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.

[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.

Integrins α2ß1 and α11ß1 regulate the survival of mesenchymal stem cells on collagen I.

Although mesenchymal stem cells (MSCs) are the natural source for bone regeneration, the exact mechanisms governing MSC crosstalk with collagen I have not yet been uncovered. Cell adhesion to collagen I is mostly mediated by three integrin receptors - α1ß1, α2ß1 and α11ß1. Using human MSC (hMSC), we show that α11 subunit exhibited the highest basal expression levels but on osteogenic stimulation, both α2 and α11 integrins were significantly upregulated. To elucidate the possible roles of collagen-binding integrins, we applied short hairpin RNA (shRNA)-mediated knockdown in hMSC and found that α2 or α11 deficiency, but not α1, results in a tremendous reduction of hMSC numbers owing to mitochondrial leakage accompanied by Bcl-2-associated X protein upregulation. In order to clarify the signaling conveyed by the collagen-binding integrins in hMSC, we analyzed the activation of focal adhesion kinase, extracellular signal-regulated protein kinase and serine/threonine protein kinase B (PKB/Akt) kinases and detected significantly reduced Akt phosphorylation only in α2- and α11-shRNA hMSC. Finally, experiments with hMSC from osteoporotic patients revealed a significant downregulation of α2 integrin concomitant with an augmented mitochondrial permeability. In conclusion, our study describes for the first time that disturbance of α2ß1- or α11ß1-mediated interactions to collagen I results in the cell death of MSCs and urges for further investigations examining the impact of MSCs in bone conditions with abnormal collagen I.

What mouse mutants teach us about extracellular matrix function.

For many years the extracellular matrix was viewed as a benign scaffold for arranging cells within connective tissues, but it is now being redefined as a dynamic, mobile, and flexible key player in defining cellular behavior. Gene targeting, transgene expression, and spontaneous mutations of extracellular matrix proteins in mice have greatly accelerated our mechanistic view of the structural and instructive functions of the extracellular matrix in developmental and regenerative processes. This review summarizes the phenotypes of genetic mouse models carrying mutations in extracellular matrix proteins, with specific emphasis on recent advances. The application of reverse genetics has demonstrated the multifunctionality of matrix proteins in a biological context and, in addition, has brought a novel perspective to the understanding of human pathologies.

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.

Distribution of the collagen-binding integrin alpha10beta1 during mouse development.

We have previously identified and characterised the collagen type II-binding integrin subunit alpha10, which is a member of the beta1 family and is expressed by chondrocytes. In the present study, we examined the expression of the alpha10 integrin in various mouse tissues. Immunohistochemical analysis of alpha10 on cryosections from 3-day-old mice demonstrated that alpha10beta1 was present in the hyaline cartilage of joints, vertebral column, trachea and bronchi. In addition, alpha10 was found in the ossification groove of Ranvier, in the aortic and atrioventricular valves of the heart and in the fibrous tissue lining skeletal muscle and ligaments. Overall, the distribution was distinct from that of the collagen-binding integrins alpha1beta1 and alpha2beta1. We also found that alpha10beta1was the dominating collagen-binding integrin during cartilage development. Expression of alpha10 appeared at embryonic day 11.5 (E11.5) at the same time as chondrogenesis started as judged by collagen type II expression. At E13.5, alpha10 was present throughout the anlage as well as in the perichondrium and in mesenchyme just outside the perichondrium, where it localised with collagen type I. Four weeks after birth, alpha10 was prominent both at the articular surface and in the growth plate. In conclusion, we found that integrin alpha10beta1 was a major collagen-binding integrin during cartilage development and in mature hyaline cartilage. In addition, we found that alpha10beta1 was present in some fibrous tissues.

The role of collagen II and cartilage fibril-associated molecules in skeletal development.

OBJECTIVE: The extracellular matrix (ECM) of hyaline cartilage contains an elaborated collagen fibrillar network, which is essential for the mechanical stability and the proper function of the tissue. Cartilage collagen fibrils consist of collagen II, the quantitatively minor collagens IX and XI, and several non-collagenous fibril-associated proteins. To understand the role some of these molecules in skeletal development, we have generated transgenic mouse strains harboring ablated genes for collagens II and IX, and matrilin-1. DESIGN: Mice lacking collagen II, collagen IX and matrilin-1 have been established earlier in our laboratory using standard techniques. To determine the consequences of the null mutations we used skeletal staining, histochemical and immunohistochemical assays, in situ hybridization and ultrastructural analysis. RESULTS: Transgenic mice deficient in collagen II (Col2a1-/-) die at birth and display a severely malformed skeleton characterized by abnormal endochondral ossification and impaired intervertebral disc development. Mice lacking collagen IX (Col9a1-/-) are viable and develop an osteoarthritis-like phenotype in knee joints between 9-12 months of age. To test the possibility that the reduction in collagen II content has an influence on the onset of degenerative changes of articular cartilage, we have generated mice, which are heterozygous for the collagen II null mutation and homozygous for the collagen IX null mutation. Col2a1+/- Col9a1-/- mice show no accelerated development of osteoarthritis compared with the collagen IX knockout animals. Finally, mice lacking matrilin-1, a non-collagenous glycoprotein that binds to both collagen fibrils and aggrecan, develop normally without detectable abnormalities in their skeleton. CONCLUSIONS: Our transgenic mouse strains carrying null mutations in genes encoding cartilage ECM proteins demonstrate that these proteins have different roles during skeletal development. Collagen II is important for cartilage formation, collagen IX for cartilage maintenance and matrilin-1 is redundant.

