Biomedical subjects

R Fässler

Publications and source records attributed to R Fässler.

At least 91 records · Page 5Linked to original sources

PubMed1998

Mutational analysis of the potential phosphorylation sites in the cytoplasmic domain of integrin beta1A. Requirement for threonines 788-789 in receptor activation.

To investigate the role of the potential phosphorylation sites in the cytoplasmic domain of integrin beta1A, point mutated variants of the protein were stably expressed in the beta1-deficient cell line GD25. Mutants T777A, Y783F, S785A, and Y795F were fully active in promoting cell adhesion, de novo formation of focal contacts, formation of fibronectin fibrils, and activation of focal adhesion kinase. Thus, phosphorylation of these residues is not required for several basic functions of integrin beta1A. On the other hand, the TT788-9AA mutant, was defective in mediating cell attachment and did not contribute to fibronectin fibril formation. The conformation of the extracellular domain was shifted towards an inactive state as measured by binding of the monoclonal antibody 9EG7. Antibody induced clustering of beta1ATT788-9AA demonstrated that the mutant cytoplasmic part was functional in mediating activation of focal adhesion kinase. Therefore, we conclude that threonines 788-789, which are conserved among most integrin beta subunits, are of critical importance for integrin function due to effects on the extracellular conformation of the receptor.

Amino Acid Sequence↗

PubMed1997

Targeted inactivation of plectin reveals essential function in maintaining the integrity of skin, muscle, and heart cytoarchitecture.

Previous studies suggest that plectin, a versatile cytoskeletal linker protein, has an important role in maintaining the structural integrity of diverse cells and tissues. To establish plectin's function in a living organism, we have disrupted its gene in mice. Plectin (-/-) mice died 2-3 days after birth exhibiting skin blistering caused by degeneration of keratinocytes. Ultrastructurally, hemidesmosomes and desmosomes appeared unaffected. In plectin-deficient mice, however, hemidesmosomes were found to be significantly reduced in number and apparently their mechanical stability was altered. The skin phenotype of these mice was similar to that of patients suffering from epidermolysis bullosa simplex (EBS)-MD, a hereditary skin blistering disease with muscular dystrophy, caused by defects in the plectin gene. In addition, plectin (-/-) mice revealed abnormalities reminiscent of minicore myopathies in skeletal muscle and disintegration of intercalated discs in heart. Our results clearly demonstrate a general role of plectin in the reinforcement of mechanically stressed cells. Plectin (-/-) mice will provide a useful tool for the study of EBS-MD, and possibly other types of plectin-related myopathies involving skeletal and cardiac muscle, in an organism amenable to genetic manipulation.

Animals↗

PubMed1997

Mapping of a defined neurocan binding site to distinct domains of tenascin-C.

Neurocan is a member of the aggrecan family of proteoglycans which are characterized by NH2-terminal domains binding hyaluronan, and COOH-terminal domains containing C-type lectin-like modules. To detect and enhance the affinity for complementary ligands of neurocan, the COOH-terminal neurocan domain was fused with the NH2-terminal region of tenascin-C, which contains the hexamerization domain of this extracellular matrix glycoprotein. The fusion protein was designed to contain the last downstream glycosaminoglycan attachment site and was expressed as a proteoglycan. In ligand overlay blots carried out with brain extracts, it recognized tenascin-C. The interaction was abolished by the addition of EDTA, or TNfn4,5, a bacterially expressed tenascin-C fragment comprising the fourth and fifth fibronectin type III module. The fusion protein directly reacted with this fragment in ligand blot and enzyme-linked immunosorbent assay procedures. Both tenascin-C and TNfn4,5 were retained on Sepharose 4B-linked carboxyl-terminal neurocan domains, which in BIAcore binding studies yielded a KD value of 17 nM for purified tenascin-C. We conclude that a divalent cation-dependent interaction between the COOH-terminal domain of neurocan and those fibronectin type III repeats is substantially involved in the binding of neurocan to tenascin-C.

Amino Acid Sequence↗

PubMed1997

Beta 1 integrin is essential for teratoma growth and angiogenesis.

Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of beta 1 integrin during teratoma formation, we compared teratomas induced by normal and beta1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, beta 1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of beta 1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in beta1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface. Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell-derived endothelial cells. In contrast, beta 1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore, endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although beta 1- deficient endothelial cells were absent in teratomas, beta 1-null ES cells could differentiate in vitro into endothelial cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in beta1-null embryoid bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive branching of blood vessels in normal embryoid bodies, it had no effect in beta 1-null embryoid bodies.

Animals↗

PubMed1997

Sequence and chromosomal localization of the mouse brevican gene.

Brevican is a brain-specific proteoglycan belonging to the aggrecan family. Phage clones containing the complete mouse brevican open reading frame of 2649 bp and the complete 3'-untranslated region of 341 bp were isolated from a mouse brain cDNA library, and cosmid clones containing the mouse brevican gene were isolated from a genomic library using a PCR-generated DNA fragment as probe. The obtained genomic sequence of 13,700 nucleotides revealed that the murine gene has a size of approximately 13 kb and contains the sequence of the mRNA for the secreted brevican isoform on 14 exons. The exon-intron structure reflected the structural organization of the multidomain protein brevican. No consensus TATA sequence was found upstream of the first exon, and RNase protection experiments revealed multiple transcriptional start sites for the brevican gene. The first part of the sequence of intron 8 corresponded to an alternative brevican cDNA, coding for a GPI-linked isoform. Single strand conformation polymorphism analysis mapped the brevican gene (Bcan) to chromosome 3 between the microsatellite markers D3Mit22 and D3Mit11.

Amino Acid Sequence↗

PubMed1997

Absence of the alpha1(IX) chain leads to a functional knock-out of the entire collagen IX protein in mice.

Cartilage fibrils contain collagen II as well as smaller amounts of collagens IX and XI. The three collagens are thought to co-assemble into cartilage-specific arrays. The precise role of collagen IX in cartilage has been addressed previously by generating mice harboring an inactivated Col9a1 gene encoding the alpha1(IX) chain, i.e. one of the three constituent chains of collagen IX (Fässler, R., Schnegelsberg, P. N. J., Dausman, J., Shinya, T., Muragaki, Y., McCarthy, M. T., Olsen, B. R., and Jaenisch, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5070-5074). The animals did not produce alpha1(IX) mRNA or polypeptides and were born with no conspicuous skeletal abnormality but post-natally developed early onset osteoarthritis. Here we show that the deficiency in alpha1(IX) chains leads to a functional knock-out of all polypeptides of collagen IX, whereas the Col9a2 and Col9a3 genes were normally transcribed. Therefore, synthesis of alpha1(IX) polypeptides is essential for the assembly of heterotrimeric collagen IX molecules. Surprisingly, cartilage fibrils of all shapes and banding patterns found in normal newborn, adolescent, or adult mice were formed in transgenic animals, although they lacked collagen IX. Therefore, collagen IX is not essential, and may be functionally redundant, in fibrillogenesis in cartilage in vivo. The protein is required, however, for long term tissue stability, presumably by mediating interactions between fibrillar and extrafibrillar macromolecules.

Animals↗

PubMed1997

Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta.

Expression of AP-2 transcription factors has been detected previously in embryonic renal tissues. We show here that AP-2beta -/- mice complete embryonic development and die at postnatal days 1 and 2 because of polycystic kidney disease. Analyses of kidney development revealed that induction of epithelial conversion, mesenchyme condensation, and further glomerular and tubular differentiation occur normally in AP-2beta-deficient mice. At the end of embryonic development expression of bcl-X(L), bcl-w, and bcl-2 is down-regulated in parallel to massive apoptotic death of collecting duct and distal tubular epithelia. Addressing the molecular mechanism we show that transfection of AP-2 into cell lines in vitro strongly suppresses c-myc-induced apoptosis pointing to a function of AP-2 in programming cell survival during embryogenesis. The position of the human AP-2beta gene was identified at chromosome 6p12-p21.1, within a region that has been mapped for autosomal recessive polycystic kidney disease (ARPKD). Sequence analyses of ARPKD patients and linkage analyses using intragenic polymorphic markers indicate that the AP-2beta gene is located in close proximity to but distinct from the ARPKD gene.

