BMP2/7异源二聚体调控CIZ的表达与自身活性的关系

BMP2/7异源二聚体的活性显著高于BMP2同源二聚体,但其机制并不清楚。采用哺乳动物细胞表达的BMP2/7异源二聚体处理成骨细胞MC3T3-E1,细胞化学染色发现BMP2/7的活性显著高于BMP2,报告载体p3GC2-LUX检测发现BMP2/7能够明显上调BMP/Smad通路的活性(P<0.05)。但在成骨细胞中过表CIZ（Casinteracting zinc finger protein）,能够显著抑制BMP2/7上调ALP与Osteocalcin的作用,并阻断BMP2/7对BMP/Smad通路的激活。同时发现BMP蛋白能够上调CIZ的表达,但BMP2/7的作用明显低于BMP2同源二聚体。可以认为BMP2/7能够诱导CIZ的表达,但由于作用较弱,所以对自身活性的反馈抑制作用也较弱,这可能是BMP2/7有着较强生物活性的关键所在。     

Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development

In the developing limb, Bmp4 is expressed in the apical ectodermal ridge (AER) and underlying mesoderm. Insight into the function of Bmp4 in limb development has been hampered by the early embryonic lethality of Bmp4 null embryos. We directly investigated Bmp4 using a conditional null allele of Bmp4 and the Prx1(cre) transgene to inactivate Bmp4 in limb bud mesoderm. The limb bud mesoderm of Prx1(cre);Bmp4 mutants was defective in production of Bmp4 but still competent to respond to Bmp signaling. Prx1(cre);Bmp4 mutant embryos had defective digit patterning including hindlimb preaxial polydactyly with posterior digit transformations. The Prx1(cre);Bmp4 mutants also had postaxial polydactyly with digit five duplications. Bmp4 mutant limbs had delayed induction and maturation of the AER that resulted in expanded Shh signaling. Moreover, the AER persisted longer in the Bmp4 mutant limb buds exposing the forming digits to prolonged Fgf8 signaling. Our data show that Bmp4 in limb mesoderm regulates AER induction and maturation and implicate signaling from the AER in regulation of digit number and identity.     

Long-term study of bone remodelling after femoral stem: a comparison between dexa and finite element simulation

A hip replacement with a cemented or cementless femoral stem produces an effect on the bone called adaptive remodelling, attributable to mechanical and biological factors. The objective of all of cementless prostheses designs has been to achieve a perfect transfer of loads in order to avoid stress-shielding, which produces an osteopenia. In order to quantify this, the long term and mass-produced study with dual energy X-ray absorptiometry (DEXA) is necessary. Finite element (FE) simulation makes possible the explanation of the biomechanical changes which are produced in the femur after stem implantation. The good correlation obtained between the results of the FE simulation and the densitometric study allow, on one hand, to explain from the point of view of biomechanical performance the changes observed in bone density in the long-term, where it is clear that these are due to a different transfer of load in the implanted model compared to the healthy femur; on the other hand, it validates the simulation model, in a way that it can be used in different conditions and at different time periods, to carry out a sufficiently precise prediction of the evolution of the bone density from the biomechanical behaviour in the interaction between the prosthesis and femur.     

BMP4 substitutes for loss of BMP7 during kidney development

Functional inactivation of divergent bone morphogenetic proteins (BMPs) causes discrete disturbances during mouse development. BMP4-deficient embryos display mesodermal patterning defects at early post-implantation stages, whereas loss of BMP7 selectively disrupts kidney and eye morphogenesis. Whether these distinct phenotypes simply reflect differences in expression domains, or alternatively intrinsic differences in the signaling properties of these ligands remains unknown. To address this issue, we created embryos exclusively expressing BMP4 under control of the BMP7 locus. Surprisingly, this novel knock-in allele efficiently rescues kidney development. These results demonstrate unequivocally that these structurally divergent BMP family members, sharing only minimal sequence similarity can function interchangeably to activate all the essential signaling pathways for growth and morphogenesis of the kidney. Thus, we conclude that partially overlapping expression patterns of BMPs serve to modulate strength of BMP signaling rather than create discrete fields of ligands with intrinsically different signaling properties.     