Characterization of the mouse matrilin-4 gene: a 5' antiparallel overlap with the gene encoding the transcription factor RBP-l.

We have isolated and characterized the gene encoding mouse matrilin-4 (Matn4), an extracellular matrix protein present in a broad spectrum of tissues. The gene spanned 16 kb, consisted of 12 exons, and localized to chromosome 2. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, did not follow the GT-AG rule and belonged to the subgroup of introns having AT-AC at the ends. Matn4 contained two exons in the 5' UTR that could be alternatively spliced. We localized a major and a minor transcription start site to two different untranslated exons: exon 0a and exon 0b. Matn4 divergently overlapped 5' with the gene encoding RBP-L (for recombining binding protein suppressor of hairless-like; Rbpsuhl), a transcription factor with homology to RBP-JK. Exon 1 of Rbpsuhl was located in the second intron of Matn4, whereas exon 0a, the first exon of Matn4, was located in the second intron of Rbpsuhl. The second exons of the respective genes overlapped in an antisense orientation. We mapped the major transcription start of Rbpsuhl to a position approximately 150 nt upstream of the splice acceptor site of the first intron, leading to the synthesis of a truncated variant of RBP-L probably missing the amino-terminal 121 amino acid residues. We analyzed the expression of the different Matn4 and Rbpsuhl transcripts by quantitative RT-PCR; this showed the highest expression for both genes in lung and brain. In situ hybridization of brain sections showed a partially overlapping expression pattern for the two genes.

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.

A novel gene, tendin, is strongly expressed in tendons and ligaments and shows high homology with chondromodulin-I.

Chondromodulin-I (CHM1) was identified recently as an angiogenesis inhibitor in cartilage. It is highly expressed in the avascular zones of cartilage but is absent in the late hypertrophic region, which is invaded by blood vessels during enchondral ossification. Blast searches with the C-terminal part of CHM1 in available databases led to the identification of human and mouse cDNAs encoding a new protein, Tendin, that shares high homology with CHM1. Based on computer predictions, Tendin is a type II transmembrane protein containing a putative proteinase cleavage and two glycosylation sites. Northern assays with mouse RNAs demonstrated strong expression of a 1.5-kb tendin transcript in the diaphragm, skeletal muscle, and the eye and low levels of expression in all other tissues investigated. In 17.5-day-old mouse embryos, in situ hybridization revealed high levels of tendin transcript in tendons and ligaments. Additional signals were detected in brain and spinal cord, liver, lung, bowels, thymus, and eye. Cartilage, where CHM1 is found, revealed low levels of tendin m-RNA. In adult mice, tendin is expressed in neurons of all brain regions and the spinal cord. The tendin gene is localized in the human Xq22 region, to which several human diseases have been mapped.

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.

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.

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 characteristics of urinary tract smooth muscles in mice lacking cGMP protein kinase type I.

Nitric oxide (NO)-mediated smooth muscle relaxation is mediated by cGMP through activation of cGMP-dependent protein kinase I (cGKI). We studied the importance of cGKI for lower urinary tract function in mice lacking the gene for cGKI (cGKI-/-) and in litter-matched wild-type mice (cGKI+/+) in vitro and in vivo. cGKI deficiency did not result in any changes in bladder gross morphology or weight. Urethral strips from cGKI-/- mice showed an impaired relaxant response to nerve-derived NO. The cGMP analog 8-bromo-cGMP (8-BrcGMP) and the NO-donor SIN-1 relaxed the wild-type urethra (50-60%) but had only marginal effects in the cGKI-deficient urethra. Bladder strips from cGKI-/- mice responded normally to electrical field stimulation and to carbachol but not to 8-BrcGMP. In vivo, the cGKI-deficient mice showed bladder hyperactivity characterized by decreased intercontraction intervals and nonvoiding bladder contractions. Loss of cGKI abolishes NO-cGMP-dependent relaxations of urethral smooth muscle and results in hyperactive voiding. These data suggest that certain voiding disturbances may be associated with impaired NO-cGKI signaling.

