Charactrazation Of TGFβ Receptor In BMP9 Induced Osteogenic Differentiation

BMPs(Bone Morphogenetic Proteins) belong to the transforming growth factorb (TGFβ) superfamily, Originally isolated as proteins that induce bone and cartilage formation in vivo, BMPs are multifunctional regulators of proliferation and differentiation during development.. BMPs can regulate the differentiation of mesenchymal stem cells into cartilage, bone, tendon/ligament, and fat lineages. More than 20 BMPs have been identified, Prior work show that BMP2, BMP5, BMP7 has potent osteogenic activity, and are currently being investigated in human clinical trials of fracture healing and spine fusion,but the result is not satisfied. Therefore, it very important to look for more efficacious factor which can stimulate osgenetic defferentiation of cells,.In our lab's previous study, we elucidate the distinct ability of 14 BMPs (BMP2~BMP15) to induce osteogenic differentiation of osteoblast progenitor cells by using adenovirus that eapress these 14 BMPs. Using mesenchymal stem cell line C3H10, we have demonstrated that, besides BMP2 and BMP7, BMP4, BMP6, BMP9 exhibit the osteogenic activity both in vitro cell experiment and in vivo nude models, and osteogenic activity of BMP9 are more than BMP2 and BMP7 which have been used in clinial trails, so BMP9 are the most strong factor to induce bone formation. BMP9 are the most efficacious factor for potentail clinical applications involving bone formation.But the mechanism under the bone formation induced by BMP9 reamins unclear. Belong to the TGFβsuperfamily, all BMPs mainly transduct their signal by TGFβsignal traduction patheay, and then control bone formation. In the first step,BMPs initiate their signaling cascade by binding to a dimeric complex of two transmembrane serine–threonine kinase receptors, termed TGFβtype I and type II. receptor,and then, the activated receptor subsequently phosphorylate transcriptional factors, called Smads, which activate the expression of target genes in concert with coactivators. Therefore, in this signal pathway, TGFβreceptor act as a key point which can bind BMPs and activate Smads, TGFβreceptor are most important molecular in early signal transduction of BMPs, and involve in osteogenic activity of BMPs. Unlike other osteogenic BMPs such as BMP2 and BMP7 whose TGFβreceptor involve bone formation have been characterized, TGFβreceptor of BMP9 in osteogenesis are unknow, this also is a limited factor for elucidating mechenism of BMP9 induced bone formation.Based on this background, in this research, we applied comprehensive techniques in molecular biology, cell biology and experimental zoology such as cell culture, gene clone, gene transfection, recominant adenovirus techniques, cell differantiation techniques, real time PCR, xenograft animal model, Micro-CT, histology techniques, and mainly focu on chracterization of TGFβreceptor in BMP9 induced bone formation, from this research, theoreetic evdence can been find to elucidate the mechanism under BMP9 induced osteogenesis. The total research is divided into two parts and the main research results are as follows:In the first part, we use PCR to amplify fragment containing extracellular domain and transmemrane domain of TGFβreceptor after analyising nucleotide of 7 TGFβtypeⅠreceptors and 4 TGFβtypeⅡreceptors, and then clone these fragments into adenovural shuutle vector pAdtrace-TO4. By using colony PCR, Restriction Endonucleases and DNA sequencing, dominant negative mutant (dn-receptor) of total 11 TGFβreceptors were constructed successfully, then, recombinant adenovirus vectors of 11 dn-receptors were generated in Bjadeasy competent cells. Recombinant adenovirus vectors were transfected into 293 packaging cell. Recombinat adenovirus were packaged and amplified in 193 packaging cell, and finally we