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Biomedical subjects

D Boettiger

Publications and source records attributed to D Boettiger.

At least 19 recordsLinked to original sources
PubMed2001

Distinct ligand-binding modes for integrin alpha(v)beta(3)-mediated adhesion to fibronectin versus vitronectin.

Cell surface integrins can adopt distinct conformations in response to ligand binding and intracellular signals. Several integrins including alpha(v)beta(3) can bind to multiple ligands. The binding of alpha(v)beta(3) to fibronectin and vitronectin was used as a model to determine whether the same or distinct forms of the receptor were utilized in strong binding to the two different ligands. A spinning-disc device was used to measure the relative strength of the alpha(v)beta(3)-ligand bonds. The initial binding reaction for both ligands occurred in the absence of metabolic energy and resulted in a strong adhesion to fibronectin but a weak adhesion to vitronectin. Increases in the strength of the alpha(v)beta(3)-vitronectin bond required phosphorylation of the beta(3) cytoplasmic domain, intracellular signals, and the binding of cytoskeletal proteins to cytoplasmic domains of beta(3) controlled by Tyr-747 and Tyr-759. In contrast, alpha(v)beta(3)-mediated adhesion to fibronectin was unaffected by phorbol 12-myristate 13-acetate, mutations of Tyr-747 and Tyr-759 to phenylalanine, or availability of metabolic energy. This suggests that strong adhesion to fibronectin used the initial binding conformation, whereas strong binding to vitronectin required signaling-induced changes in the conformation of alpha(v)beta(3).

Actins↗
PubMed2001

Activation of alpha(v)beta3-vitronectin binding is a multistage process in which increases in bond strength are dependent on Y747 and Y759 in the cytoplasmic domain of beta3.

Integrin receptors serve as mechanical links between the cell and its structural environment. Using alpha(v)beta3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between alpha(v)beta3 and vitronectin develops was analyzed by measuring the resistance of these bonds to mechanical separation. Three distinct stages of activation, as defined by increases in the alpha(v)beta3-vitronectin binding strength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the beta3 subunit. Stage 2 is characterized by a 4-fold increase in binding strength and is dependent on stage1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction with cellular proteins. The Y747F mutant blocked the transition from stage 1 to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of alpha(v)beta3 integrin.

Antigens, CD↗
PubMed2000

Insertional mutagenesis as a route to identifying genes involved in self renewal of haemopoietic stem cells.

The genes controlling self renewal in the haemopoietic system are still unknown. Using retroviral insertional mutagenesis we have established multipotent haemopoietic stem cell lines (FDCP-mix) that possess an increased self renewal capacity in vitro. To identify genes involved in the regulation of self renewal, proviral integration sites were cloned from FDCP-mix cells and used as probes to screen independently isolated FDCP-mix cell lines for a common proviral insertion site. So far, two common integration sites have been identified, A25 and M4. A25 is rearranged in 50% of the FDCP-mix cell lines and M4 in 10%. Genes located at or near these sites are likely candidates for the control of self renewal of haemopoietic stem cells.

Cell Differentiation↗
PubMed1999

Integrin-fibronectin interactions at the cell-material interface: initial integrin binding and signaling.

Integrin receptors mediate cell adhesion to extracellular matrices and provide signals that direct proliferation and differentiation. Integrin binding involves receptor-ligand interactions at the cell-substrate interface and assembly and reorganization of structural and signaling elements at the cytoplasmic face. Using a cross-linking/extraction/reversal method to quantify bound integrins, we demonstrate that the density of alpha5beta1 integrin-fibronectin bonds increases linearly with ligand density, as predicted by simple receptor-ligand equilibrium. This linear relationship is consistent with linear increases in cell adhesion strength with receptor and ligand surface densities. Furthermore, we show that phosphorylation of FAK, a tyrosine kinase involved in early integrin-mediated signaling, increases linearly with the number of integrin-Fn bonds. These linear relationships suggest the absence of cooperative effects in the initial stages of mechanical coupling and adhesion-mediated signaling.

Cell Adhesion↗
PubMed1999

Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation.

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound alpha5 and beta1 integrin subunits but not alphav or beta3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of alpha5beta1 integrin bound to Fn, and differentiation was inhibited by anti-alpha5, but not anti-alphav, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications.

