骨髓基质干细胞成骨分化过程中Wnt、骨形态发生蛋白2信号通路相关因子与辛伐他汀的作用

背景：辛伐他汀可促进体外培养的人或鼠骨髓基质干细胞向成骨细胞分化,但作用机制尚不清楚。 目的：观察辛伐他汀对大鼠骨髓基质干细胞向成骨细胞分化过程中Wnt与骨形态发生蛋白2信号途径中相关因子表达的影响。 方法：取6周龄雌性SD大鼠双侧股骨、胫骨全骨髓进行体外成骨细胞诱导培养。实验分为对照组及SIM组。SIM组加入浓度为10-7 mol/L辛伐他汀,对照组加入等量无水乙醇和PBS。培养14 d,行碱性磷酸酶染色,28 d时,行von Kossa染色观察细胞外基质矿化情况;培养14,21 d,免疫荧光细胞化学染色观察成骨细胞中β-catenin,Smad1/5,Cbfa1的表达及分布。  结果与结论：大鼠骨髓基质干细胞经体外诱导后可分化为具有碱性磷酸酶活性和矿化细胞外基质能力的成熟成骨细胞。辛伐他汀可显著上调骨髓基质干细胞成骨分化过程中碱性磷酸酶的表达。同时,与对照组比较,SIM组β-catenin,Smad1/5,Cbfa1表达明显增多(P < 0.05),且呈现明显的核内聚集趋势。说明辛伐他汀促进骨髓基质干细胞向成骨细胞分化的作用可能与调控Wnt与骨形态发生蛋白2信号通路中相关因子的表达及细胞内分布有关。     

#br# rhTGF-β1对SD大鼠MSCs骨向分化的影响及机制研究

 目的：通过观察重组人转化生长因子 β1(rhTGF-β1)对大鼠骨髓间充质干细胞(MSCs)增殖和骨向分化能力的影响,以及对骨形态发生蛋白2(BMP-2)、Smad4及核心结合因子α1(Cbfa1)的作用,阐释其对MSCs骨向分化影响以及可能的作用机制。方法：用全骨髓贴壁法分离、纯化SD大鼠MSCs;用MTT法检测0、5、10、20、40、80和100 μg/L rhTGF-β1对MSCs增殖活性的影响;以碱性磷酸酶(ALP)活性及ALP染色阳性率确定rhTGF-β1的最佳促MSCs骨向分化浓度,并以该浓度对MSCs骨向分化进行干预。按是否添加经典成骨诱导液将实验分为：正常组、经典组、rhTGF-β1组和rhTGF-β1+经典组。通过检测ALP、I型胶原、骨钙素表达和钙化结节的数目,评价各组骨向分化能力;通过检测BMP-2、Smad4和Cbfa1 mRNA的表达,评价各组促MSCs骨向分化的可能作用机制。结果：rhTGF-β1最佳促MSCs增殖浓度为10 μg/L,最佳促MSCs骨向分化浓度为5 μg/L。经典组、rhTGF-β1组和rhTGF-β1+经典组均能促进MSCs骨向分化,刺激BMP-2分泌,并上调Smad4和Cbfa1 mRNA的表达,且rhTGF-β1对MSCs成骨分化的早期、中期效果好,而rhTGF-β1+经典组对MSCs成骨分化的晚期效果更为明显。结论:经典组、rhTGF-β1组和rhTGF-β1+经典组均有促MSCs骨向分化的作用,其机制可能是促进BMP-2的分泌,通过TGF-β超家族/Smads信号通路调控骨向分化。     

Sesamin Stimulates Osteoblast Differentiation Through p38 and ERK1/2 MAPK Signaling Pathways

