Delineating The Regulatory Roleof TAp63in BMP9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

BackgroundBone graft is commonly used to treat fracture nonunion, bone defectand spinal fusion. However, bone regeneration caused by traditional bonegraft is in general slow. Moreover, fracture nonunion, vertebralpseudarthrosis sequelae and bone defectmay may develop, which affectsthe outcomes of bone regeneration and fracture healing. Effective boneregeneration requires at least three components, including bone scaffoldmaterials, bone seed (progenitor) cells and biofactors that can promoteosteogenicdifferentiation.Type I collagen sponge and hydroxyapatite basedbone scaffolds have been used rather successfully. Mesenchymal stem cells(MSCs) have been frequently used as seed cells for bone regeneration.However, because mesenchymal stem cells have multi-lineage potential, itis critical to identify factors that can effectively direct mesenchymal stemcells to differentiate into osteoblast lineage and eventually matureosteocytes.Mesenchymal stem cells are multipotent stem cells and presented in bone marrow stromal tissue and the connective tissues of multiple organs.Mesenchymal stem cells can differentiate into osteoblasts with properstimulations. Bone morphogenetic proteins (BMPs) can induce themesenchymal stem cells to differentiate into osteoblasts.There are at least14BMPs in mammalian cells. BMP2, BMP4, BMP6, BMP7and BMP9have been shown to induce the mesenchymal stem cell differentiating intoosteoblasts. Our lab previously demonstrated that BMP9is one of the mostpotent osteogenic BMPs. We and others have shown that BMP9can inducethe mesenchymal stem cells to differentiateinto osteoblasts through theclassical BMP-Smad pathway, which activatesits downstream targets totransduce BMP9signaling. It has been reported that Smad-independentnon-classical pathway (e.g. MAPK, Wnt, PI3K/Akt) may also participate inBMP9-regulated mesenchymal stem cell differentiation into osteoblasts. Tothe best of our knowledge, little is known about the upstream regulatorymechanism of BMP9functions. The current investigation focuses on therole of p63transcription regulation factor of the p53tumor suppressorfamily in regulating BMP9-induced osteogenic differentiation of MSCs.We found that p63upregulates the expression of BMP9andaugments theBMP9-induced differentiation of MSCs. Our findings significantly expandour current understanding of the molecular mechanism through whichBMP9regulates osteogenic differentiation of MSCs. Objectives(1) to investigate if there is a crosstalk between BMP9and p63, and ifBMP9is a direct downstream target of p63;(2) to investigate the effect ofTAp63on BMP9-induced osteogenic differentiation of mesenchymal stemcells;(3) to investigate the mechanisms through which TAp63exerts itsregulatory effect on BMP9-induced osteogenic differentiation of MSCs.MethodsIn the first part of this study, we test if p63protein is expressed ingrowth plates of long bones in2-week old C57mice. We constructed theadenovirus vectors expressing TAp63and ΔNp63, AdTAp63andAdΔNp63, using the AdEasy system. Using these adenovirus vectors,we infected iMEFs and Tap63-/-MEFs with AdTAp63to determine thechange in the expression of BMP9.Moreover,we analyzed if TAp63canbind to the promotor of BMP9to regulate BMP9expression.In the second part, we tested if TAp63and ΔNp63can affectBMP9-induced ALP activity both in wildtype MEFs and TAp63-/-MEFs.We also tested if TAp63and ΔNp63affect the expression of OPN、OCNand the calcium deposition,which are the late makersof osteogenicdifferentiation.Moreover,we sought to determine if TAp63and ΔNp63can affect the expression of Osterix and Runx2, both of which areimportant regulators of osteogenics differentiation. For in vivo studies, theiMEFs cells co-infected with AdBMP9/RFP, AdBMP9/TAp63orAdBMP9/ΔNp63were subcutaneously andintramuscularly injected into athymic BALB/c nude mice respectively to investigate the effect ofTAp63and ΔNp63on BMP9induced osteogenic differentiation of MSCsin vivo.In the third part,we analyzed if TAp63iMEFs can affect theproliferation of iMEFs cells. The proliferation of iMEFs treated with BMP9and the iMEFs was assessed by MTT assay. Subsequently, cell cycleanalysis of iMEFs stimulated with BMP9and/or TAp63was carried out todetermine if TAp63can affect the cell cycle prolife of iMEFs transducedwith BMP9. Furthermore, the phosphorylation of R-Smads,MAPKp38andAKT was analyzed by ICC(immunocytochemistry) in BMP9-inducedMSCs. The localization of Smad1/5/8was also determined using ICC onBMP9-induced MSCs in the presence of TAp63.Results1. AdTAp63and AdΔNp63were constructed successfully. TAp63can promote the expression of BMP9in wildtype MEFs andTAp63-/-MEFs at mRNA level. Using ChIP assay,we found TAp63canbind to promotor of BMP9directly, suggesting that there may becross-regulation between p63and BMP9. The expression of p63can befound in growth plates of long bones.2. To determine the effect of TAp63on BMP9-induced differentiation,we found that TAp63can enhance alkaline phosphatase (ALP) activity wasinduced by BMP9in wildetype MEFs and TAp63-/-MEFs. The expression of OPN、OCN、Osterix and Runx2was increased in the iMEFs co-infectedwith AdBMP9and AdTAp63. Conversely, forced expression of ΔNp63exhibited opposite effect..The in vivo results showed that TAp63canpromote BMP9-induced bone formation and maturation in bothsubcutaneous masses and muscle masses. The ΔNp63inhibitedBMP9-induced bone formation and maturation in both subcutaneousmasses and muscle masses，which is consistent with the in vitro findings.3. To understand the mechanisms through which TAp63regulatesBMP9-induced osteogenic differentiation of MSCs,we found TAp63inhibited the proliferation of BMP9-induced MSCs.The proportion ofG0/G1cells were increased significantly in the MSCs expressing bothBMP9and TAp63. Our above results suggest that TAp63may inhibit theproliferation of MSCs to pomote differentiation. Furthermore, we foundTAp63can promote the phosphorylation of Smad1/5/8、p38MAPK andAKT and the nuclear translocation of Smad1/5/8to promote signaltransduction.ConclusionsIn summary, our studies strongly suggest that TAp63may function asan upstream regulator of BMP9expression; and that TAp63expressioncan promote BMP9-induced osteogenic differentiation through theregulation of cell cycle and signal transduction pathways.