The Matrilin-3 T298M mutation predisposes for post-traumatic osteoarthritis in a knock-in mouse model.

OBJECTIVE: The human matrilin-3 T303M (in mouse T298M) mutation has been proposed to predispose for osteoarthritis, but due to the lack of an appropriate animal model this hypothesis could not be tested. This study was carried out to identify pathogenic mechanisms in a transgenic mouse line by which the mutation might contribute to disease development. METHODS: A mouse line carrying the T298M point mutation in the Matn3 locus was generated and features of skeletal development in ageing animals were characterized by immunohistology, micro computed tomography, transmission electron microscopy and atomic force microscopy. The effect of transgenic matrilin-3 was also studied after surgically induced osteoarthritis. RESULTS: The matrilin-3 T298M mutation influences endochondral ossification and leads to larger cartilage collagen fibril diameters. This in turn leads to an increased compressive stiffness of the articular cartilage, which, upon challenge, aggravates osteoarthritis development. CONCLUSIONS: The mouse matrilin-3 T298M mutation causes a predisposition for post-traumatic osteoarthritis and the corresponding knock-in mouse line therefore represents a valid model for investigating the pathogenic mechanisms involved in osteoarthritis development.

Fiberoptic microindentation technique for early osteoarthritis diagnosis: an in vitro study on human cartilage.

In this study, the capability of a fiber optic microindenter sensor to discriminate between healthy and slightly degenerated human articular cartilage samples is demonstrated. The purely optical indenter sensor is characterized by extremely reduced dimensions (0.125 mm in diameter and 27 mm in length) in comparison to existing indenter probes offering advantages for endoscopic deployment. The indenter sensor is intended to assist the surgeon in the identification of damaged articular cartilage. From each of seven specimens of human tibia plateau three samples showing different Outerbridge grading were extracted. On each sample stress-relaxation measurements were performed with eight indentation steps, each step being 40 µm and the relaxation of the material was observed for 240 s after each step. A viscoelastic model was used to fit the relaxation and to extract the characteristic parameters according to the model. A highly significant difference in stiffness (p value <0.01) was observed between the native (grade 0) and early diseased (grade 1) human cartilage samples demonstrating the potential of the fiber optic indenter for the diagnosis of cartilage breakdown.

Collagen II is essential for the removal of the notochord and the formation of intervertebral discs.

Collagen II is a fibril-forming collagen that is mainly expressed in cartilage. Collagen II-deficient mice produce structurally abnormal cartilage that lacks growth plates in long bones, and as a result these mice develop a skeleton without endochondral bone formation. Here, we report that Col2a1-null mice are unable to dismantle the notochord. This defect is associated with the inability to develop intervertebral discs (IVDs). During normal embryogenesis, the nucleus pulposus of future IVDs forms from regional expansion of the notochord, which is simultaneously dismantled in the region of the developing vertebral bodies. However, in Col2a1-null mice, the notochord is not removed in the vertebral bodies and persists as a rod-like structure until birth. It has been suggested that this regional notochordal degeneration results from changes in cell death and proliferation. Our experiments with wild-type mice showed that differential proliferation and apoptosis play no role in notochordal reorganization. An alternative hypothesis is that the cartilage matrix exerts mechanical forces that induce notochord removal. Several of our findings support this hypothesis. Immunohistological analyses, in situ hybridization, and biochemical analyses demonstrate that collagens I and III are ectopically expressed in Col2a1-null cartilage. Assembly of the abnormal collagens into a mature insoluble matrix is retarded and collagen fibrils are sparse, disorganized, and irregular. We propose that this disorganized abnormal cartilage collagen matrix is structurally weakened and is unable to constrain proteoglycan-induced osmotic swelling pressure. The accumulation of fluid leads to tissue enlargement and a reduction in the internal swelling pressure. These changes may be responsible for the abnormal notochord removal in Col2a1-null mice. Our studies also show that chondrocytes do not need a collagen II environment to express cartilage-specific matrix components and to hypertrophy. Furthermore, biochemical analysis of collagen XI in mutant cartilage showed that alpha1(XI) and alpha2 (XI) chains form unstable collagen XI molecules, demonstrating that the alpha3(XI) chain, which is an alternative, posttranslationally modified form of the Col2a1 gene, is essential for assembly and stability of triple helical collagen XI.

Perlecan maintains the integrity of cartilage and some basement membranes.

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

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.

Discoidin domain receptor 1 tyrosine kinase has an essential role in mammary gland development.

Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor tyrosine kinases, DDR1 and DDR2. Here, we used a recombinant fusion protein between the extracellular domain of DDR1 and alkaline phosphatase to detect specific receptor binding sites during mouse development. Major sites of DDR1-binding activity, indicative of ligand expression, were found in skeletal bones, the skin, and the urogenital tract. Ligand expression in the uterus during implantation and in the mammary gland during pregnancy colocalized with the expression of the DDR1 receptor. The generation of DDR1-null mice by gene targeting yielded homozygous mutant animals that were viable but smaller in size than control littermates. The majority of mutant females were unable to bear offspring due to a lack of proper blastocyst implantation into the uterine wall. When implantation did occur, the mutant females were unable to lactate. Histological analysis showed that the alveolar epithelium failed to secrete milk proteins into the lumen of the mammary gland. The lactational defect appears to be caused by hyperproliferation and abnormal branching of mammary ducts. These results suggest that DDR1 is a key mediator of the stromal-epithelial interaction during ductal morphogenesis in the mammary gland.

Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage?

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.

Profilin II is alternatively spliced, resulting in profilin isoforms that are differentially expressed and have distinct biochemical properties.

We deduced the structure of the mouse profilin II gene. It contains five exons that can generate four different transcripts by alternative splicing. Two transcripts encode different profilin II isoforms (designated IIa and IIb) that have similar affinities for actin but different affinities for polyphosphoinositides and proline-rich sequences. Profilins IIa and IIb are also present in humans, suggesting that all mammals have three profilin isoforms. Profilin I is the major form in all tissues, except in the brain, where profilin IIa is most abundant. Profilin IIb appears to be a minor form, and its expression is restricted to a limited number of tissues, indicating that the alternative splicing is tightly regulated. Western blotting and whole-mount in situ hybridization show that, in contrast to the expression of profilin I, the expression level of profilin IIa is developmentally regulated. In situ hybridization of adult brain sections reveals overlapping expression patterns of profilins I and IIa.

Functional consequences of integrin gene mutations in mice.

Integrins are cell-surface receptors responsible for cell attachment to extracellular matrices and to other cells. The application of mouse genetics has significantly increased our understanding of integrin function in vivo. In this review, we summarize the phenotypes of mice carrying mutant integrin genes and compare them with phenotypes of mice lacking the integrin ligands.

[Isolation and Characterization of Multipotent Precursor Cells from Murine Adipose Tissue using a Clinically Approved Cell Separation System]. / Isolation und Charakterisierung von multipotenten Vorläuferzellen aus murinem Fettgewebe mithilfe eines klinisch zugelassenen Aufbereitungssystems.

INTRODUCTION: Recent studies underscored the clinical potential of adipose-derived multipotent stem-/precursor cells (ASPCs). One of the main hurdles en route to clinical application was to isolate cells without having to perform expansion cultures outside the OR. A new generation of clinically approved, commercially available cell separation systems claims to provide ASPCs ready for application without further expansion cultures. However, it is unclear if the new systems yield sufficient cells of adequate quality for the use in autologous murine models. The aim of this study was to isolate and characterize adipose-derived precursor cells taken from the inguinal fat pat of wistar rats using InGeneron's clinically approved ARC™-cell separation system. MATERIALS AND METHODS: We isolated cells from the inguinal fat pad of 3 male Wistar rats according to the manufacturer's protocol. In order to reduce the influence of the atmospheric oxygen on the multipotent precursor cells, one half of the cell suspension was cultivated under hypoxia (2% O2) simulating physiological conditions for ASPCs. As a control, the other half of the cells were cultivated under normoxia (21% O2). Cell surface markers CD90, CD29, CD45 and CD11b/c were analyzed by FACS, and osteogenic and adipogenic differentiation of the ASPCs was performed. Finally, cellular growth characteristics were assessed by evaluation of the cumulative population doublings and CFU assay, and metabolic activity was evaluated by WST-1 assay. RESULTS: Processing time was 90 (± 12) min. 1 g of adipose tissue yielded approximately 60 000 plastic adhering cells. Both groups showed a high expression of the mesenchymal stem cell markers CD90 and CD29 while they were negative for the leucocyte markers CD45 and CD11b/c. A strong osteogenic differentiation and a sufficient adipogenic differentiation potential was proven for all ASPCs. Under hypoxia, ASPCs showed increased proliferation characteristics and CFU efficiency as well as a significantly increased metabolic activity. CONCLUSION: This study showed that sufficient multipotent ASPCs of appropriate quality can be isolated from the inguinal fat pad of Wistar rats using the ARC™-cell separation system. As shown in previous studies, cultivation of cells under hypoxic conditions increased their stemness. Our findings will enable future studies that focus on autologous transplantation of ASPCs in a rat model, which most closely resembles a possible clinical application.

Global fold determination from a small number of distance restraints.

