Supplementation of mesenchymal stem cells (MSCs) at sites of bone resorption is required for bone homeostasis because of the non-proliferation and short lifespan properties of the osteoblasts. Calcium ions (Ca2+) are released from the bone surfaces during osteoclast-mediated bone resorption. However, how elevated extracellular Ca2+ concentrations would alter MSCs behavior in the proximal sites of bone resorption is largely unknown. In this study, we investigated the effect of extracellular Ca2+ on MSCs phenotype depending on Ca2+ concentrations. We found that the elevated extracellular Ca2+ promoted cell proliferation and matrix mineralization of MSCs. In addition, MSCs induced the expression and secretion of osteopontin (OPN), which enhanced MSCs migration under the elevated extracellular Ca2+ conditions. We developed in vitro osteoclast-mediated bone resorption conditions using mouse calvaria bone slices and demonstrated Ca2+ is released from bone resorption surfaces. We also showed that the MSCs phenotype, including cell proliferation and migration, changed when the cells were treated with a bone resorption-conditioned medium. These findings suggest that the dynamic changes in Ca2+ concentrations in the microenvironments of bone remodeling surfaces modulate MSCs phenotype and thereby contribute to bone regeneration.
Epidermal growth factor-like repeats and discoidin I-like domain 3 (Edil3) is an extracellular matrix protein containing an Arg-Gly-Asp (RGD) motif that binds integrin. Recently, Edil3 has been implicated in various biological processes, including angiogenesis and cellular differentiation. It can inhibit inflammatory bone destruction. The objective of this study was to explore the role of Edil3 in osteoblast differentiation and its underlying molecular mechanisms. In wild-type mice, high expression levels of Edil3 mRNA were observed in isolated calvaria and tibia/femur bones. Immunohistochemical analysis showed that Edil3 protein was localized along periosteum and calcified regions surrounding bone tissues. When murine calvaria-derived MC3T3-E1 cells were cultured in osteogenic medium containing 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate, Edil3 mRNA and protein expression levels were increased. Treatment with Edil3 protein in growth media increased expression levels of alkaline phosphatase and osteocalcin gene and phosphorylation level of extracellular signal-regulated kinase (ERK). Edil3 treatment with osteogenic medium induced mineralization. Treatment with a neutralizing antibody against α5β1 and MEK inhibitor U0126 inhibited Edil3-enhanced osteogenic marker gene expression and mineral deposition. Edil3 increased protein expression levels of transcription factor runt-related transcription factor2 (Runx2). Edil3-induced Runx2 protein expression was suppressed by pretreatment with U0126. Taken together, these results suggest that Edil3 may stimulate osteoblast differentiation and matrix mineralization by increasing expression of Runx2 through α5β1 integrin /ERK pathway.
Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan nuclear receptor that regulates many key biological processes, including organ development and cell fate determination. Although the biological functions of COUP-TFII have been studied extensively, little is known about what regulates its gene expression, especially the role of inducible extracellular factors in triggering it. Here we report that COUP-TFII expression is regulated specifically by fibroblast growth factor 2 (FGF2), which mediates activation of the MEK1/2 pathway in mesenchymal lineage C3H10T1/2 cells. Although FGF2 treatment increased cell proliferation, the induction of COUP-TFII expression was dispensable. Instead, FGF2-primed cells in which COUP-TFII expression was induced showed a low potential for osteoblast differentiation, as evidenced by decreases in alkaline phosphatase activity and osteogenic marker gene expression. Reducing COUP-TFII by U0126 or siRNA against COUP-TFII prevented the anti-osteogenic effect of FGF2, indicating that COUP-TFII plays a key role in the FGF2-mediated determination of osteoblast differentiation capability. This report is the first to suggest that FGF2 is an extracellular inducer of COUP-TFII expression and may suppress the osteogenic potential of mesenchymal cells by inducing COUP-TFII expression prior to the onset of osteogenic differentiation.
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