Spinally upregulated noggin suppresses axonal and dendritic plasticity following dorsal rhizotomy

Bone morphogenetic proteins (BMPs) and their antagonists, including noggin, are required for nervous system development, but their potential roles in the reactions of the adult central nervous system to injury are unknown. Here we have examined the expression of noggin and BMPs in the spinal cord following dorsal rhizotomy. Through the use of a function-blocking antibody, we have also investigated the role of endogenous noggin in the neuritic plasticity which follows rhizotomy. Dorsal rhizotomy resulted in the upregulation of BMPs 2/4, 7 and noggin in the superficial white matter and in the dorsal neuropil of the spinal cord. These co-localized with glial fibrillary acidic protein, indicating their expression by astrocytes. Because BMPs induce dendritic sprouting and synaptogenesis in some neuronal populations in vitro, we hypothesized that administration of a noggin function-blocking antibody (FbAb) in vivo would augment rhizotomy-induced sprouting in the spinal cord. Topical application of noggin-FbAb to the dorsal surface of the spinal cord following rhizotomy resulted in significant increases in the density of microtubule-associated protein 2 (MAP-2) and substance P (SP)-positive processes within the lateral spinal nucleus. In the deafferented dorsal horn, noggin-FbAb treatment induced significant increases in the density of SP, calcitonin gene-related peptide (CGRP)- and 5-hydroxytryptamine (5-HT)-positive axons. These results suggest a novel mechanism by which endogenous plasticity of spared axons is suppressed following dorsal rhizotomy, and which might be exploited to improve the outcome of spinal cord injury and other CNS trauma.     

Amelogenin binds to both heparan sulfate and bone morphogenetic protein 2 and pharmacologically suppresses the effect of noggin

Enamel matrix derivative (EMD) is widely considered useful to promote tissue regeneration during periodontal treatment. It has been reported that the main constituent of EMD is amelogenin and that the BMP-like and TGF-beta-like activity of EMD promotes osteogenesis. However, it remains unclear whether those activities are dependent on amelogenin or another growth factor contained in EMD. We performed two-dimensional SDS-PAGE analysis of EMD, as well as Western blot analyses using anti-amelogenin, anti-BMP2/4, and anti-TGF-beta1 antibodies, and amino acid sequencing. Our results revealed that a large number of splicing forms of amelogenin, BMP2/4, and other unknown molecules were involved in EMD, though TGF-beta1 was not. In addition, we have evaluated intracellular signaling of ERK1/2 and Smad1/5/8, binding potential and alkaline phosphatase activity and have explored the potential regulatory relationship between amelogenin and BMP. Amelogenin bound to BMP2 as well as heparin/heparan sulfate. Thus, it was suggested that BMP2/4 carried over in EMD during processing promote binding activity and phosphorylate Smad1/5/8 in osteoblasts. On the other hand, amelogenin did not phosphorylate Smad1/5/8, but rather ERK1/2. Further, high-density amelogenin reduced the inhibition of alkaline phosphatase activity by noggin, though amelogenin did not have antagonistic properties against BMP. Together with the above findings, our findings suggest that the BMP2/4 contaminated during the purification process of EMD because of the avidity of amelogenin plays an important role in signaling pathway of calcification.     

Noggin蛋白对小鼠脑缺血再灌注损伤后学习和记忆能力与齿状回结构的影响

目的 探讨侧脑室注射Noggin蛋白对小鼠脑缺血再灌注损伤(CIRI)后学习、记忆能力及齿状回(DG)结构的影响,以期为临床缺血性脑血管疾病的预防与治疗提供新思路。 方法 240只小鼠随机分为:假手术组(n=80)、缺血再灌注损伤组(I/R组,n=80)、缺血再灌注损伤+Noggin治疗组(Noggin组,n=80),每组再分为1、3、7、14 d共 4个亚组;取材前1天采用Y型迷宫检测小鼠学习记忆能力(n=5);然后比色法检测缺血侧脑组织超氧化物歧化酶(SOD)与丙二醛(MDA)含量(n=5);2,3,5-氯化三苯基四氮唑(TTC)染色检测脑梗死面积(n=5); 尼氏染色与免疫荧光检测DG神经元与胶质原纤维酸性蛋白(GFAP)阳性细胞变化(n=5);Western blotting法检测骨形态发生蛋白4(BMP4)蛋白表达(n=5)。 结果 随着损伤时间的增加,I/R组、Noggin组与假手术组相比,小鼠神经功能评分升高(F组别=21.19, P<0.001; F时间=25.13, P<0.001),学习、记忆能力均下降［(F组别=216.10, P<0.001; F时间=260.10, P<0.001)、(F组别=114.40, P<0.001; F时间=184.60, P<0.001)］,脑梗死面积增加(F组别=2374, P<0.001; F时间=3 292, P<0.001),SOD活性降低(F组别=1 426, P<0.001; F时间=1 723, P<0.001),MDA含量升高(F组别=2.22, P<0.001; F时间=6.33, P<0.001),神经元数量减少(F组别=148.90, P<0.001; F时间=485.50, P<0.001),GFAP阳性细胞和BMP4蛋白表达量增加［(F组别=40.18, P<0.001; F时间=141.90, P<0.001)、(F组别=426.70, P<0.001; F时间=1 329, P<0.001)］。在各个相同时间点,与I/R组相比,Noggin各组小鼠神经功能评分降低(P<0.001),学习、记忆能力提高(P<0.001),脑梗死面积减小(P<0.001),SOD活性升高及MDA含量降低(P<0.001),神经元数量增加及染色变浅(P<0.001),GFAP阳性细胞和BMP4 蛋白表达量降低(P<0.001)。 结论 Noggin蛋白对小鼠脑缺血再灌注损伤后学习、记忆能力提高与DG组织损伤修复有改善作用,其作用机制可能与阻断BMP4蛋白表达和抑制胶质细胞的反应性增生有关。     