Mammalian skeletogenesis and extracellular matrix: what can we learn from knockout mice?

Formation of the vertebrate skeleton and the proper functions of bony and cartilaginous elements are determined by extracellular, cell surface and intracellular molecules. Genetic and biochemical analyses of human heritable skeletal disorders as well as the generation of knockout mice provide useful tools to identify the key players of mammalian skeletogenesis. This review summarises our recent work with transgenic animals carrying ablated genes for cartilage extracellular matrix proteins. Some of these mice exhibit a lethal phenotype associated with severe skeletal defects (type II collagen-null, perlecan-null), whereas others show mild (type IX collagen-null) or no skeletal abnormalities (matrilin-1-null, fibromodulin-null, tenascin-C-null). The appropriate human genetic disorders are discussed and contrasted with the knockout mice phenotypes.

High-throughput functional annotation of novel gene products using document clustering.

Gene products differentially expressed in healthy vs. diseased tissues may be considered drug targets since the change in their expression level can be related to the cause and progression of the disease studied. A significant portion of the proteins produced by these genes will be unknown and consequently their function must be characterised. The experimental elucidation of biochemical function must be supported by computational tools which can help predicting the possible function of a given protein from its amino acid sequence. We have designed a high-throughput system which automatically analyses amino acid sequences deduced from differentially represented cDNA clones. The system attempts to assign a biological function to protein sequences by carrying out searches in sequence databanks and by locating functionally relevant motifs in the query sequences. The results delivered by the various prediction methods consist of the annotations of matching sequences and/or motifs, which are free-format texts written by humans and therefore may describe the same concept with synonymous words. It is desirable to present the results in such a way that the annotations describing the same biological function are grouped together. To this end we devised an algorithm that enables the hierarchical clustering of free-format documents based on their contents. The system is capable of detecting and flagging conflicting annotations, and will speed up the interpretation of the function prediction results.

Impaired relaxation of stomach smooth muscle in mice lacking cyclic GMP-dependent protein kinase I.

1. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-dependent kinase I (cGKI) is a major receptor for cyclic GMP in a variety of cells. Mice lacking cGKI exhibit multiple phenotypes, including severe defects in smooth muscle function. We have investigated the NO/cGMP- and vasoactive intestinal polypeptide (VIP)/adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-signalling pathways in the gastric fundus of wild type and cGKI-deficient mice. 2. Using immunohistochemistry, similar staining patterns for NO-synthase, cyclic GMP- and VIP-immunoreactivities were found in wild type and cGKI-deficient mice. 3. In isolated, endothelin-1 (3 nM - 3 microM)-contracted, muscle strips from wild type mice, electrical field stimulation (1 - 16 Hz) caused a biphasic relaxation, one initial rapid, followed by a more slowly developing phase. In preparations from cGKI-deficient mice only the slowly developing relaxation was observed. 4. The responses to the NO donor, SIN-1 (10 nM - 100 microM), and to 8-Br-cyclic GMP (10 nM - 100 microM) were markedly impaired in strips from cGKI-deficient mice, whereas the responses to VIP (0.1 nM - 1 microM) and forskolin (0.1 nM - 1 microM) were similar to those in wild type mice. 5. These results suggest that cGKI plays a central role in the NO/cGMP signalling cascade producing relaxation of mouse gastric fundus smooth muscle. Relaxant agents acting via the cyclic AMP-pathway can exert their effects independently of cGKI.

Structure and mapping of the mouse matrilin-3 gene (Matn3), a member of a gene family containing a U12-type AT-AC intron.

The gene for murine matrilin-3, an extracellular matrix protein present in cartilage, was isolated and further characterized. The gene spans 23.4 kb and comprises 8 exons; with one exception, this reflects the modular structure of the protein. The major and a minor transcription start site were determined by RNase protection assays to positions approximately 72 nt and 87 nt upstream of the ATG codon, respectively. The promoter contains a TATA-like box 32 bp upstream of the main transcription start as well as several potential binding sites for eukaryotic transcription factors. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, does not follow the GT-AG rule and belongs to the subgroup of introns having AT-AC at the ends that are spliced by the U12-type spliceosome. The mouse matrilin-3 gene does not contain hidden exon sequences coding for the second vWFA-like domain present in all other matrilins. The intron that could possibly contain such sequences instead shows 75% repetitive sequences, indicating an evolutionary process that has led to the loss of sequences coding for vWFA2. Single-strand conformation polymorphism analysis was used to map the Matn3 gene to the proximal end of Chr 12, linked to the genes Synd1, Apob, Dntb, and Kif3c.