Terminal differentiation and mineralization are the final events in endochondral bone formation and allow the replacement of cartilage by bone. Retinoic acid (RA) stimulates these events, including upregulation of expression and activity of alkaline phosphatase (APase), expression of annexins II, V, and VI proteins, which bind to membranes and form Ca(2+) channels, expression of osteocalcin and runx2, another mineralization-related protein and terminal differentiation-related transcription factor, and ultimately mineralization. Chelating cytosolic Ca(2+) with BAPTA-AM, interfering with annexin Ca(2+) channel activities using K-201, a specific annexin Ca(2+) channel blocker, or suppression of annexin V expression using siRNA inhibited these events. Overexpression of annexin V in embryonic chicken growth plate chondrocytes resulted in an increase of cytoplasmic Ca(2+) concentration, [Ca(2+)](i) similar to [Ca(2+)](i) increase in RA-treated cultures. Overexpression of annexin V also resulted in upregulation of annexin II, annexin VI, osteocalcin, and runx2 gene expression, expression and activity of APase, and ultimately stimulation of mineralization. K-201 inhibited upregulation of osteocalcin and runx2 gene expression, APase expression and activity, and mineralization in annexin V-overexpressing growth plate chondrocytes. These findings indicate that annexins II, V, and VI alter Ca(2+) homeostasis in growth plate chondrocytes thereby regulating terminal differentiation and mineralization events. Overexpression of annexin V is sufficient to stimulate these terminal differentiation events in growth plate chondrocytes, whereas suppression of annexin V expression inhibits these events.
"Annexin-V protein builds Ca2+ channels, leading to mineralization of bone matrix in the growth plates during osteogenesis . An increased expression of annexin-V enhances these processes of mineralization . Such mineralization processes were observed in a prior study, whereas periosteal cells underwent long-term mechanical stimulation in bioreactors . "
[Show abstract][Hide abstract] ABSTRACT: It is commonly accepted that bone marrow-derived stem cells (BMSCs) have to be expanded in vitro, but a prolonged time in culture decreases their multilineage potential. Mechanical and biological stimuli have been used to improve their osteogenic potential. While long-term stimulation has been shown to improve osteogenic differentiation, it remains to be seen whether short-term stimulation is also sufficient.
We investigated the influence of 24 hours' cyclic loading (0.05Hz, 4kPa) on gene expression of human BMSCs in three-dimensional fibrin-DMEM constructs (n=7) in a compression bioreactor using DNA-array technology. Expression of the following genes showed a significant increase after mechanical stimulation: 2.6-fold osteopontin (OPN) and integrin-β1 (ITGB1), 2.2-fold transforming growth factor-β-receptor 1 (TGF-β-R1) and 2.4-fold SMAD5 expression, compared to controls without mechanical stimulation (p<0.05 each). Platelet-derived growth factor-α (PDGF-α ) and annexin-V were also significantly overexpressed, the mechanical stimulation resulting in a 1.8-fold and 1.6-fold expression (p<0.05).
Cells were identified as osteoblast precursors with a high proliferative capacity. Given the identical in-vitro environment for both groups, the increase in gene expression has been interpreted as a direct influence of cyclic mechanical stimulation on osteogenic differentiation. It may be postulated that short-term mechanical stimulation results in an improved osseous integration of tissue engineered grafts in bone defect healing.
The Open Orthopaedics Journal 01/2011; 5:1-6. DOI:10.2174/1874325001105010001
"After transfection, cells were cultured for up to 12 days in differentiation medium. Real-time PCR analysis using SYBR Green was performed as described previously [Wang et al., 2005]. The 18S RNA was amplified at the same time and used as an internal control. "
[Show abstract][Hide abstract] ABSTRACT: The progressive ankylosis gene (ank) is a transmembrane protein that transports intracellular pyrophosphate to the extracellular milieu. Human mutations of ank lead to craniometaphyseal dysplasia, a disease which is characterized by the overgrowth of craniofacial bones and osteopenia in long bones, suggesting that ANK plays a regulatory role in osteoblast differentiation. To determine the role of ANK in osteoblast differentiation, we suppressed ANK expression in the osteoblastic MC3T3-E1 cell line using siRNA and determined the expression of osteoblastic marker genes and the transcription factors osterix and runx2. In addition, we determined the osteoblastic differentiation of bone marrow stromal cells isolated from the bone marrow of ank/ank mice, which express a truncated, nonfunctional ANK protein, or wild-type littermates. Suppression of ANK expression in MC3T3-E1 cells led to a decrease in bone marker gene expression, including alkaline phosphatase, bone sialoprotein, osteocalcin and type I collagen. In addition, osterix gene expression was decreased in ANK expression-suppressed MC3T3 cells, whereas runx2 expression was increased. Bone marrow stromal cells isolated from ank/ank mice cultured in the presence of ascorbate-2-phosphate for up to 35 days showed markedly reduced mineralization compared to the mineralization of bone marrow stromal cells isolated from wild-type littermates. In conclusion, these findings suggest that ANK is a positive regulator of differentiation events towards a mature osteoblastic phenotype.
"The all-trans retinoic acid (ATRA), an active metabolite of vitamin A, has been recognized as a key inducer of growth plate maturation, mineralization and apoptosis, by activating the expression of multiply genes associated with cell proliferation and formation of mineral phase    . Furthermore, retinoid antagonists prevent chondrocyte hypertrophy , suggesting that retinoids are required for chondrocyte maturation. ATRA may also affect annexins during the mineralization process by modulating annexin ion channel activity . "
[Show abstract][Hide abstract] ABSTRACT: Vitamin A (all-trans retinol) and all-trans retinoid acid (ATRA) interacted with human annexin A6 (AnxA6) as evidenced by AnxA6-induced blue shift of retinoid absorption maxima, by AnxA6-Trp fluorescence quenching and by a fluorescence resonance energy transfer from a Trp residue of AnxA6 to retinol. In addition, both retinoids stimulated the calcium-dependent binding of AnxA6 to liposomes, accompanied by oligomerization of AnxA6. Up to our knowledge, it is a first report supporting the hypothesis of a direct implication of AnxA6 in vitamin A-dependent tissue mineralization.
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