BMP9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells Reguires Notch Signalling

Bone is one of the organ that retain the potential for regeneration intoadult life and the only tissue that can undergo continual remodelingthroughout life.There are two different bone formation pathways,intramembranous ossification and endochondral ossification.Boneregeneration is similar to endochondral ossification, starting withchemotaxis proliferation of mesenchymal stem cells. Efficacious boneregeneration would play an important impact on the clinical managementof many bone and musculoskeletal disorders such as segmental bone loss,fracture nonunion, osteoporotic fracture,failed spinal fusion and so on.Understanding the molecular mechanisms of bone formation is pivotal forstudying the pathogenesis of bone diseases.Bone formation in the embryo and repair and turnover in the adultinvolve the progeny of a small number of cells called mesenchymal stemcells. Mesenchymal stem cells (MSCs) are adult stem cells traditionallyfound in the bone marrow. However, mesenchymal stem cells can also beisolated from other tissues including cord blood, peripheral blood, fallopian tube, fetal liver, lung and so on. MSCs are pluripotent and capable ofdifferentiating into osteogenic, chondrogenic, myogenic or adipogeniclineage. Several signaling pathways have been implicated in regulatingstem cell selfrenewal and lineage commitment.We are just interested inelucidating the molecular mechanisms through which regulate theproliferation and differentiation of osteoblasts.BMPs(Bone Morphogenetic Proteins) are multifunctional regulatorsof proliferation and differentiation during development.BMPs can regulatethe differentiation of mesenchymal stem cells intocartilage,bone,tendon/ligament,and fat lineages.BMPs belong to thetransforming growth factor β (TGF-β) superfamily and consist of at least20members．In our lab’s previous study,we tested the distinct ability of14BMPs(BMP2-BMP15) to induce osteogenic differentiation of osteoblastprogenitor cells and found that BMP9exhibit the osteogenic activity bothin vitro cell experiment and in vivo nude models.BMP9is the most strongfactor to induce bone formation.BMP9(also known as GDF-2，growth differentiation factor2) is amember of the transforming growth factor (TGF)-β/BMP superfamily. Andwas first identified in the developing mouse liver.Its roles include inducingand maintaining the cholinergic phenotype of embryonic basal forebraincholinergic neurons, inhibiting hepatic glucose production and inducingthe expression of key enzymes of lipid metabolism, and stimulating murine hepcidin1expression. However, the functional role of BMP9in theskeletal system is largely unknown.Our lab have further demonstrated that BMP9regulates a distinct setof downstream that maybe a role in regulating BMP9-induced osteoblastdifferentiation of MSCs.The potent osteogenic activity of BMP9suggeststhat it may be used as an efficacious bone regeneration agent.Among thepossible targets of BMP9,Notch is the most valuable and less studied inosteogenesis.We just want to know the mechanism of cross-talk and signalintegration between the two systems.The aim of our study is:(1) to identify whether Notch signalingpathway is a necessary pathway in regulating BMP9-induced osteoblastdifferentiation of MSCs;(2) to investigate that Notch signaling play animportant role in BMP9-induced osteogentic differentiation;(3) toinvestigate the machanism that BMP9regulate the downstreampathway-Notch signaling to induce osteoblast differentiation of MSCs.In the first part of this study,the specific γ-secretase inhibitorsCompound E was used to inhibit the activity of γ-secretase．ALP activity ofthe early osteoblast marker was detected on osteogenic differentiation ofMSCs induced by BMP9combined with treatment of γ-secretaseinhibitors.The results demonstrated that the inhibitor of γ-secretaseinhibited BMP9-induced osteogenic differentiation of MSCs.Then，calciumdeposition of the later osteoblast marker was detected on BMP9-induced osteogenic differentiation of MSCs with the γ-secretase inhibitors byAlizarin Red S staining．The results showed that inhibition of the activity ofγ-secretase reduced calcium deposition of BMP9-induced osteogenicdifferentiation of MSCs in the later of bone formation.These data suggestthat Notch signaling play an important role in BMP9-induced osteogenicdifferentiation of MSCs．Then， the endogenic expression of Notch were detected byRT-PCR.And each Notch showed different expressive trend，Notch1wasthe most expressive one，but there was no Notch4or DLL4expressed.Andcell total RNA of iMEFs treated with dnNotch1adenovirus wasextracted，the inhibitive function of dnNotch1adenovirus was validate byRT-PCR.In vitro,all the results of ALP activity assay, expression ofosteopontin (OPN) and osteocalcin (OCN) by IHC, Alizarin Red S stainingshowed that dnNotch1can inhibit BMP9-induced early and late osteogenicdifferentiation of MSCs.In vivo,iMEFs were co-infected by dnNotch1andBMP9adenovirus,then were subcutaneous injected into nude mice toinvestigate the effection of dnNotch1in BMP9-induced bone formation.The result showed that:(1) for4weeks，cells that inoculated subcutaneouslyformed hard masses，indicating bone tissue formation;(2) The size of thebone tissues obviously decreased with inhibition of Notch1;(3) HE stainingshowed that inhibition of dnNotch1reduced the quantity and quality of thebone trabecular;(4) Alcian blue staining showed that inhibition of dnNotch1increased the immature cartilage matrix；(5) Masson’s Trichromeshow that dnNotch1reduced the mature and mineralized osteoid matrices.In short，inhibition of Notch1activity reduced the quantity and of quality ofBMP9-induced bone formation in vivo.Those results were consistent withthose observations in vitro．