Adsorption↗
PubMed1998

Two-stage activation for alpha5beta1 integrin binding to surface-adsorbed fibronectin.

By analyzing the functional binding of alpha5beta1 integrin to adsorbed fibronectin in intact cells, we demonstrate that integrin activation results in linear increases in adhesion strength as a function of ligand density, suggesting that modulation of the receptor-ligand interaction is the dominant mechanism for adhesion during the initial stages of adhesion and that cooperative binding contributes little to initial adhesion strength. Using this experimental framework, we show the existence of three distinct activation states for alpha5beta1 integrin binding to adsorbed fibronectin for both passive, antibody-induced and active, cell-controlled activation. During the initial phase of adhesion, alpha5beta1 integrin is activated in an energy-dependent process from the nonbinding ground state to an intermediate state in which the receptor binds fibronectin and provides significant mechanical coupling. In later stages of adhesion maturation, alpha5beta1 integrin is activated to a higher binding state, which provides significant increases in adhesion strength compared with the intermediate state. These multiple binding states most likely result from different integrin conformations and reflect distinct interactions between alpha5beta1 and sites on adsorbed fibronectin. Multiple activation states for alpha5beta1 suggest the existence of distinct stages in adhesion signaling and strengthening and can provide a versatile mechanism for the regulation of adhesive interactions.

Adsorption↗
PubMed1998

Force required to break alpha5beta1 integrin-fibronectin bonds in intact adherent cells is sensitive to integrin activation state.

Binding of integrin receptors to extracellular ligands is a complex process involving receptor-ligand interactions at the cell-substrate interface, signals activating the receptors, and assembly of cytoskeletal and adhesion plaque proteins at the cytoplasmic face. To analyze the contribution of these elements to overall cell adhesion, we have developed a model system that characterizes the functional binding characteristic for adhesion receptors as the force required to separate the integrin-ligand bond. A spinning disk device was used to apply a range of controlled hydrodynamic forces to adherent cells. The adhesion of K562 erythroleukemia cells, a cell line expressing a single fibronectin receptor, integrin alpha5beta1, which was uniformly activated with the monoclonal antibody TS2/16, to defined fibronectin surface densities was examined. Cell adhesion strength increased linearly with receptor and ligand densities. Based on chemical equilibrium principles, it is shown that adhesion strength is directly proportional to the number of receptor-ligand bonds. This analysis provides for the definition of a new physical parameter, the adhesion constant psi, which is related to the bond strength and binding equilibrium constant and has units of force-length2. This parameter can be measured by the experimental system presented and is governed by the activation state of integrin receptors. This simplified model isolates the integrin receptor-ligand binding parameters and provides a basis for analysis of the functions of signaling and cytoskeletal elements in the adhesion process.

Cell Adhesion↗
PubMed1998

Effect of surface reaction stage on fibronectin-mediated adhesion of osteoblast-like cells to bioactive glass.

Bioactive glasses and ceramics enhance bone formation and bond directly to bone, and have emerged as promising substrates for bone tissue engineering applications. Bone bioactivity involves physicochemical surface reactions and cellular events, including cell attachment to adsorbed extracellular matrix proteins. The effects of fibronectin (Fn) adsorption and glass surface reaction stage on the attachment of osteoblast-like cells (ROS 17/2.8) to bioactive glass were analyzed. Bioactive glass disks were pretreated in a simulated physiologic solution to produce three reaction layers: unreacted glass (BG0), amorphous calcium phosphate (BG1d), and carbonated hydroxyapatite (BG7d). Synthetic hydroxyapatite (sHA) and nonreactive borosilicate glass (CG) were used as controls. A spinning disk device which applied a linear range of forces to attached cells while maintaining uniform chemical conditions at the interface was used to quantify cell adhesion. The number of adherent cells decreased in a sigmoidal fashion with applied force, and the resulting detachment profile provided measurements of adhesion strength. For the same amount of adsorbed Fn, cell adhesion was higher on surface-reacted bioactive glasses (BG1d and BG7d) than on BG0, CG, and sHA. For all substrates, cell attachment was primarily mediated by the RGD binding site of Fn, as demonstrated by blocking experiments with antibodies and RGD peptides. Cell adhesion strength increased linearly with adsorbed Fn surface density. Analysis of this fundamental relationship revealed that improved adhesion to reacted bioactive glasses resulted from enhanced cell receptor-Fn interactions, suggesting substrate-dependent conformational changes in the adsorbed Fn.

