TGFβ/BMP Type I Receptors ALK1 and ALK2 Are Essential for BMP9-induced Osteogenic Signaling in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.     

BMP2 sensitizes glioblastoma stem-like cells to Temozolomide by affecting HIF-1�� stability and MGMT expression

Glioblastoma multiforme (GBM) is the most common brain tumour, characterized by a central and partially necrotic (i.e., hypoxic) core enriched in cancer stem cells (CSCs). We previously showed that the most hypoxic and immature (i.e., CSCs) GBM cells were resistant to Temozolomide (TMZ) in vitro, owing to a particularly high expression of O6-methylguanine-DNA-methyltransferase (MGMT), the most important factor associated to therapy resistance in GBM. Bone morphogenetic proteins (BMPs), and in particular BMP2, are known to promote differentiation and growth inhibition in GBM cells. For this reason, we investigated whether a BMP2-based treatment would increase TMZ response in hypoxic drug-resistant GBM-derived cells. Here we show that BMP2 induced strong differentiation of GBM stem-like cells and subsequent addition of TMZ caused dramatic increase of apoptosis. Importantly, we correlated these effects to a BMP2-induced downregulation of both hypoxia-inducible factor-1α (HIF-1α) and MGMT. We report here a novel mechanism involving the HIF-1α-dependent regulation of MGMT, highlighting the existence of a HIF-1α/MGMT axis supporting GBM resistance to therapy. As confirmed from this evidence, over-stabilization of HIF-1α in TMZ-sensitive GBM cells abolished their responsiveness to it. In conclusion, we describe a HIF-1α-dependent regulation of MGMT and suggest that BMP2, by down-modulating the HIF-1α/MGMT axis, should increase GBM responsiveness to chemotherapy, thus opening the way to the development of future strategies for GBM treatment.     

BMP—2和BMP—4基因结构及调控的研究进展

ＢＭＰ－４基因组包括５个外显子,不同组织来源的ＢＭＰ－４ＣＤＮＡ采用不同的启动子,ＢＭＰ－４基因上有ＣＯＵＰ－ＴＦ１的调节作用位点。ＢＭＰ－２基因组有２个外显子,不同组织来源的ＢＭＰ－２ＣＤＮＡ亦采用不同的启动子,ＢＭＰ－２基因上有Ｐ５３的作用位点。     

Is chordin a long-range- or short-range-acting factor? Roles for BMP1-related metalloproteases in chordin and BMP4 autofeedback loop regulation

Diffusible morphogen models have been used widely to explain regional specification of tissues and body axes during animal development. The three-signal model for patterning the dorsal-ventral axis of the amphibian embryo proposes, in part, that a factor(s) secreted from Spemann's organizer is responsible for converting lateral marginal zone into more dorsal cell fates. We examine the possibility that chordin, a secreted inhibitor of bone morphogenetic protein (BMP) signaling and candidate "dorsalizing signal," is a long-range-acting factor. We show that chordin can, when overexpressed, act directly over distances of at least 450 microm in the early Xenopus embryo to create a gradient of BMP signaling. However, since lower levels of chordin can still induce secondary axes and these amounts of chordin act only locally to inhibit a BMP target gene, we suggest that chordin likely acts as a short-range signal in vivo. Furthermore, BMP1, a secreted metalloprotease that cleaves chordin protein in vitro, inhibits chordin's axis-inducing effects, suggesting that BMP1 functions to negatively regulate chordin's action in vivo. A dominant-negative mutant BMP1 blocks the in vitro cleavage of chordin protein by wild-type BMP1 and induces secondary axes when injected ventrally. We argue that BMP1 and Xolloid are probably functionally redundant metalloproteases and may have two roles in the early Xenopus embryo. One role may be to inhibit the action of low-level chordin protein expressed throughout the entire embryo and a possible second role may be to inhibit activation of a juxtacrine cell relay, thereby confining chordin's action to the organizer region preventing chordin from functioning as a long-range-acting factor.     

TGF-β and BMP signaling in osteoblast differentiation and bone formation

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation.     

Comprehensive analysis of TGF-β and BMP receptor interactomes

An immense number of cellular processes are initiated by cell surface serine/threonine kinase receptors belonging to the TGF-β/BMP family. Subsequent downstream signalling cascades, as well as their crosstalk results in enormous specificity in terms of phenotypic outcome, e.g. proliferation, differentiation, migration or apoptosis. Such signalling diversity is achieved by the ability of receptors to interact with distinct proteins in a spatio-temporal manner. Following the cloning of the TGF-β/BMP receptors a variety of different technologies were applied to identify such interacting proteins. Here we present a comprehensive survey of known interactome analyses, including our own data, on these receptors and discuss advantages and disadvantages of the applied technologies.     

