Role of Zfp521 in bone formation and anabolic responses

描述(申请人提供)：保持骨量对于预防老年人骨质疏松和骨折至关重要。除了抗吸收治疗外，唯一被批准的合成代谢治疗是间歇性甲状旁腺素，其作用机制仍不完全清楚。我们已经确定Zfp521是一种含有30个锌指的蛋白质，在促进骨形成的转录过程中发挥着重要的作用。Zfp521在培养的成骨细胞中的表达随时间的延长而增加，并受BMP-2和PTH/PTHrP的调节。在体内，转基因小鼠成熟OBS中靶向过表达Zfp521导致骨形成增加(2)。相比之下，以成骨细胞为靶点的Zfp521-/-小鼠骨量减少，成骨细胞成熟受损，骨形成减少。此外，Zfp521结合Runx2和EBF1并抑制它们的转录活性以及CREB对RANKL启动子的转录活性，从而抑制OB诱导的骨吸收。因此，我们的初步数据表明，Zfp521在小鼠体内1)与几个关键的OB转录因子相互作用，2)对几个关键的骨形成调节因子做出反应，3)在体内过表达或缺失时影响骨形成和骨量。这使Zfp521成为调节骨形成和动态平衡的一个新的和重要的贡献者，值得进一步研究。因此，我们建议进一步探讨Zfp521影响OB分化和骨稳态的机制，重点是它与Runx2、EBF1和CREB的相互作用。由于我们已经生成了重要的初步数据，并且已经生成了本次研究所需的大多数遗传模型(OG2-Zfp521；2.3kb-Col1a1-Zfp521；全局Zfp521-/-和Floating Zfp521，以及OC-Cre-Zfp521和OSX-Cre-Zfp521系，EBF1-/-和Floted EBF1，2.3kb-Col1a1-EBF1)，因此本研究建议的具体目的是：进一步分析Zfp521的完全和OB靶向性、有条件的、阶段特异性的缺失对体内和体外OB分化和骨形成的影响；目的：进一步研究Zfp521作用于OBS转录因子、影响骨形成的分子机制，特别是它与Runx2、EBF1和CREB在靶基因上的相互作用。目的：通过全基因组筛选Zfp521新的伴侣、结合部位和靶基因，对这种新发现的骨形成辅助调节因子进行功能鉴定，将促进我们对调节骨稳态的关键生理过程的理解，最终探索治疗骨质疏松症和其他低骨量综合征的药物发现的新途径。

DESCRIPTION (provided by applicant): The maintenance of bone mass is essential for the prevention of osteoporosis and fractures in the elderly. Besides anti-resorptive therapies, the only approved anabolic therapy is intermittent PTH, the mechanism of action of which is still incompletely understood. We have identified Zfp521, a 30 zinc-finger containing protein, as a prominent player in the transcriptional processes that contribute to bone formation (2). Zfp521 expression in cultured osteoblasts (OBs) increases with time, and is regulated by BMP-2 and PTH/PTHrP. In vivo, targeted over-expression of Zfp521 in mature OBs in transgenic mice resulted in an increase in bone formation (2). In contrast, osteoblast-targeted Zfp521-/- mice are osteopenic with impaired maturation of OBs, and decreased bone formation. Furthermore Zfp521 binds Runx2 and Ebf1 and repress their transcriptional activity as well as that of CREB on the Rankl promoter, repressing OB-induced bone resorption. Thus, our preliminary data shows that Zfp521 1) interacts with several key OB transcription factors, 2) responds to several key regulators of bone formation and 3) affects bone formation and bone mass when over-expressed or deleted in vivo in mice. This establishes Zfp521 as a novel and important contributor to the regulation of bone formation and homeostasis, and one that deserves further exploration. Accordingly, we propose to further explore the mechanism by which Zfp521 affects OB differentiation and bone homeostasis, focusing on its interplay with Runx2, Ebf1 and CREB. Since we have generated significant preliminary data and have already generated most of the genetic models required for this investigation (OG2-Zfp521; 2.3kb-Col1a1-Zfp521; both global Zfp521-/- and floxed Zfp521, as well as the OC- Cre-Zfp521 and Osx-Cre-Zfp521 lines, Ebf1 -/- and floxed Ebf1, 2.3kb-Col1a1-Ebf1), the specific aims of this research proposal are: Aim1: Further analyze the effects of full and OB-targeted conditional, stage-specific deletion of Zfp521 on OB differentiation and bone formation in vivo and in vitro; Aim2: Further characterize the molecular mechanisms by which Zfp521 exerts its effects on transcription factors in OBs, affecting bone formation, with particular emphasis on its interplay with Runx2 , Ebf1 and CREB on target genes. Aim3: Identify Zfp521 new partners, binding sites and target genes by genome-wide screening Functional characterization of this newly discovered co-regulator of bone formation will advance our understanding of key physiological processes that regulate bone homeostasis, ultimately allowing the exploration of novel pathways for drug discovery in osteoporosis and other low bone mass syndromes.