The ephrin-B2/EphB4 system is required in musculoskeletal development and protects the articulation during osteoarthritis: a research highlight

Ephrin ligands and their Eph receptors have been implicated in the control of extracellular matrix of some tissues. Although ephrin-B2 and its specific receptor EphB4 were found to be involved in postembryonic control of bone homeostasis, their roles were unclear in musculoskeletal growth and development as well as in osteoarthritis pathology. The role of this ephrin system in musculoskeletal growth and development was delineated in vivo using a cartilage-specific ephrin-B2 knockout mouse model. Its role in osteoarthritis in vivo was explored in mice using a bone-specific overexpression of EphB4 in which osteoarthritis was induced, and in vitro in human osteoarthritic subchondral bone osteoblasts and chondrocytes. In vivo, ephrin-B2 demonstrated to be essential for normal long bone growth and development and its absence in cartilage led to knee and hip osteoarthritis features in aged mice. In vitro data showed that the ephrin-B2-induced EphB4 receptor positively impacted the abnormal metabolism of both osteoarthritic subchondral bone osteoblasts and chondrocytes. The bone?specific EphB4 overexpression in mice validated the in vitro data in that it had beneficial effects not only on the osteoarthritic subchondral bone but also on the cartilage and synovial membrane, and further substantiated the hypothesis that by prophylactically protecting the subchondral bone, the genesis of osteoarthritis could be, at least in part, inhibited. In the context of identifying new candidates targeting osteoarthritis progression, this ephrin system is extremely attractive as a potential novel therapeutic avenue, as therapies having a more global articular approach may prove to be the most successful to arrest or slow the progression of this disease

Ablation of the Sam68 RNA Binding Protein Protects Mice from Age-Related Bone Loss

The Src substrate associated in mitosis of 68 kDa (Sam68) is a KH-type RNA binding protein that has been shown to regulate several aspects of RNA metabolism; however, its physiologic role has remained elusive. Herein we report the generation of Sam68-null mice by homologous recombination. Aged Sam68(−/−) mice preserved their bone mass, in sharp contrast with 12-month-old wild-type littermates in which bone mass was decreased up to approximately 75%. In fact, the bone volume of the 12-month-old Sam68(−/−) mice was virtually indistinguishable from that of 4-month-old wild-type or Sam68(−/−) mice. Sam68(−/−) bone marrow stromal cells had a differentiation advantage for the osteogenic pathway. Moreover, the knockdown of Sam68 using short hairpin RNA in the embryonic mesenchymal multipotential progenitor C3H10T1/2 cells resulted in more pronounced expression of the mature osteoblast marker osteocalcin when differentiation was induced with bone morphogenetic protein-2. Cultures of mouse embryo fibroblasts generated from Sam68(+/+) and Sam68(−/−) littermates were induced to differentiate into adipocytes with culture medium containing pioglitazone and the Sam68(−/−) mouse embryo fibroblasts shown to have impaired adipocyte differentiation. Furthermore, in vivo it was shown that sections of bone from 12-month-old Sam68(−/−) mice had few marrow adipocytes compared with their age-matched wild-type littermate controls, which exhibited fatty bone marrow. Our findings identify endogenous Sam68 as a positive regulator of adipocyte differentiation and a negative regulator of osteoblast differentiation, which is consistent with Sam68 being a modulator of bone marrow mesenchymal cell differentiation, and hence bone metabolism, in aged mice

The ephrin-B2/EphB4 system is required in musculoskeletal development and protects the articulation during osteoarthritis: a research highlight

