Linkage exclusion and mutational analysis of the noggin gene in patients with fibrodysplasia ossificans progressiva (FOP)

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and disabling genetic disorder characterized by congenital malformation of the great toes and by progressive heterotopic endochondral ossification in predictable anatomical patterns. Although elevated levels of bone morphogenetic protein 4 (BMP4) occur in lymphoblastoid cells and in lesional cells of patients with FOP, mutations have not been identified in the BMP4 gene, suggesting that the mutation in FOP may reside in a BMP4-interacting factor or in another component of the BMP4 pathway. A powerful antagonist of BMP4 is the secreted polypeptide noggin. A recent case report described a heterozygous 42-bp deletion in the protein-coding region of the noggin gene in a patient with FOP. In order to determine if noggin mutations are a widespread finding in FOP, we examined 31 families with 1 or more FOP patients. Linkage analysis with an array of highly polymorphic microsatellite markers closely linked to the noggin gene was performed in four classically-affected multigenerational FOP families and excluded linkage of the noggin locus to FOP (the multipoint lod score was -2 or less throughout the entire range of markers). We sequenced the noggin gene in affected members of all four families, as well as in 18 patients with sporadic FOP, and failed to detect any mutations. Single-strand conformation polymorphism (SSCP) analysis of 4 of these patients plus an additional 9 patients also failed to reveal any mutations. Among the samples analyzed by SSCP and DNA sequencing was an independently obtained DNA sample from the identical FOP patient previously described with the 42-bp noggin deletion; no mutation was detected. Examination of the DNA sequences of 20 cloned noggin PCR products, undertaken to evaluate the possibility of a somatic mutation in the noggin gene which could be carried by a small subset of white blood cells, also failed to detect the presence of the reported 42-bp deletion. We conclude that mutations in the coding region of noggin are not associated with FOP.     

noggin基因修饰神经干细胞移植对衰老小鼠学习记忆能力的改善

本研究旨在探讨重组腺病毒介导的noggin基因修饰的小鼠神经干细胞(NSCs)移植入D-半乳糖致衰小鼠海马后的迁移及对小鼠学习记忆能力的影响。分别以含noggin基因的重组腺病毒(pAdEasy-1-noggin-GFP)及对照病毒(pAdEasy-1-GFP)转染体外培养的NSCs,获取神经干细胞克隆(NSCs-noggin;NSCs-GFP),移植入由D-半乳糖诱导的衰老模型小鼠右侧海马CA3区,术后4周采用Y-电迷宫检测小鼠的学习记忆行为,并对各组小鼠脑组织海马段行石蜡冠状切片、免疫组织化学和BrdU免疫荧光化学方法检测。结果显示:基因修饰的NSCs移植对小鼠的学习记忆行为的改善比pAdEasy-1-GFP组更为明显,其记忆保持率持续时间更长;NSCs移植后能在宿主海马内迁移,基因修饰的NSCs移植组在海马内的BrdU阳性细胞数较多(P<0.05)。以上结果表明noggin基因不但可能参与衰老小鼠的学习记忆行为,而且能够促进NSCs的迁移。     

腺病毒介导noggin基因修饰神经干细胞

目的:以重组腺病毒介导的noggin基因修饰小鼠海马源性神经干细胞,为基因修饰的神经干细胞移植治疗中枢神经系统疾病提供依据。方法:以重组腺病毒介导的noggin基因转染体外培养的神经干细胞,MTT法检测转染前、后神经干细胞的生长活性,应用免疫细胞化学及Western blot检测转染后Noggin蛋白在神经干细胞中的表达及noggin基因对神经干细胞定向分化的影响。结果:重组腺病毒pAdEasy-1-noggin转染神经干细胞后,神经干细胞中Noggin蛋白表达持续增加;转染后的NSCs在含血清的培养液中能定向分化为神经元、星形胶质细胞和少突胶质细胞,并且其分化为神经元的数量明显增加。结论:重组腺病毒介导的noggin基因在细胞中可持续稳定表达,noggin基因能够促进神经干细胞定向分化为神经元,抑制其向星形胶质细胞方向分化,为下一步进行神经干细胞体内移植治疗提供了实验依据。     

携带小鼠noggin基因的重组腺病毒载体对人骨髓间充质干细胞的神经样诱导作用

目的观察noggin基因能否单独将人骨髓间充质干细胞诱导为神经细胞以成为挽救神经退行性病变的"种子细胞"。方法原代培养人骨髓间充质干细胞,分为对照组、空白载体转染的Ad组和含有noggin基因的腺病毒载体转染的Ad-noggin组,观察转染后48h、4、7、10d等时相点各组细胞的形态学变化并行统计学分析。结果2种载体皆可成功转染人骨髓间充质干细胞使其自发绿色荧光,但Ad组形态无显著变化,而Ad-noggin组细胞在转染后48h即可见少量细胞分化为神经样细胞,转染后4d可见神经样细胞数目增多且伸出少量神经细胞样突起,10d时分化的细胞明显增多。与转染后7d相比,Ad-noggin组转染10d时胞体直经和树突直径明显增加(P0.05),但形态无改变。结论单独以含noggin基因的腺病毒载体转染人骨髓间充质干细胞可以使其向神经样细胞分化。     

Interplay between electrical activity and bone morphogenetic protein signaling regulates spinal neuron differentiation

A gradient of bone morphogenetic proteins (BMPs) along the dorsoventral axis of the spinal cord is necessary for the specification of dorsal neurons. Concurrently, a gradient of calcium-mediated electrical activity is present in the developing spinal cord but in an opposing ventrodorsal direction. Whether BMPs and electrical activity interact in embryonic spinal neurons remains unknown. We show that BMP decreases electrical activity by enhancing p38 MAPK-mediated negative modulation of voltage-gated sodium channels. In turn, electrical activity affects the phosphorylation status and nuclear level of activated Smads, the canonical components of BMP signaling. This interaction between calcium spike activity and BMP signaling regulates the specification of the dorsal commissural spinal neuron phenotype. The present study identifies an unexpected interplay between BMPs and electrical activity that is critical for decoding the morphogen gradient during spinal neuron differentiation.     