We have designed a distance geometry-based method for obtaining the tertiary fold of a protein from a limited number of structure-specific distance restraints and the secondary structure assignment. Interresidue distances were predicted from patterns of conserved hydrophobic amino acids deduced from multiple alignments. A simple model chain representing the protein was then folded by projecting its distance matrix into Euclidean spaces with gradually decreasing dimensionality until a final three-dimensional embedding was achieved. Tangled conformations produced by the projection steps were eliminated using a novel filtering algorithm. Information on various aspects of protein structure such as accessibility and chirality was incorporated into the conformation refinement, increasing the robustness of the algorithm. The method successfully identified the correct folds of three small proteins from a small number of restraints, indicating that it could serve as a useful computational tool in protein structure determination from NMR data.

Type IX collagen is crucial for normal hearing.

cDNA microarray analysis indicated that COL9A1 and COL9A3 are highly expressed in the human inner ear, suggesting that type IX collagen has a crucial functional role in the inner ear. This study further confirmed, by means of real-time PCR, the presence of collagen type IX genes in the mouse inner ear. Immunocytochemical analysis also revealed that type IX collagen is distributed in the tectorial membrane, where it co-localizes with type II collagen, indicating that type IX collagen may contribute to the three-dimensional integrated structure of type II collagen molecules. Mice with targeted disruption of the col9a1 gene were shown through assessment by auditory brain stem response to have hearing loss, suggesting an important role of type IX collagen in maintaining normal hearing. At the light microscopic level, the tectorial membrane of knock-out mice was found to be abnormal in shape, and electron microscopy confirmed disturbance of organization of the collagen fibrils. An antibody against type II collagen failed to detect type II collagen in the tectorial membrane of type IX collagen knock-out mice, suggesting that a lack of type IX collagen may affect the three-dimensional structure of type II collagen molecules. These findings indicate that genes encoding each chain of type IX collagen may fulfill an important function associated with the tectorial membrane in the auditory system.

Mouse models for extracellular matrix diseases.

Mutations of a number of genes encoding for extracellular matrix (ECM) proteins in mice have provided new insights regarding their role during development and disease. Many mouse strains have helped to verify the link between mutation and disease in humans, and others have produced unexpected phenotypes and identified new functions for ECM proteins. Finally, some null mutations in ECM genes provide no phenotypic alterations in mice, confronting the scientific community with a new challenge to search for their functions. This review lists all mouse strains with spontaneous and experimentally induced mutations in ECM genes. The phenotypes of these mice are discussed in comparison with the human diseases.

Sequence, structure and chromosomal localization of Crtm gene encoding mouse cartilage matrix protein and its exclusion as a candidate for murine achondroplasia.

The mouse cartilage matrix protein gene (Crtm) was isolated from a cosmid library using a mouse Crtm cDNA fragment as probe. Crtm spans 12.2 kb from the start of translation to the polyadenylation signal sequence and comprises eight exons. Sequencing of the 1.9 kb 5' flanking region revealed a TATA-like box 72 bp upstream from the initiator Met codon as well as several cis-acting motifs known to bind eukaryotic transcription factors. Analysis of the exon-intron junctions demonstrated that the last intron does not follow the gt/ag rule but belongs to the minor class of pre-mRNA introns that contain "at" and "ac" at their 5'and 3' ends, respectively. Single-strand conformation polymorphism analysis was used to map Crtm to the distal part of chromosome 4 between the microsatellite markers D4Mit16 and D4Mit339. Achodroplasia (cn), a recessive skeletal disorder in mice, has already been mapped to this region. Immunostaining for CMP and sequence of Crtm in cn/cn mice failed to reveal any disease-specific mutations, suggesting that mutations in Crtm do not cause achondroplasia.

The zonal expression of chicken cartilage matrix protein gene in the developing skeleton of transgenic mice.

Cartilage matrix protein (CMP) is a major noncollagenous glycoprotein of hyaline cartilage with a molecular mass of about 148 kDa. It has been proposed to be involved in matrix organization by its interactions with proteoglycan and type II collagen. The 54-kDa monomers form homotrimers stabilized by disulfide bonds. The gene for chicken cartilage matrix protein was isolated, and its regulation has been studied recently in transient expression experiments. To learn more about the spatial and temporal expression of the gene during ontogenic development, we created transgenic mice via microinjection of a 21.8-kb genomic fragment, encoding the chicken cartilage matrix protein. None of the founder animals exhibited any abnormal phenotype. The developmental stage-specific expression of the transgene was examined by immunostaining with a chicken CMP specific antiserum at different stages of embryonic development in cartilage from different sources: lower and upper limb, vertebrae, ribs and nasal septum. The level of transgene expression showed marked differences in various zones of cartilage. Briefly, high levels were found in the zones of proliferating chondrocytes, while little if any transgene product was detected in the very early and hypertrophic stage of chondrogenesis. The expression pattern of the transgene correlated with the endogenous mouse CMP and did not cause any morphological changes detectable by microscopic analysis of cartilage. These data indicate that the injected CMP gene with its flanking sequences contained all the information necessary for cell type-specific expression in transgenic mice.