Ephrin ligands and their Eph receptors have been implicated in the control of extracellular matrix of some tissues. Although ephrin-B2 and its specific receptor EphB4 were found to be involved in postembryonic control of bone homeostasis, their roles were unclear in musculoskeletal growth and development as well as in osteoarthritis pathology. The role of this ephrin system in musculoskeletal growth and development was delineated in vivo using a cartilage-specific ephrin-B2 knockout mouse model. Its role in osteoarthritis in vivo was explored in mice using a bone-specific overexpression of EphB4 in which osteoarthritis was induced, and in vitro in human osteoarthritic subchondral bone osteoblasts and chondrocytes. In vivo, ephrin-B2 demonstrated to be essential for normal long bone growth and development and its absence in cartilage led to knee and hip osteoarthritis features in aged mice. In vitro data showed that the ephrin-B2-induced EphB4 receptor positively impacted the abnormal metabolism of both osteoarthritic subchondral bone osteoblasts and chondrocytes. The bone‑specific EphB4 overexpression in mice validated the in vitro data in that it had beneficial effects not only on the osteoarthritic subchondral bone but also on the cartilage and synovial membrane, and further substantiated the hypothesis that by prophylactically protecting the subchondral bone, the genesis of osteoarthritis could be, at least in part, inhibited. In the context of identifying new candidates targeting osteoarthritis progression, this ephrin system is extremely attractive as a potential novel therapeutic avenue, as therapies having a more global articular approach may prove to be the most successful to arrest or slow the progression of this disease

Cartilage-specific deletion of ephrin-B2 in mice results in early developmental defects and an osteoarthritis-like phenotype during aging in vivo

Abstract Background Ephrins and their related receptors have been implicated in some developmental events. We have demonstrated that ephrin-B2 (EFNB2) could play a role in knee joint pathology associated with osteoarthritis (OA). Here, we delineate the in vivo role of EFNB2 in musculoskeletal growth, development, and in OA using a cartilage-specific EFNB2 knockout (EFNB2Col2KO) mouse model. Methods EFNB2Col2KO was generated with Col2a1-Cre transgenic mice. The skeletal development was evaluated using macroscopy, immunohistochemistry, histomorphometry, radiology, densitometry, and micro-computed tomography. Analyses were performed at P0 (birth) and on postnatal days P15, P21, and on 8-week- and 1-year-old mice. Results EFNB2Col2KO mice exhibited significant reduction in size, weight, length, and in long bones. At P0, the growth plates of EFNB2Col2KO mice displayed increased type X collagen, disorganized hyphertrophic zone, and decreased mineralization. At P15, mutant mice demonstrated a significant reduction in VEGF and TRAP at the chondro-osseous junction and a delay in the secondary ossification, including a decrease in bone volume and trabecular thickness. At P21 and 8 weeks old, EFNB2Col2KO mice exhibited reduced bone mineral density in the total skeleton, femur and spine. One-year-old EFNB2Col2KO mice demonstrated OA phenotypic features in both the knee and hip. By P15, 27 % of the EFNB2Col2KO mice developed a hip locomotor phenotype, which further experiments demonstrated reflected the neurological midline abnormality involving the corticospinal tract. Conclusion This in vivo study demonstrated, for the first time, that EFNB2 is essential for normal long bone growth and development and its absence leads to a knee and hip OA phenotype in aged mice

Ex Vivo Activity of Sam68^+/+ and Sam68^−/− Osteoblasts and Osteoclasts

<div><p>Marrow stromal cells were isolated from the long bones of juvenile mice and maintained under conditions that promote osteoblast differentiation.</p><p>(A) Cultures were fixed in 4% paraformaldehyde after 6 or 18 days and stained in situ for ALP activity and with silver nitrate (von Kossa) to detect mineralized nodules. Sam68^−/− cultures stained more intensely for ALP at early and late time points and produced significantly more mineralized nodules after 18 days. Asterisks represent <i>p</i> < 0.01.</p><p>(B) Primary osteoclasts were isolated from the crushed long bones of the same mice and plated on glass coverslips or on dentin slices to quantify numbers and activity, respectively. Osteoclasts were identified as cells with three or more nuclei that stained positive for TRAP activity (upper) and excavated pits in dentin slices, as demonstrated by SEM (lower, bar = 20 μm). No statistical differences were observed either in the number of TRAP-positive cells or in their resorptive activity.</p></div