ABSTRACT: BACKGROUND: Osteoporosisis a worldwide health problem predominantly affecting post-menopausal women. Therapies aimed at increasing bone mass in osteoporetic patients lag behind comparable investigation of therapeutic strategies focusing on the bone resorption process. Sesamin, a major lignan compound found in Sesamun indicum Linn., has a variety of pharmacological effects, though its activity on bone cell function is unclear. Herein we examine the effect of this lignan on osteoblast differentiation and function. METHOD: Cell cytotoxicity and proliferative in hFOB1.19 were examined by MTT and alamar blue assay up to 96 hour of treatment. Gene expression of COL1, ALP, BMP-2, Runx2, OC, RANKL and OPG were detected after 24 hour of sesamin treatment. ALP activity was measured at day 7, 14 and 21 of cultured. For mineralized assay, ADSCs were cultured in the presence of osteogenic media supplement with or without sesamin for 21 days and then stain with Alizarin Red S staining. MAPK signaling pathway activation was observed by using western blotting. RESULTS: Sesamin promoted the expression COL1, ALP, OCN, BMP-2 and Runx2 in hFOB1.19. On the other hand, sesamin was able to up-regulate OPG and down-regulate RANKL gene expression. ALP activity also significantly increased after sesamin treatment. Interestingly, sesamin induced formation of mineralized nodules in adipose derived stem cells (ADSCs) as observed by Alizarin Red S staining; this implies that sesamin has anabolic effects both on progenitor and committed cell stages of osteoblasts. Western blotting data showed that sesamin activated phosphorylation of p38 and ERK1/2 in hFOB1.19. CONCLUSIONS: The data suggest that sesamin has the ability to trigger osteoblast differentiation by activation of the MAPK signaling pathway (p38 and ERK) and possibly indirectly regulate osteoclast development via the expression of OPG and RANKL in osteoblasts. Therefore, sesamin may be a promising phytochemical that could be developed for osteoporotic therapy.     

Involvement of p38MAPK/NF-κB Signaling Pathways in Osteoblasts Differentiation in Response to Mechanical Stretch

Bone morphogenetic proteins (BMPs) are known to be important in osteoblasts' response to mechanical stimuli. BMPs/Smad signaling pathway has been demonstrated to play a regulatory role in the mechanical signal transduction in osteoblasts. However, little is currently known about the Smad independent pathway in osteoblasts differentiation in mechanical loading. In this study, MC3T3-E1 cells were subjected to mechanical stretch of 2000?micro-stain (με) at 0.5?Hz, in order to investigate the involvement of p38MAPK and NF-κB signaling pathways in mechanical response in osteoblasts. We found BMP-2/BMP-4 were up-regulated by mechanical stretch via the earlier activation of p38MAPK and NF-κB signaling pathways, which enhanced osteogenic gene expressions including alkaline phosphatase (ALP), collagen type I (Col I) and osteocalcin (OCN), and the expressions of these osteogenic genes were remarkably decreased with Noggin (an inhibitor for BMPs signals) pretreatment. Furthermore, BMP-2/BMP-4 expressions were suppressed by PDTC, an inhibitor of NF-κB pathway and SB203580, an inhibitor of p38MAPK pathway, respectively, leading to the declined levels of ALP, Col I and OCN. Interestingly, blocking in p38MAPK pathway can also cause the inactivation of NF-κB pathway in mechanical stretch. Collectively, the results indicate during mechanical stretch p38MAPK and NF-κB signaling pathways are activated first, and then up-regulate BMP-2/BMP-4 to enhance osteogenic gene expressions. Moreover, p38MAPK and NF-κB signals have cross-talk in regulation of BMP-2/BMP-4 in mechanical response.     