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目的探讨牙齿移动过程中骨形态发生蛋白（BMP）-1与Noggin在牙周组织中的表达及作用。方法2010年7月至2011年3月在大连医科大学附属第二医院分别建立以大鼠切牙及种植体为支抗的正畸牙齿移动模型,测量磨牙移动距离,用苏木精-伊红（HE）染色观察被移动磨牙牙周组织形态学变化,应用免疫组化方法检测BMP-1与Noggin在牙周组织改建过程中的表达情况。结果以种植体为支抗的正畸牙齿移动模型,其磨牙的移动距离和速度优于以切牙为支抗组。BMP-1与Noggin在牙周组织的分布及表达在空间和时间上具有明显的一致性：均表现为实验组表达明显强于对照组,张力侧表达强于压力侧;二者在牙周组织中的表达水平均在第7天组最强,到第28天组便基本接近对照组的水平。结论以种植体为支抗的正畸牙齿移动模型优于传统方法。BMP-1与Noggin均参与了牙周组织的改建过程,且可能在该过程中发挥重要的调节作用。     

Re-evaluating the induction of bone formation in primates

The molecular cloning of the osteogenic proteins of the transforming growth factor-β (TGF-β) supergene family and the results of numerous pre-clinical studies in several mammalian species including non-human primates, have prematurely convinced molecular biologists, tissue engineers and skeletal reconstructionists alike to believe that single recombinant human bone morphogenetic/osteogenic proteins (hBMPs/OPs) would result in tissue induction when translated in clinical contexts. This theoretical potential has not been translated to acceptable clinical results. Clinical trials in craniofacial and orthopedic applications such as mandibular reconstruction and sinus-lift operations have indicated that supra physiological doses of a single recombinant human protein are needed to induce unacceptable tissue regeneration whilst incurring significant costs without achieving equivalence to autogenous bone grafts. The acid test for clinically relevant bone tissue engineering should now become the concept of clinically significant osteoinduction, whereby the regenerated bone is readily identifiable on radiographic examination by virtue of its opacity and trabecular architecture. The need for alternatives to the hBMPs/OPs is now felt more acutely following reported complications and performance failure associated with the clinical use of hBMP-2 and hOP-1 (BMP-7). Because of the often substandard regeneration of clinical defects implanted with hBMPs/OPs, we now need to finally deal with the provocative question: are the hBMPs/OPs the only initiators of the induction of bone formation in pre-clinical and clinical contexts? The rapid induction of bone formation by the hTGF-β₃ isoform in heteropic intramuscular sites of the Chacma baboon Papio ursinus together with TGF-β₁, TGF-β₃, BMP-2, BMP-3, OP-1, RUNX-2 and Osteocalcin up-regulation and expression, hyper cellular osteoblastic activity, osteoid synthesis, angiogenesis and capillary sprouting are the molecular and morphological foundation for the induction of bone formation in clinical contexts. The induction of bone as initiated by hTGF-β₃ when implanted in the rectus abdominis muscle of P. ursinus is via the BMPs/OPs pathway with hTGF-β₃ controlling the induction of bone formation by regulating the expression of BMPs/OPs via Noggin expression, eliciting the induction of bone formation by up-regulating endogenous BMPs/OPs and it is blocked by hNoggin, providing insights into performance failure of hBMPs/OPs in clinical contexts. Physiological expression of BMPs/OPs genes upon implantation of hTGF-β₃ may escape the antagonist expression of Noggin and other inhibitors, whereas direct application of hBMPs/OPs, representing a later by-product step of the bone induction cascade as set by the TGF-β₃ master gene in primates, sets into motion Noggin' antagonist action, as shown by the limited effectiveness of hBMPs/OPs in clinical contexts. The unprecedented induction of bone formation by 250 μg hTGF-β₃ when combined with coral-derived macroporous constructs is the novel molecular and morphological frontier for the induction of bone formation in man. The induction of bone by hTGF-β₃ has been thus translated in clinical contexts to treat a large mandibular defect in a pediatric patient; 30 months after implantation of 250 μg hTGF-β₃ per gram of human demineralized bone matrix, radiographic analyses show the reconstruction of the avulsed large mandibular segment including the induction of the avulsed coronoid process.     

