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H Furthmayr

Publications and source records attributed to H Furthmayr.

At least 37 records · Page 2Linked to original sources
PubMed1997

Laminin-5 inhibits human keratinocyte migration.

Laminin-5 (previously known as kalinin, epiligrin, and nicein) is an adhesive protein localized to the anchoring filaments within the lamina lucida space of the basement membrane zone lying between the epidermis and dermis of human skin. Anchoring filaments are structures within the lamina lucida and lie immediately beneath the hemidesmosomes of the overlying basal keratinocytes apposed to the basement membrane zone. Human keratinocytes synthesize and deposit laminin-5. Laminin-5 is present at the wound edge during reepithelialization. In this study, we demonstrate that laminin-5, a powerful matrix attachment factor for keratinocytes, inhibits human keratinocyte migration. We found that the inhibitory effect of laminin-5 on keratinocyte motility can be reversed by blocking the alpha3 integrin receptor. Laminin-5 inhibits keratinocyte motility driven by a collagen matrix in a concentration-dependent fashion. Using antisense oligonucleotides to the alpha3 chain of laminin-5 and an antibody that inhibits the cell binding function of secreted laminin-5, we demonstrated that the endogenous laminin-5 secreted by the keratinocyte also inhibits the keratinocyte's own migration on matrix. These findings explain the hypermotility that characterizes keratinocytes from patients who have forms of junctional epidermolysis bullosa associated with defects in one of the genes encoding for laminin-5 chains, resulting in low expression and/or functional inadequacy of laminin-5 in these patients. These studies also suggest that during reepithelialization of human skin wounds, the secreted laminin-5 stabilizes the migrating keratinocyte to establish the new basement membrane zone.

Antigens, CD↗
PubMed1997

Actin-binding membrane proteins identified by F-actin blot overlays.

Actin and associated proteins at the cytoskeleton-plasma membrane interface stabilize the membrane bilayer, control cell shape, and delimit specialized membrane domains. To identify membrane proteins that bind directly to F-actin, we have developed a blot overlay assay with 125I-labeled F-actin. In the soil amoebae, Dictyostelium discoideum, the major proteins reactive in this assay are p30a, a 34-kD peripheral membrane protein that is concentrated in filopodia and at sites of cell-cell adhesion, and ponticulin, a 17-kD transmembrane glycoprotein required for efficient chemotaxis and for control of pseudopod dynamics. Proteins with apparent molecular masses of approximately 34- and approximately 17-kD also are observed on F-actin blot overlays of many mammalian cell lines. However, in mammalian cells, the most prominent F-actin binding proteins in this assay exhibit apparent molecular masses of 78-, 80-, 81-, approximately 120-, and 205-kD. Bovine neutrophils contain the 78-, 81-, and 205-kD proteins, all of which co-isolate with a plasma membrane-enriched fraction. We have previously identified the 78-, 80-, and 81-kD proteins as moesin, radixin, and ezrin, respectively. These proteins, which are members of the protein 4.1 superfamily, colocalize with actin in cell surface extensions and have been implicated in the protrusion of microvilli, filopodia, and membrane ruffles. The 205-kD protein (p205) appears to be absent from current databases, and its characteristics are still under investigation. We here report that the 120-kD protein is drebrin, a submembranous actin-binding protein originally identified as a developmentally regulated brain protein. Thus, it appears that F-actin blot overlays provide an efficient assay for simultaneous monitoring of a subset of F-actin binding proteins, including p30a, ponticulin, moesin, radixin, ezrin, p205, and drebrin.

3T3 Cells↗
PubMed1997

Ascending aortic aneurysm with or without features of Marfan syndrome and other fibrillinopathies: new insights.

More than 70 unique fibrillin-1 mutations have been identified in individuals with a variety of phenotypic changes. These range from severe neonatal lethal forms of Marfan syndrome to adult onset manifestations, mitral valve prolapse syndromes to isolated features such as ectopia lentis, Marfanoid body habitus and ascending aortic aneurysm and/or dissection. Fibrillin-1 mutations result in structurally and functionally defective fibrillin-1 molecules and microfibrils. Recent molecular genetic and fibrillin-1 biosynthesis studies suggest that individuals with fibrillin-1 abnormalities can be further subdivided into groups that are associated with distinct differences in severity and prognosis. In recognition of the expanding scope of related connective tissue disorders, we propose the terms microfibrillar disorder for disorders affecting fibrillin-containing microfibrils, and the more narrow concept of fibrillinopathy for clinical entities associated with abnormalities of fibrillin-1 or fibrillin-2. This latter category includes the previously defined disorders Marfan syndrome, congenital contractual arachnodactyly, and forms of ascending aortic aneurysm and/or dissection.

