BMP9通过MAPKs通路调控间充质干细胞成骨分化及其机制的初步研究
摘要
间充质干细胞(mesenchymal stem cells,MSCs)是属于中胚层的一类多能干细胞,其具有分化为成骨细胞、肌细胞、肝细胞、软骨细胞、脂肪细胞和基质细胞等多种细胞的能力。因此MSCs可以作为种子细胞,修复、重建受伤或发生病变的多种组织器官,目前在骨再生研究中得到广泛应用。但是,由于间充质干细胞具有多向分化潜能,因此寻找促进间充质干细胞定向成骨分化的细胞因子是目前制约骨再生研究发展的瓶颈。
骨形态发生蛋白(bone morphogenetic proteins,BMPs)是研究最早和最有潜力的诱导间充质干细胞成骨分化的细胞因子,其属于转化生长因子β(transform growth factorβ,TGF-β)超家族的成员之一,目前共分离和鉴定了20余种BMPs,其中已报道的具有诱导成骨活性的BMPs主要为BMP2、4、6、7、9等,其中本课题组研究发现BMP9诱导成骨的作用远强于传统应用的BMP2和BMP7,因此其有希望作为一种更强的促进细胞成骨分化的细胞因子候选者而应用于临床。BMP9(也称GDF-2,growth differentiation factor 2)是BMPs中的一种,其具有多种生物学功能,但是对于BMP9在骨形成及骨再生中的作用及其机制了解很少。
以往的研究表明,BMPs主要是通过经典的BMPs-Receptor-Smad信号转导通路传递信号,并发挥诱导成骨等生物学功能。但是最近越来越多的研究发现,BMPs也可通过非Smad的转导通路(如MAPKs、PI3K等通路)进行信号转导,这些信号转导通路不直接依赖转录因子Smad的激活,被称为BMPs的非经典(non-canonical)通路,其中BMPs-MAPKs通路是非经典通路中重要的一环。MAPKs(mitogen-activated protein kinases)即丝裂原活化蛋白激酶信号转导通路是真核细胞将胞外信号转导至胞内引起细胞应答的一类重要信号系统。在各种刺激因素(如生长因子、炎症因子等)的作用下,MAPKs信号转导通路通过一系列激酶的磷酸化级联反应,影响基因的转录和调控,参与调节细胞的增殖、分化、转化以及凋亡等一系列基本生命活动。研究表明,BMP2、BMP4和BMP7等均可以通过MAPKs信号转导通路传递信号,而且BMPs-MAPKs信号转导通路对于BMPs发挥诱导成骨作用也极为重要。因此,我们推测BMP9作为一个诱导间充质干细胞成骨分化的强效因子,其除了通过BMPs/Smad经典的信号转导通路诱导干细胞/前体细胞的成骨分化,可能还通过其它的非经典信号转导通路调节干细胞/前体细胞的成骨分化。
本研究的目的是:(1)BMP9是否能够激活MAPKs信号转导通路;(2)MAPKs信号通路在BMP9诱导的间充质干细胞成骨分化中的作用;(3)BMP9通过MAPKs信号通路调控间充质干细胞成骨分化的可能作用机制。
在本研究的第一部分,首先通过检测碱性磷酸酶(ALP)活性的方法验证BMP9对间充质干细胞成骨分化的影响,结果表明:BMP9能够诱导间充质干细胞向成骨方向分化。这为后续研究BMP9诱导间充质干细胞的成骨分化的相关信号转导通路及其机制奠定了基础。接下来,采用Western Blot的方法检测BMP9刺激后MAPKs信号转导通路中的关键蛋白激酶p38MAPK、ERK1/2和JNK磷酸化水平的改变,结果显示:BMP9的刺激均能够增加p38MAPK、ERK1/2和JNK磷酸化的水平,表明BMP9可以激活MAPKs信号转导通路。但是,BMP9对MAPKs信号转导通路的激活是否对其诱导的间充质干细胞的成骨分化具有影响,是接下来需要阐明的问题。因此,使用p38MAPK的特异性抑制剂SB203580和ERK1/2的特异性抑制剂PD98059分别抑制p38MAPK和ERK1/2的活性后,通过检测成骨早期标志物碱性磷酸酶(ALP)活性研究其对BMP9诱导成骨分化的影响,结果显示:(1)抑制p38MAPK的活性可降低BMP9诱导的C3H10T1/2、C2C12和MC3T3-E1细胞的早期成骨分化,且SB203580的这种抑制作用呈剂量依赖性;(2)抑制ERK1/2的活性能够增强BMP9诱导的C3H10T1/2、C2C12和MC3T3-E1细胞的早期成骨作用。随后,通过茜素红S染色的方法检测成骨晚期标志钙盐沉积的改变,结果显示:抑制p38MAPK的活性可降低BMP9诱导的C3H10T1/2细胞的晚期成骨作用,而抑制ERK1/2的活性则能够增强BMP9诱导的C3H10T1/2细胞的晚期成骨作用。最后,通过免疫细胞化学的方法检测成骨晚期标志物骨桥蛋白(OPN)和骨钙素(OCN)表达的变化,同样结果显示,抑制p38MAPK的活性可降低BMP9诱导的C3H10T1/2细胞的成骨标志物OPN和OCN的细胞内表达,而抑制ERK1/2的活性能够增强BMP9诱导的C3H10T1/2细胞的成骨标志物OPN和OCN的细胞内表达。综上所述,抑制p38MAPK的活性可降低BMP9诱导的间充质干细胞的早期及晚期成骨分化,而抑制ERK1/2的活性则能够促进BMP9诱导的间充质干细胞的早期及晚期成骨分化。
在本研究的第二部分,采用RNA干扰的方法抑制p38MAPK、ERK1和ERK2 mRNA的表达,检测其对BMP9诱导的间充质干细胞在体外及体内成骨分化的影响。首先,使用Real time PCR的方法分别检测将p38MAPK、ERK1和ERK2的基因沉默后其mRNA表达的改变,结果显示,使用的干扰片段可以有效地降低目的基因mRNA的表达。随后,分别将p38MAPK、ERK1和ERK2的基因沉默后,通过检测碱性磷酸酶(ALP)活性研究其对BMP9诱导的间充质干细胞成骨分化的影响,结果显示:(1)降低p38MAPK的表达同样可抑制BMP9诱导的C3H10T1/2细胞的ALP活性;(2)减少ERK1和ERK2的表达能够增强BMP9诱导的C3H10T1/2细胞的ALP活性。但采用基因沉默的方法降低目的基因mRNA的表达对BMP9诱导的间充质干细胞成骨分化的影响小于使用蛋白激酶抑制剂后其对BMP9诱导的间充质干细胞成骨分化的影响。接下来,将携带有sip38MAPK、siERK1和siERK2的腺病毒和表达BMP9的腺病毒共同感染C3H10T1/2细胞后进行裸鼠皮下接种,检测将p38MAPK、ERK1和ERK2的基因沉默后其对BMP9诱导的间充质干细胞体内成骨的影响,结果表明:(1)细胞皮下接种5周后,各组均形成了质地坚硬的包块,表明各组均形成了骨质组织;(2)降低p38MAPK的表达对形成的骨组织的体积影响不大;而减少ERK1或ERK2的表达可增加形成的骨组织的体积。(3)HE染色结果显示,减少p38MAPK表达能够抑制形成的骨小梁数量、降低骨小梁形成的质量,并且可见软骨细胞存在;而降低ERK1或ERK2的表达可明显增加形成的骨小梁的数量,且骨小梁结构更加完整。(4)Alcian blue染色结果显示,减少p38MAPK的表达能够促进被染成蓝色的软骨基质的形成;而降低ERK1或ERK2的表达可明显减少被染成蓝色的软骨基质的形成。(5)Masson’s Trichrome三色染色结果显示,降低p38MAPK的表达能够使染成红色的骨基质减少,而染成蓝色的软骨基质增多;而减少ERK1或ERK2的表达可使染成红色的骨基质增多,而染成蓝色的软骨基质减少。总之,减少p38MAPK的表达后可降低BMP9诱导的间充质干细胞体内成骨分化的成熟程度,而降低ERK1或ERK2的表达后能够增加BMP9诱导的间充质干细胞体内成骨分化的成熟程度,这与之前的体外实验结果相一致。
