Role of Twisted Gastrulation in Osteoclastogenesis

扭曲原肠胚形成在破骨细胞生成中的作用

• 批准号: 7888320
• 负责人: RAJARAM GOPALAKRISHNAN
• 金额: $ 33.49万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-10 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7888320
• 关键词: Animal Model; Area; BMP2 gene; Binding Proteins; Biology; Bone Marrow; Bone Morphogenetic Proteins; Bone Resorption; Bone neoplasms; Bone remodeling; Cell fusion; Cells; Clinic; Coculture Techniques; Collaborations; Complement component C1s; Connecticut; Cyclophosphamide; Data; Defect; Development; Disease; Dose; Drosophila genus; Environment; Genes; Goals; Hand; In Vitro; Knockout Mice; Knowledge; Label; Lead; Macrophage Colony-Stimulating Factor; Marrow; Mediating; Minerals; Minnesota; Modeling; Molecular; Mus; Osteoblasts; Osteoclasts; Osteogenesis; Osteolytic; Osteopenia; Osteoporosis; Pathogenesis; Pharmaceutical Preparations; Phenotype; Physiological; Population; Protein Binding; Protein Binding Domain; Proteins; Protocols documentation; Regulation; Reporting; Research; Role; School Dentistry; Serum; Signal Pathway; Signal Transduction; Skeletal Development; Staging; Strategic Planning; Stromal Cells; TNFSF11 gene; Testing; Tetracyclines; Transplantation; Treatment Efficacy; Universities; Up-Regulation; Wild Type Mouse; Work; Xenopus; Zebrafish; bone; bone cell; cellular targeting; expectation; extracellular; gastrulation; genetic regulatory protein; inhibitor/antagonist; innovation; medical schools; novel; osteoclastogenesis; overexpression; professor; progenitor; protein function; public health relevance; regenerative; research study

DESCRIPTION (provided by applicant): Twisted gastrulation (Twsg1) is a key extracellular regulator of bone morphogenetic protein (BMP) signaling. BMPs are potent inducers of bone formation and any dysregulation of BMP signaling can lead to defects in bone remodeling. To examine whether Twsg1 regulates bone remodeling, we generated a Twsg1-null mouse that showed profound osteopenia with significantly reduced histomorphometric parameters compared to wild type (WT) mice. Tetracycline labeling studies showed no significant decrease in mineral apposition rate in Twsg1-null mice compared to WT mice, indicating that osteopenia in Twsg1-/- mice are not due to reduced osteoblast function. On the other hand, Twsg1-/- mice showed increased bone resorption compared to WT mice characterized by larger and increased numbers of osteoclasts, increase in the area of resorption pits and increased serum CTX and TRAP levels. Enhanced osteoclastogenesis in vitro was associated with an increase in cell fusion and upregulation of key genes involved in osteoclast differentiation (NFATc1) and cell- cell fusion (DC-STAMP). We also show higher levels of phosphorylated Smad1/5/8 in Twsg1-/- osteoclasts and that the enhanced in vitro osteoclastogenesis can be reversed by increasing doses of Noggin, a BMP- specific antagonist. Further, we show that exogenous BMP2 increases pSmad1/5/8 levels in WT osteoclasts and also enhances RANKL stimulated osteoclast differentiation. These results provide compelling evidence for our hypothesis that Twsg1 inhibits osteoclast formation and function through regulation of BMP signaling. Thus in specific aim 1, we will determine if osteoclast precursors rather than stromal cell/osteoblasts are direct targets of Twsg1-/- disruption using (1A) reciprocal co-culture experiments with osteoblasts and osteoclast precursors from both WT and Twsg1-/- mice, and (1B) chimeric mice in which either WT or Twsg1-/- marrow will be transplanted into lethally irradiated Twsg1-/- or WT mice. In specific aim 2, we will evaluate whether enhanced osteoclastogenesis in Twsg1-/- mice is mediated through increased BMP signaling and elaborate the function of Twsg1 and BMP in osteoclastogenesis by (2A) determining if increased BMP signaling mediates the osteoclast phenotype in Twsg1-/- mice, and determine if BMPs can regulate RANKL stimulated osteoclastogenesis, and (2B) determining the function of Twsg1 as an inhibitor of osteoclastogenesis. In specific aim 3, we will elucidate molecular and cellular mechanisms mediating increased osteoclastogenesis in Twsg1-/- mice by (3A) recapitulating the osteoclast phenotype of Twsg1-/- mice using better defined osteoclast progenitor population from the bone marrow, and determining if osteoclast precursors are already primed to RANKL and/or M-CSF, (3B) determining whether Twsg1 disruption leads to altered RANK-mediated signaling pathways; and (3C) evaluating if NFAT-c1 is a target of Twsg1-deficiency . Completion of these aims will enable us to unequivocally determine the roles of Twsg1 in osteoclastogenesis and bone resorption. The impact of our work may not only provide an understanding of the mechanisms by which Twsg1 inhibit osteoclastogenesis but also first steps towards development of novel antiresorptive drugs that can be used in the treatment of osteoporosis and osteolytic bone tumors. PUBLIC HEALTH RELEVANCE: Twisted gastrulation (Twsg1) is a bone morphogenetic protein-binding protein whose function in skeletal development and remodeling is not known. Towards understanding its significance, we developed Twsg1-null mice by deleting part of its BMP-binding domain. Preliminary data show that Twsg1-/- mice show severe osteopenia due to enhanced osteoclastogenesis leading to increased bone resorption. The overall objective of this project is to identify the cellular targets of Twsg1-disruption and characterize the signaling and molecular mechanisms by which Twsg1 inhibits osteoclastogenesis.

