TG/WT). Although there were only mild and transient differences in absolute bone mass and strength, once normalized to body mass, the tibial dry mass (79.1% TG/WT females), ash mass (78.5% TG/WT females), and femoral strength in torsion (71.6% TG/WT females) were reduced in TG mice compared to WT mice at 90 days of age. Similar effects of genotype on bone mass and curvature were observed in 1-year-old retired breeders, indicating that these phenotypic differences between TG and WT mice were stable well into adulthood. Effects of genotype on histomorphometric indices of cancellous bone turnover were minimal and evident only transiently during growth, but when present they demonstrated differences in osteoblast rather than osteoclast parameters. Together, these results suggest that integrin signals generated during growth enhance the acquisition of a skeletal mass, structure, and strength to withstand the mechanical loads generated by weight-bearing.

Animals↗
PubMed1996

The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins.

Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind beta1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to alpha4beta1 and alpha5beta1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit beta1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4(+) T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits beta1, alpha4, and alpha5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin.

Adhesins, Bacterial↗
PubMed1996

Critical residues for ligand binding in an I domain-like structure of the integrin beta1 subunit.

Several integrin alpha subunits have an inserted sequence of about 200 residues (the I or A domain) that is critical for ligand interactions. The presence of an I domain-like structure within the integrin beta subunit has been proposed based on the similarity of the hydropathy profiles and the homology of sequences between the alpha and beta subunits. This study was designed to determine whether the region of the beta1 subunit that includes residues 101-335 has the characteristics of an I domain. We found novel critical residues for ligand binding (Ser-132, Asn-224, Asp-226, Glu-229, Asp-233, Asp-267, and Asp-295, in addition to the previously reported Asp-130) using site-directed mutagenesis. The critical residues for ligand binding are located in several of loop structures of the region (or in a potential loop between an alpha helix and a beta strand), which have been predicted using multiple secondary structure prediction methods. The data suggest that the beta subunit has multiple disrupted critical oxygenated residues for ligand binding similar to those found in the alpha I domain.

Amino Acid Sequence↗
PubMed2002

In vitro study of intracellular IL-1beta production and beta1 integrins expression in stimulated chondrocytes--effect of rhein.

The purpose of the present study was to investigate the intracellular IL-1beta production and beta1 integrins (alpha4/beta1 and alpha5/beta1) expression on chondrocytes. Chondroytes monolayer (human chondrosarcoma cell line HEM-C55) were incubated for 12, 24 and 48 hours in the presence of Tumor Necrosis Factor-alpha (TNF-alpha, Sigma, France) or recombinant human IL-1alpha (rh-IL1alpha, Becton Dickinson, France). After direct immunolabelling, cells were either analyzed on FACScan flow cytometer (Becton Dickinson, France), or observed under an epi-fluorescence inverted microscope equipped with the CellScan EPR optical scanning acquisition system (IPLab-Scanalytics, USA). We found that the IL-1beta mean fluorescence intensity in flow cytometry and in 3D microscopy was increased in the presence of TNF-alpha or rh-IL-1alpha, and alpha4/beta1 or alpha5/beta1 expression was higher on stimulated cells than on control cells. On the other hand, we have evaluated the in vitro effects of rhein (10(-5) M, Negma, France), an active metabolite of diacerein, on the intracellular IL-1beta and beta1 integrins expressed by stimulated or no-stimulated chondrocytes. The results indicated that rhein leads to a reduction of IL-1beta synthesis whereas a weak decrease of beta1 integrins receptors expression is observed. From this study, it seems that rhein partially reduce cytokine-induced intracellular IL-1beta production, and it has a weak action on alpha4/beta1 or alpha5/beta1 receptors.

Anthraquinones↗
PubMed1997

Differential signaling after beta1 integrin ligation is mediated through binding of CRKL to p120(CBL) and p110(HEF1).

CRKL is an SH2-SH3-SH3 adapter protein that is a major substrate of the BCR/ABL oncogene. The function of CRKL in normal cells is unknown. In cells transformed by BCR/ABL we have previously shown that CRKL is associated with two focal adhesion proteins, tensin and paxillin, suggesting that CRKL could be involved in integrin signaling. In two hematopoietic cell lines, MO7e and H9, we found that CRKL rapidly associates with tyrosine-phosphorylated proteins after cross-linking of beta1 integrins with fibronectin or anti-beta1 integrin monoclonal antibodies. The major tyrosine-phosphorylated CRKL-binding protein in the megakaryocytic MO7e cells was identified as p120(CBL), the cellular homolog of the v-Cbl oncoprotein. However, in the lymphoid H9 cell line, the major tyrosine-phosphorylated CRKL-binding protein was p110(HEF1). In both cases, this binding was mediated by the CRKL SH2 domain. Interestingly, although both MO7e and H9 cells express p120(CBL) and p110(HEF1), beta1 integrin cross-linking induces tyrosine phosphorylation of p120(CBL) (but not p110(HEF1)) in MO7e cells and of p110(HEF1) (but not p120(CBL)) in H9 cells. In both cell types, CRKL is constitutively complexed to C3G, SOS, and c-ABL through its SH3 domains, and the stoichiometry of these complexes does not change upon integrin ligation. Thus, in different cell types CRKL and its SH3-associated proteins may form different multimeric complexes depending on whether p120(CBL) or p110(HEF1) is tyrosine-phosphorylated after integrin ligation. The shift in association of CRKL and its SH3-associated proteins from p120(CBL) to p110(HEF1) could contribute to different functional outcomes of "outside-in" integrin signaling in different cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Homing and engraftment defects in ex vivo expanded murine hematopoietic cells are associated with downregulation of beta1 integrin.

