The Role Of Growth Hormone In BMP9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable ofdifferentiating into osteogenic, chondrogenic, adipogenic, and myogenic lineages.Osteogenic differentiation is a complicated and well-regulated process results from thecontrol of a series of endogenous and environmental factors as well as various signalingpathways. A better understating of molecular mechanisms underlying osteogenicdifferentiation will lead to the development of novel and targeted therapies for bonediseases.Bone morphogenesis Proteins (BMPs) play an important role in the early stage ofosteogenesis of MSCs. BMPs belong to the transforming growth factor β (TGF-β)superfamily， and they are a group of highly conserved, secreted multifunctionalproteins which are significant for cell proliferation，adhesion，differentiation, skeletaldevelopment and bone formation. Currently, at least15types of BMPs have beenidentified in humans. Among these members, BMP2, BMP4, BMP6, BMP7, and BMP9can effectively induce osteogenic differentiation of MSCs in vitro and in vivo.Furthermore, BMP9is one of the most osteogenic and yet least studied BMPs.In our previous study, total RNA of C3H10T1/2infected with BMP9adenovirus(Ad-BMP9) was collected for microarray analysis. The data showed that40genes weremost significantly regulated in MSCs upon BMP9stimulation, and growth hormone(GH) gene was among the most significantly up-regulated genes induced by BMP9.This result indicated that GH may associate with the regulation of osteoblastdifferentiation in response to BMP9. Growth hormone is a proteohormone secreted bythe pituitary gland. GH fulfills its function by binding to the GH receptor and insulin-like growth factorⅠ (IGF-Ⅰ). In order to determine the functional role of GH inBMP9-induced osteogenic differentiation as well as the mechanism involved in it, thevector expressing GH was constructed, and the adenovirus was generated. Subsequently,the effect of GH on BMP9-induced bone formation was investigated on three levels: invitro, organ culture and in vivo. In addition, chromatin immunoprecipitation and smallmolecule inhibitors were used to explore the molecular mechanism. Our studies mainlyinclude:①Vector named pCMV-SPORT6.1which contained human GH gene waspurchased from Thermo. After digestion reactions, GH gene was cloned into an adenoviral shuttle vector. Verified the constructs by DNA sequencing, and thentransferred the constructs into BJAdEsay bacterial cells for making adenoviralrecombinants. PacⅠ digestion reaction was performed to liberate the adenoviralgenome, followed by transfection of HEK293cells with lipofectAMINE to generateadenovirus which named AdR-GH.②C3H10T1/2cells are commonly used MSCs, obtaining from ATCC. Theexpression of MSC markers was characterized using immunofluorescence staining. Ourdata showed that C3H10T1/2cells expressed MSC markers, including CD73, CD44,CD90/Thy-1, CD117/c-kit, CD29/Integrinβ1, CD133/Prom1, BMPR Ⅱ, CD105/Endoglin and CD166/ALCAM.③C3H10T1/2cells were infected with Ad-BMP9, and total RNA was isolated ondays1,3,5,7after infection for RT-PCR reactions. Using RT-PCR, we found that theexpression of GH obviously induced by BMP9at the four time points.Immunohistochemistry (IHC) staining evaluated of juvenile long bones reveals that GHis highly expressed in the osteoblasts of the growth plate. Four adenoviruses infectionstrategies were designed for in vitro experiment: Ad-GFP/Ad-RFP（control group）,Ad-GFP/AdR-GH, Ad-BMP9/Ad-RFP and Ad-BMP9/AdR-GH. C3H10T1/2cells wereinfected with these four group viruses, and then the osteogenic markers, such as alkalinephosphatase, osteocalcin, osteopontin and mineralization were evaluated. GH alonecould not initiate osteogenic differentiation. However, a combination of GH and BMP9significantly induced the expression of osteogenic markers compared with other threegroups. These results indicated that GH promoted the BMP9-induced osteogenicdifferentiation of MSCs in vitro.④The skinned hindlimbs of fetal mouse were dissected for organ culture.Hindlimbs were infected with the four group viruses as described before, and the greenfluorescence dye calcein was added to the medium to trace the new bone formation.Tibia’s cross section area was calculated on days2,4,8,12after infection. The datashowed that more new bone formation found in BMP9/RFP group compared withGFP/RFP control group, whereas the BMP9/GH group had the highest level of newbone formation, as judged by the calcein incorporation. Haematoxylin and eosin (H&E)staining image at a higher magnification showed that BMP9/GH stimulation expandedthe hypertrophic chondrocyte zone contrasted with that of the control group. Theseorgan culture studies suggest that GH may enhance BMP9-induced endochondralossification. ⑤Subconfluent C3H10T1/2cells were infected with the four combinations ofadenoviruses mentioned above for36hours. Cells were collected for subcutaneousinjection into the flanks of athymic nude mice. Four weeks after implantation, micewere sacrificed and the implantation sites were retrieved for microcomputedtomography (MicroCT) analysis.3D data were obtained using Amira5.3software. OnlyMSCs infected with BMP9/RFP or BMP9/GH adenoviruses could generate ectopicbones. Quantitative analysis data showed that more bone masses and higher bonedensity was found in BMP9/GH treating group. On H&E and IHC staining examination,ectopic bones formed by BMP9-transduced cells had some mature bone matrices andtrabeculae as well as numerous undifferentiated mesenchymal progenitor cells. On theother hand, cells stimulated by both BMP9and GH generated more mature bonematrices and thicker trabeculae, with minimal undifferentiated mesenchymal progenitorcells. Masson’s trichrome staining confirmed that GH enhanced BMP9-induced matrixmineralization.⑥Two pairs of primers were designed for ChIP assays according to the putativeSmad binding sites in GH promoter region. Subconfluent C3H10T1/2cells wereinfected with Ad-GFP or Ad-BMP9for30hours. Cells were cross linked. GenomicDNA was sonicated for immunoprecipitation with anti-Smad1/5/8. The retrievedgenomic DNA was subjected to PCR reaction using the two pairs of primers. The PCRresults showed that anti-Smad1/5/8antibody pulled down genomic fragmentscontaining the GH promoter region, suggesting that GH may be a direct target ofBMP9-induced Smad signaling in MSCs. C3H10T1/2cells were infected with Ad-BMP9or Ad-BMP9/AdR-GH, followed by administration of STAT3, STAT5and JAKinhibitors. ALP activity was determined on day5and7after infection andadministration. Our results demonstrated that BMP9-stimulated ALP activity wassignificantly inhibited. Furthermore, these inhibitors were shown to effectively inhibitthe synergistic ALP activity induced by both BMP9and GH. In addition, RT-PCRresults indicated that GH-induced IGF1expression could be significantly inhibited byJAK and STAT5.In summary, we demonstrated that GH synergizes with BMP9in osteogenicdifferentiation by activating the JAK/STAT/IGF1pathway in mesenchymal stem cells.