Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells

Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation of mesenchymal stem cells, the molecular mechanism involved remains to be fully elucidated. In this study, we showed that BMP9 simultaneously promotes the activation of Smad1/5/8, p38 and ERK1/2 in C3H10T1/2 cells. Knockdown of Smad4 with RNA interference reduced nuclear translocation of Smad1/5/8, and disrupted BMP9-induced osteogenic differentiation. BMP9-induced osteogenic differentiation was blocked by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. SB203580 decreased BMP9-activated Smads singling, and yet PD98059 stimulated Smads singling in C3H10T1/2 cells. The effects of inhibitor were reproduced with adenovirus expressing siRNA targeted p38 and ERK1/2, respectively. Taken together, our findings revealed that Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation. Also, it is noteworthy that p38 and ERK1/2 may play opposing regulatory roles in mediating BMP9-induced osteogenic differentiation of C3H10T1/2 cells.     

BMP9经JNK激酶途径调控间充质干细胞C3H10T1/2成骨分化

为了证实JNK激酶在骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9) 诱导间充质干细胞C3H10T1/2成骨分化中的作用,利用重组腺病毒将BMP9导入间充质干细胞C3H10T1/2. 通过碱性磷酸酶(ALP)活性测定、钙盐沉积实验、荧光素酶报告基因检测、Western印迹和组织化学染色等方法,检测BMP9是否可经JNK激酶途径调控间充质干细胞C3H10T1/2向成骨分化.动物实验验证在RNA沉默JNK蛋白激酶后,对BMP9诱导间充质干细胞C3H10T1/2向成骨分化的影响．结果发现,BMP9可以增强JNK 激酶的磷酸化;利用JNK抑制剂SP600125抑制JNK激酶活性后,BMP9诱导的间充质干细胞C3H10T1/2的早期成骨指标ALP活性和晚期指标钙盐沉积均受到抑制,而且经典SMAD信号的活化也相应受到抑制;RNA干扰沉默JNK基因表达后,同样也可抑制BMP9 诱导的C3H10T1/2细胞的ALP活性和裸鼠皮下异位成骨．因此表明,BMP9可活化JNK激酶途径从而诱导间充质干细胞C3H10T1/2向成骨分化．     

RhoGDIβ Inhibits Bone Morphogenetic Protein 4 (BMP4)-induced Adipocyte Lineage Commitment and Favors Smooth Muscle-like Cell Differentiation

The integration of signals involved in deciding the fate of mesenchymal stem cells is largely unknown. We used proteomics profiling to identify RhoGDIβ, an inhibitor of the small G-protein Rho family, as a component that regulates commitment of C3H10T1/2 mesenchymal stem cells to the adipocyte or smooth muscle cell lineage in response to bone morphogenetic protein 4 (BMP4). RhoGDIβ is notably down-regulated during BMP4-induced adipocytic lineage commitment of C3H10T1/2 mesenchymal stem cells, and this involves the cytoskeleton-associated protein lysyl oxidase. Excess RhoGDIβ completely prevents BMP4-induced commitment to the adipocyte lineage and simultaneously stimulates smooth muscle cell commitment by suppressing the activation of Rac1. Overexpression of RhoGDIβ induces stress fibers of F-actin by a process involving phosphomyosin light chain, indicating that cytoskeletal tension regulated by RhoGDIβ contributes to determining adipocyte versus myocyte commitment. Furthermore, the overexpression of RacV12 (constitutively active form of Rac1) totally rescues the inhibition of adipocyte commitment by RhoGDIβ, simultaneously preventing formation of the smooth muscle-like phenotype and disrupting the stress fibers in cells overexpressing RhoGDIβ. Collectively, these results indicate that RhoGDIβ functions as a novel BMP4 signaling target that regulates adipogenesis and myogensis.     

p38蛋白激酶参与BMP9诱导的C3H10T1/2细胞成骨分化

目的:初步分析丝裂原活化蛋白激酶p38在BMP9诱导间充质干细胞C3H10T1/2成骨分化过程中的作用.方法:利用BMP9重组腺病毒感染C3H10T1/2细胞,Western blot检测p38激酶总蛋白表达水平和磷酸化水平.p38的特异性抑制剂SB203580抑制p38活性或RNA干扰抑制p38表达后,分析ALP活性...     