Animals↗

PubMed1997

Absence of integrin alpha 7 causes a novel form of muscular dystrophy.

Integrin alpha 7 beta 1 is a specific cellular receptor for the basement membrane protein laminin-1 (refs 1,2), as well as for the laminin isoforms -2 and -4 (ref. 3). The alpha 7 subunit is expressed mainly in skeletal and cardiac muscle and has been suggested to be involved in differentiation and migration processes during myogenesis. Three cytoplasmic and two extracellular splice variants that have been described are developmentally regulated and expressed in different sites in the muscle. In adult muscle, the alpha 7A and alpha 7B subunits are concentrated in myotendinous junctions but can also be detected in neuromuscular junctions and along the sarcolemmal membrane. To study the potential involvement of alpha 7 integrin, during myogenesis and its role in muscle integrity and function, we generated a null allele of the alpha 7 gene (Itga7) in the germline of mice by homologous recombination in embryonic stem (ES) cells. Surprisingly, mice homozygous for the mutation are viable and fertile, indicating that the alpha 7 beta 1 integrin is not essential for myogenesis. However, histological analysis of skeletal muscle revealed typical symptoms of a progressive muscular dystrophy starting soon after birth, but with a distinct variability in different muscle types. The observed histopathological changes strongly indicate an impairment of function of the myotendinous junctions. These findings demonstrate that alpha 7 beta 1 integrin represents an indispensable linkage between the muscle fibre and the extracellular matrix that is independent of the dystrophin-dystroglycan complex-mediated interaction of the cytoskeleton with the muscle basement membrane.

Animals↗

PubMed1997

The subcellular distribution of the high molecular mass protein, HD1, is determined by the cytoplasmic domain of the integrin beta 4 subunit.

The high molecular mass protein, HD1, is a structural protein present in hemidesmosomes as well as in distinct adhesion structures termed type II hemidesmosomes. We have studied the distribution and expression of HD1 in the GD25 cells, derived from murine embryonal stem cells deficient for the beta 1 integrin subunit. We report here that these cells possess HD1 but not BP230 or BP180; two other hemidesmosomal constituents, and express only traces of the alpha 6 beta 4 integrin. By immunofluorescence and interference reflection microscopy HD1 was found together with vinculin at the end of actin filaments in focal contacts. In OVCAR-4 cells, derived from a human ovarian carcinoma which, like GD25 cells, only weakly express alpha 6 beta 4, HD1 was also localized in focal contacts. Upon transfection of both GD25 and OVCAR-4 cells with cDNA for the human beta 4 subunit the subcellular distribution of HD1 changed significantly. HD1 is then no longer present in focal contacts but in other structures at cell-substrate contacts, colocalized with alpha 6 beta 4. These junctional complexes are probably the equivalent of the type II hemidesmosomes. Transfection of GD25 cells with beta 1 cDNA did not affect the distribution of HD1, which indicates that the localization of HD1 in focal contacts was not due to the absence of beta 1. Moreover, in GD25 cells transfected with cDNA encoding a beta 4/beta 1 chimera, in which the cytoplasmic domain of beta 4 was replaced by that of beta 1, the distribution of HD1 was unaffected. Our findings indicate that the cytoplasmic domain of beta 4 determines the subcellular distribution of HD1 and emphasize the important role of alpha 6 beta 4 in the assembly of hemidesmosomes and other junctional adhesive complexes containing HD1.

Animals↗

PubMed1997

Genetic analysis of beta1 integrin function: confirmed, new and revised roles for a crucial family of cell adhesion molecules.

Integrins are heterodimeric cell adhesion proteins connecting the extracellular matrix to the cytoskeleton and transmitting signals in both directions. These integrins are suggested to be involved in many different biological processes such as growth, differentiation, migration, and cell death. Of more than 20 known integrins, 10 contain the nearly ubiquitously expressed beta1 integrin subunit. Disruption of the beta1 integrin gene by homologous recombination allows us to assess the supposed functions of beta1 containing integrins in vivo in a new way. This review will present and discuss recent findings derived from such studies concerning the biological roles of beta1 integrins in early development, differentiation and migration, hematopoiesis, tumorigenesis, and supramolecular assembly of extracellular matrix proteins. While several former results were confirmed, others were contradicted and new functions found, significantly changing the previous view of beta1 integrin function in vivo.

