Role of Twisted Gastrulation in Osteoclastogenesis

描述（由申请人提供）：扭曲原肠胚（Twsg1）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，任何BMP信号的失调都可能导致骨重塑的缺陷。为了研究Twsg1是否调节骨重塑，我们制造了一只Twsg1缺失的小鼠，与野生型（WT）小鼠相比，它表现出严重的骨质减少，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，Twsg1-/-缺失小鼠的矿物质附着率没有显著降低，这表明Twsg1-/-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，Twsg1-/-小鼠骨吸收增加，其特征是破骨细胞数量增加，吸收窝面积增加，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和参与破骨细胞分化（NFATc1）和细胞-细胞融合（DC-STAMP）的关键基因的上调有关。我们还发现，在Twsg1-/-破骨细胞中磷酸化Smad1/5/8的水平较高，并且通过增加Noggin（一种BMP特异性拮抗剂）的剂量，体外增强的破骨细胞生成可以逆转。此外，我们发现外源性BMP2增加了WT破骨细胞中pSmad1/5/8的水平，也增强了RANKL刺激的破骨细胞分化。这些结果为我们的假设提供了强有力的证据，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，在特定目标1中，我们将通过（1A） WT和Twsg1-/-小鼠的成骨细胞和破骨细胞前体的互惠共培养实验，以及（1B）嵌合小鼠，将WT或Twsg1-/-骨髓移植到致命辐照的Twsg1-/-或WT小鼠中，来确定破骨细胞前体而不是基质细胞/成骨细胞是否是Twsg1-/-破坏的直接靶标。在具体目标2中，我们将评估Twsg1-/-小鼠破骨细胞生成的增强是否通过BMP信号的增加来介导，并通过（2A）确定BMP信号的增加是否介导Twsg1-/-小鼠的破骨细胞表型，并确定BMP是否可以调节RANKL刺激的破骨细胞生成，以及（2B）确定Twsg1作为破骨细胞生成抑制剂的功能来阐述Twsg1和BMP在破骨细胞生成中的功能。在具体目标3中，我们将阐明介导Twsg1-/-小鼠破骨细胞生成增加的分子和细胞机制，通过（3A）利用更好定义的骨髓破骨细胞祖群概括Twsg1-/-小鼠的破骨细胞表型，并确定破骨细胞前体是否已经被RANKL和/或M-CSF引物，（3B）确定Twsg1破坏是否导致rank介导的信号通路改变；（3C）评估NFAT-c1是否是twsg1缺乏的目标。完成这些目标将使我们能够明确地确定Twsg1在破骨细胞发生和骨吸收中的作用。我们的工作不仅提供了对Twsg1抑制破骨细胞发生机制的理解，而且还为开发可用于治疗骨质疏松症和溶骨性骨肿瘤的新型抗骨吸收药物迈出了第一步。

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.