Retinoic Acids Potentiate BMP9-Induced Osteogenic Differentiation of Mesenchymal Progenitor Cells

Abstract

BackgroundAs one of the least studied bone morphogenetic proteins (BMPs), BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs).Methodology/Principal FindingsBoth primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARα and RXRα into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRα or RARα significantly increases trabecular bone and osteoid matrix formation.Conclusion/SignificanceOur results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture.

BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells

“…Using our previously established stem implantation assay Huang et al, 2012a;Huang et al, 2012b;Luo et al, 2010;Zhang et al, 2010), we injected the iMEFs transduced with BMP9 and HIF1a, simHIF1a, or RFP subcutaneously into the flanks of athymic nude mice for 4 weeks. The iMEFs transduced with RFP, HIF1a or simHIF1a alone failed to form any detectable masses (data not shown).…”

“…The first strand cDNA products were further diluted five-to tenfold and used as PCR templates. Semiquantitative RT-PCR (sqPCR) was carried out as described Rastegar et al, 2010;Su et al, 2011;Zhang et al, 2010;Zhu et al, 2009). PCR primers (supplementary material Table S1) were designed by using the Primer3 program to amplify the genes of interest (,150-180 bp).…”

“…Alkaline phosphatase (ALP) activity assays ALP activity was assessed by a modified Great Escape SEAP Chemiluminescence assay (BD Clontech, Mountain View, CA) and/or histochemical staining assay (using a mixture of 0.1 mg/ml napthol AS-MX phosphate and 0.6 mg/ml Fast Blue BB salt) as described Cheng et al, 2003;Kang et al, 2009;Kang et al, 2004;Luo et al, 2004;Luo et al, 2008;Peng et al, 2004;Sharff et al, 2009;Tang et al, 2009;Zhang et al, 2010). For the chemiluminescence assays, each assay condition was performed in triplicate.…”

BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment

“…(4) Importantly, the presence of other soluble growth factors produced locally may potentiate the signaling of BMPs, such as insulin-like growth factor 2 (IGF-2) or retinoic acid, which were shown recently to potentiate the osteogenic effect of BMP-9. (54,55) …”

Characterization of skeletal alterations in a model of prematurely aging mice

“…1a). Furthermore, this age-related diminution also occurred for the gene expression of OC, a late osteoblast differentiation marker (Pockwinse et al 1992;Zhang et al 2010). It has been demonstrated that PTHrP production-an important modulator of bone formation and remodeling in adult bone (Bisello (Martinez et al 2002).…”