通过第一部分和第二部分的结果初步了解到:(1)抑制p38MAPK信号通路后能够降低BMP9诱导的间充质干细胞体外及体内的成骨分化;(2)抑制ERK1/2信号通路后能够促进BMP9诱导的间充质干细胞体外及体内的成骨分化。因此,在第三部分将从以下两方面探讨其可能的分子机制:(1)抑制MAPKs信号通路对BMP9促进间充质干细胞成骨分化进程中的细胞分化、增殖和细胞周期的影响及其相互关系;(2)在BMP9诱导间充质干细胞成骨分化过程中MAPKs信号通路对BMPs经典信号通路的影响。首先,通过使用p38MAPK和ERK1/2的特异性抑制剂在抑制p38MAPK和ERK1/2活性的情况下,在2-9天连续监测BMP9诱导的C3H10t1/2细胞ALP活性的变化,结果显示,抑制ERK1/2的活性后ALP的活性在第4天开始增加,并在第5-6天出现了一个急剧的升高,但是并没有如之前假设的抑制ERK1/2的活性后将BMP9诱导的间充质干细胞的ALP活性最高峰出现的时间点提前,而表现为ALP的活性随着时间的增加而存在一个持续性的增高,这表明抑制ERK1/2的活性后使BMP9诱导的间充质干细胞的分化持续增强。接下来,通过MTT实验检测在BMP9诱导的间充质干细胞分化进程中各组细胞增殖的改变,结果显示,抑制ERK1/2的活性后细胞在第4天后的增殖明显增高,提示抑制ERK1/2的活性后其ALP活性的增强有可能是由于其促使细胞增殖的增加而导致的。随后,用流式细胞检测技术检测在BMP9诱导的间充质干细胞分化进程中各组细胞的细胞周期的变化情况,结果显示:各组细胞间的不同细胞周期时相的变化在第1-3天间以及7-10天间变化没有明显区别,而在4-6天期间各组细胞间的不同时相出现了不同程度的差异,并且这种差异主要表现在抑制ERK1/2的活性后促使其S期细胞比例明显增多。这提示我们,抑制ERK1/2的活性后其对BMP9诱导的间充质干细胞成骨分化的促进作用可能与其能增加分化进程中S期细胞的比例并促进细胞增殖相关。另外,在第三部分通过Western Blot的方法检测了使用p38MAPK和ERK1/2的特异性抑制剂抑制p38MAPK和ERK1/2活性的情况下,其对Smad1(ser463/465)/Smad5(ser463/465)/Smad8(ser426/428)C末端的丝氨酸位点的磷酸化影响进行了检测,结果显示,在BMP9刺激30min后Smad1/5/8 C末端丝氨酸的磷酸化水平明显增加,并且这一作用可持续至120min;但是抑制p38MAPK的活性后,其Smad1/5/8 C末端丝氨酸的磷酸化水平依然在30min的时候增加,但其在120min时即出现了明显的降低;而抑制ERK1/2的活性后,Smad1/5/8 C末端的磷酸化水平的变化与BMP9处理组相似。接下来,采用激光共聚焦的方法检测Smad1/5/8在细胞内定位的改变,结果显示,BMP9能明显增加Smad1/5/8的核内转移;但是抑制p38MAPK的活性后Smad1/5/8的核内转移相对BMP9组有所减弱;而抑制ERK1/2的活性后Smad1/5/8的核内转移增加,但与BMP9组区别不大。最后,采用Real time PCR的方法检测BMPs/Smad经典信号通路的下游靶基因Smad6和Smad7 mRNA的表达情况,结果表明,抑制p38MAPK的活性后,能够使BMP9激活的Smad6 mRNA的表达降低42.34%,但仅使Smad7的mRNA表达降低14.46%;但抑制ERK1/2的活性后,其对Smad6和Smad7的mRNA表达影响均不明显。这提示,抑制p38MAPK的活性后其对BMP9诱导的间充质干细胞成骨分化的抑制作用可能与其对BMPs/Smad信号通路的下调相关。
通过第三部分结果,初步得知:(1)抑制ERK1/2的活性后其对BMP9诱导的间充质干细胞成骨分化的促进作用可能与其能增加分化进程中S期细胞的比例并促进细胞增殖相关,其具体的分子机制有待进一步研究。(2)抑制p38MAPK的活性能够减少Smad1/5/8 C末端丝氨酸的磷酸化水平维持的时间、减弱Smad1/5/8的核内转移并下调BMPs/Smad经典信号通路靶基因的表达,从而对BMP9诱导的间充质干细胞的成骨分化产生影响。
综上所述,BMP9能够激活MAPKs信号通路;抑制p38MAPK信号通路后能够降低BMP9诱导的间充质干细胞在体外及体内的成骨分化,而抑制ERK1/2信号通路后可促进BMP9诱导的间充质干细胞在体外及体内的成骨分化;抑制ERK1/2的活性后其对BMP9诱导的间充质干细胞成骨分化的促进作用可能与其对细胞增殖及细胞周期的影响相关;抑制p38MAPK的活性能够减少Smad1/5/8 C末端丝氨酸的磷酸化水平进而减弱BMPs/Smad经典信号通路的功能发挥,最终抑制BMP9诱导的间充质干细胞的成骨分化。
Mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, myogenic, hepatocyte, chondrogenic, and adipogenic lineages. It can be used as seed cells to repair and rebuild injured or diseased organs and tissues in regenerative medicine that recently find practical usage in bone regeneration therapy. Mesenchymal stem cells possess multi-directional differentiation capacity, it is vital to explore some new cytokine that can promote commitment of MSC to produce preosteoblasts followed by differentiation preosteoblasts to produce osteoblasts.