Structure of Smad1 MH1/DNA complex reveals distinctive rearrangements of BMP and TGF-β effectors

Smad1 is a downstream effector of the BMP signaling pathway that binds regulatory DNA to execute gene expression programs leading to, for example, the maintenance of pluripotency in mice. On the contrary, the TGF-β-activated Smad3 triggers strikingly different programs such as mesodermal differentiation in early development. Because Smad1 and Smad3 contain identical amino acids at the DNA contact interface it is unclear how they elicit distinctive bioactivities. Here, we report the crystal structure of the MH1 domain of Smad1 bound to a palindromic Smad binding element. Surprisingly, the DNA contact interface of Smad1 is drastically rearranged when compared to Smad3. The N-terminal helix 1 of Smad1 is dislodged from its intramolecular binding site and adopts a domain swapped arrangement with a symmetry-related molecule. As a consequence, helix 2 kinks away from the double helix disabling several key phosphate backbone interactions. Thermal melting analysis corroborates a decompacted conformation of Smad1 and DNA binding assays indicate a lower overall affinity of Smad1 to DNA but increased cooperativity when binding to palindromic DNA motifs. These findings suggest that Smad1 and Smad3 evolved differential qualities to assemble on composite DNA elements and to engage in co-factor interactions by remodeling their N-termini.     

BMP2基因远程调控元件的功能分析

近年来数项基于遗传家系的研究表明,骨形态发生蛋白2(bone morphogenetic protein 2, BMP2)基因下游的远端增强子元件调控区域的异常重复是A2型短指(趾)症(brachydactyly type A2, BDA2)的致病原因,但是这段调控区域的确切分子功能尚不明确,甚至出现有相互矛盾的结果。本研究在生物信息学分析的基础上,通过PCR技术扩增了该调控区域的不同长度的目的片段,包括高度保守的2.1 kb核心序列和3个能够完全覆盖该2.1 kb片段的不同长度的截短体片段,进而构建基因重组载体,采用双荧光素酶报告基因检测方法对这些片段的生物学功能进行分析。结果发现,高度保守的2.1 kb片段并不具有增强子活性,而3个截短体片段均表现出强烈的增强子活性。这表明BMP2基因的表达调控模式非常复杂,对其下游的这段调控区域而言,选取不同长度的片段,其对BMP2表达调控的效果可能并不一致,这很可能是由于不同长度的片段所携带的调控元件数量或种类不同所致。此项研究初步揭示了BMP2基因调控元件的复杂性,为后续深入探究BDA2的分子致病机制提供了新的线索和方向。     

BMP9、BMP12的开发

山之内制药公司与美国Genetics Institute公司(GI公司,马萨诸塞州Cambridge)合作开发骨形成因子(BMP)9和BMP12。两公司90年5月成立合并公司、CI山之内,现在临床试验作为骨再生促进药的BMP2。这次签订的合同,扩大到新的BMP。 目前BMP有十几种类缘蛋白。主要是GI公司克隆的。BMP群的结构都类似于IGF。所以估计除骨形成以外,还有很多生物活性。 这次山之内制药公司下决心合作开发的理由是发现BMP9有特异地促进软骨生长的作用,另外发现BMP12有特异地使韧带和腱生长的生物活性。所以将作为医药品开发的基础研究委托给GI公司。希望B-     

BMP2 rescues deficient cell migration in Tgfbr3−/− epicardial cells and requires Src kinase

During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. The type III transforming growth factor-β receptor (TGFβR3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Bone Morphogenic Protein-2 (BMP2) is a driver of epicardial cell migration. Utilizing a primary epicardial cell line derived from Tgfbr3^+/+ and Tgfbr3^?/? mouse embryos, we show that Tgfbr3^?/? epicardial cells are deficient in BMP2 mRNA expression. Tgfbr3^?/? epicardial cells are deficient in 2-dimensional migration relative to Tgfbr3^+/+ cells; BMP2 induces cellular migration to Tgfbr3^+/+ levels without affecting proliferation. We further demonstrate that Src kinase activity is required for BMP2 driven Tgfbr3^?/? migration. BMP2 also requires Src for filamentous actin polymerization in Tgfbr3^?/? epicardial cells. Taken together, our data identifies a novel pathway in epicardial cell migration required for development of the coronary vessels.     

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．     

Interaction of TGFβ and BMP signaling pathways during chondrogenesis

TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. Although the crosstalk between BMP and TGFβ signaling pathways is well established in bone development, the relationship between these two pathways is less well defined during cartilage development and postnatal homeostasis. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We further used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. We found that conditional deletion of Smad1 in chondrocytes resulted in a shortening of the growth plate. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components. In contrast, in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. In summary, our data demonstrate that during endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo. We also found evidence of direct interaction between the two signaling pathways in a cell model of chondrogenesis in vitro.     

