生长激素在BMP9介导的间充质干细胞向成骨分化中的作用
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摘要
间充质干细胞（Mesenchymal Stem Cells，MSCs）是具有多项分化潜能的成体干细胞，能够分化为成骨细胞、软骨细胞、脂肪细胞和肌肉细胞谱系。干细胞的成骨分化是一个复杂、调控严格的过程，这个过程受一系列内源性和各种环境因子以及信号通路的影响。对间充质干细胞成骨分化机制的研究，有望为临床上治疗骨缺损、骨折及骨不连等病症提供新方案。
在间充质干细胞成骨分化早期，骨形态发生蛋白（Bone MorphogenesisProteins， BMPs）发挥着至关重要的作用。 BMPs属于转化生长因子（Transforming Growth Factor, TGF）β超家族成员，是一组具有类似结构的高度保守的分泌型多功能蛋白，在细胞黏附、增殖、分化，骨骼发育和骨组织形成中发挥重要作用。目前发现人BMP家族至少存在15种成员，其中BMP2，BMP4，BMP6，BMP7和BMP9能有效地在体内和体外诱导间充质干细胞向成骨分化。BMP9是骨源性最强的BMP之一，同时也是目前了解得最少的BMP之一。
我们的前期研究发现，用BMP9腺病毒（Ad-BMP9）感染MSCs后，收集细胞总RNA进行微阵列分析，有大约40个基因的表达发生明显变化。其中，生长激素（Growth Hormone，GH）的表达上调最为显著。这提示生长激素参与了BMP9诱导的间充质干细胞分化过程，并且可能在其中发挥重要作用。生长激素是由垂体分泌的一种蛋白质激素，通过与GH受体结合，或与胰岛素样生长因子Ⅰ（Insulin-like Growth FactorⅠ，IGF-Ⅰ）相互作用，影响骨骼的发育和生长。为了揭示生长激素在BMP9诱导的间充质干细胞成骨分化中扮演的角色，探讨其中的分子机制，本研究首先构建了GH过表达的质粒，并包装相应的腺病毒，其次，在体外、器官培养和体内三个层面上研究GH和BMP9对间充质干细胞成骨分化的影响，最后通过染色质免疫沉淀技术（Chromatin Immunoprecipitation，ChIP）和小分子抑制剂的使用探索其中的调控机制。我们主要的研究内容包括：
①从Thermo公司购买含有人GH基因片段的质粒，酶切后将GH基因片段连接到腺病毒穿梭质粒上。测序鉴定后，将质粒转入BJAdEasy细胞中进行重组，并筛选阳性克隆。PacⅠ酶切将重组子线性化、纯化，通过脂质体转染到HEK293细胞中进行包装。将病毒命名为AdR-GH。
②C3H10T1/2细胞购买自ATCC公司，是常用的MSC细胞株。免疫荧光检测C3H10T1/2间充质干细胞标志物的表达。结果显示，C3H10T1/2表达MSC标志物CD29/Integrinβ1，CD44，CD73， CD90/Thy-1，CD117/c-kit，BMPRⅡ，CD133/Prom1， CD105/Endoglin和CD166/ALCAM。
③利用Ad-BMP9感染C3H10T1/2细胞，分别于感染后1、3、5、7天收集细胞总RNA，RT-PCR检测GH的表达。结果显示这4个时间点GH的表达均显著增强。免疫组织化学染色(Immunohistochemistry，IHC)结果表明，GH在发育中长骨生长板的成骨细胞和软骨细胞中表达。体外实验设置4个组：Ad-GFP/Ad-RFP（对照组），Ad-GFP/AdR-GH，Ad-BMP9/Ad-RFP，Ad-BMP9/AdR-GH，用这4组病毒分别刺激MSCs，并检测成骨细胞分化标记物的表达。这些标志物包括碱性磷酸酶（Alkaline Phosphatase，ALP），骨钙素，骨桥蛋白和基质的矿化作用(茜素红染色)。结果表明，GH本身不能启动MSCs的成骨分化，而当GH和BMP9共同作用时，成骨分化标志物的表达明显强于其他3组，说明GH在体外能促进BMP9介导的MSCs成骨分化。
④在器官培养的实验中，我们截取胚胎期E18.5天的小鼠后肢进行培养，并利用上述4组病毒感染组织。将钙黄绿素作为荧光指示剂加入培养基中追踪骨发育过程，在病毒感染后2、4、8、12天测量记录胫骨横截面荧光面积。结果显示，与对照组相比，单独的BMP9能有效促进骨生成，但BMP9和GH组合的促进效果更为显著。苏木精-伊红（Haematoxylin and eosin，H&E）染色结果显示，Ad-BMP9/AdR-GH组中肥大软骨区域大于对照组。这些结果表明，GH能促进BMP9介导的软骨内成骨。
