Regulation Of BMP Signaling By FBXO30 And Its Correlation With Neural Tube Defects

Neural tube defects(NTDs)are a serious congenital birth defect of the central nervous system caused by impaired embryonic neural tube closure.Its etiology is complex and the pathogenesis is not yet clear.Therefore,further investigation in pathogenesis of NTDs can help us gain more understanding in its causes.NTDs are caused by the external environment and genetic factors.The imbalance of nutrient intake during peri-pregnancy is a very important risk factor.Retinoic acid(RA),the major bioactive metabolite of retinol or vitamin A,has an important regulating effect in embryonic development.RA binds to retinoic acid receptors(RAR)as ligand when it enters cells and participates in many biological processes such as cell growth,differentiation,and embryonic development.RAR is a key factor in the retinoid signaling pathway and cooperate with FGF,Wnt,and BMP signal pathways to regulate the development of the nervous system.RAR defective embryonic animal model shows NTDs.The regulation of RAR include transcriptional and protein levels.It has been reported that RAR can be degraded by the ubiquitin-proteasome pathway.Therefore,it is relatively important to search for ubiquitin-protein ligase(E3)of RAR.IP/MS results showed that the FBXO30 and RARy may interact with each other.F-box proteins family are involved in many biological processes such as cell cycle regulation,signal transduction,nutrient metabolism,immune response and development.Whether FBXO30 regulates RARy protein stability through ubiquitin-proteasome pathway?Whether it affects the embryonic neural development-related pathways,and then cause NTDs?In order to answer these questions,we have identified the following research objectives:To study the mechanism of FBXO30 as E3 regulating RARy protein stability and regulation of BMP pathway;From the RA interventional cell model and mouse NTDs model,we studied the regulation of BMP pathway related gene expression by FBXO30,further verified the association of FBXO30 and BMP pathway related gene expression in human NTDs samples.We have demonstrated that RARy interacts with FBXO30 in vivo through internal and external co-immunoprecipitation(Co-IP)and subcellular localization.Through truncation interaction experiments,it was revealed that FBXO30 interacts with RARy via its F-box domain.In the aspect of rugulation mechanism,we verified that FBXO30 can regulate protein stability by mediating degradation of the ubiquitin-proteasome pathway of RARy by western blotting,qPCR,and in vivo ubiquitination experiments.In terms of physiological function,RNA-seq in cell lines with FBXO30 knock down found that they were associated with neural development.Because RARy is a negative regulator of the BMP pathway and FBXO30 negatively regulates the stability of RARy,we speculated that FBXO30 should be a positive regulator in BMP pathway.The BMP signal pathway penetrates in the development of the neural tube at all times,and its effect has temporal and spatial specificity.To this end,we first confirmed from the aspects of BMP reporter gene activity,Smad phosphorylation level,and BMP pathway target genes that FBXO30 antagonized the inhibitory effect of RARy on BMP pathway.Then using RA-treated cell model and mouse NTDs model confirmed that the protein level of FBXO30 decreased and the BMP pathway was inhibited under the intervention of RA.Finally,in the human NTDs samples,we used the methods of Nanostring and western blotting to further verified the FBXO30 and BMP pathway related gene changes and their relevance,suggesting that FBXO30 may participate in the occurrence of NTDs.We demonstrated the significance of the FBXO30 regulation of BMP pathways from the molecular,cellular,animal and human NTDs smaples,and revealed the importance of FBXO30 in NTDs,extending the functional scope of the F-box family.In summary,this study has revealed that the ubiquitin protein ligase FBXO30 negatively regulated the protein stability of RARy,elucidated its regulation in the BMP signaling pathway,and is validated in NTDs mouse models and human NTDs.It suggested that FBXO30 may participate in the development of neural development and occurrence of neural tube defects.