Adult↗
PubMed1996

Phosphorylation of 558T of moesin detected by site-specific antibodies in RAW264.7 macrophages.

To determine, whether 558Thr in the carboxyl-terminal domain of moesin is phosphorylated in cells other than platelets, rabbit phosphorylation state-specific antibodies were made to the chemically phosphorylated synthetic hexapeptide KYKpTLR of the moesin sequence, as well as to the unphosphorylated form. The affinity-purified antibody populations were specific for either the phosphorylated or the unmodified peptide conjugated to BSA. Site-specific phosphorylation of moesin is detected in RAW macrophages by Western blot analysis, and immunofluorescence studies demonstrate that phosphorylated moesin is localized in filopodial protrusions. After pretreatment with the phosphatase inhibitor calyculin A, a similar effect to that seen in platelets in found, namely a substantial increase in moesin phosphorylation at 558Thr and redistribution of phospho-moesin together with F-actin into one or more ring-like structures in the cytoplasm, presumably due to binding of phosphorylated moesin to F-actin.

Amino Acid Sequence↗
PubMed1996

The cytoskeletal linking proteins, moesin and radixin, are upregulated by platelet-derived growth factor, but not basic fibroblast growth factor in experimental mesangial proliferative glomerulonephritis.

The expression of the two cytoskeletal linking proteins, moesin and radixin, was examined in experimental mesangial proliferative nephritis in rats (anti-Thy1 model). Moesin and radixin mRNA and protein are constitutively expressed in all cell types of normal rat glomeruli, except podocytes. In the anti-Thy1 model the expression of moesin and radixin was increased in infiltrating macrophages and in activated, alpha-smooth muscle actin-positive mesangial cells and was concentrated in the cellular extensions of mesangial cells in areas of glomerular remodelling. Studies using neutralizing antibodies demonstrated that the increase in moesin and radixin expression by mesangial cells is mediated by PDGF, but not bFGF. The increase in these cytoskeletal proteins appears to be regulated primarily (radixin) or partially (moesin) posttranscriptionally. The data suggest that PDGF mediated upregulation of the cytoskeletal proteins, moesin and radixin, is important for cell migration and other changes that accompany the coordinated restoration of glomerular architecture after injury.

Actins↗
PubMed1996

Mutant fibrillin-1 monomers lacking EGF-like domains disrupt microfibril assembly and cause severe marfan syndrome.

Marfan syndrome (MFS), a heritable connective tissue disorder, is caused by mutations in the gene coding for fibrillin-1 (FBN1), an extracellular matrix protein. One of the three major categories of FBN1 mutations involves exon-skipping. To rapidly detect such mutations, we developed a long RT-PCR method. Either three segments covering the entire FBN1 coding sequence or a single 8.9 kb FBN1 coding segment were amplified from reverse-transcribed total fibroblast RNA. Restriction fragment patterns of these RT-PCR products were compared and abnormal fragments were directly sequenced. Six exon-skipping mutations were identified in a panel of 60 MFS probands. All skipped exons encode calcium binding epidermal growth factor (EGF)-like domains and maintain the reading frame. In five probands, exon-skipping was due to point mutations in splice site sequences, and one had a 6 bp deletion in a donor splice site. Pulse-chase analysis of labelled fibrillin protein revealed normal levels of synthesis but significantly reduced matrix deposition. This dominant-negative effect of the mutant monomers is considered in the light of current models of fibrillin assembly. Probands with this type of FBN1 mutation include the most severe forms of MFS, such as neonatally lethal presentations.

Epidermal Growth Factor↗
PubMed1996

Dermal fibroblast culture as a model system for studies of fibrillin assembly and pathogenetic mechanisms: defects in distinct groups of individuals with Marfan's syndrome.