⑤利用上述4组病毒感染C3H10T1/2细胞36小时后，收集细胞注射到无胸腺裸鼠皮下。注射4周后，处死裸鼠，收集移植部位进行微型计算机断层扫描（Microcomputed Tomography，MicroCT）。通过Amira5.3软件将扫描结果进行三维重建并进行统计分析。仅感染Ad-BMP9/Ad-RFP或Ad-BMP9/AdR-GH病毒的MSCs能生成异位骨，且BMP9和GH共同诱导的MSCs形成的异位骨骨量更多，骨密度更大。H&E染色以及骨钙素、骨桥蛋白的免疫组织化学染色结果显示，BMP9诱导生成的异位骨中形成部分成熟的骨基质和骨小梁，同时可观察到未分化的间充质祖细胞。而由BMP9和GH共同诱导生成的异位骨中存在更多的成熟骨基质和更大，更厚的骨小梁，未分化的间充质祖细胞相对较少。Masson’sTrichrome染色结果显示， GH增强了BMP9诱导的基质矿化作用。
⑥在GH启动子区域寻找假定的Smad结合位点，据此设计两对PCR寡核苷酸序列用于ChIP分析。亚融合的C3H10T1/2细胞被AdGFP或AdBMP9感染30个小时。交联细胞，超声破碎基因组DNA，然后用抗Smad1/5/8抗体进行免疫共沉淀。分别用两对引物与获得的基因组DNA进行PCR反应。结果发现，抗-Smad1/5/8抗体能拖下含有GH启动子区域的基因组片段。表明GH是MSCs中BMP9诱导的Smad信号的一个下游靶点。在BMP9或BMP9/GH诱导培养的MSCs中分别加入STAT3、STAT5、JAK的小分子抑制剂后，于第5、7天检测ALP活性。结果表明，这几种抑制剂均能有效地抑制BMP9介导的ALP活性，同时，BMP9和GH协同诱导的ALP活性也受到明显抑制。此外，RT-PCR结果显示，GH能诱导IGF1的表达，并且GH诱导的IGF1表达能被JAK和STAT5抑制剂抑制。
综合以上研究结果，我们认为生长激素与BMP9存在协同作用，通过激活JAK/STAT/IGF1信号通路促进间充质干细胞的成骨分化。
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.
在间充质干细胞成骨分化早期，骨形态发生蛋白（Bone MorphogenesisProteins， BMPs）发挥着至关重要的作用。 BMPs属于转化生长因子（Transforming Growth Factor, TGF）β超家族成员，是一组具有类似结构的高度保守的分泌型多功能蛋白，在细胞黏附、增殖、分化，骨骼发育和骨组织形成中发挥重要作用。目前发现人BMP家族至少存在15种成员，其中BMP2，BMP4，BMP6，BMP7和BMP9能有效地在体内和体外诱导间充质干细胞向成骨分化。BMP9是骨源性最强的BMP之一，同时也是目前了解得最少的BMP之一。
我们的前期研究发现，用BMP9腺病毒（Ad-BMP9）感染MSCs后，收集细胞总RNA进行微阵列分析，有大约40个基因的表达发生明显变化。其中，生长激素（Growth Hormone，GH）的表达上调最为显著。这提示生长激素参与了BMP9诱导的间充质干细胞分化过程，并且可能在其中发挥重要作用。生长激素是由垂体分泌的一种蛋白质激素，通过与GH受体结合，或与胰岛素样生长因子Ⅰ（Insulin-like Growth FactorⅠ，IGF-Ⅰ）相互作用，影响骨骼的发育和生长。为了揭示生长激素在BMP9诱导的间充质干细胞成骨分化中扮演的角色，探讨其中的分子机制，本研究首先构建了GH过表达的质粒，并包装相应的腺病毒，其次，在体外、器官培养和体内三个层面上研究GH和BMP9对间充质干细胞成骨分化的影响，最后通过染色质免疫沉淀技术（Chromatin Immunoprecipitation，ChIP）和小分子抑制剂的使用探索其中的调控机制。我们主要的研究内容包括：
①从Thermo公司购买含有人GH基因片段的质粒，酶切后将GH基因片段连接到腺病毒穿梭质粒上。测序鉴定后，将质粒转入BJAdEasy细胞中进行重组，并筛选阳性克隆。PacⅠ酶切将重组子线性化、纯化，通过脂质体转染到HEK293细胞中进行包装。将病毒命名为AdR-GH。
