Flavonoids of Herba Epimedii regulate osteogenesis of human mesenchymal stem cells through BMP and Wnt/β-catenin signaling pathway

Herba Epimedii is one of the most commonly used Chinese herbs for treating osteoporosis. In the present study, the flavonoids of Herba Epimedii (HEF) have shown to promote the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. They were noted to enhance the mRNA expression of BMP-2, BMP-4, Runx2, β-catenin and cyclinD1, all of which are BMP or Wnt-signaling pathway related regulators. The osteogenic effect was inhibited by the introduction of noggin and DKK-1, which is classical inhibitor of BMP and Wnt/β-catenin signaling, respectively. These results suggest that HEF exerts promoting effect on osteogenic differentiation, which plausibly functions via the BMP and Wnt/β-catenin signaling pathways. Considering the therapeutic efficiency and economical issues, HEF may be a potential candidate for promoting bone regeneration. On the other hand, osteogenic differentiation of MSCs may also be a promising and attractive tool to apply in bone repair.     

PI3K／Akt信号通路在骨髓间充质干细胞增殖及成骨分化调控中的作用

目的：研究PI3K/Akt信号通路抑制剂LY294002对骨髓间充质干细胞（mesenchymal stem cells,MSCs）增殖及分化的影响。方法采用贴壁法体外分离人骨髓间充质干细胞（hMSCs）,加入PI3K抑制剂LY294002（1、10μmol/L）,应用MTT法测定细胞增殖,常规成骨诱导分化培养3或7d,采用碱性磷酸酶（ALP）染色观察成骨分化水平,化学比色法测定ALP活性,茜素红染色后观察矿化钙结节数量并定量分析,Westernblot检测磷酸化Akt蛋白表达,应用Realtime-PCR检测各组细胞BMP2、Runx2、OPN及Osterix等成骨分化标记物的基因表达水平。结果从24至72h,LY294002对hMSCs增殖均产生显著抑制,随时间推延,可见抑制增殖效果增强（P＜0.05）。ALP染色和定量测定提示10μmol/L的ALP活性最强,在不同时间显著高于对照组和1μmol/L组（P＜0.05）。成骨诱导培养3和7d,1、10μmol/L组矿化量都显著高于对照组（P＜0.05）。10μmol/L组矿化量在成骨诱导7d也显著高于1μmol/L组（P＜0.05）。Westernblot检测结果证实成骨诱导可激活Akt磷酸化蛋白表达,但LY294002可抑制该蛋白磷酸化。成骨诱导分化7d,1、10μmol/L均明显促进BMP2、Runx2、OPN、Osterix4种基因mRNA表达（均P＜0.05）。结论PI3K/Akt信号通路参与hMSCs增殖和分化过程。成骨分化伴随下游Akt蛋白表达。PI3K抑制剂可抑制hMSCs增殖,但同时促进其向成骨分化和矿化。     

Peroxisome proliferator-activated receptor activity is involved in the osteoblastic differentiation regulated by bone morphogenetic proteins and tumor necrosis factor-α