Most patients with Marfan's syndrome (> 95%) and 75% of patients with uncertain diagnosis can be classified into four groups (Aoyama et al, 1994, 1995) based on abnormal patterns of synthesis, intracellular transport, and/or matrix deposition of fibrillin-1 in fibroblast cultures. Herein we report a systematic study of fibrillin assembly in normal and Marfan's syndrome fibroblasts and correlations between pulse-chase, immunofluorescence, and immunoelectron microscopic data. Normal control fibroblasts were grown at confluent conditions from 2 to 10 days before passage and then maintained at hyperconfluent cell densities for an additional period of 1 to 6 days before assaying. Maximum deposition in the extracellular matrix of pulse-labeled fibrillin required at least 6 days of confluent and 4 to 5 days of hyperconfluent culture. This result is explained by immunofluorescence studies with fibrillin-1-specific antibodies, because 1 day after seeding cells at hyperconfluency, patches of regular immunostained structures were already present. Within these patches, fluorescence intensity and fibrillar material increased over 3 to 4 days, and after only 5 days, fibrillar networks extended throughout the culture. We propose that fibrillin-containing microfibrillar material is passaged together with the cells, newly synthesized fibrillin molecules are deposited onto preexisting microfibrillar assemblies, and several additional days of culture at high cell density are necessary for the cells to construct a sufficient microfibrillar network binding and detection of pulse-labeled fibrillin molecules in insoluble form during a 20-hour chase period. This fraction is decreased to a varying extent in fibroblast cultures of four biosynthetically distinct groups of Marfan's syndrome patients, but only Groups II and IV clearly showed reduction in immunostainable microfibrils. In long-term cultures, immunoelectron microscopy of the extracellular matrix with fibrillin antibodies also detected differences among these groups and in comparison to normal controls with respect to the arrangement of fibrillin-containing microfibrils, thickness of microfibrillar bundles, and the presence of amorphous material. The data support the idea of different pathogenetic mechanisms for each biosynthetically defined group of Marfan's syndrome, which depends on the nature of fibrillin-1 mutations.

Antibodies↗
PubMed1995

Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets.

The phosphorylation and localization of the membrane-linking protein moesin was analyzed during early activation of platelets with thrombin. Activated platelets elaborate filopodia and spread to assume flat pancake-like shapes, and moesin is localized in filopodia and cell body. In resting platelets, approximately 25% of moesin molecules are phosphorylated as shown by metabolic labeling with 32P(i) and by isoelectric focusing. Within seconds after exposure to thrombin, phosphorylation increases, reaching a maximum of 35% labeled molecules by 1 min, followed by a decrease to a new basal level within 5 min. This modification affects a single residue, Thr558, which is located within or close to a binding site for F-actin. Rapid shifts (0-100%) in the number of phosphorylated molecules are observed in the presence of inhibitors of serine/threonine kinases and phosphatases. Inhibitors affecting tyrosine phosphorylation also modulate phosphorylation at this site suggesting that the enzymes involved in the modification of Thr558 are regulated by tyrosine phosphorylation. Platelets respond to both extremes of modification by forming extremely long filopodia and the absence of spreading on glass. Completely phosphorylated moesin is concentrated together with F-actin in the center of the cell. The rapid modification of moesin at or near its actin-binding domain suggests a model for regulated membrane-cytoskeleton interaction during cell activation.

Actins↗
PubMed1995

Fibrillin abnormalities and prognosis in Marfan syndrome and related disorders.

Marfan syndrome (MFS), a multisystem autosomal-dominant disorder, is characterized by mutations of the fibrillin-1 (FBN1) gene and by abnormal patterns of synthesis, secretion, and matrix deposition of the fibrillin protein. To determine the sensitivity and specificity of fibrillin protein abnormalities in the diagnosis of MFS, we studied dermal fibroblasts from 57 patients with classical MFS, 15 with equivocal MFS, 8 with single-organ manifestations, and 16 with other connective tissue disorders including homocystinuria and Ehlers-Danlos syndrome. Abnormal fibrillin metabolism was identified in 70 samples that were classified into four different groups based on quantitation of fibrillin synthesis and matrix deposition. Significant correlations were found for phenotypic features including arachnodactyly, striae distensae, cardiovascular manifestations, and fibrillin groups II and IV, which included 70% of the MFS patients. In addition, these two groups were associated with shortened "event-free" survival and more severe cardiovascular complications than groups I and III. The latter included most of the equivocal MFS/single manifestation patients with fibrillin abnormalities. Our results indicate that fibrillin defects at the protein level per se are not specific for MFS, but that the drastically reduced fibrillin deposition, caused by a dominant-negative effect of abnormal fibrillin molecules in individuals defined as groups II and IV, is of prognostic and possibly diagnostic significance.