Biocompatible Materials↗
PubMed1997

Quantification of cell adhesion using a spinning disc device and application to surface-reactive materials.

Quantitative analysis of cell adhesion is essential in understanding physiological phenomena and developing biotechnological applications. Electrochemical measurements demonstrated that the transport patterns associated with a spinning disc device approximate the fluid flow and mass transport fields for a disc spinning in an infinite fluid. Therefore, this device applies a linear range of forces to attached cells under uniform and constant chemical conditions at the interface. The application of this apparatus for examining cell adhesion to surface-active materials was illustrated by investigating the attachment of osteoblast-like cells to fibronectin adsorbed onto bioactive and non-reactive glasses for different chemical environments. Cells were seeded on fibronectin-coated substrates for 15 min and then subjected to detachment forces for 10 min. The number of adherent cells decreased non-linearly with applied force and the detachment profile was accurately described by a sigmoidal curve fit, as expected for a cell population with normally distributed adhesion properties.

Adsorption↗
PubMed1997

Epitopes of adhesion-perturbing monoclonal antibodies map within a predicted alpha-helical domain of the integrin beta 1 subunit.

Several recent studies have demonstrated the involvement of various domains of the beta 1 integrin subunit in ligand binding. Thus, specific amino acids have been shown to be important in divalent cation binding, and others have been implicated by peptide crosslinking to play an intimate role in integrin-ligand interactions. Added to these data are previous observations that a group of adhesion-blocking anti-chicken beta 1 antibodies mapped within the first 160 amino acid residues of the subunit. These observations suggested that this region plays a critical role in integrin ligand recognition. In order to further define the domain in which the epitopes for these antibodies are clustered, a series of mouse/chicken chimeric beta 1 constructs were examined for their reactivity with each of these antibodies. Most of the antibodies recognize a region between residues 124 to 160 of the chicken beta 1 subunit. Computer modeling predicted a possible amphipathic alpha-helical configuration for the region between residues 141 to 160. Consistent with this prediction, circular dichroism and NMR analysis revealed a tendency for a synthetic peptide containing these residues to form an alpha-helix. The significance of this structural characteristic was demonstrated by a mutation at residue 149 that disrupted the alpha-helix formation and resulted in a loss of the ability to form heterodimers with alpha subunits, localize to focal contacts, or be transported to the cell surface. The direct involvement of residues 141 to 160 in ligand binding was supported by the ability of a peptide with this sequence to elute integrins from a fibronectin affinity column. Thus, our data suggest that residues 141 to 160 of the integrin beta 1 subunit, when arranged in an alpha-helix configuration, participate in ligand binding.

3T3 Cells↗
PubMed1997

Involvement of alpha5beta1 integrin in matrix interactions and proliferation of chondrocytes.

Integrins are cell surface receptors involved in cellular processes including adhesion, migration, and matrix assembly. In the present study, we analyzed the possible involvement of alpha 5 beta 1 integrin in the regulation of chondrocyte adhesion, spreading, and proliferation. We found that rabbit growth plate chondrocytes were able to attach to substrates coated with type I collagen, type II collagen, or fibronectin within 24 h of culture. During this time period, attachment to fibronectin appeared to be dependent on alpha 5 beta 1 integrin, whereas adhesion to collagens was not. By day 3 of culture, chondrocytes spread onto all the substrates tested. We found that regardless of the nature of the substrate, cell spreading was reversed by treatment with RGD peptide or antibodies against alpha 5 beta 1 or fibronectin, indicating that cell spreading involved alpha 5 beta 1 and fibronectin endogenously produced and deposited by the chondrocytes themselves. Colony formation by chondrocytes in soft agar was inhibited by treatment with RGD peptides or BIIG2, an antibody that interferes with alpha 5 beta 1 integrin-ligand interactions. Furthermore, DNA content was decreased by treatment with anti-fibronectin antibody in micromass culture of chondrocytes. Immunohistochemical analysis on tissue sections revealed that the alpha 5 subunit was particularly abundant in the proliferative and hypertrophic zones of growth plate. The results of the study indicate that alpha 5 beta 1 integrin plays multiple roles in chondrocyte behavior and function and appears to be involved in the regulation of both chondrocyte-matrix interactions and proliferation.

