Fgfr2 is required for limb outgrowth and lung-branching morphogenesis.
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The aim of this study was to clarify the role of Fgfr2 during later stages of embryonic development. Of two previously reported gene-targeting experiments, the more extensive Fgfr2 deletion was lethal shortly after implantation, because of trophoblast defects, whereas the less extensive one survived until midgestation with placental insufficiency and defective limb outgrowth [Xu, X., Weinstein, M., Li, C., Naski, M., Cohen, R. I., Ornitz, D. M., Leder, P. & Deng, C. (1998) Development (Cambridge, U.K.) 125, 753-765]. Fgfr2 in the early embryo is expressed in the trophectoderm, and this extra-embryonic localization persists into mid- and late gestation, when Fgfr2 also is expressed in multiple developing organs. To gain insight into the later functions of Fgfr2, fusion chimeras were constructed from homozygous mutant embryonic stem cells and wild-type tetraploid embryos. This allowed survival until term and revealed that Fgfr2 is required for both limb outgrowth and branching lung morphogenesis. The use of fusion chimeras demonstrated that early lethality was indeed because of trophectoderm defects and indicated that in the embryonic cell lineages Fgfr2 activity manifests in limb and lung development. Highly similar lung and limb phenotypes were detected recently in the loss of function mutation of Fgf10, a ligand of Fgfr2. It is likely, therefore, that whereas during early development Fgfr2 interacts with Fgf4, in limb and lung development interactions between Fgf10 and Fgfr2 may be required. Possible epithelial-mesenchymal interactions between the splicing alternatives of Fgfr2 and their specific ligands will be discussed. (+info)
MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover.
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MT1-MMP is a membrane-bound matrix metalloproteinase (MT-MMP) capable of mediating pericellular proteolysis of extracellular matrix components. MT1-MMP is therefore thought to be an important molecular tool for cellular remodeling of the surrounding matrix. To establish the biological role of this membrane proteinase we generated MT1-MMP-deficient mice by gene targeting. MT1-MMP deficiency causes craniofacial dysmorphism, arthritis, osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a collagenolytic activity that is essential for modeling of skeletal and extraskeletal connective tissues. Our findings demonstrate the pivotal function of MT1-MMP in connective tissue metabolism, and illustrate that modeling of the soft connective tissue matrix by resident cells is essential for the development and maintenance of the hard tissues of the skeleton. (+info)
Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage?
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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. (+info)
Cleavage of aggrecan at the Asn341-Phe342 site coincides with the initiation of collagen damage in murine antigen-induced arthritis: a pivotal role for stromelysin 1 in matrix metalloproteinase activity.
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OBJECTIVE: The destruction of articular cartilage during arthritis is due to proteolytic cleavage of the extracellular matrix components. This study investigates the kinetic involvement of metalloproteinases (MMPs) in the degradation of the 2 major cartilage components, aggrecan and type II collagen, during murine antigen-induced arthritis (AIA). In addition, the role of stromelysin 1 (SLN-1) induction of MMP-induced neoepitopes was studied. METHODS: VDIPEN neoepitopes in aggrecan and collagenase-induced COL2-3/4C neoepitopes in type II collagen were identified by immunolocalization. Stromelysin 1-deficient knockout (SLN1-KO) mice were used to study SLN-1 involvement. RESULTS: In AIA, the VDIPEN epitopes in aggrecan appeared after initial proteoglycan (PG) depletion. The collagenase-induced type II collagen neoepitopes colocalized with VDIPEN epitopes. Remarkably, cartilage from arthritic SLN1-KO mice showed neither the induction of VDIPEN nor collagen cleavage-site neoepitopes during AIA, suggesting that stromelysin is a pivotal mediator in this process. PG depletion, as measured by the loss of Safranin O staining, was similar in SLN1-KO mice and wild-type strains. Furthermore, in vitro induction of VDIPEN epitopes in aggrecan and COL2-3/4C epitopes in type II collagen, on exposure of cartilage to interleukin-1, could not be accomplished in SLN1-KO mice, whereas intense staining was achieved for both epitopes in cartilage of wild-type strains. CONCLUSION: This study emphasizes that SLN-1 is essential in the induction of MMP-specific aggrecan and collagen cleavage sites during AIA. It suggests that SLN-1 is not a dominant enzyme in PG breakdown, but that it activates procollagenases and is crucial in the initiation of collagen damage. (+info)
Premature termination codon in the aggrecan gene of nanomelia and its influence on mRNA transport and stability.