描述（由申请人提供）：扭曲原肠胚形成（Twsg 1）是骨形态发生蛋白（BMP）信号传导的关键细胞外调节因子。BMP是骨形成的有效诱导剂，BMP信号的任何失调都可能导致骨重建的缺陷。为了检查Twsg 1是否调节骨重建，我们产生了一个Twsg 1-null小鼠，显示出严重的骨量减少，与野生型（WT）小鼠相比，组织形态学参数显著降低。四环素标记研究显示，与WT小鼠相比，Twsg 1-null小鼠的矿物质沉积率没有显著降低，表明Twsg 1-/-小鼠的骨质减少不是由于成骨细胞功能降低。另一方面，与WT小鼠相比，Twsg 1-/-小鼠表现出骨吸收增加，其特征在于破骨细胞数量增加，吸收陷窝面积增加，血清CTX和TRAP水平升高。体外破骨细胞生成的增强与细胞融合的增加以及参与破骨细胞分化（NFATc 1）和细胞-细胞融合（DC-STAMP）的关键基因的上调有关。我们还显示了在Twsg 1-/-破骨细胞中更高水平的磷酸化Smad 1/5/8，并且增强的体外破骨细胞生成可以通过增加剂量的Noggin（一种BMP特异性拮抗剂）来逆转。此外，我们发现外源性BMP 2增加WT破骨细胞中的pSmad 1/5/8水平，并且还增强RANKL刺激的破骨细胞分化。这些结果为我们的假设提供了令人信服的证据，即Twsg 1通过调节BMP信号抑制破骨细胞的形成和功能。因此，在具体目标1中，我们将使用（1A）WT和Twsg 1-/-小鼠的成骨细胞和破骨细胞前体的相互共培养实验，以及（1B）嵌合小鼠（其中WT或Twsg 1-/-骨髓将被移植到致死辐射的Twsg 1-/-或WT小鼠中），确定破骨细胞前体而不是基质细胞/成骨细胞是否是Twsg 1-/-破坏的直接靶点。在具体目标2中，我们将评估Twsg 1-/-小鼠中破骨细胞生成的增强是否通过增加的BMP信号传导介导，并通过（2A）确定增加的BMP信号传导是否介导Twsg 1-/-小鼠中的破骨细胞表型，并确定BMP是否可以调节RANKL刺激的破骨细胞生成，和（2B）确定Twsg 1作为破骨细胞生成抑制剂的功能。在具体目标3中，我们将阐明介导Twsg 1-/-小鼠中破骨细胞生成增加的分子和细胞机制，通过（3A）使用来自骨髓的更好定义的破骨细胞祖细胞群重现Twsg 1-/-小鼠的破骨细胞表型，并确定破骨细胞前体是否已经引发RANKL和/或M-CSF，（3B）确定Twsg 1破坏是否导致RANK介导的信号传导途径改变;和（3C）评估NFAT-c1是否是Twsg 1缺陷的靶标。这些目标的完成将使我们能够明确确定Twsg 1在破骨细胞生成和骨吸收中的作用。我们工作的影响不仅可以提供Twsg 1抑制破骨细胞生成的机制的理解，而且也是开发可用于治疗骨质疏松症和溶骨性骨肿瘤的新型抗吸收药物的第一步。 公共卫生相关性：扭曲原肠胚形成（Twsg 1）是一种骨形态发生蛋白结合蛋白，其在骨骼发育和重塑中的功能尚不清楚。为了理解其意义，我们开发了Twsg 1-null小鼠通过删除其BMP结合域的一部分。初步数据显示，Twsg 1-/-小鼠表现出严重的骨质减少，这是由于破骨细胞生成增强导致骨吸收增加。该项目的总体目标是确定Twsg 1破坏的细胞靶点，并表征Twsg 1抑制破骨细胞生成的信号传导和分子机制。