老年大鼠骨组织中c-fos和BMP表达的变化

目的：研究某些调节因子对骨组织老化过程的影响,方法：应用免疫细胞化学方法,检测大鼠骨组织骨形态发生蛋白（BMP）和细胞转录调节基因蛋白c-fos的表达和相互关系,以及BMP和c-fos蛋白表达的年龄关性变化。结果青年和老年大鼠股骨组织周围骨膜下的成骨细胞以及骨组织中的某些骨细胞BMP和c-fos蛋白呈阳性表达。而老年大鼠骨组织中,BMP和c-fos蛋白的表达明显下降。结论：老年大鼠BMP和c-fos蛋白的增龄性表达变化。可能会影响老年时期大鼠骨组织的形成和改建过程。     

A preliminary study of trace elements in plasma protein by gel chromatography combined with SXRF

Fractions of plasma protein of male Kunming mice (body weight 24.2±0.3g), treated with Cisplatin i.p. injection in dose of 10mg/kg, were obtained by separation on Sephadex-G-50 columns, buffered with ammonium acetate to pH 5.7. The SXRF experiments were performed at the BEPC (Beijing Electron Positron Collider) synchrotron radiation facility. The elements (Pt, S, Ca, Fe, Ni, Cu, Zn, Se, Br and Sr) in the fraction of the plasma proteins (< 22KD) were assayed using highly sensitive SXRF. The relative concentrations of elements were calculated by a normalization of Compton scattering intensity around 22 keV, after the normalization for collecting time of X-ray spectrum and the counting of the ion chamber, and subtracting the contribution of the polycarbonate film used for supporting the samples. The determination could prove that the element Pt in plasma was bound with macro-molecularprotein. Cu and S were present in the fraction of the protein in mice treated with Cisplatin and exhibited an increase, the ratio of treated/control were 1.66±0.06 and 1.78±0.33 respectively, whereas Zn decreased to a ratio of 0.78±0.09. Our results are in agreement with others which showed that Cisplatin exposure leads to a marked loss of kidney copper, and a moderate rise in kidney zinc. However, this work mainly focussed on the implementation of this analytical procedure, but not on the results of the investigations of the effect of Cisplatin on trace elements in plasma protein.     

Changes in bone morphogenetic protein receptor-IB localisation regulate osteogenic responses of human bone cells to bone morphogenetic protein-2

Cell responses to bone morphogenetic proteins (BMP) depend on the expression and surface localisation of transmembrane receptors BMPR-IA, -IB and -II. The present study shows that all three antigens are readily detected in human bone cells. However, only BMPR-II was found primarily at the plasma membrane, whereas BMPR-IA was expressed equally in the cytoplasm and at the cell surface. Notably, BMPR-IB was mainly intracellular, where it was associated with a number of cytoplasmic structures and possibly the nucleus. Treatment with transforming growth factor β1 (TGF-β1) caused rapid translocation of BMPR-IB to the cell surface, mediated via the p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) pathways. The TGF-β1-induced increase in surface BMPR-IB resulted in significantly elevated BMP-2 binding and Smad1/5/8 phosphorylation, although the receptor was subsequently internalised and the functional response to BMP-2 consequently down-regulated. The results show, for the first time, that BMPR-IB is localised primarily in intracellular compartments in bone cells and that TGF-β1 induces rapid surface translocation from the cytoplasm to the cell surface, resulting in increased sensitivity of the cells to BMP-2.     

骨形态发生蛋白诱导骨髓间充质干细胞成骨分化的研究进展

目前从骨髓中成功分离、鉴定BMSCs的方法较为成熟。新发现一些物质能诱导BMSCs向成骨细胞分化因子,其中对BMP研究较多。其机制可能是通过结合Ⅰ、Ⅱ型BMP受体后激活Smad信号通路诱导成骨。其诱导方法主要包括直接应用天然BMP或者将BMP及其协同基因转入BMSCs,通过靶细胞的持续表达BMP促进新骨形成。本文将近10年BMP诱导BMSCs向成骨分化的研究现状及发展趋势做一综述。     

Adeno-associated virus-mediated bone morphogenetic protein-4 gene therapy for in vivo bone formation

Adeno-associated virus (AAV) is so far the most valuable vehicle for gene therapy because it has no association with immune response and human disease. The present study was conducted to investigate the feasibility of AAV-mediated BMP4 gene transfer for bone formation. In vitro study suggested that AAV-BMP4 vectors could transduce myoblast C2C12 cells and produce osteogenic BMP4. In vivo study demonstrated that new bone formation could be induced by direct injection of AAV-BMP4 into the skeletal muscle of immunocompetent rats. Histological analysis revealed that the newly formed bone was induced through endochondral mechanism. Immunohistochemical staining further demonstrated that AAV-BMP4 gene delivery could mediate long-term transduction, and the involvement of BMP4 expression was responsible for the endochondral ossification. This study is, to our knowledge, the first report in the field of AAV-based BMP gene transfer and should be promising for clinical orthopaedic applications.     