敲低低密度脂蛋白受体相关蛋白1抑制C3H10T1/2多潜能干细胞定向成脂

C3H10T1/2多潜能干细胞成脂过程分为定向和分化两个阶段,骨形成蛋白4(BMP4)可以诱导其定向成前脂肪细胞．已有的研究表明,脂肪组织特异性敲除低密度脂蛋白受体相关蛋白1(Lrp1)的小鼠体重减轻,脂肪组织含量减少,揭示此基因对成脂具有重要作用．然而,目前尚不清楚Lrp1是否在成脂定向过程中发挥作用．采用小干扰RNA技术(RNAi),在体外水平研究低密度脂蛋白Lrp1对C3H10T1/2多潜能干细胞成脂定向的作用．分别在C3H10T1/2成脂的定向期和脂滴成熟期敲低Lrp1,通过显微镜下观察、油红O染色、Western blotting等实验证实,定向期而非脂滴成熟期敲低Lrp1显著抑制C3H10T1/2多潜能干细胞成脂．BMP4通过激活下游Smad1/5/8信号通路发挥作用,而敲低Lrp1显著抑制BMP4诱导的Smad1/5/8磷酸化．这些结果说明：敲低Lrp1通过下调Smad信号通路,抑制BMP4诱导的C3H10T1/2多潜能干细胞成脂定向．     

BMP2具有诱导C3H10T1/2细胞成脂肪分化的能力

探讨骨形态发生蛋白2（BMP2）诱导鼠胚胎间充质干细胞C3H10T1/2成脂肪分化能力,为临床脂肪代谢疾病的治疗提供理论基础．培养多潜能的间充质干细胞C3H10T1/2,用20 μg/ml BMP2对其诱导一定时间后,RT-PCR检测是否存在BMP信号通路中关键分子BMP受体BMPR I, BMPR Ⅱ及Smad 1/5/8的表达.Western印迹检测Smad 蛋白及MAPK 信号通路中p38磷酸化水平变化,QRT PCR检测成脂肪标志基因aP2以及成脂肪相关转录因子PPARγ,C/EBPα,C/EBPβ表达水平,同时用油红O染色,观测C3H10T1/2细胞成脂肪分化情况．经BMP2诱导后,C3H10T1/2细胞成脂肪分化标志(油红O染色)显著增加,Smad 蛋白及p38磷酸化水平有所上升,同时成脂肪标志基因aP2以及成脂肪相关转录因子PPARγ,C/EBPα,C/EBPβ表达水平各有一定程度提高．BMP2具有诱导C3H10T1/2细胞成脂肪分化能力,其成脂肪分化呈现对BMP2作用的时间依赖性．     

Shh信号参与调控BMP9诱导的间充质干细胞成骨分化

目的：观察sonic hedgehog（Shh）信号通路在骨形态发生蛋白9（BMP9）诱导的小鼠间充质干细胞（MSCs）C3H10T1/2和C2C12成骨分化中的作用,并初步探讨其作用机制。方法：Shh信号通路抑制剂Cyclopamine和激活剂Purmorphamine以及过表达Shh腺病毒分别作用于BMP9处理的C3H10T1/2和C2C12细胞,碱性磷酸酶（ALP）检测早期成骨指标ALP,茜素红S染色检测晚期成骨指标钙盐沉积,RT-PCR检测Shh信号相关基因以及成骨关键转录因子的表达,Western blot检测Shh的表达,荧光素酶报告基因检测Smad1/5/8的转录调控活性。结果：BMP9促进Shh信号相关基因的表达,激活Shh信号可增强BMP9诱导的C3H10T1/2和C2C12细胞早晚期成骨分化并促进了BMP9诱导的Smad荧光素酶活性,抑制Shh信号后作用相反。结论：激活Shh信号通路可促进BMP9诱导的小鼠MSCs成骨分化,抑制其活性后作用相反。     