Animals↗

PubMed1996

Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II.

Cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinases (cGKs) mediate cellular signaling induced by nitric oxide and cGMP. Mice deficient in the type II cGK were resistant to Escherichia coli STa, an enterotoxin that stimulates cGMP accumulation and intestinal fluid secretion. The cGKII-deficient mice also developed dwarfism that was caused by a severe defect in endochondral ossification at the growth plates. These results indicate that cGKII plays a central role in diverse physiological processes.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Differentiation of embryonal stem cells into keratinocytes: comparison of wild-type and beta 1 integrin-deficient cells.

beta 1 Integrins are known to regulate terminal differentiation and morphogenesis in the adult epidermis. We have investigated their role in the embryonic development of keratinocytes by comparing the differentiation of wild-type and beta 1-null mouse embryonal stem (ES) cells. By 12-15 days in culture, differentiation of embryonic or simple epithelial cells occurred in both ES cell populations, as detected by expression of keratins 8, 18, and 19. From 21 days, expression of keratins 10 and 14 and of the cornified envelope precursor involucrin indicated that some of the wild-type cells had differentiated into keratinocytes. In contrast, keratinocyte markers were not expressed in beta 1-null cultures. The beta 1-null cells failed to express the alpha 2 and alpha 3 integrin subunits on the cell surface, consistent with the association of these a subunits with beta 1. Furthermore, alpha 6 and beta 4 expression was reduced in the beta 1-null cultures. Although beta 1-null ES cells failed to undergo differentiation into keratinocytes in vitro, they did form keratinocyte cysts expressing alpha 6 beta 4, keratins 1 and 14, and involucrin when allowed to form teratomas by subcutaneous injection in mice; furthermore, beta 1-null keratinocytes were found in the epidermis of a wild-type/beta 1-null chimeric mouse. As judged by immunofluorescence microscopy, extracellular matrix assembly was severely impaired in beta 1-null ES cell cultures, but not in the teratomas or chimeric mouse skin. We therefore speculate that the failure of beta 1-null cells to differentiate into keratinocytes in vitro may reflect an inability to assemble a basement membrane.

Animals↗

PubMed1996

Skin wounds and severed nerves heal normally in mice lacking tenascin-C.

A large number of functions have been demonstrated for tenascin-C by antibody perturbation assays and in vitro cell culture experiments. However, these results contrast sharply with the lack of any apparent phenotype in mice with a genetic deletion of tenascin-C. A possible explanation for the lack of phenotype would be expression of some altered but functional tenascin-C in the mutant. We report the generation of an independent tenascin-C null mouse and conclude that the original tenascin-C knockout, which is genetically very similar to ours, is also a true null. As found previously, the absence of tenascin-C has no influence on development, adulthood, life span, and fecundity. We have studied in detail two models of wound healing. After axotomy, the regeneration of the sciatic nerve is not altered without tenascin-C. During healing of cutaneous wounds, deposition of collagen I, fibulin-2, and nidogen is identical in mutant and wild-type mice. In contrast. fibronectin appears diminished in wounds of tenascin-C-deficient mice. However, the lack of tenascin-C together with the reduced amount of fibronectin has no influence on the quality of the healing process.

Animals↗

PubMed1996

Impaired migration but not differentiation of haematopoietic stem cells in the absence of beta1 integrins.

Adhesive interactions mediated by integrins of the beta1 subfamily are thought to be critical in controlling differentiation and migration of blood cell precursors. Here we report that chimaeric mice generated with beta1-integrin-deficient embryonic stem (ES) cells lack beta1(-/-) cells in blood and in haematopoietic organs such as spleen, thymus and bone marrow. Chimaeric embryos contain beta1-null haematopoietic cells in the yolk sac and in fetal blood but not in fetal liver. We show that such beta1(-/-) haematopoietic stem cells derived from yolk sac of 10.5-day-old chimaeric embryos readily generate erythroid and myeloid colonies and that beta1(-/-) ES cells can differentiate into mature B lymphocytes in vitro. Our results indicate that haematopoietic stem cells lacking beta1 integrins can form and differentiate into different lineages but cannot colonize the fetal liver.