OBJECTIVE: Hematopoietic progenitors generated by ex vivo expansion "home" less efficiently to the bone marrow (BM) after intravenous transplantation than fresh cells. To explore the underlying cause of this transplantation defect, we examined the homing and engraftment properties in vivo of fresh and cultured marrow cells differing in beta1 integrin expression. MATERIALS AND METHODS: Fresh murine BM cells, or the expanded progeny of enriched Sca-1(+) c-kit(+)Lin(-) stem cells, were fractionated into beta1(-/lo) and beta1(+) subpopulations by cell sorting. These populations were assayed for their content of in vitro colony-forming cells (CFCs), cells able to provide radioprotection, and early and long-term multilineage hematopoietic reconstitution following transplantation into myeloablated recipients. These endpoints were correlated with the homing properties of beta1(-/lo) and beta1(+) cells that were labeled with 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) and tracked to hematopoietic organs 24 hours after injection into lethally irradiated mice. RESULTS: Most normal stem and progenitor cells express high levels of beta1 integrin. In contrast, most clonogenic cells generated in vitro are beta1(-/lo). Consequently, expanded beta1(-/lo) progenitors failed to provide radioprotection or repopulate the hematopoietic system following intravenous transplantation. Defective engraftment of expanded cells was associated with reduced homing of beta1(-/lo) cells to the bone marrow. CONCLUSION: Downregulation of beta1 integrin on primitive hematopoietic cells during ex vivo expansion reduces their homing efficiency and negatively impacts hematopoietic reconstitution in vivo. Strategies directed at preserving beta1 integrin expression during culture may improve the clinical utility of expanded hematopoietic cells.

Animals↗
PubMed1999

Beta1-integrins partly mediate binding of ovarian cancer cells to peritoneal mesothelium in vitro.

Ovarian cancer cells frequently metastasize by implanting onto the peritoneal mesothelial surface of the abdominal cavity. Although the CD44 molecule expressed by ovarian cancer cells is known to partly mediate this process, the role of other adhesion proteins such as beta1-integrins has been previously difficult to demonstrate using the 4B4 anti-beta1 neutralizing antibody. In view of the widespread expression of beta1-integrins in ovarian cancer, however, we have further examined the potential role of this class of molecules in ovarian cancer cell implantation through the use of an alternative anti-beta1 neutralizing antibody, MAB13. We now report that MAB13 is capable of inhibiting the binding of three separate human ovarian cancer cell lines (36M2, CAOV-3, SKOV-3) to mesothelium (mean 37 +/- 4% inhibition, n = 21, P < 0.001). An additive inhibitory effect was observed when MAB13 was combined with anti-CD44 antibody (clone 515) (mean 63 +/- 3% inhibition, n = 19, P < 0.001), suggesting that binding occurs through two independent pathways involving both beta1-integrins and CD44. Because fibronectin is an extracellular matrix ligand recognized by many types of integrins and is abundantly expressed on mesothelial cells, the inhibitory effects of MAB13 and 4B4 on ovarian cancer cell binding to fibronectin were directly compared. MAB13 inhibited ovarian cancer cell binding to fibronectin to a significantly greater degree than did 4B4, suggesting that the discordant effects of these two antibodies on mesothelial adhesion may be partly related to their differential ability to neutralizing fibronectin-mediated binding. Studies using anti-alpha5 neutralizing antibody demonstrated that the alpha5beta1 fibronectin receptor contributes to approximately 50% of integrin-mediated binding of 36M2 and CAOV-3 cells (which express the alpha5 chain in 54 and 58% of cells, respectively). Since the RGD sequence of fibronectin is a known recognition site for many types of integrins, including alpha5beta1 and alphanubeta3, we performed binding in the continued presence of both anti-alpha5 and RGD peptide in order to exclude other types of fibronectin-integrin interactions. The addition of RGD peptides at concentrations known to be capable of blocking fibronectin binding resulted in no additional inhibitory effect over that observed with anti-alpha5 antibody alone, suggesting that alpha5beta1 was the major receptor responsible for fibronectin-mediated ovarian cancer binding to mesothelium. These data demonstrate that ovarian cancer cell binding to peritoneal mesothelium is mediated by several adhesion pathways and that simultaneous inhibition of both beta1-integrin and CD44 function may be necessary in order to significantly limit the intraabdominal spread of this tumor in vivo.

Cell Adhesion↗