BMPs (bone morphogenetic proteins, BMPs) play important roles in regulating osteoblast differentiation and subsequent bone formation. BMPs belong to the transforming growth factorβ(transform Growth Factorβ, TGF-β) superfamily and consist of at least 20 members. BMP-2,-4,-6,-7 and -9 were shown to effectively induce osteogenic differentiation of MSCs in vitro and in vivo, in which BMP9 has far stronger function to induce osteogenic differentiation than that of BMP2 and BMP7. So it is possible to be applied in clinical treatment as a stronger and effective cytokine to induce osteogenic differentiation. BMP9 (also known as GDF-2, growth differentiation factor 2) have many biological functions belong to BMPs. However, the biological effects of BMP9 on osteoblast commitment and terminal differentiation of MSCs has not been comprehensively analyzed.
BMPs transmit signal and play the biological functions through the classical BMPs-Receptor-Smad signaling pathway. However, more and more studies found that BMPs can also transmit signal by other pathways such as MAPKs, PI3K than Smad signaling pathway, which means that transmit signal through activating Smad-independent signal pathway, called as BMPs non-classical pathway. MAPKs (mitogen-activated protein kinases) are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens, osmotic stress, heat-shock and proinflammatory cytokines) and regulate various cellular activities, such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis. BMP2, BMP4 and BMP7 can activate MAPKs signal pathway and induce osteogenic differentiation. Therefore, we provide that BMP9 as a cytokine inducing mesenchymal stem cells into osteoblasts can activate MAPKs signal pathway and induce osteogenic differentiation of MSCs.
The aim of this study is: (1) to identify whether BMP9 can activate MAPKs signal transduction pathway; (2) to investigate the effect of inhibition of MAPKs signaling pathway on BMP9 induced osteogenic differentiation of mesenchymal stem cell; (3) to investigate the mechanisms of mesenchymal stem cell induced into osteogenic differentiation by BMP9 -MAPKs signal pathway.
In the first part of this study, the expressions of ALP in BMP9 induced mesenchymal stem cell into osteogenic differentiation were detected by alkaline phosphatase (ALP) activity. The results showed that BMP9 can induce MSCs into osteogenic differentiation. Then, the phosphorylation of p38MAPK, ERK1/2 and JNK were detected by Western blot assay in cells treated by BMP9. The results showed that BMP9 can induce the phosphorylation of p38MAPK, ERK1/2 and JNK. Given the above-results, we propose to clarify that the effect of inhibition of MAPKs signaling pathway on osteogenic differentiation of mesenchymal stem cell. Therefore, the specific p38MAPK inhibitors SB203580 and the specific ERK1/2 inhibitor PD98059 were used to inhibit the expression of p38MAPK and the activity of ERK1/2. ALP activity of the early osteoblast marker was detected on osteogenic differentiation of MSCs induced by BMP9 combined with treatment of p38MAPK inhibitor and ERK1/2 inhibitor The results demonstrated that:: (1) Dose-dependent manner of the inhibitor of p38MAPK inhibited BMP9-induced osteogenic differentiation of MSCs, indicating that the differentiation of MSCs into osteogenesis can be promoted by BMP9-p38MAPK pathway. (2) Dose-dependent manner of the ERK1/2 inhibitor promoted BMP9-induced osteogenic differentiation of MSCs, indicating that the differentiation of MSCs into osteogenesis can be inhibited by BMP9-ERK1/2 pathway. These data suggest that activation of MAPKs by BMP9 affect BMP9-induced early osteogenic differentiation of MSCs. Then, calcium deposition of the later osteoblast marker was detected on BMP9-induced osteogenic differentiation of MSCs with the p38MAPK inhibitor and the ERK1/2 inhibitor by Alizarin Red S staining. The results showed that inhibition of the expression of p38MAPK reduced calcium deposition of BMP9-induced osteogenic differentiation of MSCs in the later of bone formation, while inhibition of the activity of ERK1/2 enhanced BMP9-induced calcium deposition of BMP9 induced osteogenic differentiation of MSCs in the later phase of bone formation. Finally, the expression of osteopontin (OPN) and osteocalcin (OCN) were detected on BMP9-induced osteogenic differentiation of MSCs with the p38MAPK inhibitor and the ERK1/2 inhibitor by immunocytochemistry. The results showed that the expression of osteopontin (OPN) and osteocalcin (OCN) were decreased by treatment of the p38MAPK inhibitor, but they were increased by treatment of the ERK1/2 inhibitor. In summary, the activation of p38MAPK signaling pathway by BMP9 leads to promote the osteogenic differentiation of mesenchymal stem cell in vitro, while the activation of ERK1/2 signaling pathway by BMP9 can reduce the osteogenic differentiation of mesenchymal stem cells in vitro.