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AIM: To analyze the influence of the premature termination codon on mRNA transport and stability METHODS: Chondrocyte mRNA was isolated from homozygous and heterozygous nanomelic 17-days old embryos and examined by RT-PCR analysis. To analyze aggrecan mRNA stability, mRNA synthesis was inhibited with DRB [5,6 dichloro-1-(-D-ribofuranosyl benzimidazole)], a specific inhibitor of RNA polymerase II. Visualization of the aggrecan alleles was performed by in situ hybridization. RESULTS: The level of mutant aggrecan mRNA within the nucleus was equal to that of the control, but no mutant mRNA was observed in the cytoplasm. RT-PCR revealed that the mutant transcript was only detectable in the nucleus, compared with house-keeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene or collagen type II. A restriction site induced by premature termination codon TAA allowed the distinction of normal and mutant transcripts in chondrocytes derived from embryos heterozygous for the nanomelic mutation. After the treatment with DRB, identical decay rates were demonstrated for both transcripts within the heterozygous nucleus. In situ hybridization showed no abnormal mRNA accumulation. CONCLUSION: This is the first evidence suggesting that the transcript of the mRNA with the premature termination codon within an exon does exit the nucleus. (+info)
Degradation of cartilage type II collagen precedes the onset of osteoarthritis following anterior cruciate ligament rupture.
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OBJECTIVE: To determine if degradation of cartilage matrix in primary osteoarthritis (OA) or in OA secondary to rupture of the anterior cruciate ligament (ACL) is a gradual response to excessive loading or an early, initiating event in the disease process. METHODS: Biopsy samples were obtained from the low-weight-bearing articular cartilage of the intercondylar notch, in patients undergoing knee arthroscopy (ACL injury) or arthroplasty (late-stage primary OA) or in controls. In some cases, biopsy samples were also removed from the high-weight-bearing articular cartilage of the femoral condyles. Biopsy specimens were extracted and assayed for total and denatured type II collagen (CII) by inhibition enzyme-linked immunosorbent assay and for proteoglycan using a colorimetric method. All patients were assessed radiographically for cartilage erosion. In addition, the cartilage of patients with ACL injury was assessed at arthroscopy, and the knee function of patients with primary OA was assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). RESULTS: Increased CII degradation was detected in the low- as well as the high-weight-bearing cartilage of patients with late-stage OA, and there was a positive correlation between the percentage denatured collagen and the WOMAC score. Most of the patients with ACL injury had no clinical signs of OA or macroscopic cartilage erosion. However, the low-weight-bearing articular cartilage from these patients showed a significant increase in CII degradation, similar to that observed in late-stage OA. The proteoglycan content of articular cartilage did not change significantly in patients with OA or ACL injury compared with controls. CONCLUSION: CII degradation is an early event following ACL injury and is unlikely to be a direct result of mechanical loading, since it was observed in cartilage obtained from a low-weight-bearing site. (+info)
Apocynin, a plant-derived, cartilage-saving drug, might be useful in the treatment of rheumatoid arthritis.
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OBJECTIVE: To investigate whether apocynin, 1-(4-hydroxy-3-methoxyphenyl)ethanone, is able to diminish inflammation-induced cartilage destruction in rheumatoid arthritis (RA), studied in a human in vitro model. METHODS: Apocynin was added to cultures of RA peripheral blood mononuclear cells (PBMNC). Cartilage-destructive activity was determined by addition of culture supernatant to tissue samples of human articular cartilage. In addition, the proliferation of PBMNC, their production of tumour necrosis factor alpha (TN-Falpha), interleukin (IL)-1 and IL-10, and T-cell production of interferon gamma (IFN-gamma) and IL-4, as measures for T1 and T2 cell activity, were determined. RESULTS: Apocynin was able to counteract RA PBMNC-induced inhibition of cartilage matrix proteoglycan synthesis, while no effect on inflammation-enhanced proteoglycan release was found. The effect was accompanied by a decrease in IL-1 and TNF-alpha production by the MNC. No effect on T-cell proliferation was found, but the production of IFN-gamma, IL-4 and T-cell-derived IL-10 was strongly diminished. Most important, apocynin did not show any direct adverse effects on chondrocyte metabolism; on the contrary, it diminished the release of proteoglycans from the cartilage matrix. CONCLUSION: Apocynin in vitro inhibits inflammation-mediated cartilage destruction without having adverse effects on cartilage. The latter may be an advantage of apocynin over many other non-steroidal anti-inflammatory drugs. Therefore, apocynin might have an added beneficial effect in protecting RA patients from joint destruction. (+info)
Morphological analysis of the mammalian postcranium: a developmental perspective.
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The past two decades have greatly improved our knowledge of vertebrate skeletal morphogenesis. It is now clear that bony morphology lacks individual descriptive specification and instead results from an interplay between positional information assigned during early limb bud deployment and its "execution" by highly conserved cellular response programs of derived connective tissue cells (e.g., chondroblasts and osteoblasts). Selection must therefore act on positional information and its apportionment, rather than on more individuated aspects of presumptive adult morphology. We suggest a trait classification system that can help integrate these findings in both functional and phylogenetic examinations of fossil mammals and provide examples from the human fossil record. (+info)