Cardiovascular Diseases↗
PubMed1995

Subcellular localization of moesin in dynamic filopodia, retraction fibers, and other structures involved in substrate exploration, attachment, and cell-cell contacts.

Moesin, a member of the talin-4.1 superfamily, is a linking protein of the submembraneous cytoskeleton. It is expressed in variable amounts in cells of different phenotypes such as macrophages, lymphocytes, fibroblastic, endothelial, epithelial, and neuronal cell lines. In this report we show that moesin is not randomly distributed throughout the cortical cytoskeleton, but rather that it is concentrated in specialized microdomains. It is localized in the intracellular core of microextensions known as filopodia, microvilli, microspikes, and retraction fibers. This subcellular distribution follows closely the dynamic changes in cell shape that take place when cells attach, spread, and move spontaneously or in response to extracellular signals. This suggests a similar function for moesin in diverse cell types related to the dynamic restructuring of domains of the plasma membrane and underlying membrane skeleton. Support for this comes from studies on PC-12 cells, which respond to NGF by extending neurites and moesin is redistributed from a diffuse localization to growth cone filopodia. In fibroblastic (NIH3T3) or macrophage (RAW264.7) cell lines, moesin is found in filopodia appearing at random on the cell surface soon after the cells are placed in culture, begin to attach, and spread. In polarized epithelial cells (LLC-PK1), moesin is associated with peripheral filopodia and apical microvilli. The cellular microextensions containing moesin are devoid of microtubules, focal contact proteins such as vinculin, and cortical cytoskeletal elements such as protein 4.1, but they do contain varying amounts of actin microfilaments. This localization of moesin in microextensions is not influenced by cytochalasin B. Treatment of cells with phorbolester (PMA) causes rapid cell spreading, disappearance of filopodia and retraction fibers, and moesin does not accumulate in the actin-rich lamellae that form at the cellular edges. After removal of PMA, cells retract and moesin again becomes concentrated in filopodia and retraction fibers. These studies support the hypothesis that filopodia, retraction fibers, and other microextensions of the plasma membrane are unique cellular microdomains with characteristic submembraneous components. Moesin could be involved in the dynamic restructuring of such microdomains by regulating binding interactions between the plasma membrane and the actin cytoskeleton.

3T3 Cells↗
PubMed1995

Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein.

Amino Acid Sequence↗
PubMed1995

Expression of a mutant human fibrillin allele upon a normal human or murine genetic background recapitulates a Marfan cellular phenotype.

The Marfan syndrome (MFS) is a connective tissue disorder inherited as an autosomal dominant trait and caused by mutations in the gene encoding fibrillin, a 350-kD glycoprotein that multimerizes to form extracellular microfibrils. It has been unclear whether disease results from a relative deficiency of wild-type fibrillin; from a dominant-negative effect, in which mutant fibrillin monomers disrupt the function of the wild-type protein encoded by the normal allele; or from a dynamic and variable interplay between these two pathogenetic mechanisms. We have now addressed this issue in a cell culture system. A mutant fibrillin allele from a patient with severe MFS was expressed in normal human and murine fibroblasts by stable transfection. Immunohistochemical analysis of the resultant cell lines revealed markedly diminished fibrillin deposition and disorganized microfibrillar architecture. Pulse-chase studies demonstrated normal levels of fibrillin synthesis but substantially reduced deposition into the extracellular matrix. These data illustrate that expression of a mutant fibrillin allele, on a background of two normal alleles, is sufficient to disrupt normal microfibrillar assembly and reproduce the MFS cellular phenotype. This underscores the importance of the fibrillin amino-terminus in normal microfibrillar assembly and suggests that expression of the human extreme 5' fibrillin coding sequence may be sufficient, in isolation, to produce an animal model of MFS. Lastly, this substantiation of a dominant-negative effect offers mutant allele knockout as a potential strategy for gene therapy.