In the second part of this study, RNA interference was applied to knockdown the p38MAPK, ERK1 and ERK2 expression, and the effect of inhibition of MAPKs signaling pathway on BMP9-induced osteogenic differentiation of mesenchymal stem cell were investigated in vitro and in vivo. First, the effect of knockdown of p38MAPK, ERK1 and ERK2 with RNAi were detected with by Real time PCR. The results showed that the expressions of target genes were effectively and successfully reduced with RNAi technology. Subsequently, alkaline phosphatase (ALP) activity was detected on BMP9-induced osteogenic differentiation of mesenchymal stem cell with knockdown of MAPK-p38, ERK1 and ERK2, respectively. The results showed that inhibition of the expression of p38MAPK reduced calcium ALP activity in BMP9-induced osteogenic differentiation of MSCs, while inhibition of the expression of ERK1/2 enhanced BMP9-induced ALP activity in BMP9-induced osteogenic differentiation of MSCs. Likewise, knockdown p38MAPK、ERK1 and ERK2 expression with RNAi partially affect on ALP activity in BMP9-induced osteogenic differentiation of MSCs. Next, C3H10T1/2 co-infected by AdR-sip38MAPK/Ad-BMP9, AdR-siERK1/Ad-BMP9 and AdR-siERK2/Ad-BMP9 were subcutaneously injected to BALB/c nude mice respectively to investigate the effect of knockdown of p38MAPK、ERK1 and ERK2 on BMP9 induced osteogenic differentiation of MSCs in vivo. The results showed: (1) for 5 weeks, cells that inoculated subcutaneously formed a hard mass, indicating bone tissue formation; (2) The size of the bone tissues have little change with knockdown of p38MAPK; while the size of the bone tissues have obviously increased with ERK1/ERK2 RNAi treatment to knockdown ERK1/ERK2 expression; (3) HE staining showed that knockdown of MAPK-p38 reduced the quantity and quality of the bone trabecular, and cartilage cells were found, while knockdown of ERK1/ERK2 significantly increased the amount of bone trabecular and the formation of bone trabecular was more complete. (4) Alcian blue staining showed that knockdown of p38MAPK increased the cartilage matrix, whereas knockdown of ERK1/ERK2 significantly reduced the cartilage matrix. (5) Masson's Trichrome three-color staining showed that knockdown of p38MAPK leaded to decrease of bone matrix with stained red and increase of the cartilage matrix with stained blue. Oppositely, knockdown of ERK1/ERK2 increased bone matrix with stained red while reduced cartilage matrix with stained blue. In short, knockdown of p38MAPK expression reduced BMP9-induced mesenchymal stem cell into osteogenic differentiation, while knockdown of ERK1/ERK2 expression promoted BMP9-induced mesenchymal stem cell into osteogenic differentiation. Those results were consistent with those observations in vitro.
Based on the results of the first part and second part, those data showed that: (1) The depression of p38MAPK signaling pathway leads to decrease the osteogenic differentiation of mesenchymal stem cell by BMP9; (2) ERK1/2 signaling pathway depletion leads to promote mesenchymal stem cells into osteogenic differentiation by BMP9. But the mechanisms of induced-mesenchymal stem cell into osteogenic differentiation by BMP9-MAPKs signaling pathway are under exploration. Therefore, the following studies would investigate those mechanisms from these two perspectives: (1) the relationship of differentiation and proliferation in BMP9-induced osteogenic differentiation of MSCs with the p38MAPK inhibitor and the ERK1/2 inhibior. (2) The influence of activated MAPKs pathway on BMPs/Smad pathway will be studied in BMP9-induced mesenchymal stem cells into osteogenic differentiation. Firstly, after treating with the p38MAPK inhibitor and the ERK1/2 inhibitor, the expressions of ALP in BMP9 induced mesenchymal stem cell into osteogenic differentiation were detected by alkaline phosphatase (ALP) activity from 2 to 9 days, which is aimed to verify whether the peak of ALP activity occurred earlier with the ERK1/2 inhibitor. The results showed that ALP activity on BMP9-induced MSCs into osteogenic differentiation with the ERK1/2 inhibitor was starting to rise at 4 days and dramatically increase from 5 to 6 days, and continuously increase until 9 days. The results indicated that the differentiaton of MSCs induced on BMP9 continuously promoted by the ERK1/2 inhibitor. Then, the proliferation of MSCs treated with BMP9 and the MAPKs inhibitors were detected by MTT assay. The results showed that the proliferation of BMP9-induced MSCs into osteogenic differentiation with the ERK1/2 inhibitor significantly increased at 4 days, suggesting that the increasing of ALP activity with the ERK1/2 inhibitor may be due to promotion of cell proliferation. Subsequently, the cell cycle of MSCs with BMP9 and the MAPKs inhibitor were determined by FCM. The results showed that there was no significant difference of cell cycle phase between treatment by BMP9 or MAPKs inhibitor in the first 1-3 days or in 7-10 days, but the distribution of cell cycle in every treatment group was changed among the groups which mainly happened in the S phase cells increased on BMP9-induced MSCs into osteogenic differentiation with the ERK1/2 inhibitor during the period of 4-6 days. . Therefore, we speculate that the promotion of MSCs into osteogenic differentiation with the ERK1/2 inhibitor may relate to the change of cell proliferation and cell cycle. In addition, in the third part of the study, the phosphorylation of Smad1 (ser463/465)/Smad5 (ser463/465)/Smad8 (ser426/428) C terminal were tested by Western Blot in BMP9-induced MSCs into osteogenic differentiation with the p38MAPK inhibitor and the ERK1/2 inhibitor. The results showed that the phosphorylation of Smad1/5/8 significantly increased after 30 minutes treatment with BMP9 and the ERK1/2 inhibitor, and it sustained until 2 hours, which was similar with treated by BMP9 alone. But the phosphorylation of Smad1/5/8 obviously decreased at 120min treatment with BMP9 and the p38MAPK inhibitor. Following, the localization of Smad1/5/8 was detected using Laser Scanning Confocal Microscope on BMP9-induced MSCs into osteogenic differentiation with the p38MAPK inhibitor and the ERK1/2 inhibitor. The results showed that BMP9 significantly promoted the translocation of Smad1/5/8 into the nuclear with the ERK1/2 inhibitor, which was similar to with BMP9 group, but the location of Smad1/5/8 in the nuclear showed weaker staining compared with that of BMP9 treatment alone with the p38MAPK inhibitor. Finally, the expression of Smad6 and Smad7 mRNA, which are the downstream target genes of BMPs/Smad signaling pathway, were detected by Real time PCR in BMP9-induced MSCs into osteogenic differentiation with the p38MAPK inhibitor and the ERK1/2 inhibitor. The results showed that the mRNA expression of Smad6 and Smad7 decreased respectively 42.34% and 14.46% on BMP9- induced MSCs into osteogenic differentiation with the p38MAPK inhibitor, but the mRNA expression of Smad6 and Smad7 was not significantly changed on BMP9-induced MSCs into osteogenic differentiation with the ERK1/2 inhibitor. The results suggested that the inhibition of MSCs into osteogenic differentiation with the p38MAPK inhibitor was related to down-regulation of BMPs/Smad signal pathway.
The results of the third part of indicated that: (1) the depression of p38MAPK can decrease phosphorylation of Smad 1/5/8 C-terminus and then exert influence on the biological function of classical BMPs/Smad signaling pathway, and finally affect the osteogenic differentiation of BMP9-treated MSCs. (2) the depression of ERK1/2 promotes BMP9-induced MSCs differentiation into osteogenesis may be related to the alteration of cell proliferation and cell cycle distribution caused by depletion of ERK1/2, which needs further study to clarify the molecular mechanisms.
Taken together, BMP9 can activate the MAPKs signaling pathway; the depletion of p38MAPK expression by specific siRNA inhibited the osteogenic differentiation of MSCs by BMP9 induction in vitro and in vivo; the inhibition of ERK1/2 expression promoted MSCs into osteogenic differentiation by BMP9 induction in vitro and in vivo; That the depression of ERK1/2 expression and activity enhances MSCs osteogenic differentiation induced by BMP9 may be related to the alteration of cell proliferation and cell cycle caused by ERK1/2 changes.
