Osteoblast differentiation in vitro and in vivo promoted by Osterix

Abstract

C3H10T1/2/Osx, a stably transfected cell line with Osterix (Osx), was produced and chondrocytic and osteoblastic differentiation were studied in vitro. Osx promoted osteoblastic lineage that was dexamethasone dependent. Furthermore, in vivo, Osx induced ectopic mineralization in a heterotopic mouse muscle model. Skeletogenesis involves a cascade of molecular activities sequentially performed by osteoblasts and chondroblasts. A transcriptional factor gene Osx appears to influence cell disposition toward the chondrocytic or osteoblastic phenotype and therefore may be an important signaling cue for bone formation. Understanding the molecular conditions involved with Osx promoted osteoblast differentiation will facilitate therapeutic applications of Osx. Consequently, the objective of this study was to investigate chondrocytic and osteoblastic phenotype differentiation using an Osx plasmid DNA exploiting both in vitro and in vivo methodologies. In vitro, a stably transfected C3H10T1/2/Osx cell line was established and promotion of either an osteoblast or chondroblast phenotype was performed by selectively introducing dexamethasone (dex) and assaying mRNA content and phenotype markers. In vivo, a mouse muscle model was used to determine heterotopic ossification using designated Osx plasmid DNA doses incorporated in a (50:50 Poly (D,L-lactide-co-glycolide) (i.e., PLGA) 3D scaffold. Histological assessment was used to determine implant responses. Quantitative real-time polymerase chain reaction (q-RT-PCR) showed a significant increase in mRNA expression of osteocalcin (Ocn), Runx2, osteonectin (On) and osteopontin (Op) (p < 0.05) in the C3H10T1/2/Osx cells compared to the empty vector transfected cell control. At day 21, mineralization was demonstrated in the cultures of C3H10T1/2/Osx exposed to dex, but neither in cultures lacking dex nor controls. In the absence of dex, C3H10T1/2/Osx cells revealed a significantly higher expression of Sox9 and Aggrecan (Agc). In vivo, 80 microg of Osx plasmid DNA induced heterotopic mineralization 4 weeks following implantation in a mouse muscle model and the effect was dependent on the Osx plasmid DNA dose delivered in the PLGA scaffold. Using a non-committed cell line (C3H10T1/2), cell differentiation to an osteoblast phenotype appears to be dependent upon an interaction between intracellular events initiated by the transcriptional factor Osx and the presence of dex. The in vivo findings suggest Osx may promote osteoblast differentiationand mineralization at a heterotopic site.

Quercetin reversed lipopolysaccharide-induced inhibition of osteoblast differentiation through the mitogen-activated protein kinase pathway in MC3T3-E1 cells

“…Osx, downstream of Runx2, is an osteoblast-specific transcription factor essential for osteoblast differentiation and bone formation (26). Forced expression of Osx in vitro induces the expression of several osteoblastic genes, including Col1α1 and osteocalcin (27,28). The present study demonstrated that quercetin restored the expression of Osx and Runx2, which were suppressed by LPS in MC3T3-E1 cells.…”

Protective Effects of Pretreatment with Quercetin Against Lipopolysaccharide-Induced Apoptosis and the Inhibition of Osteoblast Differentiation via the MAPK and Wnt/β-Catenin Pathways in MC3T3-E1 Cells

“…Runx2 (Cbfa1) and ALP have been suggested to be involved in the early-stage molecular events of osteoblast differentiation [31, 32], whereas OSX and OCN are involved in the late-stage molecular events [23, 33, 34]. In this study, pretreatment with quercetin significantly restored the LPS-suppressed mRNA and protein expression of ALP, Runx2, OSX and OCN in MC3T3-E1 cells in a dose-dependent manner.…”

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

“…5). Interestingly, the most sensitively upregulated gene expression in response to BMP-7 stimulation was the transcription factor SP7, which was recently reported to promote osteogenic differentiation in vitro and in vivo using the gene overexpression approach in mouse models [Fu et al, 2007]. In contrast to its upstream gene RUNX2, SP7 expression was largely and stably enhanced by BMP-7 supplementation up to 14 days, which suggests a potential mechanism of BMP-7 function in the osteogenic differentiation of BM MSCs.…”