Recent studies have suggested possible adverse effects of thiazolidinediones on bone metabolism. However, the detailed mechanism by which the activity of PPAR affects bone formation has not been elucidated. Impaired osteoblastic function due to cytokines is critical for the progression of inflammatory bone diseases. In the present study, we investigated the cellular mechanism by which PPAR actions interact with osteoblast differentiation regulated by BMP and TNF-α using mouse myoblastic C2C12 cells. BMP-2 and -4 potently induced the expression of various bone differentiation markers including Runx2, osteocalcin, type-1 collagen and alkaline phosphatase (ALP) in C2C12 cells. When administered in combination with a PPARα agonist (fenofibric acid) but not with a PPARγ agonist (pioglitazone), BMP-4 enhanced osteoblast differentiation through the activity of PPARα. The osteoblastic changes induced by BMP-4 were readily suppressed by treatment with TNF-α. Interestingly, the activities of PPARα and PPARγ agonists reversed the suppression by TNF-α of osteoblast differentiation induced by BMP-4. Furthermore, TNF-α-induced phosphorylation of MAPKs, NFκB, IκB and Stat pathways was inhibited in the presence of PPARα and PPARγ agonists with reducing TNF-α receptor expression. In view of the finding that inhibition of SAPK/JNK, Stat and NFκB pathways reversed the TNF-α suppression of osteoblast differentiation, we conclude that these cascades are functionally involved in the actions of PPARs that antagonize TNF-α-induced suppression of osteoblast differentiation. It was further discovered that the PPARα agonist enhanced BMP-4-induced Smad1/5/8 signaling through downregulation of inhibitory Smad6/7 expression, whereas the PPARγ agonist impaired this activity by suppressing BMPRII expression. On the other hand, BMPs increased the expression levels of PPARα and PPARγ in the process of osteoblast differentiation. Thus, PPARα actions promote BMP-induced osteoblast differentiation, while both activities of PPARα and PPARγ suppress TNF-α actions. Collectively, our present data establishes that PPAR activities are functionally involved in modulating the interaction between the BMP system and TNF-α receptor signaling that is crucial for bone metabolism.     

淫羊藿总黄酮对去卵巢大鼠骨髓间充质干细胞成骨和成脂分化中DKK1蛋白动态表达的影响

目的观察DKK1在淫羊藿总黄酮调控去势雌性大鼠骨髓间充质干细胞成骨和成脂分化平衡过程中的动态表达,为进一步阐明淫羊藿总黄酮治疗绝经后骨质疏松症的作用机制提供实验依据。方法体外分离培养去势雌性大鼠来源骨髓间充质干细胞,分别在成骨诱导液和脂肪诱导液条件下连续培养15 d,并在此基础上添加剂量为10μg/mL的淫羊藿总黄酮。采用ALP染色、ALP活性测定、油红O染色以及荧光定量PCR技术,观察淫羊藿总黄酮对骨髓间充质干细胞成骨和成脂分化的影响。用酶联免疫法(ELISA)检测淫羊藿总黄酮处理过程中DKK1蛋白的动态表达,观察DKK1蛋白在淫羊藿总黄酮调控去势雌性大鼠骨髓间充质干细胞成骨和成脂分化平衡过程中的作用。结果淫羊藿总黄酮能显著增加骨髓间充质干细胞ALP的表达以及成骨早期分化因子Runx2 mRNA的表达,显著下调骨髓间充质干细胞中脂肪形成关键基因PPARγ-2mRNA的表达,从而抑制脂滴的形成。在成骨诱导条件下,EFs呈时间依赖性下调DKK1的表达;在脂肪诱导条件下,EFs呈时间依赖性抑制DKK1蛋白的上调。结论通过抑制DKK1蛋白的表达调控去势雌性大鼠BMSCs成骨和成脂分化平衡,可能是淫羊藿总黄酮治疗绝经后骨质疏松症的分子机制之一。     

Rosiglitazone stimulates adipogenesis and decreases osteoblastogenesis in human mesenchymal stem cells

Thiazolidinediones (TZD) are widely prescribed for the treatment of Type 2 diabetes. Increased loss of bone mass and a higher incidence of fractures have been associated with the use of this class of drugs in post-menopausal women. In vitro studies performed in rodent cell models indicated that rosiglitazone (RGZ), one of the TZD, inhibited osteoblastogenesis and induced adipogenesis in bone marrow progenitor cells. The objective of the present study was to determine for the first time the RGZ-dependent shift from osteoblastogenesis toward adipogenesis using a human cell model. To this purpose, bone marrow-derived mesenchymal stem cells were characterized and induced to differentiate along osteogenic and adipogenic lineages. We found that the exposure to RGZ potentiated adipogenic differentiation and shifted the differentiation toward an osteogenic phenotype into an adipogenic phenotype, as assessed by the appearance of lipid droplets. Accordingly, RGZ markedly increased the expression of the typical marker of adipogenesis fatty-acid binding protein 4, whereas it reduced the expression of Runx2, a marker of osteoblastogenesis. This is the first demonstration that RGZ counteracts osteoblastogenesis and induces a preferential differentiation into adipocytes in human mesenchymal stem cells.     

BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells

Vascular calcification is associated with increased risk of cardiovascular events that are the most common cause of death in patients with end-stage renal disease. Clinical and experimental studies indicate that hyperphosphatemia is a risk factor for vascular calcification and cardiovascular mortality in these patients. Our previous studies demonstrated that phosphate transport through the type III sodium-dependent phosphate cotransporter, Pit-1, was necessary for phosphate-induced calcification and osteochondrogenic phenotypic change in cultured human smooth muscle cells (SMC). BMP-2 is a potent osteogenic protein required for osteoblast differentiation and bone formation that has been implicated in vascular calcification. In the present study, we have examined the effects of BMP-2 on human SMC calcification in vitro. We found that treatment of SMC with BMP-2 enhanced elevated phosphate-induced calcification, but did not induce calcification under normal phosphate conditions. mRNAs for BMP receptors, including ALK2, ALK3, ALK6, BMPR-II, ActR-IIA and ActR-IIB were all detected in human SMCs. Mechanistically, BMP-2 dose-dependently stimulated phosphate uptake in SMC (200 ng/ml BMP-2 vs. vehicle: 13.94 vs. 7.09 nmol/30 min/mg protein, respectively). Real-time PCR and Western blot revealed the upregulation of Pit-1 mRNA and protein levels, respectively, by BMP-2. More importantly, inhibition of phosphate uptake by a competitive inhibitor of sodium-dependent phosphate cotransport, phosphonoformic acid, abrogated BMP-2-induced calcification. These results indicate that phosphate transport via Pit-1 is crucial in BMP-2-regulated SMC calcification. In addition, BMP-2-induced Runx2 and inhibited SM22 expression, indicating that it promotes osteogenic phenotype transition in these cells. Thus, BMP-2 may promote vascular calcification via increased phosphate uptake and induction of osteogenic phenotype modulation in SMC.     

Osteoactivin的研究进展

Osteoactivin(OA)是一种最先在骨硬化动物模型中发现的新型糖蛋白.近年来研究表明,OA在骨、肝脏、肾脏等组织中均有不同程度的表达,同时具有多种生物学功能,如成骨细胞分化以及破骨细胞生长、成纤维细胞分化等.OA可诱导成骨细胞及破骨细胞分化,提示其在骨合成代谢方面具有重要作用.但只有少量研究报道了OA作用的可能机制,例如在成纤维细胞中通过细胞外信号调节激酶信号通路,诱导基质金属蛋白酶(MMP)-3的表达以及作为一个下游调节剂,参与调节骨形态发生蛋白(BMP)-2对成骨细胞分化与基质矿化的作用.对OA各种生物学功能的进一步研究将为诊断及治疗多种临床疾病提供新的靶点.     

Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling

Although the multilineage potential of human adipose-derived adult stromal cells (ADAS) has been well described, few published studies have investigated the biological and molecular mechanisms underlying osteogenic differentiation of mouse ADAS. We report here that significant osteogenesis, as determined by gene expression and histological analysis, is induced only when mouse ADAS are cultured in the presence of retinoic acid with or without recombinant human bone morphogenetic protein (BMP)-2 supplementation. Furthermore, a dynamic expression profile for the BMP receptor (BMPR) isoform IB was observed, with dramatic up-regulation during osteogenesis. Western blot analysis revealed that retinoic acid enhanced levels of BMPR-IB protein during the first 7 days of osteogenic differentiation and that RNAi-mediated suppression of BMPR-IB dramatically impaired the ability of ADAS to form bone in vitro. In contrast, absence of BMPR-IA did not significantly diminish ADAS osteogenesis. Our data therefore demonstrate that the osteogenic commitment of multipotent mouse ADAS requires retinoic acid, which enhances expression of the critical BMPR-IB isoform.