While the depression of p38MAPK can affect biological function of classical signaling pathway of BMPs/Smad and then influence BMP9-induced MSCs into osteogenic differentiation.
骨形态发生蛋白(bone morphogenetic proteins,BMPs)是研究最早和最有潜力的诱导间充质干细胞成骨分化的细胞因子,其属于转化生长因子β(transform growth factorβ,TGF-β)超家族的成员之一,目前共分离和鉴定了20余种BMPs,其中已报道的具有诱导成骨活性的BMPs主要为BMP2、4、6、7、9等,其中本课题组研究发现BMP9诱导成骨的作用远强于传统应用的BMP2和BMP7,因此其有希望作为一种更强的促进细胞成骨分化的细胞因子候选者而应用于临床。BMP9(也称GDF-2,growth differentiation factor 2)是BMPs中的一种,其具有多种生物学功能,但是对于BMP9在骨形成及骨再生中的作用及其机制了解很少。
以往的研究表明,BMPs主要是通过经典的BMPs-Receptor-Smad信号转导通路传递信号,并发挥诱导成骨等生物学功能。但是最近越来越多的研究发现,BMPs也可通过非Smad的转导通路(如MAPKs、PI3K等通路)进行信号转导,这些信号转导通路不直接依赖转录因子Smad的激活,被称为BMPs的非经典(non-canonical)通路,其中BMPs-MAPKs通路是非经典通路中重要的一环。MAPKs(mitogen-activated protein kinases)即丝裂原活化蛋白激酶信号转导通路是真核细胞将胞外信号转导至胞内引起细胞应答的一类重要信号系统。在各种刺激因素(如生长因子、炎症因子等)的作用下,MAPKs信号转导通路通过一系列激酶的磷酸化级联反应,影响基因的转录和调控,参与调节细胞的增殖、分化、转化以及凋亡等一系列基本生命活动。研究表明,BMP2、BMP4和BMP7等均可以通过MAPKs信号转导通路传递信号,而且BMPs-MAPKs信号转导通路对于BMPs发挥诱导成骨作用也极为重要。因此,我们推测BMP9作为一个诱导间充质干细胞成骨分化的强效因子,其除了通过BMPs/Smad经典的信号转导通路诱导干细胞/前体细胞的成骨分化,可能还通过其它的非经典信号转导通路调节干细胞/前体细胞的成骨分化。
本研究的目的是:(1)BMP9是否能够激活MAPKs信号转导通路;(2)MAPKs信号通路在BMP9诱导的间充质干细胞成骨分化中的作用;(3)BMP9通过MAPKs信号通路调控间充质干细胞成骨分化的可能作用机制。
在本研究的第一部分,首先通过检测碱性磷酸酶(ALP)活性的方法验证BMP9对间充质干细胞成骨分化的影响,结果表明:BMP9能够诱导间充质干细胞向成骨方向分化。这为后续研究BMP9诱导间充质干细胞的成骨分化的相关信号转导通路及其机制奠定了基础。接下来,采用Western Blot的方法检测BMP9刺激后MAPKs信号转导通路中的关键蛋白激酶p38MAPK、ERK1/2和JNK磷酸化水平的改变,结果显示:BMP9的刺激均能够增加p38MAPK、ERK1/2和JNK磷酸化的水平,表明BMP9可以激活MAPKs信号转导通路。但是,BMP9对MAPKs信号转导通路的激活是否对其诱导的间充质干细胞的成骨分化具有影响,是接下来需要阐明的问题。因此,使用p38MAPK的特异性抑制剂SB203580和ERK1/2的特异性抑制剂PD98059分别抑制p38MAPK和ERK1/2的活性后,通过检测成骨早期标志物碱性磷酸酶(ALP)活性研究其对BMP9诱导成骨分化的影响,结果显示:(1)抑制p38MAPK的活性可降低BMP9诱导的C3H10T1/2、C2C12和MC3T3-E1细胞的早期成骨分化,且SB203580的这种抑制作用呈剂量依赖性;(2)抑制ERK1/2的活性能够增强BMP9诱导的C3H10T1/2、C2C12和MC3T3-E1细胞的早期成骨作用。随后,通过茜素红S染色的方法检测成骨晚期标志钙盐沉积的改变,结果显示:抑制p38MAPK的活性可降低BMP9诱导的C3H10T1/2细胞的晚期成骨作用,而抑制ERK1/2的活性则能够增强BMP9诱导的C3H10T1/2细胞的晚期成骨作用。最后,通过免疫细胞化学的方法检测成骨晚期标志物骨桥蛋白(OPN)和骨钙素(OCN)表达的变化,同样结果显示,抑制p38MAPK的活性可降低BMP9诱导的C3H10T1/2细胞的成骨标志物OPN和OCN的细胞内表达,而抑制ERK1/2的活性能够增强BMP9诱导的C3H10T1/2细胞的成骨标志物OPN和OCN的细胞内表达。综上所述,抑制p38MAPK的活性可降低BMP9诱导的间充质干细胞的早期及晚期成骨分化,而抑制ERK1/2的活性则能够促进BMP9诱导的间充质干细胞的早期及晚期成骨分化。
在本研究的第二部分,采用RNA干扰的方法抑制p38MAPK、ERK1和ERK2 mRNA的表达,检测其对BMP9诱导的间充质干细胞在体外及体内成骨分化的影响。首先,使用Real time PCR的方法分别检测将p38MAPK、ERK1和ERK2的基因沉默后其mRNA表达的改变,结果显示,使用的干扰片段可以有效地降低目的基因mRNA的表达。随后,分别将p38MAPK、ERK1和ERK2的基因沉默后,通过检测碱性磷酸酶(ALP)活性研究其对BMP9诱导的间充质干细胞成骨分化的影响,结果显示:(1)降低p38MAPK的表达同样可抑制BMP9诱导的C3H10T1/2细胞的ALP活性;(2)减少ERK1和ERK2的表达能够增强BMP9诱导的C3H10T1/2细胞的ALP活性。但采用基因沉默的方法降低目的基因mRNA的表达对BMP9诱导的间充质干细胞成骨分化的影响小于使用蛋白激酶抑制剂后其对BMP9诱导的间充质干细胞成骨分化的影响。接下来,将携带有sip38MAPK、siERK1和siERK2的腺病毒和表达BMP9的腺病毒共同感染C3H10T1/2细胞后进行裸鼠皮下接种,检测将p38MAPK、ERK1和ERK2的基因沉默后其对BMP9诱导的间充质干细胞体内成骨的影响,结果表明:(1)细胞皮下接种5周后,各组均形成了质地坚硬的包块,表明各组均形成了骨质组织;(2)降低p38MAPK的表达对形成的骨组织的体积影响不大;而减少ERK1或ERK2的表达可增加形成的骨组织的体积。(3)HE染色结果显示,减少p38MAPK表达能够抑制形成的骨小梁数量、降低骨小梁形成的质量,并且可见软骨细胞存在;而降低ERK1或ERK2的表达可明显增加形成的骨小梁的数量,且骨小梁结构更加完整。(4)Alcian blue染色结果显示,减少p38MAPK的表达能够促进被染成蓝色的软骨基质的形成;而降低ERK1或ERK2的表达可明显减少被染成蓝色的软骨基质的形成。(5)Masson’s Trichrome三色染色结果显示,降低p38MAPK的表达能够使染成红色的骨基质减少,而染成蓝色的软骨基质增多;而减少ERK1或ERK2的表达可使染成红色的骨基质增多,而染成蓝色的软骨基质减少。总之,减少p38MAPK的表达后可降低BMP9诱导的间充质干细胞体内成骨分化的成熟程度,而降低ERK1或ERK2的表达后能够增加BMP9诱导的间充质干细胞体内成骨分化的成熟程度,这与之前的体外实验结果相一致。
