ABSTRACT
Osteoclasts differentiate from hematopoietic precursors of the monocyte/macrophage lineage to mononuclear preosteoclasts and multinuclear mature osteoclasts. In the present study, we report on the establishment of macrophage like cell lines from mouse bone marrow by coculturing bone marrow cells with mouse chondrocytes. Isolated clones (MLC-6 and MLC-7 cells) expressed fully differentiated osteoclast markers, such as calcitonin receptors, vitronectin receptors, tartrate-resistant acid phosphatase and vacuolar H+ -ATPase, in the absence of osteoclast differentiation factor/osteoprotegerin ligand/RANKL/TRANCE, which was essential for osteoclast differentiation. Most clones also maintained expression of a macrophage-associated protein, namely non-specific esterase. Both MLC-6 and MLC-7 cells released cathepsin K into the culture medium. Both clones resolved dentine slices when cocultured with the osteoblast cell line ST2. The cloned cell lines are considered to be useful tools in the study of osteoclast differentiation.
Subject(s)
Bacterial Proteins , Macrophages/cytology , Macrophages/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , Animals , Base Sequence , Biomarkers , Cell Differentiation , Cell Line , Coculture Techniques , DNA Primers/genetics , Gene Expression , Glycoproteins/genetics , Immunohistochemistry , Matrix Metalloproteinase 9/genetics , Mice , Microscopy, Electron, Scanning , Osteoprotegerin , RNA-Binding Proteins/genetics , Receptors, Calcitonin/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Tumor Necrosis Factor , Receptors, Vitronectin/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
Ultrastructural aspects of hypertrophic chondrocytes in hamster and mouse epiphysial cartilage were examined in relation to their metabolic activities. With the hypertrophic change, cytoplasmic vacuolization proceeded leaving the partially intact endoplasmic reticulum (ER). In the hypertrophic cells, cytoplasmic hyaluronan was stained with the biotinylated hyaluronan-binding region (b-HABR) of aggrecan, and mRNAs of hyaluronan synthase (Has 1, Has 2 and Has 3) were detected by in situ hybridization. When the epiphysial cartilage was cultured in the presence of 35S, 3H-GlcNAc, 3H-proline or 14C-palmitic acid, vacuolated late hypertrophic chondrocytes were labeled with these radioactive precursors. The evidence indicates that late hypertrophic chondrocytes are metabolically active, which appears to be essential for the enlargement of chondrocytes.
Subject(s)
Chondrocytes/metabolism , Chondrocytes/ultrastructure , Glucuronosyltransferase/genetics , Glucuronosyltransferase/metabolism , Glycosyltransferases , Membrane Proteins , Transferases , Xenopus Proteins , Animals , Cartilage/metabolism , Cartilage/ultrastructure , Cattle , Cell Death , Cell Differentiation , Cells, Cultured , Cricetinae , Gene Expression , Hyaluronan Synthases , Hypertrophy , Immunohistochemistry , In Situ Hybridization , Mesocricetus , MiceABSTRACT
The secondary ossification center of 14- to 16-day-old hamster tibiae was examined immunohistochemically with active and inactive Cls-specific antibodies, RK5 and RK4, respectively. At the ossification center, chondrocytes differentiate from proliferating and hypertrophic to degenerating stages, and their site is occupied by the bone marrow. Cls was strongly immunostained in hypertrophic chondrocytes. In order to discover whether Cls is activated at a particular site, the cartilage was immunostained with RK5 and RK4. RK5 mainly reacted with degrading matrix around invading vessels. In contrast, RK4 strongly stained hypertrophic chondrocytes. Immunoelectron microscopy revealed Cls on degrading fragments of chondrocytes and fibers of cartilage matrix. Decorin, one of the major matrix proteoglycans, was dose and time dependently degraded by Cls. Type II collagen and type I gelatin were also degraded. Articular cartilage from patients with rheumatoid arthritis was positively immunostained (11/12 cases) with an anti-Cls monoclonal antibody (mAb) PG11, whereas normal articular cartilage (5/5 cases) was negative, suggesting Cls participation in the etiology of rheumatoid arthritis.