Hmox1促进BMP9诱导C3H10T1/2细胞向成骨分化

目的:研究血红素加氧酶1(hemeoxygenase 1,Hmox1)在骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9)诱导下间充质干细胞C3H10T1/2向成骨分化过程中发挥的作用。方法:用Ad-BMP9感染C3H10T1/2,分别用Q-PCR和Western blot检测Hmox1mRNA和蛋白水平的变化;Hmox1激动剂COPP处理BMP9诱导的C3H10T1/2细胞,碱性磷酸酶(ALP)染色和活性测定检测早期成骨指标ALP的变化;过表达Hmox1的重组腺病毒(Ad-Hmox1)处理BMP9诱导的C3H10T1/2细胞,ALP染色和活性测定早期成骨指标ALP,茜素红染色检测晚期成骨指标钙盐沉积,Western blot检测成骨相关基因COL1A1。结果:Ad-BMP9感染C3H10T1/2后,Hmox1的mRNA及蛋白水平均升高;BMP9与Hmox1激动剂COPP联用与BMP9组相比ALP的活性增强;Ad-Hmox1可以增强BMP9诱导下C3H10T1/2细胞的ALP活性和钙盐沉积,以及成骨相关基因COL1A1表达。结论:Hmox1可以促进BMP9诱导下C3H10T1/2细胞的成骨分化。     

Single Bout Short Duration Fluid Shear Stress Induces Osteogenic Differentiation of MC3T3-E1 Cells via Integrin β1 and BMP2 Signaling Cross-Talk

Fluid shear stress plays an important role in bone osteogenic differentiation. It is traditionally believed that pulsed and continuous stress load is more favorable for fracture recovery and bone homeostasis. However, according to our clinical practice, we notice that one single stress load is also sufficient to trigger osteogenic differentiation. In the present study, we subject osteoblast MC3T3-E1 cells to single bout short duration fluid shear stress by using a parallel plate flow system. The results show that 1 hour of fluid shear stress at 12 dyn/cm² promotes terminal osteogenic differentiation, including rearrangement of F-actin stress fiber, up-regulation of osteogenic genes expression, elevation of alkaline phosphatase activity, secretion of type I collagen and osteoid nodule formation. Moreover, collaboration of BMP2 and integrin β1 pathways plays a significant role in such differentiation processes. Our findings provide further experimental evidence to support the notion that single bout short duration fluid shear stress can promote osteogenic differentiation.     

Biological Function of the Cellular Lipid BMP—BMP as a Key Activator for Cholesterol Sorting and Membrane Digestion

Bis(monoacylglycero)phosphate (BMP) is one of the most intriguing classes of lipids discovered to date. Its biosynthetic pathway is still not fully known, even though studies point to cardiolipin and PG as sources. BMP has an unusual stereoconfiguration which is responsible for its slow catabolism. It is localized exclusively in the inner membranes of late endosomes, and mostly, lysosomes. BMP is negatively charged at the acidic pH of lysosomes, and these charges are central to its role in the degradation of lipids and membranes in the lysosome. In effect, these negative charges facilitate the adhesion of the soluble positively charged hydrolases and activator proteins, thus allowing them to degrade the lipids at the interface of the inner membranes of the lysosome. In this review, we will summarize the different properties of BMP and showcase its importance in the catabolism of lipids and membranes in the lysosome.     

藏系绵羊BMP2基因克隆及组织表达分析

本研究旨在克隆藏系绵羊BMP2基因序列,并获得其生物学特征,同时阐明其组织表达规律。提取藏系绵羊皮肤组织的总RNA,利用RT-PCR技术克隆BMP2基因序列,同时利用荧光定量PCR技术检测该基因在不同组织中的表达情况。结果表明,获得藏系绵羊BMP2基因序列1 217 bp,其中CDS区长度为1 188 bp,编码395个氨基酸。BMP2蛋白有36个磷酸化位、6个糖基化位点和1个跨膜结构,属于不稳定不溶性碱性蛋白,主要在细胞核和线粒体中发挥生物学作用。藏系绵羊BMP2氨基酸序列与山羊、绵羊和牛等的氨基酸同源性很高。BMP2基因在藏系绵羊肺脏组织中表达水平最高,极显著高于其他组织(p0.01)。本研究结果将为进一步研究藏系绵羊BMP2基因的结构和功能提供参考资料。     

BMP2基因在脂肪形成过程中的功能研究进展

骨形态发生蛋白2（BMP2）属于转化生长因子β（TGF-β）超家族成员,是一种分泌性蛋白,具有多重生物学功能。BMP2基因不仅可以诱导骨细胞的形成,还可以促进间充质干细胞向脂肪细胞分化,在脂肪的形成过程中发挥着重要作用。就该基因的结构、表达及其在诱导脂肪细胞形成方面的功能等进行综述。