Methotrexate And Triptolide Regulate Notch Signaling Pathway By Targeting The Nedd4-Numb Axis

Objective:Methotrexate(MTX)is commonly used in the treatment of rheumatoid arthritis,acute leukemia and psoriasis.MTX may cause some side effects such as myelosuppression,but the exact mechanism of myelosuppression is unknown.The Notch signaling pathway is considered to be an important pathway regulating the regeneration and dynamic homeostasis of hematopoietic stem cells(HSC),which plays an important role in promoting bone marrow hematopoietic function.The current reports on MTX-induced myelosuppression are mainly based on clinical observations.In this study,we investigated the regulation of Notch signaling pathway by MTX,with the aim of providing new ideas and strategies to alleviate MTX-induced myelosuppression in clinical treatment.Methods:(1)MTX-treated primary bone marrow cells were used to detect the effect of MTX on cytotoxicity using the CCK8(Cell counting kit 8 assay).After MTX treatment,the effect of MTX on the mRNA levels of several key members of the most common hematopoieticrelated signaling pathways was analyzed by RT-qPCR,and the signaling pathways regulated by MTX were screened compared with the control group.The effect of MTX on the level of Notch 1 protein was analyzed by Western Blot,and the influence that MTX has on other members of Notch signaling pathway was further analyzed by RT-qPCR.(2)After the cells were treated with MTX,the effect of MTX on the level of Numb protein,a negative regulator of Notch signaling pathway,was analyzed by Western Blot.The effect of MTX on the level of Numb mRNA was detected by RT-qPCR.(3)After overexpression of Flag-Numb,MTX was analyzed using CHX(Cycloheximide,an inhibitor of protein synthesis)to determine whether MTX affects Numb stability.Immunoprecipitation and Western Blot were used to analyze the effect of MTX on the ubiquitination level of Numb protein and the type of ubiquitination.(4)Using PR-619-treated cells,we analyzed whether MTX regulates Numb through the E3 or DUBs pathway by the Western Blot method.Combined with the Ubibrowser E3 prediction database to analyze potential ubiquitin ligases,we used immunoprecipitation(coIP)technique as well as Western Blot method to probe the screened E3 on Numb.The interaction between them was further analyzed.Cells were treated with MTX and analyzed by Western Blot to determine whether MTX interfered with the interaction between E3 ubiquitin protein ligase and the substrate Numb.(5)Cells were treated with seven clinically common anti-inflammatory herbal monomers,respectively,and the drug Triptolide(TPL),which down-regulates the level of Numb protein,was screened by Western Blot.The effect of TPL on the expression level of endogenous Notch1,and Numb were analyzed and identified.The CCK8 assay was used to investigate the effect of TPL on mouse bone marrow primary.The effect of TPL on other members of the Notch signaling pathway was further analyzed by RT-qPCR.The combined effect of MTX and TPL was examined by Western Blot and RT-qPCR to determine whether TPL could slow down the influence of MTX on the Notch signaling pathway.The impact of the combination of the two drugs on cellular toxicity was investigated by CCK8 assay.Results:(1)The results of CCK8 experiments showed that MTX had significant toxic effects on cells.Different concentrations of MTX treatment did not affect endogenous Smad2 and Ptch1mRNA levels,but markedly decreased endogenous Notch2 mRNA levels.Further studies revealed that MTX down-regulated Notch signaling pathway receptor,ligand and downstream target gene expression which inhibited Notch signaling pathway.(2)In mouse primary bone marrow cells and RAW264.7 cells,MTX significantly upregulated the level of Numb protein,a negative regulator of Notch signaling pathway,in a dose-dependent and time-dependent manner compared with the control group.RT-qPCR results showed that MTX did not affect Numb transcript expression.(3)MTX slowed down the degradation rate of Numb.The level of polyubiquitination of Numb was down-regulated by MTX,which regulates the K48-linked polyubiquitination of Numb protein.(4)MTX regulated Numb protein levels through the E3 pathway.E3 ubiquitin ligase Nedd4 interacted with Numb and degraded Numb protein by promoting Numb protein K48linked polyubiquitination modifications.MTX restricted the Nedd4-mediated downregulation of Numb protein levels.MTX significantly inhibits Nedd4-induced upregulation of Numb protein ubiquitination levels.MTX blocked the interaction of Nedd4 with Numb and inhibited their binding.(5)TPL,a drug that down-regulates Numb protein levels,was screened from seven clinically used anti-inflammatory herbal monomers by Western Blot.TPL up-regulated Notch1 protein levels.TPL was not toxic to mouse bone marrow cells.TPL upregulated the expression of Notch signaling pathway receptors,ligands and downstream target genes,and activated Notch signaling pathway.The combination of MTX and TPL alleviated the promotive effect of MTX on Numb protein and the inhibitory impact on Notch signaling pathway.The combination of TPL and MTX attenuated the cytotoxicity caused by MTX.Conclusions:The study provides evidence that MTX strongly inhibits the Notch signaling pathway.MTX inhibited the interaction between Nedd4 and Numb,thereby limiting the K48-linked polyubiquitination of Numb.This in turn inhibited Notch signaling by reducing Notch1 protein levels.Interestingly,we identified a monomeric drug,TPL,which attenuates the inhibitory effect of MTX on Notch signaling pathway.This study further deepens our understanding of MTX-mediated regulation of Notch signaling and therefore may provide a potential idea for alleviating MTX-induced myelosuppression in clinical treatment.