Animals↗
PubMed1995

Regulation of integrin alpha 5 beta 1 affinity during myogenic differentiation.

The antigen recognized by U1 alpha, a monoclonal antibody to the alpha chain of a chicken integrin fibronectin receptor, was identified as alpha 5. It identifies the same polypeptide as antisera raised to a sequence from the alpha 5 cytoplasmic domain. The U1 alpha antibody has the unusual functional property for alpha chain antibodies of enhancing the binding of alpha 5 beta 1 for its ligand fibronectin. U1 alpha was used to examine the function of alpha 5 beta 1 during myogenic differentiation. As myogenic cells differentiated from replicating myoblasts to bipolar myocytes there was a decrease in their adhesion to the substrate caused by inactivation of alpha 5 beta 1, which could be reversed by treatment of the cells with U1 alpha. The U1 alpha induced increased adhesion to fibronectin but did not inhibit the differentiation process as measured by formation of myotubes. However, U1 alpha did interfere with both cell migration and morphogenesis of myotubes. The resulting myotubes were smaller, more branched, and showed less regular alignment of nuclei. The results suggest that the ability of the cell to regulate alpha 5 beta 1 affinity is critical to myogenic morphogenesis.

Animals↗
PubMed1995

Developmental expression and molecular cloning of REMP, a novel retinal epithelial membrane protein.

The retinal pigment epithelium (RPE), like other transport epithelia, has a polarized distribution of membrane and cytoskeletal proteins. The establishment of a polarized phenotype is an essential step in the differentiation of the RPE and the development and maintenance of visual function. Using a monoclonal antibody (MAb 3C4) we have identified a novel membrane protein that is uniquely expressed in chick RPE. We have referred to this protein as REMP for retinal epithelial membrane protein. In these studies we characterized the expression and distribution of this protein during embryonic development and determined its primary structure by cDNA cloning. The developmental expression of REMP was examined by immunocytochemical localization. REMP was first detected in the chick RPE at Embryonic Day 5 (E5) in both apical and basolateral membranes. By E14 the distribution of REMP was restricted to the basolateral surface of the RPE cells. Biochemical fractionation and surface labeling of RPE cells suggested that REMP was an integral protein. The gene encoding REMP was isolated from an E15 chick RPE cDNA library, cloned into lambda gt11, and screened with MAb 3C4. The cDNA was sequenced and found to contain one 1350-bp open reading frame encoding for a 450-amino-acid protein. The deduced amino-acid sequence of REMP shares 32.9% identity with MCT1, a monocarboxylate transporter (Garcia, Goldstein, Pathak, Anderson, and Brown, Cell, 76, 865-873, 1994). By Northern blot analysis, REMP mRNA was detected only in RPE cells. There was an increase in the expression REMP transcript during development but when RPE cells were grown in primary culture the expression of REMP was turned off. The unique expression of REMP in the RPE in vivo would suggest a role for this protein in development and maintenance of normal retinal function.

Amino Acid Sequence↗
PubMed1994

Expression of v-src alters the expression of myogenic regulatory factor genes.

Inactivation of v-src in transformed chicken myoblasts induced changes in expression of the myogenic regulatory factors (MRF's), MyoD, myogenin and Myf5. Both MyoD and Myf5 were expressed in the transformed cells. The inactivation of v-src by a temperature up-shift resulted in the loss of Myf5 expression, a transient increase in MyoD, followed by a rise in myogenin mRNA levels. The increase in transcripts for the later muscle proteins, myosin heavy and light chains, alpha-actin and troponin T followed the sequence of changes in MRF expression suggesting that their induction was the result of altered MRF expression. Thus, v-src appears to operate through effects on MRF's and not directly on the expression of the late muscle-specific proteins. When v-src was reactivated in mature myotubes by a temperature down-shift, the pattern of expression was quite different, the late muscle-specific transcripts decreased before the loss of myogenin transcripts and before the rise of Myf5 transcripts.