阻断ERK1/2激酶可增强骨形态发生蛋白9诱导的间充质干细胞成骨分化

骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)具有很强的诱导间充质干细胞定向成骨分化的能力.但对于其所涉及的相关分子机理了解并不深入.利用BMP9重组腺病毒感染间充质干细胞,Western blot检测ERK1/2激酶的磷酸化,ERK1/2的特异性抑制剂PD98059阻断ERK1/2活性,或以RNA干扰抑制ERK1/2表达,通过体外细胞实验和体内动物实验,初步分析和揭示ERK1/2对于BMP9诱导的间充质干细胞成骨分化的调控作用及其可能机制.结果发现:BMP9可以促进ERK1/2激酶的磷酸化,ERK1/2抑制剂PD98059可增强由BMP9诱导的碱性磷酸酶(alkaline phosphatase,ALP)活性、骨桥蛋白(osteopontin,OPN)表达和钙盐沉积,并促进由BMP9诱导的Runx2基因的表达和转录活性,以及Smad经典途径的活化;而RNA干扰导致ERK1/2基因沉默同样也可进一步促进BMP9诱导的ALP活性和钙盐沉积,并促进BMP9诱导的间充质干细胞在裸鼠皮下异位成骨.因此,BMP9可以促进ERK1/2蛋白激酶的活化,而阻断ERK1/2蛋白激酶可进一步增强BMP9诱导的成骨分化,ERK1/2极可能对于BMP9诱导的间充质干细胞成骨分化起着负向调控作用.     

Inhibition of CK2 binding to BMPRIa induces C2C12 differentiation into osteoblasts and adipocytes

BMP2 is a growth factor that regulates the cell fate of mesenchymal stem cells into osteoblast and adipocytes. However, the detailed signaling pathways and mechanism are unknown. We previously reported a new interaction of Casein kinase II (CK2) with the BMP receptor type-Ia (BMPRIa) and demonstrated using mimetic peptides CK2.1, CK2.2 and CK2.3 that the release of CK2 from BMPRIa activates Smad signaling and osteogenesis. Previously, we showed that mutation of these CK2 sites on BMPRIa (MCK2.1 (476S-A), MCK2.2 (324S-A) and MCK2.3 (214S-A)) induced osteogenesis. However, one mutant MCK2.1 induced osteogenesis similar to overexpression of wild type BMPRIa, suggesting that the effect of this mutant on mineralization was due to overexpression. In this paper we investigated the signaling pathways involved in the CK2-BMPRIa mediated osteogenesis and identified a new signaling pathway activating adipogenesis dependent on the BMPRIa and CK2 association. Further the mechanism for adipogenesis and osteogenesis is specific to the CK2 interaction site on BMPRIa. In detail our data show that overexpression of MCK2.2 induced osteogenesis was dependent on Caveolin-1 (Cav1) and the activation of the Smad and mTor pathways, while overexpression of MCK2.3 induced osteogenesis was independent of Caveolin-1 without activation of Smad pathway. However, MCK2.3 induced osteogenesis via the MEK pathway. The adipogenesis induced by the overexpression of MCK2.2 in C2C12 cells was dependent on the p38 and ERK pathways as well as Caveolin-1. These data suggest that signaling through BMPRIa used two different signaling pathways to induce osteogenesis dependent on CK2. Additionally the data supports a signaling pathway initiated in caveolae and one outside of caveolae to induce mineralization. Moreover, they reveal the signaling pathway of BMPRIa mediated adipogenesis.     

The effects of myostatin on adipogenic differentiation of human bone marrow-derived mesenchymal stem cells are mediated through cross-communication between Smad3 and Wnt/beta-catenin signaling pathways