柚皮苷在促大鼠骨髓间充质干细胞骨向分化过程中对MAPK信号通路的影响

目的: 研究中药单体柚皮苷(NG)对体外培养的大鼠骨髓间充质干细胞(MSCs)向成骨细胞分化过程中MAPK信号通路的影响。方法: 观察在正常、加入p38、ERK和JNK通路抑制剂SB203580、PD98059、SP600125及3种抑制剂全部加入的情况下,各组碱性磷酸酶(ALP)、骨钙素(BGP)、I型胶原(Col I)等骨向分化指标的差异。用Western blotting技术检测各组p38、ERK1/2和JNK蛋白的磷酸化水平,用荧光定量PCR技术检测细胞因子转化生长因子β₁(TGF-β₁)、骨形成蛋白2(BMP-2)和核心结合因子α1(Cbfα1) mRNA的表达。结果: (1)10^-7mol/L为本实验中NG的最佳促骨向分化浓度。(2) NG最佳浓度组的ALP和BGP含量比其它各组都高(P<0.05),Col I含量无明显差异(P>0.05);与NG组相比,加入不同抑制剂组的ALP、BGP和ColⅠ表达量出现不同程度的降低。(3)与空白组相比,NG组JNK蛋白的磷酸化水平升高(P<0.05),p38蛋白的磷酸化水平降低(P<0.01),ERK1/2蛋白的磷酸化水平无明显差异(P>0.05)。与NG组相比,加入不同抑制剂组的p38、ERK1/2和JNK蛋白的磷酸化水平有升高也有降低。(4) NG组上调BMP-2的表达(P<0.05),下调Cbfα1的表达(P<0.05),而对TGF-β₁的表达无明显影响(P>0.05)。与NG组相比,加入不同抑制剂组的TGF-β₁、BMP-2和Cbfα1 mRNA表达量出现不同程度的降低。结论: NG主要通过激活MAPK信号通路中ERK通路、JNK通路以及上调BMP-2的表达,促进MSCs的骨向分化。NG上调BMP-2的表达受MAPK通路中p38通路的影响较大。     

Short-term exposure to tumor necrosis factor-alpha enables human osteoblasts to direct adipose tissue-derived mesenchymal stem cells into osteogenic differentiation

Tumor necrosis factor-alpha (TNF-α) is one major inflammatory factor peaking at 24?h after bone fracture in response to injury; its role in bone healing is controversial. The aims of this study were to investigate whether the duration of exposure to TNF-α is crucial for the initiation of bone regeneration and to determine its underlying mechanism(s). We demonstrated that 24?h of TNF-α treatment significantly abrogated osteocalcin gene expression by human primary osteoblasts (HOBs). However, when TNF-α was withdrawn after 24?h, bone sialoprotein and osteocalcin gene expression levels in HOBs at day 7 were significantly up-regulated compared with the HOBs without TNF-α treatment. In contrast, continuous TNF-α treatment down-regulated bone sialoprotein and osteocalcin gene expression. In addition, in an indirect co-culture system, HOBs pretreated with TNF-α for 24?h induced significantly greater osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ASCs) than the HOBs without TNF-α treatment. TNF-α treatment also promoted endogenous bone morphogenetic protein 2 (BMP-2) production in HOBs, while blocking the BMP-2 signaling pathway with Noggin inhibited osteogenic differentiation of ASCs in the co-culture system. Furthermore, activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway after TNF-α treatment occurred earlier than BMP-2 protein expression. BMP-2 production by HOBs and osteogenic differentiation of ASCs in the co-culture system with HOBs was significantly decreased when HOBs were pretreated with TNF-α in combination with the p38 MAPK-specific inhibitor (SB203580). Taken together, we provide evidence that exposure duration is a critical element in determining TNF-α's effects on bone regeneration. We also demonstrate that the p38 MAPK signaling pathway regulates the expression of BMP-2 in osteoblasts, which then acts through a paracrine loop, to direct the osteoblast lineage commitment of mesenchymal stem cells.     

