Role of Zfp521 in bone formation and anabolic responses

描述（由申请人提供）：骨量的维持对于预防老年人骨质疏松和骨折至关重要。除抗吸收治疗外，唯一被批准的合成代谢治疗是间歇性甲状旁腺激素，其作用机制尚不完全清楚。我们已经确定了Zfp521，一种含有锌指的蛋白，在促进骨形成的转录过程中起着重要作用(2)。Zfp521在体外培养成骨细胞（OBs）中的表达随时间增加而增加，并受BMP-2和PTH/PTHrP的调控。在体内，转基因小鼠成熟ob中Zfp521的靶向过表达导致骨形成增加(2)。相反，以成骨细胞为靶点的Zfp521-/-小鼠骨质减少，ob成熟受损，骨形成减少。此外，Zfp521结合Runx2和Ebf1，抑制它们的转录活性以及CREB在Rankl启动子上的转录活性，抑制ob诱导的骨吸收。因此，我们的初步数据表明，Zfp521 1)与几个关键的OB转录因子相互作用，2)响应骨形成的几个关键调节因子，3)在小鼠体内过度表达或缺失时影响骨形成和骨量。这表明Zfp521是骨形成和体内平衡调控的一个新的重要贡献者，值得进一步探索。因此，我们建议进一步探索Zfp521影响OB分化和骨稳态的机制，重点研究其与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，并且已经生成了本研究所需的大多数遗传模型(OG2-Zfp521; 2.3kb-Col1a1-Zfp521；无论是全球Zfp521-/-和floxed Zfp521，以及OC- Cre-Zfp521和Osx-Cre-Zfp521系，Ebf1 -/-和floxed Ebf1, 2.3kb-Col1a1-Ebf1)，本研究方案的具体目的是：ai1：进一步分析Zfp521完全缺失和OB靶向的条件性、阶段特异性缺失对OB分化和骨形成的体内和体外影响；目的2：进一步表征Zfp521对OBs中转录因子影响骨形成的分子机制，重点研究其与靶基因Runx2、Ebf1和CREB的相互作用。目的3：通过全基因组筛选确定Zfp521新的合作伙伴、结合位点和靶基因。对这一新发现的骨形成共同调节因子的功能表征将促进我们对调节骨稳态的关键生理过程的理解，最终为骨质疏松症和其他低骨量综合征的药物发现开辟新的途径。

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. PUBLIC HEALTH RELEVANCE: The maintenance of bone mass is essential for the prevention of osteoporosis and fractures in the elderly and aside from anti-resorptive therapies, the only approved anabolic therapy is Parathyroid Hormone (PTH), the mechanism of action of which is still incompletely understood. We have identified Zfp521, a protein rich in Zinc Fingers, as a prominent player in bone formation and bone homeostasis and propose to explore the mechanism by which Zfp521 affects bone density, studying cells and genetically engineered mice. Functional characterization of this new regulator of bone formation may allow the discovery of novel pathways for anabolic drug discovery in osteoporosis and other low bone mass syndromes.