Generation of Sam68-Deficient Mice

<div><p>(A) The genomic organizations of the wild-type and targeted <i>sam68</i> alleles after homologous recombination are depicted. The location of the DNA fragment used as a probe for the Southern blot analysis is shown, as well as the sizes of the two BglII fragments detected for wild-type and targeted <i>sam68</i> alleles. The targeted allele replaces exon 4 and part of exon 5 of <i>sam68</i> with a PGK-neomycin cassette.</p><p>(B) Southern-blot analysis of genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. DNA fragments corresponding to wild-type (4.5 kb) and the targeted (5.5 kb) alleles are illustrated.</p><p>(C) Western blot analysis of Sam68 expression. Protein extracts from wild-type, heterozygous, and homozygous cells subjected to immunoblot analyses using normal rabbit serum, anti-Sam68 AD1 antibody, the peptide antibody AD1 preabsorbed with the immunogenic peptide corresponding to amino acids 330–348 of mouse Sam68, anti-Sam68 Sc333 antibody that recognizes the C-terminal 20 amino acids of Sam68, and anti-actin antibodies as loading control. The migration of the molecular mass markers is known on the left in kDa.</p></div

Old Sam68^−/− Mice Are Protected from the Development of Fatty Bone Marrow

<p>Sections of undecalcified bone were stained with von Kossa and toluidine blue and images captured at original magnifications of ×2 (A), ×40 (B and C) to evaluate mineralized tissue (A, black), marrow adipocytes (B, white), and the mineralization fronts (C, yellow and green). The 12-month-old Sam68^+/+ bone demonstrated a significant reduction in bone (A) and increase in marrow adipocytes (B) and a decrease in the distance between two consecutive fluorochrome labels (C). Magnification at source was ×40. Micrographs are representative of four to six screened in each group of animals.</p

Enhanced Osteogenic Differentiation of the C3HT101/2 Embryonic Cell Line Depleted of Endogenous Sam68

<div><p>(A) C3HT101/2 cells transfected with an empty vector (pSuper-retro) or a vector containing an shRNA (Sam68 shRNA) were selected with puromycin, and knockdown populations depleted of Sam68 were identified. The reduction in Sam68 protein was analyzed by immunoblotting with anti-Sam68 (AD1) antibody and anti–β-actin antibodies as loading controls.</p><p>(B) Osteogenic differentiation was carried out with conditioned medium containing BMP-2 for the indicated times. To assess the level of osteogenic differentiation in these cells, expression of late osteoblast marker, osteocalcin (OCN), was analyzed by RT-PCR and compared with β-actin and GAPDH controls. The DNA fragments were visualized by agarose gel stained with ethidium bromide.</p></div

Immunohistochemical Localization of Sam68 in Embryonic Mice

<div><p>(A) Embryonic mice were removed from pregnant dams at E14.5 and E16.5, fixed in 4% paraformaldehyde, and embedded in paraffin. The entire embryo was immunostained with the AD1 anti-Sam68 antibody and counterstained with methyl green, and the image was captured at ×1.2 magnification.</p><p>(B) Embryonic soft tissues from the brain, heart and gut were stained with hematoxylin (left) and immunostained with anti-Sam68 antibody (right), and images were captured at ×20 magnification.</p><p>(C) Intense anti-Sam68 immunoreactivity was seen in chondrocytes in the nasal septum (panels A–D), in developing vertebra (panels E–H), and in the femoral epiphysis (panels I–K), as well as in diaphyseal osteoblasts (panel L). Adjacent sections were stained with hematoxylin and eosin (panels A, E, and I) or with antibody preadsorbed with the immunizing peptide (panels B, F, and J). Sam68 was localized primarily in the nucleus of cells in a variety of tissues but was also found occasionally in the cytoplasm. Magnification at source ×20, except for panels D, H, and L, which were ×40. Staining patterns are representative of three to five embryos.</p></div