The effects of myostatin on adipogenic differentiation are poorly understood, and the underlying mechanisms are unknown. We determined the effects of human recombinant myostatin protein on adipogenesis of bone marrow-derived human mesenchymal stem cells (hMSCs) and adipose tissue-derived preadipocytes. For both progenitor cell types, differentiation in the presence of myostatin caused a dose-dependent reduction of lipid accumulation and diminished incorporation of exogenous fatty acid into cellular lipids. Myostatin significantly down-regulated the expression of adipocyte markers PPARgamma, C/EBPalpha, leptin, and aP2, but not C/EBPbeta. Overexpression of PPARgamma, but not C/EBPbeta, blocked the inhibitory effects of myostatin on adipogenesis. Myostatin induced phosphorylation of Smad3 in hMSCs; knockdown of Smad3 by RNAi or inhibition of its upstream kinase by an Alk5 inhibitor blocked the inhibitory effect of myostatin on adipogenesis in hMSCs, implying an important role of Smad3 activation in this event. Furthermore, myostatin enhanced nuclear translocation of beta-catenin and formation of the Smad3-beta-catenin-TCF4 complex, together with the altered expression of a number of Wnt/beta-catenin pathway genes in hMSCs. The inhibitory effects of myostatin on adipogenesis were blocked by RNAi silencing of beta-catenin and diminished by overexpression of dominant-negative TCF4. The conclusion is that myostatin inhibited adipogenesis in human bone marrow-derived mesenchymal stem cells and preadipocytes. These effects were mediated, in part, by activation of Smad3 and cross-communication of the TGFbeta/Smad signal to Wnt/beta-catenin/TCF4 pathway, leading to down-regulation of PPARgamma.     

Involvement of ERK in BMP-2 induced osteoblastic differentiation of mesenchymal progenitor cell line C3H10T1/2

The signaling mechanisms responsible for bone morphogenetic protein (BMP) induced osteoblast differentiation remains poorly understood. Previous research demonstrated that Smad proteins are the substrates and the mediators of BMP bound serine/threonine receptor kinase. In the present study, we examined the possible involvement of extracellular signal-regulated kinase (Erk) in the BMP induced osteoblast differentiation of mesenchymal progenitor cell C3H10T1/2. Our results indicate that BMP-2 inducement increased MAP kinase activity in mesenchymal progenitor cell line C3H10T1/2. Contrary to previous reports, this increased MAP kinase activity showed a latent but sustained pattern. Elevation of Erk1 and Erk2 protein levels was observed simultaneously. RT-PCR results demonstrated that the elevation of Erk protein level in BMP-2 induced cells was from the upregulation of mRNA expression. Furthermore, upregulated Erk proteins present enhanced phosphorylation. By using a dominant-negative Erk2 cell line, we demonstrated that nonfunctional Erk2 partially eliminated BMP-2 induced cell proliferation and ALP activity in the C3H10T1/2 cell. These results indicate that Erk is involved in BMP-2 induced osteoblast differentiation. The results also demonstrate that a latent and sustained signaling pattern exists in BMP induced signaling cascade.     

MiR-21协同BMP9促进间充质干细胞C3H10T1/2成骨分化

目的:探讨miR-21与BMP9之间的关系,明确miR-21在BMP9诱导间充质干细胞成骨分化中的作用。方法:(1)Ad-BMP9感染C3H10T1/2细胞,Real-time-PCR检测miR-21表达。RT-PCR检测ALP的表达。(2)MiR-21转染C3H10T1/2细胞,Real-time-PCR检测miR-21和BMP9表达。(3)MiR-21和BMP9-CM处理C3H10 T1/2细胞,ALP活性和染色实验检测C3H10 T1/2细胞早期成骨能力。茜素红S染色实验检测钙盐沉积情况。(4)MiR-21和BMP9-CM处理C3H10 T1/2细胞,Real-time-PCR检测成骨分化相关因子ALP,OCN的表达。(5)MiR-21和BMP9-CM处理C3H10T1/2细胞,Western blot检测p-Smad1/5蛋白水平的表达。结果:(1)BMP9暂时降低miR-21的表达。MiR-21也可以暂时降低BMP9的表达。(2)MiR-21可以协同BMP9增强ALP和钙盐沉积。(3)MiR-21协同BMP9增加了p-Smad1/5蛋白水平的表达。结论:MiR-21与BMP9存在相互关系,两者可以互相调节表达。MiR-21可以协同BMP9促进间充质干细胞C3H10T1/2细胞成骨分化,这一过程与增强BMP9/Smad信号的激活程度有关。