The Effect And Mechanism Of All-trans-retinoic Acid Modulates Wnt3A-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

[OBJECTIVE] Osteogenic differentiation of mesenchymal stem cells(MSCs)is a vital process for bone remodeling and plays important roles in the tissue repair and cell growth.MSCs are multipotent progenitors with the potential to differentiate into osteogenic,chondrogenic,and adipogenic lineages,depending on both the extracellular milieu and intracellular signalling.Canonical Wnt signalling is essential for MSC osteogenic differentiation,and it interacts with several nuclear receptors,including the retinoic acid receptor,vitamin D receptor,and glucocorticoid receptor.In this study,we aimed to analyse the effect of All-trans-retinoic acid(ATRA)on Wnt3A-induced MSC osteogenic differentiation and elucidate the underlying molecular mechanisms.[METHODS] 1、The effects of ATRA on Wnt3 A induced osteogenic differentiationof MSCs.ALP activity was measured using a modified Great EscAPe SEAP Chemiluminescence assay.The results were normalized to total cellular protein concentrations among the samples.Histochemical staining was performed with a BCIP/NBT Alkaline Phosphatase staining assay kit.2、We next examined whether ATRA and Wnt3 A synergistically regulate developing bone by using the foetal limb culture approach.To confirm these results,we performed in vivo stem cell implantation.Our previous studies demonstrated that MSC implantation is a reliable and effective approach to investigating ectopic bone formation.3、Crosstalk between ATRA and the canonical Wnt signalling pathway in the regulation of MSC osteogenic differentiation.We analysed the effect of ATRA on Wnt3A-induced Tcf/Lef reporter activity and the nuclear and cytoplasmic expression of β-catenin.Western blotting analysis of Cyp26a1 protein expression 4、The effect of PI3K/AKT/GSK3β pathway on ATRA and Wnt3 A induced MSC osteogenic differentiation.C3H10T1/2 cells and MEFs were treated with Wnt3 A and ATRA.Seventy-two hours after treatment,protein levels were analysed by Western blotting with the indicated antibodies.C3H10T1/2 cells were pre-treated with different LY294002 doses and treated with Wnt3 A and ATRA.Seventy-two hours post treatment,Western blotting analysis was performed with the indicated antibodies.[RESULTS] 1 、 ATRA and Wnt3 A act synergistically to induce osteogenic differentiation of MSCs.ATRA induced earlier and stronger ALP activity than Wnt3 A,and the ALP activity was further enhanced by co-treatment with both ATRA and Wnt3 A.ATRA potentiates Wnt3A-induced late osteogenic marker expression and matrix mineralization.Upon ATRA treatment,Wnt3A-infected C3H10T1/2 cells showed increased osteocalcin and osteopontin expression compared to Wnt3 A infection alone,while ATRA alone failed to induce noticeable expression of these markers.ATRA also enhanced Wnt3A-induced formation of calcium mineral deposits indicative of matrix mineralization 2、ATRA augments hypertrophic chondrocyte zone expansion in Wnt3A-induced foetal limb explants.The total area of new bone formation was increased compared to the control group,and Wnt3A/ATRA treatment had the largest effect.Histologically,ATRA or Wnt3 A alone exhibited hypertrophic zone expansion,while co-treatment further increased the hypertrophic zone.Exogenous Retinoic acid receptors α(RARα)and Wnt3 A expression synergistically to induce ectopic bone formation.To confirm these results,we performed in vivo stem cell implantation.RARα overexpression alone or with Wnt3 A formed detectable bony masses,while masses did not form in mice injected with cells infected with Wnt3 A or RFP alone.3、ATRA and canonical Wnt signalling crosstalk in MSC osteogenic differentiation.We found that ATRA only mildly induced pTOP-LUC reporter activity,whereas ATRA enhanced Wnt3A-induced reporter activity.Although total β-catenin protein levels did not change between groups,ATRA/Wnt3 A co-treatment increased nuclear β-catenin localization.We examined whether Wnt3 A affected RA signalling through Cyp26a1.qPCR analysis indicated that Wnt3 A overexpression significantly suppressed Cyp26a1 transcription.In addition Wnt3 A attenuated ATRA-induced Cyp26a1 expression.Consistent with the mRNA results,Western blotting analysis showed that Wnt3 A significantly diminished ATRA-induced Cyp26a1 expression.Furthermore,β-catenin silencing elevated Cyp26a1 expression at both the transcriptional and translation levels.4、 ATRA cooperates with Wnt3 A to promote β-catenin activity in MSCs through the PI3K/AKT/GSK3β pathway.Although total AKT protein levels were similar in all of the groups,AKT phosphorylation at Ser473 significantly increased in the Wnt3A/ATRA group.Furthermore,phosphorylation of GSK3β at Ser9,which results in its inactivation,was highest in the ATRA/Wnt3 A group.To extend these observations to other cell types,we tested MEFs,in which we observed similar results.We next investigated the important role of PI3 K in this model.The broad-spectrum PI3 K inhibitor LY294002 blocked Wnt3A/ATRA-induced AKT and GSK3β phosphorylation in a dose-dependent manner.Accordingly,ALP activity was largely abolished by 30 μM LY294002 pre-treatment,suggesting that the PI3K/AKT/GSK3β pathway at least partially contributes to MSC osteogenic differentiation.Increased AKT phosphorylation led to β-catenin translocation from cell-cell contacts to the cytosol and nucleus.We next prepared site-specific antibodies to assess the β-catenin phosphorylation status.Treatment with ATRA/Wnt3 A synergistically enhanced β-catenin phosphorylation at ser552,while ATRA or Wnt3 A alone only slightly affected the phosphorylation of this residue.[CONCLUSION] 1、ATRA potentiates the Wnt3A-induced early and late osteogenic markers expression.2、ATRA enhanced Wnt3A-induced Tcf/Lef activity and nuclear β-catenin translocation in MSCs.3、Wnt3A attenuates the ATRA-induced Cyp26a1 expression,inhibiting the degradation of ATRA into its oxidative forms.4、ATRA and Wnt3 A at least partially promote osteogenic differentiation via activating the PI3K/AKT/GSK3β signalling pathway.