Bone morphogenetic protein regulation of enteric neuronal phenotypic diversity: relationship to timing of cell cycle exit

The effects of bone morphogenetic protein (BMP) signaling on enteric neuron development were examined in transgenic mice overexpressing either the BMP inhibitor, noggin, or BMP4 under control of the neuron specific enolase (NSE) promoter. Noggin antagonism of BMP signaling increased total numbers of enteric neurons and those of subpopulations derived from precursors that exit the cell cycle early in neurogenesis (serotonin, calretinin, calbindin). In contrast, noggin overexpression decreased numbers of neurons derived from precursors that exit the cell cycle late (gamma-aminobutyric acid, tyrosine hydroxylase [TH], dopamine transporter, calcitonin gene-related peptide, TrkC). The numbers of TH- and TrkC-expressing neurons were increased by overexpression of BMP4. These observations are consistent with the idea that phenotypic expression in the enteric nervous system (ENS) is determined, in part, by the number of proliferative divisions neuronal precursors undergo before their terminal mitosis. BMP signaling may thus regulate enteric neuronal phenotypic diversity by promoting the exit of precursors from the cell cycle. BMP2 increased the numbers of TH- and TrkC-expressing neurons developing in vitro from immunoselected enteric crest-derived precursors; BMP signaling may thus also specify or promote the development of dopaminergic TrkC/NT-3-dependent neurons. The developmental defects in the ENS of noggin-overexpressing mice caused a relatively mild disturbance of motility (irregular rapid transit and increased stool frequency, weight, and water content). Although the function of the gut thus displays a remarkable tolerance for ENS defects, subtle functional abnormalities in motility or secretion may arise when ENS defects short of aganglionosis occur during development.     

Bone morphogenetic proteins in melanoma: Angel or devil?

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily serving multiple functions in many cell and tissue types including proliferation, apoptosis, differentiation, chemotaxis, angiogenesis, and matrix production during embryogenic development as well as in adult life. Despite the tremendous progress in delineating functional derangements of BMP pathways in carcinogenesis during the last decade, the biological significance of BMPs in human melanoma has received very little attention. It is now clear that biological responses to BMPs are cell type-specific and divergent effects, i.e., both oncogenic and tumor suppressor activities, have been described. Thus, knowledge generated in one system may not translate directly to another. In this review, we summarize the current understanding of BMP signaling in various human cancers and discuss original data pertaining to cutaneous melanoma obtained in our laboratory.     

Mutations of the NOG gene in individuals with proximal symphalangism and multiple synostosis syndrome

Proximal symphalangism is an autosomal-dominant disorder with ankylosis of the proximal interphalangeal joints, carpal and tarsal bone fusion, and conductive deafness. These symptoms are shared by another disorder of joint morphogenesis, multiple synostoses syndrome. Recently, it was reported that both disorders were caused by heterozygous mutations of the human noggin gene (NOG). To date, seven mutations of NOG have been identified from unrelated families affected with joint morphogenesis. To characterize the molecular lesions of proximal symphalangism, we performed analyses of NOG in three Japanese individuals with proximal symphalangism. We found three novel mutations: g.551G>A (C184Y) in a sporadic case of symphalangism, g.386T>A (L129X) in a familial case of symphalangism, and a g.58delC (frameshift) in a family with multiple synostosis syndrome. Characteristic genotype-phenotype correlations have not been recognized from the mutations in the NOG gene.     

Noggin blocks invasive growth of murine B16-F1 melanoma cells in the optic cup of the chick embryo

Melanoma cells originate from the neural crest and are characterized by high migratory potential and invasive growth. After transplantation into the neural tube of the chick embryo, melanoma cells spontaneously emigrate along the neural crest pathways without tumor formation or malignant growth. This emigration depends on the constitutive over-expression of bone morphogenetic protein-2 (BMP-2) and can be ablated by the BMP-antagonist noggin. When transplanted into the embryonic optic cup, melanoma cells invade the host tissue and form malignant tumors. Here, we asked if the invasive growth of melanoma cells in the optic cup could be influenced by BMP-2 or noggin. Mouse B16-F1 cells were grown as aggregates, treated with BMP-2 or noggin during aggregation and transplanted into the optic cup of 3-day chick embryos. After 3 days of subsequent incubation, embryos were evaluated for melanoma cell invasiveness. Immunohistochemical examination revealed that untreated and BMP-2-treated melanoma cells had grown malignantly into the host tissue. However, noggin pretreatment of the aggregates had blocked melanoma cell invasiveness and tumor formation. We conclude that invasive growth of melanoma cells in vivo is BMP-dependent and can be ablated by noggin, thus rendering noggin a promising agent for the treatment of BMP-over-expressing melanoma.