Alleles↗
PubMed1995

A Gly1127Ser mutation in an EGF-like domain of the fibrillin-1 gene is a risk factor for ascending aortic aneurysm and dissection.

Ascending aortic disease, ranging from mild aortic root enlargement to aneurysm and/or dissection, has been identified in 10 individuals of a kindred, none of whom had classical Marfan syndrome (MFS). Single-strand conformation analysis of the entire fibrillin-1 (FBN1) cDNA of an affected family member revealed a G-to-A transition at nucleotide 3379, predicting a Gly1127Ser substitution. The glycine in this position is highly conserved in EGF-like domains of FBN1 and other proteins. This mutation was present in 9 of 10 affected family members and in 1 young unaffected member but was not found in other unaffected members, in 168 chromosomes from normal controls, and in 188 chromosomes from other individuals with MFS or related phenotypes. FBN1 intragenic marker haplotypes ruled out the possibility that the other allele played a significant role in modulating the phenotype in this family. Pulse-chase studies revealed normal fibrillin synthesis but reduced fibrillin deposition into the extracellular matrix in cultured fibroblasts from a Gly1127Ser carrier. We postulate that the Gly1127Ser FBN1 mutation is responsible for reduced matrix deposition. We suggest that mutations such as this one may disrupt EGF-like domain folding less drastically than do substitutions of cysteine or of other amino acids important for calcium-binding that cause classical MFS. The Gly1127Ser mutation, therefore, produces a mild form of autosomal dominantly inherited weakness of elastic tissue, which predisposes to ascending aortic aneurysm and dissection later in life.

Adult↗
PubMed1994

Structure and localization on the X chromosome of the gene coding for the human filopodial protein moesin (MSN).

Moesin is a member of a recently discovered family of closely related proteins that includes ezrin, radixin, and merlin. It is widely expressed in different tissues and cells and has been localized to filopodia and other membranous protrusions that are important for cell-cell recognition and signaling and cell movement. Here, we have localized the coding gene (MSN) to Xq11.2-q12 by Southern and Western blot analyses of Chinese hamster x human somatic cell hybrids and by fluorescence chromosomal in situ hybridization. Moesin-like sequences were identified on chromosomes 5 and 6. The murine Msn locus was mapped to the X chromosome as well by studying a rodent x mouse hybrid panel. The structure of the human moesin gene has been determined. The 12 exons are distributed over > 30 kb, and the exon/intron junctions demarcate individual highly conserved domains. Primer extension analysis revealed two major start transcription sites, 184 and 133 bp upstream of the initiation codon. The 5'-flanking region is GC-rich, lacks a TATA box, and contains four SP1 and one AP1 binding sites.

Animals↗
PubMed1994

Quantitative differences in biosynthesis and extracellular deposition of fibrillin in cultured fibroblasts distinguish five groups of Marfan syndrome patients and suggest distinct pathogenetic mechanisms.

Pulse-chase studies of [35S]cysteine-labeled fibrillin were performed on fibroblast strains from 55 patients with Marfan syndrome (MFS), including 13 with identified mutations in the fibrillin-1 gene and 10 controls. Quantitation of the soluble intracellular and insoluble extracellular fibrillin allowed discrimination of five groups. Groups I (n = 8) and II (n = 19) synthesize reduced amounts of normal-sized fibrillin, while synthesis is normal in groups III (n = 6), IV (n = 18), and V (n = 4). When extracellular fibrillin deposition is measured, groups I and III deposit between 35 and 70% of control values, groups II and IV < 35%, and group V > 70%. A deletion mutant with a low transcript level from the mutant allele and seven additional patients have the group I protein phenotype. Disease in these patients is caused by a reduction in microfibrils associated with either a null allele, an unstable transcript, or an altered fibrillin product synthesized in low amounts. In 68% of the MFS individuals (groups II and IV), a dominant negative effect is invoked as the main pathogenetic mechanism. Products made by the mutant allele in these fibroblasts are proposed to interfere with microfibril formation. Insertion, deletion, and exon skipping mutations, resulting in smaller fibrillin products, exhibit the group II phenotype. A truncated form of fibrillin of 60 kD was identified with specific fibrillin antibodies in one of the group II cell culture media. Seven of the nine known missense mutations, giving rise to abnormal, but normal-sized fibrillin molecules, are in group IV.

Cells, Cultured↗