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）：扭曲的胃结构（TWSG1）是骨形态发生蛋白（BMP）信号传导的关键细胞外调节剂。 BMP是骨形成的有效诱导剂，任何BMP信号的失调都可能导致骨重塑的缺陷。为了检查TWSG1是否调节骨骼重塑，我们产生了TWSG1-NULL小鼠，该小鼠与野生型（WT）小鼠相比显示出深层骨质减少症，其组织态度学参数显着降低。与WT小鼠相比，Tetracycline标记研究显示，TWSG1-NULL小鼠的矿物质作用率没有显着降低，这表明TWSG1 - / - 小鼠的骨质减少症不是由于成骨细胞功能的降低。另一方面，与WT小鼠相比，TWSG1 - / - 小鼠的骨吸收增加，其特征是破骨细胞的数量较大和增加，吸收凹坑面积增加，血清CTX和陷阱水平增加。体外破骨细胞生成的增强与细胞融合的增加以及涉及破骨细胞分化（NFATC1）和细胞融合（DC-Stamp）的关键基因的上调有关。我们还显示了TWSG1 - / - 破骨细胞中磷酸化的SMAD1/5/8的较高水平，并且可以通过增加剂量的Noggin（BMP-特异性拮抗剂）来逆转体外破骨细胞的增强。此外，我们表明，外源性BMP2在WT破骨细胞中增加了PSMAD1/5/8的水平，并且增强了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TWSG1通过调节BMP信号传导抑制破骨细胞的形成和功能。因此，在特定的目标1中，我们将确定是否使用（1a）相互培养实验的成骨细胞/ - / - 与成骨细胞和twsg1-和Twsg1-/chim-chim和（1B）或（1B）或（1B）或（1B）或1b more（1B）的前体的（1A）相互培养实验的破坏是TWSG1 - / - 的直接靶标的是TWSG1 - / - 的直接目标。 TWSG1 - / - 骨髓将被移植到致命的受辐照的TWSG1 - / - 或WT小鼠中。 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破骨细胞生成，（2B）确定TWSG1作为骨质质发生的抑制剂的功能。在特定目标3中，我们将通过（3A）通过（3A）概括TWSG1 - / - 小鼠的骨质骨化术的骨骨术中的骨术中，并确定骨骼的占领群，并确定骨骼的近代群体，并确定骨骼或细胞的分子和细胞机制，介导了TWSG1 - / - 小鼠中造成骨质核壳发生的增加M-CSF，（3B）确定TWSG1的破坏是否导致等级介导的信号通路发生变化； （3C）评估NFAT-C1是否是TWSG1缺乏效率的目标。这些目标的完成将使我们能够明确确定TWSG1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理解TWSG1抑制破骨细胞生成的机制，而且还提供了开发新型抗吸毒药物的第一步，这些药物可用于治疗骨质疏松症和骨化骨肿瘤。 公共卫生相关性：扭曲的胃结构（TWSG1）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。为了理解其重要性，我们通过删除其BMP结合域的一部分来开发TWSG1-NULL小鼠。初步数据表明，TWSG1 - / - 小鼠由于骨质质发生增强而显示出严重的骨质减少症，从而导致骨吸收增加。该项目的总体目的是识别TWSG1干扰的细胞靶标，并表征TWSG1抑制破骨细胞生成的信号传导和分子机制。