| Aims: All-trans Retinoic acid(ATRA)is an active metabolite of vitamin A and plays an important role in bone development of vertebrate.The aim of this study is to elucidate the effects of RA on glucocorticoid-induced osteoporosis(GIOP)and the balance between bone formation and bone metabolism during the embryonic development of zebrafish,and to explore the mechanism of actions of RA on the bone remodeling.Methods:(1)Zebrafish embryos were exposed to 2 μM,4 μM,or 6 μM ATRA respectively from the fertilization till 14 days after fertilization(dpf).The hatching rate was recorded 72 hours after fertilization(hpf),and the mortality rate and malformation rate were recorded at 4-14 dpf.The concentration of ATRA used for the subsequent experiments was determined according to the results obtained in this part of research.(2)The GIOP zebrafish larvae was exposed to ATRA with three different concentrations(0.5μM,1 μM,or 2 μM)from 4 dpf to 9 dpf.The larvae were collected after 5 days of the treatment.Alizarin Red staining were conducted to observe the skeletal structure of the larvae.The homogenate of the whole fish was used for the measurement of the activity of alkaline phosphatase(AKP)and anti-tartaric acid phosphatase(TRACP).The total RNA extracted from zebrafish larvae was used for the quantitative analysis of the expression of osteogenic and osteoclastic marker genes Runx2 a,Runx2b,SP7,CSF1 a,RANKL and RBM14,a transcriptional co-activator.The whole mount in-situ hybridization was conducted on 9 dpf larvae to reveal the spacial expression pattern of Runx2 a,RANKL and RBM14.(3)To observe the effects of down-regulation of RBM14 on the expression of the above-mentioned osteogenic and osteoclastic marker and RA receptor coding genes,one-cell stage zebrafish embryos were injected with an antisense morpholino oligo,which specifically knocks down the expression of RBM14,and were incubated in embryonic medium till 48 hpf.Results:(1)High concentration(4-6 (?)M)of exogenous ATRA significantly inhibited the hatching rate,and increased the mortality and malformation rate of the embryos.(2)ATRA completely blocked the negative effects of prednisolone on bone mass and bone density detected by alizarin red staining and the activities of AKP enzyme.The antagonistic effects of ATRA against the osteoporosis induced by prednisolone is dose-dependent.(3)The q PCR data,which were consistent with the results of in situ hybridization analysis,showed that ATRA promoted the expression of osteogenic genes(Runx2a and Runx2b),while it also induced the up-regulation of osteoclastic genes(CSF1a and RANKL).(4)ATRA inhibited the expression of transcriptional co-activator RBM14 in zebrafish larvae,and blocked the up-regulation of RBM14 induced by prednisolone.(5)In morpholino knockdown experiments,RBM14 exhibited a negative regulatory effect on the expression of the four subtypes of RA receptors,key molecules in RA signaling pathway.The effects of down-regulation of RBM14 on the expression of the osteogenic and osteoclastic genes were similar to that induced by the exogenous ATRA that is promoting Runx2 expression and inhibiting the expression of RANKL.Conclusion:(1)The anti-osteoporotic effects of ATRA on GIOP is relied on its up-regulatory effects on the expression of osteogenesis-related genes;(2)There is a negative interaction between ATRA and transcriptional co-activator RBM14;(3)The down-regulation of RBM14 expression induced by ATRA mediates the anti-osteoporotic effects of ATRA in GIOP zebrafish. |
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