Sp1 promotes dental pulp stem cell osteoblastic differentiation through regulating noggin

Based on the high self-renewal ability and osteoblastic differentiation capacity, dental pulp stem cells (DPSCs) are suggested to be promising cell source for osteogenesis. Therefore, illustrating the mechanism of osteoblastic differentiation of DPSCs is required. This current study aims to illustrate the role and mechanism of Sp1 in regulating osteoblastic differentiation of DPSCs. In this study, we downregulated Sp1 in DPSCs and evaluated the osteoblastic differentiation by measuring Runx2 and OCN expression with Western blot analysis and by Alizarin red staining. Furthermore, we investigated the mechanism of Sp1 regulating noggin with Firefly luciferase reporter gene assay and ChIP assay, and correspondingly evaluated the function of noggin in Sp1-regulated osteoblastic differentiation of DPSCs. We found that knockdown of Sp1 inhibits the expression of ALP, Runx2, COL1A1 and OCN, and decreases ALP staining, Alizarin red staining. Sp1 binds to noggin promoter and inhibits noggin expression, thus correspondingly regulates DPSCs osteoblastic differentiation. In conclusion, our study revealed that Sp1 regulates DPSCs osteoblastic differentiation through noggin and that Sp1/noggin can provide new perspective for enhancing DPSCs osteogenesis.     

侧脑室注射Noggin对AD小鼠海马神经发生和学习记忆功能的影响

目的观察侧脑室给予Noggin对AD小鼠海马神经发生和学习记忆功能的影响。方法选取12月龄APP/PS1双转基因AD小鼠,实验组连续7d侧脑室注射Noggin,对照组向侧脑室注射等量生理盐水,Morris水迷宫检测其认知功能,免疫荧光检测海马齿回BrdU的表达。结果和生理盐水对照组相比,侧脑室注射Noggin组小鼠海马齿回BrdU阳性细胞数显著增加(P0.01),行为学检测显示逃逸潜伏期缩短、穿过原平台位置次数和在原平台象限探索时间百分率增加(P0.05)。结论侧脑室注射Noggin可促进AD小鼠海马神经发生并改善其学习记忆能力。     

BMP-4及其拮抗剂对PRL腺瘤细胞分泌、增殖和凋亡的影响

目的研究骨形态生成蛋白4（BMP-4）及其拮抗剂Noggin对PRL腺瘤细胞分泌、增殖和凋亡的影响。方法用不同浓度的BMP-4和Noggin对原代培养的PRL腺瘤细胞进行干预,观察不同时间的细胞形态,测量PRL的浓度。以免疫细胞化学、Western blot及RT-PCR检测BMP-4蛋白和BMP-4mRNA在PRL腺瘤细胞的表达,甲基噻唑基四唑（MTT）法计算Noggin对PRL腺瘤细胞生长的半最大抑制浓度（IC50）值,透射电镜,流式细胞仪观察检测Noggin处理后的细胞形态和周期改变,计算凋亡率。结果 5ng/ml的BMP-4培养基可促进PRL腺瘤细胞分泌,最大效应浓度为20ng/m·l MTT法测定IC50值为18μg/m·l 加入Noggin后;免疫细胞化学,Western blot显示,与对照组相比,作用后24hPRL腺瘤细胞表达BMP-4无明显差异,但作用48h和72h的表达显著降低（P〈0.05）,RT-PCR示Noggin作用24h,48h和72h后BMP-4mRNA基因扩增条带亮度逐渐减弱,电镜观察显示。Noggin作用24h、48h后PRL腺瘤细胞形态无明显改变,72h时可见典型的凋亡细胞。结论 BMP-4在一定浓度范围内呈剂量依赖性地促进PRL腺瘤细胞生长,增殖与分泌,其拮抗剂Noggin呈时间依赖性地诱导PRL腺瘤细胞的凋亡     

骨形成蛋白-2对室管膜前下区神经干细胞DLX5表达的影响

目的 探讨骨形成蛋白-2（bone morphogenetic protein-2,BMP-2）对室管膜前下区（anterior subventricular zone,SVZa）神经干细胞DLX5表达的影响。方法体外培养SVZa神经干细胞,用BMP-2及其拮抗剂Noggin诱导SVZa神经干细胞,分别用免疫荧光染色和逆转录一聚合酶链反应（RT-PCR）检测DLX5表达变化。结果BMP-2组SVZa神经干细胞DLX5蛋白表达和DLX5mRNA表达水平明显高于对照组（P〈0．05）,且该效应能被其拮抗剂Noggin特异性地抑制。结论BMP-2是DLX5上游调节基因,可促进SVZa神经干细胞DLX5的表达。