Glycosaminoglycan chains of biglycan promote bone morphogenetic protein-4-induced osteoblast differentiation

Biglycan (BGN) has been reported to promote bone morphogenetic protein-4 (BMP-4) stimulated osteoblastic differentiation. However, the underlying mechanism has yet to be fully elucidated. The glycosaminoglycan (GAG) chains of BGN have?a variety of?biological functions. In the present study, we explored the potential role of the GAG chains of BGN in promoting BMP-4-induced osteoblast differentiation. BGN knockout (KO) murine calvarial cells were transfected with adenovirus overexpressing wild-type BGN (Adv-BGN), adenovirus expressing GAG-mutant BGN (Adv-BGNm) and adenovirus without BGN (Adv-Emp). Transfected cells were treated with or without BMP-4. Subsequently, BMP-4 signaling and function were assessed by evaluating the expression of the osteoblast differentiation-related proteins, Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the binding specificity of the transfected cells to BMP-4 was also investigated using immunofluorescence staining. Our study demonstrated that a mutant BGN lacking GAG chains decreased BGN-assisted BMP-4 signaling and osteoblast differentiation and that the expression of this mutant BGN in biglycan knockout (BGN?KO) calvarial osteoblasts could not rescue its differentiation deficiency as efficiently as wild-type (WT) BGN. These results strongly suggest that the GAG chains of BGN promote BGN-assisted BMP-4 function.     

Continuous expression of Cbfa1 in nonhypertrophic chondrocytes uncovers its ability to induce hypertrophic chondrocyte differentiation and partially rescues Cbfa1-deficient mice

Chondrocyte hypertrophy is a mandatory step during endochondral ossification. Cbfa1-deficient mice lack hypertrophic chondrocytes in some skeletal elements, indicating that Cbfa1 may control hypertrophic chondrocyte differentiation. To address this question we generated transgenic mice expressing Cbfa1 in nonhypertrophic chondrocytes (alpha1(II) Cbfa1). This continuous expression of Cbfa1 in nonhypertrophic chondrocytes induced chondrocyte hypertrophy and endochondral ossification in locations where it normally never occurs. To determine if this was caused by transdifferentiation of chondrocytes into osteoblasts or by a specific hypertrophic chondrocyte differentiation ability of Cbfa1, we used the alpha1(II) Cbfa1 transgene to restore Cbfa1 expression in mesenchymal condensations of the Cbfa1-deficient mice. The transgene restored chondrocyte hypertrophy and vascular invasion in the bones of the mutant mice but did not induce osteoblast differentiation. This rescue occurred cell-autonomously, as skeletal elements not expressing the transgene were not affected. Despite the absence of osteoblasts in the rescued animals there were multinucleated, TRAP-positive cells resorbing the hypertrophic cartilage matrix. These results identify Cbfa1 as a hypertrophic chondrocyte differentiation factor and provide a genetic argument for a common regulation of osteoblast and chondrocyte differentiation mediated by Cbfa1.     

Involvement of ILK/ERK1/2 and ILK/p38 pathways in mediating the enhanced osteoblast differentiation by micro/nanotopography

The hierarchical micro/nanotextured topography (MNT) on titanium (Ti) implant surface significantly enhances osteoblast differentiation. We have demonstrated that integrin-linked kinase (ILK) is a key underlying signal molecule and β-catenin is one of its downstream mediators in MNT-regulated osteoblast behavior. Here we propose that mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun NH₂-terminal kinase (JNK), are other mediators downstream of ILK, and this study aims to confirm this. Firstly, the levels of ILK and MAPK activity in MG63 cells on MNT are examined by Western blot analysis. The ILK, ERK1/2 and p38 signals are significantly up-regulated by MNT, whereas the JNK activity is undetectable by Western blot. The MG63 cell morphology, proliferation and differentiation are studied in the absence and presence of the MAPK subgroup inhibitors to confirm their roles in cell functions on the Ti surface. The MAPK subgroup inhibitors obviously change the cell shape and depress cell proliferation. Blocking the ERK1/2 or p38 signaling, but not the JNK signaling, significantly down-regulates the cell osteogenesis-related gene expression, ALP production, collagen secretion and matrix mineralization. Afterwards, the ILK expression is down-regulated using ILK-specific siRNA (ILKsi) and then the MAPK activity is determined. ILKsi significantly attenuates the phosphorylated ERK1/2 and p38 levels on MNT, explicitly demonstrating that the ERK1/2 and p38 signalings are downstream effectors of ILK. In conclusion, these data demonstrate that both ILK/ERK1/2 and ILK/p38 pathways are involved in the mechanisms mediating the enhanced osteoblast differentiation by biomaterial surface topography, hopefully directing the biomaterial modification and biofunctionalization.     