Based on the results of first two parts，it can be sure that there must beclose connection between Notch and BMP9on inducing osteogenicdifferentiation of mesenchymal stem cells.Then, the overexpression weredetected by RT-PCR in cells treated by Jagged1or DLL1．In vitro,all theresults of ALP activity assay, expression of osteopontin (OPN) andosteocalcin (OCN) by IHC, Alizarin Red S staining showed that Jagged1and DLL1can increase BMP9-induced early and late bone formation ofMSCs,and DLL1increased the expression of OPN and OCN,Jagged1increased OPN but not OCN.In vivo,iMEFs were co-infected by bothJagged1or DLL1and BMP9adenovirus,then were subcutaneous injectedinto nude mice to investigate the function of Jagged1or DLL1inBMP9-induced bone formation. The masses’s size was measured andanalyzed by Micro-CT,osteogenic process in masses was analyzed byhistologic（HE Stainning,Alcian Blue Stainning and Masson’s TrichromeStainning）.The results show that DLL1increased the mature of BMP9induced bone formation and Jagged1just increased chondrogenesis.Inshort， overexpression of DLL1increased BMP9-induced osteogenic differentiation of mesenchymal stem cells，overexpression of Jagged1increased chondrogenesis.Further, We got two special MEFs cell lines which were prepared frompresenilins1(PS1) and2(PS2) double knocked out (DKO),one isDKO-EV（EV，empty vector）,the other is PS1-rescued line,DKO-PS1toidentify the connection between Notch and BMP9.Fristly，the expression ofPS1and nacastin were detected by western blot.The result showed that PS1was completely knock out in DKO-EV and DKO-PS1was rescused withPS1.Then in vitro，comparison with DKO-PS1，the results of ALP activityassay, expression of osteopontin (OPN) and osteocalcin (OCN) by IHC,Alizarin Red S staining showed that BMP9-induced early and lateosteogenic differentiation were obviously decreased in DKO-EV.Invivo,the result show that knockout of PS1significantly reduced the size ofthe bone tissues,the quantity and quality of the bone trabecular, the degreeof mature and the activity of osteogenetic process of BMP9-induced boneformation.In short, PS1, the key component of the-secreatase of Notchsignaling acts indispensably on BMP9-induced osteogenic differentiationwhich further indicated that Notch signaling play a key role inBMP-9-induced osteogenic differentiation of MSCs.Based on the results of the first part，those data showed that：(1) thedepression of γ-secretase leads to decrease the osteogenic differentiation ofMSCs by BMP9；(2) the depression of Notch1leads to decrease the osteogenic differentiation of MSCs by BMP9;(3) overexpression ofDLL1or Jagged1change the situation of BMP9-induced MSCs osteogenicdifferentiation;(4) knockout of PS1significantly reduced BMP-9-inducedosteogenic differentiation of MSCs.In short, Notch signaling play a keyrole in BMP-9-induced osteogenic differentiation of MSCs and Notchsignaling pathway is a necessary downstream pathway in regulatingBMP9-induced osteoblast differentiation of MSCs.So the following studywould explore the mechanisms of induced MSCs into osteogenicdifferentiation by BMP9regulating Notch signaling pathway:(1) toinvestigate the effect of inhibition of Notch1by dnNotch1,theoverexpression of Jagged1or DLL1,the knockout of PS1on proliferationand cell cycle in early stage of BMP9-induced osteogenetic differentiation;(2) to investigate the expression of NICD and Notch1under the treatmentof BMP9;(3) to investigate the change of the expression of Notch withBMP9treatment，the expression of Runx2，OPN,OCN， Hey1, Id1andCTGF.Firstly，the data of Flow Cytometer showed that after24hs treatment，the G2phase and G2+S phase with BMP9treatment were significantlydeduced by dnNotch1;Jagged1or DLL1increased the G2phase and G2+Sphase with BMP9treatment; under BMP9treatment,the G2phase andG2+S phase in DKO-PS1were obviously higher than DKO-EV.Theseresults indicated that Notch signaling act indispensably on BMP9-induced proliferation of MSCs.Secondly,the result of IFC showed that under24hs’ treatment,BMP9significantly increased the expression of NICD and Notch1and promotedNICD translocating to the nuclear.Then, by RT-PCR,the expression of Notch receptors and ligands weredetected in early stage from day1to day3of BMP9-induced MSCs intoosteogenetic differentiation.The results showed Jagged1,Notch1andNotch2were increased by BMP9, Jagged1and Notch1showed angradually increased trend,Jagged2and DLL1were decreased.On day1,BMP9increased DLL4’s expression,but decreased it’s expression fromday2.On day3,BMP9increased DLL3.BMP9could not change the Notch3’trend and the expression of Notch4could not be detected. In general,theincreased expression members were more than decreased under BMP’streatment.After the knockout of PS1, Runx2,OPN,OCN,Hey1and Id1,CTGF were significantly higher in DKO-PS1than DKO-EV with thetreatment of BMP9.The results of the second part indicated that：（1）BMP9can promotethe proliferation of cells in the early stage of osteogenetic differentiation ofMSCs,but the promotion can be decreased by inhibition of Notch1orknockout of PS1,can be increased by Jagged1and DLL1.These resultssuggested that maybe Notch involved in the BMP9-promoted proliferationof MSCs in the early phase.（2）BMP9can increase the expression of Notch1and promote NICD translocating to the nucleus（.3）BMP9induceddifferent expressive trend of different Notch. Runx2,OPN,OCN,Hey1,Id1and CTGF were reduced by the knockout of the PS1even with thetreatment of BMP9.Taken together,Notch is the necessary passway of BMP9-inducedosteogenetic differentiation of MSCs.BMP9activated Notch signaling byinducing the change of expression of different Notch’s members includingincreased expression of Notch1and then increased the expression of NICDwhich subsequently leads to transcriptional activation of downstream targetgenes, and further promote the proliferation and osteogenic differentiationof MSCs.