got 11 dn-receptor adenovirus. In orde to validate expression of dn-receptor adenovirus in target cell, mesenchymal stem cell C3H10 were infected by dn-receptor virus, after 24hour all 11 dn-receptor virus can infect C3H10 and express RFP in C3H10, after 48hour, cell total RNA was purified using trizol reagent and cDNA was produced by RT reaction. All 11 dn-receptors can express in C3H10 by using PCR to validate. In this part of research, we finally got 11 dn-receptor adenovirus and validated their expression in C3H10 cell. The dn-receptors can competed with wild receptors by binding to ligand, as a consequence, inhibit function of ligand. dn-receptor adenovirus constructed in this part can be used not only in second part of this reseach but also in other related TGFβreceptor research in TGFβsignal pathway.In the second part, we use dn-receptor virus and BMP9 to co-stimulate meshchymal stem cell C3H10, bone marrow stromal cells (BMSC), mouse embryonic fibroblasts (MEFs) and myogenic cell C2C12. Totally, 5 of 11 dn-rceptors who can inhibit BMP9 induced ALP and luciferase acitivity were identified by using ALP staing, ALP quantitative reading, luciferase reportor assay. They are dnALK1,dnALK2,dn-BMPRⅡ,dn-ActRⅡ和dnActRⅡB. The further study showed that the inhibition of dnALK1,dnALK2,dn-BMPRⅡ,dn-ActRⅡ和dnActRⅡB in BMP9 induced ALP repression was dose dependent, furthermore, dnALK1,dnALK2,dn-BMPRⅡ,dn-ActRⅡ和dnActRⅡB can also inhibit Calcification of C3H10 and MEFs induced by BMP9, and inhibit BMP9 target gene exression in C3H10 cell. C3H10 were co-infected by dnALK1,dnALK2,dn-BMPRⅡ,dn-ActRⅡ, dnActRⅡB and BMP9 adenovirus, and then were subcutaneous injieced into nude mice, the bulb's size was measured Micro-CT , osteogenetic process in bulb was anlysised by histologic staining. We found that dnALK1,dnALK2,dn-BMPRⅡ,dn-ActRⅡ和dnActRⅡB can inhibit bone formation of C3H10 induced by BMP9 in nude mice. Therefore, 5 wild receptors (ALK1, ALK2, BMPRⅡ, ActRⅡ, ActRⅡB) may involve bone formation induced by BMP9. We use pSOS system to screen efficacious siRNA for thse wild receptor knock down (we try ALK1/ALK2 first), and found that ALK1/ALK2 knock down can inhibit BMP9 induced luciferase activity.In all, we characterized the potential TGFβreceptor in BMP9 induced osteogenesis from molecular cell level to whole animal level comprehensively and the research conclusions are as follows:1. TGFβreceptor is correlative to osteogenetic activity of BMP9. Dominant negative mutant of BMP9 related TGFβreceptor inhibit cell osteogensis induced by BMP9.2. The dn-receptor adenovirus we constructed can efficaciously express in target cell, they can be used not only in research of BMP9 related receptor, but also in other research of TGFβsuperfamily.3. After test 4 cell line (C3H10, BMSC, MEFs, C2C12), we found that 2 of 7 typeⅠdn-receptor and 3 of 4 typeⅡdn-receptor can efficaciously inhibit BMP9 induced ALP and luciferase activity. The inhibition of BMP9 induced ALP activity by these dn-receptor are dose-dependent.4. Further study confirmed that dnALK1, dnALK2, dn-BMPRⅡ, dn-ActRⅡ, dnActRⅡB can efficaciously inhibit calcification induced by BMP9, and inhibit target gene expression controlled by BMP9. Therefore, the wild receptor of these 5 dn-receptors (ALK1, ALK2, BMPRⅡ, ActRⅡ, ActRⅡB) may play key role in BMP9 induced osteogensis.5. The efficacious siRNA for ALK1/ALK2 are screened by pSOS system, we found that ALK1/ALK2 knock-down by RNAi can inhibit BMP9 induced luciferase activity, these result implied that osteogentic activity of BMP9 can be inhibit by BMP9 related receptor knock down. It is meaningful to keep probing the effect of BMP9 related TGFβreceptor (ALK1, ALK2, BMPRⅡ, ActRⅡ, ActRⅡB) knock down on BMP9 induced bone formation, thses further study will be helpful to elucidate mechanism behind BMP9 induced osteogenesis.