Brg1调控重组人骨形态发生蛋白2诱导成骨细胞分化

背景：Brg1是依赖ATP的染色质改变复合物的核心催化亚基,该亚基在基因的转录调控、复制、重组,骨骼肌的分化、抑制肿瘤的发生等活动中起着重要的作用。 目的：探索Brg1基因在骨形态发生蛋白2诱导成骨细胞分化过程中的调控机制。 方法：采用胶原酶消化法进行小鼠颅骨成骨细胞的原代培养;分别用0,50,200 μg/L的重组人骨形态发生蛋白2诱导原代培养的成骨细胞的分化,摸索骨形态发生蛋白2的最佳作用剂量;实时荧光定量PCR和Western blot进行骨形态发生蛋白2对Brg1的作用时间的动力学分析;实时荧光定量PCR和钙钴染色法检测敲除Brg1对骨形态发生蛋白2诱导的成骨分化的影响;构建Dlx5腺病毒重组表达载体,实时荧光定量PCR和钙钴染色法检测Brg1在骨形态发生蛋白2诱导的成骨分化过程中对Dlx5的调控作用。 结果与结论：用自行合成的重组人骨形态发生蛋白2可诱导原代培养小鼠成骨细胞分化,200 μg/L剂量有着较好的诱导分化效果;重组人骨形态发生蛋白2可诱导Brg1基因转录水平和翻译水平表达水平上调;敲除Brg1可抑制重组人骨形态发生蛋白2诱导的成骨分化;Brg1能够调控Dlx5的表达水平。说明Brg1通过调控Dlx5的表达水平调控重组人骨形态发生蛋白2诱导的小鼠成骨细胞的分化。     

Osteogenesis on nanoparticulate mineralized collagen scaffolds via autogenous activation of the canonical BMP receptor signaling pathway

Skeletal regenerative medicine frequently incorporates deliverable growth factors to stimulate osteogenesis. However, the cost and side effects secondary to supraphysiologic dosages of growth factors warrant investigation of alternative methods of stimulating osteogenesis for clinical utilization. In this work, we describe growth factor independent osteogenic induction of human mesenchymal stem cells (hMSCs) on a novel nanoparticulate mineralized collagen glycosaminoglycan scaffold (MC-GAG). hMSCs demonstrated elevated osteogenic gene expression and mineralization on MC-GAG with minimal to no effect upon addition of BMP-2 when compared to non-mineralized scaffolds (Col-GAG). To investigate the intracellular pathways responsible for the increase in osteogenesis, we examined the canonical and non-canonical pathways downstream from BMP receptor activation. Constitutive Smad1/5 phosphorylation with nuclear translocation occurred on MC-GAG independent of BMP-2, whereas Smad1/5 phosphorylation depended on BMP-2 stimulation on Col-GAG. When non-canonical BMPR signaling molecules were examined, ERK1/2 phosphorylation was found to be decreased in MC-GAG but elevated in Col-GAG. No differences in Smad2/3 or p38 activation were detected. Collectively, these results demonstrated that MC-GAG scaffolds induce osteogenesis without exogenous BMP-2 addition via endogenous activation of the canonical BMP receptor signaling pathway.     