通过第一部分和第二部分的结果初步了解到:(1)抑制p38MAPK信号通路后能够降低BMP9诱导的间充质干细胞体外及体内的成骨分化;(2)抑制ERK1/2信号通路后能够促进BMP9诱导的间充质干细胞体外及体内的成骨分化。因此,在第三部分将从以下两方面探讨其可能的分子机制:(1)抑制MAPKs信号通路对BMP9促进间充质干细胞成骨分化进程中的细胞分化、增殖和细胞周期的影响及其相互关系;(2)在BMP9诱导间充质干细胞成骨分化过程中MAPKs信号通路对BMPs经典信号通路的影响。首先,通过使用p38MAPK和ERK1/2的特异性抑制剂在抑制p38MAPK和ERK1/2活性的情况下,在2-9天连续监测BMP9诱导的C3H10t1/2细胞ALP活性的变化,结果显示,抑制ERK1/2的活性后ALP的活性在第4天开始增加,并在第5-6天出现了一个急剧的升高,但是并没有如之前假设的抑制ERK1/2的活性后将BMP9诱导的间充质干细胞的ALP活性最高峰出现的时间点提前,而表现为ALP的活性随着时间的增加而存在一个持续性的增高,这表明抑制ERK1/2的活性后使BMP9诱导的间充质干细胞的分化持续增强。接下来,通过MTT实验检测在BMP9诱导的间充质干细胞分化进程中各组细胞增殖的改变,结果显示,抑制ERK1/2的活性后细胞在第4天后的增殖明显增高,提示抑制ERK1/2的活性后其ALP活性的增强有可能是由于其促使细胞增殖的增加而导致的。随后,用流式细胞检测技术检测在BMP9诱导的间充质干细胞分化进程中各组细胞的细胞周期的变化情况,结果显示:各组细胞间的不同细胞周期时相的变化在第1-3天间以及7-10天间变化没有明显区别,而在4-6天期间各组细胞间的不同时相出现了不同程度的差异,并且这种差异主要表现在抑制ERK1/2的活性后促使其S期细胞比例明显增多。这提示我们,抑制ERK1/2的活性后其对BMP9诱导的间充质干细胞成骨分化的促进作用可能与其能增加分化进程中S期细胞的比例并促进细胞增殖相关。另外,在第三部分通过Western Blot的方法检测了使用p38MAPK和ERK1/2的特异性抑制剂抑制p38MAPK和ERK1/2活性的情况下,其对Smad1(ser463/465)/Smad5(ser463/465)/Smad8(ser426/428)C末端的丝氨酸位点的磷酸化影响进行了检测,结果显示,在BMP9刺激30min后Smad1/5/8 C末端丝氨酸的磷酸化水平明显增加,并且这一作用可持续至120min;但是抑制p38MAPK的活性后,其Smad1/5/8 C末端丝氨酸的磷酸化水平依然在30min的时候增加,但其在120min时即出现了明显的降低;而抑制ERK1/2的活性后,Smad1/5/8 C末端的磷酸化水平的变化与BMP9处理组相似。接下来,采用激光共聚焦的方法检测Smad1/5/8在细胞内定位的改变,结果显示,BMP9能明显增加Smad1/5/8的核内转移;但是抑制p38MAPK的活性后Smad1/5/8的核内转移相对BMP9组有所减弱;而抑制ERK1/2的活性后Smad1/5/8的核内转移增加,但与BMP9组区别不大。最后,采用Real time PCR的方法检测BMPs/Smad经典信号通路的下游靶基因Smad6和Smad7 mRNA的表达情况,结果表明,抑制p38MAPK的活性后,能够使BMP9激活的Smad6 mRNA的表达降低42.34%,但仅使Smad7的mRNA表达降低14.46%;但抑制ERK1/2的活性后,其对Smad6和Smad7的mRNA表达影响均不明显。这提示,抑制p38MAPK的活性后其对BMP9诱导的间充质干细胞成骨分化的抑制作用可能与其对BMPs/Smad信号通路的下调相关。
通过第三部分结果,初步得知:(1)抑制ERK1/2的活性后其对BMP9诱导的间充质干细胞成骨分化的促进作用可能与其能增加分化进程中S期细胞的比例并促进细胞增殖相关,其具体的分子机制有待进一步研究。(2)抑制p38MAPK的活性能够减少Smad1/5/8 C末端丝氨酸的磷酸化水平维持的时间、减弱Smad1/5/8的核内转移并下调BMPs/Smad经典信号通路靶基因的表达,从而对BMP9诱导的间充质干细胞的成骨分化产生影响。
综上所述,BMP9能够激活MAPKs信号通路;抑制p38MAPK信号通路后能够降低BMP9诱导的间充质干细胞在体外及体内的成骨分化,而抑制ERK1/2信号通路后可促进BMP9诱导的间充质干细胞在体外及体内的成骨分化;抑制ERK1/2的活性后其对BMP9诱导的间充质干细胞成骨分化的促进作用可能与其对细胞增殖及细胞周期的影响相关;抑制p38MAPK的活性能够减少Smad1/5/8 C末端丝氨酸的磷酸化水平进而减弱BMPs/Smad经典信号通路的功能发挥,最终抑制BMP9诱导的间充质干细胞的成骨分化。
Mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, myogenic, hepatocyte, chondrogenic, and adipogenic lineages. It can be used as seed cells to repair and rebuild injured or diseased organs and tissues in regenerative medicine that recently find practical usage in bone regeneration therapy. Mesenchymal stem cells possess multi-directional differentiation capacity, it is vital to explore some new cytokine that can promote commitment of MSC to produce preosteoblasts followed by differentiation preosteoblasts to produce osteoblasts.