②C3H10T1/2细胞购买自ATCC公司，是常用的MSC细胞株。免疫荧光检测C3H10T1/2间充质干细胞标志物的表达。结果显示，C3H10T1/2表达MSC标志物CD29/Integrinβ1，CD44，CD73， CD90/Thy-1，CD117/c-kit，BMPRⅡ，CD133/Prom1， CD105/Endoglin和CD166/ALCAM。
③利用Ad-BMP9感染C3H10T1/2细胞，分别于感染后1、3、5、7天收集细胞总RNA，RT-PCR检测GH的表达。结果显示这4个时间点GH的表达均显著增强。免疫组织化学染色(Immunohistochemistry，IHC)结果表明，GH在发育中长骨生长板的成骨细胞和软骨细胞中表达。体外实验设置4个组：Ad-GFP/Ad-RFP（对照组），Ad-GFP/AdR-GH，Ad-BMP9/Ad-RFP，Ad-BMP9/AdR-GH，用这4组病毒分别刺激MSCs，并检测成骨细胞分化标记物的表达。这些标志物包括碱性磷酸酶（Alkaline Phosphatase，ALP），骨钙素，骨桥蛋白和基质的矿化作用(茜素红染色)。结果表明，GH本身不能启动MSCs的成骨分化，而当GH和BMP9共同作用时，成骨分化标志物的表达明显强于其他3组，说明GH在体外能促进BMP9介导的MSCs成骨分化。
④在器官培养的实验中，我们截取胚胎期E18.5天的小鼠后肢进行培养，并利用上述4组病毒感染组织。将钙黄绿素作为荧光指示剂加入培养基中追踪骨发育过程，在病毒感染后2、4、8、12天测量记录胫骨横截面荧光面积。结果显示，与对照组相比，单独的BMP9能有效促进骨生成，但BMP9和GH组合的促进效果更为显著。苏木精-伊红（Haematoxylin and eosin，H&E）染色结果显示，Ad-BMP9/AdR-GH组中肥大软骨区域大于对照组。这些结果表明，GH能促进BMP9介导的软骨内成骨。
⑤利用上述4组病毒感染C3H10T1/2细胞36小时后，收集细胞注射到无胸腺裸鼠皮下。注射4周后，处死裸鼠，收集移植部位进行微型计算机断层扫描（Microcomputed Tomography，MicroCT）。通过Amira5.3软件将扫描结果进行三维重建并进行统计分析。仅感染Ad-BMP9/Ad-RFP或Ad-BMP9/AdR-GH病毒的MSCs能生成异位骨，且BMP9和GH共同诱导的MSCs形成的异位骨骨量更多，骨密度更大。H&E染色以及骨钙素、骨桥蛋白的免疫组织化学染色结果显示，BMP9诱导生成的异位骨中形成部分成熟的骨基质和骨小梁，同时可观察到未分化的间充质祖细胞。而由BMP9和GH共同诱导生成的异位骨中存在更多的成熟骨基质和更大，更厚的骨小梁，未分化的间充质祖细胞相对较少。Masson’sTrichrome染色结果显示， GH增强了BMP9诱导的基质矿化作用。
⑥在GH启动子区域寻找假定的Smad结合位点，据此设计两对PCR寡核苷酸序列用于ChIP分析。亚融合的C3H10T1/2细胞被AdGFP或AdBMP9感染30个小时。交联细胞，超声破碎基因组DNA，然后用抗Smad1/5/8抗体进行免疫共沉淀。分别用两对引物与获得的基因组DNA进行PCR反应。结果发现，抗-Smad1/5/8抗体能拖下含有GH启动子区域的基因组片段。表明GH是MSCs中BMP9诱导的Smad信号的一个下游靶点。在BMP9或BMP9/GH诱导培养的MSCs中分别加入STAT3、STAT5、JAK的小分子抑制剂后，于第5、7天检测ALP活性。结果表明，这几种抑制剂均能有效地抑制BMP9介导的ALP活性，同时，BMP9和GH协同诱导的ALP活性也受到明显抑制。此外，RT-PCR结果显示，GH能诱导IGF1的表达，并且GH诱导的IGF1表达能被JAK和STAT5抑制剂抑制。
综合以上研究结果，我们认为生长激素与BMP9存在协同作用，通过激活JAK/STAT/IGF1信号通路促进间充质干细胞的成骨分化。
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.
引文
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