Animals↗

PubMed1996

Differential regulation of fibulin, tenascin-C, and nidogen expression during wound healing of normal and glucocorticoid-treated mice.

Expression and distribution of fibulins, nidogen, and tenascin-C were analyzed in healing skin wounds of normal and dexamethasone-treated mice. In normal mice both tenascin-C and fibulin-2 showed a marked increase in mRNA expression, which declined to normal levels after completion of skin repair. These two matrix proteins are found throughout the granulation tissue and persisted there after mRNA expression had ceased. Fibulin-1 is present in normal skin and in wounds but is not distinctly upregulated during the healing process. Nidogen, however, is expressed uniformly throughout the granulation tissue early in wound healing, has a peak expression around Day 7, and selectively localizes to basement membranes after healing is accomplished. Dexamethasone treatment led to a decreased expression of tenascin-C in healing wounds but had no effect on fibulin-2 expression. In vitro experiments revealed that growth factors like TGF-beta 1 can partly counteract glucocorticoid action. These data therefore provide some molecular interpretations for the well-known glucocorticoid suppression of wound healing. They also indicate that repair involves complex regulatory processes which are obviously different for each of the four proteins studied.

3T3 Cells↗

PubMed1996

Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice.

It has long been speculated that pro-inflammatory cytokines play an important role in wound repair. However, little is known about the temporal and spatial expression pattern of these cytokines during normal and impaired wound healing. In this study we show a strong and early induction of interleukins 1 alpha and beta (IL-alpha and beta) and of tumour necrosis factor alpha (TNF-alpha) expression after cutaneous injury. Highest levels of these cytokines were seen as early as 12-24 h after wounding. After completion of the proliferative phase of wound healing, mRNA levels of these cytokines returned to the basal level. During the early phase of wound repair, proinflammatory cytokines were predominantly expressed in polymorphonuclear leukocytes, suggesting a novel function of these cells in the initiation of wound healing. At later stages of the repair process, expression of IL-1 alpha, IL-1 beta and TNF-alpha was also seen in macrophages. Furthermore, TNF-alpha was detected in the hyperproliferative epithelium at the wound edge and IL-1 alpha was found in keratinocytes of the hair follicles. Induction of these cytokines after injury was significantly reduced during wound repair in healing-impaired glucocorticoid-treated mice. This finding demonstrates that wound healing defects are associated with impaired cytokine expression and suggests that the early induction of these genes is important for normal repair.

Animals↗

PubMed1996

Genetic analyses of integrin function in mice.

Recent mutations of most integrin genes in the mouse have provided new exciting insights into the role of these integrins in cell-extracellular matrix interactions during development. The embryonic lethal phenotypes obtained by ablating integrins which are predominantly expressed in the mesenchyme confirmed the essential function of those integrins in morphogenesis. In contrast, null alleles for several epithelial integrins which bind components of basement membranes showed milder phenotypes, suggesting the presence of novel and unexpected redundant and compensatory mechanisms.

Animals↗

PubMed1996

Beta 1 integrin-dependent and -independent polymerization of fibronectin.

The mouse cell line GD25, which lacks expression of the beta 1 family of integrin heterodimers due to disruption of the beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse beta 1 integrin subunit. In a stably transformed clone (GD25-beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-beta 1A attached to fibronectin and formed focal contacts which contained alpha v beta 3, but no detectable alpha 5 beta 1A. The presence of GRGDS peptide allowed alpha 5 beta 1A to locate to focal contacts of GD25-beta 1A cultured on fibronectin, while the beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 beta 1A and alpha v beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v beta 3 was colocalized with the fibronectin fibrils in GD25-beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 beta 1A and alpha v beta 3 are able to support adhesion to fibronectin, alpha v beta 3 dominates in the formation of focal contacts, and alpha 5 beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of beta 1 integrins.

Animals↗