BMPs (bone morphogenetic proteins, BMPs) play important roles in regulating osteoblast differentiation and subsequent bone formation. BMPs belong to the transforming growth factorβ(transform Growth Factorβ, TGF-β) superfamily and consist of at least 20 members. BMP-2,-4,-6,-7 and -9 were shown to effectively induce osteogenic differentiation of MSCs in vitro and in vivo, in which BMP9 has far stronger function to induce osteogenic differentiation than that of BMP2 and BMP7. So it is possible to be applied in clinical treatment as a stronger and effective cytokine to induce osteogenic differentiation. BMP9 (also known as GDF-2, growth differentiation factor 2) have many biological functions belong to BMPs. However, the biological effects of BMP9 on osteoblast commitment and terminal differentiation of MSCs has not been comprehensively analyzed.
BMPs transmit signal and play the biological functions through the classical BMPs-Receptor-Smad signaling pathway. However, more and more studies found that BMPs can also transmit signal by other pathways such as MAPKs, PI3K than Smad signaling pathway, which means that transmit signal through activating Smad-independent signal pathway, called as BMPs non-classical pathway. MAPKs (mitogen-activated protein kinases) are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens, osmotic stress, heat-shock and proinflammatory cytokines) and regulate various cellular activities, such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis. BMP2, BMP4 and BMP7 can activate MAPKs signal pathway and induce osteogenic differentiation. Therefore, we provide that BMP9 as a cytokine inducing mesenchymal stem cells into osteoblasts can activate MAPKs signal pathway and induce osteogenic differentiation of MSCs.
The aim of this study is: (1) to identify whether BMP9 can activate MAPKs signal transduction pathway; (2) to investigate the effect of inhibition of MAPKs signaling pathway on BMP9 induced osteogenic differentiation of mesenchymal stem cell; (3) to investigate the mechanisms of mesenchymal stem cell induced into osteogenic differentiation by BMP9 -MAPKs signal pathway.
In the first part of this study, the expressions of ALP in BMP9 induced mesenchymal stem cell into osteogenic differentiation were detected by alkaline phosphatase (ALP) activity. The results showed that BMP9 can induce MSCs into osteogenic differentiation. Then, the phosphorylation of p38MAPK, ERK1/2 and JNK were detected by Western blot assay in cells treated by BMP9. The results showed that BMP9 can induce the phosphorylation of p38MAPK, ERK1/2 and JNK. Given the above-results, we propose to clarify that the effect of inhibition of MAPKs signaling pathway on osteogenic differentiation of mesenchymal stem cell. Therefore, the specific p38MAPK inhibitors SB203580 and the specific ERK1/2 inhibitor PD98059 were used to inhibit the expression of p38MAPK and the activity of ERK1/2. ALP activity of the early osteoblast marker was detected on osteogenic differentiation of MSCs induced by BMP9 combined with treatment of p38MAPK inhibitor and ERK1/2 inhibitor The results demonstrated that:: (1) Dose-dependent manner of the inhibitor of p38MAPK inhibited BMP9-induced osteogenic differentiation of MSCs, indicating that the differentiation of MSCs into osteogenesis can be promoted by BMP9-p38MAPK pathway. (2) Dose-dependent manner of the ERK1/2 inhibitor promoted BMP9-induced osteogenic differentiation of MSCs, indicating that the differentiation of MSCs into osteogenesis can be inhibited by BMP9-ERK1/2 pathway. These data suggest that activation of MAPKs by BMP9 affect BMP9-induced early osteogenic differentiation of MSCs. Then, calcium deposition of the later osteoblast marker was detected on BMP9-induced osteogenic differentiation of MSCs with the p38MAPK inhibitor and the ERK1/2 inhibitor by Alizarin Red S staining. The results showed that inhibition of the expression of p38MAPK reduced calcium deposition of BMP9-induced osteogenic differentiation of MSCs in the later of bone formation, while inhibition of the activity of ERK1/2 enhanced BMP9-induced calcium deposition of BMP9 induced osteogenic differentiation of MSCs in the later phase of bone formation. Finally, the expression of osteopontin (OPN) and osteocalcin (OCN) were detected on BMP9-induced osteogenic differentiation of MSCs with the p38MAPK inhibitor and the ERK1/2 inhibitor by immunocytochemistry. The results showed that the expression of osteopontin (OPN) and osteocalcin (OCN) were decreased by treatment of the p38MAPK inhibitor, but they were increased by treatment of the ERK1/2 inhibitor. In summary, the activation of p38MAPK signaling pathway by BMP9 leads to promote the osteogenic differentiation of mesenchymal stem cell in vitro, while the activation of ERK1/2 signaling pathway by BMP9 can reduce the osteogenic differentiation of mesenchymal stem cells in vitro.
In the second part of this study, RNA interference was applied to knockdown the p38MAPK, ERK1 and ERK2 expression, and the effect of inhibition of MAPKs signaling pathway on BMP9-induced osteogenic differentiation of mesenchymal stem cell were investigated in vitro and in vivo. First, the effect of knockdown of p38MAPK, ERK1 and ERK2 with RNAi were detected with by Real time PCR. The results showed that the expressions of target genes were effectively and successfully reduced with RNAi technology. Subsequently, alkaline phosphatase (ALP) activity was detected on BMP9-induced osteogenic differentiation of mesenchymal stem cell with knockdown of MAPK-p38, ERK1 and ERK2, respectively. The results showed that inhibition of the expression of p38MAPK reduced calcium ALP activity in BMP9-induced osteogenic differentiation of MSCs, while inhibition of the expression of ERK1/2 enhanced BMP9-induced ALP activity in BMP9-induced osteogenic differentiation of MSCs. Likewise, knockdown p38MAPK、ERK1 and ERK2 expression with RNAi partially affect on ALP activity in BMP9-induced osteogenic differentiation of MSCs. Next, C3H10T1/2 co-infected by AdR-sip38MAPK/Ad-BMP9, AdR-siERK1/Ad-BMP9 and AdR-siERK2/Ad-BMP9 were subcutaneously injected to BALB/c nude mice respectively to investigate the effect of knockdown of p38MAPK、ERK1 and ERK2 on BMP9 induced osteogenic differentiation of MSCs in vivo. The results showed: (1) for 5 weeks, cells that inoculated subcutaneously formed a hard mass, indicating bone tissue formation; (2) The size of the bone tissues have little change with knockdown of p38MAPK; while the size of the bone tissues have obviously increased with ERK1/ERK2 RNAi treatment to knockdown ERK1/ERK2 expression; (3) HE staining showed that knockdown of MAPK-p38 reduced the quantity and quality of the bone trabecular, and cartilage cells were found, while knockdown of ERK1/ERK2 significantly increased the amount of bone trabecular and the formation of bone trabecular was more complete. (4) Alcian blue staining showed that knockdown of p38MAPK increased the cartilage matrix, whereas knockdown of ERK1/ERK2 significantly reduced the cartilage matrix. (5) Masson's Trichrome three-color staining showed that knockdown of p38MAPK leaded to decrease of bone matrix with stained red and increase of the cartilage matrix with stained blue. Oppositely, knockdown of ERK1/ERK2 increased bone matrix with stained red while reduced cartilage matrix with stained blue. In short, knockdown of p38MAPK expression reduced BMP9-induced mesenchymal stem cell into osteogenic differentiation, while knockdown of ERK1/ERK2 expression promoted BMP9-induced mesenchymal stem cell into osteogenic differentiation. Those results were consistent with those observations in vitro.