Update on the possible nutritional importance of silicon

Convincing evidence that silicon is a bioactive beneficial trace element continues to accumulate. The evidence, which has come from human, animal, and in vitro studies performed by several laboratories, indicate that silicon in nutritional and supra nutritional amounts promotes bone and connective tissue health, may have a modulating effect on the immune or inflammatory response, and has been associated with mental health. A plausible mechanism of action for the beneficial effects of silicon is the binding of hydroxyl groups of polyols such that it influences the formation and/or utilization of glycosaminoglycans, mucopolysaccharides, and collagen in connective tissue and bone. In addition, silicon may affect the absorption, retention or action of other mineral elements (e.g., aluminum, copper, magnesium). Based on findings from both animal and human experiments, an intake of silicon of near 25 mg/d would be a reasonable suggestion for an adequate intake that would assure its nutritional benefits. Increased intakes of silicon through consuming unrefined grains, certain vegetables, and beverages and cereals made from grains should be recognized as a reasonable dietary recommendation.     

Heparan sulfate is required for bone morphogenetic protein-7 signaling

Although genetic studies have suggested that heparan sulfate (HS) is involved in bone morphogenetic protein (BMP)-mediated embryonic morphogenesis, it is unclear whether HS is directly involved in BMP-mediated signaling. Here, we investigate the involvement of HS in BMP-7 signaling. We show that HS and heparin chains specifically bind to BMP-7. Digestion of cell-surface HS with heparitinase interferes with BMP-7-mediated Smad phosphorylation in ROS 17/2.8 osteoblastic cells. Inhibiting sulfation of cell-surface HS with chlorate also causes interruption of Smad phosphorylation. Addition of exogenous heparin to ROS 17/2.8 cells prevents BMP-7-mediated Smad phosphorylation rather than enhances the BMP-7 signal, suggesting that HS should be anchored on the plasma membrane for BMP signaling. Moreover, BMP-7 binding to ROS 17/2.8 cells is inhibited by chlorate treatment and exogenous application of heparin. These results demonstrate that BMP-7 specifically binds to cell-surface HS and the BMP-7-HS interaction is required for BMP-7 signaling.     

Rostral paraxial mesoderm regulates refinement of the eye field through the bone morphogenetic protein (BMP) pathway

The eye field is initially a large single domain at the anterior end of the neural plate and is the first indication of optic potential in the vertebrate embryo. During the course of development, this domain is subject to interactions that shape and refine the organogenic field. The action of the prechordal mesoderm in bisecting this single region into two bilateral domains has been well described, however the role of signalling interactions in the further restriction and refinement of this domain has not been previously characterised. Here we describe a role for the rostral cephalic paraxial mesoderm in limiting the extent of the eye field. The anterior transposition of this mesoderm or its ablation disrupted normal development of the eye. Importantly, perturbation of optic vesicle development occurred in the absence of any detectable changes in the pattern of neighbouring regions of the neural tube. Furthermore, negative regulation of eye development is a property unique to the rostral paraxial mesoderm. The rostral paraxial mesoderm expresses members of the bone morphogenetic protein (BMP) family of signalling molecules and manipulation of endogenous BMP signalling resulted in abnormalities of the early optic primordia.     

