Retinoic acid receptor γ activity in mesenchymal stem cells regulates endochondral bone, angiogenesis and B lymphopoiesis (original) (raw)
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RARγ is a negative regulator of osteoclastogenesis
The Journal of Steroid Biochemistry and Molecular Biology, 2015
Vitamin A is known to influence post-natal bone content, with excess intake being associated with reduced bone mineral density and increased fracture risk. Despite this, the roles retinoids play in regulating osteoclastogenesis, particularly in vivo, remain unresolved. This study therefore aimed to determine the effect of loss of retinoic acid receptors (RAR)a or RARg on bone mass (analyzed by histomorphometry and dual-energy X-ray absorptiometry) and osteoclastogenesis in mice in vivo. RARg null mice had significantly less trabecular bone at 8 weeks of age compared to wildtype littermates. In contrast, no change in trabecular bone mass was detected in RARa null mice at this age. Further histomorphometric analysis revealed a significantly greater osteoclast surface in bones from 8-week-old RARg null male mice. This in vivo effect was cell lineage autonomous, and was associated with increased osteoclastogenesis in vitro from hematopoietic cells obtained from 8-week-old RARg null male mice. The use of highly selective agonists in RANKL-induced osteoclast differentiation of wild type mouse whole bone marrow cells and RAW264.7 cells in vitro showed a stronger inhibitory effect of RARg than RARa agonists, suggesting that RARg is a more potent inhibitor of osteoclastogenesis. Furthermore, NFAT activation was also more strongly inhibited by RARg than RARa agonists. While RARa and RARg antagonists did not significantly affect osteoclast numbers in vitro, larger osteoclasts were observed in cultures stimulated with the antagonists, suggesting increased osteoclast fusion. Further investigation into the effect of retinoids in vivo revealed that oral administration of 5 mg/kg/day ATRA for 10 days protected against bone loss induced by granulocyte colony-stimulating factor (G-CSF) by inhibiting the pro-osteoclastogenic action of G-CSF. Collectively, our data indicates a physiological role for RARg as a negative regulator of osteoclastogenesis in vivo and in vitro, and reveals distinct influences of RARa and RARg in bone structure regulation.
PLoS ONE, 2010
It has been shown that high vitamin A intake is associated with bone fragility and fractures in both animals and humans. However, the mechanism by which vitamin A affects bones is unclear. In the present study, the direct effects of retinoic acid (RA) on human and murine osteoclastogenesis were evaluated using cultured peripheral blood CD14 + monocytes and RAW264.7 cells. Both the activity of the osteoclast marker tartrate resistant acid phosphatase (TRAP) in culture supernatant and the expression of the genes involved in osteoclast differentiation together with bone resorption were measured. To our knowledge, this is the first time that the effects of RA on human osteoclast progenitors and mature osteoclasts have been studied in vitro. RA stimulated proliferation of osteoclast progenitors both from humans and mice. In contrast, RA inhibited differentiation of the receptor activator of nuclear factor kB ligand (RANKL)-induced osteoclastogenesis of human and murine osteoclast progenitors via retinoic acid receptors (RARs). We also show that the mRNA levels of receptor activator of nuclear factor kB (RANK), the key initiating factor and osteoclast associated receptor for RANKL, were potently suppressed by RA in osteoclast progenitors. More importantly, RA abolished the RANK protein in osteoclast progenitors. This inhibition could be partially reversed by a RAR pan-antagonist. Furthermore, RA treatment suppressed the expression of the transcription factor nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and increased the expression of interferon regulatory factor-8 (IRF-8) in osteoclast progenitors via RARs. Also, RA demonstrated differential effects depending on the material supporting the cell culture. RA did not affect TRAP activity in the culture supernatant in the bone slice culture system, but inhibited the release of TRAP activity if cells were cultured on plastic. In conclusion, our results suggest that retinoic acid increases proliferation of human osteoclast progenitors and that it inhibits RANK-stimulated osteoclast differentiation by suppressing RANK.
Endocrinology, 2003
Whereas bone morphogenetic protein (BMP)-signaling events induce maturational characteristics in vitro, recent evidence suggests that the effects of other regulators might be mediated through BMP-signaling events. The present study examines the mechanism through which retinoic acid (RA) stimulates differentiation in chicken embryonic caudal sternal chondrocyte cultures. Both RA and BMP-2 induced expression of the chondrocyte maturational marker, colX, in chondrocyte cultures by 8 d. Though the RA effect was small, it synergistically enhanced the effect of BMP-2 on colX and phosphatase activity. Inhibition of either RA or BMP signaling, with selective inhibitors, interfered with the inductive effects of these agents but also inhibited the complementary pathway, demonstrating a codependence of RA and BMP signaling during chondrocyte maturation. BMP-2 did not enhance the effects of RA on an RA-responsive reporter construct, but RA enhanced basal activity and synergistically enhanced BMP-2 stimulation of the BMP-responsive chicken type X collagen reporter. A similar synergistic interaction between RA and BMP-2 was observed on colX expression. RA did not increase the expression of the type IA BMP receptor but did markedly up-regulate the expression of Smad1 and Smad5 proteins, important participants in the BMP pathway. Inhibition of RA signaling, with the selective inhibitor AGN 193109, blocked RA-mediated induction of the Smad proteins and chondrocyte differentiation. These findings demonstrate that RA induces the expression of BMP-signaling molecules and enhances BMP effects in chondrocytes. Abbreviations: BMP, Bone morphogenetic protein; BMPR, BMP receptor; DNBMPR, dominant negative BMPR; RA, retinoic acid; RAR, RA receptor; RCAS, replication-competent avian leukemia virus with splice acceptor.
FEBS Letters, 1989
Spatial and temporal expression pattern of retinoic acid receptor @AR) genes was investigated in mouse finger bones during development by an in situ hybridization method with riboprobes synthesized from a human cDNA of the RAR-a. We found that the RAR genes are expressed intensively and specifically in calcifying fronts of the mouse finger bones, whereas the expression pattern is rather uniform in the limb buds and cartilage matrices of the embryonic fingers. Our findings are consistent with the fact that vitamin A is essential for normal mammalian bone development.
Journal of Applied Oral Science
Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective: Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods: ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA ® software. Results: RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions: In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.
Developmental Biology, 2009
The retinoic acid receptors α, β and γ (RARα, RARβ and RARγ) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARα and RARγ (or RARβ and RARγ) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARα and RARβ, however, are virtually normal, suggesting that RARγ is essential. In good correlation, we find that RARγ is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and prehypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids as indicated by previous RARE reporter mice and our direct biochemical measurements and thus, RARγ is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARγ over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by co-repressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARγ in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.
Development, 2016
We have previously shown that human and zebrafish hypomorphs in the retinoic acid (RA)-metabolizing enzyme Cyp26b1 display coronal craniosynostosis, caused by an RA-induced premature transitioning of suture osteoblasts to preosteocytes inducing ectopic mineralization of the suture's osteoid matrix. In addition, we showed that human CYP26B1 null patients have more severe and seemingly opposite skull defects, characterized by smaller and fragmented calvaria, while the cellular basis of these defects remained largely unclear. Here, treating juvenile zebrafish with exogenous RA or a chemical Cyp26 inhibitor in the presence or absence of osteogenic cells or bone-resorbing osteoclasts, we demonstrate that both reduced calvarial size and calvarial fragmentation are also caused by RA-induced premature osteoblast-to-preosteocyte transitioning. During calvarial growth, the resulting osteoblast deprival leads to decreased osteoid production and thereby smaller and thinner calvaria, while c...