Based on the results of the first part and second part, those data showed that: (1) The depression of p38MAPK signaling pathway leads to decrease the osteogenic differentiation of mesenchymal stem cell by BMP9; (2) ERK1/2 signaling pathway depletion leads to promote mesenchymal stem cells into osteogenic differentiation by BMP9. But the mechanisms of induced-mesenchymal stem cell into osteogenic differentiation by BMP9-MAPKs signaling pathway are under exploration. Therefore, the following studies would investigate those mechanisms from these two perspectives: (1) the relationship of differentiation and proliferation in BMP9-induced osteogenic differentiation of MSCs with the p38MAPK inhibitor and the ERK1/2 inhibior. (2) The influence of activated MAPKs pathway on BMPs/Smad pathway will be studied in BMP9-induced mesenchymal stem cells into osteogenic differentiation. Firstly, after treating with the p38MAPK inhibitor and the ERK1/2 inhibitor, the expressions of ALP in BMP9 induced mesenchymal stem cell into osteogenic differentiation were detected by alkaline phosphatase (ALP) activity from 2 to 9 days, which is aimed to verify whether the peak of ALP activity occurred earlier with the ERK1/2 inhibitor. The results showed that ALP activity on BMP9-induced MSCs into osteogenic differentiation with the ERK1/2 inhibitor was starting to rise at 4 days and dramatically increase from 5 to 6 days, and continuously increase until 9 days. The results indicated that the differentiaton of MSCs induced on BMP9 continuously promoted by the ERK1/2 inhibitor. Then, the proliferation of MSCs treated with BMP9 and the MAPKs inhibitors were detected by MTT assay. The results showed that the proliferation of BMP9-induced MSCs into osteogenic differentiation with the ERK1/2 inhibitor significantly increased at 4 days, suggesting that the increasing of ALP activity with the ERK1/2 inhibitor may be due to promotion of cell proliferation. Subsequently, the cell cycle of MSCs with BMP9 and the MAPKs inhibitor were determined by FCM. The results showed that there was no significant difference of cell cycle phase between treatment by BMP9 or MAPKs inhibitor in the first 1-3 days or in 7-10 days, but the distribution of cell cycle in every treatment group was changed among the groups which mainly happened in the S phase cells increased on BMP9-induced MSCs into osteogenic differentiation with the ERK1/2 inhibitor during the period of 4-6 days. . Therefore, we speculate that the promotion of MSCs into osteogenic differentiation with the ERK1/2 inhibitor may relate to the change of cell proliferation and cell cycle. In addition, in the third part of the study, the phosphorylation of Smad1 (ser463/465)/Smad5 (ser463/465)/Smad8 (ser426/428) C terminal were tested by Western Blot in BMP9-induced MSCs into osteogenic differentiation with the p38MAPK inhibitor and the ERK1/2 inhibitor. The results showed that the phosphorylation of Smad1/5/8 significantly increased after 30 minutes treatment with BMP9 and the ERK1/2 inhibitor, and it sustained until 2 hours, which was similar with treated by BMP9 alone. But the phosphorylation of Smad1/5/8 obviously decreased at 120min treatment with BMP9 and the p38MAPK inhibitor. Following, the localization of Smad1/5/8 was detected using Laser Scanning Confocal Microscope on BMP9-induced MSCs into osteogenic differentiation with the p38MAPK inhibitor and the ERK1/2 inhibitor. The results showed that BMP9 significantly promoted the translocation of Smad1/5/8 into the nuclear with the ERK1/2 inhibitor, which was similar to with BMP9 group, but the location of Smad1/5/8 in the nuclear showed weaker staining compared with that of BMP9 treatment alone with the p38MAPK inhibitor. Finally, the expression of Smad6 and Smad7 mRNA, which are the downstream target genes of BMPs/Smad signaling pathway, were detected by Real time PCR in BMP9-induced MSCs into osteogenic differentiation with the p38MAPK inhibitor and the ERK1/2 inhibitor. The results showed that the mRNA expression of Smad6 and Smad7 decreased respectively 42.34% and 14.46% on BMP9- induced MSCs into osteogenic differentiation with the p38MAPK inhibitor, but the mRNA expression of Smad6 and Smad7 was not significantly changed on BMP9-induced MSCs into osteogenic differentiation with the ERK1/2 inhibitor. The results suggested that the inhibition of MSCs into osteogenic differentiation with the p38MAPK inhibitor was related to down-regulation of BMPs/Smad signal pathway.
The results of the third part of indicated that: (1) the depression of p38MAPK can decrease phosphorylation of Smad 1/5/8 C-terminus and then exert influence on the biological function of classical BMPs/Smad signaling pathway, and finally affect the osteogenic differentiation of BMP9-treated MSCs. (2) the depression of ERK1/2 promotes BMP9-induced MSCs differentiation into osteogenesis may be related to the alteration of cell proliferation and cell cycle distribution caused by depletion of ERK1/2, which needs further study to clarify the molecular mechanisms.
Taken together, BMP9 can activate the MAPKs signaling pathway; the depletion of p38MAPK expression by specific siRNA inhibited the osteogenic differentiation of MSCs by BMP9 induction in vitro and in vivo; the inhibition of ERK1/2 expression promoted MSCs into osteogenic differentiation by BMP9 induction in vitro and in vivo; That the depression of ERK1/2 expression and activity enhances MSCs osteogenic differentiation induced by BMP9 may be related to the alteration of cell proliferation and cell cycle caused by ERK1/2 changes.
While the depression of p38MAPK can affect biological function of classical signaling pathway of BMPs/Smad and then influence BMP9-induced MSCs into osteogenic differentiation.
引文
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