Anti-wrinkle effect of bone morphogenetic protein receptor 1a-extracellular domain (BMPR1a-ECD)

Bone morphogenetic proteins (BMPs) have diverse and important roles in the proliferation and differentiation of adult stem cells in our tissues. Especially, BMPs are well known to be the main inducers of bone formation, by facilitating both proliferation and differentiation of bone stem cells. Interestingly, in skin stem cells, BMPs repress their proliferation but are indispensable for the proper differentiation into several lineages of skin cells. Here, we tested whether BMP antagonists have an effect on the prevention of wrinkle formation. For this study we used an in vivo wrinkle-induced mouse model. As a positive control, retinoic acid, one of the top anti-wrinkle effectors, showed a 44% improvement compared to the non-treated control. Surprisingly, bone morphogenetic protein receptor 1a extracellular domain (BMPR1a-ECD) exhibited an anti-wrinkle effect which was 6-fold greater than that of retinoic acid. Our results indicate that BMP antagonists will be good targets for skin or hair diseases. [BMB Reports 2013; 46(9): 465-470]     

人骨形成蛋白2A活性片段在大肠杆菌中的高效表达

将编码人骨形成蛋白２Ａ（ＢＭＰ２Ａ）Ｃ端１７３个氨基酸（ＢＭＰ２３）和１３４个氨基酸（ＢＭＰ２４）的ＤＮＡ基因片段分别重组克隆进入ＰＬ启动子控制下的表达载体,构建了表达质粒ｐＢＬＢＭＰ２３和ｐＢＬＢＭＰ２４,分别转化大肠杆菌进行表达研究．ＳＤＳ－ＰＡＧＥ分析温敏诱导的表达菌,可以分别观察到分子量为２０ｋＤ和１５．５ｋＤ的高表达条带,与理论计算的分子量一致,表达量分别占细菌蛋白质总量的１０％和２０％左右。表达产物经包含体制备达到８０％以上纯度。Ｎ端序列测定的１５个氨基酸,与重组ｃＤＮＡ基因编码的序列相同．ＢＭＰ２３和ＢＭＰ２４包含体经复性处理后,得到二聚体分子蛋白质条带,与骨基质胶原重组后在大鼠体内测活,观察到ＢＭＰ２３诱导软骨细胞生成,ＢＭＰ２４刺激丰富的骨样胶原组织合成．     

帕金森大鼠黑质和纹状体BMP4及其mRNA表达的时程变化

目的：探讨帕金森（PD）大鼠模型体内脑黑质和纹状体内骨形成蛋白4（BMP4）及其mRNA表达的变化规律。方法：用六羟基多巴胺（6-OHDA）建立PD模型后,第2、4、6、8和10周时处死大鼠,取左侧黑质、纹状体,用免疫组化、酶联免疫、荧光定量PCR技术从蛋白水平和基因水平检测BMP4及其mRNA表达。结果：BMP4及其mRNA表达基本一致,其BMP4及mRNA表达均呈双峰,其蛋白表达在纹状体内第2周和6周时达高峰、在黑质内第2周和8周时达高峰,其mRNA表达在纹状体内第2周和8周时达高峰、在黑质内第4周和8周达高峰,差异均有统计学意义（P〈0.05）。结论：PD大鼠模型体内,BMP4及其mRNA不呈现稳定的低表达,而是有波动性,明确BMP4及其mRNA处于稳定低表达的时间后,为下一步的治疗实验时间点的选择上奠定了基础。     

Specific Expression of BMP2/4 Ortholog in Biomineralizing Tissues of Corals and Action on Mouse BMP Receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β superfamily, and have been identified by their ability to induce bone formation in vertebrates. The biomineral-forming process, called biomineralization, is a widespread process, present in all kingdoms of living organisms and among which stony corals are one of the major groups of calcifying animals. Here, we report the presence of a BMP2/4 ortholog in eight species of adult corals. The synthesis of such a protein by the calcifying epithelium of corals suggests that coral BMP2/4 plays a role in skeletogenesis, making BMP the first common protein involved in biomineralization among Eumetazoans. In addition we show that recombinant coral BMP2/4 is able to inhibit human BMP2-induced osteoblastic differentiation in mesenchymal C2C12 cells. We suggest that this inhibition results from a competition between coral BMP2/4 and human BMP2, indicating conservation of binding affinity of BMP and its receptor during evolution from corals to vertebrates. Further studies are needed to understand interactions between coral BMP2/4 and its receptors, and, thus, the action of BMP2/4 in adult corals. Nucleotide sequence of the coral BMP2/4 genes cloned in this study is available in the GenBank under the accession number EU785981 (Stylophora pistillata) and EU785982 (Acropora sp.).