Stage-and tissue-specific expression of a Col2a1-Cre fusion gene in transgenic mice.

To achieve chondrocyte-specific deletion of floxed genes we generated a transgenic mouse line expressing the Cre recombinase under the control of the mouse type II collagen gene (Col2a1) regulatory regions. Northern and in situ hybridization analyses demonstrated the expression of the transgene (Col2a1-Cre) in cartilaginous tissues. To test the excision efficiency of Cre, the Col2a1-Cre strain was crossed with the ROSA26 reporter strain. LacZ staining of double transgenic mice revealed Cre activity in both chondrogenic and non-chondrogenic tissues. During early embryonic development (E9.5-11.5), LacZ expression was detected in tissues where the endogenous Col2a1 transcript is expressed such as the otic capsule, notochord, developing brain, sclerotome and mesenchymal condensations of future cartilage. At later stages, Cre activity was observed in all cartilaginous tissues with virtually 100% of chondrocytes being LacZ positive. These data suggest that the Col2a1-Cre mouse strain described here can be useful to achieve Cre-mediated recombination in Col2a1 expressing cells, especially in chondrocytes.

Structure of the rabbit kappa-casein encoding gene: expression of the cloned gene in the mammary gland of transgenic mice.

The rabbit kappa-casein (kappa-Cas) encoding gene has been isolated as a series of overlapping DNA fragments cloned from a rabbit genomic library constructed in bacteriophage lambda EMBL3. The clones harboured the 7.5-kb gene flanked by about 2.1 kb upstream and 9 kb downstream sequences. The cloned gene is the most frequently occurring of two kappa-Cas alleles identified in New Zealand rabbits. Comparison of the corresponding domains in rabbit and bovine kappa-Cas shows that both genes comprise 5 exons and that the exon/intron boundary positions are conserved whereas the introns have diverged considerably. The first three introns are shorter in the rabbit, the second intron showing the greatest difference between the two species: 1.35 kb instead of 5.8 kb in the bovine gene. Repetitive sequence motives reminiscent of the rabbit C type repeat and the complementary inverted C type repeat were identified in the fourth and first introns, respectively. Transgenic mice were produced by microinjecting into mouse oocytes an isolated genomic DNA fragment which contained the entire kappa-Cas coding region, together with 2.1-kb 5' and 4.0-kb 3' flanking region. Expression of transgene rabbit kappa-Cas mRNA could be detected in the mammary gland of lactating transgenic mice and the production of rabbit kappa-Cas was detected in milk using species-specific antibodies. The cloned gene is thus functional.

MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture.

Microtubule-associated protein 2 (MAP2), a long, filamentous molecule thought to cross-link dendritic cytoskeleton, is rich in PEST sequences, putative signals for rapid proteolytic degradation. It is suggested that MAP2 is indeed highly susceptible to protease, e.g. calpain, attack, which is needed for a plastic change, but actual breakdown depends on the regulation of protease(s). Phosphorylation is expected to make the molecule longer and rigid, similarly to what was observed with the related tau protein. Such a structural transition may provide a mechanism for the putative role of MAP2 in dendritic branching.

Molecular kinetic modelling of associative learning.

Three molecular (enzyme) kinetic models have been designed that exhibit the basic properties of associative learning (classical conditioning). The enzyme systems are acted upon by an 'unconditioned' and a 'conditioned' ligand: temporally paired application of the two ligands leads to covalent enzyme modification, which serves as 'memory trace'. The behaviour of the systems has been investigated by computer simulation. Although the models are hypothetic, they do not contain biochemically inconceivable steps. The models demonstrate that already fairly simple molecular events may produce the phenomenology of associative learning.

Detecting changes in neuronal activities induced by N-methyl-D-aspartate receptor blockade using non-linear dynamics techniques.

The dynamics of N-methyl-D-aspartate receptor blockade-induced transitions between two types of intracellularly recorded spontaneous membrane potential oscillation from cat thalamic neurons have been studied using non-linear dynamics techniques. We report that, as previously predicted by theoretical studies, the number of degrees of freedom of these oscillations (the minimal number of independent variables governing the activity) is small, i.e. they are low dimensional. The N-methyl-D-aspartate receptor antagonists DL-2-amino-5-phosphono-valeric acid and ketamine, which transformed one type of oscillation into another, decreased the calculated dimension. DL-2-Amino-5-phosphono-valeric acid had no effect on the dimension when Mg2+ was present in the perfusion medium. The decrease in dimension was gradual and its time-course had a sigmoidal shape. It is suggested that the application of the machinery of dynamical systems theory might help to detect and monitor drug-induced membrane potential state transitions and to identify the factors underlying membrane potential oscillations.