Animals↗
PubMed1993

Alpha 5 integrin is a critical component of adhesion plaques in myogenesis.

We investigated the distribution and expression of alpha 5 beta 1 and alpha 3 beta 1 integrin in differentiating myogenic cells in culture. The myogenic cells expressed both alpha 5 and alpha 3 integrin subunits with the same molecular sizes as those expressed by chicken embryo fibroblasts (CEF). However, the ratio of total alpha 5 to alpha 3 was threefold higher in the muscle cultures than that in CEF cultures. A new method is described whereby adhesion plaque-associated integrin was cross-linked to its extracellular matrix ligand on the substrate using a nonpenetrating cross-linker, BS3, and integrin not involved in substrate adhesion as well as cytoskeletal proteins were removed with a zwitterionic detergent. alpha 5 and beta 1 integrin, but not alpha 3 could be cross-linked to fibronectin at adhesion plaques throughout myogenesis in culture. Alpha 5 beta 1 integrin was found only at the edge of myoblasts 4 hr after plating but became distributed under their entire surface by 1 day in culture. When the muscle cells became elongated, a morphology they express after the initiation of terminal differentiation, and as they began to fuse, alpha 5 was found redistributed in small adhesion plaques along the lateral edges of the postmitotic myocytes and early myotubes. In mature myotubes, which are large multinucleated branched structures, alpha 5 beta 1 integrin was localized to parallel streaks underneath their entire substrate surface. Throughout the different stages of myogenesis, vinculin colocalized with alpha 5 beta 1 integrin in adhesion plaques, but alpha-actinin only colocalized to the adhesion plaques in myoblasts, not in myotubes. These studies suggest that alpha 5 beta 1 integrin through its dynamic interaction with both fibronectin and the cytoskeleton is important for both the signals which initiate the differentiation process and for subsequent morphological and structural changes during the differentiation process.

Animals↗
PubMed1993

Beta 1 integrins mediate chondrocyte interaction with type I collagen, type II collagen, and fibronectin.

Chondrocytes isolated from the cephalic region of sterna from 14-day-old chick embryos used beta 1 integrins and required either Mg2+ or Mn2+ for attachment to plates coated with type I collagen, type II collagen, and fibronectin. beta 1 integrin was concentrated in adhesion plaques of the chondrocytes plated on type I collagen, type II collagen, and fibronectin substrates. Chondrocytes expressed at least 3 alpha-subunits, including alpha 3, alpha 5, and putative alpha 2. alpha 5, but not alpha 3, had a higher molecular weight in chondrocytes than in fibroblasts. Levels of alpha 3 and alpha 5 were about 25-30% of that in fibroblasts. When the chondrocytes were cultured in the presence of ascorbate in suspension, the cells aggregated into clusters. This aggregation was dependent on beta 1 integrin and type II collagen.

Amino Acid Sequence↗
PubMed1993

Evaluation of integrin molecules involved in substrate adhesion.

Integrins were cross-linked to their extracellular matrix ligands using non-penetrating chemical cross-linkers. This procedure did not disturb the distribution of integrin in the adhesion structure and adhesion plaque integrin staining remained even when the cultures were extracted with ionic detergents. 80-90% of the beta 1 integrin in the cross-linked culture was extracted with RIPA buffer and the remaining 10-20% was recovered following reversal of the cross-linking. This separated two distinct integrin pools, one which can be cross-linked to substrate bound extracellular matrix and one which is not. The specificity of this procedure for cross-linking of integrins involved in substrate adhesion was demonstrated using NIH 3T3 cells which express both alpha 5 beta 1 and alpha 6 beta 1 integrins. alpha 6 was cross-linked only in cells plated on laminin whereas alpha 5 was cross-linked when fibronectin was present. Using antisera directed to the cytoplasmic domains of either alpha 5 or beta 1 integrin, it was demonstrated that these domains can be blocked in the intact cell but the blocking can be removed using ionic detergent extraction after chemical cross-linking. The extracellular matrix associated with the substrate surface but not that associated with the media exposed surface is both cross-linked and retained on the plastic dish following cross-linking.

3T3 Cells↗