The bone anabolic carotenoid p-hydroxycinnamic acid promotes osteoblast mineralization and suppresses osteoclast differentiation by antagonizing NF-κB activation

Numerous plant derived nutritional factors including p-hydroxycinnamic acid (HCA), a member of the carotenoid family, have long been held to possess bone protective properties. Studies in animals have provided a mechanistic basis for these observations by demonstrating the capacity of HCA to promote bone formation and suppress bone resorption in vivo. However, the molecular mechanism by which HCA achieves these effects remains unclear. We have demonstrated that a centralized mechanism by which several other nutritional factors achieve similar effects is through modulation of the nuclear factor-κB (NF-κB) signal transduction pathway. NF-κB activation is essential for osteoclast formation and resorption but potently antagonizes osteoblast differentiation and mineralization. In this study we demonstrate that HCA does indeed antagonize the activation of NF-κB by the key osteoclastogenic cytokine receptor activator of NF-κB (RANKL) in RAW264.7 osteoclast precursors, suppressing their differentiation into osteoclasts. Furthermore, HCA augmented the in vitro differentiation of MC3T3 preosteoblastic cells into mineralizing osteoblasts and relieved the inhibitory action of tumor necrosis factor-α (TNF-α)-induced NF-κB signaling on transforming growth factor-β (TGF-β)- or bone morphogenetic protein-2 (BMP-2)-induced Smad activation, an important pathway in osteoblast commitment and differentiation. Our data provide a mechanism to explain the dual pro-anabolic and anti-catabolic activities of HCA.     

ERK MAP Kinase is required in 1,25(OH)2D3-induced differentiation in human osteoblasts

Expression of alkaline phosphatase(ALP)activity represents a key event during the differentiation processes of osteoblasts, and the level of ALP activity has been routinely used as a relative measure of differentiation stages of osteoblasts. In human osteoblasts, we showed that vitamin D₃ analogue, 1,25(OH)₂D₃, had a stimulatory effect on ALP activity after 3 days, compared with control. The treatment of PD098059, an ERK MAP Kinase inhibitor, had a reducing effect on ALP activity, a differentiation marker in 1,25(OH)₂D₃-treated primary human osteoblasts. However, SB203580, a potent p38 MAP Kinase inhibitor, had no effect on the differentiation in this system. This indicates that ERK, not p38, is directly related to 1,25(OH)₂D₃ -stimulated ALP activity in primary human osteoblasts. These results also show that the vitamin D₃ analogue stimulates ERK1 activation in primary human osteoblasts. This finding provides one of signaling pathways for differentiation in primary human osteoblasts.     

Intermittent Traction Stretch Promotes the Osteoblastic Differentiation of Bone Mesenchymal Stem Cells by the ERK1/2-Activated Cbfa1 Pathway

Mechanical stress plays a crucial role in bone formation and absorption. We investigated the osteoblastic differentiation of bone mesenchymal stem cells (BMSCs) affected by intermittent traction stretch at different time points and explored the mechanism of osteoblastic differentiation under this special mechanical stimulation. The BMSCs and C3H10T1/2 cells were subjected to 10% elongation for 1–7 days using a Flexcell Strain Unit, and then the mRNA levels of osteoblastic genes and the expression of core-binding factor a1 (Cbfa1) were examined. Furthermore, we focused specifically on the role of the extracellular signal-regulated kinases 1/2 (ERK1/2) and Cbfa1 in the osteogenesis of BMSCs stimulated by the stretch. The results of these experiments showed that the stretch induces a time-dependent increase in the expression of osteoblastic genes. The synthesis of osteoblastic genes was downregulated after the knockdown of Cbfa1 expression by short-interfering RNA. Furthermore, the stress-induced increase in the expression of Cbfa1 mRNA and osteoblastic genes was inhibited by U0126, an ERK1/2 inhibitor. These results indicate that long periods of intermittent traction stretch promote osteoblastic differentiation of BMSCs through the ERK1/2-activated Cbfa1 signaling pathway.